Functional Studies Of Screening Patients With Adult Onset Diabetes Chinese Youth (MODY) And GCK Mutations

PART1Screening of maturity-onset diabetes of the young in Chinese diabetic populationObjective To explore the clinical characteristics and molecular genetics of Chinese maturity-onset diabetes of the young.Methods Inclusion criteria for genetic testing in Chinese patients suspected with maturity-onset diabetes of the young（MODY） were developed. According to the criteria, we collected and retrospectively analyzed the clinical characteristcs and features of laboratory data of Chinese MODY pedigrees diagnosed in Peking Union Medical College Hospital （PUMCH） from2010to2014. Genomic DNA of related members in the pedigrees were extracted, followed by PCR amplification. Then direct sequencing were performed to identify mutations in the potential causative gene of MODY.Results1. A total of33pedigrees were collected and analyzed, in which11MODY2pedigrees and1MODY3pedigree were confirmed by genetic testing. In the12pedigrees,32cases of MODY2,2cases of MODY3and one case of permanent neonatal diabetes mellitus （PNDM） caused by compound heterozygous mutation in GCK gene were found.2. Totally11mutations （R43C、T168A、K169N、R191W, Y215X、E221K、R250H、 G261R、M235T、W257X、A379E） were discovered in GCKgene, in which5[K169N (c.507G>C、Y215X (c.645C>A、R250H （c.749G>A）、W257X （c.771G>A）、 G261R （c.781G>C）]were previously unreported. One previously reported mutation （P519L） in HNF1A gene was also detected.3. The study of the11MODY2pedigrees showed that a large span of the age at diagnosis of hyperglycemia, lack of typical clinical manifestations of diabetes at the time of onset and lower levels of plasma triglyceride were clinical characteristics of MODY2patients. The majority of MODY2patients had normal insulin secretion curve in oral glucose tolerance test（OGTT）. 4. Chinese patients with early-onset diabetes, including MODY2and others caused by unknown pathogenic gene, showed low level of hsCRP. This may be associated with the high percentage of carriers of rs1169288and/or rs2464196in both groups.Conclusion MODY2and MODY3account for33%and3%, respectively in Chinese MODY pedigrees. The causative gene are still unknown in majority of MODY cases. PART2Preliminary screening of mutations in the glucokinase gene in Chinese gestational subjects with abnormal glucose metabolismObjective To preliminarily assess the rate of glucokinase（GCK） gene mutation in Chinese gestational subjects with abnormal glucose metabolism.Methods We retrospectively analyzed Chinese gestational subjects who received oral glucose tolerance test and glycosylated hemoglobin （HbA1c） detection in Peking Union Medical College Hospital （PUMCH） from July2005to May2008. Subjects were selected for direct sequencing of GCK gene if they met the following three criteria:（1） fasting plasma glucose was between5.5and10.Ommol/L,（2） a small increase （<4.6mmol/L） in plasma glucose2hours after an oral glucose load,（3） HbAlc below8.0%.Results A total of577subjects were collected in our study, in which30subjects met the criteria for GCK gene mutation testing. Of the17subjects whose DNA samples were obtainable, one case of maturity-onset diabetes of the young type2（MODY2） with GCK gene mutation c.626C>T（NM₀00162.3） in exon6and one case with previously unreported variation in noncoding region had been found. The mutation c.626C>T（NM₀00162.3） resulted in the substitution of methionine for threonine in amino acid209（p. T209M, NP₀00153.1）. According to our finding, we estimated the minimum GCK gene mutation rate was0.27%in Chinese gestational subjects with abnormal glucose metabolism, and the minimum prevalence of MODY2was21/100,000in Chinese population. Conclusion The GCK gene mutations are not common in Chinese gestational subjects with abnormal glucose metabolism. PART3Functional studies of GCK gene mutationsObjective To explore the molecular mechanisms of missense mutations in the GCK gene resulted in hyperglycemia in patients of maturity-onset diabetes of the young type2（MODY2）（R43C, K169N, R191W, E221K, R250H, R275H and A379E） and hypoglycaemia in patients of congenital hyperinsulinism（CHI）（K90R and M197V）.Methods We constructed plasmids of wild type of human islet glucokinase （GCK） recombined with glutathione S-transferase（GST） and mutants carrying the respective mutations by site-directed mutagenesis. Wild type and mutants were bacterially expressed as fusion proteins and affinity-purified. Enzymatic kinetics and thermostability were evaluated for wild type and every mutants by enzyme-coupled analysis.Results1. Compared with wild type, mutants R43C, R191W, E221K, R250H, A379E and K90R resulted in lower protein yield, while mutants M197V had higer protein yield.2. Compared with wild type, mutants K169N, R191W and A379E had significant increased S0.5for glucose and decreased catalytic constant （Kcat）. Mutants R191W and A379E also had increased Km for ATP(ATP-Km. The relative activity index （Ia） for these mutants were<0.001,0.037�'�0.233, respectively.3. Compared with wild type, mutant E221K resulted in increased So.5and ATP-Km and dereased Kcat. Mutant R43C resulted in significant decreased in Kcat without changes in S0.5and ATP-Km. The Ia for both mutants were0.477and0.429, respectively.4. Though mutation of R250H caused mild decrease in Kcat, R275H caused mild decrease in both S0.5and Kcat, there were no siginificant differences in Ia when they compared with wild type.5. Both mutants K90R and M197V resulted in decreased S0.5, Hill coefficient and increased ATP-Km. Mutant K90R also resulted in mild decrease in Kcat. The Ia for both were1.620and4.690, respectively.6. Thermostability analysis revealed that mutants K169, R191W and A379E were thermal instability.Conclusion 1. Abnormal enzymatic kinetics, decreased catalytic activity and thermal stability are the causes of K169N, R191W and A379E mutation to develop hyperglycemia in patients of MODY2.2. Abnormal enzymatic kinetics and decreased catalytic activity involve in the pathogenesis of mutant E221K. R43C mutation promotes the development of MODY2by reduced catalytic activity.3. Abnormal enzymatic kinetics are the causes of M197V and K90R mutation to develop hypoglycaemia in patients of CHI.4. Results of enzymatic kinetics and thermostability analysis do not reveale the pathogenesis of R250H and R275H mutation to develop hyperglycemia.