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From The Clinical Research Centers |
Physical Activity Sciences Laboratory (Y.C.C., J.G., L.P.), Laval University, Ste-Foy, Québec, Canada, G1K 7P4; Department of Health and Kinesiology (J.W.), Texas A&M University, College Station, Texas 77843-4243; Washington University School of Medicine (I.B.B., D.C.R.), Division of Biostatistics, St. Louis, Missouri 61330; School of Nutrition and Public Health (G.R.C.), Deakin University, Geelong 3217, Victoria, Australia; School of Kinesiology and Leisure Studies (A.S.L.), Minnesota University, Minneapolis, Minnesota 55455; Department of Kinesiology (J.S.S.), Indiana University, Bloomington, Indiana 47405; and Pennington Biomedical Research Center (M.C., C.B.), Louisiana State University, Baton Rouge, Louisiana 70808
Address correspondence and requests for reprints to: Yvon C. Chagnon, Ph.D., Physical Activity Sciences Laboratory, Kinesiology, PEPS 0212, Laval University, Ste-Foy, Québec, Canada, G1K 7P4. E-mail: yvon.chagnon{at}kin.msp.ulaval.ca
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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P 0.0001). Males, not females,
showed differences across genotypes for the same phenotypes plus SF8
and leptin (0.03 P 0.0002). Carriers of
the R223 allele showed higher values than noncarriers for BMI (+4 U,
P = 0.0001), SF8 (+30 mm, P =
0.01), FM (+7 kg, P = 0.0004), %FAT (+5%,
P = 0.0002), and leptin (+4 ng/mL,
P = 0.0006). These results indicate a significant
effect of leptin receptor on adiposity in middle-aged Caucasian males. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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No linkages were observed between markers in the vicinity of
LEPR and adiposity in Pima Indians (13, 14) and in French
subjects (15), whereas positive linkages were observed in the
Québec Family Study (16). On the other hand, some 19
polymorphisms have been reported in the human LEPR among the 20
different exons and introns of the gene (15, 17, 18, 19, 20, 21, 22, 23, 24). The potential
effects of these polymorphisms have been evaluated in different
populations, with few positive results. In Pima Indians, allele
frequencies were shown to be different between 10 lean and 10 obese
subjects for 2 intronic and 1 exonic nucleotide changes
(P = 0.003) and for haplotypes of the same 3 allelic
variants and a Q223R substitution (P = 0.001) (20).
Association was also reported between a pentanucleotide
insertion/deletion polymorphism in the 3'untranslated region of
LEPR and insulin levels in obese subjects, particularly in females, in
the fasted state (P = 0.0004) or after an oral glucose
tolerance test (P = 0.02) (15, 24). In both cases,
carriers of the insertion allele showed lower insulin values. In the
Québec Family Study, linkages (0.004 P
0.02) were observed between LEPR and different adiposity and body
composition variables, the strongest results being observed between
Q223R and fat mass (FM; P = 0.004) and between a CTTT
repeat in intron 16 and fat free mass (FFM; P = 0.006)
(25). In the latter study, carriers of the Q223 allele had 4 kg less
FFM (P = 0.005) in males with a body mass index
(BMI) < 27 kg/m2. The same was true for
carriers of the less frequent allele at a CTTT repeat
(P = 0.005) in women with a BMI 
27
kg/m2 (25). On the other hand, negative results
were reported for Q223R and for other polymorphisms in American (17, 26); British (21), except for a weak association between a K656N
substitution and BMI in lean (BMI < 22
kg/m2) male subjects (P = 0.02);
Danish (22); Japanese (23); and French (15) populations.
