Endocrine Abstracts

Background and Aims: Understanding the genetic causes of monogenic forms of diabetes affecting pancreatic β cells is essential for accurate diagnosis and treatment of these rare diseases. Mutations in genes involved in endoplasmic reticulum (ER) stress or in ER-to-Golgi protein trafficking cause neonatal diabetes, microcephaly and epilepsy syndrome. The aim of this study was to identify novel genetic causes of neonatal diabetes and study the pathogenic mechanisms.

Material and Methods: We performed genome sequencing in 2 individuals with neonatal diabetes, microcephaly and epilepsy. Replication studies were performed using next generation sequencing in 284 patients diagnosed with diabetes <6 months without a known genetic cause; 7 also had microcephaly. We inserted by CRISPRCas9 the TMEM167A V59E mutation in human induced pluripotent stem cells (iPSCs) and differentiated the iPSCs into βcells. βcell development was evaluated by immunocytochemistry, and proinsulin ER-to-Golgi trafficking by the Retention Using Selective Hooks assay. Insulin secretion was evaluated in vitro and in Rag2-/- mice transplanted with human iPSC-β cells by measuring human C-peptide levels during intraperitoneal glucose tolerance tests. βcell apoptosis was assessed by Hoechst 33342 and propidium iodide staining, and ER stress markers by qPCR.

Results: We identified homozygous rare coding TMEM167A mutations in 2 probands and in 4 additional individuals in replication studies. All 6 patients with biallelic TMEM167A mutations were diagnosed with neonatal diabetes (median age at diagnosis 34 days, range 1 day - 22 weeks) and were insulin-treated from diagnosis (median insulin dose 1.13 U/kg/day, range 0.6 - 1.7). Four/6 individuals had low birth weight, all had severe microcephaly and 5/6 had epilepsy in the neonatal period. To study the impact of one of the mutations, iPSCs were edited to insert the homozygous TMEM167A V59E mutation, prior to differentiation into β cells. Mutant iPSC-β cells had impaired proinsulin trafficking from the ER to the Golgi. Mutant iPSC-βcells had a halved insulin content and blunted insulin secretion when stimulated by glucose, glucose plus the GLP-1 analogue exenatide and KCl. Upon exposure to ER stressors, mutant βcells had exacerbated ER stress and apoptosis, while treatment with exenatide, ISRIB and imeglimin improved βcell survival. After 4 months of in vivo maturation of iPSC-βcells transplanted into mice, human C-peptide was barely detectable in mice with mutant iPSC-βcells but present in those with healthy cells.

Conclusions: Recessive TMEM167A mutations are a novel cause of neonatal diabetes. The TMEM167A V59E mutation impacted proinsulin trafficking, markedly impaired insulin secretion and sensitized cells to ER stress. Exenatide, ISRIB and imeglimin hold therapeutic potential for ER stress-related diabetes. This disease highlights the crucial role of ER-to-Golgi trafficking and the ER stress response in βcells.

Keywords: TMEM167A, neonatal diabetes, ER stress, ER-to-Golgi protein trafficking