The transcription factor Hepatocyte nuclear factor-1α (HNF1A) is important for normal pancreas development and function. Some HNF1A genetic variants are associated with type 2 diabetes, others can cause Maturity-Onset Diabetes of the Young (MODY3). Since variations in the HNF1A gene give different clinical consequences, novel sequence variants require thorough clinical and functional analysis. Here we investigate, in vitro, the consequence of rare non-coding intronic sequence variants of HNF1A from the Norwegian MODY registry. To evaluate the effect of four potential splice site mutations, fragments of intron/exon/intron genomic DNA from patient blood was amplified and cloned between two exons in a pCAS2 splicing minigene vector. Total RNA was isolated from HeLa and HepG2 cells transfected with minigene plasmids (wt or mutant) and analyzed by RT-PCR followed by agarose gels and sanger sequencing. All selected variants showed alternative mRNA splicing compared to wt by the minigene assay. The major splicing event of mutant c.526+1G>A showed the activation of a cryptic donor site, causing loss of the last 31 nucleotides of exon 2. In mutant c.714-1G>A, the major splicing event showed loss of 38 nucleotides in exon 4, due to activation of a novel cryptic acceptor site. In the event of c.956-2A>G, sequencing showed a complete skipping of the subsequent exon 5. The c.1502-6G>A variant causes activation of a cryptic acceptor site in intron 7 which leads to a four-nucleotide retention of the intronic sequence. The consequences of all variants were additionally evaluated using in silico splice site predictions by the integrated software tool Alamut v.2.6. Predictions of cryptic sites and the in vitro nucleotide changes observed were fully in agreement. These four intronic HNF1A variants likely cause MODY3 due to abnormally spliced mRNAs. By combining in silico predictions and the pCAS2 minigene expression system, we here provide further evidence of the variants role as pathogenic MODY3 mutations.

Disclosure

M.H. Solheim: None. M. Nasrollahzadeh Khakiani: None. J. Molnes: None. I. Aukrust: None. P. Njølstad: None. S. Johansson: None.

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