HNF1A-MODY, is a common form of monogenic diabetes. HNF1A variants of unknown significance can be a challenge to pathogenic, and in turn may prevent a diagnosis of HNF1A-MODY. This means individuals with HNF1A-MODY who could benefit from sulfonylurea treatment, may not receive it. This thesis focuses on two HNF1A variants of unknown significance: the homozygous p.A251T (the world’s first observed homozygous potentially pathogenic variant) and heterozygous p.S19L. The overarching objective of this thesis was to assess the impact of these variants on the function of the HNF1A protein, β-cell insulin secretion, and the fundamental mechanisms driving the MODY phenotype. These investigations employed a comprehensive multimodal strategy, integrating a clinical study with in vitro functional assays utilising diverse cell lines, including immortalised and patient-derived β-cell-like cells. The clinical study underscores the difficulty in ascertaining the pathogenicity of an HNF1A variant of unknown significance exclusively through clinical characteristics and predictive biomarkers. In silico and in vitro assessments revealed that the homozygous p.A251T variant exerts a moderate impact on the HNF1A protein function, leading to a mild reduction in transactivation activity compared to wildtype HNF1A. In contrast, the heterozygous p.S19L variant exhibited a notable reduction in HNF1A protein function when compared to the wild-type, resulting in diminished transactivation activity and altered the subcellular localisation of the HNF1A protein. Due to the limitations of basic in vitro methodologies in elucidating the mechanism underlying the p.A251T variant's impact, β-cell-like cells were generated carrying the HNF1A p.A251T, derived from the probands fibroblasts obtained at skin biopsy. The A251T β-like cells displayed impaired insulin secretion in response to glucose and a decreased relative expression of insulin, as well as disrupted Ca2+ entry oscillation compared to iPSC β-cell-like cells derived from healthy donor. This study emphasises the significance of a comprehensive multimodal approach, combining clinical data, in silico analyses, and in vitro assessments, to accurately classify HNF1A variant pathogenicity.