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 results: We 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.

Conclusion: The 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.

Keywords: Dominant‐negative effect; HNF1A; I27L; MODY3; Q141Hfs*47.

Figure 1

Comparison of the amino acid sequences between the HNF1A and the HNF1A Q141fs*47 polypeptides. (A) Scheme of HNF1A and HNF1A Q141fs*47 proteins showing the dimerization domain (DD, amino acids 1–32), POU domains (POU_S, amino acids 91–181 and POU_H, amino acids 203–279) and the transactivation domain (TAD, residues 281–631). The HNF1A Q141fs*47 polypeptide only contains the dimerization domain and a fragment of the POU_S domain (residues 91–140). (B) Alignment of HNF1A and HNF1A Q141fs*47 polypeptides with Clustal Omega; the identical amino acids are in black boxes and have an asterisk below; the conserved amino acids are in gray boxes; DD is indicated by a rectangular frame; amino acids of the POU_S domain interacting with DNA in HNF1A polypeptide are indicated with arrows, whereas residues of the POU_H domain that interact with DNA are indicated with empty arrowheads; H141 in the mutant polypeptide is indicated with a black arrowhead; α‐helixes (Pα1 to Pα5) of the POU_S domain are indicated with double‐headed arrows below the sequences; the normal NLS is double‐underlined.

Figure 2

The HNF1A Q141fs*47 polypeptide lacked in DNA‐binding activity in vitro. (A) DNA‐binding activity of HNF1A and HNF1A Q141fs*47 polypeptides were assayed by EMSA. rHNF1A and rHNF1A Q141fs*47 proteins were expressed in vitro in a coupled transcription/translation system as described. Five μL of each reaction mixture were used in EMSA, using as a probe the [γ‐³²P] end‐labeled double‐stranded DNA oligonucleotide (40,000 cpm; 1.29 nm), containing the DNA‐binding site for HNF1A from the GLUT2 gene promoter. Lane 1 carried free probe. In lanes 2 and 5, no competitor was added. Lanes 2–4 carried rHNF1A. Lanes 5–7 carried rHNF1A Q141fs*47. Lanes 3 and 6 carried 300‐fold molar excess of unlabeled probe as specific competitor (SC). Lanes 4 and 7 carried 300‐fold molar excess of unlabeled E. histolytica TATTTAAA oligonucleotide used as unspecific competitor (UC). (B) 12% SDS‐PAGE of protein extracts from [³⁵S]‐Methionine‐labeled transcription/translation coupled reactions expressing no recombinant protein (lane 2), rHNF1A protein (lane 3), and HNF1A Q141fs*47 polypeptide (lane 4). Lane 1 contained molecular weight markers. (C) The gel showed in B was exposed to an autoradiography film and developed. Lane 2 contained no recombinant protein synthesized. Lane 3 contained rHNF1A polypeptide. Lane 4 contained rHNF1A Q141fs*47 protein.

Figure 3

The recombinant HNF1A Q141fs*47 polypeptide exerted a negative effect on the DNA‐binding activity of HNF1A. (A) rHNF1A and rHNF1A Q141fs*47 polypeptides were expressed in vitro in a coupled transcription/translation system as described. Five μL of reaction mixture of rHNF1A polypeptide synthesized in vitro were used in each assay. Then, increasing amounts of the rHNF1A Q141fs*47 polypeptide were added (lanes 3 to 6) and assayed by EMSA, using as a probe the [γ‐³²P] end‐labeled double stranded DNA oligonucleotide (40,000 cpm; 0.52 nm) containing the DNA‐binding site for HNF1A from the GLUT2 gene promoter. Lane 1 contained free probe. In lane 2, no mutant polypeptide was added; Lanes 3–6 contained 2.5, 5, 10, and 20 μL, respectively, of reaction mixture containing the rHNF1A Q141fs*47 polypeptide. (B) Radioactivity from EMSA assays was determined as described in Methods. We considered the amount of complex one shown in lane 2 as 100%.

