A New Form of Insulin Mutation Diabetes Defined by Zygosity, Stem Cell Models, and Population Data

The heterozygous INS c.16C>T (insulin p.Arg6Cys, R6C) mutation has been described to cause monogenic diabetes, but its pathogenicity has recently been questioned. R6C preproinsulin exhibits impaired translocation into the endoplasmic reticulum (ER), potentially affecting insulin secretion. We explored why "one copy isn't enough" for the R6C pathogenicity using integrative clinical, genetic, and functional approaches. Homozygous INS R6C individuals presented early-onset insulin-dependent diabetes, whereas heterozygous carriers showed variable or absent glycemic phenotypes. Population-level analysis revealed no significant enrichment of diabetes among heterozygous carriers. Heterozygous R6C induced pluripotent stem cell (iPSC)-derived ß cells exhibited minimal defects, while homozygous R6C cells displayed preproinsulin accumulation and reduced insulin content and secretion. In vivo, homozygous R6C ß cell transplants recapitulated human insulin deficiency and responded poorly to GLP-1 receptor agonist therapy. Transcriptionally, homozygous R6C ß cells presented repression of translation, translocation and ER related pathways. Our findings establish R6C as a recessive pathogenic mutation, prompting a reassessment of the clinical interpretation of heterozygous R6C carriers. This study highlights the power of iPSC-based disease modeling and multi-modal variant classification frameworks for dissecting the functional consequences of rare genetic variants in human monogenic diabetes. Overall design: We performed bulk-RNA sequencing on magnetic-activated cell sorting (MACS)-purified iPSC-derived stage 7 ß-like cells.