Genome-Wide and Fine-Mapping Linkage Studies of Type 2 Diabetes and Glucose Traits in the Old Order Amish Evidence for a New Diabetes Locus on Chromosome 14q11 and Confirmation of a Locus on Chromosome 1q21-q24 Wen-Chi Hsueh, 1 Pamela L. St. Jean, 2 Braxton D. Mitchell, 3 Toni I. Pollin, 3 William C. Knowler, 4 Margaret G. Ehm, 2 Callum J. Bell, 5 Hakan Sakul, 5 Michael J. Wagner, 2 Daniel K. Burns, 2 and Alan R. Shuldiner 3,6 We conducted a genome scan using a 10-cM map to search for genes linked to type 2 diabetes in 691 indi- viduals from a founder population, the Old Order Amish. We then saturated two regions on chromosomes 1 and 14 showing promising linkage signals with additional markers to produce a �2-cM map for fine mapping. Analyses of both discrete traits (type 2 diabetes and the composite trait of type 2 diabetes and/or impaired glucose homeostasis [IGH]), and quantitative traits (glucose levels during a 75-g oral glucose challenge, designated glucose 0 –180 and HbA1c) were performed. We obtained significant evidence for linkage to type 2 diabetes in a novel region on chromosome 14q11 (loga- rithm of odds [LOD] for diabetes � 3.48, P � 0.00005). Furthermore, we observed evidence for the existence of a diabetes-related locus on chromosome 1q21-q24 (LOD for type 2 diabetes/IGH � 2.35, P � 0.0008), a region shown to be linked to diabetes in several other studies. Suggestive evidence for linkage to glucose traits was observed on three other regions: 14q11-q13 (telomeric to that above with LOD � 1.82–1.85 for glucose 150 and 180), 1p31 (LOD � 1.28 –2.30 for type 2 diabetes and glucose 120 –180), and 18p (LOD � 3.07, P � 0.000085 for HbA1c and LOD � 1.50 for glucose 0). In conclusion, our findings provide evidence that type 2 diabetes sus- ceptibility genes reside on chromosomes 1, 14, and 18. Diabetes 52:550 –557, 2003 T ype 2 diabetes is a classic example of a complex disease; environmental variation, genetic influ- ences, and interactions among these factors all contribute to the risk of developing the disease (1– 4). Investigations targeting specific candidate genes have yielded relatively little insight into susceptibility genes for the common form of type 2 diabetes (5). More recently, researchers have turned to genome-wide ap- proaches for identifying genes linked to type 2 diabetes and serum glucose levels (4,6 –23). Promising linkage signals have been observed in several chromosomal re- gions in different study populations, with some of them overlapping (24). To date, only one susceptibility gene (calpain-10) has been cloned through genome-wide ap- proaches (25). The Old Order Amish of Lancaster County, Pennsylva- nia, are a genetically well-defined Caucasian founder pop- ulation who live in a relatively homogeneous environment and often have large sibships, thus potentially enhancing our ability to detect linkages to type 2 diabetes and related traits (26). The prevalence of type 2 diabetes in the Amish is �5%, and the phenotypic characteristics of this disease in the Amish are similar to common type 2 diabetes in other populations (26). Here, we report the results of genome-wide linkage analyses that were carried out in extended families of the Amish Family Diabetes Study (AFDS). Analyses were conducted on both discrete traits (type 2 diabetes and/or impaired glucose homeostasis [IGH]) and quantitatively distributed traits related to dia- betes (plasma glucose levels and HbA1c), and we used additional saturation markers placed in two chromosomal regions of particular interest. Our findings provide evi- dence for susceptibility genes for type 2 diabetes located on chromosomes 1q21-q24 and 18p and in a novel region on chromosome 14q11. From the 1Department of Genetics, Southwest Foundation for Biomedical Research, San Antonio, Texas; 2GlaxoSmithKline, Research Triangle Park, North Carolina; the 3Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland; the 4National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Phoenix, Ari- zona; 5Axys Pharmaceuticals, La Jolla, California; and the 6Geriatrics Re- search and Education Clinical Center, Baltimore Veterans Administration Medical Center, Baltimore, Maryland. Address correspondence and reprint requests to Alan R. Shuldiner, Division of Endocrinology, Diabetes and Nutrition, University of Maryland School of Medicine, 660 W. Redwood St., Room 494, Baltimore, MD 21201. E-mail: ashuldin@medicine.umaryland.edu. Received for publication 3 July 2002 and accepted in revised form 6 November 2002. W.-C.H. and P.L.S. contributed equally to this study. W.-C.H. is currently located at the University of California San Francisco School of Medicine, San Francisco, California; C.J.B. at EmerGen, Salt Lake City, Utah; and H.S. at Pfizer, Groton, Connecticut. P.L.S., M.G.E., M.J.W., and D.K.B. are employed by and hold stock in GlaxoSmithKline; C.J.B holds stock in Celera; and H.S. is employed by and holds stock in Pfizer. Additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org. AFDS, Amish Family Diabetes Study; AUC, area under the curve; IGH, impaired glucose homeostasis; LOD, logarithm of odds; LRT, likelihood ratio test; OGTT, oral glucose tolerance test; STR, short tandem repeat. 550 DIABETES, VOL. 52, FEBRUARY 2003 RESEARCH DESIGN AND METHODS Subjects and phenotypes. The AFDS was initiated in 1995 with the goal of identifying susceptibility genes for type 2 diabetes and related traits (26). Individuals with type 2 diabetes were identified by door-to-door interviews with the help of liaisons from the Amish community. Individuals who reported diabetes onset between 35 and 65 years of age were invited to participate. All first- and second-degree family members of the probands who were �18 years of age were contacted and invited to participate in the study. If another diabetic individual was identified in the family (e.g., aunt or uncle), then the family was expanded further to include that person’s first- and second-degree relatives (�18 years of age). Examinations were conducted at the Amish Diabetes Research Clinic in Strasburg, Pennsylvania, or in the subjects’ homes. A genome scan was performed on the initial set of 691 individuals enrolled and examined in the AFDS between February 1995 and February 