Toward Localization of the Werner Syndrome Gene by Linkage Disequilibrium and Ancestral ...

17 Pages·2007·2.85 MB·English

Am.J. Hum. Genet. 58:1286-1302, 1996
Toward Localization of the Syndrome Gene by Linkage
Werner
Disequilibrium and Ancestral Haplotyping: Lessons Learned from
Analysis of 35 Chromosome 8pl1.1 -21.1 Markers
Katrina A. B. Goddard,1 Chang-En Yu,5 Junko Oshima,s Tetsuro Mikij Jun Nakura,6
Charles Piussan,7 George M. Martin,2 Gerard D. Schellenberg,3'5
Ellen M. Wijsman,'4
and members of the International Werner's Syndrome Collaborative Group*
Departments of'Biostatistics, 2Pathology, and 3Pharmacology and Neurology and 4Division ofMedical Genetics, Department ofMedicine,
UniversityofWashington, and 5Geriatric Research, Education, and Clinical Center, Veteran's Affairs Medical Center, Seattle; 6Departmentof
Geriatric Medicine, Osaka University Medical School, Osaka; and 7Pediatric Genetics, University ofAmiens, Amiens
Summary Introduction
Werner syndrome (WS) is an autosomal recessive disorder Werner syndrome (WS) is an autosomal recessive disor-
characterized by premature onset of a number of age-re- der thatis characterized by the premature occurrence of
lated diseases. The gene forWS, WRN, has been mapped a large number of age-related diseases (Martin 1978).
tothe8p11.1-21.1 regionwithfurtherlocalizationthrough Some of these are the most common diseases of the
linkagedisequilibriummapping.Herewepresenttheresults elderly, such as diabetes mellitus, osteoporosis, a variety
of benign and malignant neoplasms, and arteriosclero-
oflinkage disequilibrium and ancestral haplotype analyses
of 35 markers to further refine the location ofWRN. We sis. Additionalcharacteristics ofWSinclude ocularcata-
racts, graying of hair, and subcutaneous-fat loss, which
identifiedanintervalinthisregioninwhich 14of18mark-
ers tested show significant evidence of linkage disequilib- is often associatedwithcutaneous ulcers ofthe legs (Ep-
riuminatleast oneofthetwopopulations tested. Analysis stein et al. 1966). There is extensive overlap between
ofextendedandpartialhaplotypescovering21ofthemark- the WS phenotype and that of "normal" aging. There-
ersstudiedsupportstheexistenceofbothobligateandprob- fore, the early onset ofthe symptoms, the early median
able ancestral recombinant events which localize WRN al- age atdeath of47years, usuallycausedbyamyocardial
infarction (Epstein et al. 1966), and the observation of
most certainly to the interval between D8S2196 and
reduced replicative potential of WS fibroblasts in cell
D8S2186,andmostlikelytothenarrowerintervalbetween
D8S2168andD8S2186.Thesehaplotypeanalysesalsosug- culture (Martinetal. 1970)allsuggestthatidentification
gestthatthere are multiple WRNmutations in each ofthe of the gene for this disorder could lead to new insights
into mechanisms common to a number of aspects of
twopopulationsunderstudy.Wealsopresentacomparison
of approaches to performing disequilibrium tests with aging.
The gene responsible for WS has been mapped to
multiallelic markers, and show that some commonly used
approximations for such tests perform poorly in compari- chromosome 8 (Goto et al. 1992; Schellenberg et al.
son to exact probability tests. Finally, we discuss some of 1992). Initial reports (Goto et al. 1992; Nakura et al.
1993) favored a location of WRN, the WS syndrome
the difficulties introduced bythe high mutation rate at mi-
gene, between D8S87 and ankyrin 1 (ANK1), two loci
crosatellitemarkerswhichinfluenceourabilitytouseances-
that are separated by -7 cM (Tomfohrde et al. 1992).
tral haplotype analysis to localize disease genes.
A location of WRN in regions flanking this interval
could not, however, be excluded. More recent analyses
with additional markers indicate that a location for
Received January 26, 1996; accepted for publication March 12, WRNnearD8S339islikely (Thomasetal. 1993).While
1996. D8S339 was originally thought to be in the interval
Address for correspondence and reprints: Dr. Ellen M. Wijsman,
DivisionofMedicalGenetics,Box357720,UniversityofWashington, between D8S87 and ANK1, a location that is telomeric
Seattle, WA 98195-7720. E-mail: [email protected] to D8S87 is now considered to be more likely than a
*Members of the International Werner's Syndrome Collaborative location between D8S87 and ANK1. This evidence is
Group:W.T. Brown, G. Burg,D. Cerimele, F. Cottini, C.J. Epstein, based on both results from linkage analysis (Nakura et
W. Fischer, M. Fraccaro, Y. Fujiwara, K.-I. Fukuchi, K. Hiwada, H. al. 1994) andthepresenceoflinkagedisequilibriumwith
Hoehn,Y. Hosokawa, A. F. Hurlimann, H.Ishawata,K. Kamino,K.
WRN with D8S339 and the tightly linked GSR locus
Kihara, S. Kiso,Y. Lin,T. Maeda,J. Matthews,T. Matsumura,J. E.
