linkage of wolfram syndrome to chromosome 4pl 6.1 and
TRANSCRIPT
Am. J. Hum. Genet. 59:855-863, 1996
Linkage of Wolfram Syndrome to Chromosome 4pl 6.1 and Evidencefor HeterogeneityDavid A. Collier,1 Timothy G. Barrett,3 David Curtis,1'2 Andrew Macleod,5 Maria J. Arranz,1J. Antonie Maassen,4 and Sarah Bundey3
'Section of Molecular Genetics and Department of Neuropathology, The Institute of Psychiatry, and 2Department of Psychological Medicine,St Bartholomews and Royal London School of Medicine and Dentistry, London; 3Department of Clinical Genetics, Birmingham MaternityHospital, Birmingham, United Kingdom; 4Department of Medical Biochemistry, Sylvius Laboratories, University of Leiden, Leiden; and4Royal Shrewsbury Hospitals NHS Trust, Shrewsbury, United Kingdom
Summary
Wolfram syndrome (DIDMOAD syndrome; MIM222300) is an autosomal recessive neurodegenerativedisorder characterized by juvenile-onset diabetes melli-tus and bilateral optic atrophy. Previous linkage analysisof multiply affected families indicated that the gene forWolfram syndrome is on chromosome 4p, and it pro-duced no evidence for locus heterogeneity. We have in-vestigated 12 U.K. families with Wolfram syndrome,and we report confirmation of linkage to chromosome4p, with a maximum two-point LOD score of 4.6 withDRD5, assuming homogeneity, and of 5.1, assumingheterogeneity. Overlapping multipoint analysis using sixmarkers at a time produced definite evidence for locusheterogeneity: the maximum multipoint LOD score un-der homogeneity was <2, whereas when heterogeneitywas allowed for an admixture a LOD of 6.2 was ob-tained in the interval between D4S432 and D4S431,with the peak close to the marker D4S3023. One familywith an atypical phenotype was definitely unlinked tothe region. Haplotype inspection of the remaining 11families, which appear linked to chromosome 4p andhad typical phenotypes, revealed crossover events duringmeiosis, which also placed the gene in the intervalD4S432 and D4S431. In these families no recombinantswere detected with the marker D4S3023, which mapswithin the same interval.
Introduction
Wolfram syndrome (MIM 222300; Wolfram and Wa-gener 1938) is an autosomal recessive disorder charac-terized by juvenile-onset diabetes mellitus and optic at-rophy, sometimes known as "DIDMOAD" (diabetes
Received August 22, 1995; accepted for publication July 24, 1996.Address for correspondence and reprints: Dr. David A. Collier, Sec-
tion of Molecular Genetics, The Institute of Psychiatry, De CrespignyPark, Denmark Hill, London SE5 8AF, United Kingdom. E-mail:[email protected] 1996 by The American Society of Human Genetics. All rights reserved.0002-9297/96/5904-0016$02.00
insipidus, diabetes mellitus, optic atrophy, and deaf-ness). Although cranial diabetes insipidus, sensorineuro-nal deafness, and renal tract and neurological abnormal-ities are seen in the majority of patients, only juvenile-onset diabetes mellitus and optic atrophy are necessaryto make the diagnosis (Kinsley et al. 1995; Barrett etal. 1995), and the diabetes mellitus is typically but notinvariably detected first. The optic atrophy is usuallyprogressive, following a period of normal vision, butdiagnosis can made on the basis of the observation ofoptic atrophy alone, based on analysis of 166/168 casesin the literature (Blasi et al. 1986).
In a recent cross-sectional case-finding study (Barrettet al. 1995), 45 patients with the syndrome were identi-fied. Nonautoimmune insulin-deficient diabetes mellituspresented at a median age of 6 years, followed by opticatrophy at 11 years. Median age at death (commonlydue to central respiratory failure with brain-stem atro-phy) was 30 years (range 25-49 years). Similar findingsrecently have been reported in a U.S. population by Kin-sley et al. (1995), who estimated 60% mortality by 30years of age.
Psychiatric disorders have also been associated withWolfram syndrome. In a series of 68 patients, 60% werereported to have episodes of severe depression, organicpsychosis, or organic brain syndrome (Swift et al. 1990),and presumed carriers appeared predisposed to psychi-atric illness (Swift et al. 1991). In the U.K. series of 45patients, 3 were treated for depression and 1 had prese-nile dementia. There was no evidence of psychiatric ill-ness in other patients, as judged by history, formal ex-amination, or hospital records (Barrett et al. 1995).The estimated prevalence of Wolfram syndrome in
the U.K. population is 1/770,000 (95% confidence inter-val 1/714,000-1/830,000), with a carrier frequency of1/354, based on the identification of all living Wolframpatients (n = 13) in two National Health Service regionsin the United Kingdom (total population 10,022,620)at midyear 1992 (Barrett et al. 1995). This is signifi-cantly lower than the reported prevalence of 1/100,000and a carrier frequency of 1/100 in a North Americanpopulation, based on the 1/175 occurrence of optic atro-phy in a juvenile-diabetes clinic (Fraser and Gunn 1977).
