Lab Works From Biology

Palacký University Olomouc
Department of Biology, Faculty of Medicine and Dentistry
Lab works from Biology

Second semester
Olomouc 2014
© Authors team, Department of Biology, Faculty of Medicine and Dentistry,
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Content
1. Chromosomal Abnormalities in Human ................................................................................ 7
1.1 Fill-in the figure of normal meiosis ................................................................................. 10
1.2 Fill-in the following figure of meiotic non-disjunction ................................................... 11
1.3 Cleavage division of the zygote and blastomeres ............................................................ 13
1.4 Mitotic non-disjunction .................................................................................................... 13
1.5 Complete the figure of mitotic non-disjunction of chromosomes ................................... 14
1.6 Complete the figure of normal meiosis and fertilization ................................................. 15
1.7 Robertsonian translocation ............................................................................................... 16
1.8 Robertsonian translocation of chromosomes 14 and 21 .................................................. 17
2. Mutations in human diseases ................................................................................................ 18
2.1 Gene mutations ................................................................................................................ 18
2.2 Numerical and structural chromosomal aberrations ........................................................ 19
2.3 Pathological karyotypes in patients with inborn developmental disabilities ................... 21
2.4 Pathological karyotype in cancer ..................................................................................... 23
2.5 Preparation of metaphase chromosome to assess the cell ploidy..................................... 23
3. Mendelian inheritance ........................................................................................................... 25
3.1 Inheritance of the eye colour ............................................................................................ 27
3.2 Single-gene disorder inheritance ...................................................................................... 27
3.3 Cherubinism ..................................................................................................................... 27
3.4 Phenylketonuria ............................................................................................................... 27
3.5 Sickle-cell disease ............................................................................................................ 28
3.6 Beta thalassemia ............................................................................................................... 28
3.7 The inheritance of eye colour and left-handness ............................................................. 28
3.8 Inheritance of the ABO blood group in humans .............................................................. 28
3.9 Rhesus-Factor (Rh) Inheritance ....................................................................................... 29
3.10 Single-gene human traits and their alleles ....................................................................... 30
4. Gonosomal Inheritance and pedigrees ................................................................................. 33
Inheritance of Sex-Linked Traits ................................................................................................. 35
4.1 The inheritance of haemophilia A .................................................................................... 35
4.2 The inheritance of colour-blindness (daltonism) ............................................................. 35
4.3 The inheritance of G6PD deficiency ................................................................................ 35
4.4 Phosphate diabetes ........................................................................................................... 35
Pedigrees – Genealogical Method................................................................................................ 36
4.5 Pedigree analyses ............................................................................................................. 38
4.6 Draw the pedigree ............................................................................................................ 40
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5. Gene Linkage and Gene Mapping ........................................................................................ 41
I. Gene Linkage in a Testcross .................................................................................................... 46
5.1 Testcross........................................................................................................................... 46
5.2 Testcross........................................................................................................................... 46
5.3 Gene Linkage in Drosophila ............................................................................................ 46
II. Gene Linkage in Human......................................................................................................... 47
5.4 Determination of Gene Linkage in a Family ................................................................... 47
5.5 Rhesus Factor and Elliptocytosis ..................................................................................... 48
5.6 Daltonism and Hemophilia A .......................................................................................... 48
5.7 Association of traits ......................................................................................................... 48
III. Localization of Human Genes ............................................................................................... 49
5.8 Localization of enzyme-encoding genes .......................................................................... 49
5.9 Localization of HLA and P loci ....................................................................................... 49
5.10 Selected Loci of Human Genome .................................................................................... 49
6. Non-mendelian Inheritance ................................................................................................... 51
I. Non-allelic Gene Interaction.................................................................................................... 51
I. A) Epistasis .............................................................................................................................. 51
6.1 Recessive epistasis in humans: the Bombay blood group................................................ 51
I. B) Duplicity ............................................................................................................................. 52
6.2 Duplicity and the inheritance of skin colour in human .................................................... 52
II. Multifactorial inheritance ....................................................................................................... 53
II. A) Quantitative traits .............................................................................................................. 53
II. B) Qualitative traits – illnesses ............................................................................................... 53
Heritability and Genetic Twin Studies ......................................................................................... 54
6.3 Congenital hip luxation .................................................................................................... 55
6.4 Hypertension heart disease............................................................................................... 55
6.5 Schizophrenia ................................................................................................................... 55
6.6 Eight diseases ................................................................................................................... 56
III. Mitochondrial Inheritance ..................................................................................................... 56
6.7 Draw a pedigree ............................................................................................................... 57
7. Genetic control of metabolism .............................................................................................. 58
7.1 Metabolism of Phenylalanine........................................................................................... 60
7.2 Lactose Digestion ............................................................................................................. 61
7.3 Glucose-6-Phosphate Dehydrogenase Deficiency ........................................................... 61
7.4 Familial Hypercholesterolemia (FH) ............................................................................... 62
7.5 Diabetes Mellitus ............................................................................................................. 62
8. Population Genetics ............................................................................................................... 64
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I. Interbreeding ............................................................................................................................ 66
I. A) Autosomal Genes in a Population ...................................................................................... 66
8.1 Heredity of Rhesus (Rh) factor ........................................................................................ 66
8.2 Heredity of Eye Colour .................................................................................................... 66
8.3 Albinism ........................................................................................................................... 66
8.4 Cystic fibrosis (CF) – mucoviscidosis ............................................................................. 67
8.5 Phenylketonuria (PKU) .................................................................................................... 67
8.6 Other autosomal recessive diseases ................................................................................. 67
8.7 Sickle Cell Anemia .......................................................................................................... 67
8.8 Large canine teeth ............................................................................................................ 68
8.9 Cleft palate ....................................................................................................................... 68
I. B) X-chromosome linked inheritance ...................................................................................... 68
8.10 Daltonism (Colour Blindness) ......................................................................................... 68
8.11 Haemophilia ..................................................................................................................... 68
8.12 Glucose-6-Phosphate-Dehydrogenase (G6PD) Deficiency ............................................. 68
II. Inbreeding, consanguineous marriages, coefficient of inbreeding ......................................... 69
II. A) Coefficient of Inbreeding (F) (coefficient of kinship) ...................................................... 70
II. B) Coefficient of relationship (coefficient of relatedness, Rxy).............................................. 71
8.13 Calculate the coefficient of inbreeding ............................................................................ 71
8.14 Inbreeding and diseases with AR inheritance .................................................................. 72
9. Genetic Prognosis ................................................................................................................... 73
9.1 Albinism ........................................................................................................................... 74
9.2 Cystic fibrosis (mucoviscidosis) ...................................................................................... 75
9.3 Deafness ........................................................................................................................... 75
9.4 Marphan's syndrome (arachnodactylia) ........................................................................... 75
9.5 Haemophilia ..................................................................................................................... 75
9.6 Daltonism ......................................................................................................................... 75
9.7 Tooth enamel defect ......................................................................................................... 76
9.8 Myopathia myotonica ...................................................................................................... 76
9.10 Progressive muscular dystrophy ...................................................................................... 76
9.11 Haemophilia ..................................................................................................................... 76
9.12 Haemolytic spherocytic anaemia ..................................................................................... 76
9.13 Cataract ............................................................................................................................ 77
9.15 Down syndrome ............................................................................................................... 77
10. Genetics of Immunity, HLA System ................................................................................. 78
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I. Characterization of the HLA System Polymorphism .............................................................. 80
10.1 Theoretical Number of Haplotypes .................................................................................. 81
10.2 Theoretical Number of Genotypes ................................................................................... 81
II. Analysis of HLA Allele Segregation in Families ................................................................... 82
10.3 Deducing Genotypes from Phenotypes ............................................................................ 83
10.4 Deducing Haplotypes in a Family .................................................................................... 83
10.5 Geneticist's Testimony in a Paternity Suit ....................................................................... 83
10.6 Expected Genotype Frequencies ...................................................................................... 83
III. Choosing a Donor for Transplantation.................................................................................. 84
10.7 Phenotypes of Compatible Donors .................................................................................. 84
10.8 Suitability of Organ Donors ............................................................................................. 84
IV. HLA and Autoimmune Disease Associations ...................................................................... 85
10.9 Calculate relative risk....................................................................................................... 85
11. Molecular Biology I: DNA, RNA Analysis....................................................................... 86
11.1 DNA diagnostics .............................................................................................................. 91
11.2 DNA sequencing .............................................................................................................. 91
11.3 PCR reaction .................................................................................................................... 91
11.4 Restriction analysis .......................................................................................................... 91
11.5 IVS-1-I (G→A) ß-globin Gene Mutation Analysis ......................................................... 91
11.6 V617F JAK2 Mutation Analysis...................................................................................... 92
11.7 PCR Detection of a Mutation in Inherited Disorder ........................................................ 92
11.8 Fusion Gene Analysis ...................................................................................................... 92
11.9 Paternity Law Suit ............................................................................................................ 93
11.10 Fill-in the table ............................................................................................................. 93
12. Molecular Biology II: Proteins, Medical Application ..................................................... 94
I. Protein Detection Used in Diagnosis ....................................................................................... 94
II. Proteins in Therapy ................................................................................................................ 95
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1. Chromosomal Abnormalities in Human
Theoretical background
Mitosis, meiosis, chromosomes.
Control questions
1. Characterize numerical chromosomal abnormalities.
2. What are the mechanisms leading to these abnormalities?
3. What may be the clinical features of diseases resulting from chromosomal abnormalities?
Introduction
Eukaryotic chromosomes are structural units of genetic information. DNA double-helix associates
with proteins to form chromosomes. Chromosomes are visible under a light microscope only in the
course of cellular division (mitosis and meiosis). Their role is to ensure that genetic information is
evenly distributed into daughter cells. Most cells in human organism are diploid (2n) and contain
2 complete sets of chromosomes (46 chromosomes). In these cells each chromosome is found
in two copies that form a “pair of homologous chromosomes” (e.g. chromosomes # 15: one comes
from the mother, the other from the father). Chromosomes belonging to different homologous
pairs are called non-homologous (e.g. chromosomes 2 and 17). Diploid somatic cells multiply
themselves through mitosis. In contrast, meiosis, which is also sometimes called reducing cellular
division, results in gamete production. Gametes are haploid (n), because they contain single set
of chromosomes (23). Meiosis comprises of two successive meiotic divisions called meiosis I and
meiosis II. Meiosis I is also called the reduction division because homologous chromosomes
segregate and total number of chromosomes is reduced to one half. Thus, the resulting gametes
contain only one homologous chromosome from each pair.
Chromosomal abnormalities
Abnormalities of chromosomes may be either numerical or structural; involving autosomes or sex
chromosomes (gonosomes: chromosomes X and Y). Structural chromosomal abnormalities change
the structure of chromosomes. We will talk about this phenomenon in the next practical.
Numerical aberrations, which are sometimes called genomic mutations, change the number
of chromosomes in a cell. In some cases the structural chromosomal aberration causes also
a change in number of chromosomes. An example of such aberration is Robertsonian translocation
(also called centric fusion) – for more detail see example 1.7.
Terminology
diploid – 2n (2 sets of chromosomes; somatic cells)
haploid – n (1 set of chromosomes; gametes)
euploid – exact multiple of haploid set
Numerical aberrations
polyploidy – abnormal multiple of the whole set of chromosomes
(cells are 3n, 4n, etc.)
aneuploidy – change in number of individual chromosomes;
monosomy – loss of one chromosome; chromosome count: 2n-1 (45)
trisomy – gain of one chromosome; chromosome count: 2n+1 (47)
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Chromosomal abnormalities are formed usually in the course of life of an individual in somatic
cells and may lead to the development of cancer.
In this seminar we will deal with inborn chromosomal aberrations, which occur during gamete
formation or in the course of zygote cleavage and are the cause of inborn developmental
disabilities.
Aneuploidy usually arises as a consequence of errors in the cell division. The primary mechanism
leading to aneuploidy is non-disjunction; failure of chromosomes to separate properly during
anaphase. Other causes such as chromosome loss due to anaphase lagging are rare.
Non-disjunction (ND) may be either meiotic or mitotic.
Most aneuploidies are lethal. Trisomy is more common than monosomy. Only three non-mosaic
autosomal trisomies and the following aneuploidies of gonosomes are viable after birth:
Autosomal trisomies:
Down syndrome 47, XX, +21 or 47, XY, +21
Edwards syndrome 47, XX, +18 or 47, XY, +18
Patau syndrome 47, XX, +13 or 47, XY, +13
Sex chromosome aneuploidies:
Trisomy X 47, XXX (female)
Klinefelter syndrome 47, XXY (male)
Extra “Y” chromosome 47, XYY (male)
The only monosomy of whole chromosome which is viable is X chromosome monosomy called
Turner Syndrome 45, X (female)
These chromosomal abnormalities occur in at least 3-4 % of all clinically recognized pregnancies.
Most of them are associated with severe physical and mental developmental disabilities.
An exception is extra “Y” chromosome (47, XYY), affected males are usually normal.
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Meiotic-non-disjunction (also called pre-zygotic)
Non-disjunction can occur during meiosis I or meiosis II. Errors in the proper segregation of the
chromosomes during meiosis I and meiosis II, are pictured below. Non-disjunction occurs more
often in meiosis I, when both chromosomes from a homologous pair fail to separate and enter the
same daughter cell. The result is two daughter cells that have two copies of the chromosome
(called disomic cells) and two cells that are missing that chromosome (called nullisomic cells).
This is shown in diagram A). In meiosis II, the error occurs when the sister chromatids of one
chromosome do not separate properly and thus enter the same daughter cell. This is shown
in diagram B).
A) ND during meiosis I B) ND during meiosis II
These diagrams illustrate meiotic non-disjunction.

What happens with the reproductive cells which are abnormal in number of chromosomes?
In the process of fertilization male and female gametes fuse together. If one of fusing gametes
lacks a chromosome, the result of fertilization is monosomic zygote (the outcome is
45 chromosomes in total). On the other hand, if one of fusing gametes possesses an extra copy
of chromosome, the result of fertilization is trisomic zygote (the outcome is 47 chromosomes
in total). In general, monosomies are less likely to survive when compared to trisomies, with the
exception of monosomy of the X chromosome. A non-disjunction error during meiosis I or
meiosis II can result in individual with an extra/missing chromosome in every cell of his/her
body.
Mitotic non-disjunction (also called post-zygotic)
This occurs early after fertilization in one of the dividing cells of a developing embryo. Only
a subset of the cells in the adult organism is, therefore, affected by aneuploidy. The resulting state
is called mosaicism: there are present at least 2 populations of the cells differing in genotypes
in single body. An affected individual (patient) is called a mosaic (see 1.5).
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1.1 Fill-in the figure of normal meiosis
Draw the segregation of chromosomes and chromatids during spermatogenesis; give the number
of chromosomes.
pair of homologous chromosomes

(e.g. # 21 chr.)
S-phase
st
1 meiotic division
nd
2 meiotic division
How many chromosomes are there in an egg and in a fertilized zygote?
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1.2 Fill-in the following figure of meiotic non-disjunction
In the figures below there are 2 examples of aberrant gametogenesis; A) a non-disjunction of Ch.
21 and B) non-disjunction of Ch. X & Y. Fill-in the total number of chromosomes, the number
of chromosomes in sperms/eggs and fertilized zygotes. Give the name of the syndromes, which
arise from the fusion of such abnormal gametes with normal gametes.
A) ND of chromosome 21 during the 2nd meiotic division in the course of oogenesis

(e.g. # 21 chr.)
S-phase
st
1 meiotic division
nd
2 meiotic division
ND
potential oocytes
+ + + +
spermatozoa
potential zygotes
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B) ND of chromosomes X & Y in the 1st meiotic division during spermatogenesis
chromosomes X & Y
S-phase
st
1 meiotic division
ND
nd
2 meiotic division + spermiogenesis
spermatozoa
+ + + +
oocytes
potential zygotes
Spermatogenesis: the whole process of sperm cell development. This comprises mitotic divisions
of spermatogonia, meiosis and differentiation of the spermatids into sperm cells.
Spermiogenesis: the haploid stage of spermatogenesis (differentiation of the spermatids into
sperm cells). It comprises the following individual processes:
 nuclear condensation
 acrosome formation
 flagellum formation
 cytoplasm reduction
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1.3 Cleavage division of the zygote and blastomeres
Blastomeres are formed during normal zygote cleavage by mitosis. Fill-in the chromosome
numbers in the cells.
zygote
st
1 cleavage division
nd
2 cleavage division
1.4 Mitotic non-disjunction

During mitotic non-disjunction sister chromatids of one of the homologous pair of chromosomes
fail to separate. One chromosome separates normally – sister chromatids are separated in anaphase
and move to opposite poles, i.e. into to 2 daughter cells. Both chromatids of the other homologous
chromosome move to only one daughter cell. The result is two daughter cells with different
number of chromosomes (one has 47 and the other 45 chromosomes); both differ from the normal
number of chromosomes in the mother cell (46).
The mitotic non-disjunction, which occurs at a developmental stage comprising of at least
2 blastomeres, gives rise to so called mosaic type of inborn developmental disabilities such as
mosaic Down syndrome. It is important to note, that only a population of cells in the body has
trisomy 21, and the rest of the cells are euploid (normal, see 1.5A). The afflicted individual has
milder clinical symptoms than non-mosaic type of the same syndrome, when all cells in the body
possess trisomy 21.

46
(e.g. # 21 chr.)
S-phase
ND during
mitosis
47 45
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1.5 Complete the figure of mitotic non-disjunction of chromosomes
Cleavage of a fertilized egg is depicted in the following figures; where a non-disjunction (ND)
occurred of A) chromosome 21 and B) chromosome X in one of the cleavage divisions. Give the
number of chromosomes in blastomeres.
A) Non-disjunction of chromosome 21 during the second cleavage division.
zygote
4
st
1 cleavage division
nd
2 cleavage division
ND
B) Non-disjunction of chromosome X during the third cleavage division
zygote
46
st
1 cleavage division
nd
2 cleavage division
rd
3 cleavage division
ND
1. What is the consequence of mitotic non-disjunction?

