Download Linkage Analysis

Markers in Gene Mapping
Linkage Analysis
 Genome p
project
j
 Marker
Marker--marker map; “framework map”
 Classes of markers
 Blood groups
 Electrophoretic mobility variants of serum proteins
 HLA types
 DNA RFLPs
 Minisatellites (DNA VNTRs)
 Microsatellites
Mi
lli
 DNA SNPs
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Informative Meiosis
Meiosis & Families in Linkage
 Assume that father has a dominant disease that he
 Phase
 The arrangement of the disease and maker alleles
 Phase known / unknown
inherited
i h i d along
l
with
i h marker
k A1
 Meiosis
 Informative / uninformative
 Phase
 Recombination
A and B are uninformative
C is informative & nonrecombinants
D is informative & recombinant
 Families in linkage studies
 Phase known / p
phase unknown
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Informative Families
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Choosing Families
 Choosing right families for a successful study
 Informative families: families with known phase (phase
phase--
known) between the marker and the disease locus
 Large families are ideal (eg.
(eg Huntington)
 Usually
U
ll families
f ili with
ith available
il bl genotypes
t
from
f
th
three
generations
 Good families for linkage studies: Large and informative
 Small families: more difficult (eg. Cystic Fibrosis)
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Parametric Linkage Analysis
Linkage Between Two Loci
 Are two loci linked? → Null hypothesis: no linkage
 To answer this we need these:
 Recombination fraction  between two loci
 If  is different from 0.5: is this deviation truly significant?
 Statistical tool : likelihood odds ratio (likelihood ratio, or odds ratio)
which is:
 Lod score (Z) = log10 [likelihood of data if loci are linked at
a particular
i l  / likelihood
lik lih d off d
data if llocii are unlinked]
li k d]
:
the
value
of

that
gives
the
greatest
value
of Z,
max
best estimate of . If  max differs from 50%, that is
evidence of linkage
 Positive values of Z (>1) suggest that the two loci are
linked
ed
 Negative values (<1) suggest that linkage is less likely
 Conventionally a combined Lod score of +3 or greater
(equivalent to greater than 1000/1 odds in favor of
linkage) is considered definitive evidence of linkage

