Download Mapping of both autosomal recessive and dominant variants of

 1997 Oxford University Press
Human Molecular Genetics, 1997, Vol. 6, No. 11 1823–1828
Mapping of both autosomal recessive and dominant
variants of pseudoxanthoma elasticum to
chromosome 16p13.1
Berthold Struk1,2,6, Kenneth H. Neldner4, Valluri S. Rao1,3, Pamela St Jean5 and
Klaus Lindpaintner1,2,3,6,*
1Cardiovascular
Division, Department of Medicine, Brigham and Women’s Hospital, Boston, MA 02115, USA,
of Cardiology, Children’s Hospital, Boston, MA 02115, USA, 3Department of Medicine, Harvard Medical
School, Boston, MA 02115, USA, 4Department of Dermatology, Texas Tech University Health Sciences Center,
Lubbock, TX 79430, USA, 5Department of Epidemiology and Biostatistics Case Western Reserve University,
Cleveland, OH 44109, USA and 6Max Delbrück Centre for Molecular Medicine, D-13122 Berlin-Buch, Germany
2Department
Received February 18, 1997; Revised and Accepted July 28, 1997
Pseudoxanthoma elasticum (PXE) is a classic inherited
disorder of the elastic tissue characterized by
progressive calcification of elastic fibers with a
pathognomonic histological appearance. The clinical
manifestations of PXE typically involve the skin, the eye
and the cardiovascular system, resulting in skin lesions,
decreased vision and vascular disease. Clinically, a more
common autosomal recessive and a less common
autosomal dominant pattern of inheritance, with high
penetrance, have been described; the estimated
prevalence of the disease is 1 in 70 000–100 000.
Previous failure to link the disease to any of several
candidate genes prompted us to conduct a
genome-wide screen on a collection of 38 families with
two or more affected siblings, using allele sharing
algorithms. Excess allele sharing was found on the short
arm of chromosome 16 and confirmed by conventional
linkage analysis, localizing the disease gene under a
recessive model with a maximum two point lod score of
21.27 on chromosome 16p13.1, an area so far devoid of
any obvious candidate genes. Under a dominant
transmission pattern linkage with a maximum two point
lod score of 14.53 was observed to the same region.
Linkage heterogeneity analysis predicted the presence
of allelic heterogeneity with different variants of a single
gene that resides in this chromosomal region
accounting for recessive and dominant forms of PXE.
INTRODUCTION
Pseudoxanthoma elasticum (PXE) was first described as an
independent disease entity in 1896 by the French dermatologist
Jean Darier, who characterized it histologically as a progressive
fragmentation of the elastic tissue of the papillary dermis (1).
Subsequently, PXE was recognized as a multisystem disorder of
the elastic tissue leading not only to skin disease but also to ocular
(angioid streaks) (2) and vascular complications (3). The typical
cutaneous manifestations of PXE (‘cobblestone’-like changes
with a predilection for flexural surfaces) (1,3,4) are mostly
cosmetic in nature; however, its cardiovascular complications,
while rare, can be life threatening (mainly gastrointestinal
hemorrhage and coronary disease) (5–8) and its ocular
manifestations (fractures of Bruchs’ membrane resulting in
so-called ‘angioid streaks’ followed by neovascularization) are
common, leading to retinal hemorrhage and consecutive central
blindness in about half of all affected individuals (2,3).
PXE represents a paradigm for disorders of heterotopic tissue
calcification, with the ultrastructural defect characterized by a
centrifugally progressive accumulation of calcium salt deposits
(CaCO3 and CaPO4) within elastic fibers, starting in the core and
leading, eventually, to their fracture and destruction (9–11).
Previous efforts to link the disease in limited numbers of families
to several potential candidate genes (such as elastin, fibrillins I and
II and lysyl oxidase) were negative or equivocal (12); likewise, no
biochemical abnormality, except for an accumulation of
glycosaminoglycans in the affected skin (13), has reproducibly
been shown to be associated with the disease. In the majority of
cases PXE appears to be inherited as an autosomal recessive trait
(horizontal pattern of inheritance) with high penetrance and early
onset (on average at 13 years of age), occurring about twice as often
in females as in males. In ∼10% of pedigrees an autosomal
dominant mode of transmission (vertical transmission pattern)
appears to be operative. The phenotypic manifestations of PXE
show considerable variance, which was found to be unrelated to
mode of apparent inheritance (14), with regard to system(s)
affected and severity of disease (3,15). Presently, the most widely
accepted gold standard for diagnosis of PXE relies on positive von
Kossa staining in biopsy material from affected skin, indicating
calcification and fracture of elastic fibers.
