Diabetes Treatments

2 diabetesrelated traits in 637 members of 143 French pedigrees ascertained diabetes or glucose intolerance (GI), as well as with each of the two phenotypes …

Am J Hum Genet 67:14701480, 2000

Genomewide Search for Type 2 DiabetesSusceptibility Genes in French Whites: Evidence for a Novel Susceptibility Locus for Early-Onset Diabetes on Chromosome 3q27-qter and Independent Replication of a Type 2Diabetes Locus on Chromosome 1q21q24
Nathalie Vionnet, El Habib Hani, Sophie Dupont, Sophie Gallina, Stephan Francke, Sebastien Dotte, Frederique De Matos, Emmanuelle Durand, Frederic Lepretre, Cecile Lecoeur, Philippe Gallina, Lirije Zekiri, Christian Dina, and Philippe Froguel
Institute of Biology-CNRS 8090, Institut Pasteur, Lille, France

Despite recent advances in the molecular genetics of type 2 diabetes, the majority of susceptibility genes in humans remain to be identified We therefore conducted a 10-cM genomewide search 401 microsatellite markers for type 2 diabetesrelated traits in 637 members of 143 French pedigrees ascertained through multiple diabetic siblings, to map such genes in the white population Nonparametric two-point and multipoint linkage analyzes–using the MAPMAKER-SIBS MLS and MAXIMUM-BINOMIAL-LIKELIHOOD MLB programs for autosomal markers and the ASPEX program for chromosome X markers–were performed with six
diabetic phenotypes: diabetes and diabetes or glucose intolerance GI, as well as with each of the two phenotypes associated with normal body weight body-mass index27 kg/m2 or early age at diagnosis 45 years In a second step, high-resolution genetic mapping 2 cM was performed in regions on chromosomes 1 and 3 loci showing the strongest linkage to diabetic traits We found evidence for linkage with diabetes or GI diagnosed at age 45 years in 92 affected sib pairs from 55 families at the D3S1580 locus on chromosome 3q27-qter using MAPMAKER-SIBS MLS p 467, P p 000004, supported by the MLB statistic MLB-LOD p 343, P p 00003 We also found suggestive linkage between the lean diabetic status and markers APOA2D1S484 MLS p 304, P p 00018; MLB-LOD p 299, P p 00010 on chromosome 1q21-q24 Several other chromosomal regions showed indication of linkage with diabetic traits, including markers on chromosome 2p21-p16, 10q26, 20p, and 20q These results a showed evidence for a novel susceptibility locus for type 2 diabetes in French whites on chromosome 3q27-qter and b confirmed the previously reported diabetes-susceptibility locus on chromosome 1q21-q24 Saturation on both chromosomes narrowed the
regions of interest down to an interval of 7 cM

Introduction Type 2 diabetes MIM 125853 is a heterogeneous disorder of glucose metabolism characterized by both insulin resistance and pancreatic b-cell dysfunction The chronic hyperglycemia is often associated with essential hypertension, obesity, and dyslipidemia in a complex metabolic syndrome leading to severe vascular complications, including blindness, end-stage renal failure, and coronary-heart disease The prevalence of type 2 diabetes in Westernized countries is 310, and epidemiological studies predict it may reach a total of 30
Received June 1, 2000; accepted for publication September 15, 2000; electronically published November 6, 2000 Address for correspondence and reprints: Dr Philippe Froguel, Institut Pasteur de Lille, 1 rue du Pr Calmette, 59000 Lille, France Email: froguel@mail-goodpasteur-lillefr Present affiliation: INSERM U525, Paris
2000 by The American Society of Human Genetics All rights reserved 0002-9297/2000/6706-00130200

in the next decade, which makes it a major health problem worldwide The familial clustering of type 2 diabetes, together with the higher concordance rates in MZ twins compared with DZ twins
Barnett et al 1981; Hopper et al 1999 and the close correspondence in hybrid populations, between genetic-admixture rates and disease prevalence Knowler et al 1988, suggest a genetic predisposition to type 2 diabetes, with a recurrence risk to first-degree relatives of 35 Rich 1990 In 10 of the cases, type 2 diabetes appears to segregate in a Mendelian fashion However, in the majority of cases, the pattern of inheritance of the disease suggests a complex genetic interaction with environmental factors Considerable effort has been made worldwide during the last decade to identify the underlying genetic determinants Studies of monogenic forms of type 2 diabetes led to the identification of several diabetogenes with major effects in rare families, including the insulin, insulin-receptor, and glucokinase genes and the four tran-

1470

Vionnet et al: Genomewide Search for Diabetes in French Sib Pairs

1471 type 2 diabetes and at least three individuals parents or additional sibs available for the study to assist the reconstruction of parental genotypes and to avoid nonfull sibs Families presenting bilineal inheritance of type 2 diabetes or families with subjects diagnosed at age 25
years ie, potentially having the Maturity-Onset Diabetes of the Young [MODY] phenotype or at age 165 years only potential phenocopy caused by environmental factors rather than genetic ones or families with mixture of type 1 and type 2 diabetes potential Latent Autoimmune Diabetes of Adults [LADA] were discarded However, in one family, one individual was diagnosed at age 23 years and had positive anti-GAD antibodies Nonwhite families or families with mutations in a known diabetes-susceptibility gene were also excluded from the study Prior to genotyping of all the markers for the genome scan, 18 markers for the X chromosome panel 28 were analyzed to check for DNA inconsistencies and Mendelian inheritance From a preliminary selection of 145 families, 10 were discarded and were replaced by 12 additional families checked in the same manner After the genome scan was completed, four families had to be discarded from the analyses because of recurrent Mendelian incompatibilities All analysis results presented in this report were thus obtained in 143 families, corresponding to 148 nuclear families Phenotypes were determined in the light of the clinical report and the results of the latest
measurements for fasting and/or oral glucose-tolerance test according to the recently established American Diabetes Association criteria for type 2 diabetes Expert Committee on the Diagnosis and Classification of Diabetes Mellitus 1997: diabetic DM when receiving oral hypoglycemic agents or insulin 1 year after the diagnosis, or when fasting glycemia was 7 mmol/l or glycemia 2 h after an oral glucose load was 111 mmol/l; glucose intolerant GI when fasting glycemia was 6170 mmol/l or when 2-h glycemia was 78111 mmol/l; normoglycemic NG when fasting glycemia was 610 mmol/l and 2h glycemia was 78 mmol/l; unknown UK when no data were available or type 1 diabetes was present Accordingly, the 637 individuals included in the study comprised 432 DM, 72 GI, 129 NG, and 4 UK subjects tables 1 and 2 We designed six qualitative traits, taking into account the diabetic status, the age at diagnosis, and the body-mass index BMI of each subject in an attempt to stratify families into clinically more-homogeneous groups table 3A A maximum number of 677 affected sib pairs were analyzed with the affection status large, and details of the family structure are given in table 3B for all six subgroups
Both parents were available in 6 families, and one parent was available in 30 more families