1997. Nearly all of these individuals share common ancestors insofar as the entire Amish community of Lancaster County (now numbering �30,000 individuals) are descendents of a small number of Amish families who emigrated to this area in the mid 1700s. These subjects can be connected into a single 14-generation pedigree (27). The study protocol was approved by the Institutional Review Board at the University of Maryland School of Medicine, and informed consent was obtained from each study participant. A 3-h oral glucose tolerance test (OGTT) was administered to all subjects without a prior history of diabetes. After collection of a fasting blood sample, a 75-g oral glucose challenge was administered (Trutol 100; Casco Nerl Diagnostics, Baltimore, MD), and additional blood samples were obtained at 30-min intervals for analysis of plasma glucose concentrations. Glucose concentrations were assayed with a Beckman glucose analyzer (Beckman Coulter, Fullerton, CA) using the glucose oxidase method (interassay coeffi- cient of variation � 1.52%). Glucose area under the curve (AUC) during the 3-h OGTT was calculated using the trapezoid method. HbA1c was measured by high-pressure liquid chromatography (interassay coefficient of variation � 4.3% for low standard and 2.5% for high standard). BMI was calculated as weight (kg) divided by height squared (m2). Criteria for the diagnosis of diabetes were adapted from American Diabe- tes Association recommendations (28). Diabetes was defined by a single fasting venous plasma glucose level (�7 mmol/l), a 2-h OGTT venous plasma glucose level (�11.1 mmol/l), or current treatment with insulin or oral hypoglycemic agents. Diabetic subjects with an age at diagnosis �35 years were reclassified as diabetes status unknown to minimize heterogeneity due to inclusion of subjects with type 1 diabetes. Nondiabetic subjects were classified as having IGH based on age-, sex-, and BMI-adjusted fasting and 2-h OGTT venous plasma glucose levels (fasting plasma glucose level between 6.1 and 7 mmol/l or 2-h OGTT plasma glucose between 7.8 and 11.1 mmol/l). A combined phenotype, type 2 diabetes/IGH, consisted of subjects with type 2 diabetes or IGH as defined above. Normoglycemia was defined as having fasting venous plasma glucose level �6.1 mmol/l and a 2-h OGTT venous plasma glucose level �7.8 mmol/l. Due to missing data, diabetes and IGH statuses could not be determined for 42 and 87 subjects, respectively. Genotypes. We initially performed linkage analysis using 373 highly polymor- phic microsatellite short tandem repeat (STR) markers on 22 autosomes and the X chromosome. These markers were part of the ABI Prism Linkage Mapping Set (Perkin-Elmer) and have been described previously (29). The mean marker heterozygosity was 0.75, with a range from 0.33 to 0.91. The marker order and sex-averaged distances between markers were estimated from our data by maximum likelihood methods using CRI-MAP (30). The average interval between markers was 9.7 cM, and the largest gap between markers was 25.4 cM, occurring on chromosome 7. The genetic maps calculated by CRI-MAP using the Amish data were on average 8.9% longer than the corresponding map reported by Marshfield (http://research.marshfieldclinic. org/genetics/MapMarkers/maps/IndexMapFrames.html). Overall, there was excellent agreement with marker order between the Amish map and the Marshfield map, with only five occasions in which marker order was not concordant. In two regions where interesting linkage signals were observed in our initial scan, we typed additional STR markers to increase the map density and information content. These included 36 markers on chromosome 1 between 90 and 180 cM (average intermarker distance 1.9 cM) and 18 markers on chromosome 14 between 0 and 51 cM (average intermarker distance 2.3 cM). These markers were integrated into the framework map by use of CRI-MAP and by inferring genetic distance through the use of physical map information when the genetic distance could not be properly estimated (Human Genome Project Working Draft at University of California, Santa Cruz, http:// genome.ucsc.edu/). Marker allele frequencies used in all analyses were estimated from the entire set of subjects. To identify Mendelian and/or pedigree errors, the 14-generation pedigree was broken down into 41 smaller pedigrees that were 6 generations in depth with marriage and inbreeding loops broken (D.M. Nielsen, personal commu- nication). Pedigree errors were identified and corrected by looking for subjects with systematic Mendelian inconsistencies in the scan markers and by use of SibError (31), a program that detects sibs, half-sibs, and monozy- gotic twins by investigating eight identity-by-state statuses using 50 unlinked markers. Mendelian inconsistencies were identified and genotypes involved in these discrepancies deleted via an algorithm based on Unknown (32), a program that detects, for each marker, the minimal set of subjects whose genotypes, when removed, result in eliminating said Mendel errors (R. Idury, personal communication). Seven pedigree errors were identified and cor- rected. Three sets of monozygotic twins were identified, and we removed one twin from each of the three sets before analysis. Overall, the completeness of genotype calls for markers used in the linkage analysis was 96.3 � 2.4% (means � SD) (range 81.9-–99.8%). Statistical analysis Qualitative trait linkage analysis. The two qualitative traits, type 2 diabetes and type 2 diabetes/IGH, were analyzed using two-point and multi- point nonparametric allele sharing methods as implemented in Genehunter- Plus (33) with the exponential model and Sall function. Multipoint allele sharing was assessed at 1-cM intervals. The single 14-generation pedigree was subdivided into smaller pedigrees fitting the size constraints of the Gene- hunter program. For the type 2 diabetes phenotype, the single 14-generation pedigree was broken down into 15 pedigrees with 54 affected subjects and 112 subjects who were either unaffected or of unknown diabetes status. For the type 2 diabetes/IGH phenotype, 31 pedigrees were analyzed with 140 affected subjects, 105 unaffected subjects, and 144 subjects with unknown type 2 diabetes/IGH status. Separate analysis