Leptin receptor gene variation and obesity: lack of association in a white British male population
Leptin receptor gene variation and obesity: lack of association in a white British male populationTakanari Gotoda1, Brian S. Manning1,3, Anthony P. Goldstone2, Helen Imrie1, Alison L. Evans1, A. Donny Strosberg3, Paul M. McKeigue4, James Scott1,2 and Timothy J. Aitman1,2,*
1Molecular Medicine Group, MRC Clinical Sciences Centre and 2Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK, 3Institut Cochin de Génétique Moléculaire, Laboratoire d'Immunopharmacologie Moléculaire, Paris 75014, France and 4Department of Epidemiology and Population Sciences, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK
Received January 30, 1997;Revised and Accepted February 27, 1997
Leptin, a hormone secreted by adipocytes, plays a pivotal role in the control of body weight. Rodents with mutations in the leptin receptor gene develop morbid obesity. It is possible, therefore, that leptin receptor gene mutations contribute to human obesity. To test this possibility, we determined the entire coding sequence of the human leptin receptor cDNA from peripheral blood lymphocytes of 22 morbidly obese patients with body-mass index (BMI) between 35.1 and 60.9 kg/m2. We identified five common DNA sequence variants distributed throughout the coding sequence at codons 109, 223, 343, 656 and 1019, one rare silent mutation at codon 986 and one novel alternatively spliced form of transcript. None of the five common variants, including the three that predict amino acid changes, are null mutations causing morbid obesity, because homozygotes for the variant sequences were also found in lean subjects. Furthermore, the frequency of each variant allele and the distribution of genotypes and haplotypes were similar in 190 obese (BMI >28 kg/m2) and 132 lean (BMI <22 kg/m2) white British males selected from a population-based epidemiological survey. In these subjects, there was no evidence for a significant effect of the common variants on obesity or obesity-related phenotypes. These results suggest that mutations in the leptin receptor gene are not a common cause of human obesity.
Obesity is the most common metabolic disorder in Westernized countries. Family studies suggest that human obesity is highly heritable (1 ,2 ), although, as yet, the genetic determinants remain largely unidentified.
Leptin, the circulating product of the ob gene, is secreted by adipose tissue (3 ) and its expression rises with increasing fat stores in animals and humans (4 ,5 ). In rodents, it acts to reduce food intake and increase energy expenditure through hypothalamic actions, as part of a feedback loop (6 -9 ). Mutations in the ob gene lead to defective leptin production and obesity in the ob/ob mouse (3 ), but no such mutations have yet been identified in humans (10 -12 ).
The leptin receptor (OB-R) is a member of the class 1 cytokine receptor family and has a widespread tissue distribution in several alternatively spliced isoforms in rodents and humans (13 -17 ). In mice, only the Ob-Rb isoform with a long cytoplasmic tail appears fully functional and is thought to mediate the hypothalamic actions of leptin (14 ). A mutation in the OB-R gene of db/db mice leads to abnormal mRNA splicing that replaces the Ob-Rb with the short Ob-Ra isoform (14 ,15 ), giving rise to abnormal signal transduction (17 ), complete leptin resistance and obesity (7 ,9 ). In the Zucker fa/fa rat, a missense mutation in a highly conserved extracellular domain of the receptor leads to elevated leptin levels and obesity (18 ,19 ). Since most obese humans also have elevated blood leptin levels (4 ,5 ), it is possible that defects in the OB-R gene contribute to obesity in humans. Four leptin receptor isoforms have been identified so far in humans-a long isoform of 1165 amino acids homologous to mouse Ob-Rb and three shorter isoforms (huB219.1-3) generated by alternative splicing (13 ,16 ) from a single gene located on chromosome 1p (20 ). Messenger RNA for the shorter isoforms has been identified in circulating haemopoietic cells and lymph glands (16 ,17 ).
In this study, we first screened the entire cDNA coding sequence of the human leptin receptor long isoform (human Ob-R) for mutations, by direct sequencing of overlapping cDNA fragments amplified by PCR from lymphocyte mRNA of 22 morbidly obese patients. To test the possible significance of identified sequence variants as a cause of obesity in the general population, we then determined the genotype frequency of five common leptin receptor gene variants in 322 white British males derived from an epidemiological survey population and examined the relationship of these variants to the distribution of obesity-associated phenotypes in this population.
We examined human OB-R cDNA, because the structure of the human OB-R gene has not yet been reported and our preliminary studies had shown expression of human OB-R mRNA in peripheral blood lymphocytes. Six overlapping cDNA fragments covering the entire coding sequence for the 1165 amino acid human Ob-R (13 ) were amplified by RT-PCR from total cellular RNA of peripheral blood lymphocytes (Fig. 1 ). These cDNA fragments were the same as those obtained by RT-PCR with the same sets of primers from mRNA of human hypothalamus, choroid plexus, liver, lung and fat cells (data not shown).