855
856
recombinationhotspot
fra4a
Am. J. Hum. Genet. 59:855-863, 1996
telomere
D4S1273
D4S412D4S432
4.1
1.4
2.3
1.61
MSX1
(HOX7)
D4S3023
D4S431
D4S3007D4S394DRD5
6
D4S1599
3.3D4S403
centromere
Figure 1 Genetic map of the region. Map distances are givenin centimorgans. "fra4a" indicates the position of aphidicolin fragilesite 4a, and the recombination hot spot is as described by Hubert et
al. (1994).
The Wolfram syndrome gene has been linked to theshort arm of chromosome 4 in a study of 11 familieswith two or more affected individuals (Polymeropouloset al. 1994). A maximum LOD score of 7.1 was foundin the interval between the microsatellite markersD4S431 and D4S394 (fig. 1). One family with an obli-gate recombinant between the disease and D4S431 was
found, indicating that the disease gene is more likely tolie in the 7-cM region between D4S412 and D4S431.No evidence for locus heterogeneity was reported. In-triguingly, linkage between the chromosome 4p markerD4S394 and bipolar affective disorder and depressionrecently has been reported (Blackwood et al. 1996). Thisregion of chromosome 4p is also close to a recombina-tion hot spot (Allitto et al. 1991) mapped by sperm
typing (Hubert et al. 1994) and contains a common
aphidicolin-sensitive fragile site, fra4a (Genome DataBase [GDB] 119193; Yunis and Soreng 1984). In thisstudy we report (a) confirmation of linkage betweenmarkers on chromosome 4p and 12 Wolfram familiesfrom the United Kingdom and (b) significant evidencefor heterogeneity, the latter of which implies that some
cases that currently meet the ascertainment criteria forWolfram syndrome are not due to a locus in this region.
Subjects and Methods
SubjectsPatients for this study were drawn from a larger co-
hort recruited nationally from major referral centers anda U.K. national DIDMOAD register (Barrett et al.1995). Minimum ascertainment criteria were juvenile-onset (i.e., at <30 years of age) diabetes mellitus andoptic atrophy, chosen as the only features consistently
present and earliest to develop in 166/168 case reports(Blasi et al. 1986). All patients were visited at home, forclinical history taking, examination, and blood samplingfor DNA extraction. Each patient's hospital record wasalso examined, then follow-up visits were arranged toinitiate further investigations. All affected patients wereexamined, with pupils dilated, by experienced ophthal-mologists. First-degree relatives of affected patients werealso examined for the presence of optic atrophy andhad a fasting blood-glucose estimation for detection ofasymptomatic hyperglycemia. This was defined as a fast-ing plasma glucose of >6.0 mmol/liter (>3 SD abovethe mean of the normal population). All unaffected first-degree relatives screened were negative for fasting hyper-glycemia; there were no symptoms of optic atrophy orsigns on fundoscopy. Nine patients were omitted fromthe sample because they had other diagnoses: two hadcongenital rubella syndrome; one had Leber hereditaryoptic neuropathy; four had thiamine-responsive anemia,deafness, and diabetes syndrome (TRADD; MIM249270); and two were unclassified (one had retinaldystrophy and diabetes mellitus, and the other presentedat 14 mo with ketoacidotic coma, hypoxia, and seizuresand then with secondary optic atrophy and anterior pi-tuitary failure). The ascertainment criteria were vali-dated by demonstration of their presence in all the sec-ondary cases (siblings); the prevalence of complicationsand ages at presentation were the same in the secondaryas in the index cases. Twelve families that had morethan one sibling affected (11 families) or were consan-guineous (1 family) were included in the panel for link-age analysis, on the basis of the criteria listed above.The clinical features of these families are described intable 1 and in the pedigree diagrams shown in figure 2.The study was granted local ethical committee approvalfor genetic and clinical analysis of the subjects.