McKay,M.I.Melaragno,M.Mitsuda,A.G.Motulsky,T.Murakami, (Yu et al. 1994) and other microsatellite loci in the re-
S. Murano, N. Niikawa, M. Poot, T. Ogihara, M. Rizzo, T. Saida, gion (Kihara et al. 1994; Ye et al. 1995), but not for
S. Scappaticci, T. C. A. Tannok, S. Tamaki, N. Utsu, B. Uyeno, A. WRN and markers in the D8S87-ANK1 interval (Yu et
Wakayama,M. Yanagawa,I. I.Yevich, S. Yoshida, andW. Zigrang. al. 1994), despite ample power to detect even moderate
C 1996byTheAmericanSocietyofHumanGenetics. Allrightsreserved.
levels of disequilibrium (Olson and Wijsman 1994).
0002-9297/96/5806-0023$02.00
1286
Goddard et al.: Localization ofWerner Syndrome 1287
The existence of linkage disequilibrium between two identification oflinkage disequilibrium between marker
loci can provide strong evidence that these loci are very and disease loci provides a valuable clue to narrowing
closely linked. Similar evidence, for example, the most probable region containing the disease locus.
was ex-
tremely important in the identification of the Hunting- However, for very small distances (<60-75 kb) there
ton disease locus (Huntington's Disease Collaborative appearstobelittlerelationshipbetweenstrengthofasso-
Group 1993) well several other recently identified ciation and distance (Jorde et al. 1994), although it is
as as
disease loci, including the for ataxia telangiectasia possible that such an association is not detectable be-
genes
(Savitsky et al. 1995; Uhrhammer et al. 1995) and cause of the high variance of disequilibrium estimates
Bloom syndrome (Ellis etal. 1994, 1995). The existence forsmalldistances (Hudson 1985).Itisalsonotstraight-
of linkage disequilibrium thus be useful in forward to define a measure ofassociation for a pair of
can nar-
rowing the genomic region that most likely contains the loci that is independent of allele frequencies (Hedrick
disease locus. Use of linkage disequilibrium to localize 1987), nor is a single measure ofstrength ofassociation
disease loci is a particularly attractive strategywhenthe easy to define formultiallelic markers (Lewontin 1988).
sample sizes that for detecting linkage dis- For multiple loci, which could provide additional infor-
are necessary
equilibrium compared with those needed in meiotic mation aboutgenelocation, measures ofassociation are
are
mapping. For localization to a region of -1 cM, the even more difficult to define (Lewontin 1988; Weir
sample sizes necessary to provide evidence of linkage 1990). While there have been attempts toward making
disequilibrium in case-control study be than use of linkage-disequilibrium data from multiple mark-
a can more
order of magnitude less (Olson and Wijsman 1994) ers to estimate disease-gene location (Hill and Weir
an
than the several hundred that for meiotic 1994), the assumptions necessary for the methods limit
are necessary
mapping to such an interval (Boehnke 1994). For a rare their usefulness to analysis of restricted populations
disease such WS, such differences in sample-size (Kaplan et al. 1995). These methods as currently imple-
as re-
quirements make the difference between feasible and mentedworkpoorlyforaccurategenelocalizationwhen
infeasible approaches to disease-gene localization. the population is not well defined and/or has multiple
Markers that are used for mapping and searching for disease mutations, as seems likelyformutationsrespon-
evidence of linkage disequilibrium typically highly sible for WS.
are
polymorphic with alleles. However, the of Even though a precise estimate of the location of
many use
such markers in the search for linkage disequilibrium WRN is thus not likely to be obtainable with current
introduces complexities into the analyses that do not methods, the joint information that can be obtained
exist for markers with relatively few alleles. For marker fromextendedhaplotypesofmany,closelylinkedmark-
loci with few alleles, likelihood ratio or contingency X2 ers could nevertheless provide additional evidence of a
tests can be used in most cases (Weir 1979, 1990). For regional localization by suggesting sites forancestralre-
sparse tables, small to moderate numbers ofalleles, and combinations. It is likely that there are relatively few of
relatively small sample sizes, the computationally themostcommonWSmutationswithineachpopulation
more
demanding Fisher exact test can be used. However, under study and that each such mutation will have oc-
when there are large numbers of alleles, the associated curred on a particular unique haplotype, with recombi-
contingency tables can be very sparse. In such cases the nation causing occasional perturbation ofthese original
computational demands of the Fisher exact test may haplotypes. Because recombination will almost always
exceed available computer resources, and the approxi- involve a haplotype bearing a normal WRN allele and
mations provided by likelihood ratio or Pearson X2 tests will occur with such haplotypes in proportion to their
may be ill behaved. Therefore, because of concerns frequency in the population, the recombinant parts of
aboutthe behaviorofthesestatistical tests insuchsitua- theWShaplotypesarelikelytoreflectthemorecommon
tions, many investigators have pooled cells containing controlhaplotypes.Ashortseriesofcloselylinkedmark-
rarer alleles, prior to performing such statistical tests, ers that appear to have escaped such apparent recombi-
in the hopes of avoiding problems introduced by small nation is a good candidate for the WRN location.
expectednumbers ofindividual alleles (cf. Andrewetal. In this paper, we present haplotype and linkage-dis-
1992; Sirugo et al. 1993). The effect of such pooling of equilibrium analyses of 35 markers in the region on
classes on the outcome of these tests compared to the chromosome 8 that contains WRN. The identification
results from an exact probability test is unknown. of a region in which multiple markers are in linkage
In principle, there should be inverse relationship disequilibrium with WRN coupled with identification
an
between the genetic distance between two loci and the of probable ancestral recombinants that can be defined
strength of association between them (Hill and Robert- fromthehaplotypesfurtherrefinestheprobablelocation
son 1968). Itis clear from empirical studies that linkage of WRN. We also present a comparison of approaches
disequilibrium is frequently detected between disease to performing the disequilibrium tests with multiallelic
a
locus and one or more marker loci that are within 1 markers and show that some commonly used approxi-
cM of the disease locus (cf. Jorde et al. 1994). Thus, mations used for testing hypotheses aboutthe existence
1288 Am.J. Hum. Genet. 58:1286-1302, 1996
oflinkage disequilibrium performpoorlyincomparison cM Marker
to the exact tests.
r-D8SI33
Subjects, Material, and Methods
7.6
Marker
Subjects
Patients and family history information were identi- -D8S136 -D8S2194/D8S2192
fied and collected as described by Nakura et al. (1994).