2. What syndrome is caused by mitotic non-disjunction in example A) and B)?
3. What will be the result of examination of X-chromatin in these individuals?
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1.6 Complete the figure of normal meiosis and fertilization
Draw the chromosomes into the empty circles and write the number of chromosomes into the
empty squares.
chr. 13
chr. 21
S-phase
st
1 meiotic division
nd
2 meiotic division + spermiogenesis
normal spermatozoa
+ + + +
oocytes
potential zygotes
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1.7 Robertsonian translocation
Robertsonian translocation (centric fusion of chromosomes) is an example of a structural
chromosomal aberration, which also leads to numerical chromosomal aberration. It represents
a fusion of the long arms of two acrocentric chromosomes [13, 14, 15, 21, 22], which will form
a single chromosome (having lost the short arms at the same time). Most often it occurs as 21/21,
13/14, and 14/21 translocation. The resulting karyotype has only 45 chromosomes. A carrier
of Robertsonian translocation is phenotypically normal, however may be plagued by infertility,
repeated miscarriages, or inborn defects in his/her children.
The problem of this carrier arises during meiosis – the segregation of chromosomes is influenced
and gametes with abnormal number of chromosomes are formed. After fertilization of these
abnormal gametes, abnormal zygotes are formed (trisomic or monosomic). The danger
of Robertsonian translocation is in possible affliction of offspring of a Robertsonian fusion
carrier.
45
chr. 13
chr. 21
S-phase
abnormal chromosome
formed by translocation 13;21
3 dif f erent possibilities of chromosome segregation during 1st meiotic division
A) B) C)
2nd meiotic division 2nd meiotic division 2nd meiotic division
+ + + + + +
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1.8 Robertsonian translocation of chromosomes 14 and 21
One parent is a carrier of Robertsonian translocation of the long arm of chromosome 21 onto
a long arm of chromosome 14. Draw segregation of chromosomes into gametes. Give all the
possible types of gametes and zygotes, which will be formed by fusion of these gametes with
normal gamete. Draw the chromosomes into the empty circles and write the number
of chromosomes into the empty squares. What is the risk of having an afflicted child?
chr. 14
chr. 21
S-phase
abnormal chromosome
formed by translocation 14;21
st
3 different possibilities of chromosome segregation during 1 meiotic division
A) B) C)
nd nd nd
2 meiotic division 2 meiotic division 2 meiotic division
+ normal spermatozoa
potential
zygotes
Control questions:
1. Which trisomies of whole chromosome are viable? Which monosomy of a whole
chromosome is not lethal? Give the names of these syndromes.
2. What is the result of non-disjunction of chromosomes during mitotic division?
3. How many chromosomes has a carrier of Robertsonian translocation?
4. Name the 3 forms of Down’s syndrome according to the mechanisms of their origin.
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2. Mutations in human diseases
Mutagenesis, transcription, translation, genetic code, karyotype.
Control questions
1. Which techniques can be used for the depiction of karyotype?
2. Explain the difference between somatic cell mutation and germ cell mutation.
3. Explain the term reciprocal translocation.
4. What is Philadelphia chromosome?
Introduction
Mutations are changes in genetic information (DNA). Mutations can lead to genetic disorders.
Mutations can be divided according to:
A) the cause – spontaneous (without any known exogenous cause)
– induced (by physical, chemical, or biological mutagens)
B) their type – genomic mutations (numerical chromosomal aberrations)

– structural chromosomal aberrations
– gene mutations
C) the cell type, where the mutation occurs:

– germ line: The mutation occurs during gamete formation and is, therefore, transmitted to the
next generation. The new offspring has the mutation in every cell of his/her body.
– somatic: The mutation occurs only in some somatic cells in the organism. Mutation is
transmitted only to daughter cells (e.g. tumour formation). The mutation is not transmitted to the
next generation.
2.1 Gene mutations

Gene mutations affect one or more nucleotides of a given gene and they do not, therefore,
influence the structure of the whole chromosome. Standard cytogenetic techniques (such as
karyotyping) do not detect them. Instead, molecular biology techniques are used to screen for these
mutations. We will talk about these methods later in the summer term.
Gene mutations:
 point mutations
 deletions
 insertions
Point mutations
They affect one nucleotide in a DNA sequence and substitute it with a different nucleotide. If the
mutation does not affect the final amino acid sequence, it is called a silent mutation (the new
triplet encodes for the same amino acid/AA). Missense mutation changes the meaning of the
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triplet (the new triplet encodes for a different AA). Nonsense mutation (stop mutation) leads to
truncation of the protein (the new triplet is a STOP codon).
A) silent mutation – no change in AA sequence of the protein
For example codon TCT encodes for AA serine. Serine is also encoded by TCA, TCG and TCC.
If the last nucleotide of thecodon TCT in a DNA sequence is changed for any other nucleotide,
it will not affect the amino acid sequence of the protein.
B) missense mutation – there is a change in AA in a protein
The nucleotide substitution leads to a new codon, which encodes a different AA. The newly
formed protein has a different AA sequence.
Example: Sickle Cell Anaemia. Substitution of A to T (in the 17th nucleotide) in beta globin gene
leads to the change of the 6th AA: glutamic acid to valine – GAG encodes for glutamic acid and
GTG encodes for valine. The newly formed hemoglobin is abnormal with different properties and
causes the resulting anaemia in these patients.
C) nonsense mutation – truncation of the protein

Nucleotide change leads to formation of a STOP codon (TAA, TAG, TGA). The protein synthesis
is, therefore, prematurely ended.
Example: Cystic fibrosis. Substitution of T to C in the 1609th nucleotide changes the codon for
glutamine (CAG) to the STOP codon (TAG). The resulting protein is not functional, because
instead of having 1480 AAs, it has only 493.
Deletions and insertions

They represent the loss or gain of a number of nucleotides from a DNA sequence of a gene. This
may result in disruption of the reading frame, or the grouping of the codons, resulting in a
completely different translation. This type of mutation is called “frame-shift” mutation.
2.2 Numerical and structural chromosomal aberrations

Numerical and structural chromosomal aberrations affect whole chromosomes or their bigger
parts, i.e. groups of genes simultaneously. They are very common in many inborn developmental
disabilities and malignant disorders.
Different cytogenetic methods are used to visualize the structure and number of chromosomes and
thus can be used to create the karyotype.
Karyotype is an organized arrangement of metaphase chromosomes, which is specific for each
species. It gives information about the number and morphology of chromosomes, constitution
of sex chromosomes, and eventually about any chromosomal abnormalities. Human karyotype
of somatic cells consists of 22 pairs of autosomal chromosomes and 1 pair of sex chromosomes.
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There is a set of international rules (ISCN) about the karyotype nomenclature (how to write it).
The standard order for presenting information of an karyotype is as follows: total number of
chromosomes followed by identification of sex chromosomes: 46,XX or 46,XY. In pathological
karyotypes, the chromosomal aberration is represented by a given abbreviation and the number
of chromosome, which is affected: 47,XX,+21 (Down’s syndrome, trisomy of chromosome 21).
Numerical aberrations
 polyploidy
 aneuploidy:
o monosomy
o trisomy
o hyperdiploidy – extra copies of particular chromosomes (the term is not used for
specific trisomies as Down syndrome, Klinefelter syndrome, etc.)
Structural aberrations
Structural aberrations lead to the change of the morphology and shape of the chromosomes. They
are formed on the basis of chromosomal breaks. The following aberrations belong to structural
chromosomal abnormalities: deletion, duplication, insertion, inversion, “ring” chromosome (forms
a circle), translocation.
Abbreviations used in karyotype nomenclature for structural aberrations:
Type Abbreviation according to ISCN
deletion del
duplication dup
insertion ins
inversion inv
ring chromosome r
translocation t
Chromosomal abnormalities of cancer cells

Chromosomal aberrations are a characteristic feature of many tumor cells. Diagnosis of leukaemia
is largely based on analysis of karyotype. Certain types of hematological malignancies are
characterized by the presence of specific chromosomal abnormalities, mostly translocations.
A proof of the presence of a specific translocation in leukemic cells of a patient is very important
for correct diagnosis and prognosis of the disease and often also for adequate choice of therapy.
Apart from translocations, massive hyperdipoidy (as much as 70 chromosomes) is also typical
feature of hematological malignancies.
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2.3 Pathological karyotypes in patients with inborn developmental
disabilities
The following 4 examples of pathological karyotypes were prepared by the conventional
cytogenetic technique. Give the correct karyotype nomenclature according to the international
rules. State what type of abnormality it is, if it affects autosomes or sex chromosomes, and which
syndrome it causes.
It is important to note, that these inborn chromosomal aberrations are present in all cells of the
organism.
I. Proband is an 18-year-old girl of a small stature, and infantile look.
II. Proband is a small boy with mental retardation and typical physiognomy. He is the youngest
of 6 siblings. His mother was 41 years old when he was born.
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III. The proband is clinically normal woman, who has an only child with Down syndrome.
Another pregnancy was terminated by an abortion.
IV. The proband is a 3-year-old boy with severe mental and motor retardation with muscle
hypotony and many other abnormalities.
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2.4 Pathological karyotype in cancer
The following figure shows a karyotype of blood cells of a patient suffering from a blood cancer –
chronic myeloid leukaemia. The arrows point to chromosomes 9 and 22, which are involved in a
reciprocal translocation.
It is important to note, that this chromosomal abnormality is present only in afflicted blood cells.
All the other cells of the organism of the patient are normal.
This karyotype was prepared by a different cytogenetic technique (called banding) compared to
those in the previous examples.
Karyotype nomenclature: 46,XX,t(9;22)
2.5 Preparation of metaphase chromosome to assess the cell ploidy
Principle
Chromosomes can be observed if cells are arrested in metaphase of mitosis. The procedure is
based on treatment of dividing cells with alkaloid colchicine. Colchicine is a microtubule inhibitor.
Treatment with colchicine disrupts spindle fibers, arresting the cells at metaphase.
Protocol
The cells were treated with colchicine for 4 hours. The cells were then pelleted and resuspended
in 15 ml of hypotonic solution. The cells in hypotonic solution were then incubated for 30 minutes
at 37°C. The cells were pelleted again and resuspended in Carnoy's fixative (1 part of glacial acetic
acid, 3 parts of methanol).
You will be given the cell suspension in Carnoy's fixative.
1. Disperse the pellet with a Pasteur pipette.
2. Chill a glass slide on ice and place it on a Petri dish.
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3. Aspirate the cell suspension into a Pasteur pipette. Release 1 to 2 drops of the suspension
from a Pasteur pipette onto the slide. The material should be spread well onto the slide –
drop the suspension onto the slide from a 50-cm level above the slide.
4. Dry the slide rapidly over a hot heater (the fixative will evaporate).
5. Stain cells with 2% Giemsa stain (5 min) in a Coplin jar and rinse in distilled water
(1 minute in another Coplin jar).
Search for the cells in metaphase under the scope (magnification 200×). Use 40× objective
to observe the metaphases. Count the chromosomes.
From what tissue are the cells derived, normal (healthy) or tumor (malignant)?
Count 100 cells randomly, assess whether they are in interphase or M phase. The percentage
of cells in M phase is called the mitotic index.
Control questions:
1. What is the main consequence of hyperdiploid number of chromosomes?
2. How does hyperdiploidy arise?
3. Why do we drop the cell suspension from a 50 cm height?
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3. Mendelian inheritance
Mendel’s laws, basic genetic terminology, interaction of allelic genes, types of inheritance.
Control questions
1. Explain the Mendel's first law – the law of segregation.
2. Explain the Mendel's second law – the law of independent assortment.
3. Explain complete dominance, incomplete dominance, codominance.
4. Is it possible to determine the genotype of a person showing a dominant phenotype?
A recessive phenotype? Why?
Introduction
Mendelian inheritance (or Mendelian Genetics or Mendelism) is a set of primary tenets relating to
the transmission of hereditary characteristics from parent organisms to their children. These rules,
which are called Mendel’s laws, are applied to inheritance of traits, which are determined by a
single gene (single-gene traits/disorders).
Basic terminology
Allele is an alternative form of a given gene, which differs from the other variants of the given
gene by different DNA nucleotide sequence. In diploid organisms, 2 alleles form the genotype of
the trait and are localized on the homologous chromosomes.
Locus is a specific position on a chromosome, where the gene (allele) is located.
Homozygote – possesses identical alleles of the gene on both homologous chromosomes.
Heterozygote – possesses different alleles of the gene on both homologous chromosomes.
Polymorphism/Multiple alleles is a state, where there are more than 2 relatively common
variants/alleles of a gene in a population (e.g. alleles for blood group ABO, alleles for HLA
system). Each individual can have only 2 alleles from this series for a given gene. It is due to
diploidy.
Relationship between alleles of the same gene is called interaction of allelic genes. The type of
gene interaction can be determined from the phenotype of the heterozygote. There are 3 following
types of gene interaction:
 Complete dominance (A>a)
The phenotype of the heterozygote (Aa) is the same as that of the dominant homozygote
(AA). Dominant allele is usually written with a capital letter – A. Recessive allele is written
with the same but lower case letter – a. Recessive allele a is not phenotypically expressed in
heterozygote Aa. The dominant allele completely suppresses the expression of the recessive
allele.
 Incomplete dominance (semidominance or blending inheritance) (A≥a)
The phenotype of the heterozygote (Aa) is in between of the phenotypes of both
homozygotes (AA and aa). In the heterozygote Aa, the expression of the dominant allele
in phenotype is weaker than in homozygotes AA. The dominant allele does not completely
suppress the expression of the recessive allele in heterozygote Aa.
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 Codominance (A = B)
The expression of both alleles (A and also B) in the phenotype of the heterozygote is equal
(both alleles are fully expressed); neither one is dominant nor recessive to the other. The
heterozygote displays the phenotypic characteristics of both alleles. A common example is
ABO blood group system or human leukocytes antigens (HLA antigens).
There are four basic patterns of a single-gene inheritance:
Dominant Recessive
Autosomal autosomal dominant autosomal recessive
X-linked X-linked dominant X-linked recessive

Monogenic disorders may have variable expressivity or variable gene penetrance.
Variable gene expressivity: the same genotype, in different individuals, can have a variable
influence on the strength of the trait in phenotype (different degree of severity of clinical
symptoms).
Variable gene penetrance means that only certain percentage of individuals with a given
genotype has the expected phenotype (some individuals carrying the mutant gene are ill, others are
without any clinical symptoms).
Most tasks you are going to solve today, deal with single-gene (monogenic) traits for which
Mendel’s laws can be applied. In order to solve the tasks, it is important to know that:
1. Each gamete receives only one allele from each pair of alleles during meiosis.
2. A homozygote produces all gametes with the same genotype. A heterozygote (Aa)
produces two types of gametes (half gametes with allele A, half with allele a). Double
heterozygote (AaBb) produces gametes of different genotypes (AB, Ab, aB, ab), if both
allele pairs (A/a, B/b) are located on different non-homologous chromosomes.
3. Zygote (and thus the offspring) receives for a given trait one allele from the mother (from
the egg) and the other allele of the same gene from the father (from the sperm).
Symbols are read in the following way:
A .................. dominant allele
a ................... recessive allele
AA ............... dominant homozygote for gene A
Aa ................ heterozygote for gene A
aa ................. recessive homozygote for gene A
How to solve the tasks

There are two basic situations:
1. We know the phenotypes in parents and want to know the phenotypes in their children:
a) Determine the parents’ genotypes according to given type of allele interaction.
b) Determine the genotypes in their gametes with the help of the law of allele segregation.
c) Determine the genotypes in the zygotes, which were formed by combination of the
individual genotypes of sperms and eggs.
d) Give the phenotypes of individual genotypes in the zygotes according to the type
of allele interaction. In this way you will get the phenotypes in children.
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2. We know the phenotypes in children and we want to know the genotypes and phenotypes
in parents.
a) Determine the genotypes in the children according to the given type of allele interaction.
b) In each allele pair in the child, one allele was received from the mother and the other
from the father. Write the symbols of children’s alleles as components of the mother’s and
father’s genotypes. You will get the genotypes in both parents.
c) Give corresponding phenotypes to genotypes in the parents according to the type of
allele interaction in the given example.
In some examples below there is more than one possible solution because we can not determine
some genotypes exactly.
3.1 Inheritance of the eye colour

Eye colour results from interaction of several allele pairs of different genes that determine the
formation and accumulation of pigment in the iris. We will simplify this inheritance to only one
allelic pair, where the allele encoding the brown colour of the eyes B is dominant over the allele b
for blue eyes colour.
a) A brown eyed man marries a woman with blue eyes and they have 8 brown eyed children.
What are the genotypes of all family members?
b) A brown eyed man whose both parents possess also brown eyes got married to a brown
eyed woman; her father has brown eyes and her mother has blue eyes. This couple has one
blue eyed child. Determine the genotypes of all family members.
3.2 Single-gene disorder inheritance

The incidence of a certain illness was observed in a cohort of 100 families. On the basis of the
results, say if the pattern of inheritance of this disorder is autosomal dominant or autosomal
recessive. Give the genotypes in the parents.
Parents’ phenotypes Number of families Children’s phenotypes

Healthy Ill
healthy × ill 51 80 37
healthy × healthy 40 90 16
ill × ill 9 0 17
3.3 Cherubinism
Cherubinism is familial fibrous dysplasia of the jaw. Clinically this disorder is expressed as tumors
of the fibrous tissue of the jaws. It leads to deformation of face bones and loss of teeth.
Cherubinism is an autosomal dominant disease with variable expressivity and 100 % penetrance in
men and 50 % penetrance in women.
Two healthy parents have had an affected son born. Determine the probable genotypes in the
parents and the son and explain.
3.4 Phenylketonuria
Phenylketonuria (PKU) is an autosomal recessive disorder. It belongs to enzymopathies and
metabolic disorders. PKU is characterized by the lack of an enzyme, which metabolizes
phenylalanine to tyrosine. Recessive homozygotes (when untreated) have severe clinical
symptoms; heterozygotes are healthy with weak symptoms detectable only by laboratory tests.
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a) What children could be born in a marriage of two healthy parents with abnormal activity
of the enzyme (defective in PKU) detected only by laboratory test?
b) What is the probability of having an afflicted and healthy child in the above mentioned
case?
Comment: Recessive homozygote can grow up with normal brain development, but only in the
case if he/she is on strict diet with very limited intake of phenylalanine for the rest of his or her
life. We will deal with PKU in more details latter (practical called Genetic control of metabolism).
3.5 Sickle-cell disease

Sickle-cell disease is an autosomal recessive (AR) disorder. Patients suffering from severe
anaemia caused by sickle-cell disease are homozygous (SS) for a defective allele (S) caused by
a point mutation in the β-globin gene. They produce abnormal haemoglobin HbS instead
of normal adult HbA in their red blood cells (RBC). These RBC become consequently sickle-
shaped. The heterozygotes for this mutation do not have any clinical features but they produce
both types of haemoglobin; HbS and normal HbA, which may lead to sickling in some of their red
blood cells.
a) Blood analysis of a healthy man revealed the presence of both HbS and HbA. His wife is
healthy and expresses only HbA. What phenotypes would you expect in their progeny?
b) Healthy parents both express HbS and HbA. What phenotypes would you expect in their
progeny?
3.6 Beta thalassemia

Beta thalassemia is an AR disorder caused by a point mutation in the ß-globin gene that leads to
quantitative reduction of ß-globin chain. Heterozygotes are mostly asymptomatic and/or with
slight clinical symptoms. In contrast homozygotes suffer from severe form of transfusion-
dependent anaemia. Give all possible genotypes of children of two heterozygote parents. Give
corresponding phenotypes.
3.7 The inheritance of eye colour and left-handness

