Allelic Association and Transmission Disequilibrium Test

1
Allelic Association and Transmission
Disequilibrium Test

• ?????
• ??????????
• ???????
• ??? (x 2641)

2
Linkage Disequilibrium (allelic association)

• Def nonrandom association of alleles at linked
  loci
• f(A, B) f(A) x f(B)

3
(No Transcript)
4
Haplotype

• Haplotype a series of alleles found at linked
  loci on a single chromosome. e.g,
• A33-Cw10-B58-DRB10301-DQB102(6.3)
  A2-B46-Cw11-DRB109-DQB10303 (orientals)
• A30-Cw5-B18(Bw6)-DRB10301-DQB102 (most frequent
  in Mediterranean)
• A1-Cw7-B8(Bw6)-DRB10301- DQB102 (commonest in
  European Caucasoids)

5
The Relationship of Disease Locus and Marker
Defined by Linkage Disequilibrium

• The stronger the linkage disequilibrium, the
  closer the marker is to the disease locus
• Mapping and/ or gene identification using LD is
  particularly useful in genetically unique or
  isolated populations (Amish and Finnish
  populations)
• LD rarely extends more than 1 cM from the
  susceptibility locus, its detection indicates a
  significant narrow down of candidate region

6
Allelic Association Studies

• How it happens?
• Direct biological action of the genetic
  polymorphism
• Linkage disequilibrium with a adjacent
  susceptibility gene
• How useful in the analysis of genetic complex
  disease?
• Evaluation of candidate gene loci
• Fine mapping of region that is indicated by
  linkage analysis for follow-up studies

7
Association vs Linkage

• Association and linkage distinct from each other
• Linkage a specific genetic relationship between
  loci
• Association Statistical statement about the
  co-occurrence of alleles or phenotypes
• Allele A is associated with disease D
• if individuals who have D also have A more often
  than would be predicted from the individual
  frequencies of D and A in the population

8
Allelic Association

• Def A significantly increased or decreased
  frequency of a marker allele with a disease trait
  and represents deviations from the random
  occurrence of the alleles regarding the disease
  phenotypes
• Linkage disequilibrium means allelic association
  caused or maintained by tight linkage

9
Linkage Disequilibrium in Genetic Analysis

• Defining the ancestral haplotype of a disease
  gene in relation to several marker loci
• Fine-mapping of the disease gene even when
  complete linkage (? 0.0) is established in the
  family studies
• The slowness of linkage disequilibrium decay make
  LD a useful mapping tool

10
Conditions for LD Mapping

• Founder population
• Geographically isolated population, traceable
  ancestry, most individuals of current population
  are descendants of a few individuals back ? 250
  years (? 20 generations)
• The prevalence of a genetic disease is derived
  from an allele for a disease gene from a common
  ancestor
• Both parents in many of the marriages within a
  founder populations are heterozygous for a
  recessive disease gene, will have a 25 chance of
  having an affected offspring

11
Rate of Decay of Linkage Disequilibrium time and
distance-dependent

• Dt Do(1-?)t
• t current generation number
• Dt current amount of disequilibrium
• Do disequilibrium at generation 0
• ? recombination fraction between loci

12
Decay in linkage disequilibrium
13
Significance of Allelic Association

• Allelic associations reflect sharing of ancestral
  chromosomes, only alleles at loci tightly linked
  to the disease susceptibility locus will still be
  shared
• For a locus showing recombination fraction (?)
  with the susceptibility locus, a proportion (? )
  of ancestral chromosome will lose the association
  each generation, and a proportion (1- ?) will
  retain it.
• (1-0.01)44 0.64, loci 1cM apart
• (1-0.03)44 0.26, loci 3 cM apart

14
Linkage Disequilibrium as a Mapping Tool-I

• Cystic fibrosis
• mapped to 7q32, the candidate region was still
  very large
• XV2.c and KM19 marker within the candidate gene
  show strong association between (XV2.c1,
  KM192) haplotype and CF