We determined the entire 3.5 kb cDNA coding sequence for human Ob-R in 22 morbidly obese patients [body-mass index (BMI) = 35.1-60.9 kg/m2]. We studied 10 British whites, six French whites, and six Pima Indians who were included because of strong genetic predisposition to premature obesity and type II diabetes (21 ). In total, seven single base differences were identified in comparison with previously published data (13 ). Among them, four DNA sequence variants occurred commonly in the patients from all of the three groups. These cause two non-conservative changes: glutamine to arginine at codon 223 (CAG -> CGG) and lysine to asparagine at codon 656 (AAG -> AAC); a conservative change: lysine to arginine at codon 109 (AAG -> AGG); and a silent G -> A transition at codon 1019. Another silent T -> C change at codon 343 was also common in both the British and French patients but not found in 12 Pima Indians examined for the variant. Figure 2 shows sequence electropherograms for these five common variants. In addition, a silent C -> T change was found at codon 986 of a single allele from a French patient; and codon 976 was not alanine (GCC) as reported previously but aspartic acid (GAC) in all cDNAs examined. The codon 986 silent change is a rare mutation because it was not detected in the remaining patients with morbid obesity or in an additional 40 obese British males. The codon 109, 223 and 1019 variants have also been found by analysis of the hypothalamic leptin receptor cDNA of black males by conformation-sensitive gel electrophoresis (22 ).
During analysis of the OB-R cDNA fragments in the 22 morbidly obese patients, an abnormal fragment was found in a single patient on amplification of cDNA fragment 5 (nucleotides 2411-3079) (Fig. 3 ). The abnormal fragment was shorter and lower in amount compared with the normal fragment that was co-amplified by RT-PCR (Fig. 3 A). DNA sequencing revealed a 182 bp deletion (nucleotides 2685-2866) in the abnormal cDNA fragment (Fig. 3 B). Partial analysis of the genomic structure of the human OB-R gene showed that the deleted region corresponded precisely to the second last and the third last exons (exons X and Y) (Fig. 3 C). DNA sequencing of the patient's OB-R gene showed that there was no sequence alteration in the exon-intron boundaries involving exons W, X, Y and Z except for an A -> C polymorphism found on both strands at position +37 at the 5' end of intron Y, for which the patient was heterozygous. The polymorphic site is not usually associated with mRNA splicing. The presence of this polymorphism in the heterozygous state demonstrates that this patient did not have a large deletion in genomic DNA at this region. Thus the skipping of exons X and Y, which predicts synthesis of a truncated, soluble Ob-R protein lacking transmembrane and cytoplasmic regions (Fig. 3 C), appears due to alternative mRNA splicing rather than to genomic mutation.
The five common sequence variants were distributed across the coding region, four being in the extracellular and one in the intracellular portion of the human Ob-R protein. These variants could be detected on genomic DNA by analysis of PCR-restriction fragment length polymorphism (RFLP) generated by gene amplification with specific PCR primers followed by digestion with restriction enzymes (Fig. 4 ). Although the codon 223 variant creates an MspI restriction site, the other four variants neither create nor abolish restriction sites. We therefore introduced a sequence mismatch in the 3' region of one of each pair of PCR primers, which created a new restriction site for HaeIII, MluI, AccII and NcoI for the variants at codons 109, 343, 656 and 1019, respectively (see Materials and Methods).
aNumbers on each haplotype from left to right correspond to alleles at codons 109, 223, 343, 656 and 1019 variants respectively [allele 1, wild-type allele (13); allele 2, variant allele]. Only haplotypes with a frequency >1% at least in one group are shown. No significant difference is observed (P >0.1) between the two groups.
All five common variants were in strong linkage disequilibrium in the white British male population. The gametic linkage disequilibrium coefficient D between variants at codons 109 and 223, 223 and 343, 343 and 656, and 656 and 1019 was 0.148 ([chi]2 = 209), 0.087([chi]2 = 78.2), -0.023([chi]2 = 8.54) and 0.095 ([chi]2 = 98.7) respectively (all equivalent to P <0.005). The distribution of genotypes for the common variants were in Hardy-Weinberg equilibrium except for the codon 223 variant which showed slight deviation ([chi]2 = 4.87; P <0.05). Haplotype frequencies were estimated and compared between the obese and lean groups (Table 2 ). All haplotype frequencies were similar in both groups. Because of the strong linkage disequilibrium, only six out of the 32 possible haplotypes account for >95% of all haplotypes in both the obese and lean groups.
