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John P. Robarts Research Institute (R.A.H., M.W.H.), London, Ontario, Canada N6A 5K8; and Department of Community Health Sciences, University of Manitoba (T.K.Y.), Winnipeg, Manitoba, Canada R3E 0W3
Address all correspondence and requests for reprints to: Robert A. Hegele, M.D., Blackburn Cardiovascular Genetics Laboratory, Robarts Research Institute, 406–100 Perth Drive, London, Ontario, Canada N6A 5K8. E-mail: robert.hegele{at}rri.on.ca
Abstract
We discovered that rare mutations in LMNA, which encodes lamins A and C, underlie autosomal dominant Dunnigan-type familial partial lipodystrophy. Because familial partial lipodystrophy is an extreme example of genetically disturbed adipocyte differentiation, it is possible that common variation in LMNA is associated with obesity-related phenotypes. We subsequently discovered a common single nucleotide polymorphism (SNP) in LMNA, namely 1908C/T, which was associated with obesity-related traits in Canadian Oji-Cree. We now report association of this LMNA SNP with anthropometric indexes in 186 nondiabetic Canadian Inuit. We found that physical indexes of obesity, such as body mass index, waist circumference, waist to hip circumference ratio, subscapular skinfold thickness, and subscapular to triceps skinfold thickness ratio were each significantly higher among Inuit subjects with the LMNA 1908T allele than in subjects with the 1908C/1908C genotype. For each significantly associated obesity-related trait, the LMNA 1908C/T SNP genotype accounted for between approximately 10–100% of the attributable variation. The results indicate that common genetic variation in LMNA is an important determinant of obesity-related quantitative traits.
AMONG THE INUIT (Eskimos) of Canada, obesity is now as prevalent as it is in the general North American population (1, 2). This new development probably reflects recent, rapid changes in physical activity, diet, and lifestyle (1, 2). Obesity in first nations people is believed to increase the risk for future development of chronic diseases, such as type 2 diabetes and hypertension (3). From a public health perspective, it is important to monitor and, if appropriate, to intervene in the changes in diet and physical activity to influence the development of obesity and associated adverse health effects. A more complete understanding of those endogenous, cultural, and environmental factors that contribute to obesity in aboriginal communities might be the first step toward developing an intervention program that uses both culturally and biologically appropriate strategies.
Obesity is a complex metabolic disorder with a strong genetic component (4). There are many candidate genes for obesity and its related phenotypes. Some genes are candidates for obesity because mutations in them cause rare genetic syndromes affecting adipocyte differentiation (4). For example, patients with autosomal dominant Dunnigan-type familial partial lipodystrophy (FPLD; OMIM 151660) are born with normal adipocyte distribution, but after puberty experience adipocyte degeneration in their extremities, trunk, and gluteal region (5, 6, 7). Subjects with FPLD have insulin resistance preceding the development of diabetes, which is often associated with dyslipidemia and atherosclerosis. Recently, we discovered that mutant LMNA underlies FPLD (8). The mechanisms through which LMNA mutations cause wasting of specific cell types and associated abnormal phenotypes are unknown. However, LMNA is clearly a candidate gene for adipose tissue metabolism.
In addition to the rare LMNA mutations in FPLD, we
identified a common single nucleotide polymorphism (SNP) in exon 10 of
LMNA, namely a silent C
T substitution at nt 1908
(1908C/T) (9), affecting the third base within codon 566,
which is the last codon shared in common between lamin A and C before
alternative splicing gives rise to the two distinct proteins
(9). We previously showed that this SNP was associated
with variation in obesity-related indexes in Canadian Oji-Cree, with
the LMNA 1908T allele being associated with increased
anthropometric measurements (9). We now report replication
of this observation in an independent, genetically distinct aboriginal
population, namely Canadian Inuit, suggesting that the impact of
LMNA 1908T may extend to other populations at risk for
obesity.
Subjects and Methods
Study subjects
The Northwest Territories are located above the 60th parallel of latitude and comprise one third of the landmass of Canada. In 1986 the population of the Northwest Territories was 52,000. Of these, 35% were Inuit (or Eskimos), 15% were Dene (or Athapaskan Indians), and 50% were predominantly migrants of European origin from other parts of Canada. The traditional Inuit territory extends from the Chukchi Peninsula Laboratories, Inc. in northeastern Asiatic Russia across Alaska and Northern Canada to Greenland. The present study involved residents of 8 communities from the Keewatin Region, mainly from the western shore of Hudson Bay between the 60th and 70th parallels of latitude (1, 2). These communities are included within a region that is now the self-governing jurisdiction called Nunavut.
