| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Original Studies |
Department of Genetics, Southwest Foundation for Biomedical Research (J.E.H., L.A., S.C., S.B., B.D.M., M.C.M., J.W.M., J.B., A.G.C.), and the Division of Clinical Epidemiology, Department of Medicine, University of Texas Health Science Center (M.P.S.), San Antonio, Texas 78245
Address all correspondence and requests for reprints to: Dr. Anthony Comuzzie, Department of Genetics, Southwest Foundation for Biomedical Research, P.O. Box 760549, San Antonio, Texas 78245-0549. E-mail: agcom{at}darwin.sfbr.org
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
MSH, ACTH, and ß-endorphin are derived.
Studies by others have shown that POMC-derived products
are involved in the regulation of appetite and obesity. We have used
polymorphisms in POMC to map its location within the
95% confidence interval of the peak for the linkage signal for the
QTL. We also constructed POMC haplotypes using these
polymorphisms and have found a significant association with normal
variation in leptin levels (P = 0.001). We conclude
that variation in POMC is associated with normal
variation in serum leptin levels, providing further evidence that
POMC may be the leptin QTL previously identified in
Mexican American families. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This region of chromosome 2 containing the major leptin QTL also
contains a strong positional candidate gene for human obesity,
POMC [previously mapped to 2p23 by in situ
hybridization (6, 7)], which codes for the prohormone POMC. POMC is
the precursor for several peptide hormones that are produced by
posttranslational processing, some of which are involved in energy
homeostasis, including MSH, ACTH, and ß-endorphin (8). POMC is
highly expressed in neuronal cells of the arcuate nucleus, a region of
the hypothalamus that is involved in the regulation of energy
homeostasis (8).
In this report, we present further studies of the major QTL for leptin levels in SAFHS families, including placement of additional microsatellite markers in this region of chromosome 2. We also examined genetic variation in POMC to determine the relationship of this strong positional candidate gene to the major leptin QTL. We identified and typed polymorphisms to more precisely map POMC relative to microsatellite markers and the major leptin QTL. In addition, we used the POMC polymorphisms for statistical analysis to directly test for association with leptin levels in the SAFHS families.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Subjects in this study included 337 Mexican Americans (153 males
and 184 females) distributed in 10 families ranging in size from 35–71
individuals. These families are all participants in the San Antonio
Family Heart Study, a broader project designed to investigate the
genetics of risk factors for atherosclerosis, noninsulin-dependent
diabetes mellitus, and obesity (9, 10). Probands and family members for
this analysis were ascertained without regard to obesity or any other
preexisting medical conditions. However, on the basis of the most
recent criteria (11), 72% of these SAFHS participants would be
classified as overweight (body mass index, >25), and 38% would be
classified as obese (body mass index, 
30). Age in this sample ranges
from 18–92 yr, with an average age of 39 yr. Although Mexican
Americans constitute an admixed population, the degree of admixture and
interindividual variation in admixture are predominantly functions of
socio-economic status, with low income individuals exhibiting higher
levels of Amerind admixture (12). We limited the study population to
low income families to minimize genetic heterogeneity due to
differential admixture. All protocols have been approved by the
institutional review board at the University of Texas Health Science
Center (San Antonio, TX).
The phenotype
We measured leptin levels by RIA in serum samples collected
after an overnight (
12-h) fast. The assay was conducted using a
commercially available RIA kit (Linco Research, Inc., St.
Louis, MO) (13). The interassay coefficient of variation for this assay
was 7.4%, a value within the range reported by the manufacturer
(13).
Genotyping
SAFHS family members were typed for 6 additional microsatellite markers (D2S423, D2S1400, D2S1352, D2S1394, D2S1777, and D2S1790) to the 15 previously typed as part of our 20-centrimorgan (cM) genome scan set located in the region of chromosome 2 that contains our peak linkage signal for leptin levels (2). We used lymphocyte DNA samples for PCR with fluorescently labeled primers from the MapPairs Linkage Screening Sets 6 and 8 (Research Genetics, Inc.).
