Juraj Stanik

Mutations in HNF1A Result in Marked Alterations of Plasma Glycan Profile

A recent genome-wide association study identified hepatocyte nuclear factor 1-α (HNF1A) as a key regulator of fucosylation. We hypothesized that loss-of-function HNF1A mutations causal for maturity-onset diabetes of the young (MODY) would display altered fucosylation of N-linked glycans on plasma proteins and that glycan biomarkers could improve the efficiency of a diagnosis of HNF1A-MODY. In a pilot comparison of 33 subjects with HNF1A-MODY and 41 subjects with type 2 diabetes, 15 of 29 glycan measurements differed between the two groups. The DG9-glycan index, which is the ratio of fucosylated to nonfucosylated triantennary glycans, provided optimum discrimination in the pilot study and was examined further among additional subjects with HNF1A-MODY (n = 188), glucokinase (GCK)-MODY (n = 118), hepatocyte nuclear factor 4-α (HNF4A)-MODY (n = 40), type 1 diabetes (n = 98), type 2 diabetes (n = 167), and nondiabetic controls (n = 98). The DG9-glycan index was markedly lower in HNF1A-MODY than in controls or other diabetes subtypes, offered good discrimination between HNF1A-MODY and both type 1 and type 2 diabetes (C statistic ≥0.90), and enabled us to detect three previously undetected HNF1A mutations in patients with diabetes. In conclusion, glycan profiles are altered substantially in HNF1A-MODY, and the DG9-glycan index has potential clinical value as a diagnostic biomarker of HNF1A dysfunction.

De novo mutations of GCK, HNF1A and HNF4A may be more frequent in MODY than previously assumed

Aims/hypothesisMODY is mainly characterised by an early onset of diabetes and a positive family history of diabetes with an autosomal dominant mode of inheritance. However, de novo mutations have been reported anecdotally. The aim of this study was to systematically revisit a large collection of MODY patients to determine the minimum prevalence of de novo mutations in the most prevalent MODY genes (i.e. GCK, HNF1A, HNF4A).MethodsAnalysis of 922 patients from two national MODY centres (Slovakia and the Czech Republic) identified 150 probands (16%) who came from pedigrees that did not fulfil the criterion of two generations with diabetes but did fulfil the remaining criteria. The GCK, HNF1A and HNF4A genes were analysed by direct sequencing.ResultsMutations in GCK, HNF1A or HNF4A genes were detected in 58 of 150 individuals. Parents of 28 probands were unavailable for further analysis, and in 19 probands the mutation was inherited from an asymptomatic parent. In 11 probands the mutations arose de novo.Conclusions/interpretationIn our cohort of MODY patients from two national centres the de novo mutations in GCK, HNF1A and HNF4A were present in 7.3% of the 150 families without a history of diabetes and 1.2% of all of the referrals for MODY testing. This is the largest collection of de novo MODY mutations to date, and our findings indicate a much higher frequency of de novo mutations than previously assumed. Therefore, genetic testing of MODY could be considered for carefully selected individuals without a family history of diabetes.

Identification of a Novel β-Cell Glucokinase (GCK) Promoter Mutation (−71G>C) That Modulates GCK Gene Expression Through Loss of Allele-Specific Sp1 Binding Causing Mild Fasting Hyperglycemia in Humans

OBJECTIVE Inactivating mutations in glucokinase (GCK) cause mild fasting hyperglycemia. Identification of a GCK mutation has implications for treatment and prognosis; therefore, it is important to identify these individuals. A significant number of patients have a phenotype suggesting a defect in glucokinase but no abnormality of GCK. We hypothesized that the GCK β-cell promoter region, which currently is not routinely screened, could contain pathogenic mutations; therefore, we sequenced this region in 60 such probands. RESEARCH DESIGN AND METHODS The β-cell GCK promoter was sequenced in patient DNA. The effect of the identified novel mutation on GCK promoter activity was assessed using a luciferase reporter gene expression system. Electrophoretic mobility shift assays (EMSAs) were used to determine the impact of the mutation on Sp1 binding. RESULTS A novel −71G>C mutation was identified in a nonconserved region of the human promoter sequence in six apparently unrelated probands. Family testing established cosegregation with fasting hyperglycemia (≥5.5 mmol/l) in 39 affected individuals. Haplotype analysis in the U.K. family and four of the Slovakian families demonstrated that the mutation had arisen independently. The mutation maps to a potential transcriptional activator binding site for Sp1. Reporter assays demonstrated that the mutation reduces promoter activity by up to fourfold. EMSAs demonstrated a dramatic reduction in Sp1 binding to the promoter sequence corresponding to the mutant allele. CONCLUSIONS A novel β-cell GCK promoter mutation was identified that significantly reduces gene expression in vitro through loss of regulation by Sp1. To ensure correct diagnosis of potential GCK-MODY (maturity-onset diabetes of the young) cases, analysis of the β-cell GCK promoter should be included.

Severe Insulin Resistance and Intrauterine Growth Deficiency Associated With Haploinsufficiency for INSR and CHN2: New Insights Into Synergistic Pathways Involved in Growth and Metabolism

OBJECTIVE Digenic causes of human disease are rarely reported. Insulin via its receptor, which is encoded by INSR, plays a key role in both metabolic and growth signaling pathways. