Download From linkage analysis to linkage disequilibrium mapping: the case of

From linkage analysis to linkage disequilibrium
mapping: the case of HRPT2
(a gene mutated in Hyperparathyroidism-jaw tumor syndrome)
by Silvano Presciuttini
Dept.of Biomedicine, University of Pisa, Italy
and IDRB, NHGRI, Baltimore, USA
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Linkage analysis is a powerful tool to locate genes of strong effect in
a chromosomal region. However, it is not very efficient for fine
mapping and has limited power to detect genes of ‘modest’ effect.
In contrast, association analysis is highly efficient for fine mapping,
as appreciable linkage disequilibrium exists in human between loci
with recombination fractions of less than 1-2%
S.P.
The classical linkage analysis method

The classical Lod score method of linkage analysis was devised to
locate genes responsible for Mendelian traits

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Typically, a Mendelian trait shows a biunivocal correspondence between a
genotype and a phenotype
In more general terms, classical linkage analysis necessitates that the
transmission model for a trait be specified

If the model is misspecified, then artefactually negative lod scores may be
produced
In classical linkage analysis the transmission model is fixed (possibly
with parameter values obtained from segregation analysis) and the
likelihoods of the disease and marker data are compared under the
null hypothesis of no linkage and the alternative hypothesis of
linkage
S.P.
Looking for Mendelian phenotypes in
Hyperparathyroidism
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Hyperparathyroidism affects 1 in 1,000 individuals in
the general population, and 85-90% of cases are
caused by excess hormone production due to adenoma
development in one of the parathyroid glands.
Approximately 5%-10% of patients have a family
history of parathyroid tumors. A proportion of such
familial cases occur in combination with other tumors,
such as in Multiple Endocrine Neoplasia 1 (due to
MEN1, located at 11q) and in Multiple Endocrine
Neoplasia 2A (due to RET, located at 10q )
However, hyperparathyroidism appears to be the sole manifestation in a subset of
families (Familial Isolated Hyperparathyroidism, FIH), and in others it is associated
with ossifying fibromas of the mandible, bilateral renal cysts, hamartomas, and
Wilms tumor (Hyperparathyroidism – Jaw Tumor syndrome, HPT-JT).
S.P.
The HPT-JT syndrome
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HPT-JT is inherited as an autosomal dominant syndrome with a high penetrance
for the presence of parathyroid tumors
Unlike MEN1 MEN2, which cause parathyroid tumors that are almost universally
benign, those in HPT-JT are malignant in 15% of cases. This is also remarkable
in comparison to sporadic parathyroid tumors, which are malignant in less than
1% of cases.
Approximately 30% of HPT-JT patients develop rare ossifying fibromas primarily
of the mandible and maxilla, which are different from the osteoclast-dominated,
parathyroid hormone responsive “brown” tumors of severe hyperparathyroidism
that can occur at any skeletal site.
Kidney manifestations may also be present in HPT-JT and include most
commonly (10%) bilateral renal cysts, but also renal hamartoma, polycystic
kidney disease, and Wilms tumor.
S.P.
HPT-JT: early linkage studies

Szabo et al., Am J Hum Genet 56:944-50 (1995)
Hereditary hyperparathyroidism-jaw tumor syndrome: the endocrine
tumor gene HRPT2 maps to chromosome 1q21-q31

Teh et al., J Clin Endocrinol Metab 81:4204-11 (1996)
Autosomal dominant primary hyperparathyroidism and jaw tumor
syndrome associated with renal hamartomas and cystic kidney
disease: linkage to 1q21-q32 and loss of the wild type allele in renal
hamartomas.

