Abstract
The E23K variant of the Kir6.2 gene has been shown to be associated with type 2 diabetes mellitus in Caucasian subjects. Because offspring of type 2 diabetic patients have a genetically increased risk of developing diabetes, we sought to identify the E23K variant of the Kir6.2 gene in offspring of Caribbean patients with type 2 diabetes and assess the contribution of this variant to impaired glucose tolerance in these subjects. Forty-six offspring of patients with type 2 diabetes and 39 apparently healthy subjects whose immediate parents were not diabetic (‘control’) were studied after an overnight fast. Anthropometric indices were measured and blood samples were collected. Fasting and 2 h plasma glucose, insulin and lipids were subsequently determined. Insulin resistance was calculated using the homeostatic model assessment technique. The offspring and control subjects had similar frequencies of the E23K polymorphism (52.6 vs 45.5%, P>0.05) and the frequency of the E23K variant did not differ significantly between gender and ethnic distributions, irrespectively of a family history of diabetes (P>0.05). There were no significant differences in biochemical risk factors for developing diabetes in offspring carriers of the E23K variant compared with offspring non-carriers of the mutation. Offspring with the E23K mutation had even significantly higher 2 h insulin concentrations when compared with control subjects. It is concluded that the presence of the Kir6.2 E23K genotype in Caribbean subjects with an immediate positive family history of diabetes does not confer significantly higher levels of biochemical risk factors for the development of type 2 diabetes.
Introduction
Several research studies in different populations have confirmed that offspring and first-degree relatives of patients with type 2 diabetes mellitus are at increased risk of developing the disorder in later life (Haffner et al. 1988, Eriksson et al. 1989, Osei 1990, Warram et al. 1990, Ezenwaka et al. 1993, 2001a,, Ezenwaka et al. b, Stewart et al. 1995, Gaillard et al. 1997, Snethalatha et al. 1998). Although the genetic component of type 2 diabetes is well established (Barnett et al. 1981, Newman et al. 1987), research on the genes associated with diabetes is still ongoing and inconclusive. Indeed, certain genetic variants that could affect insulin secretion and glucose utilisation have been identified. For example, it has been shown that a mutation in the gene encoding the sulphonylurea receptor subunit is associated with reduced insulin secretion and type 2 diabetes (‘t Hart et al. 2000, Reis & Velho 2002). Furthermore, recent studies in Caucasian subjects showed that the E23K variant of the Kir6.2 gene is associated with type 2 diabetes (Gloyn et al. 2003, Nielsen et al. 2003, Riedel et al. 2003). Mutations in the genes encoding sulphonylurea receptor and Kir6.2 have significant implications in developing diabetes, given that the two proteins play important roles in beta-cell insulin secretion (Aguilar-Bryan et al. 1995, Inagaki et al. 1995). However, it is important to note that the impact of these mutations has not been confirmed in other populations. For instance, other studies of independent populations from The Netherlands, Germany and the USA have shown that mutations in the Kir6.2 gene are not associated with beta-cell dysfunction or insulin resistance (IR) or even detectable alterations in glucose-stimulated insulin secretion (Inoue et al. 1997, ‘t Hart et al. 2002, Tschritter et al. 2002). This implies that the role of the Kir6.2 variant in the development of type 2 diabetes is still controversial and warrants further investigation in other population groups. Therefore, it appears important to conduct research on the Kir6.2 variant (or any other mutation) in all ethnic groups, especially in genetically predisposed individuals such as offspring of type 2 diabetic patients. Thus, we sought to identify the E23K variant of the Kir6.2 gene in Caribbean subjects and compare the levels of biochemical risk factors for developing diabetes in subjects with and without the Kir6.2 E23K mutation.