For simplicity, brown eye colour is a dominant trait (allele B); blue eye colour is a recessive trait
(allele b). Right-handness (allele R) is dominant over the left-handness (allele r).
A right-handed blue eyed man got married to right-handed brown eyed woman. They have two
children. The first brown eyed child is left-handed, and the second blue eyed child is right-handed.
After divorce the same man got married to another brown eyed right-handed woman. This time
they have six children. All of them have brown eyes and are right-handed. Determine the genotype
in this man and his two wives.
3.8 Inheritance of the ABO blood group in humans

A-B-O blood type is controlled by three alleles of a single gene. These alleles code for the
production of antigens on the surface of red blood cells. Every individual possesses only two
of these three alleles. Allele O is recessive, alleles A and B are dominant over allele O, alleles A
and B are codominant (A = B; A > O; B > O).
a) Determine all possible genotypes in people with A, B, AB, and O blood types.
b) Complete following tables:
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Table 1.
The blood type The blood type absent
The blood types of parents
of children in children
O × O
O × A
A × A
A × B
O × AB
B × AB
AB × AB
Table 2.
The blood type of mother The blood type of men
The blood type of child
excluded from paternity
O O
O A
A B
B O
AB A
AB AB
3.9 Rhesus-Factor (Rh) Inheritance

"Rh" is used as another marker for blood types, which can be either Rh negative (Rh-) or Rh
positive (Rh+). It is named after the Rhesus monkey, where it was originally found. This trait is
genetically controlled by 3 different strongly linked genes (C, D, and E). If at least one dominant
“D” allele is present in the genotype, the phenotype will be Rh factor positive (Rh+); irrespectively
of the status of the remaining C and E alleles.
-D- is dominant over C/c, d and E/e.
Complete the following tables:
Table 3.
Genotypes in parents Genotypes and phenotypes in children
Ccddee × CcDDEe
CCDdEe × CCddee
CCDdEE × ccDdEe
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Table 4.
A Rh+ × AB Rh-
AB Rh- × B Rh+
O Rh+ × AB Rh-
A Rh+ × O Rh-
AB Rh+ × AB Rh+
3.10 Single-gene human traits and their alleles

Go through the traits in Table 5; score your own phenotype for each trait in Table 6. Write the
symbols for your possible genotypes in the space provided (use the symbols given in Table 5).
If you know your parents' phenotypes, include them in the space provided.
Table 5
Trait (alleles) Expression
Bent pinky (B, b) Dominant allele causes the distal segment of the fifth finger to
bend distinctly inward toward the fourth (ring) finger.
Blue eyes (E, e) Blue eyed persons are homozygous recessive and lack pigment
in the iris of the eye; heterozygous or homozygous dominant
individuals have iris pigment, the colour of which is
determined by other genes.
Mid-digital hair (M, m) People lacking hair in the middle segments of the fingers are
homozygous recessive. The presence of hair on one or more
middle segments of the fingers may be governed by a series
of alleles each of which is dominant to the recessive.
Tongue rolling (R, r) Persons with a dominant allele in heterozygous
or homozygous condition can roll their tongues into a tube-
like shape; homozygous recessives are non-rollers and can
never learn to roll their tongues.
Widow's Peak (W, w) Dominant allele in heterozygous or homozygous individuals
results in a V-shaped front hairline; homozygous recessives
have straight hairlines.
Thumb crossing (C, c) In a relaxed interlocking of fingers, left thumb over right
indicates that the dominant allele is present in either
heterozygous or homozygous individuals; homozygous
recessives naturally place the right thumb over the left.
Ear lobes (A, a) Ear lobes may be either adherent or free and pendulous.
Homozygous recessives have attached ear lobes; heterozygous
or homozygous dominant individuals have detached (free) ear
lobes.
Hitchhiker's thumb (H, h) Homozygous recessive can bend the distal joint of the thumb
backward to a nearly 90° angle; heterozygous or homozygous
dominant condition yields thumbs that cannot bend backward
more than approximately 30°.
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Figure 3.1. Phenotypic expressions of single-gene human genetic traits
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Table 6. Personal inventory and class percentages of phenotypes and possible genotypes for
single-trait genes described in Table 5.
Record your phenotype and your probable genotype in the following chart and determine the
percentages in your class.
Your Your possible Parent’s % of class
Trait # in class
phenotype genotype(s) phenotypes with trait
Bent pinky
Blue eyes
Mid-digital hair
Tongue-rolling
Widow’s peak
Thumb crossing
Ear lobes
Hitchhiker’s thumb
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4. Gonosomal Inheritance and pedigrees
Gonosomes. X chromosome-linked inheritance. Sex-limited and sex-influenced traits. Pedigrees
and symbols used in pedigrees.
Goal
The aim of this practical training is to determine the type of inheritance for a given disease with
the help of pedigree analyses.
Introduction
Phenotype expression of some genes is dependent on the sex of the individual. One of the possible
causes of this phenomenon is that these genes are localized on sex chromosomes (gonosomes)
X and Y: these traits are called sex-linked. The sex chromosome in the zygote does not only
determine the sex of the individual but it also determines expression of traits/genes localized on it.
Traits, which are incompletely linked with the sex chromosomes, are found in the homologous
parts of the chromosomes Y and X (pseudoautosomal region). In the homologous part of X and Y
chromosome, crossing-over may occurs, which means that the trait is expressed in both sexes with
the same frequency according to the rules applied for autosomal inheritance. Traits, which are
completely sex-linked, are found in the heterologous parts of the gonosomes and their distribution
varies between the sexes. Practically only X chromosome is important and, therefore, we will
focus in the following examples only on genes located in the heterologous part of X gonosome.
We will talk only about X chromosome-linked traits.
The inheritance of X-linked traits is determined by special sets of rules, which differ from the rules
for autosomal inheritance.
X-linked recessive inheritance

1. All sons of an afflicted man are healthy.
2. All daughters of an afflicted man are carriers.
3. Half of the sons of a carrier woman are afflicted and half of the daughters are carriers.
X-linked dominant inheritance

1. All sons of an afflicted man are healthy.
2. All daughters of an afflicted man are afflicted.
3. An afflicted woman has in average half of the sons and half of the daughters afflicted.
These traits (XD) are quite rare.
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Sex-limited traits
They are controlled by autosomal genes. Trait appears (or develops) only in one of the sexes.
General scheme of sex-limited inheritance (+ means that the "trait is expressed"):

Genotype
sex AA Aa aa
man + + -
woman - - -
Examples: ovary development in females; sperm development in males; hypospadias.
Hypospadias is a birth defect of the urethra in the male that involves an abnormally placed urinary
opening. Instead of opening at the tip of the glans of the penis, a hypospadic urethra opens
anywhere along a line running from the tip along the underside of the shaft to the junction of the
penis and scrotum. Hypospadias occurs only in men. The women with this genotype do not have
any abnormal phenotype. One of the forms of hypospadias is inherited in this way: men with
genotype HH, Hh are afflicted; men with genotype hh are healthy.
Sex-influenced traits
They are controlled by autosomal genes. They are expressed in both sexes but the intensity of the
expression varies. The following table gives the general scheme, where the expression of the trait
depends:
1. on the presence of one or two dominant alleles
2. on the sex of the individual
General scheme of sex-influenced inheritance (+ means that the "trait is expressed"):

Genotype
sex AA Aa aa
man + + -
woman + - -
For example, the expression of the alleles may be influenced by the presence of certain hormones,
which either increase or decrease the effect of the alleles. One such gene (B) encodes for early
baldness. If a male has one dominant and one recessive allele (Bb), he will show that trait. In
order for this gene to be expressed in phenotype in women, it has to be in homozygote constitution
(BB). Men are bold even if they are heterozygotes probably because of male sexual hormones.
Even autosomal recessive illnesses can be influenced by sex. One example is an iron metabolism
defect - hemochromatosis –, which leads to iron overload of the organism. Women suffer from
this illness less frequently. The reason for this is a lower iron absorption in the gut and higher iron
loss due to menstruation.
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Inheritance of Sex-Linked Traits
4.1 The inheritance of haemophilia A

Haemophilia A is a classical X-linked recessive disorder in which the blood fails to clot normally
because of the deficiency of clotting factor VIII: a protein in the clotting cascade. One of the most
famous genetic cases involving haemophilia A goes back to Queen Victoria.
a) A healthy man got married to a woman, whose father had suffered from haemophilia. What
genotypes and phenotypes would you expect in children born to these parents?
b) Three children were born in a family: two healthy daughters and one haemophilic son. Both
daughters got married to healthy men. One gave birth to a sick son and healthy daughter,
the second gave birth to one healthy daughter and four sons; two of them were sick.
Determine the genotypes in all family members.
4.2 The inheritance of colour-blindness (daltonism)

In human, red-green colour-blindness is a recessive sex-linked trait. The corresponding gene is
located on the X chromosome, not on the Y. Because males have only one X chromosome, they
have a much greater chance of having red-green colour-blindness. Females would have to be
homozygous recessive in order to be red-green colour-blind.
a) A colour-blind son was born to a father with normal colour vision. What are the genotypes
in the family members?
b) Two healthy individuals got married. They had a colour-blind son and a healthy daughter.
This daughter got married to a healthy man and gave birth to four colour-blind sons and
three healthy sons. Determine the genotypes in all family members.
4.3 The inheritance of G6PD deficiency

Glucose-6-phosphatase dehydrogenase (G6PD) deficiency is the most common disease-
producing enzymopathy in humans. Inherited as an X-linked recessive disorder, G6PD
deficiency affects 400 million people worldwide. G6PD deficiency is known to have over
400 variant alleles. This deficiency confers protection against malaria, which probably accounts
for its high allele frequency. Normally, G6PD works to "neutralize" oxidative substances and
protect cells from what is referred to as oxidative stress. Without sufficient content of normal
G6PD to help red blood cells get rid of harmful oxidative substances, they can be damaged or
destroyed, leading to a condition known as haemolytic anaemia.
Two healthy individuals got married. Two children were born in this family, healthy daughter and
anaemic son. Determine the genotypes in all family members.
4.4 Phosphate diabetes

Phosphate diabetes (hypophosphataemia renalis) is inherited via a dominant allele, which is
localized on heterologous segment of chromosome X.
a) An afflicted woman and a healthy man got married. Can they have a healthy son?
b) A healthy woman gets married to an afflicted man. What will be the sex of their healthy
children?
c) A healthy child of the parents mentioned in (b) gets married to a healthy partner. Is it
possible that this couple will have an afflicted child?
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Pedigrees – Genealogical Method
Single-gene disorders (monogenic disorders) are characterized by typical means of inheritance in
families. In order to determine the type of inheritance, the first step usually is to get the
information about family anamnesis of the patient and summarize the information via a graphic
means with the use of standard symbols, i.e. in a pedigree (Fig. 1).
Figure 1: Symbols commonly used in pedigree charts (Thomson and Thomson, p. 52)
For our class, the models correspond to simplified real situations. We assume that:
1. The trait (disease) is encoded by one gene with two alleles; one allele is completely
dominant over the other allele.
2. There is complete penetrance of the affected allele.
3. We follow only one trait in a pedigree.
4. The trait is encoded either by an autosomal gene (A) or by a gonosomal gene
on X chromosome (X).
5. The occurrence of the trait is determined either by dominant allele or by recessive allele.
AD ... autosomal dominant
AR ... autosomal recessive
XD ... X-linked dominant
XR ... X-linked recessive
Autosomal Dominant Inheritance (AD)

The individuals with the genotypes AA and Aa are ill, the individuals with the genotype aa are
healthy.
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a) If the disease occurs in an individual, it will occur with high probability in the next
generation too (this differs from AR inheritance!). If the afflicted individual has the
genotype AA – all his/her descendants will be affected; if he/she has the genotype Aa, there
is a 50 % chance of an afflicted child.
b) The number of afflicted men and women is approximately equal (this differs from XR
inheritance!).
c) If both parents are healthy (aa × aa), all their children will be healthy. If an afflicted child is
born to two healthy parents, it is not an AD inheritance. (Exceptions: de novo mutation,
variable penetrance.)
d) If a healthy child is born to two afflicted parents, both these parents are heterozygotes (Aa
× Aa).
Examples of AD disorders: achondroplasia, familial hypercholesterolemia, Huntington's disease,
Marfan syndrome, familial adenomatous polyposis, polydactylia.
Autosomal Recessive Inheritance (AR)

The individuals with genotypes AA, Aa are healthy, the individuals aa are afflicted (express the
trait).
a) The trait may "skip" generations, i.e. the trait doesn't occur in all generations. If an affected
person gets married to a healthy partner either “aa” (1) or “Aa” (2). All children will be
healthy in the case (1) and 50 % of children will be healthy in the case (2).
b) The number of afflicted men and women is approximately the same (this is different from
XR inheritance).
c) If two afflicted individuals get married, then all their children are also afflicted (i.e. aa). (If
a healthy child is born to two afflicted parents – it is not AR inheritance.)
d) If an afflicted child is born to two healthy parents, then both parents are heterozygotes (Aa
× Aa).
Examples of autosomal recessive disorders: albinism, sickle cell anaemia, thalasemia, cystic
fibrosis, phenylketonuria.
X-linked Dominant Inheritance (XD)

The disease is controlled by the allele XA. Healthy women have the genotype XaXa, healthy men
XaY. Afflicted are women with genotype: XAXA, XAXa, and men with genotype: XAY.
a) If a healthy woman has an afflicted partner (XaXa × XAY), then all sons are healthy (XaY)
and all daughters are afflicted (XAXa).
b) If an afflicted woman has a healthy partner, then there are two possibilities:
 this woman is a homozygote (XAXA × XaY) – all children are afflicted
 this woman is a heterozygote (XAXa × XaY) – 50 % of sons and 50 % of daughters are
afflicted
Examples of XD disorders: vitamin D resistant rickets; hypophosphatemia; incontinentia
pigmenti.
X-linked Recessive Inheritance (XR)

The disease is controlled by the allele Xa. Healthy women have the genotype XAXa (carrier), and
XAXA; healthy men XAY. Afflicted are men with genotype XaY, and women with genotype XaXa.
a) The majority of afflicted individuals are men: XaY.
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b) If afflicted man gets married to a healthy woman with XAXA genotype (XaY × XAXA), then
all their children will be healthy, but all daughters will be carriers (XAXa).
c) A woman-carrier who gets married to a healthy man (XAXa × XAY) has approximately one
half of sons afflicted.
Examples of XR disorders: G6PD deficiency, daltonism, haemophilia, Duchenne muscular
dystrophy, androgenetic alopecia.
4.5 Pedigree analyses

Analyze the following pedigrees. Determine the most probable type of inheritance: AD, AR, XD,
or XR. Try to match the pedigrees with some of the following diseases: thalasemia, daltonism,
polydactylia, Leber’s hereditary optic atrophy, Hungtington’s disease, phenylketonuria.
First, look at the ratio of afflicted men and women and if all generations are affected. In some
cases there is a part of pedigree, which is unique for certain type of inheritance; try to find it.
Pedigree 1
I.
II.
III.
IV.
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Pedigree 2
I.
1 2 3 4 5
II.
1 2 3 4 5 6 7 8 9 10
III.
1 2 3 4 5 6 7
IV.
1 2 3 4
Pedigree 3
I.
1 2
II.
1 2 3
III.
1 2 3 4 5 6 7 8
IV.
1 2 3 4 5 6
Pedigree 4
I.
1 2 3 4
II.
1 2 3 4 5 6 7 8
III.
1 2 3 4 5 6 7 8 9
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Pedigree 5
The afflicted woman (IV/2) in this pedigree suffers from achondroplasia (an AD disorder).
I.
1 2 3 4 5 6 7 8
II.
1 2 3 4 5 6 7 8 9
III.
1 2 3 4
IV.
1 2 3 4
4.6 Draw the pedigree

Healthy girl and girl with severe form of anaemia (which had required repeated blood
transfusions) were born out of a consanguineous marriage of second-degree relatives (both
healthy). In addition, before the daughters the mother had 2 miscarriages and one son was born
dead. Now she is pregnant again and asking, if the child will be healthy or ill and if it will depend
on the sex of the child. Draw the pedigree and suggest the type of inheritance and try to answer her
questions.
Control questions
1. Compare the X and Y chromosomes.
2. How is the sex determined in human?
3. What is colour-blindness and what is the pattern of inheritance of this disorder?
4. What is haemophilia? What types of haemophilia do you know? What is the type
of inheritance of haemophilia A?
5. How do sex-linked traits differ from sex-limited or sex-influenced traits?
6. What does pedigree analysis mean? What symbols does it use?
7. What are the main rules for pedigree formation?
8. Characterize the simplified rules for determination of the type of inheritance in pedigrees,
i.e.
a) What is typical for autosomal dominant inheritance?
b) What is typical for autosomal recessive inheritance?
c) What is typical for X-linked dominant inheritance?
d) What is typical for X-linked recessive inheritance?
e) Give examples of diseases or traits inherited by each type of the above mentioned
patterns of inheritance.
9. What do you know about the detection of carriers and homozygotes of pathological alleles?
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5. Gene Linkage and Gene Mapping
Localization of structural genes on chromosomes. Gene linkage. Gene mapping. Direct DNA
diagnostics. Chromosome mapping by using somatic cell hybrids.
Introduction
Localization of genes
Two genes can be located on two different chromosomes (asyntenic) or on the same chromosome
(syntenic). (Synteny describes the physical co-localization of genetic loci on the same chromosome.)
Asyntenic genes enter into gametes during meiosis independently; they combine freely – this is
called random assortment:
gametogonia
A a B b
S-phase
primary gametocyte
A Aa a
B Bb b
meiotic assortment
gametes:
B b B b
A A a a
genotypes of gametes: AB Ab aB ab
1 1 1 1
frequency: /4 /4 /4 /4
ratio: 1 : 1 : 1 : 1
location of genes: on different chromosomes

genotypes: AaBb
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Syntenic genes are passed into the gametes together. They cannot combine independently. These
genes are called linked. A set of genes on a given chromosome is called linkage group. The
number of linkage groups is equal to the number of pairs of homologous chromosomes in the cell;
in diploid organism. It equals to haploid number of chromosomes (in gametes, i.e. 23).
Diagram of complete linkage:

I. II.
gametogonia
A a A a
B b b B
S-phase S-phase
primary
A A a a gametocytes A A a a
B B b b b b B B
meiotic assortment
gametes:
A A a a A A a a
B B b b b b B B
genotypes of gametes: AB ab Ab aB
1 1 1 1
frequency: /2 /2 /2 /2
ratio: 1 : 1 1 : 1
location of genes: on the same chromosome on the same chromosome
genotypes: AB/ab Ab/aB
phase: cis (coupling) trans (repulsion)
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In diploid organism, alleles of linked genes are located on the maternal and paternal chromosome
of a given homologous chromosomal pair. In double heterozygote (AaBb) they can be in two
positions (phases), depending on which allele pair is syntenic.
Linkage phase cis (coupling)

Both dominant alleles of two different genes are on one homologous chromosome, both recessive
alleles are on the other homolog. Syntenic pair of alleles is, therefore: AB and ab.
A B
cis
a b
Linkage phase trans (repulsion)

