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13' Polymorphisms and RFLPs

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nucleotide sequence variation at allelic chromosomal sites caused ... Polymorphic loci associated with phenotypic variation. ABO blood group system. Alpha1-antitrypsin ... – PowerPoint PPT presentation

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Title: 13' Polymorphisms and RFLPs


1
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)
2
  • 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

3
  • 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
4
  • 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
5
  • 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

6
  • 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
7
  • 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
8
fucose
A-transferase
galactose
N-acetylgalactosamine (GalNAc) transferase
N-acetylglucosamine (GlcNAc)
galactose
A
ceramide
B-transferase
Galactose transferase
B
H (type O)
9
  • 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
10
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
11
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
12
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
13
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
14
  • 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

15
Single Nucleotide Polymorphism (SNP)
  • The Human Genome Project has identified millions
    of SNPs
  • Microarray-based methods can rapidly screen
    individual genomes

16
GeneChip Assay
Products
Restriction Digest Genomic DNA
Adaptor Ligation
PCR Amplify
10n
Fragment End-Label

patient
control
17
Patient DNA
TCGGCATGTCAA //////////// AGCCGTACAGTT
Immobilized DNA
Control DNA
TCGGCATGTCAA //////////// AGCCGTACAGTT
Immobilized DNA
18
Patient DNA
CGTCTGCAATTC //////////// GCAGACGTTAAG
Immobilized DNA
Patient DNA
CGTCTGCAATTC //////////// GCAGACGTTAAG
Immobilized DNA
19
  • 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

20
  • 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

21
  • 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
22
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

23
  • 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
24
  • 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
25
  • 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

26
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
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