The Asian project for collaborative derivation of reference intervals: (1) strategy and major results of standardized analytes.

BACKGROUND: A multicenter study conducted in Southeast Asia to derive reference intervals (RIs) for 72 commonly measured analytes (general chemistry, inflammatory markers, hormones, etc.) featured centralized measurement to clearly detect regionality in test results. The results of 31 standardized analytes are reported, with the remaining analytes presented in the next report. METHOD: The study included 63 clinical laboratories from South Korea, China, Vietnam, Malaysia, Indonesia, and seven areas in Japan. A total of 3541 healthy individuals aged 20-65 years (Japan 2082, others 1459) were recruited mostly from hospital workers using a well-defined common protocol. All serum specimens were transported to Tokyo at -80°C and collectively measured using reagents from four manufacturers. Three-level nested ANOVA was used to quantitate variation (SD) of test results due to region, sex, and age. A ratio of SD for a given factor over residual SD (representing net between-individual variations) (SDR) exceeding 0.3 was considered significant. Traceability of RIs was ensured by recalibration using value-assigned reference materials. RIs were derived parametrically. RESULTS: SDRs for sex and age were significant for 19 and 16 analytes, respectively. Regional difference was significant for 11 analytes, including high density lipoprotein (HDL)-cholesterol and inflammatory markers. However, when the data were limited to those from Japan, regionality was not observed in any of the analytes. Accordingly, RIs were derived with or without partition by sex and region. CONCLUSIONS: RIs applicable to a wide area in Asia were established for the majority of analytes with traceability to reference measuring systems, whereas regional partitioning was required for RIs of the other analytes.

AGG interspersion analysis of the FMR1 CGG repeats in mental retardation of unspecific cause.

OBJECTIVES: To study the AGG interspersion pattern in mentally retarded patients of unspecified cause. METHODS: FMR1 CGG substructure in 104 normal and 232 mentally retarded (MR) males was determined by CGG repeat and AGG interspersion analyses. Genomic DNA of the study subjects was obtained for PCR and Southern hybridization analyses. RESULTS: All study subjects had less than 53 CGG repeats and none had fragile X syndrome of mental retardation. There was a significant difference (P < 0.006) in the AGG interspersion pattern. MR males had (1) more variable internal substructures, (2) proportionally less 2 and 3 AGG but more 0 and 1 AGG, less (CGG)(9)AGG(CGG)(9)AGG(CGG)(9) but more (CGG)(9)AGG(CGG)(19) alleles and (3) a longer pure 3' CGG repeat. CONCLUSIONS: Our results suggest that the MR alleles have a lesser number of interspersed AGG and a longer pure 3' CGG repeat than the normal population. They are thus more prone to instability and expansion to long repeat lengths as in the fragile X syndrome of mental retardation.

Pathogenic mutations of the lipoprotein lipase gene in Chinese patients with hypertriglyceridemic type 2 diabetes.

Elevated plasma triglyceride and nonesterified fatty acid concentrations may cause insulin resistance. Lipoprotein lipase (LPL) is a rate-determining enzyme in lipid metabolism. To investigate the role of the LPL gene in Chinese patients with hypertriglyceridemic type 2 diabetes, 277 patients with type 2 diabetes and 241 healthy control subjects were recruited and screened for sequence changes in the LPL gene by PCR, SSCP, restriction analysis and direct DNA sequencing. Ten mutations were identified: four missense mutations, Ala71Thr, Val181Ile, Gly188Glu and Glu242Lys; one nonsense mutation Ser447Ter; and five silent mutations. Ser447Ter was found in both patients and controls with no significant difference in frequency. The four missense mutations were located in the highly conserved exon 3, 5, and 6 regions and in highly conserved amino acid sites. They led to reduced LPL mass and enzyme activities in both post-heparin plasma and in vitro expression. The modeled structures displayed major differences between the mutant and wildtype molecules. These results indicated that the four missense mutations lead to LPL deficiency and subsequent hypertriglyceridemia. Based on our study and published data, a putative pathogenic pathway was suggested: LPL enzyme deficiency causes elevated plasma triglyceride level and subsequent insulin resistance; both increased free fatty acids and insulin resistance promote gluconeogenesis and hyperglycaemia, a vicious circle leading to type 2 diabetes.

Novel mitochondrial 16S rRNA mutation, 3200T-->C, associated with adult-onset type 2 diabetes.

