13' Polymorphisms and RFLPs

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13. Polymorphisms and RFLPs
a). Polymorphic alleles i). Definitions all
ele polymorphism ii). Polymorphic
genes RBC antigens galactose-1-phosphate
uridyl transferase iii). Significance of
polymorphisms b). Single nucleotide
polymorphisms (SNPs) c). Restriction fragment
length polymorphisms (RFLPs) i). Point
polymorphisms ii). Variable numbers of tandem
repeats (VNTRs)
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• Learning objectives
• know what polymorphisms are and their
  relationship to alleles
• understand the term haplotype
• understand the ABO polymorphism and the
  biochemical
• basis for the ABO blood group antigens
• understand the GALT polymorphism and its medical
  significance
• understand the general significance of
  polymorphic genes in
• the human genome
• understand SNPs and how they are assayed
• understand the relationship between polymorphic
  genes and RFLPs
• know the two basic types of RFLPs
• know the method for analysis and detection of
  RFLPs

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• Polymorphic alleles
• definitions
• allele
• one of the alternative versions of a DNA
  nucleotide sequence
• that may be at a given chromosomal locus
• polymorphism
• nucleotide sequence variation at allelic
  chromosomal sites caused
• by base-pair mutation, deletion, or
  insertion -- polymorphism
• usually refers to the existence in the
  population of two or more
• alternative genotypes -- a site is
  polymorphic if there are two or
• more alleles being maintained in the
  population

• there is a polymorphism on this chromosome
  involving alleles A and B.

A
B
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• there can be more than two alleles in a
  population, i.e., A,B,C,D or A,B,O
• or G,g,C,D,LA or 1,2,3,4,5,6, etc., etc.
• haplotype is the allelic constitution of
  multiple loci on a chromosome,
• i.e., for the A,B,C,D and 1,2,3,4,5,6 loci

Individual 1
Individual 2
chromosomal loci with multiple alleles
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• Polymorphic genes
• frequency in human genome
• the number of existing polymorphisms is 1 per
  100-300 bp
• there are 5 million differences per individual
  genome
• mutant alleles are the most obvious
  polymorphisms
• normal vs. abnormal
• rare variants are alleles that are present in
  the population at a
• frequency of lt1 - most deleterious mutations
  that lead
• to genetic disease are rare variants
• allelic heterogeneity
• mutant alleles at the same locus, each capable
  of producing an
• abnormal phenotype
• silent mutations are polymorphisms with no
  phenotype
• many genes exist in the population in several
• distinguishable forms -- some are clinically
  significant
• in certain combinations with other deleterious
  genes

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• Red Blood Cell antigens
• Systems Antigens
  Substrates
• ABO oligosaccharides glycosphingolipids
• Lewis oligosaccharides
• MN amino acid sequences glycophorin A
• Ss amino acid sequences glycophorin B
• Rh amino acid sequences

Carbohydrates having the A, B, and H (type O)
antigens
M
A
B
H
S
s
N
M
outside
glycosphingolipids
glycophorin-B
glycophorin-A
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• biosynthesis of the ABO antigens

ABO
Enzyme Enzymes
Antigens
A-transferase
A
H-transferase
B-transferase
H
B
no transferase
H (type O)
Precursor substrate
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fucose
A-transferase
galactose
N-acetylgalactosamine (GalNAc) transferase
N-acetylglucosamine (GlcNAc)
galactose
A
ceramide
B-transferase
Galactose transferase
B
H (type O)
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• genetics of the ABO antigens
• the ABO antigens result from three alleles at a
  single genetic locus
• the gene encodes two variant glycosyltransferases
  , and a third allele
• of the same gene produces no functional
  protein
• alleles A and B differ by four base-pairs
• A utilizes N-acetylgalactosamine
• B utilizes galactose
• allele O was derived from allele A by a single
  base-pair deletion

ABO A allele Leu-Val-Val-Thr-Pro CTC CTG
GTG ACC CCT T single base-pair
deletion CTC GTG GT- ACC CCT T ABO O
allele CTC GTG GTA CCC CTT Leu-Val-Val-Pro-Le
u altered reading frame no functional
protein
A single base-pair deletion at the ABO locus,
which encodes a glycosyltransferase, converted
A to O
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Importance of A, B, O blood group antigens in
medicine Transfusion compatibility Blood
type Donate to AB AB
A A or AB B B or AB
O O, A, B, AB Disease resistance Resistance
to cholera and other types of infant
diarrhea AB gt A gt B gt O Possible resistance
to other diseases malaria, syphillis, cancer
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Polymorphic loci associated with phenotypic
variation
ABO blood group system Alpha1-antitrypsin Alcohol
dehydrogenase Aldehyde dehydrogenase HLA
system Debrisoquin metabolism (CYP2D6
4-hydroxylase) Lactase activity Beta-globin Vitam
in D receptor CFTR
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Genetic variation at the galactosemia locus

• gene encodes galactose-1-phosphate
  uridyltransferase (GALT)
• recessive mutation results in inability to
  metabolize galactose
• causes mental retardation and death
• some protection afforded by complete removal of
  milk from the diet
• variant alleles exist in addition to several
  galactosemia (g) alleles
• spectrum of enzymatic activities indicates that
  normal individuals
• do not all have the same enzymatic activity levels