Subjects and Methods
Offspring of diabetic patients
Forty-six offspring (24 on follow-up and 22 new recruits) of patients with type 2 diabetes participated in the study. The recruitment strategy for the subjects was as previously published (Ezenwaka et al. 2001a,b, 2004). Briefly, posters and flyers were distributed to type 2 diabetic patients visiting two major lifestyle disease clinics in Trinidad for onward distribution to their children. Offspring of diabetic patients who expressed interest in participating were invited to our research laboratory at the Eric Williams Medical Sciences Complex (EWMSC), Trinidad, to sign consent forms and to register for a study date. All offspring were advised to continue with their routine lifestyle until the day of the test.
Control subjects
Thirty-nine (14 on follow-up and 25 new recruits) apparently healthy individuals whose immediate parents were not diabetic served as ‘control’ subjects and were recruited through oral or poster/flyer advertisements from within the EWMSC. Students and workers from within the EWMSC have demonstrated their understanding of the requirements of medical research and are more aware of their family medical history (Ezenwaka et al. 2001a,b, 2004). Similarly to the offspring of diabetic patients, controls who expressed interest in participating in the study were required to visit our research laboratory for a thorough explanation of the study protocol, and to register and sign voluntary consent forms. They were also instructed to refrain from smoking on the morning of the test and to continue with their routine lifestyle and diet before the study.
Study protocol
The study protocol was reviewed and approved by the Faculty of Medical Sciences Ethics Committee. The protocol was essentially the same as has been recently published (Ezenwaka et al. 2004). All subjects were studied at our laboratory after an overnight fast (10–14 h). Using a research questionnaire, information on background characteristics of the subjects was obtained and anthropometric indices were measured. Weight (kg) and height (m) (in light clothing and without shoes) was measured using a standard scale and metre rule. Using a measuring tape, waist circumference (cm) was taken at the level of the umbilicus and hip circumference (cm) at the largest projection of the buttocks. Then, a 10 ml fasting blood sample was drawn from each subject and preserved in fluoride-oxalate (for glucose estimation), EDTA (for DNA extraction) and plain tubes (for serum lipid and insulin). Subsequently, subjects orally consumed 75 g anhydrous glucose dissolved in 250 ml water over 5 min. At 2 h of the study, a 5 ml blood sample was collected for plasma glucose and serum insulin estimation. Plasma and serum specimens were separated after centrifugation within 30 min of collection and stored at − 20 °C.
DNA extraction and genotyping
Genomic DNA was isolated from whole blood samples using the QIAamp DNA blood Midi kit (Qiagen, Hilden, Germany). The DNA was amplified by the PCR technique using a forward primer (5′-GAATACGTCCTGA CACGCCT-3′) and a reverse primer (5′-GCCAGC TGCACAGGAAGGACAT-3′), which flanked the region containing the Kir6.2 gene (product size, 218 bp) (Nielsen et al. 2003). PCR was performed in a 50 μl volume containing 500 ng genomic DNA, 10 pmol of each primer, 10 mM dNTPs, 2.5 U PfuTurbo DNA polymerase (Stratagene, Heidelberg, Germany), 10 × polymerase buffer and water. The PCR conditions were an initial denaturation step at 95 °C for 3 min, followed by 35 cycles of denaturation at 95 °C for 1 min, annealing at 71 °C for 1 min and extension at 72 °C for 1 min, with a final extension of 5 min at 72 °C. The PCR product was purified using a QIAquick PCR purification kit (Qiagen) and subsequently digested with BanII enzyme (Roche Diagnostics, Mannheim, Germany) at 37 °C for 2 h and heat inactivated for 10 min at 65 °C. The digested probes were subjected to electrophoresis on a 3% agarose gel (Biozyme, Hess Oldendorf, Germany), and stained with ethidium bromide (Roth, Karlsruhe, Germany) for visualisation (Hani et al. 1998, Nielsen et al. 2003).
