Mapping diabetes-susceptibility genes. Lessons learned from search for DNA marker for maturity-onset diabetes of the young.

During our search for a marker for non-insulin-dependent diabetes mellitus (NIDDM) in a large multigenerational family with a form of NIDDM termed maturity-onset diabetes of the young (MODY), we learned a great deal that may serve to streamline the search for diabetes-susceptibility genes in other families. We describe here our experience and suggest strategies that may enhance the search for markers for other diabetes susceptibility genes with genetic linkage approaches.

Insulin-receptor and apolipoprotein genes contribute to development of NIDDM in Chinese Americans.

The frequencies of restriction-fragment-length polymorphism (RFLP) alleles as well as RFLP haplotypes at six genetic loci responsible for carbohydrate and lipid metabolism [insulin/insulin-like growth factor II complex, insulin receptor (INSR), HepG2/erythrocyte-type glucose transporter, apolipoprotein A-II, apolipoprotein B (APOB), and the apolipoprotein A-I/C-III/A-IV cluster (APOA1/C3/A4)] were compared between nondiabetic and diabetic Chinese Americans. The disease-association data suggest that genetic variation at the INSR, APOB, and APOA1/C3/A4 loci contributes to the development of non-insulin-dependent diabetes mellitus (NIDDM). The analysis of the INSR locus revealed "protective" haplotypes, and it may be possible to use two of the INSR haplotypes as genetic markers to identify individuals having a very low probability of developing NIDDM regardless of the presence of other genes conferring susceptibility to this disorder. The APOB and APOA1/C3/A4 loci appear to contribute to the development of NIDDM in individuals who are of lean/normal weight and overweight, respectively. The APOA1/C3/A4 locus may account for approximately 8% of the difference between baseline and total possible risk of NIDDM in overweight individuals.

Identification of glucokinase mutations in subjects with gestational diabetes mellitus.

Recent studies have shown that mutations in the glucokinase gene on chromosome 7 can cause an autosomal dominant form of NIDDM with a variable clinical phenotype and onset during childhood. The variable clinical phenotype includes mild fasting hyperglycemia (i.e., a plasma glucose value of > 110 mg/dl, a value that is at least 2-3 SDs above normal), impaired glucose tolerance, gestational diabetes mellitus, as well as overt NIDDM as defined using National Diabetes Data Group or World Health Organization criteria. Because gestational diabetes mellitus was a clinical feature associated with glucokinase mutations, we have screened a group of women with gestational diabetes who also had a first-degree relative with diabetes mellitus for the presence of mutations in this gene. Among 40 subjects, we identified two mutations, suggesting a prevalence of approximately 5% in this group. Extrapolating from this result, the prevalence of glucokinase-deficient NIDDM among Americans may be approximately 1 in 2500.

An approach for identifying simple sequence repeat DNA polymorphisms near cloned cDNAs and genes. Linkage studies of the islet amyloid polypeptide/amylin and liver glycogen synthase genes and NIDDM.

Genetic factors contribute to the development of NIDDM, and genes involved in regulating pancreatic beta-cell function and insulin's effects on glucose metabolism are good candidates for being NIDDM susceptibility loci. However, testing candidate genes for linkage to NIDDM depends on the identification of highly informative DNA polymorphisms in or near the candidate locus. Here we describe an approach for identifying highly polymorphic markers near candidate genes that utilizes the emerging physical map of the human genome. A sequence-tagged site from the candidate gene is used to screen the Centre d'Etude du Polymorphisme Humain megabase-insert yeast artificial chromosome library, which contains information on the physical localization of >3,000 genetically mapped simple sequence repeat DNA polymorphisms. Thus, identification of a yeast artificial chromosome containing the candidate locus will in many instances also identify a physically linked simple sequence repeat DNA polymorphism that can be used as a marker for the candidate gene in linkage studies. We have used this approach to identify a marker for the islet amyloid polypeptide gene on chromosome 12. The physical mapping of this gene to a yeast artificial chromosome showed that it was in the same yeast artificial chromosome as the gene encoding liver glycogen synthase, another possible NIDDM susceptibility gene. Affected sib pair studies using a simple sequence repeat DNA polymorphism physically linked to the islet amyloid polypeptide and liver glycogen synthase genes showed no evidence for linkage with NIDDM, indicating that they are not major genes contributing to NIDDM susceptibility.

Epidemiological study on obesity and its comorbidities in urban Chinese older than 20 years of age in Shanghai, China.

