Identification and functional analysis of CBLB mutations in type 1 diabetes

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Abstract

Casitas B-lineage lymphoma b (Cblb) is a negative regulator of T-cell activation and dysfunction of Cblb in rats and mice results in autoimmunity. In particular, a nonsense mutation in Cblb has been identified in a rat model of autoimmune type 1 diabetes. To clarify the possible involvement of CBLB mutation in type 1 diabetes in humans, we performed mutation screening of CBLB and characterized functional properties of the mutations in Japanese subjects. Six missense mutations (A155V, F328L, N466D, K837R, T882A, and R968L) were identified in one diabetic subject each, excepting N466D. Of these mutations, F328L showed impaired suppression of T-cell activation and was a loss-of-function mutation. These data suggest that the F328L mutation is involved in the development of autoimmune diseases including type 1 diabetes, and also provide insight into the structure–function relationship of CBLB protein.

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Materials and methods

Subjects. We examined 223 unrelated Japanese subjects with type 1 diabetes recruited from 6 university hospitals and affiliated hospitals located in 6 prefectures in Japan. Type 1 diabetes was diagnosed by both clinical features and laboratory data. All of the subjects were ketosis-prone, showed cessation of endogenous insulin secretion (C-peptide levels of <3.3 nmol/day), and needed more than four insulin injections per day. The clinical data on these type 1 diabetic subjects were as follows

Mutation screening of CBLB in Japanese subjects with type 1 diabetes

To detect CBLB mutations in subjects with type 1 diabetes, we first screened all of the coding regions and exon-intron boundaries of the gene in 223 Japanese subjects with type 1 diabetes. We detected 26 variants, including 23 single nucleotide polymorphisms (SNPs) and three insertion/deletion variants of one or two nucleotides (Supplementary Table S1). Among the 12 exonic SNPs, six missense mutations (A155V, F328L, N466D, K837R, T882A, and R968L) were identified (Table 1 and Fig. 1A). We then

Discussion

Our previous studies using KDP rats [6], together with studies of Cblb deficient mice [10], [11], strongly suggest that CBLB is a potential candidate for autoimmune diseases in humans, including type 1 diabetes. Although common variants of CBLB seem not to be associated with type 1 diabetes [12], [13], [14], [15], it is yet possible that rare variants are involved in the development of the disease. In the present study, we found six missense mutations (A155V, F328L, N466D, K837R, T882A, and

Acknowledgments

We are grateful to the patients, their referring physicians (especially Dr. M. Tanaka of Tenri Yorozu Hospital), and the volunteer control subjects for their cooperation. We thank Dr. G. Crabtree (Stanford University) for providing the NFAT luciferase construct and Jurkat-TAg cells. We also thank Drs. K. Yasuda (International Medical Center of Japan) and K. Sada (Fukui University) for helpful advice, and T. Hirata for technical assistance. Part of this study was conducted at the Department of

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