1 Department of Psychiatry, Center for Neurobiology and Behavior, University of Pennsylvania, Philadelphia, Pennsylvania
2 Department of Medicine, Epidemiology and Public Health, Yale University, New Haven, Connecticut
3 Department of Mathematical Sciences, Michigan Technological University, Houghton, Michigan
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ABSTRACT |
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INTRODUCTION |
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Leptin is an important hormone in body weight regulation (3). Leptin mutations in the rodent (ob/ob mice) can cause extreme obesity, but human leptin mutations are rare (4). Linkage analyses have found the human chromosome 7q region containing the leptin gene cosegregates with obesity (5,6). Because leptin coding region mutations do not account for 7q linkage, investigators have focused on the leptin flanking region. Several groups (including ours) (79) identified 16 polymorphisms in a 2.5-kb 5' region flanking leptin. Several common polymorphisms are associated with extreme obesity. Yet we do not know whether the polymorphisms in the leptin flanking region are solely responsible for the linkage on 7q or whether other obesity-related gene(s) are located in this region as well.
To seek other genes that potentially influence obesity, we examined the 40-cM region (7q22.17q35) flanking but mostly 5' of the leptin gene. Analyses evaluated both linkage and linkage disequilibrium (LD) in this region.
We sampled 1,425 European American individuals (1,020 members of 200 nuclear families and 405 individuals from 135 triads, Table 1) using 33 polymorphic markers (including markers for linkage analysis and transmission disequilibrium test [TDT]) (Table 2) across a 40-cM region of 7q22.17q35, including the leptin gene flanking region.
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Using haplotype TDT, we found LD at three locations near the leptin flanking region and a region near marker D7S692. Because of the small triad sample size, results did not reach statistical significance after correction for multiple comparisons and should therefore be considered only suggestive. In triads, we obtained the most significant result for quantitative traits by quantitative TDT (QTDT) (10) at a marker only 130 Kb upstream of leptin (D7S1875, P = 0.00006 for BMI, overall Bonferroni significance level = 0.0536, empirical P < 0.001 for 1,000 replicates by QTDT). In the nuclear family dataset, QPDT (11) gave LD on the leptin 5' flanking region (D7S2501 and D7S1875, nominal P = 0.01 and P = 0.002, respectively).
We also performed quantitative pedigree disequilibrium test (QPDT) analyses of the nuclear family dataset, from which we found LD for markers D7S2459 (close to the first linkage peak at D7S692) and D7S685 (nominal P = 0.005 and 0.05, respectively).
In the present study, we found evidence of linkage for obesity phenotypes in the D7S692D7S523 region. The results are consistent across different (but related) obesity phenotypes and in both discrete and quantitative analyses. The quantitative analysis results (MERLIN) matched the discrete results (GENEHUNTER and MERLIN-npl) very well, the most significant results came from the D7S692D7S523 region.
Our results suggest the possibility of an obesity-related gene(s) located in a region 20 cM upstream of leptin (D7S692-D7S523). So far, no linkage has been reported in this region for BMI other than by our study. Cheng et al. (12) found linkage for fasting insulin and systolic blood pressure in the D7S523-D7S3061 region. Arya et al. (13) also found linkage in D7S479 and D7S471 (94106 Mb) region for HDL and triglyceride in Mexican Americans; however, we have not found the same tendency in our data for triglycerides. Because we do not have enough quantitative data for insulin, blood pressure, and HDL, it is hard to compare with Chengs and Aryas results. Several candidate genes are close to D7S692-D7S523, including PIK3CG (phosphoinositide-3-kinase catalytic, -polypeptide), PRKAR2B (cAMP-dependent protein kinase regulatory subunit type IIß) and PPP1R3A (protein phosphatase 1, regulatory subunit 3A). Further analysis is needed in this 5-cM region, including candidate gene screening.
Compared with the D7S692-D7S523 region, the present results for the leptin region gave weak linkage only for extreme obesity (BMI 40 kg/m2). The stronger results for extreme obesity are consistent with previously reported linkage to the region (6), including the observed limitation to our extreme phenotype.
We also observed a weak linkage peak close to D7S1804-D7S2452. Because the D7S1804-D7S2452 peak is 5 cM away from leptin (based on the physical mapping), this peak may (or may not) be independent of the result of leptin variants, since the current resolution is poor. Feitosa et al. (14) found an LOD score of 4.9 at D7S1804 in a combined-sample study. Until now, we have not found obvious candidate genes in this region.
Because high-density single neucleotide polymorphism (SNP) mapping has become available, TDT (15) has become more and more popular for complex traits. However, extended pedigrees are not very useful for traditional TDT methods because triads created from extended pedigrees are not totally independent. On the other hand, using the triad samples alone may have less power, particularly in our case where small sample size limitations were compounded by multiple comparisons. During the past several years, there are several articles focused on TDT and extended pedigrees (10,11,16). We used QTDTs and QPDTs to analyze transmission disequilibrium in both nuclear family and triad samples. In triad samples, the leptin 5' flanking region (0.10.6 Mb upstream of leptin) still gave the most significant results and may indicate a "regulatory element" (or another obesity-related gene) located in that region. However, functional analyses are needed. The D7S692-D7S523 region was positive in all our linkage analyses as well as in TDT analyses (GENEHUNTER 2.0) in extreme obesity triads. Usually the 95% CI of QTL mapping is 10 cM (17) for a study with sample size similar to ours (200 families). However, we saturated the leptin gene region with highly informative markers and achieved a marker density much greater than a usual genome scan. Although we genotyped nine markers in a 0.4-Mb leptin flanking region, D7S692 still showed much more significant linkage. On the other hand, D7S692 is 20 Mb away from leptin, larger than the 10-cM theoretic CI. We found the same tendency for marker D7S2459 (1 cM upstream of D7S692) by QPDT in nuclear families. While only suggestive, these results also support the possibility of an obesity-related gene near D7S692.