scription-factor genes encoding HNF1-a, HNF4-a, HNF1-b hepatocyte nuclear factor, and IPF1 insulin promoter factor 1, as well as mutations in the mitochondrial DNA reviewed by Elbein et al [1997] However, these genes do not seem to play a major role in the more common forms of type 2 diabetes Studies of candidate genes in pathways impaired in subjects with type 2 diabetes led to the identification of genes that may increase the risk for type 2 diabetes, including the IRS1 insulin receptor substrate 1, glucagon-receptor, glycogen synthase, sulfonylurea-receptor, and IPF1 genes Elbein et al 1997 In any case, these genes contribute, to a modest or marginal extent, to genetic susceptibility to type 2 diabetes Several groups, therefore, have undertaken genomewide scans to identify loci for type 2 diabetes and related phenotypes So far, genomewide significant linkages Lander and Kruglyak 1995 for such susceptibility loci were identified on several distinct chromosomes in different populations These include loci on chromosomes 2q37 Hanis et al 1996, 15q21 Cox et al 1999, 10q26 Duggirala et al
1999, and 3p Ehm et al 2000 in Mexican Americans; 12q24 Mahtani et al 1996 and 1q21-1q23 Elbein et al 1999 in whites; and 1q211q23 and 11q23-q25 in Pima Indians Hanson et al 1998 Overall, these results underscore the genetic heterogeneity of type 2 diabetes in diverse ethnic groups and the need of performing carefully designed wholegenome scans in other populations, either to identify new diabetes-susceptibility genes and/or to replicate previous findings To search for a major susceptibility locus loci contributing to type 2 diabetes in the French white population, we have completed a genomewide search for diabetes-related traits in 143 families with affected sib pairs Here, we report evidence for a novel susceptibility locus for diabetes on chromosome 3q27-qter and independent confirmation of a locus on chromosome 1q21-q24 Subsequent fine-mapping in those regions has narrowed down the regions of interest to an interval of 7 cM
Subjects Families have been recruited since 1990 by means of a publicly advertised campaign for 200 families to overcome diabetes, and details of the recruitment have been described elsewhere Vionnet et al 1997 Among the 402 type 2diabetes families collected
with at least two affected sibs, 145 have been initially included in the study, according to criteria the aim of which was to reduce heterogeneity of the families and to maximize the power of the study A family was included if there were at least two diabetic subjects in the sibship being treated for diabetes to load the families with a severe form of

1472 Table 1
Clinical Characteristics of All Participants Sex Ratio M:F 40:89 198:234 36:36 Age at Time of Study years SD range 6187 1269 2891 6285 96 3193 6115 941 4187 Age at Diagnosis years SD range 495 1065 2383 5906 956 4182

Am J Hum Genet 67:14701480, 2000

Participant Status Nondiabetic n p 129 Diabetic n p 432 Glucose intolerant n p 72

Duration of Disease years SD range 1341 98 063 208 449 024

BMI kg/m2 SD range 251 41 14983671 2789 448 15114591 2737 470 19234177

Methods Genotyping Initial 10-cM genomewide screen–Genomic DNA was isolated from leukocytes using the Puregene DNA Isolation Kit, according to the suppliers protocol Gentra Systems Genotyping was performed using a fluorescently labeled human linkage-mapping set PELMSV2 comprising 400 highly informative microsatellite markers, with an average intermarker
spacing of 97 cM Multiplex PCR conditions were set up for each of the 28 panels in such a manner as to amplify the 400 markers in 87 PCRs PCR 95 C for 12 min, followed by 30 cycles of 94 C for 15 s, 55 C for 15 s, 72 C for 30 s, and 72 C for 10 min was performed on GeneAmp PCR system 9700 Perkin-Elmer: 10-ml reactions; 40 ng of genomic DNA, 25 mM MgCl2, 025 mM each dNTP Pharmacia, a variable amount 0215 pmol of 5 and 3 primers, and 04 U AmpliTaq Gold DNA polymerase Perkin Elmer in 1 PCR Buffer II Perkin Elmer Multiplex PCR conditions are available from the authors on request Pooled amplification products were electrophoresed through 5 polyacrylamide gels Long Ranger Singel pack [Perkin Elmer] for 15 h at 2,000 V on 24-cm plates on an ABI 377 DNA sequencer An automated 96-channel pipettor Multimek 96 Beckman was used for all the pipetting steps Semiautomated fragment sizing was performed by use of GENESCAN 30 software ABI, followed by allele calling with GENOTYPER 21 software ABI Each genotype was reviewed independently by two members of the research team to confirm the accuracy of allele calling Marker D14S68 was replaced by D14S67 because of reading difficulties All markers but
D12S310, the amplification of which failed, were included in the analysis Two additional markers D5S429 and D8S279 were added to the 399, to fill in gaps 120 cM The overall data dropout rate for the 255,437 genotypes 401637 analyzed was 4 The average heterozygosity for the 401 markers used in the first-stage genome search was 079 Incompatibilities were searched for with the PED-CHECK 11 program OConnell and Weeks 1998 and inconsistencies resolved Fine mapping genotyping–After the first-stage ge-