In this study we report mapping of the recessive and dominant
forms of PXE to the same chromosomal region, 16p13.1, an area
without any apparent candidate gene for the disease.
*To whom correspondence should be addressed at: Cardiovascular Division, Brigham and Women’s Hospital, 75 Francis Street–Thorn 1103, Boston, MA
02115-6195, USA. Tel: +1 617 732 8173; Fax: +1 617 264 6830; Email: [email protected]
1824 Human Molecular Genetics, 1997, Vol. 6, No. 11
Table 1. Study sample
Family characteristics
Sib pair analysis
Recessive-like families
Dominant-like families
Families
38
0
27
8
2
1
73
11
13
14
91
64/27
n/a
n/a
42
4
28
8
2
0
n/a
12
13
17
93
63/31
118
68/50
8
1
6
0
0
1
n/a
5
3
0
26
14/12
27
15/12
With 1 affected sibling
With 2 affected siblings
With 3 affected siblings
With 4 affected siblings
With 5 affected siblings
Sib pairs
2 Parent families
1 Parent families
0 Parent families
Affected individuals
Females/males
Unaffected individuals
Females/males
Column two indicates the number of families included in the initial genome screen analysis using SAGE, carried out in all families showing apparent recessive inheritance that were available at the initiation of molecular genetic studies (at this time the family with five affected siblings was considered autosomal recessive; this was
later revised to autosomal dominant upon availability of additional information) and using only affected sib pairs (no other family members). Columns 3 and 4 present
information on pedigrees selected for parametric linkage analysis according to apparent mode of inheritance and under inclusion of all available family members
regardless of affection status. Numbers of affected sib pairs in a family with n affected sibs are calculated as n(n – 1)/2. n/a, not applicable.
Table 2. Results of allele sharing analysis, using all available family
members and high density markers for chromosome 16p13
Marker
D16S406
D16S407
D16S748
D16S3114
D16S3069
D16S500
D16S405
D16S3079
D16S3103
D16S499
D16S3036
D16S403
APM
T statistic
P value
Mean π (SE)
SIBPAL
Z value
P value
3.93
3.24
6.90
7.71
4.52
5.36
4.73
4.31
4.80
4.81
4.36
2.69
0.00004
0.00061
<0.00001
<0.00001
<0.00001
<0.00001
<0.00001
0.00001
<0.00001
<0.00001
0.00001
0.00354
0.73(0.03)
0.70 (0.03)
0.77 (0.03)
0.84 (0.02)
0.76 (0.03)
0.83 (0.02)
0.77 (0.02)
0.80 (0.03)
0.76 (0.03)
0.73 (0.03)
0.74 (0.03)
0.69 (0.03)
8.40
6.58
9.75
14.45
9.55
13.68
11.54
11.84
9.62
9.09
9.75
5.82
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
Columns 2 and 3 indicate the T statistics and associated P values from the APM
program. In this analysis 45 families with 113 affected members were used. The
estimated proportion of alleles shared identical-by-descent, mean π, among 79
affected sib pairs obtained from SIBPAL is given in column 4, with the
corresponding Z statistic and P value in columns 5 and 6 respectively.
RESULTS
Most of the patients and families investigated in the present study
had originally been recruited for a previous clinical study of PXE
performed at the University of Colorado Health Science Center and
at Texas Tech University Health Sciences Center over a 20 year
period from 1973 to 1993 (3). Others were obtained from the
database of the National Association for PXE. Only biopsy-proven
diagnoses with positive von Kossa staining were considered as
PXE cases. Except for eight families, all showed a strictly
horizontal pattern of disease prevalence, suggestive of autosomal
recessive inheritance. In keeping with previous studies, we
observed a female-to-male ratio of ∼2:1 among our patients.
We followed a two stage strategy to map the causative gene,
designated PXE (the gene name PXE has been approved by the
HUGO Nomenclature Committee). First, a genome-wide screen
using a panel of tri- and tetranucleotide single sequence length
polymorphisms (16–18) was performed on 73 affected sib pairs
belonging to 38 families (see Table 1). Data were analyzed by
non-parametric allele sharing methods (SIBPAL) (19–21). Three
markers on the short arm of chromosome 16, D16S2619, D16S748
and D16S403, were found to show significant excess allele sharing
compared with chance (61 versus 50%, P = 0.0005, 66 versus 50%,
P = 0.0002, and 67 versus 50%, P = 0.0003, respectively).