The aim of this work was to establish the prevalence of overweight and obesity and its associated comorbidities in a Chinese population older than 20 years of age. A group of 2776 randomly selected adults (20-94 years of age) living in the Huayang Community in Shanghai, China, were investigated between 1998 and 2000. Body weight, height, waist and hip circumferences and blood pressure were measured, as were fasting blood glucose, fasting insulin and lipid profile, as well as blood glucose 2 h after a glucose load, and a 75-g glucose tolerance test was performed. The prevalence of overweight was 29.5% and obesity was 4.3%, with a greater number of women being obese than men. More than one-third of the population had abnormal lipid levels. Impaired glucose regulation (IGR) occurred in 10.8%; and 9.8% of the population were classified as having type 2 diabetes mellitus. Hypertension was present in 58.4% of this population. About 21% and 29.3% of the population suffered from high total cholesterol and high triglyceride, respectively. The prevalence of metabolic syndrome was 10.2%. The prevalence of diabetes, IGR and metabolic syndrome increased progressively in association with a body mass index (BMI) of >23 kg m(-2). Hence, although the prevalence of obesity is low in this Chinese population, higher BMI and waist circumference values are clearly associated with an increasing prevalence of comorbidities. The absolute risk of having diabetes, IGR and metabolic syndrome is high in adults with a BMI of > or =23 kg m(-2).

Genetic contribution of polymorphism of the GLUT1 and GLUT4 genes to the susceptibility to type 2 (non-insulin-dependent) diabetes mellitus in different populations.

Polymorphic variation of genes encoding the glucose transporters glycoproteins (GLUT) may contribute to the genetic susceptibility to type 2 (non-insulin-dependent) diabetes. In this study we evaluated the allele and genotype frequencies of GLUT1 and GLUT4 restriction fragment length polymorphism (RFLP), revealed by digestion with XbaI for GLUT1 and KpnI for GLUT4, in Caucasian, Chinese, Japanese, Asian Indian and American black populations. No differences of the KpnI GLUT 4 RFLP were found between control and diabetic subjects in any ethnic group or when all data are combined. In contrast, positive results were found for the XbaI RFLP: (1) most ethnic groups showed an association of allele 1 with type 2 diabetes, and this association was maintained when all groups were analysed together; (2) after stratifying for sex and obesity, this association was significant only for overweight/obese women. This joint analysis suggests that GLUT1 polymorphism may contribute to susceptibility to type 2 diabetes in some populations, and especially in overweight/obese women.

[A study of DNA fingerprinting in China].

A recombinant DNA insert which could be hybridized with variable number of tandem repeat (VNTR) sequence probe was obtained from the screening of human genomic DNA library and it was cloned into plasmid for using as a probe designated as ph 296. Highly individual specific DNA fingerprints was produced by hybridizing radioactive labelled ph 296 with restriction endonuclease Hinf I digested genomic DNA of Chinese individuals of Han origin. The number of DNA fragments greater than or equal to 4 kb per individual was 13.6 +/- 3.0; the frequency of shared DNA fragments in unrelated random-paired individuals was 0.25; and the probability that all fragments were detected in both random-paired individuals was 2.0 x 10(-9). ph 296 is an locus-specific probe. The application of DNA fingerprinting is discussed.

Epidemiological characteristics of diabetes mellitus and impaired glucose regulation in a Chinese adult population: the Shanghai Diabetes Studies, a cross-sectional 3-year follow-up study in Shanghai urban communities.

AIMS/HYPOTHESIS: To estimate the prevalence and incidence of diabetes mellitus and impaired glucose regulation (IGR) in a Chinese population aged 20-94 years. SUBJECTS AND METHODS: A group of 5,628 randomly selected adults, aged 20-94 years, living in the Huayang and Caoyang communities in Shanghai, China, were investigated between 1998 and 2001. During 2002-04, 2,666 subjects were followed up. All the participants underwent anthropometric measurements, blood biochemical analyses and a 75-g OGTT. RESULTS: Based on the 2000 census data of China, the age-standardised prevalences were 6.87% for diabetes and 8.53% for IGR at baseline. More than two in five cases with diabetes were undiagnosed. The age-adjusted prevalence of diabetes and IGR increased with age. The age-adjusted prevalences of hypertension, dyslipidaemia and overweight in males were significantly higher (p < 0.001) than in females. The 3-year cumulative incidence rates of diabetes and IGR were 4.96 and 11.10%, respectively. The relative risk of developing diabetes was significantly higher in subjects with IGR than in subjects with NGT (p < 0.001). CONCLUSIONS/INTERPRETATION: The prevalence and incidence rates for diabetes or IGR have increased dramatically over the last decades, especially in younger age groups. A large proportion of cases are undiagnosed. We strongly recommend that population-based diabetes screening programmes should be implemented and generalised for younger people.

Linkage disequilibrium in the human insulin/insulin-like growth factor II region of human chromosome II.

