Familial Early-Onset Type 2 Diabetes in Chinese Patients: Obesity and genetics have more significant roles than autoimmunity

The Prince of Wales Hospital (PWH) is a regional teaching hospital in Hong Kong. Its catchment area has a population of 1.2 million, accounting for 20% of the total population in Hong Kong. Because of the lack of long-term health care programs in Hong Kong, coverage by medical insurance is not widely available. Many patients with chronic diseases like diabetes are managed in public hospitals or clinics where they only pay a nominal fee. Hence, except for high social classes, our patients are largely representative of the diabetic population in Hong Kong. Since 1995, all patients attending the diabetes clinic of the PWH have been entered into the PWH Diabetes Registry after undergoing a structured assessment (19,20). During 1995 and 1996, a separate cohort of 150 young patients with early-onset diabetes (age ≤40 years and age at diagnosis ≤35 years) who underwent the structured assessment were recruited consecutively from the diabetes clinics at the PWH to form the Young Chinese Diabetes Database (6).

Among the 1,800 patients in the PWH Diabetes Registry recruited between 1995 and 1997 and the 150 patients in the Young Chinese Diabetes Database, 92 and 53 patients, respectively, were selected for the present study because they satisfied the enrollment criteria, which were early-onset (age and age at diagnosis ≤40 years) type 2 diabetes (1985 WHO criteria) (21) and a positive family history (at least one first degree relative with diabetes). Patients with classic type 1 diabetes (acute ketotic presentation or continuous requirement of insulin within 1 year of diagnosis) were excluded.

We have previously reported the prevalence of anti-GAD (6), mt3243 (22,23), and amylin gene mutations (24) among patients from the Young Chinese Diabetes Database. We have also reported the prevalence of mt3243 (23), amylin (24), glucokinase, HNF-1α, and HNF-4α gene mutations (25) in a separate cohort from the PWH Diabetes Registry. In this study, screening for glucokinase and HNF-1α gene mutations was extended to the 53 patients from the Young Chinese Diabetes Database. The HNF-4α gene was not screened in this cohort because of the expected low frequency of mutations; none was found in the 92 patients from the PWH Diabetes Registry (25). Screening for anti-GAD was extended to the 92 patients from the PWH Diabetes Registry.

Of these 145 young patients with familial diabetes, 19 (13%) met the minimal criteria for MODY (age at diagnosis ≤25 years and presence of diabetes in two consecutive generations). Altogether, 10 of 20 families with probands carrying putative diabetogenic gene mutations were recruited for a 75-g oral glucose tolerance test and clinical assessment. The 1999 WHO classification was used to define the glycemic status of the family members (26). For comparison of clinical characteristics of the early-onset patients, 290 sex-matched patients with late-onset diabetes (age at diagnosis >40 years) and a family history of diabetes were randomly selected from the current 1,800 patients in the PWH Diabetes Registry. A total of 100 healthy Chinese individuals (age 33 ± 10 years, 40 men and 60 women) were selected as control subjects from hospital staff and students for screening for the gene variants identified in our patients. Informed consent was obtained from each subject for a blood sample to be taken for DNA extraction and measurement of biochemical indexes. This study was approved by the Clinical Research Ethics Committee of the Chinese University of Hong Kong.

All patients underwent a structured assessment based on the Europe DiabCare Protocol (19). Their family history of diabetes, age at diagnosis, and anthropometric indexes were documented (19,20). BMI was used as an index of general obesity. Waist circumference, which is highly correlated with visceral fat accumulation measured by magnetic resonance imaging in Chinese patients (27), was used as an index of central obesity. After an overnight fast, venous blood was sampled for measurement of plasma glucose, insulin, HbA_1c, total cholesterol (TC), HDL cholesterol (HDL-C), LDL cholesterol (LDL-C) (calculated), triglyceride (TG), and anti-GAD. A morning spot urine sample was collected for assessment of albuminuria. Retinopathy and sensory neuropathy was assessed as previously described (28).

General obesity was defined as a BMI ≥25 kg/m² using the recent Asian criteria (29). Albuminuria was defined as an albumin-to-creatinine ratio (ACR) ≥3.5 mg/mmol in a spot urine sample (30). The homeostasis model assessment for insulin resistance (HOMA_IR) index (fasting plasma insulin × glucose/22.5), derived from the HOMA equation, was used to assess insulin resistance (31).

Plasma glucose, HbA_1c, lipids, urinary albumin, and creatinine were measured by routine assays in the Department of Chemical Pathology at the PWH as previously described (32). Plasma insulin was measured in non–insulin-treated patients by a radioimmunoassay (Pharmacia, Uppsala, Sweden) with intra-assay and interassay coefficients of variations of 6 and 13.8%, respectively. Anti-GAD was measured by a radioimmunoprecipitation assay (33). The upper normal limit of 18 units is applicable to Asian and European subjects (15,33).

