Ingvild Aukrust

Association of a Low-Frequency Variant in HNF1A With Type 2 Diabetes in a Latino Population

IMPORTANCE Latino populations have one of the highest prevalences of type 2 diabetes worldwide. OBJECTIVES To investigate the association between rare protein-coding genetic variants and prevalence of type 2 diabetes in a large Latino population and to explore potential molecular and physiological mechanisms for the observed relationships. DESIGN, SETTING, AND PARTICIPANTS Whole-exome sequencing was performed on DNA samples from 3756 Mexican and US Latino individuals (1794 with type 2 diabetes and 1962 without diabetes) recruited from 1993 to 2013. One variant was further tested for allele frequency and association with type 2 diabetes in large multiethnic data sets of 14,276 participants and characterized in experimental assays. MAIN OUTCOME AND MEASURES Prevalence of type 2 diabetes. Secondary outcomes included age of onset, body mass index, and effect on protein function. RESULTS A single rare missense variant (c.1522G>A [p.E508K]) was associated with type 2 diabetes prevalence (odds ratio [OR], 5.48; 95% CI, 2.83-10.61; P = 4.4 × 10(-7)) in hepatocyte nuclear factor 1-α (HNF1A), the gene responsible for maturity onset diabetes of the young type 3 (MODY3). This variant was observed in 0.36% of participants without type 2 diabetes and 2.1% of participants with it. In multiethnic replication data sets, the p.E508K variant was seen only in Latino patients (n = 1443 with type 2 diabetes and 1673 without it) and was associated with type 2 diabetes (OR, 4.16; 95% CI, 1.75-9.92; P = .0013). In experimental assays, HNF-1A protein encoding the p.E508K mutant demonstrated reduced transactivation activity of its target promoter compared with a wild-type protein. In our data, carriers and noncarriers of the p.E508K mutation with type 2 diabetes had no significant differences in compared clinical characteristics, including age at onset. The mean (SD) age for carriers was 45.3 years (11.2) vs 47.5 years (11.5) for noncarriers (P = .49) and the mean (SD) BMI for carriers was 28.2 (5.5) vs 29.3 (5.3) for noncarriers (P = .19). CONCLUSIONS AND RELEVANCE Using whole-exome sequencing, we identified a single low-frequency variant in the MODY3-causing gene HNF1A that is associated with type 2 diabetes in Latino populations and may affect protein function. This finding may have implications for screening and therapeutic modification in this population, but additional studies are required.

Ubiquitinated annexin A2 is enriched in the cytoskeleton fraction

Annexin A2 is a multifunctional protein and its cellular functions are regulated by post‐translational modifications and ligand binding. When purified from porcine intestinal mucosa and transformed mouse Krebs II cells, SDS–PAGE revealed high‐molecular‐mass forms in addition to the 36 kDa protomer. These forms were identified as poly‐/multi‐ubiquitin conjugates of annexin A2, and ubiquitination represents a novel post‐translational modification of this protein. Subcellular fractionation of mouse Krebs II cells revealed an enrichment of annexin A2‐ubiquitin conjugates in the Triton X‐100 resistant cytoskeleton fraction, suggesting that ubiquitinated annexin A2 may have a role associated with its function as an actin‐binding protein.

STUB1 mutations in autosomal recessive ataxias – evidence for mutation-specific clinical heterogeneity

BackgroundA subset of hereditary cerebellar ataxias is inherited as autosomal recessive traits (ARCAs). Classification of recessive ataxias due to phenotypic differences in the cerebellum and cerebellar structures is constantly evolving due to new identified disease genes. Recently, reports have linked mutations in genes involved in ubiquitination (RNF216, OTUD4, STUB1) to ARCA with hypogonadism.Methods and resultsWith a combination of homozygozity mapping and exome sequencing, we identified three mutations in STUB1 in two families with ARCA and cognitive impairment; a homozygous missense variant (c.194A > G, p.Asn65Ser) that segregated in three affected siblings, and a missense change (c.82G > A, p.Glu28Lys) which was inherited in trans with a nonsense mutation (c.430A > T, p.Lys144Ter) in another patient. STUB1 encodes CHIP (C-terminus of Heat shock protein 70 – Interacting Protein), a dual function protein with a role in ubiquitination as a co-chaperone with heat shock proteins, and as an E3 ligase. We show that the p.Asn65Ser substitution impairs CHIP’s ability to ubiquitinate HSC70 in vitro, despite being able to self-ubiquitinate. These results are consistent with previous studies highlighting this as a critical residue for the interaction between CHIP and its co-chaperones. Furthermore, we show that the levels of CHIP are strongly reduced in vivo in patients’ fibroblasts compared to controls.ConclusionsThese results suggest that STUB1 mutations might cause disease by impacting not only the E3 ligase function, but also its protein interaction properties and protein amount. Whether the clinical heterogeneity seen in STUB1 ARCA can be related to the location of the mutations remains to be understood, but interestingly, all siblings with the p.Asn65Ser substitution showed a marked appearance of accelerated aging not previously described in STUB1 related ARCA, none display hormonal aberrations/clinical hypogonadism while some affected family members had diabetes, alopecia, uveitis and ulcerative colitis, further refining the spectrum of STUB1 related disease.