DNA StudiesBlood samples were obtained from all available fam-
ily members after informed consent was given. Lym-phoblastoid cell lines were established for all affectedpatients (ECACC). DNA was extracted from wholeblood by means of Puregene DNA extraction kits (Gen-tra Systems) and was diluted to a stock solution of 25ng/gl. Probands were also karyotyped, and no gross ab-normalities were detected.The markers D4S127, D4S412, D4S432, D4S3023,
MSX1 (HOX7), D4S431, D4S3007, D4S394, DRD5,D4S1599, and D4S403 were analyzed as published(GDB, Johns Hopkins University; Dib et al. 1996).Primer sequences for D4S394 were obtained fromM. H. Polymeropoulos (personal communication). Oneprimer of each pair was 5' end-labeled with 32P, andPCR was performed in a total volume of 10 RI con-taining 25 ng of DNA, 200 iiM each of dATP, dGTP,TTP and dCTP, 1 x standard PCR buffer containing
Collier et al.: Wolfram Syndrome Linked to Chromosome 4p
Table 1
Clinical Features of Families Used in Linkage Analysis
AGE AT DIAGNOSIS(years)
FAMILYa AND AGE(in Years) OF Diabetes Optic Diabetes Sensorineuronal Renal NeurologicalAFFECTED SUBJECT Mellitusd Atrophycd Insipidusd Deafnessd Abnormalitiesd Abnormalitiesd
B:413232
445
97
34
1010
4640
F:3837
H:3336
I:36
712
612
2
2723
K:1716
811
1415
2118
66
2422
77
322930
W:4629
466
45
1712Yes
54
1015
1015
612
8
89
2.5
1111
1111
788
1212
1711Yes
34
3240
1212
1116
15
1211
No
1815
1811Yes
96
2311* . .
3830Yes
NoNo
NoNo
32No
3015
616
9
NoNo
44No
3433 36
20.. .
3035
18 32
No.. .
28No
NoNo
1815
NoNo
18No
1313
. . .
1824
142.5
NoNo
78
NoNo
No28No
* . .
No30
2027
3429
a Designations are as in figure 2.b Mean age at onset was 7 years.c Mean age at onset was 9 years.d Yes = symptoms present; age at onset unknown; No = symptoms excluded by investigation; and an ellipsis ( . .. ) indicates that the individual
was asymptomatic.
1.5 mM MgCI2, and Amplitaq polymerase (Perkin ElmerCetus). After 5 min denaturation at 950C, samples wereamplified for 30 cycles of 95"C for 1 min, an annealingtemperature appropriate to the marker for 1 min, andextension at 720C for 1 min, followed by a final exten-sion at 720C for 10 min. Alleles were sized on a 6%denaturing polyacrylamide gel (Sequagel 6; National Di-agnostics) exposed to X-ray film (Fuji Rx). Genotypeswere scored by two independent observers to produceallele assignments that were consistent across all fami-
lies. On detection of incompatibilities or double recom-binants between adjacent markers these genotypes werechecked, and, if necessary, the genotyping process wasrepeated.
Linkage AnalysisGenotypes were read and entered into the DOLINK
data-management system (Cook et al. 1993) to allowchecking and production of pedigree diagrams by meansof PEDRAW (Curtis 1990). Linkage analysis was per-
857
Am. J. Hum. Genet. 59:855-863, 1996
FAMILY B
D4S1277? ? ?
D48412 ? ? 35
D4S3023 1 1 5
04S431 11111
a a
D03007 I04S304 4 *D0608 7?
04 745
Inf .3 4 3 1 013 ?71 7
41114 4 4 4 14 4 114
5 1
6 6 a 6
1 3 31 3 ?
2 7 2 77 7 7 7
21111 66 116 1147 7
FAMILY K+ +
{ 2
D4127 3 n:4 2 flMSXII MII 1 14W4S4124 14 2E112DWS432 I I1 9| I1D4S3023 ? El? ? ?D4S431 I I4 3D4S3007 ? ? ?04S3946 7 8DROSS 110
D041599 ? II? ? * ?04S403 S SUS
NA1 62 113
0O4127 fW 3 2 Ei4 f13MSX14 111 1EI 4 1D4541221142214 2II4D48432111I I11 1I1ID4S3023 ? *?? ? ? ?D4S431 51 63 4548D08O 7 7 70481867 7 7 7 ID484038 885 585
FAMILY T+ +
11 12
D481271 f4 1 1f4MSXI ?1 17 4 111
D4S41251117 7 11104S4322111 1 1D4S3023 ? |? |04S431 4 4[1 2D03007 7 ? 1 ?