15
Linkage Disequilibrium as a Mapping Tool-II

• Nijmegen breakage syndrome (NBS)
• Rare autosomal recessive disease characterized by
  chromosomal breakage, growth retardation,
  microcephaly, immunodeficiency and a
  predisposition to cancer
• Genetic linkage in families mapped NBS to an 8-Mb
  regions between D8S271 and D8S270

16
Linkage Disequilibrium as a Mapping Tool-II

• Nijmegen breakage syndrome (NBS)
• 74 haplotypes related to a common ancestral
  haplotype.
• Some do not have this haplotype, possibly carry
  independent NBS mutations.
• Others share only part of haplotype, showing the
  effect of recombination in distant ancestors

17
Ancestral haplotype in patients with Nijmegen
breakage syndrome A allele attributed to AH
51 unrelated patients typed for microsatellites
18
Linkage Disequilibrium Mapping
I1
I1
I2
I3
G1
G1
G3
G2
E1
E1
E1
E2
C1
C1
C1
C3
A1
A1
A1
A1
10 generations
Mutation
90 generations
N
M
M
M
N?M
B1
B1
B1
B1
D1
D1
D1
D1
F1
F1
F1
F2
H1
H1
H1
H3
J1
J1
J3
J4
K1
K1
K4
K2
19

• Jennings 1917 first developed the concept of LD
• Richard Lewontin (1964) first used LD to measure D

20
Linkage Disequilibrium Coefficient (D)(Lewontin
1964)

• D P11- p1q1 (if D significantly differs from
  zero, LD is thought to exist)
• P11 observed frequency of the 1/1 haplotype
• p1 frequency of the allele 1 at the locus A
• q1 frequency of the allele 1 at the locus B

locus A(1,2)
locus B(1,2)
21

• p1 f(A1), p2 1- p1 f(A2)
• q1 f(B1), q2 1-q1 f(B2)
• Dmax min(p1q2, p2q1)
• Dmin max(-p1q1, -p2q2)
• D D/Dmax
• Alternatively,

22

• D h11- p1q1 , where h11 is the frequency of
  the haplotype with the rarer allele at each
  locus, and where p and q are frequencies of the
  rarer alleles at loci 1 and 2, respectively
  (Devlin and Risch, 1995).
• D is positive when the rarer alleles at each
  locus are associated and is negative when a
  common allele is associated with a rare allele
• Dmax min(pq, (1-p)(1-q), for D lt0
• Dmax min(p(1-q), q(1-p), for D gt0

23
Determination of LD Coefficient
D

• a, b, c, d are the phenotype frequencies of the
  /, /-, -/
• and -/- combinations of the alleles in each
  haplotype and
• n is the sum of the a, b, c, d.

• HFij Dij GFi x GFj

• GF 1 -

24
Measure LD by D(2)(Hill and Robertson 1968)

• R or ? v?2/N D/v(p1p2q1q2)
• ?2 statistic obtained from 2 x 2 table
• N total number of haplotypes in the sample

Genome research 10 1435-1444
25
Measure of LD-(3)(Bengtsson and Thomson 1981)

• ? D/(q1p22)
• q1 The population frequency of a disease
  allele, B1
• P22 the frequency of chromosome that contain
  marker allele A2 and normal allele, B2

26

• ? and D give more reliable estimates of
  physical distances than do D and R, because D and
  R depends on allele frequency

27
(No Transcript)
28
Quantitative Measurement of Linkage Disequilibrium

• Yule coefficient (p1,1- p1,2)/(p1,1 p1,2 - 2
  p1,1 p1,2)(used in Huntingtons disease)
• p1,1 the frequency of allele A1 on the
  chromosome carrying allele B1
• p1,2 the frequency of allele A1 on the
  chromosome carrying allele B2

B1, B2
A1, A2
locus A
locus B
29
Allelic association around the locus for
Huntington Disease
Acc I
Total Distance 2500 kb
250 kb
Mbo I
Taq I
L19ps11
BS674
30
(n1.)(n.1)/N expected values(600 x 300 /1000
180)
31
Advantages of Association Studies

• Association studies are easier to conduct than
  linkage analysis , no multicase families or
  special family structures
• LD is a short range phenomenon, if association
  exists, it defines a small candidate region in
  which to search for disease gene
• Association is more powerful than linkage for
  detecting weak susceptibility alleles

32
Possible Causes of Positive Association-I

• Direct causation having allele A make you
  susceptible to disease D possession of A is
  neither necessary nor sufficient to develop
  disease, but it increases the likelihood
• Natural selection People who have disease D
  might be more likely to survive and have children
  if they also have allele A
• Population stratification population contains
  several genetically distinct subsets. Both the
  disease D and allele A happen to be particularly
  frequent in one subset (HLA-A1 and chopsticks
  user in San Francisco bay area).