We identified and typed POMC polymorphisms in
lymphocyte DNA samples from SAFHS family members. A known polymorphic
RsaI site in the 5'-flanking region was typed using Southern
blots with a POMC complementary DNA probe (American Type Culture Collection, Manassas, VA; clone plP2) according to
the method of Feder and colleagues (14). We used a PCR-based method to
type a cryptic trinucleotide repeat polymorphism in exon 3 as
previously described (15). We found two new polymorphisms by direct
sequencing of exon 3 sequences from 20 SAFHS participants [C
T at
position 7284, CT at position 7566; numbered according to Takahashi
and colleagues (16)]. For association studies, we simultaneously typed
both polymorphisms in the SAFHS samples using direct nucleotide
sequencing. Exon 3 sequences were amplified from lymphocyte DNA samples
using PCR with a forward primer (5'-AGCCCCGCAGCGATGGT-3') and a reverse
primer (5'-TCCATGCTGCTGTTATTTGACGGCTACG-3'). PCR conditions included
denaturation at 97 C for 5 min, followed by 30 cycles of annealing at
63 C for 40 s, elongation at 74 C for 40 s, denaturation at
97 C for 40 s, and a final elongation step at 74 C for 5 min. The
PCR products were directly sequenced on an automated DNA Sequencer (ABI
model 377) using a reverse primer (5'-CTGCTGTTATTTGACGGCTACG-3') with
the ABI Prism Cycle Sequencing kit (Perkin Elmer Corp.,
Foster City, CA). The CT polymorphism at position 7566 can also be
typed by treatment with the restriction enzyme EarI.
Statistical analysis
We performed variance component linkage analysis as
described by Almasy and Blangero (17) and implemented in the program
package SOLAR. Map distances between markers were estimated using
CRI-MAP (18). We used a measured genotype analysis (19, 20) that has
been implemented in the program package SOLAR (17) to look for evidence
of association between serum leptin levels and polymorphisms in
POMC. Specifically, we compared a model in which the
genotype-specific means were forced to be equal across the
POMC genotypes (e.g. µAA =
µAa = µaa) to a model in which each of
these parameters was estimated separately. We also adjusted for the
effects of age and sex on leptin levels by simultaneously estimating
their effects in each of the models evaluated in the association
analysis. The comparison of the competing models was performed by means
of the likelihood ratio test that yields a test statistic
asymptotically distributed as a 
2 with degrees of
freedom equal to the difference in the number of independent parameters
being estimated in the two models.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To further refine the localization of the leptin QTL (2), we typed
family members for six additional microsatellite markers located in
this region of chromosome 2 and performed multipoint QTL analysis of
leptin levels in the extended families. Figure 1
shows the multipoint linkage curve for
serum leptin levels using the additional microsatellite markers. The
peak LOD score increased to 7.46 (P = 2 x
10-9) at 64 cM (95% confidence interval, 56.0–69.0 cM),
which is substantially higher than our previously reported peak LOD
score of 4.95 (2).
|
We also investigated POMC as a positional candidate
gene that may be responsible for the major QTL for leptin levels that
was originally identified using random markers. We identified and typed
polymorphisms in several regions of POMC in the SAFHS
families. Figure 2 shows a schematic of
POMC and the location of the polymorphisms. We typed a known
polymorphic RsaI site in the 5'-flanking region (14). The
frequency of the R1 allele (absence of the site) was 0.16, and the
frequency of the R2 allele (presence of the site) was 0.84. We also
examined a known cryptic trinucleotide repeat polymorphism in exon 3,
but a preliminary survey showed only limited variability in the SAFHS
population (15). In addition to these known polymorphisms, we searched
for new polymorphisms by direct sequencing of exon 3 in 20 SAFHS
participants. Exon 3 contains the coding sequences for the various
polypeptide products of POMC. We found two nucleotide
substitutions, but only a CT polymorphism at position 7566 (7566C/T)
in the 3'-untranslated region was sufficiently common for further
typing in the SAFHS population. The frequency of the C allele was 0.89,
and the frequency of the T allele was 0.11.
|
shows the location of POMC
relative to the markers and the multipoint linkage curve for
leptin levels. POMC mapped to 50.9 cM from the p-ter of
chromosome 2, with a 95% confidence interval from 45.3–59.0 cM. The
95% confidence interval for the map placement of POMC
overlaps with the 95% confidence interval for the leptin QTL,
indicating that POMC is located within the region of
chromosome 2 that shows the peak linkage signal for serum leptin levels
(Fig. 1). POMC haplotypes are significantly associated with serum leptin levels
We constructed POMC haplotypes using the
RsaI and 7566C/T polymorphisms and tested for association
with leptin levels in the SAFHS participants. The haplotypes and their
relative frequencies in the study group are given in Table 1. The POMC haplotypes were
used to test for association with serum leptin levels using maximum
likelihood-based statistical analysis. This analysis also included
adjustment for significant covariates that affect leptin levels,
including sex and age. The results showed that POMC
haplotypes were associated with significantly different serum levels of
leptin (P = 0.001; Table 1). The homozygotes for the
R1-C haplotype had the lowest leptin level (25.6 ± 2.1 ng/mL).
