MOLECULAR DIAGNOSIS OF HEREDITARY DISEASE

Arthur S. Schneider, M.D. Department of Pathology Chicago Medical School at Rosalind Franklin University of Medicine and Science

MOLECULAR PATHOLOGY
rapidly growing subspecialty area of pathology practice and investigations major applications in:
neoplasia (leukemias, lymphomas, and solid tumors) hereditary disorders and disease predispositions responsiveness to pharmacologic agents identification of infectious agents forensic applications (crime lab, paternity) others (list is growing rapidly)

HEREDITARY DISORDERS COMMONLY DIAGNOSED BY MOLECULAR METHODS

adult polycystic kidney disease achondroplasia 1-antitrypsin deficiency canavan disease Charcot-Marie-Tooth disease congenital adrenal hyperplasia cystic fibrosis

HEREDITARY DISORDERS COMMONLY DIAGNOSED BY MOLECULAR METHODS

Duchenne/Becker muscular dystrophy factor V Leiden mutation familial adenomatous polyposis familial hypercholesterolemia fragile X syndrome galactosemia

HEREDITARY DISORDERS COMMONLY DIAGNOSED BY MOLECULAR METHODS

Gaucher disease hemophilia a and b Huntington disease Marfan syndrome mitochondrial disorders myotonic dystrophy neurofibromatosis types 1 and 2

HEREDITARY DISORDERS COMMONLY DIAGNOSED BY MOLECULAR METHODS

ornithine transcarbamoylase deficiency phenylketonuria spinal muscular atrophy spinocerebellar ataxia sickle-cell disease

HEREDITARY DISORDERS COMMONLY DIAGNOSED BY MOLECULAR METHODS Tay-Sachs disease - and -thalassemia tuberous sclerosis von Hippel-Lindau disease many others

Online Mendelian Inheritance in Man

OMIM is major source for information on molecular changes in Mendelian disorders http://www.ncbi.nlm.nih.gov/omim/

TYPES OF MUTATIONS IN HUMAN GENETIC DISEASE

deletions and insertions
entire gene, entire exon, multiple bases, repeat sequences, etc.

codon deletions and insertions (number of

bases is a multiple of 3) frameshift mutations with premature termination of translation (number of bases deleted or inserted is not a multiple of 3)

SUBSTITUTIONS (POINT MUTATIONS)

nonsense mutations (e.g., TCA (serine) to TAA (stop)) missense mutations (amino acid substitutions)
conservative e.g. TCA (serine) to ACA (threonine) non-conservative e.g. TCA (serine) to CCA (proline)

SUBSTITUTIONS (POINT MUTATIONS)

silent mutation e.g. TCA (serine) to TCC (serine) mutations affecting promoter RNA splicing mutations (intron/exon splice sites or cryptic sites)

ALTERATIONS IN NON-CODING RNAS

inhibit translation of mRNAs into specific proteins (see text, pages176 and 218)
microRNAs, (miRNAs) long non-coding RNAS (lncRNAs) (very large number: greatly exceeds number of coding mRNAs Small interfering RNAs (siRNAs)

CYSTIC FIBROSIS DELETION OF AN ENTIRE CODON

one of the most common autosomal
recessive disorders

one in 22 Caucasians carries a mutation in

CFTR gene (more than 500 mutations)

defect in gene that encodes CFTR (cystic

fibrosis transmembrane conductance regulator)

 delta-F508 mutation in 70 percent of cases

deletion of three base pairs that code for phenylalanine CFTR protein made by the cell but not transferred to cell membrane

CYSTIC FIBROSIS DELETION OF AN ENTIRE CODON

DELTA F 508 MUTATION

Normal DNA

T T G T G G T T T C T A C T A

T T G T G G T T A C T A

CF DNA

Ile . Ile . Phe . Gly . Val. .ATC ATC TTT GGT GTT... Ile . Ile . Gly . Val. .. ATC ATT GGT GTT...