of pedigrees that are indeed related to one another introduces the risk of underestimating the degree with which any two individuals are related. This could result in the overestimation of the differ- ence between expected and observed allele sharing, possibly leading to inflated evidence of linkage. To address this issue, we simulated unlinked markers whose information content was equivalent to those in the observed linkage peaks. These unlinked markers were then dropped down through the entire 14-generation pedigree (D.M. Nielsen, personal communication). This single large pedigree was divided into the smaller analysis pedigrees (see above), and these were then analyzed by Genehunter-Plus. We conducted 20,000 replicates and defined the probability that the observed logarithm of odds (LOD) score is a false positive report as the proportion of replicates exceeding our observed LOD score. The P values reported for the observed LOD scores from the type 2 diabetes and type 2 diabetes/IGH analyses were obtained from this simulation. Conditional analyses were performed for the discrete trait, type 2 diabetes/ IGH, to test for interactions between genomic regions (the type 2 diabetes trait was not tested due to its small sample size). To minimize the number of comparisons, we conditioned on regions with LOD scores of �1.18 (pointwise P � 0.01 [34]) and examined the rest of the genome for further evidence of diabetes loci. Using the Genehunter NPL score, two weighting schemes were applied; the first one tests a multiplicative model or epistatic model, and the second tests for heterogeneity among families (35). To determine the empir- ical significance of LOD scores from the conditional analyses, we randomly assigned weights to the families through permutation, keeping the overall number of families with a weight of 1 for a given weighting scheme constant with the number observed with a weight of 1. These permutations were conducted 5,000 times. Quantitative trait linkage analysis. Linkage analyses were also per- formed on the quantitative traits, the plasma glucose levels obtained during a 75-g 3-h OGTT (fasting glucose levels and glucose levels obtained at 30-min intervals, noted as glucose 30, 60, 90, 120, 150, and 180), glucose AUC, and HbA1c. Sample sizes used for each trait-specific analysis ranged from 525 (for glucose AUC) to 661 (HbA1c), as described in Table 1. To eliminate confound- ing influences of diabetes treatment, subjects taking medications for diabetes were excluded from these analyses. Furthermore, to reduce skewness, extreme outliers were excluded from these analyses and HbA1c values were transformed by their natural logarithm. To reduce the computational com- plexity of these analyses, we divided the single large pedigree into 28 separate smaller pedigrees, ranging in size from 3 to 69 individuals. Even after splitting the large pedigree, the sample included a very large number of relative pairs, among these 1,384 pairs of siblings, 1,404 avuncular (aunt, uncle/niece, nephew) pairs, 110 grandparent-grandchild pairs, and 1,261 first cousin pairs. These analyses were conducted using a pedigree-based likelihood method to partition the total phenotypic variation into effects due to covariates, effects of a specific locus (or linkage), and residual additive genetic effects (or herita- bility). Statistical significance was assessed by computing the likelihood of the pedigree data under competing genetic models (linkage versus no linkage) and comparing the likelihoods using the likelihood ratio test (LRT), which W.-C. HSUEH AND ASSOCIATES DIABETES, VOL. 52, FEBRUARY 2003 551 yields a �2 statistic with (in this case) a single degree of freedom. P values obtained from the LRT were then converted to LOD scores using the formula: LOD � �2/[2 � ln(10)]. Linkage analysis for the quantitative traits was carried out using the SOLAR software program (35). Use of the LRT to evaluate evidence for linkage using variance component methods can be problematic when the multivariate normality assumption is violated (37). We therefore used simulation to empirically estimate the probability of obtaining false evidence for linkage. We derived the distribution of nominal LOD scores under the null hypothesis of no linkage by simulating 10,000 unlinked markers, dropping them through the pedigrees, and conduct- ing linkage analysis with each of the markers for each of the glucose traits and HbA1c. The probability of obtaining a false positive result was defined as the proportion of replicates for which we obtained a specified LOD score or higher. The P values obtained from the simulation study were then converted into LOD scores as described above. All LOD scores from quantitative trait locus analyses presented in this report were obtained from this simulation. RESULTS Genome-wide linkage analysis. Clinical characteristics of the 691 study subjects are shown in Table 1. The mean age was �47 years, and the mean BMI was 26.3 and 28.1 kg/m2 in men and women, respectively (P � 0.0001). Diabetes was present in 8.5% of men and 12.7% of women, and IGH was present in 14.3% of men and 20.4% of women. The greater prevalence of type 2 diabetes and IGH in women was likely due to greater BMI. Since participants were ascertained around family members with type 2 diabetes, these prevalence rates do not reflect the preva- lences of type 2 diabetes or IGH in the general Amish population, which are estimated to be 5 and 20%, respec- tively (26). The sibling relative risk(s) for type 2 diabetes in this population was previously reported to be 3.3 (95% CI 1.58 – 6.80) (26). The heritabilities for the glucose traits measured during the 3-h OGTT and HbA1c in family members of diabetic subjects not previously known to have diabetes are shown in Table 1. The heritabilities for all glucose traits were significantly greater than zero (P � 0.0001), with estimates ranging from 0.21 for glucose 0 (fasting) and glucose 180 to 0.38 for glucose 60. The heritability for the integrated measures, glucose AUC and HbA1c, were 0.44 and 0.30, respectively. Results from the initial linkage analyses based on the 10-cM screening set of 373 markers are summarized in Table 2. These analyses revealed LOD scores of �2.0 for