nome search, we observed that linkage was primarily supported with markers on 3q27-qter and 1q21-q24 across most methods and phenotypes We then saturated these regions with 16 additional genetic markers in each region, to confirm and further localize the linkage results Each of the 32 markers was amplified individually by PCR, and genotyping was performed similarly to the genomewide scan In addition, PCR and genotyping were performed a second time for the markers used in the genome scan and were localized in 3q27-qter and 1q21-q24 loci seven and five markers, respectively The overall data dropout rate for the 28,028 genotypes–16 2 additional 12 genome-scan markers 637
individuals–analyzed in this second step was of 05, thus ensuring maximal informativity for multipoint analyses The information content for markers used in the multipoint analyzes exceeded 085 for both regions Data Statistical Analysis Nonparametric two-point and multipoint analyzes were performed with the programs MAPMAKER-SIBS 20 MLS; Kruglyak et al 1995, by using the unweighted option, and MLBGH 10 Abel and MullerMyhsok 1998, for autosomal markers The maximumlikelihood binomial MLB method, which is based on the binomial distribution of parental marker alleles among affected offspring, overcomes the common problem of multiple sibs by considering the sibship as a whole Two-point and multipoint analyzes of the chromosome X markers were performed using ASPEX 1998 David A Hinds and Neil Risch Allele frequencies for the parameter file were generated from the data with DOWNFREQ 11 Terwilliger et al 1995 Marker map positions for the genomewide scan and the high-resolution map on chromosome 3q27-qter were obtained from the sex-averaged maps compiled by Ge nethon and are given in Haldane centimorgans cM The high-resolution map order, from the Genethon map, of all markers tested at
3q27-qter was consistent with the Marshfield map order and with the results of mapping in the Genebridge 3 Radiation Hybrid Panel Research Genetics data not shown Of the 16 additional markers of chromosome 1q21-q24, 5 belonged to the Marshfield sex-averaged linkage map For the high-resolution map on chromosome 1q21-q24, we used the

Vionnet et al: Genomewide Search for Diabetes in French Sib Pairs

1473 single-point analyzes data not shown There was a cluster of linked markers on chromosomes 3, 1, and 2 The positive chromosome 3 markers P p 01 spanned 138 cM on the terminal q arm and gave positive results for two of six diabetes phenotypes with both statistics Genomewide significance was observed at the D3S1580 locus when the MLS statistic was used MLS p 391, P p 00002 for the large young-onset phenotype, consistent with suggestive linkage using the MLB statistic LOD p 297, P p 00011 Linkage was still suggestive with both statistical methods when applying a conservative correction factor for the number of traits studied by multiplying the P value by 6 number of phenotypes Linkage was also suggested in this chromosomal region with the strict-young-onset phenotype, with both MLS and
MLB statistics: at D3S1580; MLS p 289, P p 00026, MLB-LOD p 224, P p 00067 table 4A When we restricted the sample to young-onset diabetes status, the sample size was reduced to 92 sib pairs, and we observed unequal sibship sizes However, simulation studies for the chromosome 3 results showed that the empirical P values were very close to the value expected under asymptotic conditions Therefore, we can rely on the P values given by the MLB method The positive chromosome 1 markers spanned 120 cM on the q arm and also gave positive results for two diabetes phenotypes Suggestive linkage was observed with the strict-lean phenotype with both the MLS and MLB statistics, and results remained potentially interesting after Bonferroni correction Positive results for chromosome 2 markers were primarily for the strict phenotype, with suggestion of linkage only with the MLB analyses, and spanned an interval of 26 cM on the p arm Second-Stage Mapping of Chromosomes 3q and 1q From the first-stage scan, the strongest and most consistent evidences for linkage were found for markers from chromosomes 3q27-qter and 1q21-q24, across different analytical methods and different phenotypes We thus added 16
more markers in the 3q27-qter and 1q21q24 regions, for totals of 23 and 21 markers with average map densities of 26 and 19 cM, respectively Figures 1A and 1B show results from the multipoint analyzes for both MLS and MLB statistics of the saturation of 3q with the large young-onset phenotype and 1q with the strict-lean phenotype These analyzes further supported and strengthened the findings from the initial 10-cM scan Evidence of linkage was confirmed in the 3q27-qter region with both the large young-onset and strict young-onset phenotypes D3S1580 ; MLS p 467 and 356 P p 000004 and 000054, respectively MLB-LOD p 343 and 265

Table 2
Medication History of Diabetic Participants Medication History Diet alone Sulfonylureas Sf Biguanides Bg Insulin Sf and/or Bg insulin Sf Bg Glucor or mediator of Diabetic Participants 107 249 140 126 53 309 16

map positions from the Marshfield map, since mapping in the Genebridge 3 Radiation Hybrid Panel was more consistent with the Marshfield map order than with the Genethon map Intermarker distances in our sample were systematically checked with a program derived from VITESSE OConnell and Weeks 1995 When we restrict the sample to young-onset
diabetes status, the sample size is reduced to 92 sib pairs, and we observe unequal sibship sizes It is then legitimate to investigate the reliability of the P values We simulated the transmission of the marker keeping the familial structure and affection status When the founders had typed parents, we used their actual genotype and simulated the allele transmission When they were unknown, their genotypes probability was determined according to the population allele frequency and their offspring genotypes A total of 70,000 replicates were simulated through Monte Carlo method and were analyzed with MLBGH Results First-Stage Genome Scan Table 4 summarizes results of the multipoint analyses of the 401 framework markers from the initial genome scan We considered any region with a Znorm of 23 and a LOD score of 117 P p 01 as potentially interesting, and we applied the criteria of Lander and Kruglyak to further define regions of significant Znorm p 411, LOD p 36, P p 00002 or suggestive Znorm p 319, LOD p 22, P p 0007 linkage Overall, 26 of the 2,298 tests using the MLB statistics for the autosomal chromosomes 383 markers tested for the six phenotypes gave LOD scores 117 P 01,
corresponding to 22 distinct markers that were distributed in 15 distinct genomic intervals table 4A The MLS statistics also gave LOD scores 117 P 01 for those genomic intervals table 4A, as well as for markers in nine additional regions table 4B Of the 108 tests performed using the MLS statistics with ASPEX program for the X chromosome, 2 showed indication of linkage table 4C Most of the multipoint results were supported by

1474 Table 3
Description of Affection Status and Structure of the 148 Nuclear Families 3A DESCRIPTION Affection Status Affected
OF

Am J Hum Genet 67:14701480, 2000

AFFECTION STATUS Large

FOR THE

SIX PHENOTYPIC GROUPSa Large-Lean Strict-Lean NIDDM with BMI 27 NA GI NIDDM with BMI 127 NG Large Young-Onset NIDDM GI with age at onset 46 NA NIDDM GI with age at onset 146 NG Strict Young-Onset NIDDM with age at onset 46 NA GI NIDDM with age at onset 146 NG

Strict NIDDM

NIDDM GI

NIDDM GI with BMI
27

Unknown

NA

NA GI

NA NIDDM GI with BMI 127 NG

Nonaffected 3B NO n 2 3 4 5 6 7 8 Total families Total sib-pairs
OF

NG FAMILIES
WITH N

NG AFFECTED
IN

EACH PHENOTYPIC GROUPb Large-Lean 37 19 5 1 3 Strict-Lean 40 11 5 1 Large Young-Onset 41 11 3 Strict
Young-Onset 45 6 3

Large 43 54 30 12 4 4 1 148 677

Strict 66 54 18 6 1 2 147 453

65 179

57 113

55 92

54 81

a Affection status was determined taking into account patients diabetic status overt diabetes [NIDDM], GI, NG, or data not available [NA], BMI kg/m2, and age at diagnosis of diabetes Three affection statuses were used for the statistical analyses: affected, nonaffected, and unknown coded 2, 1, and 0, respectively b Total number of sib pairs was calculated with the following formulae: number of families with n affected, n p [28] X[nXn 1]/2

P p 00003 and 00024, respectively Moreover, the high-resolution mapping identified an indication of linkage, which was not observed at the 10-cM initial genome scan mapping phase, with an additional phenotype that is the strict phenotype ie, type 2 diabetes regardless of the age at diagnosis 453 sib pairs, MLB-LOD p 128, P p 007 On the other hand, although they did not reach the Lander-Kruglyak statistical thresholds for significance, results in the 1q21-q24 region were highly suggestive with the strict-lean phenotype APOA2D1S484; MLB-LOD p 299, P p 00010; MLS p 304, P p 00018 This saturation step with dense genetic map allowed us to
restrict the 1-LOD-unit CI Ott 1991 from 11 to 62 cM and from 19 to 65 cM for the loci on chromosomes 3 and 1, respectively Discussion The present genome scan in French whites provides 1 strong evidence for a susceptibility locus for diabetes and impaired glucose tolerance with early onset at age 46 years on chromosome 3q27-qter and 2 suggestive evidence that there is a diabetes-susceptibility gene in French whites in a previously reported region on chromosome 1q21-q24 In our genomewide-scan design for type 2 diabetessusceptibility genes, emphasis has been focused on power to maximize the chances for success

We used a large number of sib pairs 1200 for the large and strict phenotypes, and a 10-cM marker grid in a one-stage study yielding a power of 198 for a risk ratio ls of 3 and of 170 for a risk ratio ls of 2 Holmans and Craddock 1997 Because genotyping errors lead to false-positive or false-negative results, automation and stringent quality-control criteria have been applied Families were selected from among our large collection to be as homogeneous as possible, to avoid background noise caused by bilineal inheritance of the trait DeStefano et al 1998, other genetic forms of
type 2 and/or type 1 diabetes such as MODY or LADA, and nongenetic forms caused by environmental factors Only families in which accurate inference of relationships among sib pairs was possible were included, to avoid reduced power to detect linkage caused by misclassification of half-sib pairs or unrelated individuals as full sibs Boehnke and Cox 1997 Two powerful sib pairbased methods were used for analyses: the MLS method, which was previously shown to be most sensitive Davis and Weeks 1997, and the MLB method, which takes into account sibships with several affecteds in a natural way and was shown to perform well in terms of type 1 error and power Abel and Muller-Myhsok 1998; Abel et al 1998 Our best linkage result was found between markers on chromosome 3q27-qter and diabetes and impaired glucose tolerance diagnosed at age 45 years Criteria

Vionnet et al: Genomewide Search for Diabetes in French Sib Pairs

1475

Table 4
Multipoint Results of the 10-cM Genome-Scan Initial Stage 4A RESULTS Intervala 1p21 1q21-q24 1q43 2p25 2p21-p16 2q24-q23 3q27-qter 4p16 4q31 5q31-q33 7q11 9q34 10q26
OF

MLB

AND

MLS MULTIPOINT ANALYSES Position cM 14112 178 178 27623 617 7531 17922 21797 21797
125 18599 15943 9274 16001 17465 18523 18523 1524 3692
c

FOR

SIX DIABETES-RELATED PHENOTYPES Phenotype MLB-LOD P 127 247 123 139 161 228 125 297 224 134 187 152 132 130 159 122 144 134 120 00777 00037 00877 00575 00324 0006 00826 00011 00067 00643 00166 00412 00682 00724 00342 00899 00504 00648 00948 MLS P 148 250 112 164 202 227 122 391 289 135 209 167 168 122 124 169 145 125 126 00782 00066 01911 00533 00212 00116 01507 00002 00026 01087 00175 00492 00479 01494 01427 00472 00841 01371 01358

Positive Markersb D1S206 D1S484, D1S498 D1S484 D1S2785 D2S319 D2S2259, D2S391 D2S2330 D3S1580, D3S1262, D3S1601 D3S1580, D3S1601 D4S2935, D4S412 D4S1539 D5S410, D5S436 D7S669 D9S158 D10S1655 D10S212 D10S212 D11S1338 D19S221
WITH

11p15 19p13

Strict-lean Strict-lean Strict Large young-onset Strict young-onset Strict Large-lean Large young-onset Strict young-onset Large Large-lean Large Large-lean Strict-lean Strict Large Large-lean Strict young-onset Large young-onset

4B ADDITIONAL LOCI Interval 2p25 2p23
a

MLS SCORE 1117
b

Positive Markers D2S168 D2S305 D2S305 D2S305 D8S270 D12S345 D15S1007 D16S3068 D18S478 D20S186 D20S186, D20S115 D20S178
OF

Positionc cM 293 4164 4164 4164 10472 5556
2678 146 5471 3393 3393 6695
FOR

Phenotype Strict Large Large young-onset Strict young-onset Large Large young-onset Strict young-onset Strict young-onset Large young-onset Large young-onset Strict young-onset Large

MLB-LOD P 82 116 62 80 97 84 74 109 109 114 108 05 02621 0103 04537 02707 01723 02447 03211 01253 0126 01093 01304 06295

MLS P 190 198 160 170 144 141 150 139 175 186 168 172 00278 0023 00581 00459 00865 00925 00753 00971 00401 00312 00475 00432