WS304 8 3 890D418002 8 3 6WS40368 5 4 7
+ +11:1 N:2
D4812711 1 4 nn4msx1 ? I 111I1
04S41281 8 1SDS431111 1 11D4S3023| ? ? || ?D4S4312 14 2 404S3007 ? ? ?IID4S394 9 10RD8S 4 I44S10S0 6 2 6 204S4037
7
a 4 8
FAMILY D+ +
1:1 1:2
041272 1 nD454128 5
5 11D4S432 2 *15 3 *D483023 6 6 61
043182a 5 211
0431007 ? ? ?D4S3947|| l
D4S1599?s7 ? 7s l?04S4035 5 44
L1+0-1 6:2
D0 27 6 af 6 l6
D4S41251 | 1 51 111
D45432 5116 51116D4S4318 4 40483007 1 3 1 3
048403 5
FAMILY H
+ 3 +
1:1 1:2
D4S1271 I IMSX14 4 4 2
D4S412 E5 S lS045432 1 1I 1 44S3023 1 3 2D4S431 0 S a 9D0S3007 1 3 1 2
4S394 7 S 12 7DRDS S 10 S S
D4S15066 80 6D4S403 1 _3 4 1
61 62 63 N4 65 H6
SXl 4 14 4 - 2 4 2 4 _ 2 4 404S412 S 5 S 5 1 5S 5 5 5048432 1 I 1 4 I'4 1 4 1 1D483023 S *S 5 5 5 ? ? I S048431 S 6 S 5 9 6D03007 3 1 3 1 3 2 1 204304 5 12 S 12 5 7 7 7 ? ?ORDS 10 5 10 S 10 S 5 S S S
D451500 6 I6 0 6 60 ?D4S403 3 4 3 1 4
FAMILY Q
+ +
1:1 1:2
0W41273 l21 2e 2MSXI41112 1112
04S412 3 14 31 120S432 11 16 61112D4S3023 58115 8 ll5D45431-IS1 7W43007 3 13 1WS34 5lI 3@ 17
4S18609 I 8 I054038 7 7 8
j b~~~+h+ + + d+0 1 62 03 64 68
0S127 3E2 3 2 3112 2- 2 7 IflMSXI 4 112 4 2 4112 21112 4 12
045412 3112 3 3 3113 ?111 3 1204S432 1 1112 1 6 1116 61U2 1 1112
D4S30238111 58 8118 5lI5 8111
D4S431 5 F7
8 7S
7 5 116
0483007 3 3 3 3 11104340M 7 8 3 5 3 1 3 ? ?DR0480 5 7 7 75 7 7 8 5
D4S403 S 5s S5 7 U7 ? 7 5 5s
formed by FASTLINK (Lathrop et al. 1985; Cottinghamet al. 1993; Schaffer et al. 1994), under recessive inheri-tance with a disease-gene frequency of .005. Penetrancewas set to be 95% by age 15 years, and unaffectedsubjects below this age were classified as having un-known affection status. The marker-allele frequenciesused were calculated from unrelated founder membersof the families.
Multipoint AnalysisMultipoint analysis was performed by means of the
VITESSE program (O'Connell and Weeks 1995).VITESSE allows fast multipoint analyses with highlypolymorphic markers, through the use of a computa-tionally efficient "fuzzy inheritance" algorithm. How-ever, the current version cannot analyze families withconsanguinity loops, so, in order to run VITESSE, wesimplified and removed the loops from the pedigrees.We compared the two-point disease-marker LOD scoresobtained from these simplified pedigrees with those from
the full'pedigrees, and we found only minor differences.We therefore used VITESSE to perform overlappingmultipoint analyses, using six markers at a time againstthe disease locus. The most recent genetic map fromGenethon (Dib et al. 1996), together with informationfrom earlier maps (GDB, Johns Hopkins University),was used for linkage analysis. For both the two-pointand multipoint analyses, total LOD scores were calcu-lated under homogeneity, and to test for linkage underheterogeneity we used the LOD2 statistic (Risch 1989),which is maximized over the proportion of linked fami-lies, alpha. These calculations were performed automati-cally by means of the TABLE program from the DO-LINK package (Cook et al. 1993).
Transmission-Disequilibrium AnalysisThe transmission-disequilibrium test (TDT; Spiel-
man et al. 1993) was used to test for linkage andlinkage disequilibrium between Wolfram syndromeand marker alleles, by means of the program ETDT
858
Collier et al.: Wolfram Syndrome Linked to Chromosome 4p
FAMILY E
1:2S
0S1274 2 * fl2MSX 2 14 2 E114
D4S41251115 I l ID4S432 7 14 1 lE 6D4S3023 5 | 504S431 3 a1 9
D03007 3 3 13 1DWS3"46I E 161lDRD5 3 5 1
D4SlS99 3 13 11I6104S403 6 5 4 5
A+ I+ a+11 11-2 11 3
04127 4 2 4 l2 7MS5 l12 4h1 2 4lI 4 4
D4S41253 1 1 1 1D454324 75 41
D4530235111 5Ill 5111 5
04543106 43 4 a
04S394050451599 3 3 1 3 1045403 5 5 a 4 5
FAMILY F
11 12
04S127 4 3MSX11 4
04S412 1 304S432 1 3
D4S3023 5 1D4S431 9 704S3007 3 3D45394 5
4DR43 18 104S403 5
1+ 1+ 1+1II 11:2 113 11:4
D0127 3 5 4 5 7 ?MSX1 4 2 1 2 1 4
D4S4121 1 1 1 6D4S432 3 1 3 1 3 3D4S30235 5f 5 5 1 504S431 9 7 9 7 7 704S30073 3 3 3 3 3D4S394 8 "11DRD5 6 4 e 4 1 704S403 5 5 5 5 a
FAMILY I
1 1 2
04S127 4 SMSX1 2 1
04S412 1 1I04S432 4 404S3023 6 6DWS431 3 3D4S3007 3 3D4S394 3 aDRDS 10 4
D01599 2 a04S403 5 S
111 1N2 113
FAMILY J
+ +
11 12
0S127 7 ? 7 ?MSX1 4 2 2 4
D4S412 1 1 1 5
D4S432 5 1 1 404S3023 5 5 5 S04S431 5 7 4 4
DWS3007 3 1 3 3W4S394 6 10 ? ?
