Identification and functional analysis of c.422_423InsT, a novel mutation of the HNF1A gene in a patient with diabetes

Abstract

Background HNF1A gene regulates liver‐specific genes, and genes that have a role in glucose metabolism, transport, and secretion of insulin. HNF1A gene mutations are frequently associated with type 2 diabetes. HNF1A protein has three domains: the dimerization domain, the DNA‐binding domain, and the trans‐activation domain. Some mutations in the dimerization or DNA‐binding domains have no influence on the normal allele, while others have dominant negative effects. The I27L, A98V, and S487N polymorphisms are common variants of the HNF1A gene; they have been found in T2D and non‐diabetic subjects.Methods and ResultsWe searched for mutations in the first three exons of the HNF1A gen in an Amerindian population of 71 diabetic patients. DNA sequencing revealed the previously reported I27L polymorphism (c.79A>C) in 53% of diabetic patients and in 67% of the control group. Thus, the I27L/L27L polymorphism might be a marker of Amerindians. In addition, we found the c.422_423InsT mutation in the HNF1A gene of one patient, which had not been previously reported. This mutation resulted in a frame shift of the open reading frame and a new translation stop in codon 187, leading to a truncated polypeptide of 186 amino acids (Q141Hfs*47). This novel mutation affects the DNA‐binding capacity of the mutant HNF1A protein by EMSA; its intracellular localization by fluorescence and confocal microscopy, and a dominant‐negative effect affecting the DNA‐binding capacity of the normal HNF1A by EMSA. We also studied the homology modeling structure to understand the effect of this mutation on its DNA‐binding capacity and its dominant negative effect.ConclusionThe HNF1A Q141Hfs*47 mutant polypeptide has no DNA‐binding capacity and exerts a dominant negative effect on the HNF1A protein. Therefore, it might produce severe phenotypic effects on the expression levels of a set of β‐cell genes. Consequently, its screening should be included in the genetic analysis of diabetic patients after more functional studies are performed.

Reduced calcium levels and accumulation of abnormal insulin granules in stem cell models of HNF1A deficiency

“…S11h ). Previous studies have shown that frameshift mutations in the DNA-binding domain can cause dominant-negative effects 41 .…”

Human stem cell model ofHNF1Adeficiency shows uncoupled insulin to C-peptide secretion with accumulation of abnormal insulin granules

“…Interestingly, the hESC heterozygous line harboring a frameshift mutation in the DNA-binding domain and causing a premature stop codon presented a stronger bias towards a-cell fate. Previous studies in MODY3 patients have shown that frameshift mutations in the DNA-binding domain cause dominantnegative effects affecting the DNA binding capacity of the normal HNF1A (43).…”

Identification Of Pathogenic Mutations And Application Of Polygenic Risk Scores To Differentiate MODY Patients From Other Diabetes Types

“…The GCK variant p.Gly80Ala was located within the glucokinase ATP binding site, where Gly80 residue plays a role in its formation, although not directly binding ATP (29,30). The novel missense variant p.Ala15Asp is located within the HNF1A N-terminal dimerization domain (31), and p.Cys55Tyr -within a highly conserved HNF4A zinc-nger DNA binding domain (32). Another novel missense variant, p.Glu339Val, is also expected to have a negative effect on gene function, as it occurs within the GCK gene mutational hot spot.…”