Based on this information, we proceeded to genotype all 264
affected and unaffected subjects belonging to the 42 pedigrees
with an apparently recessive inheritance pattern, as well as those
belonging to the eight families with an apparently dominant
inheritance pattern, using a set of high resolution, highly
informative markers (PIC > 0.7). Twelve microsatellite markers
fulfilling these conditions were tested. They were selected from
the collections assembled by the Cooperative Human Linkage
Center (CHLC; Extended Généthon V2 Recombination-Minimization Sex Averaged Map, v.4.0) (16–18) and by Généthon
(22). All were localized in the area of interest and had an average
spacing of 2.9 cM and an average polymorphism information
content of 0.77.
Forty five of the 50 families had affected relative pairs other
than parent–child pairs; they underwent non-parametric analysis
by both the APM (23) and SIBPAL (21) methods. Results from
these analyses were consistent with the original findings of
linkage to the region, i.e. to chromosome 16p13, and with each
other (Table 2). Both analyses provide significant evidence of
linkage for markers spanning the region D16S406–D16S403,
with a peak around D16S500.
Intermarker distances and orders were estimated using the
family data and ILINK (24) and are shown in Table 3. Parametric
two point maximum lod scores (MLS) were then calculated for
all 50 pedigrees assuming either autosomal recessive or autosomal dominant models of transmission respectively (Table 3).
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Figure 1. Multipoint linkage analysis (using the GENEHUNTER program) for all 50 pedigrees assuming (a) autosomal recessive mode of inheritance (graph AR),
(b) autosomal dominant mode of inheritance (graph AD) and (c) performing multipoint non-parametric linkage calculations (graph NPL).
An MLS of 21.27 was found for D16S500 at θ = 0.045 assuming
the recessive model and an MLS of 14.53 for the same marker at
θ = 0.011 under an assumption of dominant inheritance.
Model-based multipoint linkage analysis results using
GENEHUNTER (25) are shown in Figure 1. Under the recessive
model (Fig. 1, graph AR) an MLS of 21.8 was observed between
D16S3079 and D16S3103. Allowing for heterogeneity, this lod
score increased to 23.7 with an estimated 88% of families linked.
The MLS obtained under the dominant model (Fig. 1, graph AD)
was 16.9. Non-parametric multipoint linkage analysis using
GENEHUNTER NPL revealed similarly significant results (Fig.
1, graph NPL), with a maximum NPL score of 9.81 (P = 2.47 ×
10–25) close to D16S405. The information content of the map of
chromosome 16 markers was ≥80% across the entire region. Thus
both the model-based and model-free analyses provide highly
significant evidence of linkage between PXE and markers in this
region on chromosome 16.
To address the issue of heterogeneity across the sample in an
unbiased fashion, we conducted a number of analyses using the
program HOMOG (26) on multipoint lod scores obtained with
VITESSE (27). These analyses were designed to evaluate the
relative likelihood of: (i) the presence of linkage heterogeneity
with a fraction of the families displaying genetic linkage of the
disease to chromosome 16 and the remaining families not
showing linkage; (ii) the possibility of distinct loci coding for the
disease in different families; (iii) the presence of different modes
of inheritance at the same locus.
First, we tested, across all families, the evidence for locus
heterogeneity assuming either autosomal recessive or autosomal
dominant modes of inheritance (allowing a fraction of families
not to be linked to the chromosome 16p13 locus). As illustrated
in Table 4A, locus heterogeneity was favored over linkage with
homogeneity in both the recessive and the dominant models, with
likelihood ratios of 148.2 and 8.8, based on 90 and 95% of
families linked to chromosome 16, respectively and with an
associated shift of the maximum likelihood placement for PXE
from between D16S3079 and D16S3103 to between D16S500
and D16S405 under both models. Next, we tested for locus
heterogeneity within the 25 cM region of interest on chromosome
16p13, assuming two closely linked causative loci to account for
the disease in all families (Table 4B). As before, evidence of locus
heterogeneity was favored for both the recessive and dominant
models, with likelihood ratios of 4.30 θ 104 and 299.6 respectively over the single locus model. Imposing an autosomal recessive
mode of inheritance, 85% of families showed linkage to the
D16S3079–D16S3103 region, whereas the remainder showed
linkage to D16S406. When a dominant mode of transmission was
imposed, 70% of families showed linkage to D16S405 and the
remaining 30% to D16S3103. Last, we compared both the general
locus heterogeneity models (H2s from Table 4A) with a model of
allelic heterogeneity in which different modes of transmission
were possible in individual families, consistent with clinical
observations. This analysis favored, with a likelihood ratio of
4 × 103, the latter scenario over the former two, with 75% of
families appearing to be linked under the recessive model to a
region midway between D16S500 and D16S405 and the remaining 25% best fitting a dominant model with linkage to D16S500.