0D0 5 5 5 55d1599 5 9 9 5DS403 4 5 7 5
II 1 112 11:3
W512 7 7 fl? ?msx14 4 ? ? 4 u14
04S4121 5 ? 7 1 u1S04S4325 4 1 *1 51 1404S3023 55 ? ? 5 5
D4S4315 457 4 5 1404S3007 3 3 * 3 3DWS394 6 | 10? A?7 11°DRD55 5 5 5 S 5
D0150099 9 9 9 5 5D4S403 5 7 7 7 4 5
FAMILY W+
1:1 1:2
D4127 6 6
MSX12204412 ? ?D4S432 S 2D453023 1 7DWS431 7 3D4S3007 3 30S394 7 4
10MM1S4 ? ?04S403 8
"01 62 113 114 WS
D0127 7i3 IEE 6 36 3
MS5 4 11173 2 || ? 2 ||3 4 1113
04 5412 ? ? ? ?D4S432 211 2 5 115 2111 2
0453007045304 4 7 7 10 7 107
0005 5 5 15 5 10 5 5 5
D041500 ?045453 06 6 a 0a 6
FAMILY S+ +
I1 1:2
D0127 nf 6 3MSX1 1.1l11 13E1 11
04S412 4E113 4E11404S432 2111 1 4 14DWS3023 21 IS 21 1104S431 5 a 5 6043507 1 3 304S394 12 7 6
0D 010 10D4 1S99 91 5 1961I04S403 7 2 5 13
a+ + L+ +111 112 I13 114
D0127 mu6 6uu 60 6 60 3MSXI1 1 3 1 u3 1 u3 111111
04S412 4 4 4f 4 4 4 31 4D4S432 2 u4 2 4 2 4 11114DWS3023 2 |2 2 |2 2 |2 SI I1DWS431 SUE SE SE5 11116DWS3007 1 3 1-3 |3 1113DWS394 12 8e 1266 12 6 7 1|MDRD 10 69 106w 10 9 1011 ISNS1599 9 9 ?"1? 9 11 51115DWS403 7 NS 7 U3 7oS 2U 3
Figure 2 Pedigree structure, genotypes, and recombinants. Blackened symbols indicate affected individuals, and obligate carriers areindicated by a dot within the symbol. A plus sign (+) indicates that DNA was available for analysis. Recombinants are indicated by bars. Forfamilies D, E, H. J. K, Q. S. and T. the most probable haplotypes were reconstructed by hand, by means of the program Cyrillic 2 (CherwellScientific). The left-hand bar for each haplotype corresponds to the chromosome from the leftmost parent. No double recombinants weredetected. For families B. F. I, and W. missing parental genotypes denote that haplotypes could not be reconstructed, and bars are used toillustrate recombination between the subjects in each sibship.
(Sham and Curtis 1995), which implements an exten-sion of the TDT that is applicable to multiallelicmarkers. The ETDT program performs a geno-
typewise analysis that examines each heterozygousparental genotype separately to see whether there isdeviation from the expected 50:50 transmission ofalleles to expected offspring. These deviations are
combined into an overall X2 statistic with the numberof df equal to the number of distinct parental geno-
types. A more parsimonious allelewise analysis is alsoperformed, which applies logistic regression to ana-
lyze whether certain alleles are more likely than oth-ers to be transmitted to affected offspring, across a
number of different parental genotypes. This avoidsthe problem of multiple testing, which otherwisemight be involved if each allele were consideredagainst the rest (Sham and Curtis 1995). In the pres-
ent application, analysis of particular alleles and hap-lotypes was undertaken only when both the geno-
typewise and allelewise methods were significantlypositive.
Results
The Genetic Map of 4p16The genetic map used in the analysis is shown in figure
1. As a test of the integrity of our genotype data andthe genetic map, two point marker-marker LOD scores
were calculated. This is important for both themultipoint linkage mapping and analysis of haplotypesfor recombination events. In particular, we wished toexamine uncertainty regarding the position of themarker MSX1 (HOX7). Recent genetic maps (C4M72and C4M77; GDB, Johns Hopkins University) placeMSX1 (HOX7) distal to D4S412, 17.9 cM fromD4S403 and probably 2 cM proximal to D4S432. How-ever, a prior map places MSX1 (HOX7) 13 cM fromD4S394, a probable location 1 cM distal to D4S412.Since there were recombinants between MSX1 (HOX7)and Wolfram syndrome in our families (fig. 2), whichwould have consequences for mapping the disease gene,we used the genetic data to attempt to better map thismarker. Two-point analysis revealed a maximum LOD
859
Am. J. Hum. Genet. 59:855-863, 1996
score of 5.4 (theta = 0) with the marker D4S127, indi-cating that MSX1 (HOX7) lies close to this marker ina location more distal than that in the most recent pub-lished maps. It was not linked to any other markers. Allother markers showed linkage with one or more adja-cent markers in the map, with the exception of D4S412.
