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Human Molecular Genetics Pages 675-680

Structure and sequence variation at the human leptin receptor gene in lean and obese Pima Indians
Introduction
Results
Discussion
Materials And Methods
Acknowledgements
References

Structure and sequence variation at the human leptin receptor gene in lean and obese Pima Indians

Structure and sequence variation at the human leptin receptor gene in lean and obese Pima Indians D. Bruce Thompson*, Eric Ravussin, Peter H. Bennett and Clifton Bogardus

Phoenix Epidemiology and Clinical Research Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 4212 N 16th Street, Phoenix, AZ 85016, USA

Received November 21, 1996; Revised and Accepted February 3, 1997

The cloning of human and mouse cDNAs from brain that encode high affinity leptin receptors was recently reported. We have physically localized the human leptin receptor gene (LEPR) to a region at 1p31, between the anonymous microsatellite markers D1S515 and D1S198. The genomic structure of the human leptin receptor gene, corresponding to the published human brain cDNA sequence, spans over 70 kb and includes 20 exons. Since the leptin receptor gene is a candidate gene for obesity, and because of its proximity to D1S198, a marker previously linked to insulin secretion, the LEPR gene was sequenced in 20 non-diabetic Pima Indians chosen for extremes in percent body fat and in their acute insulin response to intravenous glucose. Seven polymorphic sites were identified. Two of these polymorphisms, Lys109Arg and Gln223Arg, are amino acid substitutions in the extracellular domain of the leptin receptor, one polymorphism is a silent substitution, and four occur in non-coding regions of the leptin receptor. Four of these sites are in linkage disequilibrium with one another. Nucleotides at three non-coding polymorphic sites were found exclusively in obese Pima Indians. This demonstrates an association between variation at the leptin receptor gene and obesity in humans.

INTRODUCTION

The Pima Indians of Arizona have the highest reported prevalence of non-insulin-dependent diabetes mellitus (NIDDM) in the world, and obesity is also prevalent (1 ,2 ). The Pimas farmed along the banks of the Gila and Salt Rivers in central Arizona for ~2000 years (3 ). In this arid environment, cycles of feast and famine were frequent, depending on the availability of water (3 ). As originally hypothesized by Neel (4 ), natural selection may have favored a metabolically efficient phenotype, based on a `thrifty genotype' that increased the chances of surviving periods when food was scarce. The resulting thrifty phenotype could be characterized by a lower rate of energy expenditure and/or hyperphagia. Either or both of these characteristics would result in positive energy balance and increase fat stores during `times of plenty' which would be advantageous for survival during periods of drought and limited food supplies. However, in westernized societies, where food is plentiful, and typically high fat, this `thrifty genotype' would be disadvantageous, resulting in marked obesity and increased risk of NIDDM.

Two mouse models of obesity, ob and db, are recessively inherited mutations which are characterized by increased rates of insulin secretion, low rates of energy expenditure, hyperphagia, obesity and diabetes (5 ). Parabiosis experiments between control, ob/ob and db/db animals led Coleman to hypothesize that the ob/ob mouse lacked a circulating satiety factor and that food regulatory centers in the brain of the db/db mouse were unresponsive to the same factor (6 ). These genes encoding this circulating satiety factor, now called leptin, and its receptor, were recently cloned, and mutations have been identified that result in absence of leptin production in fat cells in the ob/ob mouse and decreased expression of this receptor in the db/db mouse (7 ). The human leptin (LEP) and leptin receptor genes (LEPR) are therefore good candidate genes potentially resulting in thrifty phenotypes in the Pima Indians. Studies of the leptin gene in the Pima Indians did not reveal any amino acid substitutions (8 ), so we have now examined the leptin receptor gene.