8q22 12p11 15q15 16p11 18q12 20p13-p12 20q13 4C RESULTS Interval
a

ASPEX MULTIPOINT ANALYSES Positive Markers
b

SIX PHENOTYPES Phenotype Large Strict MLB-LOD P MLS P 167 00491 156 00643

Positionc cM 8934 8934

Xp11-p21

DXS991 DXS991

a Chromosomes locations were determined from the map available in the Genome Database and the CEDAR database b When several markers per line are shown, the first one is the nearest to the top of the peak c Map positions of the nearest marker to the top of the peak, obtained from sex-averaged maps compiled by Genethon

for assessment of statistical significance in genetic-linkage studies of complex traits have been controversial Specific critical values have been proposed; some investigators
have suggested that a relatively stringent threshold of Z 1 36 is needed to obtain a genomewide P 05 Lander and Kruglyak 1995, whereas others have maintained that the less stringent traditional criterion of Z 1 30 is unlikely to be a false positive Holmans et al 1997; Morton 1998; Rao 1998 In addition,

when several correlated traits have been analyzed, it is unclear how to adjust for multiple comparisons The present analysis provides strong evidence for linkage on chromosome 3 with diabetes and impaired glucose tolerance diagnosed at age 45 years Z p 467 and for diabetes diagnosed at age 45 years Z p 356 Both two-point and multipoint MLS and MLB statistics gave consistent results that were still significant after Bonferroni correction for the number of phenotypes ana-

Results of the MLB and MLS multipoint analyses for the high-resolution genetic mapping at chromosome 3q27-qter with the large young-onset phenotype A and at chromosome 1q21-q24 with the strict-lean phenotype B The thresholds for putative LOD 117; P p 01, suggestive LOD 22; P p 0007, and significant linkage LOD 36; P p 00002 are indicated by horizontal lines Marker names followed by b were also used in the first
stage 10-cM genomewide screen and were fully retyped during the high-resolution mapping step see Methods section The horizontal axis corresponds to the partial genetic map of chromosomes 3q27-qter and 1q21-q24 intervals studied in the saturation step with high-density genetic maps The vertical axis indicates the logP obtained with multipoint analyses at each point in the studied interval

Figure 1

Vionnet et al: Genomewide Search for Diabetes in French Sib Pairs

1477

lyzed In addition, the width of the peak on chromosome 3q27-qter, given by all positions providing a P value of 01 in the multipoint analysis, was broad 120 cM, which is an argument in favor of this peak being a true positive rather than a false positive Terwilliger et al 1997 We found linkage on chromosome 3 through stratification of families in clinically more-homogeneous subgroups, which is known to help in identification of susceptibility loci for a complex disease Rao 1998 The result on chromosome 3q has been observed in a subgroup of 55 non-MODY families 37 of total families with glucose intolerance or diabetes diagnosed between age 25 and 45 years, although both parents were not affected Therefore, this group
probably represents the most genetic form of nonMODY type 2 diabetes Results of simulation studies showed that the nominal P values obtained in this reduced sample were reliable On the basis of a single sample, it is difficult to unambiguously distinguish true from false linkage results However, linkage of diabetes to the chromosome 3q27-qter region is supported by 1 the consistency of results across phenotypes and analytical methods, 2 the extremely low P values, and 3 the results of our simulations Additional support comes from replication of results either in the same population or in other populations We could not perform a replication study in our population, because the huge size of the replication sample recommended by simulation studies Risch and Merikangas 1996 was incompatible with the small number of similarly ascertained families remaining in our French collection Our region of positive linkage on chromosome 3q27-qter spanned 120 cM in the first-stage genome scan and was refined to 7 cM after the fine-mapping step was conducted This region includes several reports of positive linkage with metabolic traits; we agree with Neel and colleagues 1998 that type 2 diabetes,
essential hypertension, and obesity [are] syndromes of impaired genetic homeostasis Hegele et al 1999 found an indication of linkage between D3S2418 and type 2 diabetes in a 20-cM genomewide scan in a Canadian Oji-Cree population P p 01 They did not find evidence of linkage disequilibrium between D3S2418 and diabetes; neither did we with D3S1580 in the 55 linked families using the Monte Carlobased x2 implemented in CLUMP Sham and Curtis 1995; data not shown QTLs for impaired acute insulin response, insulin resistance in nondiabetics Pratley et al 1998 and in diabetics Hanson et al 1998, and waist:hip ratio a measure for obesity Norman et al 1998 were also suggested in this chromosomal region, in the sole Pima Indian population Interestingly, a QTL for small low-density lipoprotein particle phenotype, known to be a risk factor for cardiovascular disease, has been recently mapped in the same region on chromosome 3q27-qter Rainwater et

al 1999 Several studies have reported a polymorphism in the apolipoprotein D APOD locus, in the region of the maximal peak of our linkage at this chromosomal region, to be associated with diabetes and obesity Hitman et al 1992; Vijayaraghavan et al
1994 Although none of those previously reported linkages on chromosome 3 reached statistical significance, as did the linkages in our study, they do strengthen our results, and it is tempting to speculate that the same locus is responsible for these results Saturation of the chromosome 3q27-qter region in our families reduced the 1-LOD-unit confidence interval CI to 62 cM around D3S1580, which, according to the Unified Database for Human Genome Mapping, spans 45 mb, an interval of suitable size to initiate positional cloning and disequilibrium mapping efforts This chromosomal region contains several potentially interesting genes, including the genes coding for somatostatin, apolipoprotein D, and inhibitor 2 of the synthase-activating enzyme, type 1 protein phosphatase, I-2 PPP1R2, and the bifunctional enzyme with enoylCoA-hydratase and 3-hydroxyacyl-CoA dehydrogenase activity MIM 182450, MIM 107740, MIM 601792, and MIM 261515, respectively We also found evidence of linkage between markers on chromosome 1q21-q24 and diabetes in lean patients with BMI 27 Although not reaching the stringent statistical thresholds for significance, results on chromosome 1q were highly suggestive P p
00010, and, again, the broad width of the peak in the initial genome scan 120 cM also suggested true-positive results Linkage on chromosome 1q was also found through stratification of families, in a homogeneous subgroup of 57 families 38 of families in which diabetes is enriched in lean patients Since obesity is frequently associated with diabetes and is genetically determined as well, it could be a confounding variable in genetic studies of diabetes Therefore, genetic studies of lean diabetic patients are more likely to identify true diabetes-susceptibility loci, rather than obesity-susceptibility loci Our results on chromosome 1q confirm and replicate previous reports of evidence for linkage between diabetes and markers in the same region in at least two different populations Elbein et al 1999 reported evidence of linkage between diabetes and markers CRP/APOA2 in 19 multigenerational families of northern European ancestry from Utah using a recessive model for parametric analyzes In a study of the Pima Indians, among whom diabetes and obesity are highly prevalent, Hanson et al 1998 found evidence of a diabetes locus on chromosome 1q, a region that was not linked to obesity in
their study They observed a very strong evidence for linkage with diabetes Z p 41 at D1S2127, in the 55 sib pairs who had onset of diabetes at age 25 years, supported by comparisons of sib pairs concordant for diabetes