one or more traits in only three chromosomal regions. Between 104 and 110 cM on chromosome 1, we observed linkage peaks for glucose 180 with a LOD of 2.16 and for glucose 150 with a LOD of 1.98. At position 14 –33 cM on chromosome 14, we observed LOD � 2.81 for glucose 150 at 14 cM, LOD � 2.06 for glucose 180 at 29 cM, and LOD � 2.41 for glucose AUC at 33 cM. On chromosome 18 at position 9 cM, we observed linkage peaks for HbA1c (LOD � 3.07) and glucose 0 (LOD � 1.50). LOD scores of 1.50 –1.99 for one or more traits were observed in three other chromosomal regions, all on chromosome 2: at position 36 –38 cM (LOD � 1.77 and 1.75 for glucose 120 and glucose AUC, respectively), at position 150 cM (LOD � 1.59 for glucose 120), and at position 265 cM (LOD � 1.87 for HbA1c). Eight more chromosomal regions (on chromosomes 4, 7, 8, 15–17, and 19) showed linkage signals, with LOD between 1.18 (corresponding to a point- wise P � 0.01) (Table 2) and 1.49. The complete genome scan results can be viewed at http://medschool.umaryland. edu/Endocrinology/Amish/amlinkindex.html or in an on- line appendix at http://diabetes.diabetesjournals.org. Fine mapping on chromosomes 1 and 14. Based on results from the initial linkage analyses, we genotyped additional STR markers on chromosomes 1 and 14. On chromosome 1, we typed an additional 36 markers within the 90- to 180-cM interval on our framework map (flanked by markers D1S209 and D1S484), and on chromosome 14, we genotyped an additional 18 STR markers falling within the region between 0 and 50 cM on our map (flanked by D14S261 and D14S288). Results of the linkage analyses using the extended set of markers on chromosomes 1 and 14 are shown in Fig. 1 for the discrete traits, and glucose 150 and 180 (results for other traits can be viewed at http://medschool. umaryland.edu/Endocrinology/Amish/amlinkindex.html or http://diabetes.diabetesjournals.org). On chromosome 1p, evidence for linkage to the discrete type 2 diabetes trait increased to 1.78 (P � 0.0043), occurring at 116 cM (nearest marker: D1S2766). Results for quantitative trait locus analysis remained similar to those using framework markers only. The peak signal for glucose 180 occurred at 112 cM near D1S551 (LOD � 2.31, P � 0.00055). Analyses TABLE 1 Clinical characteristics of study population by sex* Trait† N (M/F) Male Female h2‡ Age (years) 691 (308/383) 47.2 � 15.4 46.7 � 15.9 NA BMI (kg/m2) 679 (302/377) 26.3 � 3.7 28.1 � 5.6‡ 0.41 � 0.07 Diabetes (%) 649 (294/355) 8.5 12.7 NA IGH (%) 534 (244/290) 14.3 20.4 NA Plasma glucose levels (mmol/l)§ Glucose 0 629 (281/348) 5.26 � 1.13 5.25 � 1.12 0.21 � 0.06 Glucose 30 562 (250/312) 8.74 � 2.08 8.42 � 1.75� 0.34 � 0.07 Glucose 60 556 (247/309) 8.71 � 2.45 9.07 � 2.45 0.38 � 0.08 Glucose 90 557 (247/310) 7.34 � 2.60 8.22 � 2.65� 0.37 � 0.07 Glucose 120 559 (248/311) 6.05 � 2.26 7.36 � 2.50¶ 0.33 � 0.07 Glucose 150 545 (243/302) 5.02 � 1.81 6.11 � 2.24¶ 0.29 � 0.07 Glucose 180 532 (237/295) 4.46 � 1.40 4.99 � 1.83� 0.21 � 0.06 Glucose AUC (mmol � l�1 � h) 525 (236/289) 20.28 � 5.07 22.05 � 5.47� 0.44 � 0.08 HbA1c (%)§ 608 (277/331) 5.05 � 0.56 4.91 � 0.59 0.30 � 0.08 *See reference 26 for additional characteristics of the AFDS; †Age, BMI, glucose traits, and HbA1c reported as mean � SD; §Due to confounding influences of diabetes treatment on these traits, subjects with previously diagnosed diabetes were not included; ‡Heritability estimates � SE, all with P � 0.0001; �P � 0.05; ¶P � 0.0001. NA, not available. GENOME SCAN OF TYPE 2 DIABETES 552 DIABETES, VOL. 52, FEBRUARY 2003 of three other quantitative traits also provided supportive evidence for linkage, including glucose 120 (LOD � 1.28 at 106 cM near D1S216), glucose 150 (LOD � 1.90 at 113 cM near D1S551), and glucose AUC (LOD � 0.71 at 105 cM near D1S216). Since the analyses of glucose traits ex- cluded subjects with previously diagnosed diabetes, link- age of glucose traits and diabetes to the same region of chromosome 1p31 provide complimentary evidence that a gene influencing glucose homeostasis resides in this re- gion. In the region of 1q21–24, the LOD score for type 2 diabetes/IGH increased to 2.35 (P � 0.0008) at 166 cM near D1S2858. D1S2715 at 166.4 cM has the largest two-point LOD score in this region (LOD � 2.07). However, dropping this marker from the saturation map resulted in only a slight drop in the multipoint LOD score at 166 cM, from 2.35 to 2.09. On chromosome 14, we also observed increased evi- dence supporting linkage using a denser genetic map. For the analysis of diabetes, the peak LOD score increased to 3.48 (P � 0.00005) at 7 cM near D14S1023. The information content at 7 cM increased from 0.62 in the scan to 0.76 in the fine-mapping study. With an intermediate 5-cM map the LOD score for type 2 diabetes at 7 cM was 2.66. This 5-cM map did not contain D14S1023, the marker with the largest two-point LOD score (LOD � 1.57) in this region. The peak LOD score for glucose 150 (LOD � 2.16, P � 0.0008) occurred at the same position (7 cM). Again, since the analysis of glucose traits excluded subjects with previously diagnosed diabetes, linkage to both diabetes and glucose traits provide complimentary evidence for linkage in this region. Similar to the analysis with the framework markers, the denser map showed a secondary peak for glucose 150 (LOD � 1.82, P � 0.0019) that coincided at virtually the same position (at 35 cM near D14S1060) as the peak LOD for two other traits, glucose 180 (LOD � 1.86 at 35 cM), and glucose AUC (LOD � 1.84 at 44 cM). Conditional analyses for type 2 diabetes/IGH pheno- type. We examined the family-wise NPL scores for the type 2 diabetes/IGH trait at three regions that had LOD scores of �1.18 (pointwise P � 0.01) for the conditional analyses. The three regions that were conditioned on were 1q23 near D1S2858 (LOD � 2.35 at 166 cM), 4q28 near D4S1575 (LOD � 1.28 at 139 cM), and 17q24 near D17S949 (LOD � 1.48 at 109 cM). Two regions showed increased evidence of linkage under a positive weighting scheme (epistatic model). At 1q24-q31 (D1S218-D1S238, 204 cM), the LOD score for type 2 diabetes/IGH increased from 0.89 to 2.60 (P � 0.0022) when we conditioned on the linkage results at the 4q28 region. After conditioning on linkage results