RESULTS

The human leptin receptor gene is located ~1.5 Mb telomeric from D1S198 (9 ,10 ). Based on comparison of the cDNA sequence and the genomic sequence, the human brain leptin receptor gene is encoded in 20 exons and spans over 70 kb of DNA (Fig. 1 ). The first two exons are non-coding and are capable of forming several alternative secondary structures. Exon 1 has a GC content of 70% and includes two 8 bp palindromes beginning at positions 28 and 57 (11 ). Homology between the rodent and human leptin receptor begins in exon 3, which contains the initiation codon and the putative signal sequence that is also encoded in exons 3 and 4. A newly described imperfect dinucleotide repeat is located in the third intron, ~230 nucleotides (nt) downstream of exon 3 (11 ). In a sample of 88 chromosomes genotyped in the Pima, only two alleles were observed with frequencies of 0.49 and 0.51. The single putative transmembrane region lies in exon 18 and the previously described splice site for the alternate forms of the leptin receptor occurs at the exon/intron boundary of exon 19 (13 ). The intracellular domain is encoded in exons 19 and 20. Exon 20, the largest exon, spans over 900 nt and encodes the last 274 amino acids of the leptin receptor (Fig. 1 ). The intron/exon boundaries conform to the established consensus sequences and all the splice donor and acceptor sites contain GT or AG dinucleotides, respectively (14 ).


Figure 1. The genomic structure of the LEPR gene. The gene is composed of 20 exons and spans >70 kb of DNA. The exons are represented as vertical bars at double scale and are numbered above each exon. Estimated intron sizes are shown below except for intron 2 whose size is unknown. Polymorphisms in the coding region are shown below the map and are identified by their amino acid number. Polymorphisms in non-coding regions are shown above the map and identified by the number of nucleotides before or after their respective exons.

Twenty Pima Indians were chosen for sequence analysis of the leptin receptor gene. These persons were not first degree relatives, all had normal glucose tolerance, were from the upper and lower quartiles of percent body fat, adjusted for age and sex, and from the upper and lower quartile of the acute insulin response (AIR) (Table 1 ). The AIR is a measure of pancreatic [beta]-cell function in response to intravenous glucose and is decreased or lost with the onset of glucose intolerance or diabetes (15 ). PCR primer pairs developed from intron sequences of the leptin receptor gene were used to amplify the intervening exons from Pima Indian genomic DNA (Fig. 1 ). These PCR products were sequenced and compared with the known leptin receptor cDNA sequence to screen for nucleotide variation in the coding region of LEPR (16 ).

The Pima Indian consensus coding sequence did not differ from the reported Caucasian sequence except at three positions. At position 326 in exon 4 an A -> G transition in codon 109 results in a lysine to arginine amino acid substitution (Fig. 2 ). As previously reported in Caucasians, an A -> G transition in the second position of codon 223, at position 668 in exon 6 of the leptin receptor gene, results in the substitution of an arginine for a glutamine (17 ). This amino acid substitution lies 46 amino acids upstream from the missense mutation found in the fatty Zucker rat (18 ). A final mutation in the coding region was identified at position 3057 in exon 20, amino acid 1019. This silent transition does not change the proline at this position.

In addition, four polymorphic nucleotide positions in non-coding regions were identified. Thymine/adenine transversions are present 36 and 85 nt preceding the beginning of exon 17. Thirty- seven and 52 nt downstream of exon 19 are two additional non-coding polymorphic sites, an A -> C transversion at position 37 and a C -> T transition at position 52. The frequencies of these polymorphisms in Pima Indians and Caucasians are shown in Table 2 . The allele frequencies at each polymorphic site were significantly different between populations, except for the -85 and the +52 substitutions, which were observed at low frequencies in both populations.