I-D8S2196
7.4 E
Patients previously described by Nakura et al. (1994)
consisted of 17Japanese patients from consanguineous F -D8S2198
marriages and 6 Caucasian patients from consanguine- hD8SI37 G -D8S339
ous marriages. Controls and many of the cases used in 9 -8Sl31
H -D8S2204
thecurrentstudyarethose described byYuetal. (1994) D8S2202
and Nakura et al. (1994). DNA samples from the AG 6.7 J -D8S2206
series of subjects were derived from amplified skin fi-
-D8S2202/D8S339
broblast or lymphoblastoid cell lines deposited in the 1.6
-D8S278-D8S87
AgingCellRepositoryofthe Coriell Institute ofMedical K -D8S2134
Research, Camden, NJ. Both isolated cases and individ- 2.5 -FGFRI LJM ,D8S2144/D8S2156
N -D882138
uals for whom family structure information was avail- 2.8 0 -D8S2168
able were used in the analyses presented here. Not all 2.1 -D8S255 Qp -D8S2174
-D8S268/ANK1 sD8S2150
individualswere used inall analyses. Caucasian subjects R D8S2180
were ofmixed ethnic background with respect to coun- 2.8 S -D8S2162
-PLAT
try oforigin, as described by Nakura et al. (1994), and
included subjects with ancestry from several Western T -D8S2186
European countries, as well countries such as Brazil,
Syria, Turkey, and India. Control samples consisted of
11.4
100 unrelated Japanese and 100 Caucasian controls
as described by Yu et al. 1994. The studywas approved
by the University of Washington Institutional Review
1.0 -D8S165
Board. -D8S166
DNA Samples andMarkers
43.8
Genotypes were determined for 35 chromosome
8pll.1-21.1 markers between and including D8S133
D8S164
and D8S164 (fig. 1) by standard methods essentially as -
described byWeber andMay (1989) with the following Figure 1 Map of the WRN region. Markers C-T are included
modifications. The denaturing polyacrylamide gels used inhaplotypeanalysesdescribedintables3-7andareshownapproxi-
forgenotypingcontained, inaddition to 8 Murea, 12% matelytoscale,spanning 1.2-1.4cM.Markerordersanddistances
betweenmarkerstakenfromtheNationalInstitutesofHealth (NIH)/
deionized formamide. For each primer set, one primer
CEPH Collaborative Mapping Group (1992), Oshima et al. (1994),
was end-labeled using P32-ATP and T4-kinase as de- and C.-E. Yu, J. Oshima, F. Hisama, S. Matthews, B. Trask, and
scribed elsewhere (Sambrook et al. 1989, pp. 11.31- G. D. Schellenberg (unpublished data). The interval D8S278-D8S87
11.33). Primer sequences used and accession numbers includes markers D8S259 and D8S283. D8S137, D8S131, and
are described by C.-E. Yu, J. Oshima, F. Hisama, S. D8S278 are markers A, B, and U, respectively.
Matthews, B. Trask, and G. D. Schellenberg (unpub-
lished data) and Nakura et al. (1994). All allele sizes in
both Japanese and Caucasian individuals were deter- pared in addition to providinginformation aboutwhich
mined from a DNA sequencing ladder and were stan- markers showed evidence ofbeing in linkage disequilib-
dardized by comparison to allele sizes in the two CEPH riumwithWRN.AFisherexacttestwascomputedwhen
reference samples 133101 and 133102, which were run possible for each n X 2 table of n observed alleles in
concurrently as controls. cases versus controls. A Monte-Carlo Markov-chain
(MCMC) estimate of the Fisher exact test P-value was
Linkage-Disequilibrium Tests computed with methods described by Guo and Thomp-
Statisticaltests to determine whetherthere is evidence son (1992).APearsonX2wascomputedforthecomplete
oflinkage disequilibrium between the disease locus and n x 2 table without taking into account the possibility
marker lociwere done with five different approaches so thatsome expected cell sizeswill be quite low. A second
that the properties of the different tests could be com- Pearson x2 (called the "reduced X2 test") was computed
Goddard et al.: Localization ofWerner Syndrome 1289
after alleles fromthe rarestclasses werepooled, until all Haplotypes defined in both populations were exam-
expected numbers reached at least 5. Choice of alleles ined for evidence of recombination. Definitive evidence
for pooling was done by rank ordering alleles on the of recombination within families was obtained when
basis of frequency on the WRN chromosomes, after twoaffectedsiblingshaddiscordantmarkergenotype(s).
