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Brief Genetics Reports Exonic and Intronic Sequence Variation in the Human Leptin Receptor Gene (LEPR') Wendy K. Chung, Loraine Power-Kehoe, Melvin Chua, Florence Chu, Louis Aronne, Zilla Huma, Melinda Sothern, John N. Udall, Bowie Kahle, and Rudolph L. Leibel I ncreased adiposity is a major risk factor for cardiovascular disease and NIDDM (1). Genetic determinants of the degree of adiposity and body fat distribution have been demonstrated by twin and adoption studies, and the heritability (h2) of obesity has been estimated to be as high as 0.90 (2). However, the major genes underlying the heritable contribution to body fatness in humans have remained elusive. Rodent models of genetically determined obesity provide excellent candidate genes for evaluation in humans. The linkage of obesity-related phenotypes in humans to genomic regions homologous to rodent leptin (Lep) (3) and leptin receptor (Lepr) (4,5) has been recently demonstrated. The recent cloning of Lepr, which is mutant in the diabetes (Lepr^) mouse and in fatty (Leprfa) and Koletsky (Leprfqf) rats (6-9), the mapping of this gene (LEPR) to Ip32 in humans (10), and the description of the genomic structure of LEPR and two polymorphic intronic microsatellites (11) have provided the necessary reagents for the evaluation of LEPR in the genetics of human obesity. Because the phenotype associated with genetic defects in Lepr in Leprdb/Leprdb mice and Leprfa/Leprfa rats is profound early-onset obesity, we sought to identify allelic variations in LEPR, which may be responsible for the genetic variation in adiposity in humans. To maximize the likelihood of the detection of such sequence variants, we examined genomic DNA from a total of 229 obese and lean adults and children, ascertained in medical centers around the U.S. (New York, New York; New Orleans, Louisiana; and Huntington, West Virginia). The study sample was predominantly Caucasian, but also From the Laboratory of Human Behavior and Metabolism (W.K.C., L.P.-K., M.C., E C , L.A., R.L.L.), Rockefeller University, New York, New York; the Department of Pediatrics (Z.H.), Cornell University Medical College, New York, New York; the School of Medicine in New Orleans (M.S., J.N.U.), Louisiana State University Medical Center, New Orleans, Louisiana; and the Department of Biological Sciences (B.K.), Marshall University College of Science, Huntington, West Virginia. Address correspondence and reprint requests to Dr. Rudolph L. Leibel, 1230 York Ave., Box 181, New York, NY 10021. E-mail: [email protected]. Received for publication 7 January 1997 and accepted in revised form 8 May 1997. PCR, polymerase chain reaction; SSCA, single-strand conformation analysis. DIABETES, VOL. 46, SEPTEMBER 1997 included blacks and Hispanics, ascertained through several pediatric and adult endocrinology and obesity centers throughout the U.S. Adiposity was assessed by BMI (determined as weight [in kilograms] divided by height [in meters] squared). Twenty milllliters of blood was drawn for leukocyte isolation and plasma leptin determination (12). The initial study population comprised 167 obese adults and children (mean BMI, 42.0 kg/m2) and 27 lean adults and children (mean BMI, 23.2 kg/m2). We used single-strand conformation analysis (SSCA) to screen all subjects for variation in coding exons 5 and 18, the exons containing, respectively, the sequence that is mutant in the fa rat (Leprfa) (13) and the sequence that is incorrectly spliced in the db mouse (Leprdb) (Fig. 1) (7). Genomic DNA was extracted from whole blood, using standard methodology (13). Polymerase chain reactions (PCRs) and sequencing were performed as previously described (11). Primers