The homozygotes for the R2-T haplotype had the highest leptin level
(45.0 ± 5.0 ng/mL), almost 2-fold higher than that in the R1-C
homozygotes. In general, the intermediate haplotypes followed the same
pattern, so that the R1 and C polymorphisms were associated with lower
leptin levels, and the R2 and T polymorphisms were associated with
higher leptin levels.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We investigated the role of a positional candidate, POMC, which had previously been mapped to this region of chromosome 2 by in situ hybridization (7). By discovering new variants in POMC and typing known polymorphisms, we were able to apply marker to marker linkage analysis to genetically map POMC to the same region of chromosome 2 containing the maximum LOD of the leptin QTL. In general, genetic mapping is an important step in positional candidate gene analysis, because, like POMC, most such candidate genes have been mapped only by in situ hybridization, and because genetic map position may show natural variation due to different proportions of men and women among study populations. These mapping results provide further support for POMC as a positional candidate gene for the major leptin QTL on chromosome 2.
We also used the POMC polymorphisms to construct haplotypes for statistical analysis to test for direct associations with serum leptin levels in Mexican Americans. We found a significant association of POMC haplotypes with leptin levels (P = 0.001), with almost 2-fold differences between the most disparate homozygotes (R1-C/R1-C vs. R2-T/R2-T). It should be noted that this association study differs from those reported in previous candidate gene studies, because our first criterion for selection of POMC was its proximity to the leptin QTL on chromosome 2 (i.e. POMC is a positional candidate gene). Previous association studies have typically examined candidate genes based largely on a priori assumptions concerning their involvement in relevant physiological processes, and the results were often not replicated in different study populations. One reason for this inconsistency may be the reliance of association studies on linkage disequilibrium between candidate gene markers and a causative mutation that differs according to population histories (a genetic effect). Spurious associations may also arise as a statistical artifact caused by hidden stratification within study populations (a nongenetic effect). Our initial identification of the leptin QTL using linkage analysis that relies only on genetic effects indicates that this association of POMC with leptin levels is due to linkage disequilibrium rather than simply hidden stratification within the population.
In addition to its map position, POMC is a strong candidate
gene for the leptin QTL because of the central role its multiple
hormone products play in energy homeostasis. Recent studies by Krude
and colleagues (21) have shown that rare mutations in POMC
that abolish normal translation and processing of its hormone products
cause a distinct form of early-onset human obesity. We do not yet know
how normal genetic variation in POMC influences leptin
levels, but numerous physiological studies may provide some clues. For
example, ACTH, a product of POMC, acts on the adrenal cortex
to stimulate the production of glucocorticoids. Previous studies have
suggested a relationship of glucocorticoid levels with obesity (22, 23), and molecular studies have shown that glucocorticoids may directly
regulate transcription of the OB gene that encodes leptin
(24, 25). An alternative hypothesis comes from recent studies of the
catabolic role of the hypothalamic melanocortin system, with MSH
strongly implicated in the control of food intake (26, 27, 28, 29, 30, 31, 32). MSH is a
high affinity ligand for both the melanocortin 3 and melanocortin 4
receptors, and deficiency of the melanocortin 4 receptor in mice leads
to hyperphagia and obesity (27, 28, 33). Thus, this association of
POMC haplotype with leptin levels might derive from
variation in the control of food intake and body fat deposition via
variations in melanocortin expression and activity (27, 32, 34, 35, 36).
As far as we know, neither of the polymorphisms used to construct the POMC haplotypes for the association analysis has any direct impact on the expression of POMC or the function its hormone products. The RsaI substitution is located in the 5'-flanking region, but has not been localized with respect to known sequence elements that regulate POMC transcription. The 7566C/T substitution is located in the 3'-untranslated region and does not alter the amino acid sequence of POMC. It is more likely that these polymorphisms are not themselves responsible for the observed association with leptin levels, but are in linkage disequilibrium with another polymorphism that does affect POMC expression or function. Given that the chromosome 2 QTL and POMC haplotypes affect variation in levels of leptin, and changes in the POMC-coding sequences might affect multiple overlapping hormone products involved in other important physiological processes, the causative mutation seems more likely to occur in a regulatory region rather than in the coding sequence. We are now searching for such variants in the POMC regulatory elements in SAFHS subjects that could be responsible for this association with normal differences in serum leptin levels.
|
Footnotes |
|---|
Received January 29, 1999.
Revised May 7, 1999.
Accepted May 12, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-MSH. J Endocrinol. 155:73–78.