Enzyme Genotype Frequency Activity
Phenotype G/G 87.4 100 Normal
G/D 7.5 75 Normal G/LA
3.7 120 Normal G/g 0.9
50 Normal D/D 0.16 50
Normal D/LA 0.16 95 Normal
LA/LA 0.04 140 Normal D/g
0.04 25 Borderline LA/g 0.02
70 Normal g/g 0.0025
lt5 Galactosemia
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Population distribution of normal GALT
activities
G/G
Relative number of individuals
G/LA
G/g
G/D
g/g
0 20 40 60 80 100
120 140 160
Relative GALT enzyme activity
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• Significance of polymorphisms
• There is a considerable degree of individual
  diversity in the population
• Thousands of polymorphisms exist that are
  inherited independently
• Enormous numbers of combinations of genotypes
  are possible
• Many gene products interact in metabolic
  pathways
• Individuals have a unique, genetically
  determined, chemical makeup
• Medical significance of polymorphisms
• Each person will respond differently to the
  environment, to diet,
• and to pharmacological treatments

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Single Nucleotide Polymorphism (SNP)

• The Human Genome Project has identified millions
  of SNPs
• Microarray-based methods can rapidly screen
  individual genomes

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GeneChip Assay
Products
Restriction Digest Genomic DNA
Adaptor Ligation
PCR Amplify
10n
Fragment End-Label

patient
control
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Patient DNA
TCGGCATGTCAA //////////// AGCCGTACAGTT
Immobilized DNA
Control DNA
TCGGCATGTCAA //////////// AGCCGTACAGTT
Immobilized DNA
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Patient DNA
CGTCTGCAATTC //////////// GCAGACGTTAAG
Immobilized DNA
Patient DNA
CGTCTGCAATTC //////////// GCAGACGTTAAG
Immobilized DNA
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• AmpliChip CYP450 test
• Detects variations in the CYP2D6 and CYP2C19
  genes
• Provides associated predictive phenotype
• (poor, intermediate, extensive, or ultra-rapid
  metabolizer)
• 29 known polymorphisms are detected on one chip

• CYP2D6 drugs used to treat severe depression,
  schizophrenia, bi-polar
• disorder, cardiovascular disease treated with
  beta-blockers, ADHD, etc.
• CYP2C19 anti-convulsants, proton pump
  inhibitors, benzodiazepines, and
• anti-malarials
• Both CYP2D6 and CYP2C19 certain tricyclic
  antidepressant drugs

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• Restriction fragment length polymorphism (RFLP)
• a different kind of polymorphism that is useful
  for
• mapping genes (as with any genetic marker)
• predicting those at risk for a disease
• isolating genes by positional cloning

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• Restriction fragment length polymorphisms (RFLPs)
• Definitions
• polymorphism
• nucleotide sequence variation at allelic
  chromosomal sites
• caused by base-pair mutation, deletion, or
  insertion...
• RFLP
• polymorphism that can be detected as a change
  in the
• restriction fragment length pattern -- alleles
  are defined
• by the sizes of bands obtained by Southern blot
  analysis
• Two alleles A and a that differ
• from one another by the absence
• or presence of a restriction enzyme
• cut site in the chromosome

EcoRI
EcoRI
A
GAGTTC
EcoRI
EcoRI
EcoRI
a
GAATTC
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Southern blotting procedure
human genomic DNA (isolated from
many cells)
-

• gel electrophoresis
• of the DNA fragments
• gel will separate DNAs
• according to size

• restriction enzyme
• digestion

millions of DNA fragments

• hybridize membrane with
• a 32P-labeled DNA probe
• probe will base pair with the
• complementary DNA strands

• denature
• DNA into
• single-
• strands
• transfer
• DNA fragments to
• nitrocellulose membrane

• expose membrane to X-ray film
• develop film to visualize DNA band

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• Fragments detected by Southern blotting
• cut DNA by restriction enzyme digestion
• hybridize labeled probe to DNA fragments
• labeled probe will detect one restriction
  fragment as shown

• labeled hybridization probe
• restriction fragment detected
• by hybridization probe
• adjacent fragment not detected

32P
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• Restriction fragment length polymorphisms (RFLPs)
• RFLP
• polymorphism that can be detected as a change in
  the restriction
• fragment length pattern -- alleles are defined
  by the sizes of bands
• obtained by Southern blot analysis
• polymorphism due to the absence or presence of a
  single cut site

A and a are alleles
RFLP probe
AA Aa aa
32P
A
a
polymorphic site caused by the creation /
elimination of a restriction enzyme cut site
polymorphism detected by Southern hybridization
with a 32P- labeled probe
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• polymorphism due to variable number of tandem
  repeats (VNTR)

RFLP probe
A, B, and C are alleles
AA AB BC
32P
A
B
C
polymorphism detected by Southern hybridization
with a 32P- labeled probe
polymorphism caused by deletion / insertion
between restriction enzyme cut sites

• VNTRs are useful because they are frequently
  highly polymorphic--
• there are more than two alleles, i.e.,
  A,B,C,D,E
• highly polymorphic sites are more useful for
  distinguishing
• chromosomes in the population because there are
  more
• heterozygous chromosomes

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Inheritance of a hypervariable polymorphism
Southern blot using a probe for a hypervariable
VNTR Everyone in the pedigree is heterozygous at
the locus
From Thompson Thompson, Genetics in Medicine