Linkage AnalysisTwo-point LOD scores for the pedigrees are shown
in table 2, and the marker data used for the computationare shown in figure 2. Under homogeneity, the maxi-mum two-point LOD score obtained was 4.62 at theta= .05, with DRD5. When heterogeneity (alpha = .75)was taken into account, a LOD2 of 5.1 at theta = 0was obtained. For D4S431 a maximum LOD score of3.09 was obtained at theta = .1, under homogeneity,and a LOD of 3.2 was obtained under heterogeneity(theta = .05; alpha = .85). For D4S3023 a maximumLOD score of 3.2 was obtained at theta = .01 and .05(alpha = 1). These results confirm linkage but fail toprovide statistical evidence for locus heterogeneity.When we subjected the families to formal multipoint
analysis using overlapping sets of six markers at a time,the total LOD score was negative over much of theregion (fig. 3) and reached a maximum of 1.9 distal toD4S403. By contrast, when we allowed for heterogene-ity, a maximum LOD2 of 5.2 was obtained midwaybetween D4S432 and D4S431 (alpha = .85), near themarker D4S3023. MSX1 (HOX7) was not included inthe multipoint linkage analysis, both because of uncer-tainty over its exact position in the map, and because,since it was tightly linked to D4S127 in our families, itwas not expected to provide additional information onthe location of the gene.One family (family K; fig. 2) provided strong evidence
against linkage in the region between the markers MSX1(HOX7) and D4S403, with multipoint LOD scores of<-2. Two affected individuals seemed to have differenthaplotypes across the region, and one affected individualshared all alleles with an unaffected sibling who hadpassed the age of risk.
Clinical Features of the Unlinked FamilyAlthough family K met the diagnostic criteria for Wol-
fram syndrome (juvenile-onset diabetes mellitus and op-tic atrophy) and was consequently included in the study,the age at onset of the symptoms differed from those inthe other families (table 1). For the two probands in thisfamily, optic atrophy was first diagnosed at the ages of6 mo in one sibling and 2 years in the other, > 1 decadebefore the onset of diabetes mellitus. No period of nor-mal vision was recorded, and symptoms of diabetes in-sipidus, renal dysfunction, and neurological abnormali-ties are not present. In contrast, the other affectedsubjects in the linkage study developed diabetes mellituseither at the same time or before the onset of optic atro-
phy, with the exception of family J, where optic atrophyhad developed 2 years earlier in one sibling. Thus, al-though meeting our ascertainment criteria for Wolframsyndrome, family K has an atypical phenotype. Themultipoint analysis was therefore repeated without thisfamily, and the maximum LOD score under homogene-ity increased to 6.2, with a shape very similar to thatobtained when family K had been included and with thepeak in exactly the same place.
Haplotype Inspection of the PedigreesIn pedigrees with typical symptoms of Wolfram syn-
drome, recombination events suggest that the probablelocation of the disease gene is within the interval of -5.5cM between the markers D4S432 and D4S431. In familyH (person II:2; fig. 2), there is a maternal recombinationwith the marker D4S431, indicating that the diseasegene lies telomeric. In family Q (person I1:4), there is arecombinant with D4S432 between both affected sub-jects. In addition, in family B, both the genotype thatsubject IV:7 had for D4S432 and the genotype that sub-ject IV:15 had for D4S431 were different than those inthe other two affected individuals in the family. Theonly marker alleles that the three affected members ofthis pedigree had in common was D4S3023. However,only one of the four parents of these individuals wasavailable for genotyping, so it was not possible to recon-struct haplotypes.
Because multipoint linkage analysis provides signifi-cant evidence for locus heterogeneity, inferences such asthese should be treated with caution, because it mightbe that these pedigrees do not segregate a linked locusat all but that affected subjects simply share by chancesome markers in the region. However, the only familythat provided conclusive evidence against linkage hadan atypical phenotype, and data for all other familiesare consistent with linkage in the region. Since bothmultipoint linkage analysis and haplotype inspection ofthe pedigrees provide evidence for linkage to the sameinterval (i.e., D4S432-D4S431), we are confident thatthis localization is correct.
