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Atypical diabetes is considered to be different from the common forms of diabetes (types 1 and 2); however, it may share pathogenetic and phenotypic features with the common forms of diabetes. Targeted next-generation sequencing focused on genes causing monogenic diabetes has been reported to be useful for diagnosis of a few subtypes of atypical diabetes caused by genetic defects, such as MODY. In this study, considering the clinically heterogeneous nature of atypical diabetes, we developed a comprehensive gene panel containing 383 monogenic diabetes causing genes and genes associated with common forms of diabetes, and conducted targeted next-generation sequencing in 3 patients with previously confirmed and 10 patients with suspected atypical diabetes. In addition, phenotype-driven bioinformatics analysis using genetic and phenotypic information was performed to identify causal variants from sequencing data. A total of 11 mutations including 2 gross deletions were identified, which led to diagnosis of four previously undiagnosed patients and identification of potentially pathogenic variants of interest in other three patients. Of note, a novel missense mutation of Rab GAP domain of TBC1D4 was identified in a diabetic patient with severe insulin resistance. The TBC1D4 locus has been reported to be associated with type 2 diabetes and insulin resistance. In silico structural analysis suggested that the mutation would cause pathogenesis by functional and structural modification of TBC1D4, insulin-dependent phosphorylation of which is necessary for GLUT4 translocation mediated glucose uptake in skeletal muscle and adipose tissues. The results from this study indicate that comprehensive genetic screening analyzing genes associated with common and rare subtypes of diabetes can identify causal variants efficiently, thereby aiding early diagnosis of atypical diabetes. Disclosure J. Hosoe: None. H. Kadowaki: Research Support; Spouse/Partner; Astellas Pharma Inc., Daiichi Sankyo Company, Limited, Eli Lilly and Company, Kowa Pharmaceutical, Kyowa Hakko Kirin Co., Ltd., Mitsubishi Tanabe Pharma Corporation, MSD, Nippon Boehringer Ingelheim, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi, Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited. Speaker's Bureau; Spouse/Partner; Astellas Pharma Inc., AstraZeneca, Mitsubishi Tanabe Pharma Corporation, MSD, Nippon Boehringer Ingelheim, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited. F. Miya: None. M. Takakura: None. H. Waki: Research Support; Self; Astellas Pharma Inc., Kyowa Hakko Kirin Co., Ltd., MSD K.K., Nippon Boehringer Ingelheim Co Ltd, Novo Nordisk Foundation, Ono Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited, The Cell Science Research Foundation. T. Sasako: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Merck & Co., Inc., Novo Nordisk Inc. T. Kato: None. T. Tsunoda: None. N. Shojima: None. T. Yamauchi: Research Support; Self; Astellas Pharma Inc., Daiichi Sankyo Company, Limited, Eli Lilly and Company, Kissei Pharmaceutical Co., Ltd., Kowa Pharmaceutical Europe Co. Ltd., Kyowa Hakko Kirin Co., Ltd., Merck & Co., Inc., Mitsubishi Tanabe Pharma Corporation, Novartis Pharmaceuticals Corporation, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi-Aventis, Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited. Speaker's Bureau; Self; Astellas Pharma Inc., AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company, Merck & Co., Inc., Mitsubishi Tanabe Pharma Corporation, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi-Aventis, Takeda Pharmaceutical Company Limited. T. Kadowaki: Research Support; Self; Astellas Pharma Inc., Daiichi Sankyo Company, Limited, Eli Lilly and Company, Kowa Pharmaceutical, Kyowa Hakko Kirin Co., Ltd., Mitsubishi Tanabe Pharma Corporation, MSD, Nippon Boehringer Ingelheim, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi, Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited. Speaker's Bureau; Self; Astellas Pharma Inc., AstraZeneca, Mitsubishi Tanabe Pharma Corporation, MSD, Nippon Boehringer Ingelheim, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited.