We have analyzed three polymorphisms (K109R, Q223R, and K656N) located in exons 4, 6, and 14 of LEPR to test for linkage and association with adiposity and body composition variables in the HERITAGE Family Study. The HERITAGE cohort includes Caucasian subjects, but also blacks who have not been investigated yet for variation in LEPR.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The HERITAGE Family Study cohort has been previously described (27). HERITAGE included nonascertained (according to obesity) black and Caucasian nuclear families from the greater Québec City; Phoenix, AZ; Minneapolis, MN; Austin, TX; and Indianapolis, IN areas. Subjects were tested for a battery of morphometric and physiological variables before and after a 20-week exercise program. The study protocol had been previously approved by the Human Subjects Committee at each participating institution. Informed written consent was obtained from each subject. Only baseline data, i.e. before the exercise program, were used for the present study. Blood samples were obtained for various biochemical assays, and permanent lymphoblastoid cell lines were established for the extraction of DNA. A total of 319 black subjects (88 parents and 231 offspring) from 115 families and 522 Caucasians (192 parents and 330 offspring) from 99 families were available for the study. Dependent variables include BMI (weight in kg divided by height in m2) and percent body fat (%FAT) estimated from body density measurements obtained by underwater weighing and the equations of Siri (28) and Lohman (30) for Caucasian men and women, respectively, and of Schutte (31) and Ortiz (32) for black men or women, respectively. FM (kg) and FFM (kg) were calculated from %FAT body fat and body weight. Pulmonary residual volume was assessed by the helium dilution technique (33) or oxygen dilution (34, 35) techniques. Subcutaneous fat (mm) was estimated by the sum of height skinfold thicknesses (SF8 = abdominal, subscapular, suprailiac, medial calf, triceps biceps, midaxillary, and thigh). Leptin level (ng/mL) was evaluated by an RIA (Linco Research, Inc., St. Charles, MO) in which the lowest quantity detectable was 0.5 ng/mL in plasma.
Molecular analysis
Genomic DNA was prepared from permanent lymphoblastoid cells by the proteinase K and phenol/chloroform technique. DNA was dialyzed four times against TE buffer (10 mmol/L Tris, 1 mmol/L EDTA, pH 8.0) for 6 h at 4 C, and ethanol was precipitated. The three restriction fragment length polymorphisms analyzed have been described elsewhere (19). PCR was performed on a Perkin-Elmer Corp. 9600 apparatus using 100 or 200 ng genomic DNA, 300 nmol/L of each primer, 200 µmol/L deoxynucleotides, and 0.5 U Taq polymerase in PCR buffer (Roche Molecular Biochemicals, Laval, SC) with 1.5 mmol/L MgCl2, for a final vol of 10 µL. PCR cycles consisted of 40 cycles at 94 C for 30 sec, annealing at 55 C for 30 sec, and extension at 72 C for 30 sec, with a final extension of 10 min at 72 C. PCR products were digested for 12 h at 37 C with 5 U HaeIII, 5 U MspI, or 5 U Bst UI restriction enzymes, an isoschizomer of the Mvn I enzyme used originally (19), for the K109R, Q223R, and K656N polymorphisms, respectively. The resulting fragments were separated on 2.5–3% agarose gels.
Statistical analysis
Phenotypic variables were adjusted, within race, sex and age groups, with the latter defined as lower then 35, between 35 and 50, and more than 50 yr old, for age, age2, and age3 using a regression procedure in which outliers (±3 SD) were excluded for the estimation of the regression parameters. Residuals from all subjects, including outliers, were then standardized to a mean of 0 and an SD of 1. The sibpair linkage analysis was performed on nuclear families using the SIBPAL version 3.0 software from S.A.G.E. (Statistical Analysis for Genetic Epidemiology) (36) with the population allele frequencies estimated, by race, from unrelated subjects. Association studies were undertaken on all subjects from both generations, because pooling all family members in each race produced unbiased residuals, even in the presence of extremely correlated clusters (Province MA, Rice T, Rao PC, unpublished data). Phenotypes were compared between genotypes using covariance analysis with the same covariates as for linkage analysis plus clinical center of origin. A chi-square test was used to compare allele frequencies and genotype distributions between black and Caucasian subjects and to test for Hardy-Weinberg equilibrium of the genotype distribution of the polymorphisms. Statistical Analysis System (version 6.08) for PC was used for the analysis.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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. A Hardy-Weinberg equilibrium is
observed for the three polymorphisms and their haplotypes within both
races. The allele frequencies for Caucasians in the HERITAGE Family
Study are similar to those reported in the Québec Family Study,
whereas blacks differ significantly from Caucasians for genotype and
allele distributions at the K109R polymorphism (Table 2). The allele frequencies are comparable
with those observed in British (21), Danish (22), American (26), and
French (20) Caucasian populations. Both blacks and Caucasians from the
HERITAGE Family Study, as well as Caucasians from other populations,
exhibit differences for K109R with Pima Indians, and for Q223R with
Japanese and Pima Indians (Table 1). Therefore, allelic variations in
LEPR are characterized by a significant race component with
black, Japanese, and Pima Indian populations, showing specific allelic
frequency differences among them, whereas Caucasian subpopulations
exhibit homogeneous frequencies. Similar differences in haplotype
frequencies (Table 1B) are observed between blacks and Caucasians in
HERITAGE, with four out of the six observed haplotypes showing
significant differences (0.04 P 0.001). The
three polymorphisms are also strongly in linkage disequilibrium in
Caucasians (
2 = 166.38; 7 df;
P < 0.0001) and weakly in blacks
(2 = 18.07; 7 df; P < 0.02).