1826 Human Molecular Genetics, 1997, Vol. 6, No. 11
Table 3. High resolution mapping of PXE
Intermarker distance
(MLE)
cM
lod
Marker
D16S406
4.9
37.4
6.0
30.9
0.1
56.5
0.3
54.6
1.6
45.9
1.2
46.0
0.5
38.1
D16S407
Marker characteristics
n
PIC
Two point linkage analysis
MLE (rec)
θ
lod
MLE (dom)
θ
lod
11
0.80
0.139
7.69
0.068
6.36
17
0.86
0.126
7.98
0.001
7.89
9
0.80
0.064
15.93
0.014
10.66
17
0.87
0.056
19.64
0.011
14.51
16
0.72
0.064
15.51
0.015
11.60
12
0.82
0.045
21.27
0.011
14.53
8
0.73
0.045
15.90
0.013
11.16
11
0.72
0.038
19.74
0.012
12.58
10
0.76
0.068
15.18
0.012
12.11
11
0.75
0.080
12.22
0.014
11.01
11
0.82
0.094
11.67
0.001
12.16
12
0.84
0.169
5.78
0.088
5.25
D16S748
D16S3114
D16S3069
D16S500
D16S405
D16S3079
3.0
31.0
1.3
42.1
1.5
40.6
5.0
31.6
D16S3103
D16S499
D16S3036
D16S403
Twelve highly informative markers (PIC > 0.7) were typed in all available members of all families. Intermarker distances were calculated using all available pedigree
members. Intermarker distances and lod scores for the linear order of markers indicated were determined using ILINK (24) and are represented in columns 2 and
3 on the lines between markers, indicating that they pertain to the interval between two markers. Orientation of the map is represented as telomeric to centromeric,
from top to bottom. Abbreviations: MLE, maximum likelihood estimates; cM, centiMorgan (Kosambi); lod, lod score; n, number of alleles encountered; PIC, polymorphism information content; θ, recombination fraction; rec, all families analyzed under assumptions of recessive inheritance; dom, all families analyzed under
assumptions of dominant inheritance.
Table 4. Results of heterogeneity analysis
Test
Model
Hypothesis
Max lnL
LR
α1
α2
A
AR
H2
56.46
148.22
0.90
0.1
H1
51.46
H2
40.43
H1
38.26
H2
62.13
H1
51.46
H2
43.96
H1
38.26
AD
B
AR
AD
1.00
8.81
0.95
0.15
0.70
D16S405
0.85
D16S406
D16S3079-1.2-PXE-1.8-D16S3103
0.30
D16S405
D16S3103
D16S3079-1.2-PXE-1.8-D16S3103
H3
64.76
0.75
0.25
D16S500-0.7-PXE-0.5-D16S405
AR
H2A
56.46
0.90
0.00
D16S500-0.7-PXE-0.5-D16S405
AD
H2B
40.43
0.00
0.95
C
D16S3079-1.2-PXE-1.8-D16S3103
D16S3079-1.2-PXE-1.8-D16S3103
1.00
4001.8
D16S500-0.7-PXE-0.5-D16S405
0.05
1.00
299.63
Region2
D16S3079-1.2-PXE-1.8-D16S3103
1.00
4.30 × 104
Region1
D16S500
D16S405
Chromosome 16 multipoint lod scores from all 50 families were examined using HOMOG to test for linkage heterogeneity in general (A), for locus heterogeneity
assuming two linked loci on chromosome 16p13 (B) and for allelic versus locus heterogeneity (C). AD, autosomal dominant; AR, autosomal recessive; Max lnL,
maximum log likelihood; LR, likelihood ratio; α1 and α2, proportion of families linked and unlinked (A), linked to either locus on chromosome 16p13 (B) or autosomal recessive and autosomal dominant (C) respectively. In (C) the H2s are identical to those from (A) and the LR represents H3 versus H2A/H2B. This test also
allows for a certain proportion (α3) of families to be unlinked; in our data α3 was estimated to be 0. Region1 and Region2, maximum likelihood placement of the
two modeled loci on chromosome 16p13 (B) and of loci linked to phenotype in an autosomal recessive and autosomal dominant mode respectively.