33
Possible Causes of Positive Association-II

• Statistical artefact association studies often
  tests a range of loci, each with several alleles,
  for association with a disease. The raw p values
  need correction for the numbers of genes tested
• Linkage disequilibrium If LD exist between the
  disease D and the locus A, there should be a gene
  near to the A locus that has mutations in people
  with disease D.

34
Advantages and Pitfalls of Association Study

• Advantages
• Not restricted to nonmendelian genetics
• Easy to perform (case and control)
• Pitfalls
• Selection of controls is very crucial (
  representative?!)

35
Probabilities calculated from Association studies
must be corrected for the number of questions
asked
Threshold of significance is p 0.05/n
(Bonferroni correction)
36
Relative Risk (RR)/Odds Ratio(OR)

• Estimate how many times more the carrier of a
  specific allele or haplotype was likely to have a
  specific disease associated with the particular
  genetic marker studied

37
Calculation of RR

• RR a x d/ b x c
• a the number of patients positive for the allele
  or haplotype
• b the number of patients negative for the allele
  or haplotype
• c the number of controls positive for the allele
  or haplotype
• d the number of controls negative for the allele
  or haplotype

38
Calculation of RR (Relative Risk)

• 
  Sample
• Risk factor Cases Controls Total
• Present a b ab
• Absent c d cd
• Total ac bd n
  

39

• Linkage disequilibrium mapping is carried out
  after genetic linkage between a polymorphic locus
  and the disease gene is determined

40
The Genetic Distance(?) between a Marker Locus
and the Disease Gene that are in Linkage
Disequilibrium-I

• Pexcess Paffected-Pnormal/(1- Pnormal) (1- ?
  gq-1 )(1- ? )g
• ? recombination fraction between marker and
  disease loci
• ? mutation rate (1 x 10-6)
• g the number of generations after since the
  common ancestor
• q world wide frequency of the disease ( 0.001)

41
The Genetic Distance(?) between a Marker Locus
and the Disease Gene that are in LD-II

• Pnormal the proportion of the marker allele in
  normal chromosomes
• Paffected the proportion of the marker allele
  in diseased chromosomes
• Pexcess measure of disequilibrium, the fraction
  of the excess occurrence of a chromosome with the
  disease gene and a marker allele compared with
  the chromosome with the nondisease gene and the
  marker allele

42
Functional SNPs in the lymphotoxin-a gene that
are associated with susceptibility to myocardial
infarction(Nature Genetics 32 650-654, 2002)

• Linkage disequilibrium mapping
• Haplotype analysis
• D D/Dmax

43
65,671/92,788 70.8
44
A cut-off P value of 0.01 for association in
either recessive and dominant mode
45

• A SNP in intron 1 of LTA (252A-gtG) on chromosome
  6p21was associated with myocardial infraction in
  the initial screening

46
(No Transcript)
47

• Construct a high-density SNP map for LD mapping
  by sequencing 16 MI patients and 16 normal
  controls.
• 187 SNPs, 130 kb within 6p21
• Select 120 SNPs (gt10) and genotyping 94 MI
  patients and 94 subjects from general population
• 26 SNPs with minor allele frequency gt 0.25

48
ive
49
1
2
3
4
50

• Located in one extended block of intense LD, with
  D dropping off near p5-1 and AIF1
• They concluded that the gene associated with
  susceptibility to myocardial infraction was
  probably located between these two loci

51
(No Transcript)
52
(No Transcript)
53
The Disatrophic Dysplasia Gene Encodes a Novel
Suflate Transporter Positional Cloning by
Fine-Structure Linkage Disequilibrium Mapping