This article has been cited by other articles:
 |
|
 |
|
  I. S. Farooqi and S. O'Rahilly Genetics of Obesity in Humans Endocr. Rev., December 1, 2006; 27(7): 710 - 718. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. S. Farooqi, S. Drop, A. Clements, J. M. Keogh, J. Biernacka, S. Lowenbein, B. G. Challis, and S. O'Rahilly Heterozygosity for a POMC-Null Mutation and Increased Obesity Risk in Humans Diabetes, September 1, 2006; 55(9): 2549 - 2553. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. R. Patel Shared genetic risk factors for obstructive sleep apnea and obesity J Appl Physiol, October 1, 2005; 99(4): 1600 - 1606. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Baker, N. Gaukrodger, B. M. Mayosi, H. Imrie, M. Farrall, H. Watkins, J. M.C. Connell, P. J. Avery, and B. Keavney Association Between Common Polymorphisms of the Proopiomelanocortin Gene and Body Fat Distribution: A Family Study Diabetes, August 1, 2005; 54(8): 2492 - 2496. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. S. J. de Souza, A. M. Santangelo, V. Bumaschny, M. E. Avale, J. L. Smart, M. J. Low, and M. Rubinstein Identification of Neuronal Enhancers of the Proopiomelanocortin Gene by Transgenic Mouse Analysis and Phylogenetic Footprinting Mol. Cell. Biol., April 15, 2005; 25(8): 3076 - 3086. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Cai, S. A Cole, R. A Bastarrachea-Sosa, J. W MacCluer, J. Blangero, and A. G Comuzzie Quantitative trait locus determining dietary macronutrient intakes is located on human chromosome 2p22 Am. J. Clinical Nutrition, November 1, 2004; 80(5): 1410 - 1414. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Santoro, E. M. del Giudice, G. Cirillo, P. Raimondo, I. Corsi, A. Amato, A. Grandone, and L. Perrone An Insertional Polymorphism of the Proopiomelanocortin Gene Is Associated with Fasting Insulin Levels in Childhood Obesity J. Clin. Endocrinol. Metab., October 1, 2004; 89(10): 4846 - 4849. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. G. Challis, A. P. Coll, G. S. H. Yeo, S. B. Pinnock, S. L. Dickson, R. R. Thresher, J. Dixon, D. Zahn, J. J. Rochford, A. White, et al. Mice lacking pro-opiomelanocortin are sensitive to high-fat feeding but respond normally to the acute anorectic effects of peptide-YY3-36 PNAS, March 30, 2004; 101(13): 4695 - 4700. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Pomp, M. F. Allan, and S. R. Wesolowski Quantitative genomics: Exploring the genetic architecture of complex trait predisposition J Anim Sci, January 1, 2004; 82(13_suppl): E300 - 312. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. J. F. Loos, P. T. Katzmarzyk, D. C. Rao, T. Rice, A. S. Leon, J. S. Skinner, J. H. Wilmore, T. Rankinen, and C. Bouchard Genome-Wide Linkage Scan for the Metabolic Syndrome in the HERITAGE Family Study J. Clin. Endocrinol. Metab., December 1, 2003; 88(12): 5935 - 5943. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. T. K. Chow and M. C. Phoon MEASUREMENT OF SERUM LEPTIN CONCENTRATIONS IN UNIVERSITY UNDERGRADUATES BY COMPETITIVE ELISA REVEALS CORRELATIONS WITH BODY MASS INDEX AND SEX Advan Physiol Educ, June 1, 2003; 27(2): 70 - 77. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. E. Pritchard, R. L. Oliver, J. D. McLoughlin, S. Birtles, C. B. Lawrence, A. V. Turnbull, and A. White Proopiomelanocortin-Derived Peptides in Rat Cerebrospinal Fluid and Hypothalamic Extracts: Evidence that Secretion Is Regulated with Respect to Energy Balance Endocrinology, March 1, 2003; 144(3): 760 - 766. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. G. Challis, L. E. Pritchard, J. W.M. Creemers, J. Delplanque, J. M. Keogh, J.'a. Luan, N. J. Wareham, G. S.H. Yeo, S. Bhattacharyya, P. Froguel, et al. A missense mutation disrupting a dibasic prohormone processing site in pro-opiomelanocortin (POMC) increases susceptibility to early-onset obesity through a novel molecular mechanism Hum. Mol. Genet., August 15, 2002; 11(17): 1997 - 2004. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Adage, A. J. W. Scheurink, S. F. de Boer, K. de Vries, J. P. Konsman, F. Kuipers, R. A. H. Adan, D. G. Baskin, M. W. Schwartz, and G. van Dijk Hypothalamic, Metabolic, and Behavioral Responses to Pharmacological Inhibition of CNS Melanocortin Signaling in Rats J. Neurosci., May 15, 2001; 21(10): 3639 - 3645. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I S. Farooqi and S. O'Rahilly Recent advances: Recent advances in the genetics of severe childhood obesity Arch. Dis. Child., July 1, 2000; 83(1): 31 - 34. [Abstract] [Full Text]
|
|
|
|
 |
|
 R. A. HEGELE, H. CAO, S. B. HARRIS, B. ZINMAN, A. J. HANLEY, and C. M. ANDERSON Genetic variation in LMNA modulates plasma leptin and indices of obesity in aboriginal Canadians Physiol Genomics, June 29, 2000; 3(1): 39 - 44. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
MSH,