Sickle Cell Disease Notes 4/1/2014 1:44:00 PM

The first patient with Sickle Cell Disease

Walter Noel presented standard symptoms to the hospital.
o Man of African origin
o First to be identified with SCD in NY
o Sample of his blood was taken
o Recovered from initial illness but died 9 years later
o 1910: James Herrick: supervised Ernest Irons, published
paper describing the casefirst clinical description of SCD

Symptoms of Sickle Cell Disease
Lots of muscle pain when sickle shaped blood cells in their
circulation are numerous enough to impede blood flow in smaller
vessels
Reduced blood flow deprives surrounding tissues of oxygen, causing
pain and long term damage
Sickled red blood cells are easily damaged and are removed from
the circulatory system and often cant be replaced quickly enough,
leading to chronic anemia.

Heterozygous for SCD means:
About 46% of blood cells look like sickles
Incomplete penetrance
More healthy than unhealthy cells means you dont display disease

Hemoglobin Structure
Tetramers w/2 protein chains each of two different globin genes,
alpha and beta
Most common form is hemoglobin A (HbA)
Each protein in Hb carries one iron containing molecule of heme,
which reversibly binds to oxygen (carries Oxygen)

At present, nearly 500 different allelic variants of alpha and beta globin
genes are known. Nearly all are rare.

SCD is a common hereditary anemia caused by a single base pair
substitution in beta globin gene
Mutant allele is Beta
S
, while normal allele is Beta
A

Single base change that results in amino acid change
Individuals with SCD carry two B
S
alleles and so have genotype
B
S
/B
S
(homozygous)

Causes of Sickle Shape in RBC
2 abnormal Beta-globin proteins + 2 normal Alpha globin proteins
The instability of abnormal hemoglobin molecule can cause them to
collapse into linear crystal molecules causes deformation of RBCs

Individuals Heterozygous for the Mutant Gene
Carriers of SCD have genotype B
S
B
A

Some of their hemoglobin molecules carry defective B-globin
proteins but b/c most of their RBCs are normal, they dont develop
anemia
Identified as having sickle cell train b/c they may have mild
symptoms

Genetic Variation Can be detected by Examining DNA, RNA, and
Proteins
Some techniques used to analyze Bs and Ba alleles

Spoke about Plasmids/Agarose Gel Experiment
Opens up plasmid
Sticky ends
EcoR
Engineered primers to have EcoR restriction sites on them
Put them in a digest
Take plasmid + gene in tube + ligase
EcoR could go on in two different ways
Take new recombinant plasmid w/gene of interest and stick it back
You should get a band the size of the plasmid and a band for gene
of interest
Then sequence the gene

Gel Electrophoresis (GE)
James Neel(1949)
o Used transmission genetic analysis & showed SCD is a
recessive disorder
Linus Pauling
o Published first molecular description of SCD and used the
term molecular disease
They used Gel electrophoresis to separate hemoglobin molecules
(protein) of each type
With DNA we use EtBr (reaches in & attaches to DNA)
Proteins generally use reversible or irreversible stain

Technique of Gel Electrophoresis
Gel is basically a matrix made of agarose (seeweed derivative) or
polyacrylamide (neurotoxin, powder form, synthetic): both
materials dont interact w/proteins or nucleic acids
o Can separate protein, RNA, or DNA
DNA runs to red(+): smaller fragment runs faster
GE allows separation of proteins in an electric field, based on
differences in size, shape, and charge
A support matrix (the gel) is made, through which proteins will
move
How dense do you want the agarose depends on your fragment of
interest?
The sample wells serve as the origin of migration: starting point
of the proteins
Once samples are loaded, an electrical current is applied to the gel
Samples migrate through tiny pores and passages in the gel matrix,
from charge to + charge
o Rates of migration depend on characteristic of proteins
o MW: smaller molecules migrate through pores faster
o MC: greater negative charge move toward + pole more
quickly
o Mshape: tightly condensed globular molecules migrate more
quickly