Caucasian (N = 128) and Chinese (N = 84) subjects were typed for RFLPs in the insulin (INS)/insulin-like growth factor II (IGF2) region of chromosome 11. Both the analysis of extended haplotypes and the pairwise measures of linkage disequilibrium among the RFLPs indicate that there is extensive linkage disequilibrium in the INS/IGF2 region. The disequilibrium extends across the hypervariable region (HVR) located just 5' to the INS gene and encompasses a region of at least 40 kbp. Previous studies had suggested that linkage disequilibrium in the INS region was negligible and that this region may therefore contain a "recombinational hotspot" (Chakravarti et al. 1986). However, results of this and another recent study (Thompson et al. 1988) highlight the importance of the frequencies of associated alleles in the ability to detect linkage disequilibrium. Thus, the previous failure to detect disequilibrium in the INS region may have reflected a lack of power, rather than a true absence of disequilibrium in this region.

cDNA sequence and localization of polymorphic human cytosolic phosphoenolpyruvate carboxykinase gene (PCK1) to chromosome 20, band q13.31: PCK1 is not tightly linked to maturity-onset diabetes of the young.

Complementary DNA clones encoding human cytosolic phosphoenolpyruvate carboxykinase (GTP) [GTP: oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32) (PEPCK)] were isolated from a human kidney cDNA library. The nucleotide sequence of the 2.7 kb insert of one of these clones indicates that human PEPCK is a protein of 622 amino acids whose sequence shows 90% identity with that of the cognate rat enzyme. The human PEPCK gene (PCK1) was isolated by hybridization using a fragment of the hPEPCK cDNA as a probe. PCK1 was mapped to human chromosome 20 using DNA from a panel of reduced human-hamster somatic cell hybrids. This assignment was confirmed using fluorescence in situ chromosomal hybridization which localized PCK1 to chromosome 20, band q13.31. A simple tandem repeat DNA polymorphism in the 3'-untranslated region of the mRNA was characterized and used to localize PCK1 relative to the gene responsible for a form of non-insulin-dependent (Type 2) diabetes mellitus called maturity-onset diabetes of the young (MODY). Linkage studies showed that PCK1 is not tightly linked to MODY in one large pedigree and exclude this diabetes candidate gene as the cause of MODY in this family.

Increased risk for gestational diabetes mellitus associated with insulin receptor and insulin-like growth factor II restriction fragment length polymorphisms.

Gestational diabetes mellitus (GDM) is defined as glucose intolerance with onset or first recognition during pregnancy. We have examined restriction fragment length polymorphisms (RFLPs) near "candidate diabetogenic genes" as one approach to identify molecular markers for GDM genes. Genotypes for insulin hypervariable region (HVR), insulin-like growth factor II (IGF2), insulin receptor (INSR), and glucose transporter (GLUT1) RFLPs were studied in 96 GDM and 164 control subjects, matched to GDM for race, age, and gravidity. Logistic regression analysis was used to explore the relationship between genotypes at these candidate gene loci and GDM, while adjusting for the effects of potential confounding variables. Among black subjects, the INSR allele 1 (P = 0.001) and interactions between INSR allele 1 with body mass index (BMI) (P = 0.002) and history of DM in subject's mother (P = 0.004) contributed significantly to GDM risk. Among Caucasian subjects, a similar relationship between the INSR allele 1 (P = 0.007) and INSR allele 1-BMI interactions (P = 0.011) on GDM risk were observed. In Caucasians, an additional significant risk factor was determined by an INSR allele 1-IGF2 allele 2 interaction (P = 0.018). No risk factors were identified in Hispanic subjects. These data continue to support the hypothesis that GDM is a heterogeneous disorder with respect to phenotypic and genotypic features. Furthermore, our data suggest that risk for GDM in black and Caucasian subjects is not due to obesity perse but to interactions between obesity and INSR alleles. In Caucasian women, INSR and IGF2 alleles interact to confer additional risk for GDM. Thus genes underlying susceptibility to GDM in some women may be similar to genes conferring risk to NIDDM, while in others novel genes may contribute to GDM risk.

Gene for non-insulin-dependent diabetes mellitus (maturity-onset diabetes of the young subtype) is linked to DNA polymorphism on human chromosome 20q.

Maturity-onset diabetes of the young (MODY) is a form of non-insulin-dependent diabetes mellitus characterized by an early age of onset, usually before 25 years of age, and an autosomal dominant mode of inheritance. The largest and best-studied MODY pedigree is the RW family. The majority of the diabetic subjects in this pedigree has a reduced and delayed insulin-secretory response to glucose, and it has been proposed that this abnormal response is the manifestation of the basic genetic defect that leads to diabetes. Using DNA from members of the RW family, we tested more than 75 DNA markers for linkage with MODY. A DNA polymorphism in the adenosine deaminase gene (ADA) on the long arm of chromosome 20 was found to cosegregate with MODY. The maximum logarithm of odds (lod score) for linkage between MODY and ADA was 5.25 at a recombination fraction of 0.00. These results indicate that the odds are greater than 178,000:1 that the gene responsible for MODY in this family is tightly linked to the ADA gene on chromosome 20q.