| ObjectiveNeonatal diabetes mellitus is a rare monogenic diabetes, which is usually diagnosed in the first6months of life. However, there’re some patients diagnosed after6months were reported to have mutations in typical genes underlying neonatal diabetes. Up to date, more than20causal genes in total have been reported, among which the most common ones are the mutations in KCNJ11and ABCC8, encoding two subunits of potassium channels respectively. Most patients with mutaitons in postassim channels are able to switch from insulin to oral sulfonylureas with optimal glycemic control. At the meanwhile, patients’family can benefit from a definite genetic diagnosis. However, genetic basis underlying neonatal diabetes is highly heterogeous and there’re some clinical defined neonatal diabetic patients with unknown genetic cause. In addition, atypical type1diabetes is very different from the typical ones both in manifestations and disease progression, which could be attributed to a different profile of etiology.Despite that Sanger sequecing is the standard protocol for diagnosis of monogenic disease, it’s not appropriate for exploring unknown genetic cause for regular tested negative neonatal diabetes as well as atypical type1diabetes. Thanks to the next-generation sequencing introduced in2009, rapid progress has been made in terms of etiology study for monogenic diseases. In this study, we aimed to explore the genetic etiology of neonatal diabetes as well as atypical type1dabetes by means of targeted next-genration sequencing combined with the traditional ones. The unknown genetic cause will be focused.Materials and MethodsPatients recruitment. There’re30cases in accordance with our criteria included in our study. Patients’parents were given written informed consent. patients’ manifestations, associated symptoms, laboratory tests as well as family history were taken. Peripheral blood sample were taken from both patients and their parents, and suspected relatives in case.Genetic screening strategy. All included patients were divided into2subgroups based on age at diagnosis. patients from group1were dignosed before6month of age, they were tested for KCNJ11,INS and ABCC8subsequently. Targeted next-generation sequencing was introduced in negative cases. for the other group, patients were diagnosed after6months. Among which, patients with diabetes onset before1yr of age were tested for KCNJ11and if negative, INS. Negative cases together with other patients diagnosed after1yr of age were tested by next-generation sequencing.Data analysis. Fisrt of all, an evaluation for the quality of hight throughput sequencing was done. data with poor reads were filtered, the rest was analysed according to the following criteria.1) functional variants:insertion/deletions in CDS and splicing region; SNP:nonsense, splice site and missense;2) variants with an allele frequency below0.01in either of the public databases or those not included in databases.Validation. Candiate functional variants screened were retested by Sanger sequencing. Confirmed variants should be tested among families and health controls subsequently. Further research about candidate genes’ structure and function is considerable.ResultsAmong6neonatal diabetic patients,4were tested positive for KCNJ11(2/6) and INS(2/6).1/24atypical type1diabetes presented with Wolfram syndrome and resulted positive for homozygous mutaitons in WFS1. In total,there’re24regular tested negative cases screened by targeted next-generation sequecing but resulted negative.ConclusionRegular Sanger sequencing is still the first-line protocol for diagnosis of neonatal diabetes. intensive test by targeted next-generation sequencing is necessary for neonatal diabetes with unknown genetic cause. On the other hand, regular genetic test for atypical type1diabetes is of worth, but further study is required to decide whether the intensive genetic test is worth trying in such patiens. |
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