at 1q23, the LOD scores at 5q22 near D5S421 (115 cM) increased from 0.55 to 1.42 (P � 0.003). Under a negative weighting scheme (heterogeneity model), only one additional region was identified with a significant increase in LOD score. When conditioning on linkage results at 4q28, the LOD score at 14q11 (D14S1023, 6 cM) increased from 0.95 to 1.75 (P � 0.0048). The region at 14q11 is the same region showing evidence for linkage to the trait, diabetes (Fig. 1). Conditioning on 17q24 did not significantly increase evidence for linkage elsewhere in the genome. DISCUSSION Type 2 diabetes has an important genetic basis in the Amish, as it does in other populations. The sibling relative risk of type 2 diabetes was estimated to be �3.3 (26), suggesting a familial aggregation of this disease in this population. Furthermore, in nondiabetic family members of subjects with known diabetes, there are moderate yet TABLE 2 Chromosomal regions showing evidence for linkage, with a LOD � 1.18 (P � 0.01), to diabetes and related traits using 373 framework STR markers Chromosome Location (cM) Trait Peak LOD score Empirical P value LOD-1 support interval (cM) Flanking markers 1 106 Type 2 diabetes 1.44 0.0115 90–124 D1S209, D1S216 104 Glucose 120 1.63 0.0031 90–120 D1S209, D1S216 104 Glucose 150 1.98 0.0013 92–120 D1S209, D1S216 110 Glucose 180 2.16 0.0008 95–124 D1S216, D1S207 2 36 Glucose AUC 1.75 0.0023 15–45 D2S312, D2S220 38 Glucose 120 1.77 0.0022 20–59 D2S312, D2S220 150 Glucose 120 1.59 0.0034 130–170 D2S347, D2S368 265 HbA1c 1.87 0.0017 255–275 D2S206, D2S338 4 40 HbA1c 1.23 0.0087 25–65 D4S419, D4S391 139 Type 2 diabetes/IGH 1.28 0.0141 126–156 D4S1575, D4S424 7 100 Glucose 60 1.45 0.0049 50–120 D7S669, D7S657 8 160 Glucose 180 1.27 0.0078 130–165 D8S284, D8S272 14 11 Glucose 0 1.56 0.0037 0–35 D14S283, D14S80 14 Glucose 150 2.81 0.00016 0–36 D14S283, D14S80 23 Glucose 120 1.75 0.0023 10–40 D14S80, D14S70 29 Glucose 180 2.06 0.0010 11–42 D14S80, D14S70 33 Glucose AUC 2.41 0.0004 16–53 D14S70, D14S288 15 114 Glucose 120 1.30 0.0072 100–120 D15S130, D15S1014 16 120 Glucose 180 1.48 0.0045 109–120 D16S520 17 109 Type 2 diabetes/IGH 1.48 0.0081 90–128 D17S949, D17S802 18 9 Glucose 0 1.50 0.0043 0–28 D18S59, D18S452 9 HbA1c 3.07 0.000085 0–31 D18S59, D18S452 19 45 Glucose 120 1.37 0.0060 30–55 D19S226, D19S433 W.-C. HSUEH AND ASSOCIATES DIABETES, VOL. 52, FEBRUARY 2003 553 significant heritabilities for plasma glucose concentrations during an OGTT as well as for HbA1c, providing further evidence of genetic influence on type 2 diabetes. Despite the well-recognized genetic contribution to type 2 diabe- tes, little is known about the specific genetic causes of diabetes or variation in glucose levels. Most linkage stud- ies have examined diabetes or glucose levels during fast- ing and/or 2 h after a glucose challenge. To our knowledge, our study is the first to investigate the genetic influence on plasma glucose levels during the course of a 3-h OGTT, allowing us to examine whether the variation in glucose levels during the OGTT have shared genetic influences. Based on results from our genome scan, we identified several regions with suggestive evidence of linkage. Al- though none of the linkage signals reached genome-wide significance at P � 0.05, these studies provide evidence for linkage in the regions of 1p31, 1q21-q24, and 14q11. Link- age signals for several glucose concentrations measured at different times during the 3-h OGTT (glucose 120, 150, and 180 and glucose AUC) were observed in a region on FIG. 1. Multipoint linkage analysis results of glu- cose and diabetes for chromosomes 1 and 14 in the fine-mapping study. GENOME SCAN OF TYPE 2 DIABETES 554 DIABETES, VOL. 52, FEBRUARY 2003 chromosome 1p31 (between 104 and 110 cM). Further- more, analysis of the discrete trait, diabetes, also provided evidence for linkage in the same region. On chromosome 1q21-q24, the peak LOD score for type 2 diabetes/IGH was 2.35 (at 166 cM). On chromosome 14q11, we obtained much stronger evidence for linkage to diabetes, with a peak LOD score of 3.48. For the type 2 diabetes/IGH traits, the peak LOD score was 0.95 on chromosome 14 at virtually the same region. The peak signal for glucose 150 also occurred at 7 cM. Linkage to a similar region on chromosome 14q11 for the dichotomous traits (type 2 diabetes and type 2 diabetes/IGH), as well as glucose levels as quantitative traits (in which subjects with previ- ously diagnosed diabetes were not included), provides strong evidence for a novel type 2 diabetes locus in this region. We also obtained evidence for linkage to glucose traits on chromosomes 2 and 18. Notably, evidence for linkage to HbA1c on chromosome 18 at 9 cM was quite strong (LOD � 3.07, P � 0.00008) and deserving of further investigation. While evidence for linkage of more than one glucose trait in the same region was regarded as encour- aging, it should be noted that these traits were moderately correlated with each other. For example, the correla- tion coefficient was 0.87 between glucose 120 and 150, 0.87 between glucose 150 and 180, and 0.76 between glucose 120 and 180. (For correlation coefficients between other glucose traits, see http://medschool.umaryland.edu/ Endocrinology/Amish/amlinkindex.html.) By conditioning on evidence for linkage previously detected in one or more regions of the initial genome scan, we may increase the power to strengthen linkages in other regions and to detect other diabetes-related loci. Indeed, conditioning on the 4q28 region increased evidence of linkage to type 2 diabetes/IGH on both chromosomes 1 and 14. These analyses extended evidence of linkage into the 1q24-q31 region with a LOD of 2.60 at 204 cM. As seen in Fig. 2, this region still overlaps with evidence of diabetes linkage reported in other populations. While conditioning on evidence of linkage at 4q28, the LOD score for type 2 diabetes/IGH on chromosome 14 increased to 1.75 in the same region, providing further evidence for linkage to type 2 diabetes (LOD � 3.48). By contrast, no interaction occurred between 1q23 and 14q11, suggesting that for the type 2 diabetes/IGH trait, the genetic effects from loci on 1q23 and 14q11 are independent of one another. Our region of linkage on chromosome 14q11 