Five hundred and sixteen randomly selected individuals, aged 18–80 yr, participated; of these, 281 (54.4%) reported themselves as being Inuit, 112 (21.7%) reported themselves as being of mixed ethnic background, 92 (17.8%) reported themselves as being of European background (white), and 31 (6.0%) reported themselves as being of an ethnic background other than Inuit, mixed, or white. At the time of the study these communities continued to adhere to a more traditional lifestyle, including the consumption of arctic fish at least three times per week. Blood samples were obtained with informed consent. The first exclusion criterion was self-reported non-Inuit ancestry. The second exclusion criterion was an inadequate blood sample for genetic determinations. This left 186 subjects (36.0% of the initial sample), who were similar to the nonincluded subjects in all measured traits (data not shown). The project was approved by the institutional review board of the University of Manitoba.
Clinical and anthropometric assessment
The survey consisted of an interviewer-administered questionnaire, clinical examination, and laboratory tests. The questionnaire was adapted from existing health survey protocols (1, 2). Standardized procedures were used in performing blood pressure and anthropometric measurements (1, 2). Field staff were trained by instructors from the Canada Heart Health Survey (1, 2). Subjects were measured without shoes in cotton examination gowns and underclothes. Each measurement was performed twice, and their average was used in the analysis. Height was measured to the nearest 0.1 cm using a tape measure with heels together and buttocks, back, shoulders, and head touching the wall. Weight was measured to the nearest 0.1 kg using a standard hospital balance beam scale (Health-O-Meter, Bridgeview, IL). Body mass index (BMI) was defined as weight (kilograms)/height (meters)2. Waist and hip girths were measured with a tape measure, with the umbilicus and iliac crest serving as the anatomical landmarks, respectively. Large calipers (Cambridge Scientific Instruments, Cambridge, MD) were used to measure subscapular and triceps skinfold thickness. The waist to hip circumference ratio (WHR) and subscapular to triceps skinfold thickness ratio (STR) were derived for each subject. Concentrations of fasting plasma leptin were determined by quantitative enzyme-linked immunoabsorbent assay (Quantikine Human Leptin, R\|[amp ]\|D Systems, Inc., Minneapolis, MN). The leptin assay had a minimal detectable concentration of 0.5 ng/mL, a limit of linearity of 100 ng/mL, and an interassay coefficient of variation of 8.3%
Genetic analyses
The LMNA 1908C/T SNP genotype was determined from leukocyte DNA using amplification with primers LMNASNP1908F (5'-GCA AGA TAC ACC CAA GAG CC-3') and LMNASNP1908R (5'-ACA CCT GGG TTC CCT GTT C-3') over 30 amplification cycles and an annealing temperature of 60 C. The 1069-bp amplification product was then digested with PmlI and electrophoresed in 1.5% agarose gels. Digestion of the 1908C allele gave 2 fragments of 887 and 182 bp, whereas digestion of the 1908T allele gave a single fragment of 1069 bp.
Statistical analyses
Statistical analyses were performed using SAS statistical
software, version 6.12, as previously described (9, 10).
Between-sex and between-genotype differences in baseline clinical and
biochemical traits were assessed using t tests with
Bonferroni adjustment for multiple comparisons. Deviation of genotype
frequencies from Hardy Weinberg equilibrium was assessed using
2 analysis. The association of LMNA
genotype with quantitative traits was tested by ANOVA, using a general
linear model, with levels of significance computed from the type III
sums of squares. This approach is most appropriate for an unbalanced
study design and reports significance after all covariates are taken
into account.
The LMNA 1908C/T SNP genotype was introduced as a
dichotomous variable in the analyses; subjects who carried at least one
1908T allele were compared with subjects who were homozygous for
1908C/1908C (i.e. a dominant model for 1908T). Although
continuous dependent variables were not normally distributed,
transformations resulted in variables with distributions that did not
deviate significantly from normal by Wilk’s test of normality.
Logarithmic (loge) transformation of WHR,
subscapular skinfold thickness, STR, and plasma leptin gave
distributions that were not significantly different from normal.
