The HNF1A mutant Ala180Val: Clinical challenges in determining causality of a rare HNF1A variant in familial diabetes.

AIMS: Heterozygous mutations in hepatocyte nuclear factor-1A (HNF1A) cause maturity-onset diabetes of the young type 3 (MODY3). Our aim was to compare two families with suspected dominantly inherited diabetes and a new HNF1A variant of unknown clinical significance. METHODS: The HNF1A gene was sequenced in two independently recruited families from the Norwegian MODY Registry. Both familes were phenotyped clinically and biochemically. Microsatellite markers around and within the HNF1A locus were used for haplotyping. Chromosomal linkage analysis was performed in one family, and whole-exome sequencing was undertaken in two affected family members from each family. Transactivation activity, DNA binding and nuclear localization of wild type and mutant HNF-1A were assessed. RESULTS: The novel HNF1A variant c.539C>T (p.Ala180Val) was found in both families. The variant fully co-segregated with diabetes in one family. In the other family, two subjects with diabetes mellitus and one with normal glucose levels were homozygous variant carriers. Chromosomal linkage of diabetes to the HNF1A locus or to other genomic regions could not be established. The protein functional studies did not reveal significant differences between wild type and variant HNF-1A. In each family, whole-exome sequencing failed to identify any other variant that could explain the disease. CONCLUSIONS: The HNF1A variant p.Ala180Val does not seem to cause MODY3, although it may confer risk for type 2 diabetes mellitus. Our data demonstrate challenges in causality evaluation of rare variants detected in known diabetes genes.

Structure-function studies of HNF1A (MODY3) gene mutations in South Indian patients with monogenic diabetes.

Maturity-onset diabetes of the young (MODY) is a genetically heterogeneous monogenic form of diabetes characterized by onset of diabetes below 25 years of age, autosomal dominant mode of inheritance and primary defect in insulin secretion. Mutations in the gene (HNF1A) encoding transcription factor hepatocyte nuclear factor 1A (HNF-1A) results in one of the most common forms of MODY (MODY3). HNF-1A is mainly enriched in pancreatic ß-cells and hepatocytes and important for organ development and normal pancreatic function. We here report on the functional interrogation of eight missense HNF1A mutations associated with MODY3 in South Indian subjects, and the contributing effect of common variant (S487N) within HNF1A. Of the eight mutations, three mutations (p.R171G, p.G245R and p.R263H), in particular, affected HNF-1A function in transfected HeLa cells by reducing both transcriptional activity and nuclear localization, possibly due to disruption of the integrity of the three dimensional structure. The common variant p.S487N contributed further to the loss-of-function of p.R271Q (p.R271Q+p.S487N double mutant), in vitro, on both activity and localization. Our data on the first functional study of HNF1A mutations in South India subjects confers that the defect of the HNF-1A mutant proteins are responsible for MODY3 diabetes in these patients.

MODY associated with two novel hepatocyte nuclear factor-1alpha loss-of-function mutations (P112L and Q466X).

Maturity-onset diabetes of the young (MODY) is an autosomal dominant form of diabetes characterized by early onset of pancreatic dysfunction. MODY type 3 is caused by mutations in the hepatocyte nuclear factor (HNF)-1alpha. During a screening of Norwegian patients with suspected MODY we identified two novel HNF-1alpha mutations, P112L and Q466X. The molecular mechanisms underlying the disease were studied by analyzing the DNA binding properties, transcriptional activation, and subcellular localization of HNF-1alpha P112L and Q466X compared to wild type HNF-1alpha. P112L had reduced ability to bind an HNF1 consensus sequence and to activate transcription. Q466X did not differ from wild type HNF-1alpha in DNA binding activity. Transactivation, however, was markedly reduced. When both mutants were coexpressed with wild type HNF-1alpha in HeLa cells, transcriptional activity appeared unaffected, suggesting that a dominant-negative mechanism was not present. Immunolocalization experiments showed that P112L HNF-1alpha was correctly targeted to nuclei in HeLa cells. In contrast, some Q466X HNF-1alpha protein was retained in the cytoplasm, which indicated that the mechanism for nuclear localization was disturbed. Thus, the HNF-1alpha mutations P112L and Q466X both seem to impair pancreatic beta-cell function by loss-of-function mechanisms; P112L by reduced DNA binding and reduced ability to transactivate, and Q466X by reduced transactivation and incomplete nuclear targeting.

A new candidate region for the positional cloning of the XLP gene.

X-linked lymphoproliferative disease (XLP) is an inherited immunodeficiency characterised by selective susceptibility to Epstein-Barr virus and frequent association with malignant lymphomas chiefly located in the ileocecal region, liver, kidney and CNS. Taking advantage of a large bacterial clone contig, we obtained a genomic sequence of 197620 bp encompassing a deletion (XLP-D) of 116 kb in an XLP family, whose breakpoints were identified. The study of potential exons from this region in 40 unrelated XLP patients did not reveal any mutation. To define the critical region for XLP and investigate the role of the XLP-D deletion, detailed haplotypes in a region of approximately 20 cM were reconstructed in a total of 87 individuals from 7 families with recurrence of XLP. Two recombination events in a North American family and a new microdeletion (XLP-G) in an Italian family indicate that the XLP gene maps in the interval between DXS1001 and DXS8057, approximately 800 kb centromeric to the previously reported familial microdeletion XLP-D.

The interaction between human Fc gamma RI and the gamma-chain is mediated solely via the 21 amino acid transmembrane domain of Fc gamma RI.

We have established a biological assay to investigate the nature of the non-covalent interaction between two integral type I membrane proteins, Fc gamma RI and gamma-chain. Fc gamma RI, the human high affinity receptor for immunoglobulin G (IgG), is expressed on the surface of macrophages and monocytes and mediates a broad range of important immunological functions. Fc gamma RI relies on a functional interaction with a second integral type I membrane protein, gamma-chain, to mediate many of these functions. For example, Fc gamma RI can only mediate phagocytosis of IgG-coated particles in COS cells when co-expressed with gamma-chain. We have previously shown that the cytoplasmic domain of Fc gamma RI is not necessary for this functional interaction. In this study we have used the phagocytosis assay to investigate the role of the transmembrane region of Fc gamma RI in mediating this functional interaction with gamma-chain by using mutant and chimeric forms of the receptor. Three mutants, which introduce or remove charged residues from a conserved 10 amino acid stretch of amino acids in the proximal transmembrane region of Fc gamma RI, were able to mediate phagocytosis of IgG-coated particles. In contrast, two chimeric receptors, In which 21 of the amino acids in the distal transmembrane region of Fc gamma RI were replaced with the transmembrane region of the related receptors CD2 or LFA3, were expressed but failed to interact functionally with gamma-chain to mediate phagocytosis. Thus, these mutants demonstrate that the interaction between human Fc gamma RI and gamma-chain is mediated solely via these 21 amino acids in the transmembrane domain of Fc gamma RI.