for all 18 coding exons were as previously described (11) or divided into smaller fragments (primer sequences available by request). Of the PCR reaction, 5 ul was heat denatured, immediately placed on ice, and electrophoresed on a 6% polyacrylamide gel with or without 10% glycerol at 5 W at room temperature for 24 h. We also screened all 18 coding exons of the "long" form of LEPR ("Rb") containing the membrane spanning domain (6,14) in 32 morbidly obese individuals (BMI, >50 kg/m2), including five children, by SSCA. In addition, we screened 14 morbidly obese adults (mean BMI, 66.5 kg/m2) by measuring fasting plasma leptin concentrations with a solid-phase sandwich enzyme immunoassay (12). These plasma leptin concentrations were compared with regression plots (by sex and menopausal status) of plasma leptin versus BMI for a group of 67 lean and obese individuals (12). Two female subjects were identified with plasma leptin concentrations that were 36 and 98% above those predicted for BMI. In these two individuals, all 18 coding exons of the long form of LEPR were directly sequenced and compared with a normal weight control subject. No unique exonic sequence variants were detected in the two obese subjects with relative elevations in plasma leptin concentrations. As indicated in Fig. 1, using PCR primer pairs located in sequences flanking the 18 coding exons of LEPR (11), we identified by SSCA and direct sequencing three allelic variants associated with amino acid changes (LyslO9Arg, Gln223Arg, 1509 SEQUENCE VARIATION IN LEPR Lys109Arg Exonic variants A-?G 519 Qln223Arg Lys 666 Asn A-5>G G-3>C 861 1222 2161 <-31 bp <r-A2 bp Intronic variants Location of rodent mutations 1 <-37bp A-S-G Lepr* 5 2 Lepr1 Lepr' 6 10 11 12 13 14 15 16 17 18 Coding exon FIG. 1. Summary of all of the nucleic acid variants detected in the human leptin receptor gene (LEPR*). The number of each coding exon is indicated along the bottom of the figure. Exons 5,14, and 18, which are altered in Leprfa (Zucker/attf/ rat), LeprfaS (Koletsky rat), and Lepr db (dft mouse), respectively, are indicated. Nucleotides are numbered according to the LEPR long form (GenBank accession number U43168) (6). Allelic variants within exons are indicated above the genomic structure, and variants within introns are indicated below the genomic structure. Locations of intronic variants are indicated as the number of base pairs from the indicated splice junction. Amino acids alterations are indicated for nonsilent allelic variants. and Lys656Asn), three silent mutations (nt 1222 T^C, nt 3217 A-*G, and nt 3250 G-»A), and four intronic sequence variants (Fig. 1). Thus, a total of six differences from the originally published LEPR cDNA sequence (6) were detected, three of which have been previously reported (nt 519/LyslO9Arg, nt 861/Gln223Arg, and nt 3250) (15). Of the three nucleotide alterations that produce amino acid changes, Gln223Arg and Lys656Asn result in changes in charge (neutral to positive and positive to neutral, respectively) and are, therefore, the mostly likely to have functional consequences. In addition, all three amino acids (Lys 109, Gln223, and Lys656) are conserved among rat, mouse, and human species (6,16). It will be necessary to test each amino acid variant alone and in combination, using in vitro binding and signal transduction assay systems to determine their effects on leptin binding and signal transduction (8). Although many polymorphisms were observed in obese patients, notably absent were any mutations that would result in an obvious loss of function such as nonsense, frameshift, or deletion/insertion mutations. Although mutations in specific splice variants as observed in the Lepr^ mouse (7,14) are more