Globin Protein Variants can be Separted by GE
Pauling said Hb proteins showed that proteins from individuals
w/different b-globin genotypes have different electrophoretic
mobilitythe characteristic rate of migration
o Based on electrophoretic mobility, each type of protein
migrating through a gel forms a distinctive band
Two different versions of alleles (different alleles in the
genes make proteins)
Mutant allele (Bs) has lower electrophoretic ability than
Ba
Pauling used densitometry to show homozygous individuals have B-
globin proteins of just one type.
o Measures how much light is blocked from passing through the
gel by the protein in a band
o Demonstrated that Hb variation explains inheritance of SCD
as molecular disease

Hb peptide fingerprint analysis
1957: Vernon ingram published molecular basis of SCD based on
Hb protein
at the time, it was not known that Hb is a tetramer, composed of 2
types of globin proteins
Used peptide fingerprint analysis
o Hb protein broken into many fragments
o Then fragments separated by gel electrophoresis
o Fragments are next separated in a 2
nd
dimension
perpendicular to the first using, chromatography.

Chromatography:
Solvent used to separate fragments w/different amino acid
composition to different final positions
At the end of the 2 separations, locations of the protein spots
provide a fingerprint of the protein
Ingram identified ONE peptide fragment that differed b/w SCD Hb
and normal Hb

Analysis of peptide fragment of mutant and normal Hb showed 1
amino acid difference
Amino acid Val is found at position 6 of the B-globin protein instead
of Glu
Heterozygous SCD had 1 Glu containing spot and one Val containing

Scientists compare sequences from different organisms by aligning them
side by side noting number, location, and type of nucleotide differences
Single nucleotide polymorphisms(SNPs): single nucleotide
difference
o Genetic markers, tools in genetic mapping
o Transmitted genetically like any alleles
o Usually occur in unexpressed regions of genomes, w/no
detectable effect on genotype
o Sometimes they occur in expressed regions of genes, where
variation can affect phenotype

Restriction endonucleases can be used for genetic analyses of SNPs.
Cleave DNA molecules only at specific sequences.
The enzyme recognizes the short restriction sequences and then
cuts the DNA symmetrically.
w/multiple restriction sequences cut w/restriction enzyme, many
DNA fragments produced
inherited variability in the # or length of restriction fragments is
called restriction fragment length polymorphism (RFLP)

Restriction enzymes:
Each enzyme recognizes its own 5 to 3 nucleotide sequence on
DNA strand
Restriction sequences are palindromes
Cuts each sequence in the same way to produce either blunt ends
or sticky ends
Sticky ends: short, ss ends; fragments w/the same sticky ends can
bae pair w/one another
Blunt ends: no ss ends


SNP variation: sequence change that can either destroy or create a
restriction sequence
When SNP variation affects a restriction site, the length of restriction
fragments may also change (kb)=1000 nucleotide bases

EtBr allows detection of DNA or RNA fragments in gels, but isnt specific to
any particular gene.
Intercalates into DNA or RNA molecules
Exposure to UV light causes EtBr to emit fluorescence

General protein stains:
Can bind to any protein
Like EtBr, they are NONSPECIFIC
Used to pinpoint the locations of proteins in an electrophoresis gel

Methods for blotting involve transfer of nucleic acids/proteins from gel to
membrane
Southern blotting: DNA transfer
Northern blotting: RNA transfer
Western blotting: protein transfer

Molecular probe: antibodies or ss nucleic acids that specifically bind to
target molecules; used to identify specific molecules from a large pool of
heterogeneous molecules in an electrophoresis gel.

Ss nucleic acid probes used to detect RNA/DNA in northern or southern blots
Pairing of complementary sequences b/w probe & target nucleic acid is
hybridization.

**Check out Restriction fragment length polymorphism animation**


DNA sequence changechange in mRNAchange in protein sequence
Result of a SNP that alters the sixth DNA triplet of the coding
sequenceGLU replaced w/VAL
Gag to gug

Mutation that produces sickle cell disease destroys a restriction
sequencedetected by southern blot analysis

The b-globin gene has either 2 or 3 restriction sequences for the restriction
endonuclease, DdeI, depending on whether its SCD or WT allele

Normal and SCD alleles of the B-globin gene each produce mRNA of the
same size so mRNAs have the same electrophoretic mobility
Northern blot analysis of mRNA is note useful in detecting variation

However, amino acid difference in the 2 proteins causes electrophoretic
mobility differenceswestern blot analysis will detect this
4/1/2014 1:44:00 PM

4/1/2014 1:44:00 PM