1478

Am J Hum Genet 67:14701480, 2000

versus discordant sib pairs at D1S1677 The finding of linkage in three different populations strengthens the evidence for a diabetes-susceptibility locus in this region Saturation of the chromosome 1q21-q24 region in our families reduced the 1-LOD-unit CI to 65 cM around APOA2/CRP, which, according to the Unified Database for Human Genome Mapping, corresponds to 6 mb Indeed, positional cloning and disequilibrium-mapping efforts have already been initiated in diabetic Pima Indians to identify the chromosome 1q locus, and several of the potential candidate genes have been screened Baier et al 1998; Wolford et al 1999a These identified a linkage disequilibrium between diabetes and single-nucleotide polymorphisms near the KCNJ9 gene coding for the G-protein-coupled inward rectifier K channel homolog GIRK3, suggesting that the diabetes-susceptibility gene on chromosome 1q21q24 is closely linked to the KCNJ9 locus Wolford et al
1999b Linkage disequilibrium between diabetes and chromosome 1 markers in the same region was also found in the Old Order Amish St Jean 1999a, 1999b The possible role for those genes in the French families that contributed to our linkage results is under investigation We identified 22 other regions showing at least nominal indication of linkage P 01 When looking very closely at the published data, we found similar results in other studies in most of our regions Of particular interest is our linkage result on chromosome 10q26 that largely overlaps the region of linkage with diabetes and age at onset of diabetes identified in Mexican Americans Duggirala et al 1999 We were therefore able to replicate their findings independently in our population Our suggestive linkage results on chromosome 2p21p16 with the diabetic phenotype are supported as well by several reports for metabolic traits in that region, mostly obesity-related phenotypes, including QTLs for leptin levels, fat mass, or BMI in different populations Comuzzie et al 1997; Hager et al 1998; Rotimi et al 1999 It is therefore possible that a genetic defect localized on chromosome 2p determines the occurrence of both obesity
and type 2 diabetes It has already been reported that some loci, such as the one on chromosome 11q23-q25, harbor diabetes and obesity genes Hanson et al 1998 As far as chromosome 20 is concerned, we were the first to report positive linkage on chromosome 20q in another selection of French diabetic families Hani et al 1996; Zouali et al 1997 Afterward, several studies confirmed our findings Bowden et al 1997; Ji et al 1997; Ghosh et al 1999, including results of a metaanalysis of all data available worldwide for this chromosome Boehnke et al 1998 Obesity-related traits have also been mapped on this chromosome Lembertas

et al 1997; Norman et al 1998; Lee et al 1999 From our studies and others, it seems possible that there are two diabetes- and/or obesity-susceptibility genes on chromosomes 20p and 20q Further fine mapping and sequencing of selected candidates HNF3-b, MC3R, CEBP-b, and Nkx22 are under way to elucidate the role of chromosome 20 genes in the French diabetics Because emphasis has been placed on power in our genomewide-scan design for type 2 diabetessusceptibility genes, in this study, we were able to exclude linkage at the diabetes loci on chromosomes 2q37, 15q21, and
12q24–identified in Mexican-American and Finnish subjects, respectively–as well as at the diabetes-and-obesity locus on chromosome 11q23-q25 that was identified in Pima Indians or the diabetes locus recently mapped on chromosome 3p in Mexican Americans It is therefore very likely that genes that play an important role in isolated or recently admixed populations have a marginal role, if any, in a representative admixed population such as these French whites In conclusion, we report the first evidence for a novel diabetes-susceptibility locus gene on chromosome 3q27qter Several results of positive, although not significant, linkage at this locus that were previously reported in the literature support our findings In addition, we independently confirm linkage with diabetes in the 1q21q24 interval Hanson et al 1998; Elbein et al 1999 Other loci on chromosomes 2p21-p16, 10q26, and 20 are potentially interesting but await further studies to be confirmed

Acknowledgments
We are indebted to all families who participated to this study We thank Dr Teng for technical assistance We thank Drs Graeme I Bell, Jacqui Beckman, and Florence Demenais for useful comments on the manuscript We thank
Valerie Delan noy for her help with radiation-hybrid mapping This research was supported in part by Eli Lilly and by Region Nord-Pas de Calais to SD

Electronic-Database Information
Accession numbers and URLs for data in this article are as follows: CEDAR, http://cedargeneticssotonacuk/pub for chromosome locations Genethon, ftp://ftpgenethonfr/pub/Gmap/Nature-1995 for marker maps Genome Database, http://wwwgdborg for chromosome locations Marshfield, http://researchmarshfieldclinicorg/genetics/ for marker map Online Mendelian Inheritance in Man OMIM, http://www ncbinlmnihgov/Omim for type 2 diabetes [MIM 125853], somatostatin [MIM 182450], apolipoprotein D