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DISCUSSION
Our present data provide robust evidence that PXE maps to
chromosome 16p13.1, within a 4.8 cM region between D16S500
and D16S3103. A review of integrated map data available for
chromosome 16 (28) reveals that the region of interest (16p13.1)
has been saturated with ordered mega-YAC contigs as well as
with cosmid and flow-sorted mini-YAC contigs. So far only two
genes, MRP and MHY11, which code for a multiple drug
resistance-associated protein and for smooth muscle myosin
heavy chain-11 respectively, have been mapped to this region,
which also represents the breakpoint for a pericentric inversion,
in(16)(p13q22), associated with acute myeloid leukemia. The
homologous region in the mouse, which maps to mouse
chromosome 16, likewise does not contain any apparent candidate genes for PXE. Publicly accessible EST databases have also
so far failed to point to a candidate disease gene.
Whereas our analyses provide strong evidence either for locus
heterogeneity with two closely linked, but distinct, disease loci or
for allelic heterogeneity, they do not presently allow a distinction
between the two (although the results tend to favor the latter) and
they may even be compatible with both. If, indeed, we are
witnessing allelic heterogeneity with both recessive and dominant forms of PXE resulting from molecular variations of the
same gene, then this raises intriguing questions about the nature
of the gene product and of its respective mutations. By analogy
to the hypothesis advanced for rhodopsin-linked retinitis pigmentosa, one of the few similar examples of allelic heterogeneity
giving rise to dominant and recessive variants of the same disease
(29,30), one might suspect the presence of unstable or non-translated ‘null’ mutants in the recessive and of structurally abnormal
(dominant negative acting) gene products in the dominant form
of the disease.
Our localization of the causative gene is sufficiently precise to
allow genetic diagnosis of affection or carrier status by linkage
analysis in families with at least one affected member for both
dominant and recessive forms of PXE. We expect that a continued
search for additional informative meioses, coupled with ongoing
efforts at targeted marker development and positional cloning,
will lead us to the discovery of the causative gene and, in due
course, open up specific approaches to prevention or therapy of
PXE and its complications. Perhaps even more importantly, the
status of PXE as a model for disorders of aberrant tissue
calcification may give the present results potentially broader
implications for our understanding of a number of similar disease
states characterized by abnormal tissue calcification.
MATERIALS AND METHODS
Collection of PXE families and establishment of a PXE
DNA repository
Recruitment of patients and their family members was done by
K.H.N. at the Clinical Coordinating Center in Lubbock, based on
this investigator’s clinical database. Clinical data and blood
samples were collected over a 2 year period from families with
two or more affected individuals. Affection status was confirmed
based on positive von Kossa staining in biopsy material in all
patients. Only individuals older than 30 years who showed no
evidence of cutaneous and/or retinal lesions were considered as
unaffected family members for linkage analyses. All participants
in the study provided written, informed consent using a form that
was approved by the Institutional Review Board at Texas Tech
University Health Sciences Center.
High molecular weight genomic DNA was extracted from 1 ml
EDTA-anticoagulated whole blood using a commercially available, adsorption-based method (QIAmp; Qiagen), and stored in
multiple aliquots in 0.1× TE at –80C.
Genetic markers and genotyping
A subset of 169 CHLC microsatellite tri- and tetranucleotide
markers (Weber 6a) with an average heterozygosity of 78% and
an average spacing of 24.2 cM was purchased from Research
Genetics (Huntsville, AL). Sequence information for additional
microsatellites used for high resolution mapping of chromosome
16p was retrieved from either The Genome Data Base (GDB)
(http://gdbwww.gdb.org/) or the CHLC (http://www.chlc.org/)
database. Primers were designed such that amplification products
for up to four markers could be co-electrophoresed without
interference, to optimize the genotyping program. Primers were
designed using the Oligo program (31).