• Cell 78 1073-1087, 1994

54
Diastrophic dysplasia (DTD) (??????)

• One form of Osteochondrodysplasia
• Constitutional disorders of skeletal system
  result in disturbed growth and/or density of bone
• First described by Lamy and Maroteaux in 1960
• Autosomal recessive

55
Diastrophic dysplasia)(DD)(??????)

• Short-limbed short stature, kyphoscoliosis(?????),
  generalized dysplasia of the joints, peculiar
  flexion limitation of the finger joints,
  hitchhiker thumbs, metatarsus adductus (???)
  deformity of the feet, and deformation of the ear
  lobes and cleft palate
• Joint changes are progressive in nature
• Osteoarthroses(???????) and contractures develop
  at an early age

56
Diastrophic dysplasia)(DD) (??????)

• Patients are severely physically handicapped and
  need repeated corrective orthopedic surgery
• Increased mortality in infancy due to respiratory
  difficulties and spine anomalies
• Intelligency and sexual development are normal
• Life expectancy is not clearly shortened
• Abnormal cartilage, number and morphology of
  chondrocytets, the organization of collagen
  fibrils, and the deficiency of sulfated
  proteoglycans

57
Diastrophic dysplasia and D5S72 is Genetically
Closely Linked
No recombination between DD and D5S72 (Z(?) 7.37)
58
Principle of Linkage Disequilibrium (LD) Mapping

• Among a collection of chromosomes carrying the
  same ancestral mutation, genetic markers nearest
  the disease gene will have recombined least often
  and thus should show the highest degree of
  allelic association with the disease on such
  chromosomes

59
How to Do Linkage Disequilibrium Mapping

• Determine the haplotypes of disease-bearing
  chromosomes for an extremely dense collection of
  genetic markers
• Use these haplotypes to identify a subset of
  chromosomes likely to carry a common ancestral
  mutation
• Find the genetic markers that show the strongest
  allelic association with the disease on these
  chromosomes

60
(No Transcript)
61
Genetic Linkage studies of DTD

• 18 multiplex and 59 singleton families (a total
  of 144 DTD-baring chromosomes)
• DTD was initially localized to the interval
  between GRL and SPARC on chromosome 5q
• CSF1R gene 1-1 haplotype at these two marker
  loci was present on 95 of DTD-baring
  chromosomes, but only 4 of normal chromosomes
  (RFLP analysis)

62
Physical map shows locations and direction of
transcription

• AC3 STS from the 3 end of CSF1R
• BT1 CA-repeat (SSR)
• STS content mapping of the clones
• Known genomic organization of PDGFRB-CSF1R

63
(No Transcript)
64
Genetic map ordered by recombinational mapping

• DTD was estimated to lie about 0.064 cM from
  CSF1R
• Genetic map order of each pair of adjacent loci
  supported by obligate crossovers with exchange of
  flanking markers either in CEPH pedigrees or DTD
  families
• GRL-ADRB2-D5S413-D5S372-BT1-CSF1R-PDGRRB-RPS14-SPA
  RC-D5S72

65
Physical map shows locations and direction of
transcription

• AC3 STS from the 3 end of CSF1R
• BT1 CA-repeat (SSR)
• STS content mapping of the clones
• Known genomic organization of PDGFRB-CSF1R

66
Number of Normal and DTD chromosomes carrying
Alleles from Finnish Ancestral DTD Haplotype
67

• Most DTD-bearing chromosomes carrying a single
  predominant allele at each of the 11 markers
  studied
• 95 of DTD-bearing chromosomes with haplotype
  1-1-1-1-1-1-1-1-1-1-1

68
Linkage Disequilibrium Mapping

• P excess (P affected P normal)/(1-P normal)
• ?a(1-?)g
• P directly related to proximity to the disease
  locus
• a proportion of disease-bearing chromosomes
• g the number of generations since the
  introduction of ancestral chromosome

69
Linkage Disequilibrium in DTD Region
70
Clues Regarding the Location of DTD Proximal to
CSF1R

• P excess falls rapidly a the PDGFRb-CSF1R, but
  remains high at the marker BT1 proximal to CSF1R.
• Haplotype pattern of 144 DTD chromosomes
• Single DTD chromosome lack ancestral allele at
  the genetic markers within the PDGFRB and CSF1R
  and present at the more proximal genetic markers
  BT1 and D5S372.