Mutations of the insulin receptor (INSR) gene cause severe insulin resistance, resulting in several syndromes with various abnormal phenotypes associated with variable severity of INSR dysfunction. As a member of the insulin receptor family, the IGF-I receptor (IGF1R) shows the same domain structure as INSR containing the fibronectin type III (FnIII) domains where there are an α-β cleavage site and a part of the ligand binding site. Mutations of IGF1R can cause intrauterine and postnatal growth retardation. We compiled 3 mutations of the FnIII-2 and FnIII-3 domains in IGF1R registered in HGMD, ClinVar and UniProt databases as causing growth retardation as well as the SNP with >1% allele frequency in IGF1R from the 1000 Genomes Project data, ESP6500, HGVD and 3.5KJPN, a reference panel of Japanese individuals. We conducted in silico structural analysis of the mutations of the FnIII domains in IGF1R which revealed that the pathogenic mutations were predicted to severely impair stability of the FnIII domains and hydrophobic core formation or affect processing site, while the SNP was predicted not to affect folding of the domains. In addition, IGF-1 proreceptor processing was impaired in cells expressing mutations of FnIII compared to cells expressing WT form. Previously, we performed structural analysis of mutations of the FnIII domains in INSR, which revealed that missense mutations causing the most severe Donohue syndrome were predicted to severely impair hydrophobic core formation and stability of the FnIII domains, while those causing less severe Rabson-Mendenhall syndrome were predicted to lead to localized destabilization and not to affect folding of the FnIII domains. Genotype-structure-phenotype correlations of variants of the FnIII domains in INSR and IGF1R identified in these analyses will enable early diagnosis of pathogenic mutations in INSR and IGF1R and provide a valuable insight into the molecular mechanism of severe insulin or IGF-1 resistance. Disclosure J. Hosoe: None. H. Kadowaki: None. F. Miya: None. M. Takakura: None. T. Tsunoda: None. N. Shojima: None. T. Yamauchi: Research Support; Self; Novo Nordisk Inc., Daiichi Sankyo Company, Limited, Sanofi, Takeda, Tanabe-Mitsubishi. T. Kadowaki: Consultant; Self; Novo Nordisk A/S, AstraZeneca, Merck Sharp & Dohme Corp.. Research Support; Self; Kissei Pharmaceutical Co., Ltd., Taisho Pharmaceutical Co., Ltd., Sanofi, Kyowa Hakko Kirin Co., Ltd., Novo Nordisk A/S, Astellas Pharma, Daiichi Sankyo Company, Limited, Takeda, Mitsubishi Tanabe Pharma Corporation, Ono Pharmaceutical Co., Ltd., Merck Sharp & Dohme Corp., Nippon Boehringer Ingelheim Co. Ltd.. Speaker's Bureau; Self; Astellas Pharma, AstraZeneca, Merck Sharp & Dohme Corp., Ono Pharmaceutical Co., Ltd., Takeda, Eli Lilly and Company, Nippon Boehringer Ingelheim Co. Ltd., Novo Nordisk A/S.

The insulin receptor (INSR) gene was analyzed in four patients with severe insulin resistance, revealing five novel mutations and a deletion that removed exon 2. A patient with Donohue syndrome (DS) had a novel p.V657F mutation in the second fibronectin type III domain (FnIII-2), which contains the α-β cleavage site and part of the insulin-binding site. The mutant INSR was expressed in Chinese hamster ovary cells, revealing that it reduced insulin proreceptor processing and impaired activation of downstream signaling cascades. Using online databases, we analyzed 82 INSR missense mutations and demonstrated that mutations causing DS were more frequently located in the FnIII domains than those causing the milder type A insulin resistance (P = 0.016). In silico structural analysis revealed that missense mutations predicted to severely impair hydrophobic core formation and stability of the FnIII domains all caused DS, whereas those predicted to produce localized destabilization and to not affect folding of the FnIII domains all caused the less severe Rabson-Mendenhall syndrome. These results suggest the importance of the FnIII domains, provide insight into the molecular mechanism of severe insulin resistance, will aid early diagnosis, and will provide potential novel targets for treating extreme insulin resistance.