Vionnet et al: Genomewide Search for Diabetes in French Sib Pairs

1479
Nguyen Q, Reisman M, Lai EH, Joslyn G, Shepherd NS, Bell C, Wagner MJ, Burns DK 2000 Genomewide search for type 2 diabetes susceptibility genes in four Americans populations Am J Hum Genet 66:18711881 Elbein SC 1997 The genetics of human noninsulin-dependent type 2 diabetes mellitus J Nutr 127:1891S1896S Elbein SC, Hoffman MD, Teng K, Leppert MF, Hasstedt SJ 1999 A genome-wide search for type 2 diabetes susceptibility genes in Utah Caucasians Diabetes
48:11751182 Expert Committee on the Diagnosis and Classification of Diabetes Mellitus, The 1997 Report of the expert committee on the diagnosis and classification of diabetes mellitus Diabetes Care 20:11831197 Ghosh S, Watanabe RM, Hauser ER, Valle T, Magnuson VL, Erdos MR, Langefeld CD, et al 1999 Type 2 diabetes: evidence for linkage on chromosome 20 in 716 Finnish affected sib pairs Proc Natl Acad Sci USA 96:21982203 Hager J, Dina C, Francke S, Dubois S, Houari M, Vatin V, Vaillant E, Lorentz N, Basdevant A, Clement K, Guy-Grand B, Froguel P 1998 A genome-wide scan for human obesity genes reveals a major susceptibility locus on chromosome 10 Nat Genet 20:304308 Hani EH, Zouali H, Philippi A, Beaudoin JC, Vionnet N, Passa P, Demenais F, Froguel P 1996 Indication for genetic linkage of the phosphoenolpyruvate carboxykinase PCK1 gene region on chromosome 20q to non insulin dependent diabetes mellitus Diabete Metab 22:451454 Hanis CL, Boerwinkle E, Chakraborty R, Ellsworth DL, Concannon P, Stirling B, Morrison VA, et al 1996 A genomewide search for human non-insulin-dependent type 2 diabetes genes reveals a major susceptibility locus on chromosome 2 Nat Genet 13:161166 Hanson RL,
Ehm MG, Pettitt DJ, Prochazka M, Thompson DB, Timberlake D, Foroud T, Kobes S, Baier L, Burns DK, Almasy L, Blangero J, Garvey WT, Bennett PH, Knowler WC 1998 An autosomal genomic scan for loci linked to type II diabetes mellitus and body-mass index in Pima Indians Am J Hum Genet 63:11301138 Hegele RA, Sun F, Harris SB, Anderson C, Hanley AJ, Zinman B 1999 Genome-wide scanning for type 2 diabetes susceptibility in Canadian Oji-Cree, using 190 microsatellite markers J Hum Genet 44:1014 Hitman GA, McCarthy MI, Mohan V, Viswanathan M 1992 The genetics of non-insulin-dependent diabetes mellitus in South India: an overview Ann Med 24:491497 Holmans P, Craddock N 1997 Efficient strategies for genome scanning using maximum-likelihood affected-sib-pair analysis Am J Hum Genet 60:657666 Hopper JL 1999 Is type II non-insulin-dependent diabetes mellitus not so genetic after all? Diabetologia 42:125127 Ji L, Malecki M, Warram JH, Yang Y, Rich SS, Krowlewski AS 1997 New susceptibility locus for NIDDM is localized to human chromosome 20q Diabetes 46: 876881 Knowler WC, Williams RC, Pettitt DJ, Steinberg AG 1988 Gm3,5,13,14 and type 2 diabetes mellitus: an association in American Indians with
genetic admixture Am J Hum Genet 43:520526 Kruglyak L, Lander ES 1995 Complete multipoint sib-pair analysis of qualitative and quantitative traits Am J Hum Genet 57:439454

[MIM 107740], type 1 protein phosphatase I-2 [PPP1R2; MIM 601792], bifunctional enzyme with enoyl-CoA-hydratase and 3-hydroxyacyl-CoA dehydrogenase activity [MIM 261515] Unified Database for Human Genome Mapping, http:// bioinformaticsweizmannacil/udb/

References
Abel L, Alcais A, Mallet A 1998a Comparison of four sibpair linkage methods for analyzing sibships with more than two affecteds: interest of the binomial maximum likelihood approach Genet Epidemiol 15:371390 Abel L, Muller-Myhsok B 1998b Robustness and power of the maximum-likelihood-binomial and maximum-likelihood-score methods, in multipoint linkage analysis of affected-sibship data Am J Hum Genet 63:638647 Baier L, Wiedrich C, Dobberfuhl A, Traurig M, Thuillez P, Bogardus C, Hanson R 1998 Sequence analysis of candidate genes in a region of chromosome 1 linked to type 2 diabetes Diabetes 47:A171 Barnett AH, Eff C, Leslie RDG, Pyke DA 1981 Diabetes in identical twins: a study of 200 pairs Diabetologia 20:8793 Boehnke M, Cox NJ 1997 Accurate inference
of relationships in sib-pair linkage studies Am J Hum Genet 61:423429 Boehnke M, Cox NJ, International Type 2 Diabetes Linkage Analysis Consortium 1998 Lessons learned in a combined linkage analysis 24 datasets, 12000 families of type 2 diabetes on chromosome 20 Paper presented at the 48th annual meeting of American Society of Human Genetics, Denver, October 2731 Bowden DW, Sale M, Howard TD, Qadri A, Spray BJ, Rothschild CB, Akots G, Rich SS, Freedman BI 1997 Linkage of genetic markers on human chromosomes 20 and 12 to NIDDM in Caucasian sib-pairs with a history of diabetic nephropathy Diabetes 46:882886 Comuzzie AG, Hixson JE, Almasy L, Mitchell BD, Mahaney MC, Dyer TD, Stern MP, MacCluer JW, Blangero J 1997 A major quantitative trait locus determining serum leptin levels and fat mass is located on human chromosome 2 Nat Genet 15:273276 Cox NJ, Frigge M, Nicolae DL, Concannon P, Hanis CL, Bell GI, Kong A 1999 Loci on chromosomes 2 NIDDM1 and 15 interact to increase susceptibility to diabetes in Mexican Americans Nat Genet 21:213215 Davis S, Weeks DE 1997 Comparison of nonparametric statistics for detection of linkage in nuclear families: singlemarker evaluation Am J Hum Genet
61:14311444 DeStefano AL, Nicolaou M, Cupples LA 1998 Effect of bilineal inheritance on the power of affected sib-pair linkage analysis Paper presented at the Seventh Annual Meeting of the International Genetic Epidemiology Society, Arcachon, France, September 810 Duggirala R, Blangero J, Almasy L, Dyer TD, Williams KL, Leach RJ, OConnell P, Stern MP 1999 Linkage of type 2 diabetes mellitus and of age at onset to a genetic location on chromosome 10q in Mexican Americans Am J Hum Genet 64:11271140 Ehm MG, Karnoub MC, Sakul H, Gottschalk K, Holt DC, Weber JL, Vaske D, Briley D, Briley L, Kopf J, McMillen P,