POINT MUTATION IN PROMOTER

Factor IX promoter
start of transcription

CTAATCGACCTTACCACTTTCACAATCTGCA
start of transcription

G mutation

POINT MUTATION AFFECTING SPLICE-SITE

normal splice site Normal HEXA allele CCAGGCTCTG gtaagggt. Tay-Sachs allele CCAGGCTCTG ctaagggt. no splicing

NONSENSE MUTATION

Normal allele

Asp - Asp - Ala - Lys -Arg -Gln GAT GAT GCC AAA CGA CAA

NF1 allele

GAT GAT GCC AAA TGA CAA Asp - Asp - Ala - Lys -Stop

FRAME-SHIFT WITH PREMATURE TERMINATION OF TRANSLATION

ABO A allele Leu - Val - Val - Thr - Pro .. CTC GTG GTG ACC CCT T ABO O allele CTC GTG GTA CCC TT Leu - Val - Val - Pro altered reading frame

SICKLE CELL ANEMIA

most common hereditary anemia in

persons of African lineage

change in codon 6 of -globin gene
GAG (glu) --> GTG (val)
abolishes a restriction site for MstII readily diagnosed by PCR or Southern blot

PCR reaction can be used

SICKLE CELL ANEMIA

MISSENSE MUTATION SICKLE CELL ANEMIA

A 1 2 3 4 5 6 7 beta Val - His - Leu - Thr - Pro -Glu -Glu globin GTG CAC CTG ACT CCT GAG GAG

S beta GTG CAC CTG ACT CCT GTG GAG Val - His - Leu - Thr - Pro -Val -Glu globin

Hemoglobin S Loss of restriction site

Pro . Glu . Glu CCT GAG GAG Pro . Val . Glu CCT GTG GAG A beta-globin

1.15 kb

0.2kb

S beta-globin

1.35 kb

HUNTINGTON DISEASE
autosomal dominant inheritance severe neurological disorder motor, cognitive, and psychiatric manifestations characterized by involuntary movements, mental deterioration, and death after 5-20 years

HUNTINGTON DISEASE
progressive neurodegeneration with

neuronal depletion mainly in striatum

(caudate nucleus and putamen) and frontal cortex delay of clinical abnormalities until age 30-40

HUNTINGTON DISEASE
increased numbers (more than 11-34)

of CAG trinucleotide repeats in HD

(huntingtin) gene on tip of short arm of chromosome 4 (4p16.3)

HUNTINGTON DISEASE
paternal transmission results in

increased number of CAG repeats and

earlier onset of disease in successive generations (anticipation)

HUNTINGTON DISEASE
CAG trinucleotide repeat expansion

codes for expanded polyglutamine tract

HUNTINGTON DISEASE
gain of toxic function as a result of an expanded polyglutamine tract can cause the protein huntingtin to interact abnormally with a variety of proteins, resulting in the complex

of neuropathological changes seen in

Huntington's disease

HUNTINGTON DISEASE
several huntingtin-interacting proteins

might be associated with normal

function of huntingtin and/or involved in the pathology of Huntington's disease

HUNTINGTON DISEASE
CAG repeats common to at least nine

inherited neurodegenerative diseases.

HUNTINGTON DISEASE
Since these diseases show distinct

neuropathological changes, it has been

suggested that protein environment and protein-protein interactions may play an important role in the specific neuropathology of these diseases

PRENATAL DIAGNOSIS
techniques
cytogenetics FISH for trisomies, etc. molecular analysis

specimen sources
amniotic fluid cells chorionic villus sampling umbilical cord blood

MOLECULAR TECHNIQUES
List of new techniques is rapidly increasing

PROCEDURES UTILIZING GENOMIC DNA

electrophoretic and blotting techniques
restriction analysis and southern blotting

amplification techniques
polymerase chain reaction, ligase chain reaction, several others

DNA sequencing methods

manual and automated sequence analysis

SOURCES OF HUMAN GENOMIC DNA

blood and other body fluids oral swabs and washings amniotic fluid cells, chorionic villus sampling, and umbilical cord blood urine sediment freshly isolated tissue samples in frozen surgical specimens preserved tissue samples (including formalinfixed/paraffin-embedded tissues) cultured cells forensic samples (clotted blood, hair, semen)

SOUTHERN BLOT
identifies gene fragments and demonstrates molecular size of fragment multistep procedure
digestion electrophoresis southern transfer hybridization detection