The minimal promoter regions and exons of the glucokinase (β-cell form), HNF-1α, and HNF-4α (HNF-4α2 form) genes were screened for mutations by direct sequencing of polymerase chain reaction (PCR) products as described previously (34–36). The occurrence of putative mutations in other family members and control subjects was determined by PCR–restriction fragment–length polymorphism (RFLP). Mt3243A→G and amylin gene S20G mutations were determined by PCR-RFLP as described (10,22).

Normally distributed data are expressed as means ± SD. Data with skewed distributions were normalized by logarithmic transformation. The resultant means were antilogarithmically transformed and expressed as geometric means together with 25th and 75th percentiles. χ² tests and Student’s unpaired t tests were used for between-group comparisons. A P value <0.05 (2-tailed) was considered to be significant. All statistical analyses were performed using the Statistical Package for Social Sciences (SPSS for Windows, version 9.0).

Among the 145 patients with familial early-onset diabetes, there were 20 (14%) with putative mutations; of these, 7 (5%) involved the HNF-1α gene, 6 (4%) involved the glucokinase gene, 4 (3%) involved mt3243, and 3 (2%) involved amylin S20G. Anti-GAD was positive in six (4%). No mutation in the HNF-4α gene was found in the 92 patients from the PWH Diabetes Registry. All mutations identified in the HNF-1α and glucokinase genes were novel (Table 1). The HNF-1α G20R and glucokinase Q239R mutations were found in four unrelated patients. None of these mutations were found in 100 healthy control subjects.

Among the 20 patients carrying putative gene mutations, 10 had families who were recruited for cosegregation study (Fig. 1). Cosegregation of a mutation with clinical diabetes or glucose intolerance were observed in the families of the following four patients: HK10 with HNF-1α IVS2nt-1G→A, YDM142 with glucokinase V101 mol/l, HK84 with glucokinase I110T, and HK50 with mt3243. Segregation was inconclusive in the other six families. For the families of three patients (HK54 with HNF-1α R203H, YDM83 with mt3243, and CX216 with amylin S20G mutations), the probands carried the gene mutations, but none of the diabetic or nondiabetic family members who presented for screening carried the mutations. For the families of three other patients (YDM67 with glucokinase Q239R, HK61 with mt3243, and YDM99 with amylin S20G mutations), the mutations were found in both diabetic and nondiabetic family members. Among the three families with glucokinase mutations, all mutation carriers from the families of YDM142 and HK84 had higher fasting plasma glucose concentrations (5.8–8.9 mmol/l) than those with no mutation (4.2–5.3 mmol/l). On the other hand, the four mutation-carrying siblings of proband YDM67 had normal fasting plasma glucose concentrations (4.0–5.6 mmol/l), irrespective of their glycemic status.

Although 26 of the patients with early-onset diabetes carried putative gene mutations or the autoimmune marker, the causes of diabetes in the other 119 patients remain to be determined. These young patients with diabetes of unknown cause (age at diagnosis 30 ± 6 years) differed clinically from the 290 late-onset patients (age at diagnosis 52 ± 8 years) (Table 2). Thus, despite a positive family history of diabetes in all patients in both groups, those with early-onset diabetes more frequently had a father with diabetes (39 vs. 22%) and a mother with diabetes (63 vs. 41%), but less frequently a sibling with diabetes (30 vs. 53%) (P < 0.001). The early-onset patients had a higher BMI but lower blood pressure (BP) and lower prevalence of retinopathy and neuropathy than the late-onset patients. The early-onset patients had better glycemic control (glucose and HbA_1c) as well as higher fasting insulin concentrations than the late-onset patients. Notwithstanding a similar mean disease duration of only 4 years, both the early- and late-onset patients had a disproportionately high prevalence of albuminuria, 40 and 38%, respectively, compared with the prevalence rates of other microangiopathic complications. Insulin resistance, as assessed by the HOMA_IR index, was similar between the two groups of non–insulin-treated patients. The proportion of patients treated with insulin was similar in both groups (8 vs. 7%), but fewer patients with early-onset diabetes were treated with oral drugs (33 vs. 61%, P < 0.001) when compared with the late-onset group.

Because of the high prevalence of general obesity in both early-onset patients of unknown cause and late-onset patients (55 and 46%, respectively), we further analyzed the association of obesity with cardiovascular risk factors and complications in these patients (Table 2). Among the early-onset patients, the obese patients had worse glycemic control (HbA_1c) as well as higher systolic BP levels, a more adverse lipid profile (higher TG, lower HDL-C, and higher TC/HDL-C), and higher fasting insulin than the nonobese patients. They were also more insulin-resistant (HOMA_IR index) and had a higher prevalence of retinopathy and albuminuria than the nonobese patients. Among the late-onset patients, the obese patients had better glycemic control (glucose and HbA_1c) than the nonobese patients. However, they had a higher systolic and diastolic BP, and a higher fasting insulin than the nonobese patients. The degree of insulin resistance and prevalence of complications were similar in the two groups.