One dominant allele of one gene is on one homologous chromosome with recessive allele of the
second gene; with the opposite being the true for the other homolog. Syntenic pair of alleles is
therefore: Ab and aB.
A b
trans
a B
Linked genes in phase cis (coupling) are written as: AB/ab, in phase trans (repulsion): Ab/aB.
Let us imagine that the fraction stands for the pair of homologous chromosomes: each
denominator and numerator has one pair of alleles, which together enter the same gamete. If there
is no crossing-over, the cis double heterozygote, therefore, forms two types of gametes AB, ab
with the same probability (0.5); the trans double heterozygote forms gametes Ab, aB with the
same probability (0.5).
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Change of the linkage phase of double heterozygote by genetic recombination
Crossing-over may occur between two allele pairs in the course of gamete formation. This will
cause their recombination and change of phase cis to trans and vice versa. The offspring, who has
linked genes in a recombined phase, is called a recombinant.
crossing-over
linkage phase cis st
(during 1 meiotic prophase)
A A a a A A a a
B B B b
b b B b
st
1 meiotic division is finished
linkage phase trans
A A a a A A a a
B b B B
B b 2
nd
meiotic b b
division
recombinant
gametes
The probability that recombination will occur is expressed as frequency of this event (%). The
probability is growing with the distance between the two allele pairs on chromosomes. If the two
genes are very distant to each other (located at the ends of the same chromosome), they may be
freely recombined (the frequency of recombination may be near 50 %).
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The strength of gene linkage
Complete linkage of two genes means that there is no crossing-over. Double heterozygote for
these two genes will not form recombined gametes. Complete linkage is a rare event.
If there is incomplete linkage, recombined gametes may be formed. Genes, which are
incompletely linked, can recombine. More often crossing-over occurs and more recombinants are
formed the strength of linkage is weaker. More distant allele pairs will recombine more often than
closer allele pairs. The frequency of recombination (expressed in percentage of recombinants) is,
therefore, dependent on the distance between the genes and it gives the linkage strength.
1 % of recombination ⇒ 1 % of recombinant gametes ⇒ 1 % of recombinants among the
offspring coming from a cross between a double heterozygote (cis or trans) with double
homozygote recessive ⇒ 1 cM (centimorgan, map unit). In this case the recombination occurred in
1 % of cells in the course of meiotic division. We can also say that these 2 genes are 1 cM away
from each other. Maximum of gametes with recombined genotype is 50 % (these genes combine
freely). If the distance between 2 genes is greater than 50 cM (which is true for the distant genes
on the long arms of chromosomes) the percentage of recombinant gametes does not augment
anymore.
The strength of linkage between two different genes can vary from 0 to 50 %.
The relationship between the frequency of recombination (in %) and the strength of gene linkage is
as follows:
0 % ………….......... 1-5 % ………......... 30-40 % ……....……........ 50 %
complete linkage strong linkage weak linkage free combination
The strength of gene linkage can be determined by a testcross

The table below shows how the frequency (%) of individual gametes genotypes changes in a cis
and trans heterozygote with different % of recombination. The original parental genotypes (non-
recombined) and their frequencies are underlined.
Genotype (phase)
recombination AB/ab (cis) Ab/aB (trans)
frequency (%)
gamete frequency (%) gamete frequency (%)
AB Ab aB ab AB Ab aB ab
0 50 0 0 50 0 50 50 0
...
10 45 5 5 45 5 45 45 5
50 25 25 25 25 25 25 25 25
As you can see the increase in recombination frequency between two allele pairs A/a, B/b leads to
an increase in the relative frequency (%) of gametes with recombined genotype and decrease in the
frequency of gametes with parental (non-recombined) genotype. This principle is used in test cross
of cis or trans double heterozygote with double recessive homozygote for the same genes. By this
cross the linkage phase and the strength of gene linkage can be determined by the ratios of
genotypically different offspring.
Explanation
If the recombination frequency is lower than 50%, the double heterozygote will form more non-
recombined gametes than recombined gametes. There will be more offspring with alleles in the
same phase as their heterozygous parents; the frequency of recombinants will be lower. The phase
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of alleles in the tested heterozygous parent can be determined based on the most frequent genotype
in offspring. The frequency of recombinants (%) coming out of this cross gives the strength
of linkage between the two genes. If expressed in cM, it also gives the distance between the two
genes on a chromosome (in a linkage group).
In the following examples, we assume that the probability of recombination during oogenesis and
spermatogenesis is the same (in fact it is higher during oogenesis).
I. Gene Linkage in a Testcross
5.1 Testcross
Complete the cross of AaBb × aabb with the assumption that both allele pairs are completely
linked a) in cis phase, b) in trans phase.
5.2 Testcross
Do the same cross of AaBb x aabb with the assumption that both allele pairs are a) incompletely
linked in cis phase, p=5 cM, b) incompletely linked in trans phase, p=5 cM. Give the frequency
of genotypes of the offspring from both crosses. Compare and explain the differences.
5.3 Gene Linkage in Drosophila

Alleles B/b control the body colour. The allele B (brown body) is completely dominant over the
allele b (black body). The allele Vg for normal wing development is completely dominant over the
allele vg for dwarfish wings.
The following individuals were obtained from a real hybridization experiment after cross of double
heterozygous fruit-flies with double recessive homozygous fruit-flies:
91 individuals with brown body and normal wings,
411 individuals with black body and normal wings,
413 individuals with brown body and dwarfish wings,
and 84 individuals with black body and dwarfish wings.
Determine their genotypes. Are the allelic pairs B/b and Vg/vg in one linkage group? Decide and
explain. Write the phase of the linked alleles. Calculate the number of map units between the
genes B and Vg.
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II. Gene Linkage in Human
5.4 Determination of Gene Linkage in a Family

We are able to determine gene linkage in families if:
1. one parent is a double heterozygote
2. the phase of the linked genes can be recognized from the family tree
3. we have information about at least three generations (minimum).
The disease in studied family is encoded by a rare dominant allele with complete penetrance (A).
The diseased locus is linked with gene locus for MN blood type, which possesses two codominant
alleles: M and N. Find possible recombinant(s) in the third generation and calculate the
approximate number of map units between both genes (with corresponding alleles A/a and M/N).
MN M
I.
MN M
II.
MN M M MN MN
III.
The principle:
1. Afflicted man II,1 received from his mother I,2 aM. He got AN from his father I,1. His
genotype is, therefore, AN/aM.
2. He passed AN onto his sons III,1 and III,5 and aM to his children III,2 and III,3.
3. Individual III,4 is very interesting. He could get only aM from his mother II,2. But he is
not afflicted (he cannot have A) and his blood group is MN. He had to get aN from his
father II,1 instead of expected AN.
4. The father II,1 did not have genes in phase aN. We assume that phase aN was formed by
recombination. The individual III,4 is, therefore, a recombinant.
5. The rest of the family members are non-recombinants, because the phase of the allele pairs
is the same as in their parents.
Give genotypes of all family members. What is the ratio of recombinants to non-recombinants in
children in this family? (1 out of 5). Give the linkage strength for both loci in the percentage
of recombinants. (20 %) This type of information is reliable only if obtained from a larger number
of families.
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5.5 Rhesus Factor and Elliptocytosis
Elliptocytosis is an inherited disorder or red blood cells. A mutation in a gene encoding for one
protein of cytoskeleton leads to an abnormal ellipsoid shape of red blood cells. Both Rh-positivity
and elliptocytosis are inherited as autosomal dominant traits. A Rh-negative healthy man II,1 got
married to a Rh-positive woman II,2 suffering from elliptocytosis. The woman's father I,1 is Rh-
positive and he suffers from elliptocytosis as well. Her mother I,2 is Rh-negative and healthy.
What is the prognosis concerning children's health if:
1. the D/d and E/e genes are completely linked
2. the genes are recombined in 3 % of the cases.
Are there any differences in (1) and (2) concerning the prognosis? Are there any differences in (1)
and (2) concerning the possibility to predict a child's genotype if the Rhesus factor of the fetus is
known?
I.
II.
III.
5.6 Daltonism and Hemophilia A

Colour-blindness (alleles C/c) and hemophilia A (alleles H/h) in one family.
Both of these disorders show X-linked recessive pattern of inheritance. Mother is a heterozygous
carrier of both diseases; alleles for both genes are in cis phase. Father has the recessive allele c and
dominant allele H on the X chromosome, i.e. he is colour-blind. They have one colour-blind son,
one colour-blind and hemophilic son, and a colour-blind daughter. Explain.
I.
Daltonism
Haemophilia
II.
5.7 Association of traits

Patients suffering from pernicious anemia have often blood group A. Is it enough to find
association (coupling) of two traits – disease and certain blood group – to say that the genetic loci
are linked? How can you prove the genetic linkage?
The use of gene linkage

Gene linkage is used for indirect DNA diagnostics. It can be used if it is impossible to detect the
causative mutation directly (direct DNA diagnostics) or if this mutation is not yet known. The
indirect diagnostics is based on the analysis of so called markers. Markers are tightly linked with
the mutant allele or with the causative allele.
For indirect DNA diagnostics the following conditions have to be fulfilled:
1. strong linkage between the mutation and marker, so that recombination is not probable
2. the family is informative, i.e. important family members are accessible and are
heterozygous for the markers
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3. the linkage phase is known or can be easily determined
4. no recombination occurs between the marker and the mutant allele
III. Localization of Human Genes
5.8 Localization of enzyme-encoding genes

Human somatic cells were fused with mouse cells. The following hybrid cell clones were isolated:
Clone 1 contains human chromosomes 1, 2, and 14,
produces human enzyme isocitratedehydrogenase 1
produces the human enzyme peptidase B
produces both of the above mentioned enzymes.
What can be said about the chromosomal location of the genes determining the production of the
above-mentioned enzymes?
5.9 Localization of HLA and P loci

Determine and explain chromosomal localization of human genes for histocompatibility (HLA)
and blood group P, if the cellular hybrid clones possess these features:
Clone 1 contains human chromosomes 5, 12, and 20 and
has neither the HLA antigen nor P.
Clone 2 contains human chromosomes 5, 6, and 17 and
has trait HLA and trait P.
Clone 3 contains human chromosomes 9 and 17 and
does not have trait HLA, has trait P.
Clone 4 contains human chromosomes 6, 12 and 20 and
has trait HLA and does not have trait P.
5.10 Selected Loci of Human Genome

Many databases with maps of human chromosomes describing loci and their alleles, gene linkage
and the precise location of genes associated with genetic diseases exist on the web. One of the
most extensive sites is National Center for Biotechnical Information (USA,
http://www.ncbi.nlm.nih.gov/). Here you can find database called Online Mednelian Inheritance in
Man (http://www.ncbi.nlm.nih.gov/sites/entrez?db=omim), where you can search for localization
of genes implied in human genetic diseases. The search is done either by the name of the genetic
disorder or by the name of the gene.
In addition, on the same website, there are data from the project, which mapped the human
genome (http://www.ncbi.nlm.nih.gov/science96/). There are figures of human chromosomes with
graphical demonstrations of selected important loci on human chromosomes.
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Example:
Search for: galactosemia
Location Symbol Title MIM # Disorder Comments Method Mouse
Galactose-1-
Galactosemia,
9p13 GALT phosphate 606999
230400 (3)
uridyltransferase
Search for: thalassemia
Location Symbol Title MIM # Disorder Comments Method Mouse
Sickle cell anemia (3);

Thalassemias, beta-
(3); Erythremias, beta-
(3); pseudogene
Methemoglobinemias, HBBP1
Hemoglobin
11p15.5 HBB 141900 beta- (3); Heinz body between
beta
anemias, beta- (3); HBG and
HPFH, deletion type HBD loci
(3); Thalassemia-beta,
dominant inclusion-
body, 603902 (3)
Control questions
1. Why are alleles of genes, which are localized on different chromosomes, assorted into
gametes independently during meiosis?
2. Explain the difference in segregation into gametes if alleles are located on the same
chromosome and if they are on different chromosomes.
3. Explain process, which is able to change this linkage.
4. Explain the coupling and repulsion phase.
5. Is there any correlation between a probability of recombination affecting certain alleles and
localization of those loci on a chromosome?
6. How can this relation be used in gene mapping?
7. Explain problems of this method in humans.
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6. Non-mendelian Inheritance
Interaction of non-allelic genes, multifactorial inheritance, heritability, twin studies, mitochondrial
inheritance.
In the past classes we have talked about inheritance of monogenic traits, where the Mendel’s laws
are applicable.
In this class we will concern on some examples of Non-Mendelian inheritance. For the inheritance
of these traits/disorders Mendel’s laws either can be applied in a modified form – non-allelic gene
interaction or can not be applied – multifactorial and mitochondrial inheritance.
I. Non-allelic Gene Interaction

Some qualitative traits can be determined by one or more genes (multigenes). The inheritance is
therefore sometimes called as multigenic inheritance. As well as for the monogenic inheritance the
expression of a trait encoded by multigenes is not influenced by environmental factors. The
relationship between two alleles of different loci is called the interaction of non-allelic genes or
intergenic interaction. Mendel’s laws of independent assortment and segregation are applied
without any changes. However the phenotypic ratios may differ from the phenotypic ratios
predicted by Mendel depending on the type of intergenic interaction.
There are several types of interaction of non-allelic genes, which are foremost known in animal
and plant genetics. It is highly probable, that some human traits that are considered to be
monogenic have a multigenic basis. To prove this statement, it is necessary to study segregation of
traits/disorders in large family cohorts.
I. A) Epistasis
Epistasis takes place when the action of one gene is modified by one or several other genes.
Dominant epistasis – dominant allele of 1 gene (epistatic gene) masks the phenotype encoded by
another gene (hypostatic gene).
Recessive epistasis – 2 recessive alleles of 1 gene (the epistatic gene) prevent expression of the
phenotype encoded by another gene (hypostatic gene).
6.1 Recessive epistasis in humans: the Bombay blood group

The ABO blood group is a single-gene trait with three basic alleles: A, B, and O. The phenotypic
expression of alleles A and B is influenced by the allelic pair H/h of another gene which is called
Bombay, because it was first described in this Indian city. The allele h in homozygous constitution
(hh) will not allow the phenotypic expression of alleles A and B (recessive epistasis). Allele H
encodes sugar polymer called antigen H, which is a precursor of polysaccharide antigens A and B.
If an individual is not able to make the “H” antigen (hh), even if the “A” or “B” enzymes are
present, they cannot make “A” or “B” antigen since there is no precursor for the antigens to act
upon.
An individual who cannot produce the "H" antigen (hh) will appear to have blood type “O”
even if A and/or B are present at ABO locus. The relative frequency of allele h in the population
is very low therefore this recessive epistasis is a very rare event. It is most common in tribal
populations in India.
a) A woman (blood group O) got married to a man (blood group A). The woman’s father who
has blood group O and woman’s mother who has blood group B were blood related. One
child (blood group AB) was born from this marriage. Give possible explanation.
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b) A man with blood group O (genotype OO) got married to a woman with blood group B
(genotype BB). Twins were born in this family with a blood group O. Give possible
genotypes of all family members and explain.
I. B) Duplicity
Duplicity means that a trait is determined by two or more pairs of alleles. One allelic pair is
enough for the expression of this trait; still the interaction with other allelic pairs influences the
intensity of the trait.
6.2 Duplicity and the inheritance of skin colour in human

The skin colour in humans is determined by several allelic pairs. We will simplify this situation
and use the Davenport’s hypothesis. According to Davenport’s hypothesis the inheritance of skin
colour is determined by two allelic pairs S/s, T/t. These two allelic pairs affect the trait additively.
The intensity of the skin colour depends on the total sum of dominant alleles on these two loci:
SSTT ... black skin
SSTt, SsTT ... deep brown skin
SsTt, SStt, ssTT ... brown skin
Sstt, ssTt ... light brown skin
sstt ... white skin
The parents: SSTT × sstt, SsTt × Sstt, ssTT × SStt. Predict the phenotypes of their children.
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II. Multifactorial inheritance
Traits and diseases determined by genotype and environmental factors exhibit multifactorial
inheritance. The trait/disease originates in people only with certain genotype (on one or more loci)
but with contribution of certain environmental factors with variable strength (e.g. diabetes
mellitus). The inheritance of these traits/disorders will not follow Mendel’s laws. Diseases such as
hypertension, ulcus duodeni, and asthma which are triggered by environmental factors but develop
only in people with genetic predisposition also belong into this group.
Quantitative traits as well as some qualitative traits (complex diseases) are determined by this type
of inheritance.
II. A) Quantitative traits

 are measurable characteristics
 e.g. height, blood pressure
 their variability in the population is continuous
 the frequency in the population follows the Gauss’ distribution
II. B) Qualitative traits – illnesses

 are not measurable characteristics
 e.g. diabetes, lip cleft
 their variability is not continuous – there are only two possibilities: ill, healthy
 Gauss distribution applies to disease predisposition
 limit model is applied: trait/disease will show in phenotype only if the predisposition will get
over a certain limit
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Diseases with multifactorial inheritance are very common and they are the most common causes
of human morbidity and mortality. These diseases occur in families, but only the predisposition to
the disease (degree of susceptibility to the disease) is being inherited. Even in one family the
severity of clinical symptoms of the disease will vary according to the influence of outer
environmental factors. It is also very common to have differences in the percentage of afflicted
men and women. In the following examples you will learn how to determine the influence
of genetic and environmental factors on the development of multifactorial inheritance.
Heritability and Genetic Twin Studies

Very appropriate method of discerning genetic influences from environmental influences is the
genetic study of twins. The method of genetic analyses of twins makes possible to estimate genetic
and non-genetic portion of variability of a trait.
Monozygotic (MZ) twins have the same genotype because they originated during cleavage
division from one zygote.
Dizygotic (DZ) twins have different genotypes as any brothers and sisters, because they originated
by fertilization of two eggs by two sperms.
The twins may be either concordant or discordant in terms of having or not having certain trait.
The concordance means both twins have the trait. The discordance means that one twin has the
trait and the other does not.
Concordance means the frequency of phenotypically same twin pairs in a given cohort in
percentage. By comparing these concordances and disconcordances we can determine the
heritability of a given trait. The pairs of the same sex must be included in compared group of DZ
twins.
If the trait is influenced predominantly by genetic factors:
 MZ twins will be phenotypically identical (the gene must have complete penetrance)
 DZ twins may differ (similarly to sib-pairs)
If the trait is modified by environmental factors and the effect of genotype is less significant, then
the size of congruence will be the same in MZ and DZ twins.
If the MZ twin concordance is high (over 80 %) and twice higher than DZ twin concordance, then
we presume high genetic influence on the trait or disease. We can say that this trait is determined
mainly genotypically.
Heritability is a value, which states to what degree the phenotypic trait is determined by genotype
or outer environmental factors. Heritability is expressed as h2.
The value of h2 varies from 0 (when the phenotype is not determined by genotype) to 1 (when the
phenotype is determined only by genotype). In order to simplify these estimations we use
parameter H, which does not directly equals to heritability, but highly positively correlates with
heritability.
The estimation of genetic and environmental influence on the origin of the trait using the
H statistic:
C MZ − C DZ
H=
100 − C DZ
CMZ ... proportion of concordant pairs of MZ twins (in %)