In the E23K point mutation of the Kir6.2 gene, substitution of G for A in the codon for glutamic acid resulted in the codon for lysine, that is GAG→AAG. Because of this substitution, BanII restriction enzyme was unable to recognise the sequence for cutting and hence no digestion products were obtained. Restriction fragment length polymorphism (RFLP) analysis of amplified wild type (+/+) probes with BanII generated two products (178 and 40 bp). However, since the E23K mutation of the Kir6.2 gene product leads to the loss of the recognition site for BanII, the digestion of heterozygous (+/−) mutated probes generated three products (218, 170 and 40 bp) and homozygous (−/−) mutated probes generated one product (218 bp) only.
Biochemical analysis
Plasma glucose, serum triglyceride, total cholesterol and high density lipoprotein (HDL)-cholesterol concentrations were measured using enzymatic methods with commercial dry slide kits in a multi-channel auto-analyser (Johnson & Johnson Vitros 250; Ortho-Clinical Diagnostics, Inc., Rochester, NY, USA). Low density lipoprotein (LDL)-cholesterol was calculated using the Friedwald equation (Friedwald et al. 1972). The serum insulin level was determined by ELISA using a Mercodia insulin ELISA kit (Mercodia AB, Uppsala, Sweden).
Statistics and calculations
The results are expressed as means ± s.e. The Statistical Package for the Social Sciences (SPSS Inc., Chicago, IL, USA) software was used in all analyses. IR, defined as the product of fasting serum insulin and plasma glucose divided by 22.5, was assessed using fasting serum insulin and plasma glucose concentrations in a homeostasis model assessment (Matthews et al. 1985). Comparisons of the mean differences in biochemical parameters in different sub-groups of subjects were performed using Student’s t-tests while chi-square was used for non-parametric tests. A P value <0.05 was considered statistically significant on two-tailed testing for all analyses.
Results
Figure 1 shows the RFLP detection of the E23K variant of the Kir6.2 gene. Of the 46 offspring of diabetic patients and 39 control subjects who participated in the study, PCR products could not be isolated in eight (17.4%) offspring and six (15.4%) control subjects. The anthropometric and biochemical parameters of these subjects (eight offspring and six controls) were excluded in the data analysis. Table 1 shows the distribution of Kir6.2 E23K variants in the 38 offspring of diabetic patients and 33 control subjects of which PCR products were isolated. The offspring and control subjects had similar frequencies of the E23K polymorphism (52.6 vs 45.5%, P>0.05) and the frequency of this variant did not differ significantly between gender and ethnic distributions, irrespectively of the family history of diabetes (Table 1, P>0.05). With the exception of plasma HDL-cholesterol concentration, there were no significant differences in biochemical risk factors for developing diabetes in all subjects with the heterozygous gene (E23K) mutation compared with carriers of the wild type gene (E23E) (Table 2, all P>0.05). After an oral glucose tolerance test (OGTT), control subjects with the E23K variant showed a tendency towards lower 2 h insulin concentrations than control carriers of the wild type (E23E), which did not reach statistical significance. However, offspring with the E23K mutation had significantly higher 2 h insulin and 2 h glucose concentrations compared with corresponding control subjects (Table 3, P<0.05). Importantly, the fasting and 2 h plasma glucose levels in offspring and control carriers of the E23K variant were not significantly higher compared with their respective wild type subjects. Furthermore, sub-analysis of the data showed that offspring carriers and non-carriers of the E23K variant had similar levels of fasting insulin and glucose, and 2 h insulin and glucose after 5 years of follow-up (Table 4, P>0.05).
Discussion
The present study identified the Kir6.2 E23K mutation in Caribbean subjects with and without an immediate family history of diabetes, and subsequently demonstrated that carriers and non-carriers of the E23K mutation had similar levels of glucose and insulin concentrations before and after an OGTT.