which rare alleles were pooled. Thus, for single-dftests, Presumptive evidence of recombination was obtained
the cells were defined as the most common allele on the when an offspring of a consanguineous marriage was
WRN chromosomes versus all other alleles. Finally, a homozygousacrossalargenumberofmarkers,followed
likelihood ratio X2 was also computed on the complete by a heterozygous marker. Because there is a small, but
n X 2 table. Withtheexception oftheMCMC analyses, nonzero, probability that such an individual might not
statistical tests were performed with SAS version 6.09 be IBD atWRN, strongpresumptive evidence ofrecom-
(SASInstitute, 1990).The35 markersforwhichdisequi- bination required the existence either of two or more
librium tests were performed were those within the such individual presumptive recombinant events or of
WRNregion, asdefinedbyresults frompreviouslinkage the existence of several heterozygous markers in the re-
analyses. gion providing evidence ofrecombination. Presumptive
Allele counting prior to the linkage-disequilibrium evidence ofancestral recombinationwas obtainedwhen
testing was done as follows: In affected offspring of alleles forseveralneighboringmarkers differedfromthe
consanguineous marriages, ifthemarkergenotypeswere consensus haplotype within the haplotypic group.
homozygous, consistentwith a highprobabilitythatthe Partial haplotype analyses.-Haplotype frequencies
two WRN alleles were derived from a single ancestral were estimated for subsets of the complete collection of
chromosome, onlyasinglemarker allelewascounted in markers forthe purpose ofcomparinghaplotype frequen-
determining the marker allele frequencies onthe disease cies in cases and controls. These subsets were chosen to
chromosomes. Forindividuals fromnonconsanguineous explore further the possibility ofrecombination in the re-
matings, or for individuals from consanguineous mat- gion between markers C and T by also accounting for
ings where the marker locus was heterozygous and thus haplotype frequencies in controls. A maximum of six
not identical by descent (IBD), bothmarker alleles were markers at a time were used to construct partial haplo-
counted. Only one affected individual from each pedi- types, because ofcomputational constraints in estimating
gree was used to estimate marker allele frequencies on haplotype frequenciesimposedbythepresenceofmultiple
the WS chromosomes (the chromosome with mutations allelespermarkerlocusandphaseambiguities inindividu-
in WRN). Control-allele frequencies were obtained by als from nonconsanguineous marriages. Haplotype fre-
genecounting, ontheassumptionoftwoallelesperlocus
quencieswereestimatedwithanEMalgorithm (Ceppelini
per control individual. Allele frequencies in both cases
et al. 1955; Dempster et al. 1977) under the assumption
and controls were estimated separately for theJapanese
of Hardy-Weinberg equilibrium of haplotype frequencies
and Caucasian samples.
within each population. Frequencies were estimated sepa-
Haplotype Analyses rately for cases and controls for both the Japanese and
Extended-haplotypeanalyses.-Haplotypeswerecon- Caucasian populations. Only a single haplotype was
structed for the 21 markers between D8S137 and counted for all individuals from consanguineous matings
D8S278 (markers A-U in fig. 1) by inspection of the who were homozygous for all markers included in the
data for WS cases. For the Japanese cases, haplotypes haplotype reconstructions. Onlycases forwhom no more
were constructed for all cases who were homozygous than one marker genotype was missing and only controls
across most ofthe region. Haplotypes were grouped for for whom no marker genotypes were missing were used
the Japanese cases by apparent similarity, and within inestimatinghaplotypefrequencies, againbecauseofcom-
each group a consensus haplotype was defined by the putational difficulties associated with the high degree of
mostcommon allele foreachmarker. Forthe Caucasian polymorphism for the systems used.
cases, becausetherewerefarfewerhomozygouspatients Haplotypes were grouped into sets that appeared to
from consanguineous marriages and because ofthe eth- be descended from common ancestral haplotypes under
nic heterogeneity of the sample, cases were either the assumption that a small number ofindependentWS
grouped by apparent similarity or by country oforigin, mutations is responsible for the majority of the haplo-
after which a consensus haplotype was defined for each types in the population. Evidence of possible ancestral
suchgroup. Thepossibilityofregionalclusteringofhap- recombinant events was then evaluated by inspection.
lotypes in the Japanese was investigated by looking for Theassumptionwasmadethatthemostfrequenthaplo-
evidence of clustering of haplotypes by geographic re- types represented nonrecombinant haplotypes, those
gion inJapan. The origin ofthe haplotype was taken to that were similar to these over a large portion of their
be the town or prefecture of the case, if known, the length but then diverged may represent recombinant
parents or grandparents if the location of the case was haplotypes and those that are divergent at only one
unknown,or,ifnoinformationonbirthplaceswasavail- marker locus and only by 2 bp may represent mutation
able, thehospitalormedicalcenterreferringthepatient. at the divergent marker locus.
1290 Am.J. Hum. Genet. 58:1286-1302, 1996
Table 1 Results
Markers Included in Partial Haplotype Analyses Patients and Pedigrees
A total of 68 WS probands were identified and used
MARKERS
for at least some of the analyses presented here. There
SET E G H I J K M P Q R S were 26 consanguineous and 1 nonconsanguineousJap-
anesefamiliesand 13 consanguineous and7nonconsan-
I ... X ... X ... X ... X X X guineous Caucasian families. Cases used in the current
II X X X X X
III ... .X.. ...... .X.. .X.. .X.. X study that were not previously described by Nakura et
... ... ... ... al. (1994) are described in table 2. The JV pedigree in
NoTE.-Marker labels as in table 3. the Caucasian data set (Nakura et al. 1994) had an
unusual and complex pedigree structure (fig. 2). For a
few families, additionalaffectedpedigreememberswere
sampled, including four Japanese and four Caucasian
Three sets of markers were used for comparing case
pedigrees (two nonconsanguineous and two consan-
andcontrolhaplotype frequencies (table 1). The first set
guineous pedigrees foreach population). The additional
of markers, set I, spanned the entire region between
affected pedigree members were all siblings, with the
markers H and S, a region of <800 kb (C.-E. Yu, J.