Hobbs et al., Am J Hum Genet 70:1376-7 (2002)
Revised 14.7-cM locus for the hyperparathyroidism-jaw tumor
syndrome gene, HRPT2.
S.P.
An international consortium to map HRPT2
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
An international consortium was
established In 1999, with the
goal of sharing DNA samples of
HPT-JT families from several
countries (Sweden, U.K.
Netherlands, USA) and to
perform a fine mapping study to
narrow down the region of
HRPT2
John Carpten, at the NHGRI of
Bethesda, leaded the group that
performed the new genotyping
John Carpten
S.P.
Results of the fine mapping study
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We began our study with 22 kindreds, which were genotyped with
26 microsatellite markers in the 1q24-q32 interval.
Of these families, 18 were informative for genetic analysis.
Affected haplotypes were constructed and a recombination map was
obtained.
A novel proximal recombination between D1S238 and D1S461 in
Kindred-02 reduced the candidate interval by 3 cM. On the contrary,
the distal boundary did not change.
Therefore, the genetic interval was reduced from 15 cM to 12 cM,
including a chromosome segment of 14 Mb.
S.P.
Genetic analysis of HPT-JT kindreds and partial
transcript map of the critical region
S.P.
Linkage Disequilibrium (Allelic Association) methods
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Linkage analysis is a powerful tool for detecting ‘major’ genes
which does not require a candidate and is, therefore, a means of
genome screening. However, its main limitation is its low-resolution
mapping of the linked chromosomal interval (usually some cM),
which could contain tens, or hundreds, of genes.
One way to perform fine mapping and confirm linkage of a
susceptibility locus is to test for allele association due to linkage (i.e.
"linkage disequilibrium") between particular genetic markers and the
disease.
Linkage disequilibrium of a particular marker allele will occur when
the disease locus and the marker locus are so closely positioned that
recombination events between them are very rare and a certain
marker allele is associated with the disease gene. A popular method
to analyze linkage disequilibrium is the Transmission Disequilibrium
Test (TDT).
S.P.
A modified transmission distortion test applied to the
HPT-JT haplotype data
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Our approach involved the selection of trios (mother, father, child), each
trio from a different family, both the child and one of the parents being
affected. The affected chromosomes (cases) were defined as those
inherited by the child from the affected parent, and the unaffected
chromosomes (controls) were those inherited by that child from the
unaffected parent.
This procedure yields two set of chromosomes of equal size, which are
contrasted to each other in terms of their genetic composition. In the
presence of linkage disequilibrium, the affected chromosome set will show
a reduced level of haplotype diversity, or an excess of haplotype sharing.
We considered sliding-window haplotypes of 2, 3, 4, … contiguous
markers, and determined the haplotype frequency spectra (the distribution
of the number of haplotypes observed in 1, 2, ..., n individuals) in the two
chromosome sets for each n–marker haplotype. Heterogeneity between the
two sets of chromosomes was tested by chi-square.
S.P.
Design of a modified L.D. mapping method
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Affected
chromosomes
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Unaffected
chromosomes
The main advantage of our
method derives from
selecting trios in which both
the child and one of the
parents are affected. This
allows us to determine which
of the two chromosomes in
the child is the "case", so that
we do not have to use all the
four founding chromosomes
to look for transmission
disequilibrium.
S.P.
2.2
1.8
1.4
1
1
2
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4
5
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14
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23
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6
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0
2
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0
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2
4
2
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3
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1
D1S477
2
2
4
3
2
4
4
3
2
5
4
2
3
3
2
2
4
3
D1S2622
D1S518
4 4
5 9
6 9
4 4
5 10
3 6
4 10
4 4
5 9
5 1
3 7
4 6
7 9
6 7
7 8
5 9
6 2
5 7
D1S1632
D1S202
4
5
3
4
4
3
3
4
5
3
3
4
3
3
3
5
4
5
A319WH1
D1S2138
1
3
3
1
3
2
2
1
4
3
3
3
4
3
3
4
1
4
D1S1660
D1S2848
2
4
2
5
4
4
3
5
4
3
3
3
5
4
3
4
4
4
D1S413
D1S191
7
8
9
3
3
7
7
3
9
3
7
7
5
3
3
3
7
5
D1S2840
D1S444
2
1
1
1
2
1
4
2
1
6
1
1
1
2
2
4
4
2
A207WB12
D1S254
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
A329VC1
9
D1S2794
8
D1S412
7
D1S384
6
D1S542
5
D1S461
4
D1S238
3
1 7
3 5
1 6
1 14
1 9
5 11
1 8
5 8
1 7
3 5
1 8
1 7
6 8
1 7
1 5
6 7
1 1
6 11
6
3
3
3
6
6
1
6
8
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6
6
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3
3
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3
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3
2
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5
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1
2
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1
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1
1
2
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1
1
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1
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2
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3
2
2
2
2
2
2
2
2
5
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4
5
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5
4
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4
4
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4
4
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3
1
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5
1
4
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2
3
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5
1
5
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6
1
5
1
3
1
1
4
5
7
5
4
4
1
1
3
1
5
1
5
5
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4
4
4
7
1
2
3
2
6
5
5
5
6
5
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3
4
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3
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7
3
6
3
3
4
3
3
3
4
6
3
3
6
3
4
2
2
5
2
3
4
6
2
3
3
2
3
2
3
2
8
9
2
10
7
8
5
8
11
10
8
10
14
2
10
2
8
3
4
2
7
7
7
7
2
7
2
5
9
2
7
6
9
5
7
7
7
5
D1S222
2
D1S2127
FAMILY ID
Kindred-01
Kindred-02
Kindred-03
Kindred-04
Kindred-05
Kindred-06
Kindred-07
Kindred-08
Kindred-09
Kindred-10
Kindred-11
Kindred-12
Kindred-13
Kindred-14
Kindred-15
Kindred-16
Kindred-17
Kindred-18
1
D1S466
0.6
D1S215
Ratio of unique haplotypes in
case to control chromosomes
A peak of haplotype sharing among the 18 “affected”
chromosomes
The peak
points to the
interval
between
D1S384 and
D1S412, the
location where
HRPT2 was
actually
identified
S.P.
Conclusions
On the basis of our haplotype-sharing analysis, we
expected to identify some identical mutations in
some of the families
 This was not the case, as the final report included
data on 11 different HRPT2 mutations identified in
12 probands, out of 26 screened families.
 The reasons of this result have still to be explained.
 However, our study shows the usefulness of
following up linkage analysis with fine-mapping
intrafamilial linkage disequilibrium analysis

S.P.