OBJECTIVE: To investigate the role of a potential diabetes-related mitochondrial region, which includes two previously reported mutations, 3243A-->G and 3316G-->A, in Chinese patients with adult-onset type 2 diabetes. METHODS: A total of 277 patients and 241 normal subjects were recruited for the study. Mitochondrial nt 3116 - 3353, which spans the 16S rRNA, tRNA(leu(UUR)) and the NADH dehydrogenase 1 gene, were detected using polymerase chain reaction (PCR), direct DNA sequencing, PCR-restriction fragment length polymorphism and allele-specific PCR. Variants were analyzed by two-tailed Fisher exact test. The function of the variants in 16S rRNA were predicted for minimal free energy secondary structures by RNA folding software mfold version 3. RESULTS: Four homoplasmic nucleotide substitutions were observed, 3200T-->C, 3206C-->T, 3290T-->C and 3316G-->A. Only the 3200T-->C mutation is present in the diabetic population and absent in the control population. No statistically significant associations were found between the other three variants and type 2 diabetes. The 3200T-->C and 3206C-->T nucleotide substitutions located in 16S rRNA are novel variants. The 3200T-->C caused a great alteration in the minimal free energy secondary structure model while the 3206C-->T altered normal 16S rRNA structure little. CONCLUSIONS: The results suggest that the 3200T-->C mutation is linked to the development of type 2 diabetes, but that the other observed mutations are neutral. In contrast to the Japanese studies, the 3316G-->A does not appear to be related to type 2 diabetes.

[Mutation-function analysis in the lipoprotein lipase gene of Chinese patients with hypertriglyceridemic type 2 diabetes].

OBJECTIVE: To investigate the role of lipoprotein lipase (LPL) gene on Chinese patients with hypertriglyceridemic type 2 diabetes. METHODS: Three subject groups, including hypertriglyceridemic group, normalipidemic type 2 diabetes group and healthy controls, were recruited and screened for sequence changes in LPL gene with PCR, SSCP, restriction analysis and direct DNA sequencing. LPL mass and activity in post-heparin plasma and in in vitro expression were investigated. Comparative modeling was performed via Swiss-PDB Viewer to provide the potential 2-D structures of wildtype and mutant proteins. RESULTS: Four missense mutations, Ala71Thr, Val18Ile, Gly188Glu and Glu242Lys, were identified in patients with hypertriglyceridemic type 2 diabetes, and not in both normalipidemic diabetes and the control subjects. The four missense mutations were located in the highly conserved amino acid sites, which are involved in highly conserved exon 3, 5, or 6 regions. They led to reduced LPL mass and enzyme activities in both post-heparin plasma and in vitro expression. The modeled structures displayed the differences to a great extent between the mutant and wide-type molecules. CONCLUSION: These results indicated that the 4 missense mutations lead to LPL deficiency and subsequent hypertriglyceridemia. The LPL deficiency predispose a progressive diabetic pathway to those affected individuals. LPL gene is one of susceptibility gene for hypertriglyceridemic type 2 diabetes.

Sources of variation of commonly measured serum analytes in 6 Asian cities and consideration of common reference intervals.

BACKGROUND: In a previous study to determine the feasibility of common reference intervals in Asia, we found significant differences among populations from 6 cities. In this study, we attempted to define the sources of these differences. METHODS: We enrolled 580 healthy volunteers (279 men, 301 women, 20-62 years old), after a selection process that was based on the Clinical and Laboratory Standards Institute guidelines, and used a lifestyle questionnaire. All sera were obtained at a basal state and frozen at -80 degrees C until the collective assay was done. We measured 21 basic chemical analytes and 10 serum proteins. RESULTS: We used 3-level nested ANOVA to separate the variation (SD) into between-city (SD-city), between-sex (SD-sex), between-age (SD-age), and between-individual (SD-indiv) components. SD-indiv corresponds to one-quarter of the "pure" reference interval obtained after removing variations due to city, sex, and age. The SD-sex to SD-indiv ratio was >0.8 for creatinine, urate, retinol-binding protein, and transthyretin. We observed high SD-city to SD-indiv ratios, ranging from 0.4 to 0.7, for 11 analytes including lactate dehydrogenase (LDH), electrolytes, IgG, and complement components and SD-age to SD-indiv ratios >0.4 for LDH, alkaline phosphatase, and total cholesterol. Multiple regression analysis demonstrated several other relevant sources of variation, including body mass index, alcohol consumption, and cigarette smoking, although their contributions were generally smaller than those for sex, region, or age. CONCLUSION: We observed unacceptably large regional differences in measured values of some analytes even after adjustment for age, sex, and lifestyle variables. Genetic and environmental factors may account for the residual differences.