Table 1 Phenotypes and genotypes of Pima subjects
 

 Sex

 %Fat

 AIR

 326

 668

 -36

 +37

 3057
1

 F

 high

 high

 G/G

 G/A

 A/T

 A/C

 A/G
2

 M

 high

 high

 G/A

 G/A

 A/A

 A/A

 A/A
3

 F

 high

 high

 G/G

 G/G

 A/A

 A/A

 A/A
4

 M

 high

 high

 A/A

 G/A

 A/A

 A/A

 A/A
5

 F

 high

 high

 A/A

 G/A

 A/T

 A/C

 A/G
6

 M

 high

 low

 G/A

 G/A

 A/T

 A/C

 A/G
7

 M

 high

 low

 A/A

 A/A

 T/T

 C/C

 G/G
8

 M

 high

 low

 G/G

 G/G

 A/A

 A/A

 A/A
9

 F

 high

 low

 G/G

 G/A

 A/T

 A/C

 A/G
10

 M

 high

 low

 G/G

 G/G

 A/A

 A/A

 A/A
11

 M

 low

 high

 G/A

 G/G

 A/A

 A/A

 A/A
12

 F

 low

 high

 G/A

 G/G

 A/A

 A/A

 A/A
13

 F

 low

 high

 A/A

 G/G

 A/A

 A/A

 A/A
14

 M

 low

 high

 G/G

 G/G

 A/A

 A/A

 A/A
15

 M

 low

 high

 G/A

 G/G

 A/A

 A/A

 A/A
16

 M

 low

 low

 G/A

 G/G

 A/A

 A/A

 A/A
17

 M

 low

 low

 G/A

 A/A

 A/A

 A/A

 A/A
18

 M

 low

 low

 G/G

 G/G

 A/A

 A/A

 A/A
19

 M

 low

 low

 -/-

 G/G

 A/A

 A/A

 A/A
20

 M

 low

 low

 G/A

 G/G

 A/A

 A/A

 -/-
P value (lean versus obese)

 0.79

 0.235

 < 0.003

 < 0.003

 < 0.003
Genotypes at five polymorphic sites identified in the leptin receptor gene in Pima Indians. The groups are divided on the basis of percent body fat and and the AIR. The obese group (high % fat) averaged 40 +- 5 % body fat (individuals 1-10) and the lean group (low % fat) averaged 23 +- 5 % (individuals 11-20). Persons with a high log10 AIR averaged 2.8 +- 0.2 [mu]U/ml (individuals 1-5 and 11-15) and persons with low AIR averaged 1.7 +- 0.1 [mu]U/ml (individuals 6-10 and 16-20) (31). The nucleotides found at each polymorphic site are in the following columns. The sites are identified by their position in the cDNA relative to the start of translation, for substitutions within the coding region, 326, 668 and 3057. Polymorphic sites that are found in non-coding regions are identified by the number of nucleotides either upstream (-, upstream of exon 17) or downstream (+, downstream of exon 19) of that exon, -36 and +37. Underlined nucleotides represent the two haplotype alleles. The bold nucleotides including position 668 further define a larger haplotype. Polymorphisms at frequencies <= 0.05 are not shown. (-/-), Genotypes not determined.The [chi]2 analysis of the distribution of alternate nucleotides at each polymorphic site in lean and obese groups are: position 326, P = 0.79; position 668, P = 0.235; positions -36, +37 and 3057, P = 0.003.

Table 2 . Allele frequencies of polymorphic sites in Pima Indians and Caucasians
 

 326

 668

 -36

 -85

 +37

 +52

 3057
Pima Indians

 G = 0.58

 G = 0.75

A = 0.85

T = 0.95

A = 0.85

C = 0.97

A = 0.85
 

 A = 0.42

 A = 0.25

 T = 0.15

 A = 0.05

 C = 0.15

 T = 0.03

 G = 0.15
Caucasians

 G = 0.28

 G = 0.44

A = 0.36

T = 0.89

A = 0.35

C = 1.00

A = 0.38
 

 A = 0.72

 A = 0.55

 T = 0.64

 A = 0.11

 C = 0.65

 T = 0.00

 G = 0.62
Allele frequencies in Pima Indians and Caucasians for the seven polymorphic sites in the leptin receptor gene. In the Pima, frequencies are based on the sample of 20 individuals. In Caucasians, the frequencies are based on a subset of the CEPH pedigrees including 56 individuals (26). Allele frequencies at each of the nucleotide positions were significantly different between Pima Indians and Caucasians (P = 0.001) except nucleotide positions -85 and +52.

Table 3 Frequency of haplotype alleles in Pima Indians and Caucasians
 

 Haplotype

 Pima

 Caucasian
1

 GAAA

 0.81 (26)

 0.16 (9)
2

 AAAA

 0.125 (4)

 0.11 (6)
3

 GTCG

 0.00 (0)

 0.28 (16)
4

 ATCG

 0.0625 (2)

 0.45 (25)
Each haplotype is based on the nucleotides at the polymorphic posititons 668, -36, +37 and 3057. The frequency (number) of each haplotype is based on 32 unambiguous chromosomes in the Pima and on 56 chromosomes in Caucasians. Haplotypes 2 and 3 would result from intragenic recombination between alleles 1 and 4. Haplotype differences between groups are statistically significant (P < 0.001).


