1466-P: Screening and Functional Analysis of Mutations and Variations in NEUROD1 Gene in Pedigrees of Maturity-Onset Diabetes of the Young

Introduction & Objective: To screen the mutations of NEUROD1 gene in maturity-onset diabetes of the young (MODY) families, and to investigate the association of mutations and variants with the onset of MODY6 in Chinese, and its potential pathogenesis.

Methods: PCR-direct sequencing was used for screening NEUROD1 mutations and variations from 96 MODY probands who were negative for mutations in the GCK/MODY2, HNF1A/MODY3, and HNF1B/MODY5 genes. A de novo modeling method was used to predict the three-dimensional (3D) structures of wild type (WT) and mutated NEUROD1 proteins. Transcriptional activities of both WT and mutant of NEUROD1 on insulin gene were detected using dual luciferase reporter gene system. Moreover, the genotypic frequency of NEUROD1 variations were compared between the 96 MODY probands and 100 nondiabetic control subjects.

Results: Glu59Gln (NM_002500.5, c.175G>C), a heterozygous missense mutation in the NEUROD1 gene, was identified in a MODY pedigree. 3D structural analysis showed that the mutation transformed the negatively charged Glu59 of WT into uncharged mutation Gln59, leading to the loss of Glu59-Arg54 and Glu59-Lys88, two salt bridge bonds, and the formation of Gln59-Arg54, one new hydrogen bond. Transcriptional activity of Glu59Gln mutant for insulin gene was reduced 36.3% when compared with that of WT (P<0.01). A common variation Ala45Thr (G-A) was identified, and AA+GA genotypic frequency of the variation was significantly elevated in the 96 MODY probands in comparison to 100 nondiabetic control subjects (P=0.002).

Conclusion: Glu59Gln mutation altered the N-terminal molecular conformation of NEUROD1 protein, resulting in decreased transcriptional activity of insulin gene, which was the cause of the defective insulin secretion in mutation carriers of the MODY6 pedigree. In addition, the Ala45Thr variation was associated with earlier age of onset of diabetes in MODY6 probands.