RESTRICTION ENZYMES

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RestrictionEnzymes.html

Southern Blot

Restriction enzyme DNA of various sizes Electrophorese on agarose gel gel Denature - transfer to filter paper.
blot

http://www.asip.org/edu/hs/Power%20of%20MBT.ppt

Denature- transfer to filter paper.

blot

Hybridize to probe

Visualize

http://www.asip.org/edu/hs/Power%20of%20MBT.ppt

Southern Blot

http://www.asip.org/edu/hs/Power%20of%20MBT.ppt

southern blot -- DNA

major deletions, insertions mutations affecting restriction sites

northern blot -- RNA

measure of gene expression

western blot -- proteins

specific antibody used as probe

ALLELE SPECIFIC OLIGONUCLEOTIDE HYBRIDIZATION

detects single nucleotide alterations useful for mutations that do not cause alterations of restriction sites

ALLELE SPECIFIC OLIGONUCLEOTIDE HYBRIDIZATION

oligonucleotide probes minor mismatch detected by destabilization of complex of probe and target DNA
vary temperature or salt concentration to detect destablization

ALLELE-SPECIFIC OLIGONUCLEOTIDE HYBRIDIZATION

ASOH IN CYSTIC FIBROSIS

Normal allele CF allele

IN-SITU HYBRIDIZATION
visual identification of sequences in tissue sections localization of genes on chromosome important widely used variation is fluorescent in situ hybridization (FISH)

RESTRICTION FRAGMENT LENGTH POLYMORPHISMS (RFLP's)

genetic linkage to DNA "marker" sequences

based on DNA polymorphisms

approximately one in every 200 to 500 bp is a

heritable polymorphism
most in non-coding areas create or abolish restriction sites

RESTRICTION FRAGMENT LENGTH POLYMORPHISMS (RFLP's)

fragments of differing lengths reflect

polymorphisms (RFLP's)
proper combination of restriction endonuclease and probe for a sequence physically "near" gene permits gene "tracking"

RESTRICTION FRAGMENT LENGTH POLYMORPHISMS (RFLP's)

useful for diagnosis facilitates localization of unknown genes

RESTRICTION FRAGMENT LENGTH POLYMORPHISMS (RFLP's)

requires samples from individuals displaying the genetic phenotype and key family members reliability increased the nearer the marker sequences to the gene of interest

RFLP ANALYSIS

VARIABLE NUMBER OF TANDEM REPEATS

Restriction site

Restriction site

Restriction site

Restriction site

Restriction site

SINGLE NUCLEOTIDE POLYMORPHISMS (SNPs)

much more common than RFLPs occur throughout genome used in similar fashion to RFLPs now considered to be marker of choice

DNA SEQUENCE ANALYSIS

chemical degradation or chain termination multiple fragments of DNA terminating alternatively at either G, A, T, or C gel resolution of fragments differing by only one nucleotide

DNA SEQUENCE ANALYSIS

four separate lanes each representing one of the G, A, T, or C termination sites read nucleotide sequence directly definitive tool for identifying sequence variations

DIDEOXY CHAIN TERMINATION SEQUENCING

Enzymatic Elongation Reactions
C G G A T C G A T A T

ddGTP dNTPs

ddATP dNTPs

ddCTP dNTPs

ddTTP dNTPs

POLYMERASE CHAIN REACTION (PCR)

oliogonucleotide primers bracket desired area of amplification

POLYMERASE CHAIN REACTION (PCR)

synthesis of new complementary strands mediated by heat resistant DNA polymerase multiple cycles of annealing, extension, and denaturation
controlled by sequentially raising and lowering temperature exponential amplification, twenty-five cycles theoretically amplifies DNA segment 106 fold

POLYMERASE CHAIN REACTION (PCR)

permits use of extremely small samples prior DNA purification not required facilitates use of molecular techniques in clinical setting

RT-PCR
variation of PCR using RNA as starting material RNA is first converted to c-DNA (complementary DNA) by treatment with reverse transcriptase Then PCR is performed

PCR
3

5 primers
3

DNA synthesis
3

5
5
3 3

5
3