Download Exonic and Intronic Sequence Variation in the Human Leptin

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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,
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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.
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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.
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