Analysis of variance was used to examine the association between each genotypic variant and BMI, skinfold thickness, and obesity-related metabolic phenotypes including plasma insulin, glucose and triglycerides [fasting and 2 h oral glucose tolerance test (O-GTT)] in the 322 white British males. Since the frequency of the homozygous genotype for each variant allele (genotype 2/2) was similar in both the obese and lean groups and was relatively low (Table 1 ), analysis was performed comparing subjects with the genotype homozygous for the wild-type allele (genotype 1/1) and those with the other genotypes (1/2 and 2/2). No significant association was observed between OB-R genotypes and obesity related-phenotypes (data not shown) or obesity except between BMI and variation at codon 656 (Table 3 ). Within the lean group, mean BMI was 0.58 kg/m2 lower in subjects with genotype 1/1 for codon 656 variation (P = 0.017). The significance of any of these results was unchanged after adjusting for age in multiple regression analysis. When we compared genotype frequencies in the lower and upper halves of the lean group (BMI <20.9 or 20.9-22.0 kg/m2), genotype 1/1 for codon 656 was significantly overrepresented in the lower half [P = 0.015; odds ratio = 2.72 (95% CI = 1.21-6.11)]. Comparison of genotype frequencies in the upper and lower halves of the obese group (BMI >31.5 or 28.0-31.4 kg/m2) revealed no significant differences.
In this study, we have shown that the OB-R cDNA sequence can be obtained from human peripheral blood lymphocytes and, using this approach, have identified five common DNA sequence variants in the OB-R cDNA coding sequence of 22 morbidly obese subjects of Northern European or American Indian origin. It is unlikely that any of these variants are null mutations causing morbid obesity, because homozygotes for the variant sequences were found at a similar frequency in obese and lean subjects. Considering the results of the haplotype analysis and the linkage disequilibrium observed between the five variants, it is also unlikely that there is a common OB-R gene haplotype or mutation associated with obesity in the general white British male population. Finally, the results of the present association study provided no evidence for a relationship between the five OB-R gene variants and either obesity or obesity-related phenotypes. Taken together, these findings indicate that mutations in the OB-R gene are not a common cause of human obesity, at least in Northern European males, although this does not exclude the possibility that OB-R gene mutations are a cause of rare familial forms of inherited obesity or of obesity in females. In this regard, a recent sib-pair analysis in Pima Indians showed no evidence for linkage of obesity to polymorphic microsatellite markers flanking the human OB-R gene (24 ).
Association studies are very powerful for detecting the phenotypic effects of genetic variants, but hidden population stratification is generally claimed to cause false positive associations. This possibility is minimized in our study, because the sample was drawn from a representative population-based survey and restricted to a homogeneous group. Furthermore, the comparison of extremes increased the power of our analysis. We therefore conclude that the overall contribution of OB-R gene variants to the development of obesity is minor, if any, in the white British male population. The significant association of codon 656 with BMI (P = 0.017) that we observed only within the lean group may, however, suggest a possible association of variation at codon 656 not with development of obesity, but with maintenance of low body weight. Because this was the only significant result among 50 tests, it may have arisen by chance alone. Confirmation of this observation must therefore await detailed family studies and association studies in other populations.
Our results indicate that it is difficult to explain human obesity on the basis of common mutations in the human OB-R gene. However, abnormalities in the post-receptor signalling mechanisms or aberrant splicing of OB-R mRNA may be present in obese subjects. For example, several shorter isoforms that appear defective in mediating leptin action have been shown to be generated by alternative splicing in mice and humans (13 -17 ). Therefore cis- or trans-acting factors that influence splicing may reduce expression of functional leptin receptor and hence lead to leptin resistance and obesity. In this context, the novel alternative splicing event observed in one obese patient (Fig. 3 ) is of interest. We do not have evidence that the same splicing event occurs in the hypothalamus of this patient. However, if this is the case, obesity in this patient may in part be a consequence of alternative splicing of OB-R mRNA.
Despite the recent discovery of genetic mutations in the leptin and leptin receptor system as a cause of obesity in several rodent models, no such genetic abnormality has thus far been found in this system in obese humans. Elucidation of the genetic component of human obesity is an important step toward development of novel and safe therapeutic strategies for treating patients with obesity.