exception of the consanguineous LRV and nonconsan-
Oshima, F. Hisama, S. Matthews, B. Trask, and G. D.
guineous SYR pedigrees. In the LRV pedigree, the two
Schelleberg, unpublished data). This set ofmarkers was
affected relatives are fromtwo branches ofthe pedigree,
chosen for the purpose of exploring the possibility of
each of which represents a consanguineous marriage.
detection of ancestral recombination between the two
However, the specific relationship between the branches
ends of the region. The second set of markers, set II,
is unclear (Nakura et al. 1994). In the SYR pedigree,
spanned the telomeric region between markers H and
one of the three affected individuals was a half-sibling
P.aregionof<400 kb (C.-E. Yu,J. Oshima, F. Hisama,
of the other two affected full siblings. In 12 pedigrees,
S. Matthews, B. Trask, and G. D. Schellenberg, unpub-
additional unaffected individuals were sampled (table
lished data). Marker set II shared two markers in com-
2). In addition to the 46 individuals for whom family
monwith set I: markers H andJ. Marker setIIIcovered
structureinformationwas available, therewere 10Japa-
the centromeric end ofthe region withthe 5 markers K,
nese and 12 Caucasian unrelated cases without such
P, Q, R,and S. spanning <350 kb (C.-E. Yu,J. Oshima,
familyinformation.Alltheseweretreatedasnonconsan-
F. Hisama, S. Matthews, B. Trask, and G. D. Schelle-
guineous pedigrees in the analyses. Among the 32 Cau-
berg, unpublished data). This set was used to look for casian WS probands, there were 4 each with German,
evidence ofrecombination between markers in the cen- Italian (all from Sardinia), and French ancestry. These
tromeric region. were the only samples of Caucasians that were suffi-
Marker-locus mutation.-Evaluation of evidence of ciently large for use in grouping haplotypes by country
ancestral recombinant events between WRN and of origin.
marker loci is complicated bythe high mutation rates
that are typical of dinucleotide repeat markers (We- Approaches to Disequilibrium Testing
ber andWong 1993). There are a number ofWS hap- A comparison of the results from the different pair-
lotypes thatwere identicaltoeachother across a large wise tests used to search for markers in linkage disequi-
numberofmarkers butwhere one ortwo loci differed librium withWRN is given in figure 3. Seventy marker-
between pairs of haplotypes or where a patient from disease comparisons were made, 35 in each population.
a consanguineous marriage is homozygous across a Of these comparisons, all but seven of the tests could
large stretch ofmarkers in the region, with the excep- be performed with the Fisher exact test, and all but one
tion ofone that is heterozygous. The different alleles withthereducedX2test. D8S166 hadtoo smallasample
for such loci on the relevant haplotypes were gener- sizeinthe Caucasians topoolalleles andretainexpected
ally nearly identical in size. Because ofthis there was cell sizes of at least five alleles, and the seven compari-
concern that such differences among haplotypes sons for which the Fisher exact test could not be com-
might have been caused by mutation rather than re- puted were limited by available computer memory on
combination, consistent with a model of mutation the Sparcstation20/51 used forthese analyses. Itisclear
through slipped-strand mispairing (Levinson and that, in comparison to the Fisher exact test, the MCMC
Gutman 1987). Mutation at marker loci was consid- approach does outstandingly well, with a correlation of
eredto bealikelyexplanation fordifferences between .999 between P-values obtained with the two ap-
two haplotypes when (1) there was a single marker proaches. The standard Pearson X2 test, without any
that differed between the haplotypes, and (2) the dif- attempt to adjust for small cell sizes, did the next best,
ference was 2 bp (a one-step mutation). with a correlation of .983. The likelihood-ratio x2 test,
Goddard etal.: Localization ofWerner Syndrome 1291
Table 2
Cases Used in PresentStudy, in Addition toThose Described by Nakuraetal. (1994)
Ethnic Affecteds Total
Subject Groupa CountryofOrigin Consanguinityb Sampled Sampled
AGO7 C Second
1 1
AG3 CC No
1 1
AG33 C No
1 1
AG4 C No 1 1
AG41 C No
1 1
AG52 J Japan Yes
1 1
AG6 C No
1 1
AG78 C First 1 1
CP1 C France No
C 1 4
CP3 C France No
C 2 3
CTA C India/East Africa No 1 1
DJG Germany No
C 1 1
EKL Switzerland (German) No
J 1 1
FES No
J 1 1
FJ First
Japan 1 1
FNH Japan No
1 1
FNJ J Japan No 1 1
FNK J Japan No
c 1 1
HA J Japan No
c 1 1
HE c France No
C 1 1
HK c Turkey First
C 1 1
HM J Japan First
1 1
IB J Japan First
1 1
IND2 India First 6
IND3 India No
1 4
J02 J Japan Second
1 1
KKY JC Japan No
1 1
KM Japan No
1 1
KO Jc Japan First
1 1
KUN J Japan No
1 1
LGS C Germany, England No
1 9
MCI Japan No
J 2 2
MIMi C Japan First
J 1 1
OB Jc Japan No
1 1
PIR Jc Italy First
2 2
SUG Germany, Latvia (Jewish) No
1 1
SY JC Japan First
1 4
SYR c Syria No 3 23
TH Japan No
1 1
TK Japan First
1 1
TUR Turkey No
1 6
UH No
1 1
WMI J Japan First
1 1
aC = Caucasian; J = Japanese.