CDZ ... proportion of concordant pairs of DZ twins (in %)
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The H parameter varies from 0 to 1. If the value is near 0, environmental factors influence the trait
variability predominantly. If the value is close to 1 (the number of congruent MZ twins is close to
100 %), the genotype influences the trait variability predominantly.
H = 0..........………………………………….........1
effect of environmental factors effect of inherited factors
Nota bene:
Even though the twin studies are a very useful tool for analysis of genetic and environmental factors influences, it is
important to be careful with interpretations because of the following reasons:
1. MZ female twins will not have the same expression of X-linked genes in different tissues due to random
X chromosome inactivation even if they have absolutely the same genotype. Heterozygous women can even
have a different phenotype.
2. Most recent studies have shown that the genetic information of MZ twins differs on the epigenetic level i.e.
not in DNA sequence, but in DNA modifications (e.g. by base methylation), which does not change the
primary structure, but influences the gene expression.
3. An extreme case is that the disease is not genetically based, rather there are nongenetic phenocopies.
(A phenocopy is an individual whose phenotype (generally referring to a single trait), under a particular
environmental condition, is identical to the one of another individual whose phenotype is determined by the
genotype. In other words the phenocopy mimics the phenotype produced by a gene. An example of a
phenocopy is a person whose anti-psychotic medication causes them to manifest the same symptoms as the
genetically determined Parkinson disease).
Table of concordance of some diseases

Concordance (%)
Disease
MZ DZ
Non-traumatic epilepsy 70 6
Multiple sclerosis 17.8 2
Diabetes mellitus 40 4.8
Schizophrenia 53 15
Osteoarthrosis 32 16
Rheumatoid arthritis 12.3 3.5
Psoriasis 72 15
Lip cleft (not palate) 30 5
Systemic lupus erythematodes 22 0
6.3 Congenital hip luxation

The congenital hip luxation was examined in newborns. In a set of 290 pairs of MZ twins,
concordance existed in 120 pairs. In addition, concordance existed in 9 pairs in a set of 327 DZ
twin pairs. Evaluate the influence of genetic factors and non-genetic factors on this disease.
6.4 Hypertension heart disease

The concordance in hypertension heart disease existed in 120 MZ twin pairs in a cohort of 340
twin pairs. The concordance in DZ twin pairs was 12 in a cohort of 436 twin pairs. Evaluate the
influence of genetic and non-genetic factors to the development of this disease.
6.5 Schizophrenia
In a cohort of 378 MZ twin pairs, the concordance in schizophrenia was found in 283 pairs.
In a cohort of 985 DZ twin pairs the concordance existed in 147 pairs. What can you say about the
influence of genetic and environmental factors on the development of this disease?
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6.6 Eight diseases
Concordance rates in MZ twins and DZ twins found in some diseases are noted in the following
table. Arrange these diseases according to contribution of genetic factors to the disease
development. Evaluate the practical meaning of this finding in prevention of the diseases
development.
Concordance (%)
Disease
MZ DZ
Congenital hip luxation 41 3
Hypertension heart disease 53 23
Schizophrenia 53 15
Basedow’s disease 47 3
Diabetes mellitus 40 4.8
Rheumatic fever 33 10
Tuberculosis 54 27
Asthma bronchiale 63 38
III. Mitochondrial Inheritance

The inheritance of a trait encoded in the mitochondrial genome is called mitochondrial inheritance.
Mitochondrial DNA is transmitted in a matrilinear manner (meaning from mother to children),
because all of the mitochondria in the fertilized egg come from the egg. Persons with a
mitochondrial disease may be male or female but they are always related in the maternal line and
no male with the disease can transmit it onto his children. The daughters will pass the
mitochondrial DNA onto their children. The sons will not.
 affected women transmit the disease to all offspring
 affected men transmit the disease to none of their offspring
Example of a pedigree of a family suffering from a disease caused by a defect in

mitochondrial DNA:
I.
II.
III.
IV.
All diseases resulting from changes in mitochondrial DNA are fundamentally the result of
malfunctions of the respiratory chain for oxidative phosphorylation. The phenotypic effects of
mitochondrial mutations reflect the extent to which a tissue relies on oxidative phosphorylation;
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the central nervous system is the most sensitive, followed by skeletal muscle, heart muscle,
kidney, and liver. Some typical syndromes are encephalopathy, myopathy, ataxia, retinal
degenerations and loss of function of outer oculomotor muscles.
Examples of mitochondrial diseases:

 Leber's Hereditary Optic Neuropathy (LHON) – quick necrosis of optical nerve leading to
loss of vision in early adulthood, cardiac dysrhythmia
 Myoclonic Epilepsy and Ragged Red Fiber Disease (MERRF) – central nervous system
abnormalities and deficiencies of skeletal and cardiac muscle function
 Kearns-Sayre Syndrome – neuromuscular symptoms including paralysis of eye muscles,
dementia, and seizures
6.7 Draw a pedigree

A women suffering from certain mitochondrial disease got married to a healthy man. They had one
son. This son got married to a healthy woman and they had 2 sons and 2 daughters. Draw the
pedigree and determine healthy and afflicted family members.
Control questions
1. What is heritability?
2. What information do we get by determining the heritability?
3. Describe the origin of monozygotic (MZ) and dizygotic (DZ) twins. Compare possible
differences in genotypes.
4. What pairs of twins are concordant and what pairs of twins are discordant?
5. What is the “H parameter”? What information does it give us?
6. Describe the procedure of calculation of the parameter H.
7. Explain the purpose of genetic analyses of twins.
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7. Genetic control of metabolism
Metabolic disorders, inborn errors of metabolism, enzymopathies, hemoglobinopathies.
Introduction
The chemical reactions in metabolic pathways are enabled by enzymes. The protein part of each
enzyme is encoded by at least one structural gene. Genetic control of metabolism is based on the
fact that enzymes, structural, and regulatory proteins are being synthesized based on the genetic
information. These proteins then influence the process of synthesis and decomposition of chemical
parts of cell and organism.
Genes and metabolic pathways

Individual structural genes (e.g. G1 to G4) encode specific enzymes (E1 to E4), which are required
for individual steps in chemical transformations in a given metabolic pathway:
genes: G1 G2 G3 G4
enzymes: E1 E2 E3 E4
metabolic
substrate M1 M2 M3 final product
pathway:
The results of activity of enzymes are metabolic products – metabolites (M1-M4) which are
formed in characteristic order in individual pathways. There are many different metabolic
pathways in an organism. They are connected in a harmoniously and efficiently working network,
which represents the biochemical background of an organism.
Metabolic disorders
1. Enzymopathies. They arise due to a specific mutation of a structural gene encoding for a
specific enzyme. The result is decreased or completely lost activity of that given enzyme in the
body. Clinical symptoms of the lack of an enzyme leading to a block of metabolic pathway are
most often caused by the lack of the product of this pathway, excess of the substrate and its
deposition in cells, or by toxic metabolite, which is produced by an alternative metabolic pathway
(Fig.1).
Figure 1: Possible outcomes of enzymopathies
A
Substrate
B × C
Product
deficiency
excess
Toxic
D metabolite
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Enzymopathies exhibit recessive inheritance, i.e. only recessive homozygotes will be afflicted. The
heterozygotes for an enzymopathy can be determined on the biochemical level. Heterozygote,
though healthy, has a half of the activity of the affected enzyme than dominant homozygote. The
heterozygote has only one functional allele able to produce a functional enzyme, homozygote
(AA) has two. The determination of heterozygote (healthy carrier, Aa) can be of utmost
importance for genetic prediction of affliction of children in a family with history of enzymopathy.
The most well-known enzymopathies are:
 phenylketonuria (PKU) – defect of metabolism of aminoacid phenylalanine
 galactosemia – defect of galactose metabolism
 glucose-6-phosphate dehydrogenase deficiency (G6PD) – defect in NADPH production in
pentose cycle.
2. Metabolic disorders can also arise due to mutation, which does not directly influence any
enzyme production. An example is a defect in lipoprotein metabolism – familial
hypercholesterolemia (FH). Genetic basis for this disease is a mutation in the gene encoding
receptor for plasmatic low density lipoprotein (LDL-R, Low Density Lipoprotein Receptor). This
receptor plays a key role in maintaining cholesterol homeostasis in the body. FH inheritance is
autosomal semidominant.
3. Diabetes mellitus (DM) is the most well known metabolic syndrome. As far as etiology is
concerned, it is a heterogeneous group of disorders, with a common feature of hyperglycemia
(abnormally high sugar concentration in blood). The glucose levels in blood are controlled by a
hormone: insulin, which is produced by beta-cells in pancreas.
Two most common forms of diabetes, diabetes mellitus type 1 (DM type 1, insulin dependent)
and diabetes mellitus type 2 (DM type 2, non-insulin dependent) display multifactorial
inheritance.
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7.1 Metabolism of Phenylalanine
Phenylketonuria is one of the most common inherited disorders (AR). It occurs in babies who
inherit two mutant alleles for the enzyme phenylalanine hydroxylase (PAH). This enzyme
normally breaks down molecules of the amino acid phenylalanine that are in excess of the body's
needs for protein synthesis. Because we inherit two copies of the gene for the enzyme, both must
be defective to produce the disease. The person with one “healthy” allele is perfectly healthy
because the non-mutated allele produces sufficient level of the enzyme. However, these
heterozygous individuals are “carriers” of the disease.
A laboratory test that measures how quickly an injection of phenylalanine is removed from the
blood can distinguish a person who has one PKU allele (heterozygote or carrier) from a person
who has none (healthy homozygous individual).
a) In Figure 2, you can see results from the phenylalanine tolerance test in a group of people
with no clinical symptoms. What do curves A and B stand for?
Figure 2: Phenylalanine tolerance test
b) A child inherits two recessive alleles for phenylketonuria (one from mother and one from
father). He/she is a recessive homozygote (aa). Is it possible to protect the child from
falling ill by phenylketonuria? Which metabolites will be accumulating (see Figure 3)?
Which will be missing? Why has the ill child excessively fair hair and skin? What are the
treatment options for the child?
Figure 3: Phenylalanine metabolic pathway
gene PAH
tyrosine melanine
enzyme PAH
phenylalanine
enzyme A phenylpyruvic acid
gene A
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7.2 Lactose Digestion
Lactose (milk sugar) is digested to glucose and galactose by an enzyme lactase in small intestine.
Lactase activity is most important in children in nursing age (because of high content of lactose
in maternal milk) and it diminishes with the age. Both monosaccharides are then metabolized by
other enzymes.
The lack of enzyme galactose-1-phosphate-uridyl-trasferase (GALT), which metabolizes
galactose, causes galactosemia. Pathological metabolites (galactitol, galactose-1-phosphate) are
accumulating in blood and tissues because of abnormal function of GALT. This leads to damage
of different organs: liver, kidney, etc., and to cataract. Restriction of lactose in diet (as a main
source of galactose) stops the production of excess of metabolites and thus protects many organs
from damage. Galactose is, as other carbohydrates, essential for each cell function and for the
function of the whole organism. Each cell in the body is therefore able to manufacture galactose
by itself by so called endogenous galactose production.
Figure 4: Lactose digestion and galactose metabolic pathway

lactose
lactase lactose intolerance
glucose + galactose
GALT galactosemia
uridin diphosphogalactose
(UDPG)
Galactosemia can sometimes be mistaken for a much milder metabolic disorder: lactose
intolerance (Fig. 4). This is a pathology caused by lower production or complete lack of lactase.
Lactase enables the digestion of lactose to monosaccharide. Milk consumption in the affected
individuals leads to floating and flatulency. These symptoms quickly subside if milk and milk
products are restricted from the diet.
1. Even very firmly restricted diet cannot prevent the organ damage in galactosemia. Try to
explain it. Compare to PKU.
2. Why are some individuals with lactose intolerance able to digest some types of yogurt and
other fermented milk products?
7.3 Glucose-6-Phosphate Dehydrogenase Deficiency

Glucose-6-phosphate dehydrogenase (G6PD) is an enzyme, which catalyses the initial step in
pentose phosphate cycle, i.e. in the process of NADPH production. NADPH production is required
for reduction of glutathion. Reduced form of gluthation protects the cells against oxidative stress
(it removes H2O2). This process is very important for red blood cells (RBC), because it is the sole
protection for RBC against the oxidative damage. Excessive production of H2O2 is associated with
consumption of certain drugs and food (most notably broad beans) as well as with some illness
(e.g. severe infections). People with lower capacity of G6PD under these conditions lack the
necessary protection of RBC against oxidative damage, which leads to damage of RBC integrity
and their hemolysis. Typical drugs causing oxidative stress are: antimalaric drugs, sulfonamide
antibiotics, nitrofurantoin, chloramphenicol, vitamin K, chinidine etc.
1. Why is this disease more common in malaria countries?
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2. What is the mode of inheritance of this deficiency? Can an afflicted father have a healthy
child?
7.4 Familial Hypercholesterolemia (FH)

Cholesterol is transported in the bloodstream by lipoproteins. One type of lipoproteins is called
Low Density Lipoprotein – LDL. In order to get into the cells the complex of LDL and cholesterol
requires a specific LDL receptor (LDL-R). LDL-R mutations stop the entry of LDL-cholesterol
from plasma to cells and lead to increase of its concentration in plasma, which is one of the factors
contributing to the development of atherosclerosis.
1. What are the consequences of higher levels of LDL cholesterol in blood?
2. Can you state the diagnosis of FH only based on the increased levels of cholesterol in the
bloodstream?
3. Explain semidominant inheritance.
7.5 Diabetes Mellitus

Two most common types of diabetes are at least partly inherited, more precisely; the genetic
predisposition to the development of this disease is inherited.
1. DM type 1 (insulin-dependent) is caused by decreased insulin production. The ultimate
cause is the damage of beta cells of Langerhans islets of pancreas due to autoimmune
inflammation.
2. DM type 2 (non-insulin-dependent) is caused by decreased ability of an organism to react
to insulin (so called insulin resistance). This type represents about 90% of all DM cases.
DM type 1
Today, there are about 20 candidate loci identified, which are associated with increased risk
of development of this disease. The most well known is the association with some HLA antigens
(HLA DR3, HLA DR4). Polymorphisms of insulin gene, mutations in TAP1 and TAP2 genes
(which encode for cellular transporters) also belong to this group.
Environmental factors play a key role in the development of DM type 1; foremost viral infections,
stress, some chemicals (nitrosamines from smoked food), premature consumption of cow’s milk
products and overall structure of the diet.
DM type 2
The influence of genetic factors is stronger than in DM type 1. Candidate genes can be divided
into 2 groups:
a) genes that influence the function of beta cells in pancreas: ABCC8 and KCNJ11 genes
encoding proteins, which are important for potassium channel function; SLC2A2 gene
encoding a glucose transporter; and insulin gene
b) genes that influence insulin activity: insulin receptor gene (INSR); genes encoding
proteins, which are important in insulin signaling pathway (PIK3R1 and SOS1)
Some of the risk environmental factors are: obesity, inappropriate diet, not enough physical
activity.
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Diabetes with monogenic inheritance
About 1-5 % of DM cases are single-gene disorders (with monogenic inheritance). There is a clear
proof of mutation of a concrete gene causing the development of diabetes without any effect
of environmental factors. These mutations are in genes encoding proteins, which influence insulin
production or insulin ability to lower the glucose levels in blood.
1. MODY („Maturity Onset Diabetes of the Young“) displays autosomal dominant
inheritance. The patients are not dependent on insulin. There are 6 MODY types
according to the causative mutation. Five types are caused by mutation in transcription
factors: MODY 1 (HNF-4 alpha), MODY 3 (HNF-1 alpha), MODY 4 (IPF-1), MODY 5
(HNF-1 beta), and MODY 6 (NEUROD1). One type, MODY 2, is caused by a mutation
in glucokinase.
2. Neonatal Diabetes Mellitus – NDM is a very rare form of diabetes with the onset of the
disease during the first 6 months of life. Patients do not produce enough insulin and NDM
may be mistaken for DM type 1. There are different types of NDM according to causative
mutations. These mutations can exhibit autosomal dominant, autosomal recessive or X-
chromosome linked pattern of inheritance.
Questions
1. Can we generally say that diabetes is inherited in families? Explain.
2. Can we influence the development of diabetes?
3. Genetic twin studies have shown that concordance of DM type 2 is in monozygous twins
nearing 90 % (in DM type 1 it is < 50 %). One of the monozygous twins suffers from DM
type 1. What is the probability, that the other twin will also develop DM? What is the
answer, if the first twin suffers from DM type 2?
Simplified summary of the most common DM forms

DM type 1 DM type 2 MODY
onset youth adulthood youth
multifactorial multifactorial
genetics AD
(predisposition) (predisposition)
autoimmune
yes no no
process
insulin resistance no yes no
connected with
no yes no
obesity
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8. Population Genetics
Genetic structure of population. Hardy-Weinberg (HW) law. HW law application to real
populations. Direct estimation of allele frequencies. Evolution factors. Inbreeding.
Introduction
Population genetics is the study of the allele frequency distribution and change under the influence
of the four evolutionary forces: natural selection, mutation, migration, and genetic drift.
Population in Hardy-Weinberg equilibrium

The distribution of allele frequencies to genotypes follows the Hardy-Weinberg law in an ideal
population, which fulfils the following criteria:
1. panmixia (panmixy: interbreeding without limitations)
2. sufficient size of population
3. no evolution factor is active
If a population is in Hardy-Weinberg equilibrium, then there is a simple interrelation of the
frequencies of alleles and genotypes:
(p + q)2 = p2 + 2pq + q2
p: frequency of the dominant allele A, or XA
q: frequency of the recessive allele a, or Xa
p2: frequency of dominant homozygotes (AA, or XAXA)
2pq: frequency of heterozygotes (Aa, or XAXa)
q2: frequency of recessive homozygotes (aa, or XaXa)
where p+q = 1
This panmictic combination of alleles stabilizes the population in an unchanging state of HW
equilibrium and it does not allow evolution to occur.
Population with genetic fond evolution

As mentioned above, the evolutionary forces act against the HW equilibrium. This can lead to
distribution of new alleles, loss of other alleles and formation of new genotypes. The genetic
structure of population is thus changed.
How to solve the tasks?