In summary, we found suggestive linkage for obesity and related phenotypes in a 40-cM region on 7q22.17q35. The most significant result was from the D7S692-D7S523 interval. Positive linkage disequilibrium results suggest a locus is very close to D7S692. It is suggested that at least two obesity-related genes lie in chromosome region 7q22.135.
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RESEARCH DESIGN AND METHODS |
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A partially overlapping set of 135 extreme obesity European American triads (405 subjects, 63 of 135 triads were selected from nuclear families) were studied using the TDT analyses. Probands in triads had a BMI 40 kg/m2 and at least one parent with normal weight (BMI <27 kg/m2). All subjects gave informed consent, and the protocol was approved by the Committee on Studies Involving Human Beings at the University of Pennsylvania.
Phenotypes.
BMI was calculated based on measured height and weight: BMI = weight (in killograms)/height (in meters) (2). Percent fat was measured by bioelectric impedance (Tanita TBF310 Pro Body Composition Analyzer; Tanita, Arlington Heights, IL). Fasting serum glucose and triglycerides were assayed by Quest Diagnostic. Plasma leptin levels were measured using the radioimmunoassay method with the Human Leptin RIA Kit (Linco Research). Waist and hip circumferences were measured when blood samples were collected. All quantitative variables were adjusted for linear effects of age, within generation and sex using SPSS 6.1.
Clinical characteristics of nuclear families and triads, including trait-specific coefficients of skewness and kurtosis, are shown in Table 1.
DNA preparation and genotyping.
DNA was extracted using a high-salt method (20). A total of 23 polymorphic markers (including two SNPs) for nuclear families and 27 markers (including 8 SNPs) were genotyped (Table 2). Map distances were taken from the Human Genome Working Draft (Human Genome Browser, http://genome.ucsc.edu). Genotyping of microsatellite markers was performed as previously described (18). The -2,548 and -633 polymorphisms were detected by PCR-SSCP; selected samples with shifted bands were reamplified, and PCR products were purified using QIAquick PCR Purification Kit (Qiagen). The Genetics Core Facility at the University of Pennsylvania sequenced this DNA with an ABI 377 automatic sequencer. PCR for the -2,548 polymorphism was performed using 1.6 mmol/l MgCl2. We used special conditions for -188 and +19 SNPs. For -188, we used -dCTP body labeling, 0.8 units Taq/10 µlPCR, and Bss H II 50°C digestion for >3 h. For +19 polymorphism, we used
-33P-dATP body labeling, 1.2 mmol/l MgCl2, and Msp AI1 digestion (37°C) for >3 h. PCR products were separated using 10% PAGE sequencing gel, 1x TBE, 60 W for 1 h. Six samples from each 96-well set were chosen and put on a common plate, which was scored first as a key for all plate genotyping. All band patterns had two independent scorers who were blind to phenotype.
Discrete data analysis.
We performed nonparametric multipoint linkage analyses using GENEHUNTER version 1.3 (21) for BMI. We used dichotomous obesity affection status: BMI 27,
30,
35, and
40 kg/m2. Gene frequencies were estimated by allele counting using all individuals who provided DNA. This approach gives asymptotically unbiased estimates of the allele frequencies (22). All Mendelian inheritance and haplotype errors were checked and resolved using GENEHUNTER 1.3. Any genetically unrelated parents and siblings were excluded, as were all half siblings. Using the same affection status (BMI 27, 30, 35, and 40), we performed multipoint NPL analyses by MERLIN (23).
Pedigree regression analysis.
We carried out the pedigree-wide regression analyses (24) using MERLIN for quantitative BMI. The trait mean, variance, and heritability were estimated according to epidemiology studies (25). We set trait mean (BMI) = 25.5, variance = 4, and heritability = 50%. Varying these parameter values had little effect on results.
Simulation.
Using MERLIN, we simulated 500 replicates of our datasets for NPL and family regression analyses. Empirical P values (Table 3) were computed by dividing the number of replicates that exceeded the observed Z score or LOD score by the number of replicates (500).
TDT.
We performed TDT analysis by GENEHUNTER 2.0. Up to four marker haplotypes were constructed and analyzed using GENEHUNTER 2.0. We used QTDT (10) to analyze quantitative traits in triads. Empirical P values were computed by QTDT based on a 1,000-replicates simulation. We also used the QPDT (11) to analyze BMI values in the nuclear families.
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ACKNOWLEDGMENTS |
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We acknowledge the cooperation of our subjects. We thank Guang Ming Yuan, Richard Joseph, Dr. Balasahib Shinde, Elizabeth Joe, Jan Merideth, Amy Morrison, Melissa Rosato, Alison Bernstein, Kenneth Luguya, and Kye Yun for technical assistance. We also thank Dr. Chuanhui Dong for suggestions.
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FOOTNOTES |
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Received for publication18 September 2002 and accepted in revised form 18 February 2003.
LD, linkage disequilibrium; LOD, logarithm of odds; NPL, nonparametric linkage; SNP, single nucleotide polymorphism; TDT, transmission disequilibrium test; QPDT, quantitative pedigree disequilibrium test; QTDT, quantitative transmission disequilibrium test.
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REFERENCES |
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