Figure 2. The locations of the seven polymorphisms described in this study. Polymorphisms in exons 4, 6 and 20 are shown with the published cDNA sequence. The codon number is underneath, followed by the corresponding amino acid. The alternative nucleotide at each polymorphic site is shown above the cDNA sequence and the substituted amino acid is shown on the last line. Polymorphisms located within introns 16 and 19 are shown as the cDNA sequence with the alternative nucleotide above each polymorphic site.

The Lys109Arg substitution at position 326 is not associated with either percent body fat, the acute insulin response nor any of the other nucleotide substitutions in this sample of 20 Pima Indians. Except for a single individual, the Gln223Arg substitution at position 668 was found exclusively in obese persons, but the allele frequency was not significantly different between the obese and lean groups (P = 0.235), nor was there any association with AIR (Table 1 ). The nucleotide substitutions at each of the polymorphic sites -36, +37, and 3057, however, were associated with differences in percent body fat ([chi]2, lean versus obese individuals, P = 0.003) but not with differences in the AIR. The non-coding nucleotide substitutions at -85 and +52 were in low frequency and not associated with obesity or AIR.

Nucleotide substitutions at four positions (668, -36, +37 and 3057) display linkage disequilibrium (P <0.001) and identify haplotypes at the leptin receptor, two of which are shown in bold in Table 1 , e.g. individuals 7 and 8. The nucleotides adenine, thymine, cytosine, and guanine (ATCG) define one haplotype and another is defined by the nucleotides guanine, adenine, adenine, adenine (GAAA). A recombination event between position 668 (codon 223) and -36 has disrupted the two haplotypes and produced the additional genotype AAAA, Table 1 , individual 17. Based on the frequency in Pima Indians, the two ancestral haplotypes are GAAA and ATCG. These four sites also display linkage disequilibrium in a sample of CEPH individuals (P <0.001), although the frequencies of the different haplotypes differ significantly between Pima and Caucasians (Table 3 ). When the smaller three site haplotypes are considered (underlined genotypes in individuals 7 and 8, Table 1 ), all lean individuals are characterized by the AAA haplotype, while the obese group shares the other haplotype combinations. The TCG haplotype was genotyped in the parents of individual 7 and was inherited in a Mendelian fashion.

DISCUSSION

The structure of the leptin receptor gene spans 70 kb and includes 20 exons. The full length mRNA in humans, which corresponds to Ob-Rb in mice, is ~5.1 kb in length (16 ). The other previously identified alternative mRNAs match well with the genomic structure of the leptin receptor (13 ). Murine Ob-Ra mRNA corresponds to a loss of splicing at the intron/exon boundary at the end of exon 19, followed by translation of the intron. The conceptional translation of intron 19 agrees with the published amino acid sequence for Ob-Ra. Ob-Rc and Ob-Rd are also both spliced at the boundary of exon 19, but small exons are spliced onto the end of these messages. These small exons should lie somewhere within the 14 kb intron that follows exon 19. Ob-Re, a proposed secreted form of the leptin receptor, would result from loss of splicing of intron 16 and translation of intron 16.

The LEPR initiation sequence poorly matches the consensus Kozak sequence for eukaryotic transcription start sites; only the ATG and the purine at the -3 position are present (12 ). The fidelity and frequency of translation depend upon 5' sequences in the mRNA (19 ,20 ). The poor Kozak consensus sequence at the ATG of the cDNA open reading frame might interfere with translation. There are an additional two potential start sites upstream, both of which are out of frame. This unusual feature, found in less than 10% of vertebrate mRNAs, might also be part of the initiation complex. The first of these additional potential start sites lies within one of the palindromic sequences. The two palindromes have [Delta]Gs of approximately -23 kcal/mol, well within the ability of the 40S ribosomal subunit to melt; this could slow the 40S subunit allowing the use of the first AUG as an initiation site. This would predict some 5' variability in the leptin receptor messages produced.

Previously we reported genetic linkage between the acute insulin response (AIR) and the microsatellite marker D1S198 (22 ). Due to the close physical distance between the leptin receptor locus and a putative genetic element controlling the AIR, the leptin receptor is also a candidate for the AIR genetic element. The earliest observable diabetic phenotype in the db/db mouse is hyperinsulinemia followed by obesity and [beta]-cell failure (23 ). Recently it was reported that the leptin receptor is expressed on [beta]-cells, indicating that leptin may directly effect insulin secretion (24 ). However, the lack of any association between any of the identified polymorphisms and the AIR in the 20 subjects studied indicates that genetic variation in the coding region of the leptin receptor gene is unlikely to explain the previously identified linkage between this genomic region and insulin secretory function in Pima Indians.

