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Introduction The aim of this study was to perform familial co-segregation analysis and functional trial in vivo during mixed meal tolerance test (MMTT) of novel variants in diabetes candidate genes.Research design and methods It is a continuation of the project “Genetic diabetes in Lithuania” with the cohort of 1209 patients with diabetes. Prior screening for autoimmune markers confirmed type 1 diabetes (T1D) diagnosis in 88.1% (n=1065) of patients, and targeted next-generation sequencing identified 3.5% (n=42) pathogenic variants in MODY genes. Subsequently, 102 patients were classified as having diabetes of unknown etiology. 12/102 were found to have novel variants in potential diabetes genes (RFX2, RREB1, SLC5A1 (3 patients with variants in this gene), GCKR, MC4R, CASP10, TMPRSS6, HGFAC, DACH1, ZBED3). Co-segregation analysis and MMTT were carried out in order to study beta-cell function in subjects with specific variants.Results MMTT analysis showed that probands with variants in MC4R, CASP10, TMPRSS6, HGFAC, and SLC5A1 (c.1415T>C) had sufficient residual beta-cell function with stimulated C-peptide (CP) >200 pmol/L. Seven individuals with variants in RFX2, RREB1, GCKR, DACH1, ZBED3 and SLC5A1 (c.1415T>C, and c.932A>T) presented with complete beta-cell failure. No statistical differences were found between patients with sufficient CP production and those with complete beta-cell failure when comparing age at the onset and duration of diabetes. Nineteen family members were included in co-segregation analysis; no diabetes cases were reported among them. Only in patient with the variant c.1894G>A in RFX2 gene, none of the family members were affected by proband’s variant.Conclusions Functional beta-cell study in vivo allowed to select five most probable genes for monogenic diabetes. Familial co-segregation analysis showed that novel variant in RFX2 gene could be a possible cause of diabetes. Future functional analysis in vitro is necessary to support or rule out the genetic background as a cause of diabetes.