1480
Lander ES, Kruglyak L 1995 Genetic dissection of complex traits: guidelines for interpreting and reporting linkage results Nat Genet 11:241247 Lee JH, Reed DR, Li WD, Xu W, Joo EJ, Kilker RL, Nanthakumar E, North M, Sakul H, Bell C, Price RA 1999 Genome scan for human obesity and linkage to markers in 20q13 Am J Hum Genet 64:196209 Lembertas AV, Perusse L, Chagnon YC, Fisler JS, Warden CH, Purcell-Huynh DA, Dionne FT, Gagnon J, Nadeau A, Lusis AJ, Bouchard C 1997 Identification of an obesity quantitative trait locus on chromosome 2 and evidence of linkage to body fat and
insulin on the human homologous region 20q J Clin Invest 100:12401248 Mahtani MM, Widen E, Lehto M, Thomas J, McCarthy M, Brayer J, Bryant B, Chan G, Daly M, Forsblom C, Kanninen T, Kirby A, Kruglyak L, Munnelly K, Parkkonen M, ReeveDaly MP, Weaver A, Brettin T, Duyk G, Lander ES, Groop LC 1996 Mapping of a gene for type 2 diabetes associated with an insulin secretion defect by a genome scan in Finnish families Nat Genet 14:9094 Morton NE 1998 Significance levels in complex inheritance Am J Hum Genet 62:690697 Neel JV, Weder AB, Julius S 1998 Type II diabetes, essential hypertension, and obesity as syndromes of impaired genetic homeostasis: the thrifty genotype hypothesis enters the 21st century Perspect Biol Med 42:4474 Norman RA, Tataranni PA, Pratley R, Thompson DB, Hanson RL, Prochazka M, Baier L, Ehm MG, Sakul H, Foroud T, Garvey WT, Burns D, Knowler WC, Bennett PH, Bogardus C, Ravussin E 1998 Autosomal genomic scan for loci linked to obesity and energy metabolism in Pima Indians Am J Hum Genet 62:659668 OConnell JR, Weeks DE 1995 The VITESSE algorithm for rapid exact multilocus linkage analysis via genotype set-recoding and fuzzy inheritance Nat Genet 11:402408 —— 1998
PedCheck: a program for identification of genotype incompatibilities in linkage analysis Am J Hum Genet 63:259266 Ott J 1991 Analysis of human genetic linkage Revised ed Johns Hopkins University Press, Baltimore Pratley RE, Thompson DB, Prochazka M, Baier L, Mott D, Ravussin E, Sakul H, Ehm MG, Burns DK, Foroud T, Garvey WT, Hanson RL, Knowler WC, Bennett PH, Bogardus C 1998 An autosomal genomic scan for loci linked to prediabetic phenotypes in Pima Indians J Clin Invest 101: 17571764 Rao DC 1998 CAT scans, PET scans and genomic scans Genet Epidemiol 15:118 Rainwater DL, Almasy L, Blangero J, Cole SA, Vandeberg JL, MacCluer JW, Hixson JE 1999 A genome search identifies major quantitative trait loci on human chromosomes 3 and 4 that influence cholesterol concentrations in small LDL particles Arterioscler Thromb Vasc Biol 19:777783

Am J Hum Genet 67:14701480, 2000

Rich SS 1990 Mapping genes in diabetes: genetic epidemiological perspective Diabetes 39:13151319 Risch N, Merikangas K 1996 The future of genetic studies of complex human diseases Science 273:15161517 Rotimi CN, Comuzzie AG, Lowe WL, Luke A, Blangero J, Cooper RS 1999 The quantitative trait locus on chromosome 2 for serum
leptin levels is confirmed in AfricanAmericans Diabetes 48:643644 Sham PC, Curtis D 1995 Monte Carlo tests for associations between disease and alleles at highly polymorphic loci Ann Hum Genet 59:97105 St Jean PL, Hsueh WC, Mitchell B, Pollin T, Burns D, Bell C, Wagner M, Shuldiner AR 1999a Linkage disequilibrium between chromosome 1 markers and type 2 diabetes in the Old Order Amish Paper presented at the Second Research Symposium on the Genetics of Diabetes, San Jose, October 1719 St Jean PL, Mitchell BD, Hsueh WC, Burns DK, Ehm MG, Wagner MJ, Bell C, Aburomia R, Nanthakumar E, Shuldiner AR 1999b Type 2 diabetes loci in the old order Amish Diabetes 48:A46 Terwilliger JD 1995 A powerful likelihood method for the analysis of linkage disequilibrium between trait loci and one or more polymorphic marker loci Am J Hum Genet 56: 777787 Terwilliger JD, Shannon WD, Lathrop MG, Nolan JP, Goldin LR, Chase GA, Weeks DE 1997 True and false positive peaks in genomewide scans: applications of length-biased sampling to linkage mapping Am J Hum Genet 61:430 438 Vijayaraghavan S, Hitman GA, Kopelman PG 1994 Apolipoprotein-D polymorphism: a genetic marker for obesity and hyperinsulinemia J Clin
Endocrinol Metab 79:568570 Vionnet N, Hani EH, Lesage S, Philippi A, Hager J, Varret M, Stoffel M, Tanizawa Y, Chiu KC, Glaser B, Permutt MA, Passa P, Demenais F, Froguel P 1997 Genetics of non insulin dependent diabetes mellitus NIDDM in France: studies with 19 candidate genes in affected sib-pairs Diabetes 46:10621068 Wolford JK, Bogardus C, Hanson R, Prochazka M 1999a Molecular analysis of a type 2 diabetes-linked region on chromosome 1q21-q23 Diabetes 48:A47 Wolford JK, Bogardus C, Prochazka M 1999b Linkage disequilibrium mapping of a region on 1q21-23 linked with type 2 diabetes mellitus in Pima Indians Paper presented at Second ADA Research Symposium on the Genetics of Diabetes, San Jose, October 1719 Zouali H, Hani EH, Philippi A, Vionnet N, Beckmann J, Demenais F, Froguel P 1997 A susceptibility locus for earlyonset non-insulin-dependent type 2 diabetes mellitus maps to chromosome 20q, proximal to the phosphoenolpyruvate carboxykinase gene Hum Mol Gen 6:14011408

Source:kazzoom.com