showed a strong linkage signal for diabetes (LOD � 3.48). This region contains a cluster of immune function genes, e.g., interleukin 17E 18 (IL17E), interferon-stimulated gene transcription factor 3 (ISGF3), cytotoxic T-cell–associated serine esterase-1 (CTLA1), T-cell antigen receptors (TCRA) and (TCRD), and the leukotriene b4 receptor (LTB4R). Although none of these genes have been previ- ously considered as candidate genes for type 2 diabetes, there is an increasing body of evidence that the inflamma- tory process may be primarily involved in type 2 diabetes pathogenesis (38,39). In addition, there are several genes in this region that may influence glucose and/or lipid metabolism through their effects on signal transduction or gene transcription, including protein kinase C � (PRKCM), adenylate cyclase-4 (ADCY4), CCAAT/enhancer-binding protein � (CEBPE), and the estrogen receptor-2 (ESR2). We observed modest (LOD � 1.28 –2.30) evidence for linkage to four traits (diabetes and glucose 120, 150, and 180) on chromosome 1p31. Although no other groups have reported evidence of linkage to diabetes on 1p, Norman et al. (40) observed a LOD score of 2.6 for 24-h respiratory quotient between D1S1728 and D1S551 (�113 cM on the Amish map), and Thompson et al. (39) found linkage to acute insulin release at D1S198 (�98 cM on the Amish map). The ratio of carbohydrate to fat oxidation is a predictor of weight gain, and obesity is a known risk factor for type 2 diabetes. Similarly, decreased acute insulin release is an early indicator of glucose intolerance and type 2 diabetes. This region is known to harbor the leptin receptor. Linkage signals for diabetes to the region of chromo- some 1q21-q24 have been reported in four other popula- tions (Fig. 2). In a genome scan conducted in the Pima Indians (9), allele sharing of sib-pairs concordant (defined as onset before age 45 years) and discordant (defined as nondiabetic at age 45 years), revealed evidence for linkage of diabetes to D1S1677 on chromosome 1q (LOD � 2.5, at �185 cM on the Amish map). Moreover, when 55 sib-pairs with age of diabetes onset of �25 years were analyzed separately in an affected sib-pair analysis, very strong evidence for linkage was detected near D1S2127 (LOD � 4.1, at �208 cM on the Amish map). In Utah Caucasians (12), linkage to diabetes was observed in the region between CRP and APOA (�176 cM on the Amish map) using both parametric analysis (LOD � 4.30) and model- free affected sib-pair analysis (LOD � 2.96). More recently, in a French study (14), affected sib-pair analysis using 113 lean (BMI �27 kg/m2) diabetic sib-pairs found a linkage peak near D1S484 (at 179 cM on the Amish map) with an LOD of 3.04. In British affected sib-pairs, Wiltshire et al. (20) observed evidence for linkage to diabetes on 1q24 near D1S2799, �199 cM on the Amish map (LOD � 1.98 after saturation markers were placed). It is often difficult to evaluate whether a putative gene underlying the linkage signal in one study (e.g., the Amish) represents the same gene as that detected by linkage in other studies (e.g., Pima Indians and Utah, French, and British Caucasians). Simulation studies have suggested FIG. 2. Regions showing linkage signals to diabetes and its related traits on chromosome 1q from five populations (traits shown in parentheses). *Results from analysis conditioning on linkage signal to type 2 diabetes/IGH on chromosome 4q28. DM, diabetes mellitus. W.-C. HSUEH AND ASSOCIATES DIABETES, VOL. 52, FEBRUARY 2003 555 that even under an ideal scenario (i.e., sufficient power and identical phenotype definition, ascertainment strategy, pedigree structure, genetic map, genetic and environment heterogeneity, and statistical approach), difference in sam- pling alone may cause the variation in location estimates of the linkage peaks to be as wide as 20 cM (42– 44). When we compared results from our linkage analysis with these four earlier studies showing linkage signals to diabetes on chromosome 1q (9,12,14,20), we found the genetic signals observed from these studies cluster in a 50-cM region on chromosome 1q21-q24 (Fig. 2). It is thus possible that these linkages in several populations may represent the same gene. Alternatively, there may be more than one type 2 diabetes susceptibility gene in this region. Chromosome 1q21-q24 contains at least 133 known genes, 145 putative transcripts with homology to known genes, and as many as 393 other potentially expressed sequences (20). Several of these genes are potentially associated with lipid or glucose metabolism, including lamin A/C (LMNA) (45,46), phosphoprotein enriched in astrocytes 15 (PEA15) (47,48), potassium inwardly recti- fying channel, subfamily J, member 9 (KCNJ9) (49), pre– B-cell leukemia transcription factor 1 (PBX1) (50), solute carrier family 19 (thiamine transporter), member 2 (SLC19A2) (51), retinoid X receptor (RXRG), insulin receptor–related receptor (INSRR), and others. Several other regions identified in our study with LOD scores of �1.18 have been reported by other researchers. These include chromosomes 2p24-p23 (14), 4q24 (18,19), 5q22 (18), 8q24.2- q24.3 (20), 13q32-q33 (9), 17q24.1-q24.3 (18,40), and 18p11.32-p11.31 (12,18). By contrast, the link- ages we observed in the Amish on chromosomes 2q14-q22, 2q37.3, 4p16.2- p15.2, 4q11.2, 7q11.2-q21.1, 15q26.3, 16q24.3, and 19p13.1-p12 appear to be novel. In summary, we obtained significant evidence for link- age to type 2 diabetes with a novel locus on chromosome 14q11, as well as suggestive linkage signals to its related traits on several other novel chromosomal regions. Fur- thermore, we observed evidence for the existence of diabetes-related loci on chromosome 1q21-q24, a region previously linked to diabetes by several other groups. Our findings provide the rationale for positional cloning of type 2 diabetes susceptibility genes on chromosomes 1 and 14. ACKNOWLEDGMENTS This study was supported in part by a research grant from GlaxoWellcome and Axys Pharmaceuticals, National Insti- tutes of Health grants DK54361 and DK02673, and an American Diabetes Association Research Award to A.R.S. We thank Wendy Warren, Mary Ann Drolet, Denis Massey, Mary Morrissey, Janet Reedy, and our Amish liaisons for their energetic