Inverse transformation for BMI, weight, and waist gave distributions
that were not significantly different from normal. Square root
transformation of triceps skinfold thickness gave a distribution that
was not significantly different from normal. We also created a variable
defined as the ratio of leptin to BMI (leptin/BMI ratio), as previously
reported (9), to intrinsically correct for variation in
leptin that was related to BMI. ANOVA was performed for weight, BMI,
waist circumference, WHR, subscapular and triceps skinfold thickness,
STR, plasma leptin, and leptin/BMI ratio using the transformed value
for each as the dependent variable and the LMNA genotype,
age, and sex as the independent variables. Confirmatory
post-hoc analyses of between-genotype differences were
conducted with the nonparametric Kruskal-Wallis
2 approximation test of the Wilcoxon rank sums
for nontransformed variables, as previously reported (9).
Post-hoc parametric analyses were also conducted for each
sex separately, using LMNA genotype and age as independent
variables. All parametric analyses were conducted using transformed
variables, but untransformed mean values are shown in Tables 1
and 4.
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Results
Clinical and biochemical attributes
The clinical and biochemical attributes of 186 adult Inuit
subjects are shown in Table 1
. None of the subjects had diabetes,
muscular dystrophy, cardiomyopathy, or conduction system disease. None
of the study subjects was taking oral hypoglycemic, antihypertensive,
or antihyperlipidemic medications. We noted between-sex differences in
most obesity-related traits (Table 1). Women were found to have higher
BMI, subscapular and triceps skinfold thickness, plasma leptin
concentration, and leptin/BMI ratio than men. In contrast, women had
lower weight, WHR, and STR than men. Mean age and waist were not
significantly different between the sexes. The observed 6-fold
between-gender difference in plasma leptin concentration was
particularly striking and was consistent with low adipose tissue mass
in male Inuit.
Allele and genotype frequencies
The allele and genotype frequencies in the overall study sample
and in each sex separately are shown in Table 2. None of the genotype frequencies
deviated significantly from expectations of the Hardy-Weinberg
equation. There were no significant differences between allele and
genotype frequencies between the sexes.
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In the 186 nondiabetic Inuit subjects, ANOVA (Table 3) revealed significant associations
between LMNA 1908C/T SNP genotype and transformed weight,
BMI, waist circumference, WHR, subscapular skinfold thickness, and STR
(P = 0.016, 0.0031, 0.019, 0.024, 0.0036, and 0.029,
respectively). These associations were each confirmed in an independent
post-hoc nonparametric ANOVA (data not shown). There was no
significant association of LMNA 1908C/T SNP genotype with
triceps skinfold thickness, plasma leptin, or leptin/BMI ratio. Among
nongenetic covariates, age was significantly associated with
transformed BMI, waist circumference, and WHR, whereas sex was
significantly associated with transformed weight, BMI, WHR, subscapular
and triceps skinfold thickness, plasma leptin, and leptin/BMI ratio.
When males and females were examined separately, there was no
difference in the associations of quantitative traits with
LMNA genotype (data not shown).
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; each P < 0.05, by
Bonferroni test).
Multivariate regression analysis (Table 5) revealed that LMNA 1908C/T
genotype accounted for 33.4%, 22.4%, 11.4%, 7.1%, 19.9%, and
100%, respectively, of the attributable variation in transformed
weight, BMI, waist circumference, WHR, subscapular skinfold thickness,
and STR (P = 0.007, 0.008, 0.016, 0.008, 0.0016, and
0.016, respectively). Sex accounted for 54.4%, 15.5%, 18.7%, 80.1%,
100%, 100%, and 100%, respectively, of the attributable variation in
transformed weight, BMI, WHR, subscapular and triceps skinfold
thicknesses, plasma leptin, and leptin/BMI ratio (P =
0.0008, 0.025, <0.005, <0.0001, <0.0001, <0.0001, and <0.0001,
respectively).
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We found that common LMNA 1908C/T SNP is associated with obesity-related anthropometric quantitative traits in Inuit. In particular, Inuit with the LMNA 1908T allele had significantly higher weight, BMI, waist circumference, WHR, subscapular skinfold thickness, and STR compared with Inuit who did not carry this allele. In addition, the LMNA genotype accounted for about 10% to almost 100% of the attributable variation in these traits in multivariate regression models. The findings are consistent with our previous observation of similar associations of the LMNA 1908T allele with increased obesity indexes in Oji-Cree. The replication of these results in samples taken from two independent, genetically distinct aboriginal populations suggests that the common LMNA 1908C/T SNP genotype may have a more general association with variation in indexes of obesity.