Since 1990, our group has documented the high prevalence of diabetes and the pattern of this disease in the Hong Kong Chinese population (1,5,32,37). Using carefully phenotyped patient cohorts, we have ascertained the high prevalence of young-onset diabetes and its phenotypic heterogeneity, with obesity and family history as important features. In agreement with our previous studies (6,16,38) and other studies from Asia (14,39), only 6 (4%) of 145 young patients with familial diabetes with type 2 presentation were positive for anti-GAD, indicative of the rarity of the autoimmune form of diabetes in Chinese populations.

Our present study further demonstrates that young patients with familial early-onset diabetes are genetically heterogeneous. Up to 14% of patients might carry mutations in one of the diabetes-associated genes. Each of the novel mutations identified in the HNF-1α and glucokinase genes alter conserved amino acids in different species (35,40), suggesting that they might have functional significance. Two such mutations, HNF-1α G20R and glucokinase Q239R, were found in four unrelated patients, but haplotype analysis, which tests for a common founder effect in these mutations, was not possible because only one of the families was accessible.

Although putative genetic or autoimmune causes of diabetes were identified in nearly 20% of our familial early-onset patients, a phenotypic characterization of different types of diabetes was not feasible because of the small number of families being identified and the uncertainty of the diabetogenic role of each mutation. In this respect, despite an in vitro study demonstrating a pathogenic role of amylin S20G mutation (9), cosegregation findings in family studies were inconclusive (10, 11, present study). Similarly, whereas carriers of glucokinase variants from the families of YDM142 and HK84 had high fasting plasma glucose compatible with glucokinase deficiency (41), carriers of the glucokinase Q239R mutation from the YDM67 family did not have fasting hyperglycemia. In light of these findings, the prevalence of diabetogenic mutations could not be directly deduced in our population, and further functional studies are required to demonstrate their effects directly. Nevertheless, it is still plausible that these genetic mutations might represent rare variants or were affected by incomplete penetrance, phenocopies, and/or environmental factors.

Despite the familial nature of their diabetes, the remaining young patients with diabetes of unknown cause exhibited different phenotypic patterns compared with the late-onset patients. Cardiovascular risk factors (adverse lipid profiles and BP) and diabetic complications (retinopathy and neuropathy) were more frequent in late-onset patients, despite matching for sex and disease duration, perhaps because of delayed diagnosis, older age, and poorer glycemic control (42). In both early- and late-onset patients, general (BMI) and central (waist circumference) obesity clustered with cardiovascular risk factors and hyperinsulinemia. These findings were consistent with the important role of obesity in insulin-resistant and hyperinsulinemic states (43,44). The high rate of having a parental history of diabetes and obesity with its associated cardiovascular and complication risks in the early-onset patients prompts further search for other MODY- or obesity-associated genes. Our findings in early-onset patients with familial diabetes are in accordance with a recent report in Caucasian subjects, which described a high prevalence of obesity and resemblance to the metabolic syndrome in MODYx families (45). In addition, despite a mean disease duration of only 4 years, the young patients had a disproportionately higher prevalence of albuminuria (40%) compared with diabetic retinopathy and neuropathy, both of which had a <10% prevalence rate. These findings have also been reported in our previous analyses in a separate cohort of young diabetic patients (28). The clustering of obesity and albuminuria in these young patients warrants further investigation (46).

Based on these findings, we argued that if all the putative mutations found in the HNF-1α, glucokinase, mt3243, and amylin S20G genes were diabetogenic, genetic factors were more significant than autoimmune markers in the pathogenesis of familial early-onset type 2 diabetes in Chinese individuals. The high prevalence of obesity, often in association with multiple cardiovascular risk factors, suggests that genetic causes for obesity require further investigation in young Chinese subjects with type 2 diabetes. Early identification of such genetic factors and an understanding of gene-gene and gene-environment interactions are important for both the diagnosis and treatment of these high-risk patients and their families (47).

This work was supported by grants from the Hong Kong Research Grants Committee (4289/97M and 4292/99M), the Chinese University of Hong Kong Strategic Grant Program (SRP9902), the Innovation and Technology Support Fund (ITS/33/00), and an educational grant from Armedic Servier.

We thank Betty Yip for technical support, Kelvin Yu and Patricia Pinna for management of our diabetes database, and Cherry Chiu and Maggie Lau for the recruitment of young onset patients and their family members. We are also indebted to the late Professor Robert Turner, Oxford University, for his collaboration in our family studies in Chinese diabetic patients. We thank all nursing and medical staff at the PWH Diabetes and Endocrine Center for their dedication and professionalism.