For simplicity, we will assume that population is in HW equilibrium and that allele frequencies
follow the binomial distribution for all given examples.
A) Autosomal Inheritance
In a population there are two alleles for a certain gene, i.e. A and a with a complete dominance and
recessivity. Let us assume that individuals with dominant phenotype (i.e. with genotype AA or Aa)
are present in a population with 84% frequency, and individuals with recessive phenotype
(genotype aa) with 16% frequency. Calculate the frequencies of individual alleles and the
frequencies of dominant homozygotes and heterozygotes.
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Solution:
1. The frequency of recessive homozygotes is already given: q2 = 16 % = 0.16.
2. The best way to approach this task is to calculate the frequency of recessive allele a with
value q. You can calculate it from the frequency of recessive homozygotes (q2):
q = √0.16 = 0.4.
3. Because there is a rule that p + q = 1, then the frequency of the second allele A is equal to:
p = 1 – 0.4 = 0.6.
4. The frequency of dominant homozygotes and heterozygotes can be calculated as follows:
Genotype: AA Aa
Frequency: p2 2pq
Calculation: (0.6)2 = 0.36 = 36 % 2 × 0.6 × 0.4 = 0.48 = 48 %
B) X-chromosome linked inheritance

There are two alleles XA, Xa located on the heterologous region of X chromosome, which are
defined with complete dominance and recessivity. The frequencies of genotypes in a population
are:
Genotype XAXA XAXa XaXa XAY XaY
2 2
Frequency p 2pq q p q
For X-linked recessive traits it is possible to calculate the frequency of women carriers (XAXa),
who are phenotypically indistinguishable from women with constitution XAXA. The easiest way
how to evaluate the Xa allele frequency is to look at the frequency of men with genotype XaY,
which can be phenotypically distinguished. Men XaY are hemizygotes (possess only one allele
of a given gene). Therefore if they posses one recessive allele Xa in the genotype, they express the
recessive phenotype. This means that the frequency of recessive phenotype in males equals to the
frequency of recessive allele (Xa) = q.
Simplified application of HW law in case of rare alleles

This approach simplifies the estimation of frequency of heterozygotes in a population. It is used
only with rare alleles with frequency lower than or equal to 0.01. If q = 0.01, then p = 0.99,
therefore p can be rounded up to 1. There are three basic rules for the limited form of HW law.
The following rules are modified for rare alleles with AR diseases.
1. Most rare recessive alleles are present in heterozygotes in a given population, where they
are not expressed in phenotype.
2. If we evaluate randomly chosen sample from a population (100s of individuals), we will
only come across healthy individuals (AA, Aa). The possibility to find an afflicted
individual is very low, because the frequency of the afflicted recessive homozygotes (aa) is
lower or equal to 0.012.
3. The frequency of heterozygotes (Aa) is two times the frequency of recessive allele, i.e.
2q.
The frequency of dominant homozygotes (AA) is equal to 1 – 2q.
Example: The frequency of afflicted individuals with phenylketonuria is (q2) = 3×10-5,
q = 0.007, the frequency of heterozygotes is 2q = 0.014.
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I. Interbreeding
I. A) Autosomal Genes in a Population
8.1 Heredity of Rhesus (Rh) factor

The Rh factor belongs to a group of antigens found on the surface of red blood cells. There are
16% of Rh- people (dd) in the Czech Republic.
a) What is the frequency of Rh+ dominant homozygotes (DD) and Rh+ heterozygotes (Dd)?
b) What is the frequency of a marriage of Rh+ (DD) man and Rh- (dd) woman in this
population? (The fetus and the mother are Rh factor incompatible. In this case all children
are in danger of fetal erythroblastosis.)
c) Answer the same question if there are 0.9% of Rh- people in the population. Which
population possesses higher risk of Rh factor incompatibility?
Comment: Erythroblastosis Fetalis (Haemolytic Disease of the Newborn)
If blood of an Rh+ person enters the body of an Rh- person then the host immune system recognizes the entering red
blood cells as foreign material which has to be destroyed. The immune system will form anti-Rh antibodies, the
sensitivity of the host immune system towards Rh+ material increases and any new entrance of Rh+ blood induces
immune response resulting in a powerful lytic reaction of the entering cells. If an Rh+ fetus is carried by an Rh-
mother, some of the Rh+ blood cells may enter the mother's circulatory system and induce reaction of the mother’s
immune system. The immune reaction starts if a minimal amount of the fetal blood passes the placental barrier, which
separates the fetal and the mother's vessels. The fetal cells that enter the maternal circulation will be destroyed. This
happens only in a few cases and usually in later stages of pregnancy due to the movements of the fetus or in the course
of delivery. If it is so, there is not enough time for the mother's immune system to produce significant amount of
antibodies before the birth of the child. A possible subsequent pregnancy is in higher risk if sensitization took place in
the first Rh+ pregnancy, as the mother's immune system is often already induced (sensitized) and the antibodies are
able to pass the placenta and attack and lyse the red blood cells of the fetus. This may result either in delivery of a
child with anemia and erythroblastosis (the erythroblasts are precursors of red blood cells) or even in a miscarriage or
a birth of dead child. Because of this, all pregnant women should be tested for Rh factor. If a pregnant woman is Rh-,
then the father is tested. If he is Rh+, the pregnancy needs to be followed carefully by the physician to prevent
possible induction of the mother's immune system and to save the fetus if this happens. Rh immunoglobulin (RhIg) is
a blood product that can prevent an Rh- mother from being sensitized. It prevents her body from responding to Rh+
blood cells of the fetus.
8.2 Heredity of Eye Colour

For simplicity we assume that only one allelic pair controls the eye colour. The allele for brown
eye colour (B) is dominant over allele for blue eye colour (b). There are 36 % people with blue
eyes and the rest with brown eyes in a given population.
a) What is the frequency of the brown eye colour allele in this population?
b) How many people with brown eyes are homozygotes and heterozygotes?
8.3 Albinism
Albinism is an autosomal recessive disorder of melanin synthesis. It occurs in one out of 10,000
individuals in a population. What is the frequency of albinism causing allele and albinism carriers
in this population?
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8.4 Cystic fibrosis (CF) – mucoviscidosis
In the Czech Republic each 3250th newborn suffers from CF; in Finland each 25000th newborn is
affected. CF is inherited as an autosomal recessive disorder.
a) What are the frequencies of the defective allele in the Czech and in the Finnish population?
b) What are the frequencies of the heterozygotes (healthy but in danger to give birth to sick
children) in these populations?
Comment: CF causes the body to produce thick, sticky mucus that clogs the lungs, leads to infection, and blocks the
pancreas, which stops digestive enzymes from reaching the intestine.
In the case of the respiratory tract glands, the ciliary system on the cells surface is unable to remove the viscous
recrement from the lungs, this stays in the lungs and bacteria with inhaled particles are concentrated in the lungs
instead of being removed into the gullet, swallowed and killed by the stomach acid. The consequences are chronic
inflammatory processes and degeneration of the lungs. The pancreas is also affected, and undergoes degeneration.
Degeneration of these two essential systems limits the survival of the affected people. The sooner the therapy is
initiated the longer and better the life can be. Today, the disease gene is known and is used for prenatal diagnosis.
8.5 Phenylketonuria (PKU)

PKU is an autosomal recessive disorder. In a population there is about one afflicted individual out
of 10,000 healthy people.
a) Give the allele frequency for the causative allele of this disorder in this population.
b) What percentage of population possesses the defective allele in a heterozygous
constitution?
c) Why is this information (b) important for healthcare?
8.6 Other autosomal recessive diseases

The frequency of the recessive allele in a population is:
a) glaukom................................0.014
b) galactosemia ........................0.004
c) retinitis pigmentosa .............0.005
Calculate the frequencies of corresponding dominant alleles, heterozygotes and afflicted
individuals in this population.
8.7 Sickle Cell Anemia

Allele A encodes the synthesis of normal beta-globin chain; allele S encodes the synthesis
of abnormal beta-globin chain. The sickle cell anemia is inherited in the following way:
SS ... ill (abnormal Hb, anemia)
AS ... without any clinical symptoms (normal and abnormal Hb)
AA ... a healthy individual (normal Hb).
In different regions of Africa, two different populations were studied. The frequency of allele S=
0.10 in a population I. The allele frequency S = 0.41 in population II.
a) What percentage of population suffers from sickle cell anemia in population I and II?
b) What percentage of population does not have any signs of anemia but produces normal and
abnormal Hb in population I and II?
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8.8 Large canine teeth
Large canine teeth can be found in 2 % of Germans. The inheritance of this trait is autosomal
dominant. What are the frequencies of alleles and genotypes?
8.9 Cleft palate

In a population each 2500th child is born with cleft palate. The defect is inherited in an autosomal
dominant fashion. What can be said about the allele and genotype frequencies? What can be said
about the conditions under which the Hardy-Weinberg law is applicable in this case? Is there any
difference between the situation 100 years ago and now (medical care)? Compare the answers with
the case of large canine teeth.
I. B) X-chromosome linked inheritance
8.10 Daltonism (Colour Blindness)

Daltonism is an X-linked recessive disorder. It occurs in 8 % of males in a certain population.
a) Determine the carrier frequency in the population.
b) What is the frequency of women affected by daltonism in this population?
8.11 Haemophilia
In haemophilia A, 2 out of 10,000 men suffer from the disease, in haemophilia B, 4 out
of 100,000 men are sick.
a) What are the carrier frequencies for haemophilia A and B?
b) What are the frequencies and real numbers of women suffering from haemophilia A or B
in the population of the Czech Republic (10,000,000 inhabitants)?
8.12 Glucose-6-Phosphate-Dehydrogenase (G6PD) Deficiency

In a Black African population there are 7 % of men suffering from enzymopathy (G6PD
deficiency). In white Caucasian race there were only 1.5 % of afflicted men. Defect is XR.
a) Count the frequency of women carrier in both populations.
b) What percentage of women suffering from this disorder can you expect in these
populations?
Control questions
1. What is the definition of genotype frequencies, and allele frequencies in a population?
2. What is “random mating” in a population?
3. Explain the Hardy-Weinberg law.
4. What is the interrelation of the frequencies of alleles and genotypes according to Hardy-
Weinberg law?
5. What are the conditions under which the Hardy-Weinberg law is applicable?
6. Explain the specificities of Hardy-Weinberg law application to X-linked genes.
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II. Inbreeding, consanguineous marriages, coefficient of inbreeding
Inbreeding is breeding between close relatives and leads to a reduction in genetic diversity and to
an increase in homozygosity (the same allele at the same locus on both chromosomes in a
chromosome pair). The inbred individuals are more likely to show physical and health defects,
including:
 loss of immune system function
 reduced fertility both in litter size and sperm viability
 increased genetic disorders
 fluctuating facial asymmetry
 lower birth rate
 higher infant mortality
 slower growth rate
 smaller adult size
(Inbreeding is also used in animals and plants. E.g. livestock breeders often practice inbreeding to “fix” desirable
characteristics within a population. However, they must then cull unfit offspring, especially when trying to establish
the new and desirable trait in their stock.)
Inbreeding in humans
If two individuals with a common ancestor have children, the children have higher degree
of homozygosity. This is particularly important because of recessive disorders.
In consanguineous marriages, there is higher risk that the children will suffer from diseases
with recessive inheritance, because there is higher probability that both parents will carry
a mutated deleterious allele inherited from the common ancestor.
The possibility of inbreeding in humans may be augmented by genetic isolates: linguistic,
religious, and geographical barriers.
Incest is a sexual intercourse between closely related individuals. In most countries it is prohibited
by law although the definition of prohibited degree of kinship varies.
Diseases and royalty

The royal and noble families of Europe have close blood ties which are strengthened by royal
intermarriage, very often for example in the House of Habsburg. This may lead to acute inbreeding
and phenotypic manifestation of recessive traits. Famous in this case is the Habsburger (Unter)
Lippe (Habsburg jaw/Habsburg lip/ “Austrian lip”), typical for many Habsburg relatives over a
period of six centuries. The condition progressed through the generations to the point that the last
of the Spanish Habsburgs, Charles II of Spain, could not properly chew his food (mandibular
prognathism / recessive trait controlled by multiple genes).
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II. A) Coefficient of Inbreeding (F) (coefficient of kinship)
It is the probability that homozygote has received both alleles from the same ancestral source at a
given locus (follow allele A1m in Fig. 1); it is also the proportion of loci at which a person is
homozygous or identical by descent. The coefficient of inbreeding is a way of judging how close
two people are genetically related to one another. In cases of common ancestors, coefficient of
inbreeding explains the homozygosity for deleterious alleles. These alleles are replicated and
transmitted through both lines of descent (p=paternal, m=maternal) to the offspring with certain
probability.
Figure 1
A1mA2m A1pA2p
I.
A2A2 A1mA2p A1mA1p A1A2
II.
A1mA2 A1mA1
III.
A1mA1m
IV.
Let’s say, that the common ancestor (I.1) has genotype A1mA2m (m as maternal). The product of
consanguineous union (IV.1) between III.1 and III.2 has homozygous genotype A1mA1m. Both of
these two alleles are originally from I.1. The transmission of allele A1m is depicted in bold) If
there are two common ancestors, there is a maximum of four different possible homozygous
genotypes in the offspring IV.1: A1mA1m, A2mA2m, A1pA1p, or A2pA2p (p as paternal).
The calculation of coefficient of inbreeding:

F = S × (1/2)P + M
S stands for the highest possible number of different homozygous constitutions, which could arise
in the offspring. If there is one common ancestor S=2, if there are two common ancestors S=4 etc.
Formally we can say that the value of S is two times the number of common ancestors.
P is the number of generations from the common ancestor to the offspring on the father’s side.
M is the number of generations from the common ancestor to the offspring on the mother’s side.
1/2 is the probability that the offspring will get a concrete allele from the parent (e.g. A1m)
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II. B) Coefficient of relationship (coefficient of relatedness, Rxy)
This can be calculated from coefficient of inbreeding. Rxy gives the degree of genetic relatedness
(kinship) between two individuals (X a Y).
Rxy = 2 × F
See Figure 1. Rxy expresses the probability that a randomly chosen allele A from locus L of an
individual X (III.1) and an allele from the same locus of an individual Y (III.2) are coming from
the same common ancestor (I.1). Rxy is used to calculate the specific risk of affliction of a child
with genetic inherited disease (see example 2).
8.13 Calculate the coefficient of inbreeding
Table: Coefficients of inbreeding and relationship in the most common mating of close
relatives
Relatives Coefficient of Inbreeding Coefficient of Relationship
parent – child
1/4 1/2
brother – sister
uncle – niece
1/8 1/4
aunt – nephew
first cousins 1/16 1/8
cousin –
1/32 1/16
the child of a cousin
second cousins 1/64 1/32
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8.14 Inbreeding and diseases with AR inheritance
Phenylketonuria (PKU): In a population there is approx. 1 afflicted individual out of 10,000
healthy individuals. This gives the probability of having an afflicted child in a marriage of non-
related parents. How will this probability change, if the child is born out of a marriage of two
cousins? Both cousins are healthy.
Risk calculation
Disease risk = population risk (PR) + specific risk (SR)
PR = q2
 population risk is the disease frequency in the population
 gives the probability of two healthy persons having an afflicted child
 mutant alleles do not come from a common ancestor
SR = Rxy × 2pq × 1/4

 specific risk gives the probability of two healthy relatives having an afflicted child as a result
of inbreeding
 mutant alleles do come from a common ancestor
 Rxy ... coefficient of relationship of the parents
 2pq ... frequency of heterozygotes in a population (carriers of the mutant allele)
... probability that the parents are heterozygotes
1
 /4 ... probability that two heterozygous parents will have an afflicted child
... Aa × Aa → AA, Aa, Aa, aa
Solution:
q2 = 10-4
q = 10-2
p=1
2pq = 2q = 2×10-2
Rxy = 1/8
Disease risk = PR + SR
= q2 + Rxy × 2pq × 1/4
= 0,0001 + 1/8 × 0,02 × 0,25
= 0,000725 = 7×10-4
The result tells us, that the risk of having a child suffering from PKU in a marriage of two
cousins is 7 times higher than in the rest of the population.
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9. Genetic Prognosis
Control questions
1. Explain: prior probability, conditional probability, Bayesian analysis (see pp. 379-382
Thompson and Thompson).
2. How can we evaluate recurrence risks? (Explain the favourable and unfavourable
prediction.)
3. Name the data necessary for the risk assessment.
Introduction
Genetic counselling is a process intended to allow individuals/families with hereditary disorder
understand the recurrence risk in children and other family members.
Genetic Risk Assessment

During each pregnancy there is a 3 % risk of having a child affected by a congenital disorder or
developmental defect – taken for a normal population average. In cases of genetic counseling, the
risk under 5 % is considered as low, the 5-10 % risk is considered as intermediate and the risk
higher than 10 % is considered as high.
When a genetic risk is smaller than 10 % – it is considered as favourable genetic prediction.
Higher than 10 % – it is unfavourable genetic prediction.
It is important to realize that genetic prognosis can only predict the likelihood and not the
certainty that an individual will be affected and will develop disease.
60 % of all children with inborn defects have no readily apparent cause, i.e. family history.
Recall:
 assumptions: large population, random mating, no selection/migration
Autosomal genes
A) Mendelian ratios for monohybrid crosses (Punnett square analysis)
B) Population genetics: p + q =1 and p2 + 2pq + q2 = 1
p ... frequency of dominant allele
q ... frequency of recessive allele
AA = p2
Aa = 2pq
aa = q2
p + q = 1 and p2 + 2pq + q2 = 1
X-linked diseases
XAXA XAXa XaXa XAY XaY
2 2
p 2pq q p q
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Information for Risk Assessment
The correct solution and the reliability of the prognosis require the following objective
information:
1. The exact diagnosis of the disease.
2. The type of its heredity in the examined family.
3. The penetrance of the pathological gene.
4. The frequency of heterozygous carriers (2pq) of the pathological allele in population.
Procedure of Estimating Recurrence Risks in Autosomal Recessive Disorders

Situation characterization
1. Husband and wife are both healthy. What is the probability that they will have an affected
child?
The probability P = (P1 that the first partner is a heterozygous carrier) × (P2 that the second
partner is a heterozygous carrier) × (P3 that two heterozygous carriers will have an affected
child born)
P = 2pq × 2pq × 1/4
2. One of the partners has a sick brother or sister. What is the chance for this couple of having
a sick child born? The man or woman with an affected brother or sister has certainly both
heterozygous parents. The chance of being a carrier for the child born to two heterozygotes
is 2/3.
P = (P1 that the first partner is a heterozygous carrier) × (P2 that the second partner is
a heterozygous carrier) × (P3 that two heterozygotes will have an affected child born)
P = 2/3 × 2pq × 1/4
3. Both partners have one sick brother or sister. What is the chance of having an affected
child?
P = (P1 that the first partner is a heterozygous carrier) × (P2 that the second partner is
a heterozygous carries) × (P3 that two heterozygotes will have an affected child born)
P = 2/3 × 2/3 × 1/4 = 11.1 %
4. Assume that one from the couple is affected, the other one is healthy. What is the chance
of having an affected child?
P = (P1 that the healthy partner is a heterozygous carrier) × (P2 that the sick partner is
homozygous recessive) × (P3 that the heterozygous partner and the sick homozygous
partner will have an affected child born)
P = 2pq × 1 × 1/2
Apply your knowledge from previous lectures to determine recurrence risks