Figure 4

The HNF1A Q141Hfs*47 mutant polypeptide is inefficiently translocated to the nucleus. (A) Diagram of the HNF1A‐EGFP and HNF1A c.422_423InsT‐EGFP fusion genes showing the location of primers used in expression assays by RT‐PCR. (B) RT‐PCR products analyzed by electrophoresis in a 1% agarose gel stained with ethidium bromide. Lane 1 contained molecular size markers. Lanes 2–5 contained RT‐PCRs performed with RNA isolated from COS‐7 cells transfected with the pEGFP‐N1 plasmid. Lanes 6–8 contained pEGFP‐N1/HNF1A. Lanes 9–11 contained pEGFP‐N1/HNF1A_c.422_423InsT. Lanes 2, 3, 6, and 9 contained RT‐PCR amplifications for B2M (β‐2‐microglobulin). Lanes 4, 7, and 10 contained HNF1A‐EGFP. Lanes 5, 8, and 11 contained HNF1A_c.422_423InsT‐EGFP. RT, reverse transcriptase. (C–H) confocal microscopy of COS‐7 cells transfected with plasmid pEGFP‐N1/HNF1A (C–E) or pEGFP‐N1/HNF1A_c.422_423InsT (F–H). Nuclei stained with Hoechst 33342 (C, F, blue channel). Fluorescence of EGFP fusion proteins (D, G, green channel). (E) Merge of fluorescence signals of C and D. (H) Merge of fluorescence signals of F and H. Scale bar, 30 μm.

Figure 5

Structural comparison of the molecular models of HNF1A Q141fs*47 and HNF1A polypeptides. The molecular structure of the HNF1A Q141Hfs*47 polypeptide was theoretically determined with the Modeller program using as a template the structure of the HNF1A bound to DNA (PDB ID 1IC8). Both structures were superimposed with the Chimera program. The mutant HNF1A Q141Hfs*47 polypeptide is displayed as green ribbons and with its surface as a green mesh. The normal HNF1A protein is shown as gray ribbons. (A) The α‐helixes Pα1 to Pα5 of the POU_S domain are indicated with arrows. Encircled is the POU_H domain of the HNF1A polypeptide. Only the Pα4 helix involved in DNA‐binding activity is shown. (B) Structures shown in A were rotated 270° about the horizontal axis.

Figure 6

Comparison of the surface electrostatic potential of HNF1A and HNF1A Q141Hfs*47 polypeptides. The electrostatic potential for each molecule was calculated as described in Methods. Different faces of the normal HNF1A polypeptide are shown in A, B, C, G, H, and I. Different faces of the HNF1AQ141Hfs*47 polypeptide are shown in D, E, F, J, K, and L. Residues located in the POU_S domain of wt HNF‐1α polypeptide involved in interactions with DNA and pointed by arrows are shown in A. Mutated residues located in the POU_S domain from the HNF1A Q141Hfs*47 polypeptide are shown in D.

Figure 7

Structural comparison of the DNA–protein interface of the POU_S domains in HNF1A and HNF1A Q141Hfs*47 polypeptides. (A–C) Residues of normal HNF1A that contacted the major groove of DNA (N140, Q141, S142, H143, Q146, and N149), as well as those that did not (L144, S145, H147, and L148). (D–F) Residues of HNF1A Q141Hfs*47 protein that contacted DNA (N140, H141, V142, P143, P146, and Q149), and those that did not (P144, V145, T147, and P148). The color codes for HNF1A and HNF1A Q141Hfs*47 polypeptides were: N140 – yellow for both proteins; Q141 and H142 – magenta; S142 and V142 – orange; H143 and P143 – cyan; L144 and P144 – white; S145 and V145 – brown; Q146 and P146 – green; H147 and T147 – red; L148 and P148 – purple; N149 and Q149 – blue. Molecule rotated nearly 90° (B, E, H) and 180° (C, F, I) along the DNA alpha helix. (G–I) Superimposed HNF1A and HNF1A Q141Hfs*47 POU_S domain structures. HNF1A – cyan; HNF1A Q141Hfs*47 – green.