71
(No Transcript)
72
Gene Identification of Region 100 Kb Proximal to
CSF1R

• Exon trapping of P1-1013, P1-1014
• Direct cDNA selection
• Analysis of genomic fragments from this region
• Clones (JH10140B, 0.8 kb fragment from
  centromeric end of P1-1014, show strong aa
  similarity to the 5 end of rat gene (sat-1,
  sulfate transporter) from BLAST program

73
Expression Profile of Candidate DTD Gene
Northern blot analysis
Probe 0.8 Kb JH10140B
74
DTD 8.4 kb mRNA is Missing from DD Patients
75
DTDST gene contains two exons separated by 1.8
kb intron
76
DRA gene (down-regulated adenoma) from
subtractive hybridization between colon and colon
carcinoma
77
DTDST Shows 12 Transmembrane Helices (hydropathy
analysis)
Two potential N-glycosylation sites at the
N-terminal extracellular loop
78
Confirmation of Mutation
Normal
SP Family
Normal
FV Family
JD IM
SA(AG?AC, BsaAI created)
Codon 575 (AAG?AG, Dde I eliminated)
1000 bp? 2 x 500 bp
100 bp(Dde I) ? 40 bp 60 bp
79
Summary of Mutations
80
(No Transcript)
81
Transmission disequilibrium test (TDT)

• Association studies with internal controls
• Couples who have one or more affected offspring,
  it is irrelevant wheterh either parents is
  affected or not
• To test whether marker alleles M1 is associated
  with the disease
• Those parents who are heterozygous for M1

82

• Let a be the number of times a heterozygous
  parent transmits M1 to the affected offspring,
  and b be the number of times the other allele is
  transmitted.
• TDT test statistic is (a-b)2/(ab)

83
Stevens-Johnson Syndrome (SJS) or Toxic Epidermal
Necrosis (TEN)

• In 1922, Stevens and Johnson described two
  children with fever and stomatitis, severe
  disseminated conjunctivitis and cutaneous
  eruptions
• The reaction develops within 1-4weeks from the
  beginning of drug therapy
• Mucosal lesion includes lesions of the mouth,
  eyes, GI, respiratory tract, anus and vagina

84
Drugs associated with SJS and TEN

• Sulfonamides,
• anticonvulsant agents
• Allopurinol, are the most consistently associated
  with SJS and TEN
• Nonsteroidal antiinflammatory drugs (NSAIDs)
• Analgestic agents
• Nonsulfonamides antibiotics, controversial?

85
Typical pattern of Stevens-Johnson syndrome (SJS)
Blisters develop on widespread purpuric macules
N Engl J Med 1333 1600-1607, 1995
86
Typical pattern of toxic epidermal necrosis (TEN)

• Severe forms of SJS
• Extensive skin detachment and a poor prognosis
  (death rate 3040)

Blisters and wrinkled areas result from
full-thickness necrosis of the epidermis
87
A marker for Stevens-Johnson Syndrome

• carbamazepine, treatment for seizures(??)
• 44 CBZ-SJS patients
• 101 CBZ-tolerant (93 Normal controls)
• Diagnostic criterion of SJS/TEN were based on
  clinical morphology
• SJS skin detachment of ?10,
• TEN skin detachment of ?30,
• Overlapping SJS/TEN skin detachment of 10-30

Nature 428 486, 2004
88
Genotyping methods

• HLA-A, -B, -C, DRB1(sequence-specific
  oligonucleotide probe, SSOP)
• cytochrome p450 (157 Single nucleotide
  polymorphism)
• What is single nucleotide polymorphism ?
  ATG(M)?ATA(I) TTC(F) ?ATC(I))

89
Nature 428 486, 2004
90
Nature 428 486, 2004