The two amino acid substitutions located at the 5' end of the leptin receptor gene and in the extracellular domain of the leptin receptor were not associated with differences in body fat in this sample (Table 1 ). In contrast, the two non-coding polymorphisms and the silent substitution which cluster at the 3' end of the leptin receptor gene were only found in obese Pima Indians. This suggests that the putative mutation in obese Pima Indians could lie in the 3' end of the LEPR gene. Since the nucleotide substitutions comprising these three sites do not alter the coding region of the leptin receptor, they may act as a marker for another, as yet unidentified, mutation that alters some other aspect of leptin receptor function and could involve transcription, mRNA stability or translational differences. Conversely, this haplotype may act as a marker for another, as yet unidentified, gene in linkage disequilibrium with this haplotype, or the small sample size of 20 individuals may not be large enough to reveal an association between one of the amino acid substitutions and obesity or AIR. However, leptin receptor gene expression is similar in obese and lean individuals, suggesting mRNA stability is unlikely to account for the associations (21 ). Since the frequency of obesity is much less prevalent in Caucasians than Pima, the high frequency of the TCG haplotype (73%) in Caucasians indicates that the association between this haplotype and obesity is restricted to Pima Indians. The recombination event that has disrupted the larger haplotype may account for the lack of association between obesity and the missense mutation at codon 223 in Pima Indians. This observation may also explain why an earlier study failed to find an association between BMI (body mass index) and the missense mutation at codon 223 (21 ).

As part of a proposed system that regulates adipose mass, the leptin receptor is an obvious candidate for a `thrifty gene' as envisioned by Neel (4 ). Although previous evidence for linkage between percent body fat and D1S198 was weak (P = 0.05) (25 ), the identification of a haplotype within the leptin receptor associated with obesity in Pima Indians strengthens this observation and is evidence for another, as yet unidentified, mutation that plays a role in the thrifty phenotype. We speculate that this putative mutation may act to alter the expression of the leptin receptor protein, presumably in areas of the brain either decreasing rates of energy expenditure or increasing food intake that results in increased fat stores and increased chances of survival in a desert environment.

MATERIALS AND METHODS

Twenty Pima Indian volunteers who are participating in longitudinal studies of NIDDM were admitted to the Clinical Diabetes and Nutrition Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (26 ,27 ). Written informed consent was obtained from all subjects who were then given physical examinations. Subjects were healthy, taking no medications and were non-diabetic. DNA for sequencing was extracted from Epstein-Barr virus transformed lymphocytes, or from blood samples drawn in conjunction with other studies (28 ). Body fat was determined by hydrostatic underwater weighing and corrected for age and sex (29 ). AIR (the log10 of the mean plasma insulin concentration above basal, over 3, 4, and 5 min after the injection of 25 g of intravenous glucose) was determined in all individuals (30 ). The subjects were divided into four groups based on percent body fat and acute insulin response. Percent body fat +- SD averaged 40 +- 5 and 22 +- 8 in the obese and lean groups, respectively. The AIR averaged 2.7 +- 0.2 and 1.7 +- 0.1 [mu]U/ml in the high and low insulin secretion groups.

Oligonucleotide primers were designed from the cDNA sequence and used to sequence across the intron/exon boundaries of the 20 exons of the leptin receptor gene. YAC clones containing the leptin receptor gene (9 ) were obtained from Research Genetics (Huntsville, AL). XL PCR (Perkin Elmer, Norwalk, CA) was performed according to manufacturers' instructions. Alu-vector PCR and Inverse PCR were also used to isolate the ends of YAC arms (26 ). In some instances extra long PCR was combined with inverse PCR to isolate large segments of the leptin receptor. PCR products were amplified in parallel from Pima genomic DNA and the yeast artificial chromosomes 940H12, 930F4 and 895B12 containing the LEPR gene. All PCR products were sequenced to assure they were from the leptin receptor. Statistical analyses were performed using programs of the SAS institute (Cary, NC).