Linkage-Disequilibrium MappingThe markers closest to the region of tight linkage-
markers D4S412, D4S432, D4S3023, D4S431, D4S394,and DRD5-were analyzed by means of the TDT. Allmarkers provided statistically significant results, but forthree of the markers inspection of the pedigrees revealedthat these results seemed to reflect linkage rather thanlinkage disequilibrium. However, for one of the markersthere was one particular allele that demonstrated excesstransmission to affected offspring: allele 5 of D4S431(0.254 kb; GDB ID GOO-063-142) was transmitted 13times and was not transmitted 3 times (X2 = 6.25; 1 df; P= .012); for this marker, examination of possibly linkedfamilies revealed that allele 5 was transmitted 7 times
860
Collier et al.: Wolfram Syndrome Linked to Chromosome 4p 861
Table 2
Two-Point LOD Scores between Wolfram Syndrome and Markers on Chromosome 4p
LOD SCORE AT THETA =MARKER ANDSTATISTICa 0 .01 .05 .1 .2 .3 .4
D4S127:LODiLOD2Alpha
D4S412:LOD1LOD2Alpha
D4S432:LODiLOD2Alpha
D4S3023:TotalLOD2Alpha
MSX1:bLOD1LOD2Alpha
D4S431:LODWLOD2Alpha
D4S3007:TotalLOD2Alpha
D4S394:LOD1LOD2Alpha
DRD5:LOD1LOD2Alpha
D4S1599:LODiLOD2Alpha
D4S403:LOD1LOD2Alpha
-21.80.07.10
-3.90.66.40
-11.622.55.65
2.942.941
-10.65.84.40
-9.212.81.65
-4.36.60.50
-13.941.41.45
1.275.06.75
-7.43.83.55
-21.01.90.35
-7.84.07.10
-1.05.69.45
-.962.48.65
3.173.171
-1.96.81.40
-.843.00.75
.791.03.75
-3.071.39.50
3.975.00.80
-1.57.83.60
-5.56.91.40
-3.17.08.15
.50
.81
.60
1.722.36.75
3.183.181
.53
.72
.60
2.873.23.85
1.701.701
.221.34.55
4.624.70.90
.10
.80
.65
-1.00.93.50
-1.39.09.20
.92
.92
.90
2.302.34.90
2.822.821
1.18.60.90
3.093.10.90
1.711.711
1.181.33.75
4.334.331
.53
.72
.75
.45
.95
.60
.14
.10
.40
.82
.821
1.971.97
1.891.891
1.14.37
1
2.342.341
1.201.201
1.331.331
3.123.121
.55
.551
1.001.001
.14
.141
.11
.111
.46
.461
.14
.131
1.151.15
.40
.40
.97
.971
.27
.271
.66
.171
.22
.041
1.261.261
.63
.631
.39
.391
.21
.211
.82
.821
.26
.261
1.751.751
.59
.591
.31
.311
.09
.091
.66
.661
.19
.191
a LOD1 = standard LOD score; LOD2 = analysis under locus heterogeneity; and alpha =
maximized.b Also known as HOX7.
and was not transmitted 1 time, but statistical signifi-cance was not reached (two-tailed binomial test; P= .07). Because of this, we examined the allele frequencyof D4S3023, the adjacent marker in the disease-geneinterval, in one affected individual from each family.The .55 frequency of allele 5 (0.254 kb; GDB ID G00-063-142; allele 2 in GDB) in affected individuals was
proportion of families linked when LOD2 is
similar to the .50 published allele frequency. There wasno obvious haplotype of D4S431 and D4S3023 associ-ated with Wolfram syndrome.
DiscussionIn this study we present the first confirmation of the
localization of a Wolfram syndrome gene to chromo-
Am. J. Hum. Genet. 59:855-863, 1996
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40
Figure 3 Results of multipoint analysis using VITESSE. Thedashed line indicates the LOD score under homogeneity, and the dot-ted line indicates the LOD score under heterogeneity (LOD2). The X-axis scale is in LOD-score units, and the Y-axis, according to publishedmaps, is in centimorgans of genetic distance. The position of the ge-
netic markers used in the multipoint analysis is drawn to scale on thebar above the graph.
some 4p. Furthermore, multipoint linkage mapping pro-
vides statistically significant evidence for locus heteroge-neity. It is crucial to be aware of locus heterogeneity,because, if it is not allowed for, estimates of the locationof the disease gene may be seriously in error.
Examination of individual families did not provideconclusive evidence for locus heterogeneity, because ofmissing parental data or homozygosity at particularmarkers, with the exception of family K. This familywas not linked across the entire region, and inspectionof the clinical data for this family revealed that the age
at onset for optic atrophy was >1 decade before theonset of diabetes mellitus. This finding was differentfrom those seen in the other families, since optic atrophyusually developed after diabetes mellitus, although inone other subject (in family J) it developed 2 years ear-
lier. In addition, all other affected subjects had recordedperiods of normal vision before the development of opticatrophy, which was progressive in nature. It should benoted that the diagnostic criteria for Wolfram syn-
drome, on the basis of 166/168 published cases (Blasi etal. 1986), are juvenile-onset diabetes mellitus and opticatrophy; and progressive development of the latter wasnot included in the diagnosis. Consequently, at the startof this study family K met our ascertainment criteriafor Wolfram syndrome. Although the family may now
provide grounds for refinement of the phenotype, we
have not felt able to exclude them from the linkageanalysis. The diabetes mellitus and optic atrophy in thisfamily may represent a distinct syndrome or true locusheterogeneity for Wolfram syndrome, and a definitive
conclusion will have to await isolation of susceptibilitygene(s).