directly analyzed with mRNA, no alterations in splice sites were noted. Also, Considine et al. (15) found no gross alterations in the size or quantity of LEPR message, or cDNA sequence, in hypothalamic mRNA obtained from eight obese human subjects. However, not all intronic sequences were analyzed in our subjects; therefore, undetected mutations producing novel splice sites may exist. The allele frequencies for the three coding sequence variants were determined in the original 167 obese and 27 lean subjects originally used to examine exons 5 and 18. The allele frequencies, by racial group and obesity phenotype, are shown in Table 1. There is little difference in allele frequency by racial group. Formal association and sib-pair studies of earlyonset, extreme, and milder degrees of obesity with multiple phenotypic measures related to in vitro studies of receptor binding and signal transduction will be necessary to evaluate the possible clinical significance of these three amino acid variants in LEPR completely. Such studies are underway (17). The present genetic analysis was limited to the coding sequence of a single splice ("long" form) variant of LEPR. However, other splice variants and relevant regulatory sequences remain unexplored. Multiple markers, including two intronic microsatellites, are now available spanning the length of LEPR (11) and provide the necessary reagents for association studies and haplotype analysis to determine whether, in a particular population, there are likely to be sequence variants (in linkage disequilibrium with particular alleles of the polymorphic markers) that could account for a significant component of relevant phenotypic variance. Note added in proof. Since this paper was submitted, Echwald et al. (Int J Obes 21:321-326,1997) reported additional sequence variants in LEPR (Phel7Leu and ValllOMet), neither associated with juvenile-onset obesity. Thompson et al. (Hum Mol Genet 6:675-679, 1997) reported an association TABLE 1 Allele frequencies of variant alleles of LEPR producing amino acid alterations Allelic position Percentage of subjects; ARG 109/G 519 ARG 223/G 861 ASN 656/C 2161 Asian 1.0% Black 11.3% Caucasian 67.5% Hispanic 20.1% Lean 13.9% Obese 86.1% 25 50 0 21.9 37.7 11.4 21.0 33.6 16.8 22.9 36.6 14.8 26.3 42.7 19.5 20.2 32.7 14.6 A total of 167 obese (mean BMI, 42.0 kg/m2) and 27 lean (mean BMI, 23.2 kg/m2) subjects originally used for mutation detection in exons 5 and 18 were subsequently genotyped for the three amino acid variants of LEPR shown in Fig. 1. Genotypes were determined for the exon 2 variant by restriction digestion analysis using Hae III with a modified reverse primer, which introduces a Hae III site in the ARG 109 allele; for the exon 4 variant by restriction digestion analysis using Msp I, which restricts the ARG 223 allele; and for exon 12 by SSCA. Allele frequencies by race or obesity phenotype are presented. 1510 DIABETES, VOL. 46, SEPTEMBER 1997 W.K. CHUNG AND ASSOCIATES between sequence variation in noncoding regions of LEPR with obesity in Pima Indians. ACKNOWLEDGMENTS This work was supported by National Institutes of Health grants DK52431, 2P30-DK26687, DK47473, T32-DK07559, GCRC RR00102, and HG00008 and by the Nutritional Research Institute. We gratefully acknowledge the leptin assays performed by Dr. Margery Nicolson (Amgen); DNA oligonucleotide synthesis and sequencing provided by Camille Camisso, Maria Pospichil, and Renata Lee (the DNA Core facility of Rockefeller University); assistance in patient collection by Mindy Schreier, Michael Wajnrajch, and Emelia Hinds; and assistance in manuscript preparation by Sarita Whitehead. REFERENCES 1. Stern MP: Type II diabetes and its macrovascular complications. In