efforts in study subject recruit- ment and characterization; Drs. Alejandro Schaffer and Richa Agarwala for assistance in pedigree construction; and Dr. Dahlia Nielsen for supplying programs for simula- tion studies and for decomposing the 14-generation pedi- gree into subunits. We also thank Don Holt, Derek Traughber, Darrin London, Santhi Sampath, Bo Zheng, Keith Tanner, and Demian Lewis for technical assistance. This study would not have been possible without the outstanding cooperation of the Amish community. REFERENCES 1. Elston RC, Namboodiri KK, Nino HV, Pollitzer WS: Studies on blood and urine glucose in Seminole Indians: indications for segregation of a major gene. Am J Hum Genet 26:13–34, 1974 2. Rich SS: Mapping genes in diabetes: genetic epidemiological perspective. Diabetes 39:1315–1319, 1990 3. Hanson RL, Elston RC, Pettitt DJ, Bennett PH, Knowler WC: Segregation analysis of non-insulin-dependent diabetes mellitus in Pima Indians: evi- dence for a major-gene effect. Am J Hum Genet 57:160 –170, 1995 4. Stern MP, Mitchell BD, Blangero J, Reinhart L, Krammerer CM, Harrison CR, Shipman PA, O’Connell P, Frazier ML, MacCluer JW: Evidence for a major gene for type II diabetes and linkage analyses with selected candidate genes in Mexican-Americans. Diabetes 45:563–568, 1996 5. Silver K, Shuldiner AR: Candidate genes for type II diabetes mellitus. In Diabetes Mellitus: A Fundamental and Clinical Text. LeRoith D, Taylor SI, Eds. Philadelphia, Lippincott, 2000, p. 709 –719 6. 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, Reeve-Daly MP, Weaver A, Brettin T, Duyk G, Lander ES, Groop LC: Mapping of a gene for type 2 diabetes associated with an insulin secretion defect by a genome scan in Finnish families. Nat Genet 14:90 –94, 1996 7. Hanis CL, Boerwinkle E, Chakraborty R, Ellsworth DL, Concannon P, Stirling B, Morrison VA, Wapelhorst B, Spielman RS, Gogolin-Ewens KJ, Shepard JM, Williams SR, Risch N, Hinds D, Iwasaki N, Ogata M, Omori Y, Petzold C, Rietzch H, Schroder HE, Schulze J, Cox NJ, Menzel S, Boriraj VV, Chen X, et al: A genome-wide search for human non-insulin-dependent (type 2) diabetes genes reveals a major susceptibility locus on chromo- some 2. Nat Genet 13:161–166, 1996 8. 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: An autosomal genomic scan for loci linked to prediabetic phenotypes in Pima Indians. J Clin Invest 101:1757–1764, 1998 9. 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: An autosomal genomic scan for loci linked to type II diabetes mellitus and body-mass index in Pima Indians. Am J Hum Genet 63:1130 –1138, 1998 10. Duggirala R, Blangero J, Almasy L, Dyer TD, Williams KL, Leach RJ, O’Connell P, Stern MP: 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 64:1127–1140, 1999 11. Hegele RA, Sun F, Harris SB, Anderson C, Hanley AJ, Zinman B: Genome- wide scanning for type 2 diabetes susceptibility in Canadian Oji-Cree, using 190 microsatellite markers. J Hum Genet 44:10 –14, 1999 12. Elbein SC, Hoffman MD, Teng K, Leppert MF, Hasstedt SJ: A genome-wide search for type 2 diabetes susceptibility genes in Utah Caucasians. Diabetes 48:1175–1182, 1999 13. Ehm MG, Karnoub MC, Sakul H, Gottschalk K, Holt DC, Weber JL, Vaske D, Briley D, Briley L, Kopf J, McMillen P, Nguyen Q, Reisman M, Lai EH, Joslyn G, Shepherd NS, Bell C, Wagner MJ, Burns DK, American Diabetes Association GENNID Study Group: Genomewide search for type 2 diabetes susceptibility genes in four American populations. Am J Hum Genet 66:1871–1881, 2000 14. Vionnet N, Hani El-H, Dupont S, Gallina S, Francke S, Dotte S, De Matos F, Durand E, Lepretre F, Lecoeur C, Gallina P, Zekiri L, Dina C, Froguel P: Genomewide search for type 2 diabetes-susceptibility genes in french whites: evidence for a novel susceptibility locus for early-onset diabetes on chromosome 3q27-qter and independent replication of a type 2-diabetes locus on chromosome 1q21- q24. Am J Hum Genet 67:1470 –1480, 2000 15. Watanabe RM, Ghosh S, Langefeld CD, Valle TT, Hauser ER, Magnuson VL, Mohlke KL, Silander K, Ally DS, Chines P, Blaschak-Harvan J, Douglas JA, Duren WL, Epstein MP, Fingerlin TE, Kaleta HS, Lange EM, Li C, McEachin RC, Stringham HM, Trager E, White PP, Balow J Jr, Birznieks G, Chang J, Eldridge W: The Finland-United States investigation of non-insulin-depen- dent diabetes mellitus genetics (FUSION) study. II. An autosomal genome scan for diabetes-related quantitative-trait loci. Am J Hum Genet 67:1186 – 1200, 2000 16. Ghosh S, Watanabe RM, Valle TT, Hauser ER, Magnuson VL, Langefeld CD, Ally DS, Mohlke KL, Silander K, Kohtamaki K, Chines P, Balow J Jr, Birznieks G, Chang J, Eldridge W, Erdos MR, Karanjawala ZE, Knapp JI, Kudelko K, Martin C, Morales-Mena A, Musick A, Musick T, Pfahl C, Porter R, Rayman JB: The Finland-United States investigation of non-insulin- dependent diabetes mellitus genetics (FUSION) study. I. An autosomal GENOME SCAN OF TYPE 2 DIABETES 556 DIABETES, VOL. 52, FEBRUARY 2003 genome scan for genes that predispose to type 2 diabetes. Am J Hum Genet 67:1174 –1185, 2000 17. Permutt MA, Wasson JC, Suarez BK, Lin J, Thomas J, Meyer J, Lewitzky S, Rennich JS, Parker A, DuPrat L, Maruti S, Chayen S, Glaser B: A genome scan for type 2 diabetes susceptibility loci in a genetically isolated population. Diabetes 50:681– 685, 2001 18. Parker A, Meyer J, Lewitzky S, Rennich JS, Chan G, Thomas JD, Orho- Melander M, Lehtovirta M, Forsblom C, Hyrkko A, Carlsson M, Lindgren C, Groop LC: A gene conferring susceptibility to type 2 diabetes in conjunc- tion with obesity is located on chromosome 18p11. Diabetes 50:675– 680, 2001 19. Luo TH, Zhao Y, Li G, Yuan WT, Zhao JJ, Chen JL, Huang W, Luo M: A genome-wide search for type II diabetes susceptibility genes in Chinese Hans. Diabetologia 44:501–506, 2001 20. Wiltshire S, Hattersley AT, Hitman GA, Walker M, Levy JC, Sampson M, O’Rahilly S, Frayling TM, Bell JI, Lathrop GM, Bennett A, Dhillon R, Fletcher C, Groves CJ, Jones E, Prestwich P, Simecek N, Rao PV, Wishart M, Bottazzo GF, Foxon R, Howell S, Smedley D, Cardon LR, Menzel S, McCarthy MI: A genomewide scan for loci predisposing to type 2 diabetes in a U.K. population (the Diabetes UK Warren 2 Repository): analysis of 573 pedigrees provides independent replication of a