bFirst= offspringoffirst-cousinmarriage;second =offspringofsecond-cousinmarriage;Yes =offspring
ofconsanguineous marriage ofundefined degree; No = Noconsanguinity, orconsanguinity unknown.
cAlso partNative American.
which is sometimes suggested for loci with large num- for the MCMC approach was nominal, requiring <1
bers ofalleles and sparse data (Weir 1979, 1990, 1992; CPU min on an Alphastation 3000/300X, especially as
Hernandez and Weir 1989), performed somewhat less compared to the Fisher exact test, which required
well than did either the Pearson x2 or the MCMC ap- -11.25 CPUh onthe Sparcstation (-60% ofthe speed
proach, with a correlation of .913. The reduced X2 test oftheAlphastation) for analyses thatran tocompletion
performed quitepoorlyincomparisontothethreeother and 6-13.75 CPU h before memory usage problems
approaches, with a correlation of only .524 compared became apparent for analyses that could not be com-
to the Fisher exact test. The computation time needed pleted.
1292 Am.J. Hum. Genet. 58:1286-1302, 1996
equilibrium spans the region between markers F and S.
The markers in table 3 are shown in their most likely
order along the chromosome with the centromeric
markers at the top of the table, as based on meiotic
mapping, radiation hybrid mapping, and physical map-
pingmethods (Oshimaetal. 1994; C.-E. Yu,J. Oshima,
F. Hisama, S. Matthews, B. Trask, and G. D. Schellenb-
erg, unpublished data). The total size of the region be-
tween markers F and S, based on physical mapping
methods, is -1.2-1.4 Mb (C.-E. Yu,J. Oshima, F. Hi-
sama, S. Matthews, B. Trask, and G. D. Schellenberg,
unpublished data). Ofthe 17 markers in the region be-
tween markers F and S, 10 in theJapanese and 8 in the
Caucasians gave P-values <.03 with the Fisher exact
test or the MCMC approach. Eight ofthese 17markers
in the Japanese and 3 in the Caucasians gave P-values
<.005. A few additional markers that fell outside this
interval also gave statistically significant P-values with
MCMC methods: FGFR in the Caucasians and PLAT
and D8S164 in the Japanese. For PLAT, inspection of
thedataindicatesthattheassociationderivedfromthree
relatively common alleles that had lower frequencies in
Figure2 StructureoftheJVpedigree.Shadedsymbolrepresents cases than controls, which is inconsistent with a WS
Ws.
founder effect and probably represents mismatching of
cases and controls. For the other two loci, the results
The reduced X2 test appeared to give somewhat lower areconsistentwitheitherlinkagedisequilibriumorcase-
P-values than did the Fisher exact test. For the 69 com- control mismatching.
parisons in which the results of the two approaches Among the markers in this region there appear to be
could be compared, 22 had lower P-values with the two regions in which there is some evidence of linkage
Fisher exact test, and 41 had lower P-values with the disequilibrium. IntheJapanesedataset, 7ofthe 8mark-
reduced x2. The remainder produced approximately ers between markers E and L and 3 of the 5 markers
equal P-values or P-values <.001. The reduced x2 test between markers P and T gave highly significant P-val-
had somewhat higher numbers of tests with nominally ues with the Fisher exact test when the case versus con-
statistically significant results (P < .05) than did the trolmarker allele frequencies werecompared. The three
Fisher exact test or its estimate obtained with the markers in between these two regions gave nonsignifi-
MCMC approach. Of all the contrasts performed with cant results with the Fisher exact test, although marker
N gave a suggestive P-value of .068. In the Caucasian
the reduced x2 test, the number for which the P-value
<.05 17 and 11 intheJapanese and Caucasian data set, three of the eight markers between markers E
was was
data sets, respectively, as compared to 12 and 9 with and L and five of the seven markers between markers
N and T gave highly significant results. It is also worth
the Fisher exact test. There were only two contrasts in
noting that, while some markers in the center of this
which the Fisher exact test gave a P-value <.05 when
region gave nonsignificant evidence of linkage disequi-
the reducedx2 testdidnot. Ofthe 16 contrasts inwhich
librium with the Fisher exact test, most of these same
the P-value obtained by one approach was at least five-
markers gave significant (P < .05) or suggestive (P
fold greater than that obtained by the other approach,
in 11 the reduced x2 test had the lower P-value, while < .08) P-values when the reduced X2 test was used.
in only 5 did the Fisher exact test have the lower P-
Haplotype Studies
value, which suggests that the reduced x2 tends to be
Extended-haplotype analyses.-The extended hap-
too liberal.