Taken two by two, K109R and Q223R showed the strongest disequilibrium
in Caucasians (2 = 114.04; df = 3;
P < 0.001), whereas no disequilibrium were observed in
blacks (P > 0.05).
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. Mean BMI for unrelated subjects of
both sexes from the parental generation was 28.8
kg/m2 in blacks (n = 88; range, 19–43) and
28.0 kg/m2 in Caucasians (n = 192; range,
19–48 kg/m2). In parents, normal-weight
(BMI < 25 kg/m2), overweight (25
BMI < 30 kg/m2), and obese (BMI 30
kg/m2) subjects (38) are present in both blacks
(22%, 43%, and 35%, respectively) and Caucasians (27%, 42%, and
31%, respectively), with no significant difference in the distribution
between the two groups (2 = 1.099; 2 df;
P = 0.58). Similarly, no significant differences in the
leptin levels, adjusted for age and sex, were observed between black
and Caucasian parents. Black children had a lower mean BMI, of 27.7
kg/m2 (n = 231, range, 17–51), and a
different BMI distribution (42%, 27%, and 32%, respectively) than
their parents. Similarly, Caucasian children are leaner than their
parents, with a mean BMI of 24.6 kg/m2 (n =
330, range, 17–44 kg/m2) and a BMI distribution
of 63%, 24%, and 13%, respectively. Fifty seven percent (black
males) to 74% (Caucasian males) of the leptin variance is explained by
the FM of the subjects.
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), the number of sibpairs varied,
depending on marker and phenotype, with a range from 75–119 pairs in
blacks and 317–364 pairs in Caucasians. Suggestive linkages were
observed in Caucasians for K109R with BMI (P = 0.02)
and FM (P = 0.05), with borderline P values
of 0.10 for SF8 and 0.11 for FFM. No statistically significant linkage
was observed in blacks.
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). No
evidence of association was observed in blacks, and in Caucasians for
the K109R and K656N polymorphisms. Association analyses for Q223R were
also performed in Caucasians for each generation and sex separately
(Table 5). Only parents were characterized by significant associations
with BMI, FM, and %FAT (0.04 P 0.0001).
Moreover, in Caucasian parents, males (not females) showed differences
for the same phenotypes, plus SF8 and leptin (0.03
P 0.0002). In these subjects, carriers of the R223
allele showed higher values than noncarriers for BMI (+4 units,
P = 0.0001), SF8 (+30 mm, P = 0.01), FM
(+7 kg, P = 0.0004), %FAT (+5%, P =
0.0002), and leptin (+4 ng/mL, P = 0.0006). Haplotype
analysis (data not shown) yielded weak evidence of association in
Caucasians only, with carriers of the K109/Q223/K656 haplotype showing
a lower BMI (-1 unit, P = 0.04) and FM (-2 kg,
P = 0.04), and carriers of the R109/R223/K656 haplotype
a slightly higher BMI value (+1 unit, P = 0.05) than
other haplotypes.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Here, we report weak evidence of linkages for K109R in Caucasians with
BMI and FM (Table 4
) but without association (data not shown). In
contrast, no linkage was observed for Q223R, but strong associations
were found (Table 5). We obtained the same kind of nonconsistent
results in the Québec Family Study for Q223R, which showed strong
positive linkages with adiposity phenotypes but negative associations
(25). This is what is expected under linkage equilibrium. For instance,
polymorphims K109R and Q223R are in strong linkage in Caucasians
(2 = 114.04, 3df; P < 0.001)
but not in blacks (2 = 7.33, 3 df;
P > 0.05). Evidence for linkage does not depend on
which allele is shared by sibs. In contrast, associations are detected
by the apparent effects of a marker with such a functional mutation.