SUMOylation of Pancreatic Glucokinase Regulates Its Cellular Stability and Activity

Background: Glucokinase is a key player in carbohydrate metabolism, but how this enzyme is regulated by post-translational modifications is largely unknown. Results: Glucokinase is SUMO-modified in vitro and in pancreatic β-cells, increasing its activity and stability. Conclusion: SUMOylation of glucokinase is a novel form of modification, regulating its cellular stability and activity. Significance: SUMO conjugation of glucokinase may have an important regulatory function in pancreatic β-cells. Glucokinase is the predominant hexokinase expressed in hepatocytes and pancreatic β-cells, with a pivotal role in regulating glucose-stimulated insulin secretion, illustrated by glucokinase gene mutations causing monogenic diabetes and congenital hyperinsulinemic hypoglycemia. A complex tissue-specific network of mechanisms regulates this enzyme, and a major unanswered question in glucokinase biology is how post-translational modifications control the function of the enzyme. Here, we show that the pancreatic isoform of human glucokinase is SUMOylated in vitro, using recombinant enzymes, and in insulin-secreting model cells. Three N-terminal lysines unique for the pancreatic isoform (Lys-12/Lys-13 and/or Lys-15) may represent one SUMOylation site, with an additional site (Lys-346) common for the pancreatic and the liver isoform. SUMO-1 and E2 overexpression stabilized preferentially the wild-type human pancreatic enzyme in MIN6 β-cells, and SUMOylation increased the catalytic activity of recombinant human glucokinase in vitro and also of glucokinase in target cells. Small ubiquitin-like modifier conjugation represents a novel form of post-translational modification of the enzyme, and it may have an important regulatory function in pancreatic β-cells.

Targeted next-generation sequencing reveals MODY in up to 6.5% of antibody-negative diabetes cases listed in the Norwegian Childhood Diabetes Registry

Aims/hypothesisMODY can be wrongly diagnosed as type 1 diabetes in children. We aimed to find the prevalence of MODY in a nationwide population-based registry of childhood diabetes.MethodsUsing next-generation sequencing, we screened the HNF1A, HNF4A, HNF1B, GCK and INS genes in all 469 children (12.1%) negative for both GAD and IA-2 autoantibodies and 469 antibody-positive matched controls selected from the Norwegian Childhood Diabetes Registry (3882 children). Variants were classified using clinical diagnostic criteria for pathogenicity ranging from class 1 (neutral) to class 5 (pathogenic).ResultsWe identified 58 rare exonic and splice variants in cases and controls. Among antibody-negative patients, 6.5% had genetic variants of classes 3–5 (vs 2.4% in controls; p = 0.002). For the stricter classification (classes 4 and 5), the corresponding number was 4.1% (vs 0.2% in controls; p = 1.6 × 10−5). HNF1A showed the strongest enrichment of class 3–5 variants, with 3.9% among antibody-negative patients (vs 0.4% in controls; p = 0.0002). Antibody-negative carriers of variants in class 3 had a similar phenotype to those carrying variants in classes 4 and 5.Conclusions/interpretationThis is the first study screening for MODY in all antibody-negative children in a nationwide population-based registry. Our results suggest that the prevalence of MODY in antibody-negative childhood diabetes may reach 6.5%. One-third of these MODY cases had not been recognised by clinicians. Since a precise diagnosis is important for treatment and genetic counselling, molecular screening of all antibody-negative children should be considered in routine diagnostics.

Binding of ATP at the active site of human pancreatic glucokinase – nucleotide‐induced conformational changes with possible implications for its kinetic cooperativity

Glucokinase (GK) is the central player in glucose‐stimulated insulin release from pancreatic β‐cells, and catalytic activation by α‐d‐glucose binding has a key regulatory function. Whereas the mechanism of this activation is well understood, on the basis of crystal structures of human GK, there are no similar structural data on ATP binding to the ligand‐free enzyme and how it affects its conformation. Here, we report on a conformational change induced by the binding of adenine nucleotides to human pancreatic GK, as determined by intrinsic tryptophan fluorescence, using the catalytically inactive mutant form T228M to correct for the inner filter effect. Adenosine‐5′‐(β,γ‐imido)triphosphate and ATP bind to the wild‐type enzyme with apparent [L]0.5 (ligand concentration at half‐maximal effect) values of 0.27 ± 0.02 mm and 0.78 ± 0.14 mm, respectively. The change in protein conformation was further supported by ATP inhibition of the binding of the fluorescent probe 8‐anilino‐1‐naphthalenesulfonate and limited proteolysis by trypsin, and by molecular dynamic simulations. The simulations provide a first insight into the dynamics of the binary complex with ATP, including motion of the flexible surface/active site loop and partial closure of the active site cleft. In the complex, the adenosine moiety is packed between two α‐helices and stabilized by hydrogen bonds (with Thr228, Thr332, and Ser336) and hydrophobic interactions (with Val412 and Leu415). Combined with enzyme kinetic analyses, our data indicate that the ATP‐induced changes in protein conformation may have implications for the kinetic cooperativity of the enzyme.