Oligonucleotide primers for sequence analysis were designed on the basis of the intron sequence, synthesized on an Applied Biosystems oligonucleotide synthesizer (model 391, Applied Biosystems, Foster, CA), and used to PCR amplify the corresponding sequence from Pima genomic DNA. DNA sequence analysis was carried out with an ABI 377 automated sequencer (Perkin Elmer, Norwalk, CA). PCR templates for sequence analysis were separated and isolated from 0.8% agarose gels and purified using QIAquick gel extraction kits (Qiagen, Chatsworth, CA). The individual exons and intron sequence flanking each exon have been submitted to GenBank under the following accession numbers: exon 1, U59246; exon 2, U59247; exon 3, U59248; exon 4, U59249; exon 5, U59250; exon 6, U59251; exon 7, U59252; exon 8, U59253; exons 9 and 10, U59254; exon 11, U59255; exon 12, U59256; exons 13 and 14, U59257; exon 15, U59258; exon 16, U59259; exon 17, U59260; exon 18, U59261; exon 19, U59262; exon 20, U59263. The following oligonucleotides were used to amplify regions of the leptin receptor:

exon 2F, AGAAGGTTATGCAGCCATNCTACTATC;

exon 2R, CCACAAAGGGACTGTGTTCATAAACTG;

exon 3F, TAAATTNAGAGACTTATCTATAATCCC;

exon 3R, TAACTAGAAATAGGAAATTCTGTTAGC;

exon 4F, CACATTGTACAATGGAAGCACAAAGTT;

exon 4R, TGTTAAAATCATAGCCATAAGACATCT;

exon 5F, TTTTTTTTAATTCAGATGCAAACTGGA;

exon 5R, TGCAAGAGGTTTATAATCTACTTTCCGT;

exon 6F, ACCCTTTAAGCTGGGTGTCCCAAATAG;

exon 6R, AGCTAGCAAATATTTTTGTAAGCAATT;

exon 7F, TGACTTTATTTTATTCAGCTATAATTG;

exon 7R, GTAATTGCTATGGGACTTAAGAGGGTC;

exon 8F, TTTTAGTAACGGTTCCACATCAACTTG;

exon 8R, CAAGTTCAGGAAATATATTTCCCTCCC;

exon 9F, CAGAATGTTTGTCTTCATCTGATATCC;

exon 9R, CATTCAAGTTGTGGAACAAAATGAACA;

exon 10F, TCATTGAATTTTTTGGAGATTTTATGC;

exon 10R, CAGGAAGCAAATAATCTGTAAGAC;

exon 11F, GACTGCTGTTTTAAACAACAAATCAG;

exon 11R, CTGCATACAAATCTGCTAACACAAATG;

exon 12F, TGAAAATATAACACAATGTTTTTAGGC;

exon 12R, TTTATGCCAATAAAATTAATCTAATGC;

exon 13F, GGTTTAAAATAAAATGTACTTCAGGGC;

exon 14R TGGACCATGAAGTCTTTTTAAGAGTA;

exon 15F, GAGACTGTGGCAGAGGCAAACTATATC;

exon 15R, ATTGCAGGCTGCTTGAAAGATAATTTA;

exon 16F, TTCCCTTTAGTAGGTTATAAGTTCCTC;

exon 16R, TTTTTGAAGTTTTCATTAACTGGCTAT;

exon 17F, TTTGGAAACTCCCTTGATAATTTAATC;

exon 17R, CCTCACCATGAAAAATCTACAGAAGAC;

exon 18F, AGAGATTTGTGATGAATTCAGAAAATG;

exon 18R, TTAAATCAGGGTTTGAATACGCGTAAG;

exon 19F, CGGAGGCATAGTTGATCTGGTGGCTAA;

exon 19R, GCGCTTGAAATTTGTTTCTTCCTGATT;

exon 20F, CAAACTTCCATTTTCTGCCAGTATGAC;

exon 20R, CATTGGTAGGCTTATGAAGGCTTTCAC.

ACKNOWLEDGEMENTS

We wish to acknowledge the technical and analytical assistance of Warren Apel, Victoria Ossowski, Jeff Sutherland, Jennifer Biesterfeildt, Larry Grahmn and Rachel Janssen. We thank the clinical staff of the Clinical Diabetes and Nutrition Section for collection of the phenotypic information and Dr Michal Prochazka for helpful comments and discussion. Finally, we wish to thank the members of the Gila River Indian Community, without whose assistance and dedication this work would not be possible.