The identification of the Kir6.2 E23K mutation in Caribbean subjects with and without an immediate positive family history of diabetes is interesting. Trinidad and Tobago is a small country (population 1.3 million) with diabetes prevalence rate between 16 and 20% in its two major (people of African and East Indian descents) ethnic groups (Miller et al. 1996, Central Statistical Office 1998). It has been projected that by the year 2010, about 89 000 people in Trinidad and Tobago will be diagnosed with type 2 diabetes (Amos et al. 1997). Thus, research on susceptible genes that may increase the risk of developing diabetes is warranted. Although the E23K variant is widespread and has been identified in many populations (Inoue et al. 1997, ‘t Hart et al. 2002, Tschritter et al. 2002, Gloyn et al. 2003, Nielsen et al. 2003, Riedel et al. 2003), the finding of this mutation in 46–53% of subjects studied suggests that further investigation in larger cohorts is necessary in this population. Previous studies in other populations showed that the E23K polymorphism is associated with impaired glucose-induced insulin release (Gloyn et al. 2003, Nielsen et al. 2003, Riedel et al. 2003) and a trend towards this functional effect of the E23K mutation was only seen in our control subjects who have the E23K mutation, but not in offspring of diabetic patients (Table 3). However, recent evidence suggests that the impact of the E23K variant in beta-cell dysfunction and glucose metabolism is not similar in all populations (Inoue et al. 1997, ‘t Hart et al. 2002, Tschritter et al. 2002). Thus, the role of E23K polymorphism in developing diabetes remains controversial, especially as environmental factors, such as lifestyle and obesity, are relevant in the interpretation of the effect of gene mutation in different populations. For instance, sub-analysis of our data did not show any significant differences in the biochemical risk factors for developing diabetes in obese and non-obese subjects with the E23K variants (data not shown), while a previous study showed that the E23K polymorphism is associated with a significant increase in body mass index (BMI) (Nielsen et al. 2003).
Furthermore, our finding that the E23K polymorphism does not increase impaired glucose tolerance in carriers of the E23K genotype is consistent with previous reports (Inoue et al. 1997, ‘t Hart et al. 2002, Tschritter et al. 2002), and appears to suggest that the E23K variant does not essentially confer increased biochemical risk in persons with the mutation. This is in apparent contrast with the reports of Nielsen et al.(2003) and Reidel et al.(2003) in Caucasian subjects. Although the E23K gene was not identified in all the offspring of diabetic patients, there is no evidence in our study suggesting that carriers of this mutation have an increased biochemical risk for developing diabetes compared with non-carriers. However, the observed trend (P>0.05) towards a relative fasting and postprandial hyperglycaemia and hyperinsulinaemia in carriers of the E23K gene mutation could be explained by the apparently non-significantly higher IR levels (Table 3), which is consistent with our previous reports on the offspring of diabetic patients (Ezenwaka et al. 2001a,b). This is further supported by the results of the sub-analysis of offspring of diabetic patients on follow-up, which showed that carriers and non-carriers of the E23K variant had similar levels of glucose and insulin (Table 4). Therefore, there is no clear evidence in the current study to suggest that the presence of the E23K gene increases the biochemical risk of developing diabetes. It should, however, be noted that the number of subjects in the present study is relatively small and this might have reduced the statistical power of the tests. We intend to expand the present study to include subjects with overt type 2 diabetes with the aim of identifying the frequency of the E23K gene polymorphism in persons already living with diabetes. It is possible that such an enlarged study, with stronger statistical power, might be able to provide clearer evidence of the impact of the E23K variant in the antecedent biochemical markers for developing diabetes. In the meantime, the present results have demonstrated that the presence of the Kir6.2 E23K genotype in Caribbean subjects with an immediate or remote positive family history of diabetes did not increase impaired glucose tolerance in the subjects studied.
