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The ras-associated diabetes (Rad) was initially identified by subtraction cloning from the skeletal muscle of humans with non-insulin dependent diabetes mellitus (type 2 DM).［1］ Rad mRNA expression is markedly increased in the skeletal muscle of type 2 DM patients compared with normal controls. Since the last decade, it has been suggested that Rad overexpression may be involved in the pathogenesis of type 2 DM and insulin resistance.［1,2］ Rad protein levels in the skeletal muscle were found positively correlated with both BMI and percentage of body fat, while negatively correlated with a resting metabolic rate.［3］ Therefore, it is plausible that there is a synergistic effect of the Rad gene variant and obesity on the risk of type 2 DM.We conducted a molecular genetic epidemiologic study to assess if there is an association of the Rad gene with the genetic predisposition of type 2 DM with or without dependence on obesity.
METHODS
Study population and DNA sample preparation
The study consisted of 101 patients aged 55.0±8.4 years who were diagnosed with type 2 DM at the Third Affiliated Hospitals of Peking University. A total of 101 of their healthy spouses aged 55.7±9.6 years residing in Beijing were chosen as controls. All study subjects were given informed consent.
Mutation detection
Genomic DNA was extracted according to Puregene DNA isolation kits (Gentra Systems, USA) using standardized protocols. PCR was performed exactly as described.［4］ PCR products were run on 1% agarose gels and visualized by UV transillumination after staining with ethidium bromide.
The mutation screening was performed by denaturing high pressure liquid chromatography (DHPLC). Polymor-phisms were scored by the visual inspection of the chromatograms. Criteria for identifying polymorphisms include: the number of peaks, relative peak height, peak width, and altered retention times. DNA sequencing was carried out to identify polymorphic base substitutions.
Genotyping of T4681C and C4690G polymorphisms using oligonucleotide ligation assay
We genotyped two novel SNPs using the oligonucleotide ligation assay (OLA), which involved three steps: PCR amplification, PCR product purification, and linear amplification. OLA products were loaded onto an 8% denaturing polyacrylamide sequencing gel. The location and relative quantity of OLA products were automatically recorded with Genescan version 2.1 software (GeneScan, USA).
Statistical analysis
A statistical analysis was performed with SAS statistical software. The logistic regression model was utilized. Potential confounding factors were controlled by a multivariate logistic regression analysis. All P values were two-tailed and a P value less than 0.05 was considered statistically significant.
RESULTS
Molecular scanning of the Rad gene in type 2 DM patients
DHPLC analysis of the exons and their splice sites revealed two aberrant peak patterns. Subsequent DNA sequencing of the PCR products identified two polymorphic base substitutions within intron 4 of the Rad gene: C-T at position 4681, and C-G at position 4690.
Association of the Rad intron 4 polymorphism with type 2 DM
The C allele frequency of the C4681T polymorphism was 69.0% and 66.3% among cases and controls, respectively. The G allele of the C4690G polymorphism was present in 0.5% type 2 DM patients and normal controls. When stratified by the BMI status (≤25 kg/m2 vs >25 kg/m2), a CC homozygote was found associated with a mildly elevated risk of type 2 DM in both non-obese (adjusted OR=1.78; 95%CI: 0.65-4.84) and obese (adjusted OR=1.69; 95%CI: 0.67-4.25) subjects. But the results did not reach statistical significance in either subgroup. After controlling for major confounders including age, sex, cigarette smoking, and alcohol consumption, it was notable that obesity alone was significantly associated with an increased risk of type 2 DM (adjusted OR=3.80; 95%CI: 1.50-9.58; P=0.005). Further analyses undertaken to examine the joint effects of the Rad gene polymorphism and obesity showed that the combination of obesity and a CC homozygote of the Rad gene increased the risk for type 2 DM over five folds (adjusted OR=5.46; 95%CI: 1.98-15.11; P<0.001).
DISCUSSION
Our data provide the first evidence that there is a significant joint effect between a novel polymorphism of Rad gene and obesity in conferring an increased risk of type 2 DM. When the Rad gene C4681T polymorphism was examined alone, only a mildly increased risk of type 2 DM was observed. However, the disease risk was substantially elevated only when the variant was combined with obesity. It is noted that a couple of previous molecular epidemiologic studies established a synergism between genetic polymorphisms and obesity on the risk of type 2 DM.［5,6］ Our genotyping results reveal that only one case and one control were heterozygous for the C4690G polymorphism and all other study subjects were wild-type homozygotes. Thereafter, we conducted extensive analysis only for the most common C4681T polymorphism in association with type 2 DM.
Genetic heterogeneity exists when several genes are associated with the disease. Failure to correct for genetic heterogeneity of disease often leads to an inaccurate report of weak to moderate ORs depicting the association between the disease and marker alleles.［7］ The Rad gene may not be a “necessary” major gene for type 2 DM, but a “susceptibility” gene that has a major impact on type 2 DM risk only at a certain metabolic state.
Garvey et al［3］ measured Rad mRNA and protein levels in the skeletal muscle from well-characterized subjects with and without type 2 DM. Their data showed that the Rad protein levels were positively correlated with both BMI (r=0.43, P=0.033) and even more significantly correlated with percentage of body fat (r=0.55, P<0.005). According to these results, the Rad protein in the skeletal muscle has a higher degree of expression in obese subjects, and consequently, the Rad gene effect should be manifested to a greater extent among obese than non-obese individuals. These results are supported by our findings that the Rad gene polymorphism, when combined with obesity, is associated with a considerably increased risk of type 2 DM.
In conclusion, our results suggest that the novel C4681T polymorphism of the Rad gene and obesity are likely to have a joint effect on the risk of type 2 DM. More research using prospective study designs are warranted to test this hypothesis.
REFERENCES
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2.Kahn CR. Banting lecture. Insulin action, diabetogenes, and the cause of type Ⅱ diabetes. Diabetes 1994;43:1066-1084.
3.Garvey WT, Maianu L, Kennedy A, et al. Muscle Rad expression and human metabolism: potential role of the novel Ras-related GTPase in energy expenditure and body composition. Diabetes 1997;46:444-450.
4.Wang G, Qian R, Li Q, et al. The association between PPP1R3 gene polymorphisms and type 2 diabetes mellitus. Chin Med J 2001;114:1258-1262.
5.Xiang K, Wu S, Wang Y, et al. The population association of glucokinase gene with type 2 (noninsulin-dependent) diabetes mellitus in Chinese. Chin Med J 1995;108:5-10.
6.Koch M, Rett K, Maerker E, et al. The PPARγ2 amino acid polymorphism Pro 12 Ala is prevalent in offspring of type Ⅱ diabetic patients and is associated to increased insulin sensitivity in a subgroup of obese subjects. Diabetologia 1999;42:758-762.
7.Hodge SE. What association analysis can and cannot tell us about the genetics of complex disease. Am J Med Genet 1994;54:318-323.
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