All Exon Sequencing Applications In Neonatal Diabetes Research

ObjectiveNeonatal diabetes mellitus (NDM) is a rare disease, defined as a monogenic diabetes developed within the first half year of life. Till now there are nearly20genes reported responsible for NDM, which are all related with pancreatic B cells, such as KCNJ11, INS, ABCC8, GCK and so on. But there are still30%of NDM patient yet to have a genetic cause identified. The regular treatment of NDM is lifelong insulin injection, however, it has been reported that early transfer from insulin to oral sulfonylurea therapy is successful in most of NDM patients with ABCC8and KCNJ11mutations which are two genes encoding the ATP sensitive potassium channel of pancreaticBcells. So gene diagnosis may also guide the etiotropic therapy.Due to the application of Whole exome sequencing technology, the research of monogenic diseases has made a great breakthrough. The exons play a vital role in the regulation and translation of various proteins and are closely related to the phenotypes of the Mendelian diseases. So since its introduction, many unknown causative genes of Me ndelian diseases have been discovered. And it has also been used to find predisposing genes of complex diseases. The purpose of this research is to explore the unknown responsible genes of NDM.Materials and Methods1. Selection of appropriate patients for exome sequencing First we used Sanger sequencing to analyze the variations of KCNJ11, ABCC8, INS and GCK, which are four most common reasons for NDM. After excluding eight patients with mutations, we chose two sibs with typical manifestations, whose parents have normal glucose profile. We operated whole exome sequencing in this family and used two ways for further analysis.2. Data analysis1st way:two sibs from the same family developed the same disease at the similar small age, we supposed that inheritance was the most reasonable explanation (hypothesis1). Since both parents showed no symptoms, it was probably an autosomal recessive model. We did analysis in two conditions: one is that two sibs are carrying mutations in the same causative gene (hypothesis1.1), while the other condition is two different genes (hypothesis1.2). Besides, we also considered the possibility of de novo mutation (hypothesis2), but due to the low incidence we ruled it out temporarily.2nd way:Reviewing all the reported genes of NDM, all are related to B cells, which can be classified as three subgroups according to gene functions, pancreatic development related, B cell mass related and insulin secretion related. So we postulated that unknown genes are related to B cells too. We picked out all the possible B cell related genes through literature retrieval, and according to the variations of these genes in four members we found the candidate gene.3. validationAfter we got the candidate gene, we did a further research about its structure and function. Meanwhile we completed clinical manifestations and chemical examinations related to this gene.Results1st way:in the hypothesis1.1, we got none. While in the hypothesis1.2, due to the different causative genes, we separated the four-member family into two families, the brother’s family including brother, father and mother and the sister’s family including sister, father and mother. We got10genes and4genes respectively through statistics. Combining the gene function and the results of SIFT anticipation, we got the candidate gene PCSK1. But this gene can’t explain the sister’s pathology. Further validation should be carried out.2nd way:After analyzing all the variations in the B cell related genes in four members, we got three (CDKAL1,THADA,PCSK1), but only PCSK1fit the inheritance mode of Mendelian diseases. Above all, we thought PCSK1was probably the new responsible gene of NDM. Further research in NCBI revealed that this gene encodes a type1proinsulin enyme and regulates the biosynthesis of insulin. Mutation in this gene is often related with obesity and hyperproinsulinism.Conclusion This research is aimed to find unknow responsible genes of NDM using whole exome sequencing. After analyzing the data got from a four-member family with two NDM patients, we found the candidate gene PCSK1, which is involved in the process of proinsulin to inslin. But its function remains to be validated in further research. And this study suggests that whole exome sequencing is a useful tool in the research of Mendelian diseases.