lotype analyses provide evidence that there are several
Linkage Disequilibrium and WS different WS mutations in each population. Table 4
Thepairwiselinkage-disequilibriumtestsindicatethat shows extended haplotypes for the 21 markers A
there is genomic region chromosome 8 between through U. Haplotypes are grouped by similarity in the
a on
markers CandTinwhichmanymarkers showevidence Japanese data setandbycountryoforigin inthe Cauca-
of linkage disequilibrium in both populations sian data set. In both cases there are some haplotypes
one or
under study (table 3). The region inwhich both popula- that do not fit neatly into one of the subgroups (data
tions simultaneously show evidence oflinkage dis- not shown), but there appears to be three groups of
some
Goddard etal.: Localization ofWerner Syndrome 1293
a)
._
co Go
C1) 0a
C,,
cu
03 co
C, 0
C) 0 & 0
0
co Sb 0
's 0 *0
C1) 0 9~~ 0
09
0* 0 0
a) * 0. 0
(1) ._d
* 0* 0 0 ~~~R=0.913
9 00
I I I
.0 .2 .4 .6 .8 .0 .2 .4 .6 .8
Fisher's Exact Fisher's Exact
Co 0 co 0
a) @0 0,0
co 00
h._ 0*
C, 0
0
:3 0 0*00*@ 00
0 100~~0
C.) 0.0.. 00~~~
CCa)) 00 90*%~~
A-1 * ** 0
. .0 0 900 *0#
o
9 Ago
* R=0.983 0 . R=O.999
I I I I I I I I I
.0 .2 .4 .6 .8 .0 .2 .4 .6 .8
Fisher's Exact Fisher's Exact
Figure 3 P-values obtained for 70 pairs ofmarker-disease association tests. R = correlation coefficient.
highly similar haplotypes in the Japanese population, ofJapan; whether this is because the only information
and in the Caucasian population there appears to be ongeographic location formany patients is the medical
similarity among the haplotypes within both the Ger- center through which they were ascertained or because
man and the Italian groups. the mutation(s) on this predominant haplotype is rela-
IntheJapanesepopulation, thereislittleevidencethat tively widespread in Japan is unknown. There is, how-
individuals within each of the haplotype clusters are ever, some weak evidence for geographic clustering of
from a common geographic location (fig. 4). In particu- the haplotypes in the three pedigrees J02, ST-and SK:
lar, individuals who appeartohavethemajorhaplotype these three pedigrees all have a roughly central location
(Japanese-1) appear to have ancestry from most parts inJapan. It is worth notingthattheextendedhaplotype
1294 Am.J. Hum. Genet. 58:1286-1302, 1996
Table 3
P-Valuesfrom Linkage-Disequilibrium Tests between WRN and Chromosome 8 Markers
CAUCASIANS JAPANESE
LABELAND
MARKER X2-Red. %2 X2-LR MCMC Fisher X2-Red. %2 X2-LR MCMC Fisher
D8S133 .477 .472 .500 .488 .487 .436 .258 .418 .284 .270
D8S136 .504 .824 .843 .734 ... .288 .202 .225 .281 .279
AD8S137 .602 .260 .118 .324 .321 .628 .090 .042 .062 .066
B D8S131 .969 .020 .062 .102 .104 .756 .035 .052 .064 .064
C D8S2194b .005 .008 .009 .004 .005 .619 .078 .074 .075 .080
D D8S2192b .038 .342 .381 .184 .179 .004 .100 .032 .093 .097
ED8S2196 1 .547 .310 .504 .511 <.001 .004 .002 .008
FD8S2198 .094 .002 .006 .005 a <.001 <.001 <.001 <.001 a
G D8S339 .004 .025 .005 .014 .012 <.001 <.001 <.001 <.001 <.001
H D8S2204C .002 .013 .004 .011 .011 <.001 .002 .003 .003 .003
ID8S2202C .524 .267 .164 .342 .341 <.001 <.001 <.001 <.001 <.001
J D8S2206 .257 .646 .655 .604 .604 <.001 <.001 <.001 <.001 <.001
KD8S2134 .232 .210 .091 .235 .236 <.001 .006 <.001 .007 .007
LD8S2144d .034 .535 .200 .599 .588 .06 .460 .261 .552 .551
M D8S2156d .074 .285 .181 .219 .225 .059 .341 .267 .251 .261
ND8S2138 .031 .010 .001 <.001 <.001 .013 .079 .013 .068 .068
0 D8S2168 .125 .034 .009 .013 .017 .169 .226 .033 .174 ....
PD8S2174 .011 .044 .009 .018 .018 <.001 .004 <.001 .002 .002
Q D8S2150 .413 .806 .611 .828 .835 .013 .279 .084 .300 .300
RD8S2180 .107 .107 .100 .082 .127 .001 .001 <.001 <.001 <.001
S D8S2162 .023 .024 .011 .087 .032 <.001 .035 .010 .028 .028
TD8S2186 <.001 <.001 .001 <.001 <.001 .264 .536 .342 .657 .659
UD8S278 .715 .379 .497 .477 .475 .504 .792 .790 .800 .796
D8S259 .797 .296 .411 .239 .276 .242 .634 .421 .693 .691
D8S283 .516 .481 .406 .554 ...a .048 .493 .516 .482 .476
D8S87 .580 .773 .659 .856 .857 .607 .849 .854 .804 .815
D8S135 .702 .302 .267 .434 .435 .171 .359 .163 .448 .456
FGFR .015 .059 .096 .017 .018 .155 .221 .232 .139 ...a
D8S255 .842 .771 .669 .808 .812 .047 .221 .257 .232 .237
ANK1 .477 .639 .524 .666 .669 .040 .040 .041 .053 .053
D8S268 .048 .178 .228 .203 .205 .096 .329 .378 .230 .230
PLAT .310 .139 .098 .120 .121 .840 .003 .001 .001 <.001
D8S165 .251 .334 .214 .321 .316 .167 .214 .131 .146 .158
D8S166 ...' .273 .110 .182 .177 .575 .881 .769 .947 .948
D8S164 .326 .696 .586 .716 .721 .005 .016 .002 .003 .003
NoTE.-X2-Red = Reduced X2; X2-LR = Likelihood ratio X2; MCMC = Monte-Carlo Markov-chain estimate of Fisher exact test; Fisher
= Fisher exacttest. Underline denotes P-value <.05.
aInsufficient memoryto calculate the Fisher's exact test
bMarkers are unordered with respectto each other.