New Horizons in Diabetes Mellitus and Cardiocascular Disease. Schwartz CL, Born GVR, Eds. London, Current Science, 1995, p. 1-10 2. Bouchard C: The Genetics of Obesity. Boca Raton, FL, CRC Press, 1994 3. Duggirala R, Stern MP, Mitchell BD, Reinhart LJ, Shipman PA, Uresandi OC, Chung WK, Leibel RL, Hales CN, O'Connell P, Blangero J: Quantitative variation in obesity-related traits and insulin precursors linked to OB gene region on human chromosome 7. Am J Hum Genet 59:694-703, 1996 4. Thompson DB, Janssen RC, Ossowski VM, Prochazka M, Knowler WC, Bogardus C: Evidence for linkage between a region on chromosome lp and the acute insulin response in Pima Indians. Diabetes 44:478-481, 1995 5. Chagnon YC, Lamothe M, Preusse L, Chagnon M, Gagnon J, Nadeau A, Chung WK, Power-Kehoe L, Chua SC, Leibel RL, Bouchard C: Suggestive genetic linkages between markers on human Ip32-p22 and body fat and insulin levels in the Quebec Family Study. Obes Res 5:115-121, 1997 6. Tartaglia LA, Dembski M, Weng X, Deng N, Culpepper J, Devos R, Richards GJ, Carnpfield LA, Clark FT, Deeds J, Muir C, Sanker S, Moiarty A, Moore KJ, Smutko JS, Mays GG, Woolf EA, Monroe CA, Tepper RI: Identification and expression cloning of a leptin receptor, OB-R. Cell 83:1263-1271, 1995 DIABETES, VOL. 46, SEPTEMBER 1997 7. Chen H, Charlat 0, Tartaglia LA, Woolf EA, Weng X, Ellis SJ, Lakey ND, Culpepper J, Moore KJ, Breitbart RE, Duyk GM, Tepper RI, Morgenstern JP: Evidence that the diabetes gene encodes the leptin receptor: identification of a mutation in the leptin receptor gene in db/db mice. Cell 84:491-495, 1996 8. Chua SC, Chung WK, Wu-Peng XS, Zhang Y, Liu S-M, Tartaglia L, Leibel RL: Phenotypes of mouse diabetes and rat fatty due to mutations in the OB (leptin) receptor. Science 27:994-996,1996 9. Wu-Peng XS, Chua SC Jr, Okada N, Liu S-M, Nicolson M, Leibel RL: Phenotype of the obese Koletsky ( / ) rat due to Tyr763Stop mutation in the extracellular domain of the leptin receptor (Lepr): evidence for deficient plasmato-CSF transport of leptin in both the Zucker and Koletsky obese rat. Diabetes 46:513-518, 1997 10. Chung WK, Power-Kehoe L, Chua M, Leibel RL: Mapping of the OB receptor (OBR) to lp in a region of non-conserved gene order from mouse and rat to human. Genome Res 6:431-438,1996 11. Chung WK, Power-Kehoe L, ChuaM, Lee R, Leibel RL: Genomic structure of the human OB receptor (OBR) and identification of two novel intronic microsatellites. Genome Res 6:1192-1199,1996 12. Rosenbaum M, Nicolson M, Hirsch J, Heymsfield SB, Gallagher D, Chu F, Leibel RL: Effects of gender, body composition, and menopause on plasma concentrations of leptin. J Clin Endocrinol Metab 81:3424-3427, 1996 13. Chua SC Jr, White DW, Wu-Peng XS, Liu S-M, Okada N, Kershaw EE, Chung WK, Power-Kehoe L, Chua M, Tartaglia LA, Leibel RL: Phenotype offatty due to Gln269Pro mutation in the leptin receptor (Lepr). Diabetes 45:1141-1143, 1996 14. Lee G-H, Proenca R, Montez JM, Carroll KM, Darvishzadeth JG, Lee JI, Friedman JM: Abnormal splicing of the leptin receptor in diabetic mice. Nature 379:632-655, 1996 15. Considine RV, Considine EL, Williams CJ, Hyde TM, Caro JF: The hypothalamic leptin receptor in humans: identification of incidential sequence polymorphisms and absence of the db/db mouse and fa/fa rat mutations. Diabetes 45:992-994, 1996 16. Phillips MS, Liu Q, Hammond HA, Dugan V, Hey PJ, Caskey CT, Hess JF: Leptin receptor missense mutation in the fatty Zucker rat. Nahire Genet 13:18-19, 1996 17. Echwald SM, Sorensen TD, Sorensen TIA, Tybjaerghansen A, Andersen T, Chung WK, Leibel RL, Pedersen 0: Amino-acid variants in the human leptin receptor: lack of association to juvenile-onset obesity. Biochem Biophys Res Commun 233:248-252, 1997 1511