Consequently, linkage can be observed with a specific locus without
allelic association and vice-versa, particularly as here, when the two
polymorphisms are in linkage disequilibrium. Moreover, association
analysis is more sensitive than linkage analysis, and so association
can be detected without linkage. The Q223R polymorphism exhibited
evidence of an association in Caucasians only, and, more particularly,
in parents (Table 5). Caucasian adult offspring are leaner than their
parents, as reflected by mean BMI and BMI classes distribution. They
are also younger, with no overlap in age range (Table 3b). Similar
differences are observed in blacks (Table 3a). Differences in the
association results between black and Caucasian parents could come from
a race effect on LEPR expression, because biological characteristics,
such as BMI and age, are similar. The difference between blacks and
Caucasians for allele and haplotype distributions, and the presence
(Caucasians) or absence (blacks) of linkage disequilibrium between the
markers, support the hypothesis that two different gene pools have been
sampled. These differences in LEPR expression could come from
modulating or permissive factors acting on different genetic
backgrounds, as has been described in rodent models of obesity (39, 40, 41). In Caucasians, associations were observed only in males,
whereas younger and leaner subjects showed no effect of LEPR
polymorphisms.
Negative results have been reported for the Q223R polymorphism in other
Caucasian populations. In a study of American subjects, a comparison
across genotypes was performed for obese subjects only, with males and
females pooled together (26). Similarly, a negative association with
BMI was reported in British males with a BMI < 28
kg/m2 or with a BMI 28
kg/m2, analyzed separately (21). In the
present study, if Caucasian male parents are divided into the same two
BMI categories (BMI < 28 kg/m2
vs. BMI 28 kg/m2), no
associations with Q223R are observed with BMI (P = 0.23
and P = 0.21, respectively). Finally, in a Danish
study, (22) homozygotes for Q223 allele were not included in the
comparison of BMI across Q223R genotypes, and we observed here
that the main differences are precisely between carriers of the R223
allele and the Q223Q homo-zygotes.
Two duplicate cytokine domains (C domain) have been reported in LEPR (2) that represent two putative leptin binding regions. The Q223R substitution is located in exon 6 within the first C domain. The Zucker rat mutation in the LEPR (Leprfa) is located in the first C domain of LEPR (19). Leprfa involves the substitution of a glutamine (Q) residue at position 269 (270 in humans) for a proline (P) residue, which affects the functionality of the receptor (42, 43, 44, 45). The Q223R polymorphism has been shown, in the present study, to be associated with adiposity variables such as BMI, SF8, FM, %FAT, and leptin. The Q223R polymorphism was also linked to BMI, SF6, and FM in the Québec Family Study (25). It could be hypothesized that the single amino acid change, a glutamine for an arginine, observed at the residue 223 in human exon 6, changes the signaling capacity of LEPR, as is observed for the Leprfa mutation in rat (42, 43, 44, 45). This single substitution effect could be comparable with the arginine to tryptophane substitution at codon 105 in the human LEP gene, which is sufficient to impair the normal processing of leptin through the secretory pathway (7).
Associations in the HERITAGE Family Study and linkages in the
Québec Family Study were detected for the Q223R polymorphism but
without reciprocal linkages in the present study or associations in the
Québec Family Study. Differences in the proportion of lean,
overweight, or obese subjects among the two groups are observed (27%,
42%, and 31%, respectively in HERITAGE vs. 48%, 27%, and
25%, respectively in the Québec Family Study). On the other
hand, subjects from the Québec Family Study were exclusively of
French descent, from the immediate region of Québec City, whereas
Caucasians from the HERITAGE Family Study were recruited in
Québec City but also at three centers in the United States. When
the two subgroups are analyzed separately, it can be seen that similar
(BMI, P = 0.004 vs. 0.02; FM,
P = 0.07 vs. 0.05) and divergent (SF8,
P = 0.52 vs. 0.12; %FAT, P
= 0.46 vs. 0.09; leptin, P = 0.59
vs. 0.07) results are observed between subjects from the
United States and Québec. Fewer Caucasians are available from the
Québec Clinical Center, compared with the United States Clinical
Centers (
70 vs. 120) for these analyses, but higher mean values of
the different phenotypes for the R223 allele carriers are noted in both
subsamples of Caucasians (data not shown).
In conclusion, there is a significant effect of the Q223R LEPR polymorphism on adiposity in humans. The effect is observed among middle-aged male Caucasians only, with carriers of the R223 allele exhibiting higher mean adiposity values. The specific effect of the Q223R substitution on the functionality of the LEPR remains to be investigated.
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Footnotes |
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2 Partially supported by the Henry L. Taylor endowed Professorship in
Exercise Science and Health Enhancement.
3 Supported by the Donald B. Brown Research Chair on Obesity funded
by the Medical Research Council of Canada and Hoffmann-La Roche Canada.
Received December 21, 1998.
Revised June 25, 1999.
Revised August 23, 1999.
Accepted September 3, 1999.
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