Twenty nanograms of genomic DNA were amplified by PCR
in a final volume of 10 µl containing 100 nM each primer, 200 µM
dNTPs, 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris–HCl, 0.1%
Triton X-100 and 0.025 U Taq DNA polymerase. Forward
primers were labeled with [γ-32P]ATP (3000 Ci/mmol; Dupont/
NEN) using T4 polynucleotide kinase (NEB). Reactions were
processed on a MJR Thermal Cycler (PTC 100; MJ Research,
Watertown, MA) using an initial denaturation at 96C for 3 min,
followed by 35 cycles of annealing for 1 min, extension at 72C
for 1 min and denaturation at 96C for 15 s, with a final extension
step at 72C for 7 min. Optimized annealing temperatures ranged
from 55 to 65C. PCR products were multiplex-loaded in groups
of up to four markers and size fractionated on 6% polyacrylamide
sequencing gels containing 37.5% formamide, 8 M urea, 90 mM
Tris–borate and 2 mM EDTA (Sequagel; National Diagnostics). Gels were run at 60 W for 3.5–6 h, depending on the PCR
product size, and exposed to Kodak XAR-5 film at –80C for
autoradiography. All markers were scored independently by two
observers who were blind as to diagnosis and family structure.
Linkage analyses
Prior to embarking on the genome-wide screen we carried out
extensive simulations using the computer program SLINK (32),
under the assumption of recessive inheritance and (near) complete penetrance. Results suggested an expected lod score of >4.0
for markers with a polymorphism information content (PIC) of
≥0.7, given the number of families and the average marker
spacing we planned to use. For the genome-wide screen all 169
genotyped markers in 38 families were tested for linkage to
affection status using a qualitative affected sib pair method as
implemented in SIBPAL, in v.2.7 of SAGE (21). SIBPAL
provides an estimate of the proportion of alleles shared
identical-by-descent in affected sib pairs.
After excess sharing on chromosome 16 was revealed, an
additional 12 families were collected (a total of 50 families) and
12 chromosome 16 microsatellites were genotyped and used in
subsequent linkage analyses. Of the 50 families available, 45
contained affected relative pairs other than parent–child and there
were a total of 79 affected sib pairs. APM (23) and SIBPAL were
used to investigate excess allele sharing on chromosome 16
1828 Human Molecular Genetics, 1997, Vol. 6, No. 11
among affected relative pairs (APM) or affected sib pairs
(SIBPAL). The APM method makes use of identity-by-state
sharing and a weighting function for marker allele matches that
takes into account that it is more striking for affected relatives to
share rare alleles. The weighting function f(p) = 1/√p, where p is
the allele frequency, was used, as Weeks and Lange (23) have
suggested that it is the best compromise between maintaining
approximate normality with respect to the test statistic, T, and
maximizing the power to detect linkage.
Two different disease models were utilized for the parametric
analysis. For the autosomal recessive model we assumed a
disease allele frequency of 2.5 × 10–3 with 90% penetrance and
a phenocopy rate of 10–5, while for the autosomal dominant
model we assumed a disease allele frequency of 10–5 with 50%
penetrance and a phenocopy rate of 5 × 10–6. Two point linkage
analysis was conducted using ILINK (24) with the FASTLINK
program (33) for all 50 families under the two different models.
Parametric and non-parametric multipoint linkage analysis was
carried out using both GENEHUNTER (25) and VITESSE (27).
For the multipoint analyses intermarker distances and marker
order were estimated using ILINK, based on genotype data from
all available family members. Analyses for homogeneity were
conducted using the HOMOG programs (26), as detailed in
Results, to test for general locus heterogeneity, for locus
heterogeneity constrained to the region of interest and for allelic
heterogeneity.
ACKNOWLEDGEMENTS
We thank the members of the PXE families for their participation
in this study. This work was supported by Project Grant 695-0209
from the March of Dimes Birth Defects Foundation (White
Plains, NY) and by a Pilot and Feasibility Grant from the Harvard
Skin Disease Research Center at Brigham and Women’s Hospital.
K.L. is the recipient of a Research Career Development Award
(K04-HL03138-01) from the National Heart, Lung, and Blood
Institute. Some of the results were obtained using the program
package SAGE which is supported by US PHS resource grant
RR03655 from the Division of Research Resources. A preliminary report of this study was presented at the Fifth International
Workshop on Human Chromosome 16, Toronto, Canada, March
3–4, 1997.
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