Ethnicity- and gender-related distribution of Kir6.2 E23K variants in offspring and control subjects
| Offspring genotype | Controls genotype | |||||
|---|---|---|---|---|---|---|
| E23E(+/+) | E23K(+/−) | K23K(−/−) | E23E(+/+) | E23K(+/−) | K23K(−/−) | |
| All (%) | 18 (47.4) | 20 (52.6) | 0 | 17 (51.5) | 15 (45.5) | 1 (3.0) |
| Ethnicity | ||||||
| African (%) | 4 (22.2) | 5 (25.0) | 0 | 7 (41.2) | 5 (33.3) | 0 |
| East Indian (%) | 12 (66.7) | 9 (45.0) | 0 | 6 (35.3) | 5 (33.3) | 1 (100) |
| Mixed (%) | 2 (11.1) | 6 (30.0) | 0 | 4 (23.5) | 5 (33.3) | 0 |
| Gender | ||||||
| Male (%) | 8 (44.4) | 7 (35.0) | 0 | 6 (35.3) | 9 (60.0) | 1 (100) |
| Female (%) | 10 (55.6) | 13 (65.0) | 0 | 11 (64.7) | 6 (40.0) | 0 |
Selected biochemical parameters (mean ± s.e.) of all subjects with E23E and E23K genotypes
| Kir6.2 genotype | ||
|---|---|---|
| E23E (+/+) (n=35) | E23K (+/−) (n=35) | |
| *P<0.05 for comparison between E23E and E23K subjects. | ||
| Parameter | ||
| Sex (m/f) | 14/21 | 16/19 |
| Age (years) | 28.0 ± 1.3 | 29.0 ± 1.4 |
| BMI (kg/m2) | 25.6 ± 0.9 | 25.7 ± 0.9 |
| Waist circumference (cm) | 86.1 ± 2.2 | 85.8 ± 2.0 |
| Fasting serum insulin (mIU/l) | 9.2 ± 0.7 | 9.9 ± 0.8 |
| 2 h serum insulin (mIU/l) | 84.7 ± 11.5 | 74.2 ± 10.3 |
| Fasting plasma glucose (mmol/l) | 4.7 ± 0.9 | 5.0 ± 0.1 |
| 2 h plasma glucose (mmol/l) | 6.2 ± 0.4 | 6.3 ± 0.4 |
| Fasting triglyceride (mmol/l) | 1.4 ± 0.1 | 1.3 ± 0.1 |
| Fasting total cholesterol (mmol/l) | 4.5 ± 0.1 | 4.7 ± 0.2 |
| Fasting HDL-cholesterol (mmol/l) | 1.0 ± 0.1* | 1.2 ± 0.1 |
| Fasting LDL-cholesterol (mmol/l) | 3.2 ± 0.1 | 3.2 ± 0.2 |
| IR (pmol/mol/l) | 14.3 ± 1.2 | 15.8 ± 1.3 |
Comparison of anthropometric and biochemical parameters (mean ± s.e.) of offspring and control subjects according to their Kir6.2 genotype
| Offspring (with immediate FH of diabetes) | Controls (without immediate FH of diabetes) | |||
|---|---|---|---|---|
| E23E (+/+) (n=18) | E23K (+/−) (n=20) | E23E (+/+) (n=17) | E23K (+/−) (n=15) | |
| FH, family history of diabetes; *P<0.05 for differences between +/+ offspring and +/− offspring;* P<0.05 for differences between +/+ ‘controls’ and +/− ‘controls’; #P<0.05 for differences between offspring and ‘control’ subjects with +/+ genotype; ¶P<0.05, ¶ ¶P<0.01 for differences between offspring and ‘control’ subjects with +/− genotype. | ||||
| Parameter | ||||
| Sex (m/f) | 8/10 | 7/13 | 6/11 | 9/6 |
| Age (years) | 32.1 ± 1.8 | 33.0 ± 1.9¶¶ | 25.3 ± 1.4# | 23.7 ± 1.2 |
| BMI (kg/m2) | 26.5 ± 1.4 | 26.9 ± 1.3 | 24.5 ± 1.2 | 24.2 ± 1.0 |