CD8S2204 is GSR1 and D8S2202 is GSR2 fromYuetal. (1992).
dMarkers are unordered withrespect to eachother.
eSample size too small to reduce the table
(Japanese-2) in these individuals is identical to the pre- ofthis region at marker M thus places WRN either into
dominant haplotype (Japanese-1) at all markers centro- the centromeric interval between markers A and M or
merictoD8S2202, andthusitispossiblethatthesethree into the telomeric interval between markers M and U.
individuals represent a single ancestral recombinant be- The extended haplotypes also provide presumptive
tween markers I and J on the most common Japanese evidenceofrecombination (fig. 5).Severalsuchprobable
WS haplotype. recombinant events place WRN centromeric to marker
Among families with two or more affected siblings B with high probability. Others most likely placing
there are two obligate recombinants in pedigrees HW WRN centromeric to marker F. Both pedigrees KO and
and ZM that bound the interval containing WRN (fig. SEP appear to represent double recombination in the
5) to that between markers A and U. An additional region.InKO,oneoftheserecombinanteventsprobably
obligate recombinant in the SYR pedigree in the middle occurred as an ancestralrecombinant, sincethemarkers
Goddard etal.: Localization ofWernerSyndrome 1295
Table 4
Extended Haplotypes forJapaneseand Caucasian WSSubjects
MARK LQ
A B C D E F G H I J K L M N O P Q R S T U
Japanese:
Japanese-1 2 4 9 1 4 8 7 5 11 5 5 12 4 3 9 1 3 1 3 2 3
FJ 9 1 6
FUW 1 4
IU 2 3/5 5
Jo0 9 6
KY 11 3 12 6
TK 2/4 4
TO 8
ZM-25 3/4
ZM-30 4
TH 9/10
HA 7 NA 4 1/6
KO 2/5 NA 5 4 6
HW-0 2/4 4/7 9
HW-5 2/3 4/7 9
Japanese-2 2 4 ? 3 12 6 2 4 8 5 5 12 4 3 9 1 3 1 3 2 3
J02 4/7 11 NA 2
SK 2/4 NA 3/5
ST 6 9 4
Japanese-3 2 4 12 4 9 6 1 4 8 4 5 12 4 3 9 1 3 1 3 4 ?
HM 6
MH 9/12 4
NN 6 4 3
Caucasian:
German-1 4 8 3 9 7 7 5 7 1 5 12 4 ? 9 1 3 1 4 1 3
?
LGS NA NA NA NA NA NA NA
SUGjb 1 11 11 NA 2
EKL 1/3 1/8 4/9 6 3 1/2
DJGb NA NA I 4
3
German-2 2 4 ? 3 6 6 4 8 1 ? 12 4 2 9 1 3 1 3 2 5
DJG' 8 1 NA NA NA
SUGic 1 11 NA 3
Italian 3 1 9 5 15 7 6 5 7 1 5 12 5 3 9 1 3 1 3 2 3
LRV-0 1/2 8/9 9/12 NA 5/6 8/9
LRV-1 NA 8 5
MSA 1/3 1/2
SEP 4 1 16/17 4 2/4 3/5
PIR 2/3 4 6 3 3 3 4 8 4 12 6 NA
French ? 4 1 3 ? 5 1 5 7 1 2 ? 1 12 1 1 6 1 4 4 3
HE 1 9 NA 7 2 1
iv 3 1/4 9 11 1/7 1 4 1 4
CP1 2 1/8 4 4/5 7/8 5 12 4 3 7/9 3 3 1/4 2/3
CP3 1/3 8/10 5/9 1/6 4 7/8 1/5 5 12 2/4 3/13 1/6 1/4 3/4 1/4 4/8 2 2/3
Syrian ? ? 11 3 12 8 7 5 ? 2 2 1 1 ? 1 1 6 ? 4 1 ?
SYR-6 NA NA 11 3 12 8 7 S NA 2 2 1 1 NA 1 1 6 NA 4 1 NA
SYR-8d NA NA 11 3 12/15 8 7 S NA 2/6 2 1 4 NA 1 1 6 NA 4 1 NA
allele,
NoTE.-NA =notavailable;? =unknownconsensus ormultiplealleleswiththesamehighestfrequency.
aMarkerlabelsA-Urepresentmarkersequivalentlylabeledinfigure1andtable3.Blanksindicateallelesthatarethesameasthoseontheconsensushaplotype.
Underlinedallelesdifferfromthealleleontheconsensushaplotypebyonerepeatunit.Allelesarelabelednumericallyinincreasingorderofallelesize.Sequential
allelenumbersrepresentallelesdifferingby2bp. Forthe21 loci,correspondence betweenallele 1 andsmallestallele (inbp) isasfollows:A-152;B-132;C-247;
D-149;E-217;F-181;G-162;H-115;1-121;J-236;K-175;L-273;M-163;N-138;0-162;P-186;Q-140;R-130;S-136;T-137;U-232. Exceptionstoallelesizing
were0-14at187bpratherthan188bpandP-3at189bpratherthan190bp.Dashesfollowedbynumbersinpedigreenames(e.g.,SYR-6andSYR-8)distinguish
twoindividualsfromthesamepedigree.
bOnehaplotypefromacompoundheterozygote.
'Secondhaplotypefromacompoundheterozygote.
dHalfsiblingofindividualsgivingrisetoconsensushaplotype.