REFERENCES

1 Knowler W.C., Bennett P.H., Hamman, R.F. and M. Miller. (1978) Diabetes incidence and prevalence in Pima Indians: a 19-fold greater incidence than in Rochester, Minnesota. Am. J. Epidemiol. 108, 497-505.

2 Knowler, W.C., Pettitt, D. J., Saad, M. F., Charles, M. A., Nelson, R. G., Howard, B. V., Bogardus, C. and P. H. Bennett. (1991) Obesity in the Pima Indians: its magnitude and relationship with diabetes. Am. J. Clin. Nutr. 53, 1543S-1551S. MEDLINE Abstract

3 Fontana, B.L. (1979) Handbook of North American Indians. Smithsonian Institution. Volume 10, pages 125-135. Fink, T.M. The prehistoric Irrigation canals and reservoirs of southern Arizona and their possible impact on Hohokam health. Health and Disease in the Prehistoric Southwest. Edited by Charles F. Mervs and Rover J. Miller, Arizona State University Anthropological Research Papers, number 23, 359-379.

4 Neel. J.V. (1962) Diabetes mellitus: a `thrifty' genotype rendered detrimental by progress. Am. J. Hum. Genet. 14, 353-362.

5 Coleman, D.L. (1978) Obese and diabetes: Two mutant genes causing diabetes-obesity syndromes in mice. Diabetologia 14, 141-148. MEDLINE Abstract

6 Coleman, D.L. (1973) Effects of Parabiosis of obese with diabetes and normal mice. Diabetologia 9, 294-298. MEDLINE Abstract

7 Chau, S.C., Chung, W.K., Wu-Peng, S., Zhang, Y., Liu, S., Tartaglia, L. and R. L. Leibel. (1996) Phenotypes of mouse diabetes and rat fatty due to mutations in the OB (leptin) receptor. Science 271, 994-996.

8 Maffei, M., Stoffel, M., Barone, M., Dammerman, M., Ravussin, E., Bogardus, C., Ludwig, D.S., Flier, J.S., Talley, M., Auerbach, S. and J.M. Friedman. (1996) Absence of mutations in the human OB gene in obese/diabetic subjects. Diabetes 45, 679-682. MEDLINE Abstract

9 Thompson. D. B., Sutherland, J., Appel, W. and V. Ossowski. (1997) A physical map at 1p31 encompassing the acute insulin response locus and the leptin receptor. Genomics 39, 227-230.

10 Winick, J.D., Stoffel, M. and J.F. Friedman. (1996) Identification of microsatellites linked to the human leptin receptor gene on chromosome 1. Genomics 36, 221-222. MEDLINE Abstract

11 Thompson, D.B., Ossowski, V., Sutherland, J., Apel, W. and Biesterfeldt, J. (1996) Human leptin receptor (LEPR) gene, exons 1-20. GenBank Accession numbers U59246-U59263.

12 Kozak, M. (1986) Influences of mRNA secondary structure on initiation by eukaryotic ribosomes. Proc. Natl. Acad. Sci. USA 83, 2850-2854. MEDLINE Abstract

13 Lee, G.H., Proenca, R., Montez, J.M., Carroll, K.M., Darvishzadeh, J.G. and J.M. Friedman. (1996) Abnormal splicing of the leptin receptor in diabetic mice. Nature 379, 632-635. MEDLINE Abstract

14 R.A. Padgett, P.J. Grabowski, M.M. Konarska, S. Seiler and P.A. Sharp. (1986) Splicing of messenger RNA precursors. Annu. Rev. Biochem. 55, 1119-1150.

15 Yallow, R.S. and W.A. Bauman. (1990) Plasma insulin in health and disease. In Rifkin, H. and Porter, D. Jr (eds), Diabetes mellitus: Theory and Practice. New York, Elsevier p 119-142.