Using simplified pedigrees with the loops removed,we obtained, from multipoint mapping, statistical evi-dence for localization of the gene to the interval betweenD4S432 and D4S431. Using two-point LOD scores andhaplotypes, Polymeropoulos et al. (1994) suggested thatthe location of the Wolfram syndrome gene is betweenthe markers D4S412 and D4S431, and analysis ofmultipoint LOD scores provided evidence for a locationbetween D4S431 and D4S394. One obligate recombi-nant with D4S431 was reported, indicating that the genelies distal to this marker. No recombinants for distalmarkers were reported.Examining haplotypes in individual families from the
present study reveals recombinants between D4S432, infamily H, and a recombinant with D4S43 1, in familyQ, indicating that the locus lies in the interval betweenD4S432 and D4S431. This location is consistentthrough all of the families that had a typical phenotype.No recombinants were detected with the markerD4S3032, although it was not genotyped in the singlefamily (family K) that was clearly unlinked to the region.The maximum two-point LOD score obtained was
with the marker DRD5. However, evidence from bothhaplotype inspection of the families and multipoint link-age analysis indicates that the gene for Wolfram syn-drome is unlikely to lie in the interval containing DRD5and that the reason for obtaining a maximum LODscore with this marker is that two of the families (fami-lies H and I) providing statistical evidence against link-age for other markers in the region are uninformativefor DRD5. Thus we are able to refine the localizationof the Wolfram syndrome gene to the interval D4S432-D4S431, with a suggested location close to the markerD4S3023. Recombination events also occurred betweenMSX1 (HOX7) and Wolfram syndrome. Although re-cent published genetic maps indicate that this gene mapsproximal to D4S432, which would allow further re-finement of the disease-gene locus, analysis of two-pointlinkage data in our families indicates that MSX1(HOX7) may in fact map distal to D4S412; and, becauseof this, it cannot be relied on to provide better localiza-tion.
Detection of linkage disequilibrium between a markerand disease may provide additional information on theposition of a putative disease gene, through the identifi-cation of an associated allele or ancestral haplotype. Thedata were analyzed by the TDT; and, although specificalleles of MSX1 (HOX7) and D4S431 were preferen-tially transmitted, both of these markers showed recom-binants in apparently linked families. When applied tothe study of apparently linked families, the TDT cannotdistinguish the effects of linkage disequilibrium fromlinkage, and the positive results probably do not implythat the gene is especially close to these markers. The
* l
862
II. ------
"1''.. ...........................................I. --I
Collier et al.: Wolfram Syndrome Linked to Chromosome 4p 863
only completely linked marker, D4S3023, did not dem-onstrate association with the disease gene. It may bethat we do not have sufficient power to detect linkagedisequilibrium in the present family sample and that theascertainment of additional families will be required toresolve this.
In summary, we have confirmed linkage of Wolframsyndrome to chromosome 4p and provide evidence forlocus heterogeneity that corresponds to phenotypic vari-ation within the currently accepted ascertainment crite-ria for the disease. We have detected a novel recombi-nant between the disease and the marker D4S432, andconsequently we are able to refine the locus for the dis-ease to the interval D4S432-D4S431. If further markerswere available for typing in this interval, then it mightbe possible to achieve a narrower interval, although weknow of no such markers at present. Fine mapping maywell depend on the detection of linkage disequilibriumor other approaches, such as detection of additional re-combinants, although this is likely to require the ascer-tainment of additional families.
AcknowledgmentsT.G.B. is funded by a Price-Hall Fellowship from The United
Birmingham Hospitals Trust. This work was funded by theRoyal National Institute for the Blind. We are grateful to Lou-ise Brown, Paul Brown, David Bryon, and Martin Giles forDNA extraction; to Barry Chioza, Sanober Shaikh, ElisabethDawson, David Ball, and Naveed Anwar for additional geno-typing; to the HGMP Resource Centre, Hinxton Hall, Cam-bridge, for oligonucleotide primers and access to genome dataand computing facilities; and to all the medical personnel whohave cooperated with this study. We also thank the subjectsand their families for their cooperation, and we thank Dr. T.Mitchell (University of Cork, Republic of Ireland) and B. Dar-low (University of Otaga, Canterbury New Zealand) for theirvaluable assistance.
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