Total cellular RNA was isolated from peripheral blood lymphocytes of 22 subjects with BMI >35 kg/m2, defined here as morbidly obese, (range = 35.1-60.9 kg/m2) and amplified by RT-PCR as described previously (25 ) with some modifications. RNA (1 [mu]g) was reverse-transcribed in 25 [mu]l of reaction mixture with 100 pmol of random hexamer primers. After incubation for 5 min at 95oC, 1/10 of the products were amplified by PCR with each of six pairs of oligonucleotide primers (Table 4 A), which resulted in the amplification of six cDNA fragments covering the entire coding region of human OB-R cDNA (Fig. 1 ). Amplification was performed during 35 cycles of PCR (94oC 30 s, 50oC 45 s, 72oC 60 s) with 1.5 mM MgCl2 concentration, followed by an additional extension (72oC 5 min). In the PCR amplification from lymphocytes frozen for a long period, an additional preceding PCR amplification was carried out with primers external to the above PCR products (Table 4 B). PCR products (50 ng) were sequenced by cycle-sequencing with Taq polymerase FS dye-terminator sequencing kits (Perkin-Elmer) on a model 373A or 377 automated DNA sequencer (Applied Biosystems).
Several OB-R gene fragments could be amplified from leukocyte DNA by PCR using primers with exonic sequences. A 14 kb gene fragment comprising introns W, X and Y (Fig. 3 ) was amplified by a Gene Amp XL PCR kit (Perkin-Elmer) with a forward primer on exon W (5'-CCATTGAGAAGTACCAGTTC-3') and a reverse primer on exon Z (5'-AGGACCACATGTCACTGATG-3'). DNA sequencing was carried out for the 14 kb fragments from a normal control subject and the obese patient with the abnormal cDNA fragment 5, focusing on the six exon-intron boundaries shown in Figure 4 C.
Genotyping of the five common DNA sequence variants was carried out by PCR-RFLP analysis. Genomic DNA (100 ng in 50 [mu]l reactions) was amplified by PCR with a pair of oligonucleotide primers specific for each sequence variant (Table 4 C), and the PCR products were digested with an appropriate restriction endonuclease (see Fig. 4 ). Reverse primers for the codon 109, 656 and 1019 variants and a forward primer for the codon 343 variant have a sequence mismatch in their 3' region in order to create an artificial restriction site. PCR amplification was performed as described above, except for a MgCl2 concentration of 2.0 mM for the codon 109 assay, 1.25 mM for the codon 343 assay and 1.0 mM for the codon 223 assay.
White British men aged 40-64 years with both parents born in Britain were selected from the 1988-1990 population-based epidemiological survey of factory workers and Family Practitioner lists in Southall, West London (23 ). Of 1262 consecutively sampled white males from the original survey, blood samples were available for DNA extraction from 97% of the males. Because extremes in populations provide the most power to detect genetic effects on quantitative traits, and because we wished to examine differences in genotype frequencies between obese and lean individuals, white males of British descent with BMI >28 kg/m2, defined here as obese, (range = 28.0-51.2 kg/m2; n = 190) or <22 kg/m2, defined as lean, (range = 16.1-22.0 kg/m2; n = 132) were selected for genotyping. The mean age of the obese subjects was 52.4 (SD 7.9) years and of the lean subjects 52.9 (7.2) years. The study was approved by the relevant local research ethics committees. Informed written consent was obtained from all study subjects.
Allelic frequencies were estimated by gene counting. The distribution of genotypes was tested for Hardy-Weinberg equilibrium by [chi]2 analysis. Haplotype frequencies and the gametic linkage disequilibrium coefficient D were estimated by maximum likelihood using the EH program and tested for significance by a likelihood ratio [chi]2 (26 ). Differences in BMI, total skinfold thickness (as a second measure of adiposity and defined as the sum of subscapular, suprailiac, anterior thigh, suprapatellar skinfold thickness), plasma insulin, glucose and triglycerides (fasting and 2 h O-GTT) and frequency of impaired glucose tolerance and diabetes were assessed by analysis of variance according to leptin receptor genotype. Analyses of insulin, glucose and triglycerides used log-transformed values. The effects of genotype on BMI were also examined in least-squares regression models, with age and genotype as predictor variables in an F-test with two degrees of freedom. All statistical analyses were performed using the STATA program (Stata Corp). Because of the multiple tests performed on several obesity-related phenotypes, it is likely that a few results will be significant at the 5% level by chance alone.
We thank Ms. A. Thomson for assistance with sample handling, Drs E. Ravussin, P. Froguel and B. Guy-Grand for blood samples from Pima Indians and French subjects, and Drs W. Cookson and C. Shoulders for critical reading of the manuscript. T.G. was supported by a Manpei Suzuki Diabetes Foundation Fellowship. B.S.M. was a recipient of an EMBO short-term fellowship. A.P.G. is a UK MRC Clinical Training Fellow.
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*To whom correspondence should be addressed. Tel: +44 181 383 4253; Fax: +44 181 383 2028; Email: taitman@rpms.ac.uk
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Table 3
Table 4