The widely expressed LMNA gene products, lamins A and C, are important elements of the nuclear lamina. Alternative splicing at exon 10 of LMNA gives rise to lamins A and C (11), which share sequence identity for the first 566 residues, but have distinctive C-termini (11). As the LMNA 1908C/T SNP is silent at the amino acid level, it is probable that the associations were the result of linkage disequilibrium with a functional variant elsewhere at this locus. However, we observed no other LMNA coding sequence variants in the Inuit, suggesting that this is unlikely. It is also possible that there was unmeasured variation within flanking noncoding regulatory sequences of LMNA or within a nearby gene on chromosome 1q21-q22, which we have not yet ruled out. Finally, it is possible that the LMNA 1908C/T SNP may mark a DNA change that has a functional molecular consequence. The affected residue is at the third base of LMNA codon 566, which is the last codon shared in common between lamin A and C before alternative splicing gives rise to the two distinct proteins (11). Although we are unaware of a precedent for a common SNP at a crucial site affecting message splicing, the proximity of this variant to such a focal nucleotide in LMNA might be more than coincidental, especially in light of the consistent phenotypic associations.
The mechanism underlying the association between common variation in LMNA and indexes of obesity is not clear. Lamins A and C are members of the intermediate filament multigene family and are present in most differentiated cells. Lamin A and C polymerize to form part of the nuclear lamina, a structural meshwork of 10-nm filaments on the nucleoplasmic side of the inner nuclear membrane (11). Lamins A and C form dimers through their rod domains. Variation that affects splicing of LMNA could have an effect on the ratio of lamin A to C isoforms in adipose tissue, which might have consequences for the development of adipocytes over time.
In the Oji-Cree sample, LMNA 1908T was associated not only
with increased anthropometric indexes, but also with increased (by
18%) plasma leptin concentration (9). In the Inuit, we
observed a consistent, but nonsignificant, increase in plasma leptin in
carriers of LMNA 1908T compared with noncarriers (10.5
± 1.0 vs. 8.6 ± 1.8 ng/mL; P = 0.10).
The magnitude of this between-genotype difference (
20%) was the
same as the magnitude of the between-genotype difference in plasma
leptin observed in the much larger Oji-Cree sample (9).
Our failure to detect an LMNA 1908C/T SNP genotype
association with plasma leptin may simply have reflected a smaller
sample size of the Inuit compared with the Oji-Cree (9).
It is most probable that the differences in plasma leptin reflect
variation in adipocyte mass rather than an independent effect of the
LMNA variation.
Interestingly, sibling pair analyses performed in another North American aboriginal group, the Pima Indians, have suggested that there is a potential diabetes susceptibility locus on chromosome 1q (12). It would be of interest to determine whether variation in LMNA, possibly the 1908C/T variant itself, is associated with obesity-related end points in that aboriginal group and indeed in other aboriginal people. Of course, the LMNA 1908C/T variant may be in linkage disequilibrium with other variants at other nearby genes, such as ARNT, RXR, CRP, CTSS, or CTSK.
In conclusion, we report associations between the LMNA 1908C/T SNP and indexes of obesity in Canadian Inuit. The variation in these traits attributable to LMNA 1908C/T SNP ranged from approximately 10–100%, consistent with the idea that common variation in LMNA may be a more generally important contributor to variation in these traits. Further epidemiological and genetic studies of the LMNA gene locus and nearby SNPs are required to improve our understanding of the possible role of this potentially important gene in adipocyte biology and obesity.
Acknowledgments
We acknowledge the cooperation and assistance of the members of the Health Canada research team and also the technical assistance of Matthew Ban, Michael Carruthers, Ajay Prakash, Carol Anderson, Henian Cao, and Jane Edwards.
Footnotes
1 This work was supported by grants from the Canadian Institutes for
Health Research (MT13430), the Heart and Stroke Foundation of Ontario
(3628), the Canadian Diabetes Association (in honor of Reta Maude
Gilbert), and the Blackburn Group. 
2 Career Investigator (CI-2979) with the Heart and Stroke Foundation
of Ontario and Canada Research Chair in Human Genetics.
3 Senior Scientist of the Medical Research Council of Canada.
Received September 29, 2000.
Revised December 14, 2000.
Accepted February 9, 2001.
References
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