9.1 Albinism
This disease has an AR mode of inheritance; the frequency of heterozygous carriers in
a population is 1/50.
a) A healthy woman has father with albinism. Her husband is healthy with no family history
of albinism. Determine the recurrence risk for the child of this couple.
b) Is it possible that two healthy parents have an albinotic child? (The albinism did not occur
in their families.) Give reasons.
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9.2 Cystic fibrosis (mucoviscidosis)
Cystic fibrosis is inherited as an AR disease. The frequency of heterozygous carriers in population
(2pq) is 1/25. A woman is going to get married. Her brother died of cystic fibrosis, her parents are
healthy. She is afraid of having an affected child. What is the risk of having an affected child?
9.3 Deafness
In some cases the deafness is AR inherited. The frequency of heterozygotes in population is 0.04.
a) A deaf husband and deaf wife want to know, if they can hope for a healthy child or if their
children will be affected by the same disease.
b) Detailed examination of both parents was performed. A non-hereditary origin of deafness
was proved in husband, and the hereditary origin of the disease was detected in the wife.
Recalculate the probability of a child having the disease in this family.
(A man with non-inherited deafness is not “aa” like his wife in the gene for the disease. He
can be either AA or Aa: deduce genetic consequences and determine the probability
of a deaf child for both a) and b) possibilities.)
Compare these results. Realize the importance of an exact disease diagnosis and of the
genealogical examination for the genetic prognosis.
9.4 Marphan's syndrome (arachnodactylia)

This disease is controlled by an AD gene with 80% penetrance. Affected woman gets married to
a healthy man. Her mother is healthy; her father is affected; her only brother is affected.
In her husband’s family this condition did not occur. Draw the pedigree. What is the probability
that this couple will have an affected child?
9.5 Haemophilia
Two healthy individuals got married. The woman's father suffered from XR inherited haemophilia.
Both man's parents were healthy, but his grandfather (his mother's father) suffered from this
disease.
a) Draw the pedigree.
b) What is the probability that this couple will have an affected son?
c) If they already have an affected son born, what would be the risk of having the next child
affected by haemophilia?
9.6 Daltonism
A healthy woman got married to a healthy man. Both women’s parents are healthy, but her
grandfather (her mother's father) suffered from colour blindness (daltonism).
a) What is the probability that this couple will have an affected son? Daltonism is an
X-linked recessive disease.
b) If they already have an affected son born, what would be the risk of having the next son
affected by daltonism?
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9.7 Tooth enamel defect
The primary disorder of the tooth enamel matrix is X-linked dominantly inherited. The teeth are
yellow almost brown, sometimes they may be semi-translucent.
a) The women with healthy teeth got married to a man suffering from this defect. What is the
probability:
 that their daughter will exhibit this defect?
 that their son will exhibit this defect?
b) A healthy man and a woman suffering from the above mentioned disease got married. They
want to have four sons and one daughter. What is the probability that all these children will
have normal teeth?
9.8 Myopathia myotonica

A man asks whether he can be affected with myopathia myotonica (an AD disease with complete
penetrance), when his grandfather was affected by this condition and got sick when he was 40
years old.
a) Grandfather’s son (man’s father) is healthy. Draw the pedigree and explain your answer.
b) Assume that the man’s father was affected by this condition too. Determine the possibility
that the man has inherited the “disease” allele and he will get sick as his grandfather and
father?
9.9 Phenylketonuria
A son with phenylketonuria (AR inherited disease) was born to a healthy couple. The mother is
pregnant for the second time and asks, whether her next child will be affected too. Draw the
pedigree and answer her question. Explain.
9.10 Progressive muscular dystrophy

A husband and wife have a daughter affected with progressive muscular dystrophy (XD inherited
disease). The husband is affected by this disease. His healthy wife is pregnant again and they are
afraid they will have another affected child born. Draw the pedigree. Explain the principle of child
gender choice and its importance for prevention of this disease.
9.11 Haemophilia
A healthy woman has a brother with haemophilia (XR inherited disease). She asks what is the risk
of her son being haemophiliac. Draw the pedigree and answer her question.
9.12 Haemolytic spherocytic anaemia

Haemolytic spherocytic anaemia is inherited in AD fashion. An affected individual has healthy
parents and 6 healthy siblings. His only daughter is affected. Determine the risk of his next
children being affected. The man is a carrier of a new genetic mutation – explain.
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9.13 Cataract
Cataract (clouding of the crystalline lens of the eye) is an AD inherited disease. Two healthy
individuals got married. Woman’s mother is affected, woman’s father is healthy. Both brothers
of the woman are affected. The husband’s father has cataract, his mother is healthy and his sister is
affected by the disease.
a) Draw the pedigree. What is the possibility of this couple having an affected child?
b) If the affected sister of the husband gets married to a healthy man, what is the possibility
of having an affected child born?
9.14 Phenylketonuria
Phenylketonuria (PKU) has incidence of 1/10,000. A healthy couple (cousins) asks whether their
risk of having an affected child is the same or not. Explain and count the risk. Rxy of two cousins
is 1/8.
9.15 Down syndrome

Down syndrome is the most common inborn developmental defect with the incidence 1/830.
A couple inquires what is the risk of having an affected child, when husband‘s sister has a child
affected by this syndrome. Suggest a plan of genetic counselling, including eventual tests, and
discuss possible alternatives.
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10. Genetics of Immunity, HLA System
Control questions
1. What is the principle of specific recognition of a foreign antigen by the host immune
system?
2. The human immune system possesses enormous number of different antibodies. What is
the cause of this diversity?
3. What is the HLA system?
4. What genes encode the Class I and Class II Histocompatibility Molecules?
5. How is the HLA phenotype of a cell determined?
6. What type of allelic gene interaction is applied to the HLA system?
7. What is the difference between haplotype and genotype?
8. Explain the term linkage disequilibrium.
9. What are the prerequisites of a successful transplantation concerning the HLA system?
The aim of the seminar

In this chapter we will focus on the understanding of genetic control of antigens, which influence
the tissue compatibility in transplantations in humans. They are called therefore, histocompatibility
(or transplantation) antigens: abbreviated HLA antigens.
Introduction
HLA system (Human Leukocyte Antigen) is the name for Major Histocompatibility Complex or
MHC in humans. This system is responsible for production of membrane glycoprotein (antigen-
presenting proteins), which are simply called HLA antigens.
HLA antigens
1. are involved in the protection against diseases
2. are involved in the recognition of own and foreign cells
3. are responsible for graft rejection
4. are associated with some autoimmune diseases.
Genes encoding for HLA antigens are localized on chromosome 6 in so called HLA region.
HLA loci, which have been described are labelled as A, B, C, (E, F, G,); DR, DQ, DP.
Figure 1: HLA region on chromosome 6
The HLA loci can be divided into two classes:
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1. MHC class I includes “classical” genes HLA-A, HLA-B, HLA-C and
“non-classical” genes HLA-E, HLA-F, HLA-G and others.
“Classical” genes HLA-A and HLA-B are expressed by all nuclear cells with the
exception of neurons, germ cells and trophoblast.
“Non-classical” genes are expressed by embryo and trophoblast cells. They play a key role
in immune tolerance between maternal body and developing embryo (consider the fact that
embryo contains half of HLA antigens which are different from HLA antigens found on
cells of mother´s body). HLA-G antigen is also produced in soluble form. The degree
of genetic polymorphism in “non-classical” genes is lower if compared to “classical”
genes.
2. MHC class II includes the following genes: HLA-DR, HLA-DQ, HLA-DP. These
antigens are expressed in all cells which are involved in immune response.
(More information can be found in database of European Bioinformatics Institute:
http://www.ebi.ac.uk/imgt/hla/stats.html)
There are multiple alleles described for each locus (10s, 100s). HLA alleles are labelled with
a letter of the locus, where they are defined and with a number, e.g. HLA-A1. Different alleles
of the same locus are codominant. For instance A2A11 phenotype is determined by alleles A2 and
A11. A person who had detected single antigen of certain locus (e.g. A2) is either homozygote
(A2A2) or heterozygote (A2AX).
According to traditional system of HLA nomenclature different alleles were distinguished from
one another based on their reaction to antisera. With the development of molecular genetics the
nomenclature has augmented. Today the alleles are labelled with a letter of a specific locus and
with a four-digit code: the first two numbers represent the serological group, the third and fourth
number show the specific DNA-sequence of the allele. Individual alleles in a serological group can
differ from one another by one or more nucleotides. E.g. alleles HLA-A*0205 and A*0210 code
for different polypeptides, but both belong to serotype A2, i.e. both encode for epitopes which are
recognized by anti-A2 antiserum. These polypeptides differ by several aminoacids which are not
located in the epitope region. Epitope is the part of the molecule, which is recognized by immune
system.
Comment: Serotyping is commonly used for determination of HLA serotype of given locus. If single antigen
controlled by given locus is found (e.g. A2), it is assumed that person is heterozygote and that the 2nd allele of given
locus was not found (this 2nd allele is marked AX). It can be explained by the fact that there may be present higher
number of various antigens controlled by given locus in the whole population than the number of available antisera is.
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Figure 2: Class I and II MHC molecules
The HLA gene expressivity is tissue-specific: the amount of the HLA antigens is different
in different tissues.
HLA genes are closely linked. A group of alleles localized on one chromosome will be inherited
together. The entire group of alleles on one chromosome is called haplotype (derived from words
haploid genotype).
I. Characterization of the HLA System Polymorphism

HLA genes are the most polymorphic genes in the human genome. Gene polymorphism can be
characterized by a situation in a population, where there are simultaneously (i.e. in one generation)
3 or more alleles of a given gene (locus); each individual possesses only 2 alleles of the given gene
(2 different or 2 same alleles of the given gene). Theoretical number of all possible HLA
haplotypes, genotypes and phenotypes in a population can be simply calculated. In reality all
variants will not be formed due to strong gene linkage and linkage disequilibrium (some allele
combinations are more common). Therefore, it is not possible to calculate the frequency of a given
phenotype like this. The frequency is established empirically.
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The number of alleles for certain loci is given below (X/2012):
HLA locus No. of different serotypes No. of known alleles
HLA-A 1 527 2 132
HLA-B 2 110 2 798
HLA-C 1 200 1 672
HLA-E 3 11
HLA-F 4 22
HLA-G 16 50
DRB1 887 1 196
DQB1 128 179
DPB1 136 158
10.1 Theoretical Number of Haplotypes

Let us assume that a given allele at a certain locus (e.g. at locus HLA-A) can be combined with
any other allele at a different locus (e.g. at locus HLA-B). Theoretical number of all possible
haplotypes (n) can, thus, be calculated by multiplication of the number of alleles at all given loci.
Fill-in the table:
HLA loci Theoretical No. of Haplotypes
A-B 2 132 × 2 798 = 5 965 336
A-B-DRB1
A-B-DRB1-DQB1
10.2 Theoretical Number of Genotypes

Genotype of each individual consists of two haplotypes. The sum of all possible genotypes is equal
to the number of possible pairs of haplotypes from the total number of possible haplotypes (n).
The general formula is:
 (n + 1) n × (n + 1)
  =
 2  2
Fill-in the table:

HLA loci Theoretical No. of Genotypes
A-B (5 965 336 × 5 965 337)/2 = 35 585 239 558 232
A-B-DRB1
A-B-DRB1-DQB1
Both examples are given to demonstrate the enormous genetic variability of the HLA system.
In reality the number of haplotypes and genotypes are lower because of the strong gene linkage
and linkage disequilibrium. Usually they are determined empirically.
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II. Analysis of HLA Allele Segregation in Families
In the following section we will focus only on two loci: HLA-A and HLA-B. In order to simplify
calculations we will deal only with HLA loci on the level of serotypes (e.g. A1, B8 etc.). The basic
general characteristics of the HLA antigen inheritance are as follows:
1. Each individual possesses two alleles at each HLA locus and, therefore, maximum of two
different antigens encoded by the locus (result of the diploid state).
2. If one of the parents has e.g. antigens A1 and A2, then his/her child will inherit either
antigen A1 (50 % probability) or antigen A2 (also 50 % probability).
3. Child will possess only those antigens, which are present in his/hers parents.
How to solve the examples

a) HLA phenotype A11, A24, B39, Bw56 was detected in an individual. Give possible
genotypes. In each haplotype there is always one allele at HLA-A locus and one allele
at HLA-B locus. In order to state possible genotypes it is important to find out all possible
pairs of haplotypes. The given HLA phenotype can be explained by one of the two
following genotypes:
 A11, B39/A24, Bw56
 A11, Bw56/A24, B39
b) HLA phenotypes of all family members are known. Give genotypes, which may be
considered.
HLA phenotypes of the family:
Mother: A23, A31, B13, B39
Child: A31, Aw36, B7, B13
Father: A11, Aw36, B7, B4
First, you have to realize that the child received one haplotype form the mother and the
other from the father. In this case the child received from mother haplotype A31, B13, and
from father Aw36, B7. Now, it is clear what genotypes are possible:
Mother’s genotype: A31, B13/A23, B39
Child’s genotype: A31, B13/Aw36, B7
Father’s genotype: Aw36, B7/A11, B45
c) In the past, huge variability in the HLA was used to solve paternity law suits. Nowadays
there are used another methods in paternity law. Based on practice described in a) and b),
we will explain such examples concerning HLA system to better understand HLA antigens.
The phenotypes of participants of the law suits are as follows:
Mother: A23, A31, B13, B45
Child: A31, A24, B8, B45
The man under suspicion: A3, A29, B7, B51
Decide if the man under suspicion can be excluded from the fatherhood.
The child inherited haplotype (A31, B45) from mother. Haplotype (A24, B8) had to be
inherited from his/her biological father. The man under suspicion does not have antigens
A24, B8 in his phenotype; therefore he cannot possess haplotype (A24, B8). He is,
therefore, excluded from the paternity, because he could not be the biological father of the
child.
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10.3 Deducing Genotypes from Phenotypes
What possible genotypes correspond to the following phenotypes?
a) A1, A23; B7, B19
b) A2; B8, B39
c) A2; B8
10.4 Deducing Haplotypes in a Family

Deduce the haplotypes of all members of the following families:
a) mother: A11, A30; B7, B35
child: A2, A11; B7, B37
father: A2, A23; B18, B37
b) mother: A1, A2; B18, B35
child: A1, A2; B8, B18
father: A1, A2; B8, B27
10.5 Geneticist's Testimony in a Paternity Suit

During a paternity suit the following phenotypes were detected in analyzed individuals. Write
down possible genotypes and decide if the man could be the biological father of the child:
a) mother: A1, A24; B27, B35
child: A3, A24; B7, B27
man: A3, A23; B7, B49
The frequency of A3, B7 men in the population is 0.0367 %.
b) mother: A1, A11; B35
child: A2, A11; B35, B44
man: A1, A2; B8, B18
The frequency of A2, B18 men in the population is 0.05 %.
10.6 Expected Genotype Frequencies

Calculate the frequency of people with the following phenotypes:
a) A1, A2, considering that we know, that the allele A1 frequency is 14.9 % in the population
and A2 = 26.0 %. In this case of a heterozygote, use the formula 2pq.
b) A33, A30, if f(A33) = 1.7 %, and f(A30) = 2.4 %
c) A36, A36, if f(A36) = 0.3 %. In this case of a homozygote, use the formula p2
d) A1,B8/A3,B7. As there is so called “linkage disequilibrium” in the haplotype frequencies,
we have to use the haplotype frequencies established by HLA typing of large population
samples (so called empiric values). In the case of A1, B8 haplotype, the frequency is
0.0591; in the case of A3, B7 it is 0.0315. To obtain the genotype frequency, we need to
multiply 2 × 0.0591 × 0.0315
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III. Choosing a Donor for Transplantation
The degree of HLA matching between donor and recipient is very important for the success
of transplantation therapy. The donor and the recipient are perfectly compatible only if the donor
has only those HLA antigens, which are also present in the haplotypes of the recipient, nothing
more. The HLA matching is judged on different levels according to the type of transplanted organ.
For instance, the degree of HLA matching for kidney transplantation is evaluated on the level
of serotypes at loci HLA-A, HLA-B, HLA-DR. For bone marrow transplantation HLA-A,
HLA-B, HLA-DR, HLA-DQ are being considered. For heart and lung transplantation the HLA
matching is not as important because there is low level of expression of these antigens in these
tissues. Similar situation is in the case of liver transplantation.
Let us assume that a patient, who is waiting for kidney transplantation (recipient), has the
following HLA phenotype: A1, A2, B7, B12, DR3. A donor with one of the 9 following serotypes
would be compatible with this recipient:
1. A1, B7, DR3
2. A1, B12, DR3
3. A1, B7, B12, DR3
4. A2, B7, DR3
5. A2, B12, DR3
6. A2, B7, B12, DR3
7. A1, A2, B7, DR3
8. A1, A2, B12, DR3
9. A1, A2, B7, B12, DR3
In order to calculate the probability of compatible donor, we would have to calculate the frequency
of all given serotypes in the population.
10.7 Phenotypes of Compatible Donors

The following phenotypes are known for patients who are waiting for kidney transplantation.
Write all the possible phenotypes of compatible donors and calculate their frequencies in the
population.
a) A2, B8, DR7
b) A11; Aw33, B18, Bw53, DR1, DR9
The haplotype frequencies can be found in a table of empirical values, which will be available
during the seminar.
10.8 Suitability of Organ Donors

Put the following people in the order of their suitability for donorship (begin with the most suitable
one): sibling, monozygous twin, non-related person, mother, dizygous twin.
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IV. HLA and Autoimmune Disease Associations
In the table below there are few examples of diseases with positive association with certain HLA
antigens.
Antigen frequency in %
Disorder patients healthy people
Ankylosing spondylitis B27 90 8
Reiter’s disease B27 80 9
Celiac sprue DQ2, DQ8 90 22
Diabetes mellitus DR3, B8 50 21
Psoriasis vulgaris Cw6 50 23
Multiple sclerosis DR2 55 23
Relative risk (RR) informs us about the frequency of a disease in individuals with a certain antigen
in comparison with patients without that antigen. RR<1 means negative association, RR = 1 means
no association; RR>1 means positive association. The relative risk is calculated from
prospective data, using the following formula:
RR = ad/bc
where:
a ... No. of patients with the antigen
b ... No. of patients without the antigen
c ... No. of healthy people with the antigen
d ... No. of healthy people without the antigen
10.9 Calculate relative risk

Calculate the relative risk of developing a certain disease in an individual with a given antigen
in these situations:
a) Diabetes mellitus and B8, where a = 48, b = 590, c = 59, d = 1900
b) Ankylosing spondylitis and B27, where a = 57, b = 160, c = 6, d = 183
c) Celiac sprue and DQ2, DQ8, where a = 30, b = 275, c = 4, d = 750
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11. Molecular Biology I: DNA, RNA Analysis
Medical Application of Molecular Biology Methods