| Waist circumference (cm) | 89.4 ± 3.3 | 88.6 ± 2.8 | 82.6 ± 2.8 | 82.1 ± 2.9 |
| Fasting serum insulin (mIU/l) | 8.8 ± 1.0 | 9.9 ± 1.0 | 9.7 ± 0.9 | 9.9 ± 1.4 |
| 2 hour serum insulin (mIU/l) | 91.4 ± 16.6 | 92.6 ± 16.4¶ | 77.1 ± 16.2 | 49.8 ± 6.3 |
| Fasting plasma glucose (mmol/l) | 4.9 ± 0.1 | 5.1 ± 0.2 | 4.5 ± 0.1# | 4.8 ± 0.1+ |
| 2 hour plasma glucose (mmol/l) | 7.0 ± 0.6 | 7.2 ± 0.5¶ | 5.4 ± 0.3# | 5.1 ± 0.3 |
| Fasting triglyceride (mmol/l) | 1.6 ± 0.2 | 1.3 ± 0.1 | 1.2 ± 0.2 | 1.2 ± 0.2 |
| Fasting total cholesterol (mmol/l) | 4.6 ± 0.2 | 4.8 ± 0.2 | 4.5 ± 0.2 | 4.5 ± 0.3 |
| Fasting HDL-cholesterol (mmol/l) | 1.0 ± 0.04* | 1.2 ± 0.1 | 1.0 ± 0.1 | 1.3 ± 0.1 |
| Fasting LDL-cholesterol (mmol/l) | 3.2 ± 0.2 | 3.4 ± 0.2 | 3.2 ± 0.2 | 3.0 ± 0.3 |
| IR (pmol/mol/l) | 14.5 ± 2.1 | 16.1 ± 1.7 | 14.2 ± 1.4 | 15.5 ± 2.2 |
Similarities in insulin and glucose levels (mean ± s.e.) of offspring carriers and non-carriers of the E23K variant after 5 years of follow-up
| Offspring after 5 years of follow-up | ||
|---|---|---|
| E23E (+/+) (n=5) | E23K (+/−) (n=18) | |
| **P<0.01 for differences between +/+ offspring and +/− offspring. | ||
| Parameter | ||
| Fasting serum insulin (mIU/l) | 10.7 ± 2.4 | 10.0 ± 1.1 |
| 2 hour serum insulin (mIU/l) | 111.8 ± 49.5 | 90.9 ± 18.2 |
| Fasting plasma glucose (mmol/l) | 4.9 ± 0.2 | 5.0 ± 0.2 |
| 2 hour plasma glucose (mmol/l) | 7.3 ± 0.9 | 6.9 ± 0.5 |
| Fasting triglyceride (mmol/l) | 2.2 ± 0.3 | 1.3 ± 0.1** |
| Fasting total cholesterol (mmol/l) | 5.1 ± 0.4 | 4.8 ± 0.2 |
| Fasting HDL-cholesterol (mmol/l) | 0.9 ± 0.1 | 1.1 ± 0.1 |
| Fasting LDL-cholesterol (mmol/l) | 3.7 ± 0.4 | 3.4 ± 0.2 |
| IR (pmol/mol/l) | 17.6 ± 4.7 | 16.0 ± 1.8 |
RFLP detection of the E23K variant of the Kir6.2 gene. (1) 25 bp ladder, (2) undigested sample − 218 bp, (3) wild type − 178 and 40 bp, (4) homozygous mutated −218 bp, (5) heterozygous mutated − 218, 178 and 40 bp. The 40 bp fragments are not shown.
Citation: Journal of Endocrinology 185, 3; 10.1677/joe.1.06117
We are grateful to Joanna Owen and Keisha Lewis for technical assistance, Birgit Hurow for secretarial assistance and to the offspring and control subjects for their time and blood.
Funding
This study was supported by a Research Grant from the Volkswagen Foundation to J E and C E, and by the Ministerium für Wissenschaft und Forschung des Landes Nordrhein-Westfalen. We declare that there is no conflict of interest that would prejudice the impartiality of this paper.
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