Likelihood of data if loci are linked at a particular 
Likelihood of data if loci are unlinked ( = 0.50)
 Result expressed as the log10 of the ratio and called a Lod score (Z),
“logarithm of the odds”
 Use of log allows data collected from different families to be combined
by simple addition
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Example/ Phase Known Families
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Try This Example!!
 Consider this pedigree
 Phase is the disease allele
(D) with marker allele B normal allele d with marker
allele b
()0 (1 - )6
Z = log10
2
(1/2)0 (1/2)6
 Calculate Z similarly for
disease allele and marker
locus 1
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What if the Phase is Unknown?
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Lod Score in Phase Unknown Families
 In this case we have to consider two different possibilities
 So the Lod score would be
 Here the overall likelihood odd ratio of the two loci being
linked is the mean of the likelihood obtained from two
scenarios
 So the Lod score would be less for phase
phase--unknown
families
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1/2(1 - )3 + 1/2 3
Z = log10
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1/8
Try More !!
Try More !!
 Family
Family1
1: AD disease, marker alleles A, B, & C
 Family
Family2
2: same disease and marker
2
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Calculation of Lod Score
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Example of Lod Score Curves
 Family 1: Z > 3, with no recombinants
 Family
F il 2: Z > 3,  is
i 0
0.23
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 Nawdays
y Calculation of Lod score is computerized
p
 Family 3: Z < -2,  is 0.12, linkage is
excluded
 Family
F il 4:
4 data
d t iinconclusive
l i ffor allll
recombination fractions
 Exclusion mapping: excluding linkage is
also important
 Final results is the sum of Lod score
 What have you learnt from your calculation?
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obtained
obta
ed from
o d
different
e e t families
a es
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Limitations in Linkage Studies - 1
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Limitations in Linkage Studies - 2
 Requirement of a specific genetic model
 Mode of inheritance,
inheritance & penetrance of each genotype
 Non
Non--mendelian characters ???
 Computational limits (continues…)
 Many programs available
 MAKEPED, PREPLINK, LCP (Linkage Control Program),
LINKMAP, LINKLODS,
O S MLINK
 Genehunter & Merlin: good for whole
whole--genome studies
 Limits on the mapping resolution
 Mapping resolution ~ number of meiosis
 Linkage boundaries: 10 cM
 Available
A il bl families,
f ili
ffamily
il size
i
 Solution: sperm typing
 Vulnerability to errors
 Phenotype description
 Misdiagnosis, reduced penetrance
penetrance,, variable expressivity
 Genotype versus phenotype
 Genetic heterogeneity (allelic, and locus)
 Computational limits
 http://linkage.rockefeller.edu
h //li k
k f ll
d (under
( d lilinkage
k
analysis
l i
programs)
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Limitations in Linkage Studies - 3
Limitation in Linkage Studies - 4
 Problems with locus heterogeneity
 Vulnerability to errors (continues….)
 Ascertainment bias
 Error in g
genotyping
yp g
 Tuberous
T b
sclerosis
l
i
 TSC
TSC1
1 on 9q34 (hamartin),
hamartin), TSC2
TSC2 at 16p
16p13 (tuberin
tuberin))
 Information content of the p
pedigrees
g
or families
 Chromosomal phase
 Bilineal & unilineal pedigrees
 Solution:
 Defining phenotype (sub(sub-classes of disorders)
 Choosing families from isolated population
 Autozygosity mapping
 Nonpaternity
 Spurious recombinants
 Inflation of the length of genetic map
 Genehunter and Homog
Homog:: considering heterogeneity for a
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proportion of families
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Autozygosity Mapping
Application
 Autozygosity mapping:
 Application of autozygosity mapping
 Mapping genes for rare recessive conditions
 Extended inbred kindreds
 Isolated populations
 IBD & IBS
 Bateson (1902
(1902)) & Garrod (1908
1908):
): alkaptonuria
 Lander & Botstein (1987
(1987)) introduced the term
No locus heterogeneity
 Multiple people affected
 Definitions:
 Autozygosity
A
i ? (vs
(vs homozygosity
h
homozygosity)
i )
 Autozygosity mapping ?
Two or more sibships linked by inbreeding
 Examples
 Houwen et al. 1994
1994:: mapping benign recurrent intra
intra--hepatic
cholestasis
 Four affected in an isolated Dutch village
Using
U i linkage
li k
disequilibrium
di
ilib i
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Recessive Congenital Deafness
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Common Arterial Trunk (CAT)
 Chaib et al., 1996
1996.. Hum. Molec. Genet. 5, 155155-158
158..
 CAT or persistent truncus arteriosus (PTA): failure of
septation of cardiac outflow tract into the pulmonary
artery and aorta
 1% of congenital heart defect
 CAT can be a part of extended cardiac defects or in
isolated form
 Heart failure develops if untreated: usually death during
first 6 months of life
 22
22q
q11 microdeletion (DiGeorge and Velocardiofacial
syndromes) are among the causes
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CAT in a Pedigree
DNA Analysis
 Autozygosity mapping to identify a candidate gerion
gerion::
 A Kuweiti family with
 A genome screen in affected using 10 cM density set of STR markers
d bl fi
double
first-cousin
firsti
marriages and 6 affected
children
hild
with
i h CAT without
ih
any other cardiac defects
 Homozygosity in four locations
 Further analysis
y
of whole family:
y 3 loci were excluded
 Homozygosity IBD observed in 8p21
21(a
(a 14 cM region)
 Typing more markers within candidate interval: reduced the critical region of
h
homozygosity
it to
t ~ 6 Mb
 Three affected died during
 S
Searching
g for g
genes in candidate region
g
surgery
 Known genes potentially involved: FZD3
FZD3, FBX016
FBX016,, BNIP3
BNIP3L, TNFRSF
 Novel genes including: predicted human homolog to NKx2
NKx2.6
 Karyotype analysis for the
 Mutation screening of candidate genes
rest: excluded 22
22q
q11
microdeletion
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Next Steps
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Common Arterial Trunk
 Mutation screening of candidate genes
 T451
451C
C transition was found in exon 2 of NKx2
NKx2.6: all affected
homozygous , and all parents were heterozygous for this
 Substitution of phenylalanine for leucine at position 151 in the
predicted
di t d protein
t i (F151
(F151L)
L)
 Position 20 of homeodomain
 This variant was absent in 100 Kuwaiti,
Kuwaiti 100 Arab,
Arab and 250 Caucasian
control DNA samples
 Functional analysis of the mutation
 F151L
151L missense mutation impairs transcriptional activity of NKx
NKx2
2.5
and NKx2
NKx2.6
 It reduces synergistic transcriptional activation of TBX
TBX5
5 and NKx
NKx2
2.5
and removes this with GATA4
GATA4
 This mutation reduces DNA binding activity of NKx2
NKx2.5
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Thank you for listening, any comments?
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