Functional Investigations of HNF1A Identify Rare Variants as Risk Factors for Type 2 Diabetes in the General Population

Variants in HNF1A encoding hepatocyte nuclear factor 1α (HNF-1A) are associated with maturity-onset diabetes of the young form 3 (MODY 3) and type 2 diabetes. We investigated whether functional classification of HNF1A rare coding variants can inform models of diabetes risk prediction in the general population by analyzing the effect of 27 HNF1A variants identified in well-phenotyped populations (n = 4,115). Bioinformatics tools classified 11 variants as likely pathogenic and showed no association with diabetes risk (combined minor allele frequency [MAF] 0.22%; odds ratio [OR] 2.02; 95% CI 0.73–5.60; P = 0.18). However, a different set of 11 variants that reduced HNF-1A transcriptional activity to <60% of normal (wild-type) activity was strongly associated with diabetes in the general population (combined MAF 0.22%; OR 5.04; 95% CI 1.99–12.80; P = 0.0007). Our functional investigations indicate that 0.44% of the population carry HNF1A variants that result in a substantially increased risk for developing diabetes. These results suggest that functional characterization of variants within MODY genes may overcome the limitations of bioinformatics tools for the purposes of presymptomatic diabetes risk prediction in the general population.

The intronic ABCA4 c.5461-10T>C variant, frequently seen in patients with Stargardt disease, causes splice defects and reduced ABCA4 protein level

Despite being the third most common ABCA4 variant observed in patients with Stargardt disease, the functional effect of the intronic ABCA4 variant c.5461‐10T>C is unknown. The purpose of this study was to investigate the molecular effect of this variant.

High Incidence of Heterozygous ABCC8 and HNF1A Mutations in Czech Patients With Congenital Hyperinsulinism

CONTEXT Congenital hyperinsulinism of infancy (CHI) represents a group of heterogeneous disorders characterized by oversecretion of insulin from pancreatic β-cells causing severe hypoglycemia. OBJECTIVE We studied the distribution of genetic causes of CHI in a Czech population. METHODS Countrywide collection of patients with CHI included 40 subjects (12 females, median age of diagnosis, 1 wk [interquartile range, 1-612 wk]). We sequenced the ABCC8, KCNJ11, GLUD1, GCK, HADH, UCP2, SLC16A1, HNF4A, and HNF1A genes and investigated structural changes in the ABCC8 gene. We functionally tested novel variants in the ABCC8 gene by Rb(86+) efflux assay and novel variants in the HNF1A gene by transcriptional activation and DNA-binding tests. RESULTS We found causal mutations in 20 subjects (50%): 19 carried a heterozygous mutation while one patient was homozygous for mutation in the ABCC8 gene. Specifically, we detected 11 mutations (seven novel) in ABCC8, one novel mutation in KCNJ11, five mutations (two novel) in HNF1A, two novel mutations in HNF4A, and one in GCK. We showed a decrease of activation by diazoxide in mutant KATP channels with novel ABCC8 variants by 41-91% (median, 82%) compared with wild-type (WT) channels and reduced transcriptional activity of mutant HNF1A proteins (2.9% for p.Asn62Lysfs93* and 22% for p.Leu254Gln) accompanied by no DNA-binding ability compared with WT HNF1A. CONCLUSION We detected a higher proportion of heterozygous mutations causing CHI compared with other cohorts probably due to lack of consanguinity and inclusion of milder CHI forms. Interestingly, HNF1A gene mutations represented the second most frequent genetic cause of CHI in the Czech Republic. Based on our results we present a genetic testing strategy specific for similar populations.

GCK-MODY diabetes as a protein misfolding disease: the mutation R275C promotes protein misfolding, self-association and cellular degradation.

GCK-MODY, dominantly inherited mild hyperglycemia, is associated with more than 600 mutations in the glucokinase gene. Different molecular mechanisms have been shown to explain GCK-MODY. Here, we report a Pakistani family harboring the glucokinase mutation c.823C>T (p.R275C). The recombinant and in cellulo expressed mutant pancreatic enzyme revealed slightly increased enzyme activity (kcat) and normal affinity for α-D-glucose, and resistance to limited proteolysis by trypsin comparable with wild-type. When stably expressed in HEK293 cells and MIN6 β-cells (at different levels), the mutant protein appeared misfolded and unstable with a propensity to form dimers and aggregates. Its degradation rate was increased, involving the lysosomal and proteasomal quality control systems. On mutation, a hydrogen bond between the R275 side-chain and the carbonyl oxygen of D267 is broken, destabilizing the F260-L271 loop structure and the protein. This promotes the formation of dimers/aggregates and suggests that an increased cellular degradation is the molecular mechanism by which R275C causes GCK-MODY.