susceptibility locus on chromosome 1q. Am J Hum Genet 69:553–569, 2001 21. Lindgren CM, Mahtani MM, Widen E, McCarthy MI, Daly MJ, Kirby A, Reeve MP, Kruglyak L, Parker A, Meyer J, Almgren P, Lehto M, Kanninen T, Tuomi T, Groop LC, Lander ES: Genomewide search for type 2 diabetes mellitus susceptibility Loci in Finnish families: the Botnia study. Am J Hum Genet 70:509 –516, 2002 22. Busfield F, Duffy DL, Kesting JB, Walker SM, Lovelock PK, Good D, Tate H, Watego D, Marczak M, Hayman N, Shaw JT: A genomewide search for type 2 diabetes-susceptibility genes in indigenous Australians. Am J Hum Genet 70:349 –357, 2002 23. Meigs JB, Panhuysen CI, Myers RH, Wilson PW, Cupples LA: A genome- wide scan for loci linked to plasma levels of glucose and HbA1c in a community-based sample of Caucasian pedigrees: the Framingham Off- spring Study. Diabetes 51:833– 840, 2002 24. Hsueh WC, Mitchell BD, Shuldiner AR: Use of genome scans to identify susceptibility genes for type 2 diabetes. In Genetics of Diabetes Mellitus. W. Lowe, Ed. Norwell, MA, Kluwer Academic Publishers, 2001, p. 231–250 25. Horikawa Y, Oda N, Cox NJ, Li X, Orho-Melander M, Hara M, Hinokio Y, Lindner TH, Mashima H, Schwarz PE, del Bosque-Plata L, Horikawa Y, Oda Y, Yoshiuchi I, Colilla S, Polonsky KS, Wei S, Concannon P, Iwasaki N, Schulze J, Baier LJ, Bogardus C, Groop L, Boerwinkle E, Hanis CL, Bell GI: Genetic variation in the gene encoding calpain-10 is associated with type 2 diabetes mellitus. Nat Genet 26:163–175, 2002 26. Hsueh WC, Mitchell BD, Aburomia R, Pollin T, Sakul H, Gelder Ehm M, Michelsen BK, Wagner MJ, St Jean PL, Knowler WC, Burns DK, Bell CJ, Shuldiner AR: Diabetes in the Old Order Amish: characterization and heritability analysis of the Amish Family Diabetes Study. Diabetes Care 23:595– 601, 2000 27. Agarwala R, Biesecker LG, Hopkins KA, Francomano CA, Schaffer AA: Software for constructing and verifying pedigrees within large genealogies and an application to the Old Order Amish of Lancaster County. Genome Res 8:211–221, 1998 28. The Expert Committee on the Diagnosis and Classification of Diabetes Mellitus: Report of the Expert Committee on the Diagnosis and Classifi- cation of Diabetes Mellitus. Diabetes Care 20:1183–1197, 1997 29. Hsueh WC, Mitchell BD, Schneider JL, Wagner MJ, Bell CJ, Nanthakumar E, Shuldiner AR: QTL influencing blood pressure maps to the region of PPH1 on chromosome 2q31–34 in Old Order Amish. Circulation 101:2810 – 2816, 2000 30. Green P, Falls K, Crooks S: Documentation for CRI-MAP. Version 2.4. St. Louis, MO, Department of Genetics, School of Medicine, Washington University, 1990 31. Ehm MG, Wagner MJ: A test statistic to detect errors in sib-pair relation- ships. Am J Hum Genet 62:181–188, 1998 32. Ott J: Analysis of Human Genetic Linkage. Baltimore, MD, Johns Hopkins University Press, 1991 33. Kong A, Cox NJ: Allele-sharing models: LOD scores and accurate linkage tests. Am J Hum Genet 61:1179 –1188, 1997 34. Sham PC: Statistical methods in psychiatric genetics. Stat Methods Med Res 7:279 –300, 1998 35. Cox NJ, Frigge M, Nicolae DL, Concannon P, Hanis CL, Bell GI, Kong A: Loci on chromosomes 2 (NIDDM1) and 15 interact to increase suscepti- bility to diabetes in Mexican Americans. Nat Genet 21:213–215, 1999 36. Almasy L, Blangero J: Multipoint quantitative-trait linkage analysis in general pedigrees. Am J Hum Genet 62:1198 –1211, 1998 37. Allison DB, Neale MC, Zannolli R, Schork NJ, Amos CI, Blangero J: Testing the robustness of the likelihood-ratio test in a variancecomponent quanti- tative-trait loci-mapping procedure. Am J Hum Genet 65:531–544, 1999 38. Barzilay JI, Abraham L, Heckbert SR, Cushman M, Kuller LH, Resnick HE, Tracy RP: The relation of markers of inflammation to the development of glucose disorders in the elderly: the Cardiovascular Health Study. Diabetes 50:2384 –2389, 2001 39. Pradhan AD, Manson JE, Rifai N, Buring JE, Ridker PM: C-reactive protein, interleukin 6, and risk of developing type 2 diabetes mellitus. JAMA 286:327–334, 2001 40. 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: Autosomal genomic scan for loci linked to obesity and energy metabolism in Pima Indians. Am J Hum Genet 62:659 – 668, 1998 41. Thompson DB, Sutherland J, Apel W, Ossowski V: A physical map at 1p31 encompassing the acute insulin response locus and the leptin receptor. Genomics 39:227–230, 1997 42. Hauser ER, Boehnke M: Confirmation of linkage results in affected-sib-pair linkage analysis for complex trait (Abstract). Am J Hum Genet 61 (Suppl. 1):A278, 1997 43. Hsueh WC, Goring HH, Blangero J, Mitchell BD: Replication of linkage to quantitative trait loci: variation in location and magnitude of the LOD score. Genet Epidemiol 21 (Suppl. 1):S473–S478, 2001 44. Roberts SB, MacLean CJ, Neale MC, Eaves LJ, Kendler KS: Replication of linkage studies of complex traits: an examination of variation in location estimates. Am J Hum Genet 65:876 – 884, 1999 45. Cao H, Hegele RA: Nuclear lamin A/C R482Q mutation in Canadian kindreds with Dunnigan-type familial partial lipodystrophy. Hum Mol Genet 9:109 –112, 2000 46. Wolford JK, Hanson RL, Bogardus C, Prochazka M: Analysis of the lamin A/C gene as a candidate for type II diabetes susceptibility in Pima Indians. Diabetologia 44:779 –782, 2001 47. Reynet C, Kahn CR: Rad: a member of the Ras family overexpressed in muscle of type II diabetic humans. Science 262:1441–1444, 1993 48. Wolford JK, Bogardus C, Ossowski V, Prochazka M: Molecular character- ization of the human PEA15 gene on 1q21– q22 and association with type 2 diabetes mellitus in Pima Indians. Gene 241:143–148, 2000 49. Wolford JK, Hanson RL, Kobes S, Bogardus C, Prochazka M: Analysis of linkage disequilibrium between polymorphisms in the KCNJ9 gene with type 2 diabetes mellitus in Pima Indians. Mol Genet Metab 73:97–103, 2001 50. Thameem F, Wolford JK, Bogardus C, Prochazka M: Analysis of slc19a2, on 1q23.3 encoding a thiamine transporter as a candidate gene for type 2 diabetes mellitus in Pima Indians. Mol Genet Metab 72:360 –363, 2001 51. Thameem F, Wolford JK, Bogardus C, Prochazka M: Analysis of PBX1 as a candidate gene for type 2 diabetes mellitus in Pima Indians. Biochim Biophys Acta 1518:215–220, 2001 W.-C. HSUEH AND ASSOCIATES DIABETES, VOL. 52, FEBRUARY 2003 557