The knowledge of molecular pathophysiology is the basis for correct diagnosis and adequate
choice of therapy for genetic disorders. Molecular biology is a new discipline, which is also
working on discovering the causes of diseases on the molecular level. It searches for causative
associations of DNA mutations and phenotype (disease). Molecular biology uses a variety
of specialized methods, which enable us to study the molecular basis of gene functions, of their
structure on the level of DNA, RNA, and protein, and of their biological functions.
Molecular biology methods can be used for:
1. Detection of inherited mutations
a) for diagnosis of genetic disorders
b) for diagnosis of genetic disorders
c) in preclinical stage of a disease for identification of mutant allele carriers (carriers of AR
disorders) or for detection of genetic predisposition to genetic disorders
d) in prenatal diagnosis – for analysis of inherited disorders, determination of sex
2. Detection of acquired mutations
a) in oncogenetics – for analysis of somatic mutations associated with cancer
3. Detection of pathogens causing infectious diseases – viruses, bacteria or yeasts.
Brief Overview of Basic Molecular Biology Methods for DNA and RNA Analysis
1. DNA/RNA isolation
The basis for successful nucleic acid analysis is to isolate the material in sufficient quantity and
quality. The most common material for DNA/RNA isolation is peripheral blood (peripheral
leukocytes), and different tissues (skin, liver, spleen, tumours), or hair, hairs, or sperm. The first
step of isolation is disruption of all cellular structures by detergents. DNA, RNA, proteins,
polysaccharides, and lipids are freed into the solution. The subsequent steps are dealing with
DNA/RNA purification.
Gel Electrophoresis
This method is used for DNA/RNA separation in a gel in direct electrical field. (Nucleic acids are
negatively charged and move towards anode.) The molecules are separated on the basis of their gel
mobility. Smaller molecules move faster (are further from the start) than bigger molecules (Fig. 1).
Visualization is performed by ethidium bromide, which intercalates itself into DNA double helix.
After stimulation with UV light fluorescence is observed. The molecular size of nucleic acids is
expressed in number of nucleotides or base pairs (bp).
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Figure 1: Electrophoresis of genomic DNA visualized with ethidium bromide in UV light. Lines
1-4 genomic DNA isolated from peripheral blood lymphocytes, line 5 DNA marker (see below)
start
2. PCR
Polymerase chain reaction (PCR) is a method used for amplification of DNA fragments up to the
size of 10 thousand nucleotides. The given DNA sequence must be defined at the beginning and
at the end with short oligonucleotides called primers. DNA is amplified by reaction similar to
replication, where DNA Taq polymerase (a thermostable enzyme) recognizes DNA with
an annealed primer and catalyses primer elongation and thus DNA amplification. PCR consists
of repeated cycles. Each PCR cycle requires three temperature steps to complete one round
of DNA synthesis:
1. Denaturation – DNA is heated to ~95°C (30 s). The hydrogen bonds are broken and DNA
double helix is unwinding at this temperature. The result is a single-stranded DNA.
2. Primer annealing – Temperature is lowered to ~60°C. At this temperature the primers
anneal to corresponding target sequence in DNA on the basis of complementarity. The
polymerase binds to the annealed primers.
3. Elongation – Actual DNA synthesis happens at this step. The optimal activity of Taq
polymerase is at 75-80 °C. The new DNA strand, which is complementary to template
DNA, is being synthesized in the 5' to 3' direction.
PCR products are analyzed on gel electrophoresis (Fig. 2). DNA markers are used to check the
size of the PCR products. Marker consists of DNA fragments of defined lengths (Fig.2 lane 5).
Figure 2: PCR products
Today, there is a big variety of different modifications of PCR reaction. For example RT-PCR is
used for amplification of a specific region of mRNA.
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The Use of PCR in Diagnosis:
1. Detection of pathogens:
 virus: HIV, hepatitis A, B, C
 bacteria and yeasts: Mycobacterium tuberculosis (TBC), Candida species
 amplification of genes, which are specific for a given pathogen in a patient’s sample
(blood, tissue, sputum)
2. Detection of gene deletions – shortening of PCR product.
3. Detection of fusion gene on the level of mRNA by RT-PCR
 the first primer is located (P1) in the coding sequence of the first gene involved in the
fusion, the second (P2) is in opposite direction in the coding sequence of the partner
fusion gene
4. Quantification of gene expression (quantification of mRNA).

5. Preparation of material for further analyses like:
a) restriction analysis
b) sequencing
3. Restriction Analysis
Restriction endonucleases (REs) are enzymes, which specifically bind to DNA and cleave it
in specific places located inside or nearby the recognized sequence. The name of the enzymes is
derived from bacteria, which produces them (genus, species, and strain). The Greek numbers show
the order of identification of the enzyme in the bacterium (first, second etc.). Bacteria produce RE
in order to defend themselves against foreign DNA (from bacteriophages). For instance EcoRI is
RE isolated from E. coli. Fig. 3 shows the restriction site of EcoRI.
Figure 3: EcoRI restriction site
REs are used for restriction analyses. DNA or PCR products are digested by one or more REs.
The resulting DNA fragments are visualized by electrophoresis. Fig. 4 shows the restriction
analysis of a PCR product (499bp), which contains one restriction site for a specific RE. After
the restriction took place 2 fragments are produced: 268 a 231 bp. On the right you can see the
photo of gel electrophoresis: in the first lane there is the non-restricted PCR product, in the second
lane there are the 2 fragments resulting from the restriction of the PCR product. Note that the DNA
fragments move in the gel according to their size.
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Figure 4: Restriction analysis
restriction
endonuclease
Restriction analysis is used for detection of known point mutations, which abolish or create
restriction site in the wild type DNA sequence.
4. Sequencing
Sequencing is a technology, which determines the DNA primary structure (base sequence). Today
the automatic sequencing is broadly used. The output of a sequence analysis is called
chromatogram (Fig. 5). Chromatogram gives the nucleotide sequence of an analyzed sample (most
often a PCR product). The peaks correspond to individual bases; each base is labeled with
different fluorescent dye.
Fig. 5 shows sections of two chromatograms. On the left, there is a section of a gene sequence
of a healthy individual. On the right, there is a sequence of an ill person. The arrow points to
the position of a point mutation T→C. Note, that there are 2 peaks at this position. These two
peaks tell us that one allele of the patients has normal sequence and the second allele is afflicted
by the point mutation. The patient is thus a heterozygote.
Figure 5: Result of sequence analyses: chromatograms
Sequencing is used for detection of mutations, which cannot be identified by restriction analysis or
for detection of new mutations, which have not been described yet: point mutations,
substitutions, short deletions, or insertions.
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5. Hybridization – Detection of Specific Sequence with Labeled Probe
The principle of all hybridization methods is detection of a specific sequence with labeled probes.
Probes are single-stranded polynucleotides with known sequence. Probes are labeled with
radioactive elements, fluorescent dyes, or other chemical substances. The probe hybridizes (binds)
to the target DNA sequence based on the complementarity (see Figure 6), which is then visualised
by radioactivity, fluorescence or chemiluminiscence.
a) Southern blot – hybridization of genomic DNA. DNA is digested by restriction
endonuclease(s), fragments are separated according to their size by electrophoresis and
blotted onto a membrane. The membrane is hybridized with a labeled probe specific for
target sequence (Fig. 6)
Figure 6: Southern blot
1 2 3
probe hybridization
autoradiographic
digested DNA blotted detection of positive
onto a membrane sequence
radioactively
probe labelled
hybridization
Southern blot hybridization is used for detection of longer gene deletions, for
identifications of fusion genes on the level of DNA, or for DNA fingerprinting.
b) Northern blot – mRNA hybridization.
c) FISH –DNA hybridization on the cytogenetic slides.
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6. DNA fingerprinting
DNA fingerprinting (also called DNA testing, DNA typing, or DNA profiling) is a technique
which creates unique genetic profile of an individual. It is based on the analysis of non-coding
regions of genome. These regions are highly polymorphic and are composed of repetitions of short
sequences e.g. -CATG-CATG-CATG-, which are called tandem repeats. Different individuals
possess different numbers of these repetitions, i.e. VNTR (variable number of tandem repeats).
VNTR leads to length variability of the given DNA regions among individuals. Each length
variant acts as an inherited allele and is labeled with a number. The method used for DNA
fingerprinting is restriction analysis with subsequent Southern blot hybridization or more
commonly PCR analysis, which determines the length of a given repetition in a given individual.
Due to high variability among individuals in a population, there is a very small chance, that two
non-related individuals will have the same alleles of many polymorphisms. Normally, around
15 different polymorphisms are being tested.
DNA fingerprinting is used not only in criminal investigations for person identification but also
in paternity law suits. Each individual inherits one allele of a given polymorphism from the mother
and one from the father. It is assumed that the mother is always certain. Therefore alleles, which
do not come from the mother, had to be inherited from the father.
Answer following question and solve the tasks

11.1 DNA diagnostics
Which tissues can be used for DNA isolation in diagnostics: tissue biopsy, blood, cultivated cells
from embryonic fluid, erythrocytes?
11.2 DNA sequencing

What are the benefits of DNA sequencing?
11.3 PCR reaction

What does a mixture for PCR reaction consist of?
11.4 Restriction analysis

There are 2 restriction sites for the given RE in a PCR product. How many fragments will be
produced in restriction analysis? Draw the situation.
11.5 IVS-1-I (G→A) ß-globin Gene Mutation Analysis

This point mutation creates a restriction site for BsaBI restriction endonuclease. 8 individuals were
tested because of suspicion of having β-thalassaemia. A given part of ß-globin gene was amplified
by PCR. PCR product was then subjected to BsaBI restriction analysis. Explain the results of this
analysis.
What further analysis (what methods) would you suggest if the restriction analysis would be
negative but other laboratory test hint to the presence of ß-globin gene mutation?
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IVS-1-I (G→A) mutation (mutation of the first nucleotide in the first intron) is one of the most
common mutations causing ß-thalassaemia minor in the Czech population.
11.6 V617F JAK2 Mutation Analysis

Mutation V617F of JAK2 gene leads to substitution of the 617th aminoacid (valine V for
phenylalanine F) in JAK2 kinase. This point mutation abolishes a BsaXI restriction site. The given
part of JAK2 gene was amplified by PCR. The PCR product was digested by BsaXI restriction
endonuclease. Explain the following results of this analysis in 3 patients (line 1-3):
JAK2 V617F is an acquired mutation in JAK2 gene. It is present in a subset of hematopoietic cells
and it is associated with polycythemia vera (PV). PV is a malignant disorder of red blood cell
lineage, which is characterized by abnormal production of erythrocytes.
11.7 PCR Detection of a Mutation in Inherited Disorder

Healthy parents had one healthy daughter and one daughter afflicted by a congenital disease. Part
of the affected gene was amplified by PCR. Define the type of mutation!
C: control (healthy individual), F and M: father and mother, # 1 and # 2: daughters. Which sample
(# 1 or # 2) belongs to the afflicted daughter? What type of inheritance does this given disorder
possess?
C 1 2 F M
11.8 Fusion Gene Analysis

In the figure below you can see schematic representation of BCR-ABL fusion gene:
a) Which tissue/cells will you need for the detection of this fusion gene?
b) Which method would you choose for the fusion gene detection and why?
c) Draw the position and orientation of primers, which could be used for the fusion gene
detection.
d) What results would you expect in a sample from a healthy individual and in a patient with
this fusion gene?
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11.9 Paternity Law Suit
Two men are in paternity law suit. Both were tested on the level of DNA. Figure below shows
the result of PCR analysis of several VNTRs. Which of the two men is the biological father
of the child? (M: mother, C: child, m1: man number 1, m2: man number 2.)
M C m1 m2
11.10 Fill-in the table

Type of mutation Method of detection
Point mutation
Deletion
Fusion gene
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12. Molecular Biology II: Proteins, Medical Application
In order to understand the pathophysiology of diseases it is necessary to know protein structure,

function, and properties. This knowledge is today applied not only in diagnosis, but also in modern
quickly developing therapies using artificially prepared recombinant proteins or in therapies based
on the understanding of protein structure (low-molecular-weight inhibitors of protein kinases).
I. Protein Detection Used in Diagnosis

Protein Electrophoresis: Native a Denaturing
The basic method for protein detection is electrophoresis. This separation method uses different
mobility of proteins in a gel in electric field due to different size, charge or conformation of the
protein. Proteins in native (natural) state are studied by native electrophoresis. In contrast, SDS-
PAGE (sodium-dodecyl sulphate polyacrylamide gel electrophoresis) is electrophoresis under
denaturing conditions, i.e. it uses protein lysates that are denatured and all protein molecules have
uniform charge. In SDS-PAGE the proteins migrate in electric field only according to their
molecular size. Denaturing electrophoresis is used in western blotting see immunostaining.
Questions concerning native electrophoresis:
Explain the principle of native protein electrophoresis and discuss the possibilities of protein
detection in gel. What is the application of this method? Can you detect every gene mutation
causing an amino acid substitution in the protein structure by this method? Why?
This method can be used for the detection of abnormal haemoglobin variants, interpret. Which
diseases are associated with the production of abnormal haemoglobins?
Immunostaining
In general, immunostaining describes a broad range of methods used in histology, cellular and
molecular biology. All these methods are based on detection of specific protein/antigen by specific
antibodies. Explain the general principle of antigen detection by antibody. How are these
commercially available antibodies produced? What is the difference between monoclonal and
polyclonal antibody? Name different types of immunostaining.
Imunohistochemistry/ imunocytochemistry
Immunostaining of the cells – immunocytochemistry or tissue sections – immunohistochemistry
are perhaps the most commonly applied immunostaining techniques. Explain the principle.
Immunostained cells or tissue sections are visualized either using fluorescent microscope
(labelling with fluorescent dyes) or light microscope (usage of enzymes such as peroxidase and
alkaline phosphatase). Explain the differences. What is the difference between direct and indirect
detection? Explain the terms primary and secondary antibody. This method can localize cellular or
tissue proteins. Why is this useful? This method is used for instance in diagnosis of solid tumours
or it is widely used in basic research, give some examples.
Flow Cytometry
You have already been introduced to the theory of flow cytometry last term. Explain the principle
of this method. In clinical practice this method is routinely used for instance in the determination
of the number and types of leukocytes. Different leukocyte populations can be differentiated on the
basis of CD antigens (cluster of differentiation). Explain the term CD. In addition, this method is
used also for immunophenotypisation of leukaemias, e.g. in diagnosis of chronic lymphocytic
leukaemia. Give an example of flow cytometry application in medical diagnostic and give
explanation.
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ELISA (Enzyme-Linked Immunosorbent Assay)
This method also uses the reaction of an antigen with an antibody. The antigen or antibody is
bound onto a solid surface. The antibody is conjugated with an enzyme. If antibody binds to
antigen the enzyme catalyses a reaction with a production of an easily-detectable coloured product.
For instance this method determines quantitatively the concentration of a substance in serum or
urea (e.g. of hormones LH, HCG, TSH or of drugs such as cocaine, opiates, marihuana, i.e. THC).
In addition, this method is used to test the donors’ blood for viral infections (like HIV, hepatitis
B). Explain the principle of this method and give examples of its use in medicine.
Western Blot (Immunoblot)

This method detects proteins in homogenous protein lysates from cells or tissues. Protein lysate is
subjected to gel electrophoresis under denaturing conditions, which separates the proteins
according to their size (see electrophoresis). Followed electrophoresis proteins are transferred
(blotted) from the gel onto a membrane in electric field. The membrane can then be probed with
antibodies using methods similar to immunohistochemistry, but without a need for fixation.
Detection is typically performed using peroxidase linked antibodies to catalyze a
chemiluminescent reaction. This method is used for example for confirmation of positive results
from ELISA, e.g. HIV detection in serum. In addition, it is used to verify the diagnosis of BSE
(Mad Cow Disease) or in some test for Lyme disease. This method is of course widely used in
basic research. Explain the principle of this method. Give some examples of its use in medicine.
II. Proteins in Therapy

Recombinant Proteins
Recombinant protein is a protein produced by genetically-modified organism. DNA encoding this
protein is transferred into a genome of another organism. The first drug used in clinical setting,
which was prepared by genetic engineering was insulin. (Human insulin gene was transferred into
bacteria / Escherichia coli.) The use of recombinant protein eliminated allergic reactions to insulin
isolated from animals. How are recombinant proteins produced? Why cannot be insulin taken
per os? What other recombinant proteins are used in therapy of human diseases?
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© Authors team, Department of Biology, Faculty of Medicine and Dentistry,
Lab works from Biology
2014
Second semester
For students of master degree programmes General Medicine, Dentistry
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These diagrams illustrate meiotic non-disjunction.

pair of homologous chromosomes

How many chromosomes are there in an egg and in a fertilized zygote?

pair of homologous chromosomes

1.4 Mitotic non-disjunction

pair of homologous chromosomes

B) Non-disjunction of chromosome X during the third cleavage division

1. What is the consequence of mitotic non-disjunction?

3 dif f erent possibilities of chromosome segregation during 1st meiotic division

2nd meiotic division 2nd meiotic division 2nd meiotic division

B) their type – genomic mutations (numerical chromosomal aberrations)

C) the cell type, where the mutation occurs:

2.1 Gene mutations

C) nonsense mutation – truncation of the protein

Deletions and insertions

2.2 Numerical and structural chromosomal aberrations

Chromosomal abnormalities of cancer cells

2.5 Preparation of metaphase chromosome to assess the cell ploidy

X-linked X-linked dominant X-linked recessive

How to solve the tasks

3.1 Inheritance of the eye colour

3.2 Single-gene disorder inheritance

Parents’ phenotypes Number of families Children’s phenotypes

3.5 Sickle-cell disease

3.6 Beta thalassemia

3.7 The inheritance of eye colour and left-handness

3.8 Inheritance of the ABO blood group in humans

3.9 Rhesus-Factor (Rh) Inheritance

3.10 Single-gene human traits and their alleles

X-linked recessive inheritance

X-linked dominant inheritance

General scheme of sex-limited inheritance (+ means that the "trait is expressed"):

General scheme of sex-influenced inheritance (+ means that the "trait is expressed"):

4.1 The inheritance of haemophilia A

4.2 The inheritance of colour-blindness (daltonism)

4.3 The inheritance of G6PD deficiency

4.4 Phosphate diabetes

Autosomal Dominant Inheritance (AD)

Autosomal Recessive Inheritance (AR)

X-linked Dominant Inheritance (XD)

X-linked Recessive Inheritance (XR)

4.5 Pedigree analyses

4.6 Draw the pedigree

location of genes: on different chromosomes

Diagram of complete linkage:

location of genes: on the same chromosome on the same chromosome

genotypes: AB/ab Ab/aB

phase: cis (coupling) trans (repulsion)

Linkage phase cis (coupling)

Linkage phase trans (repulsion)

linkage phase trans

The strength of gene linkage can be determined by a testcross

I. Gene Linkage in a Testcross

5.3 Gene Linkage in Drosophila

5.4 Determination of Gene Linkage in a Family

5.6 Daltonism and Hemophilia A

5.7 Association of traits

The use of gene linkage

III. Localization of Human Genes

5.8 Localization of enzyme-encoding genes