16 Tartaglia, L.A., Dembski, M., Weng, X, Deng, N, Culpepper, J., Devos, R., Richards, G.J., Campfield, L.A., Clark, F.T., Deeds, J., Muir, C., Sanker, S., Moriarty, A., Moore, K.J., Smutko, J.S., Mays, G.G., Woolf, E.A., Monroe, C.A. and Tepper, R.A. (1995) Identification and expression cloning of a leptin receptor, OB-R. Cell 83, 1263-1271. MEDLINE Abstract

17 Cioffi, J.A., Shafer, A.W., Zupancic, T.J., Smith-Gbur, J., Mikhail, A., Platika, D. and Snodgrass, H.R. (1996) Novel B219/OB receptor isoforms: Possible role of leptin in hematopoiesis and reproduction. Nature Med. 2, 585-589.

18 Chau, S.C., White, D.W., Wu-Peng, X.S., Lui, S., Okada, N., Kershaw, E.E., Chung, W.K., Power-Kehoe, L., Chau, M., Tartagila, L.A. and Leibel, R.L. (1996) Phenotype of fatty due to Gln269Pro mutation in the leptin receptor (Lepr). Diabetes 45, 1141-1143.

19 Kozak, M. (1989) Circumstances and mechanisms of inhibition of translation by secondary structure in eucaryotic mRNAs. Mol. Cell. Biol. 9, 5134-5142. MEDLINE Abstract

20 Kozak, M. (1987) An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125-8148. MEDLINE Abstract

21 Considine, R.V., Considine, E.L., Williams, C.J. and Caro, J.F. (1996) The hypothalamic leptin receptor in humans-identification of incidental sequence polymorphisms and absence of the db/db mouse and fa/fa rat mutations. Diabetes 45, 992-994. MEDLINE Abstract

22 Thompson, D.B., Janssen, R.C., Ossowski, V.M., Prochazka, M., Knowler, W.C. and Bogardus, C. (1995) Evidence for linkage between a region on chromosome 1 and the acute insulin response in Pima Indians. Diabetes 44, 478-481. MEDLINE Abstract

23 Hummel, K.P., Dickie, M.M. and Coleman, D. (1996) Diabetes, a new mutation in the mouse, Science 153, 1127-1128.

24 Kieffer, T.J., Jeller, R.S. and Habner, J.F. (1996) Leptin receptors expressed on pancreatic [beta]-cells. Biochem. Biophys. Res. Comm. 224, 522-527. MEDLINE Abstract

25 Norman, R.A., Leibel, R.L., Devoto, M., Knowler, W.C., Thompson, D.B., Bogardus, C. and Ravussin, E. (1996) Linkage of obesity and energy metabolism to markers flanking homologues of rodent obesity genes is not evident in Pima Indians. Diabetes 45, 1299-1232.

26 Lillioja, S., Mott, D.M., Spraul, M., Ferraro, R., Foley, J.E., Ravussin, E., Knowler, W.C., Bennett, P.H. and Bogardus, C. (1993) Insulin resistance and insulin secretory dysfunction as precursors of non-insulin-dependent diabetes mellitus. N. Engl. J. Med. 329, 1988-1992. MEDLINE Abstract

27 Knowler, W.C., Pettitt, D.J., Saad, M.F. and Bennett, P.H. (1990) Diabetes mellitus in the Pima Indians: incidence, risk factors and pathogenesis. Diabetes Metab. Rev. 6, 1-27. MEDLINE Abstract

28 DeFronzo, R.A., Bonadonna, R.C. and Ferrannini, E. (1992) Pathogenesis of NIDDM: a balanced overview. Diabetes Care 15, 318-68. MEDLINE Abstract

29 Siri, W.E. (1961) Body composition from fluid spaces and density: analysis of methods. pp.223-244. Brozek, J and Henschel, A. In Techniques for Measuring Body Composition: Proceeding of a Conference. National Research Council. Washington DC, USA.

30 Janssen, R., Bogardus, C., Takeda, J., Knowler, W.C. and Thompson, D.B. (1994) Linkage analysis of acute insulin secretion with GLUT2 and glucokinase in Pima Indians and the identification of a missense mutation in GLUT2. Diabetes 43, 558-563. MEDLINE Abstract

31 Zoghbi, H. and Chinault, C. (1994) Generation of YAC contigs by walking. In D.L. Nelson and B.H. Brownstein(eds) YAC Libraries a Users Guide. W.H. Freeman and Company, New York pp 93-112.

*To whom correspondence should be addressed. Tel: +1 602 200 5300; Fax: +1 602 200 5335; Email: bthompson@phx.niddk.nih.gov

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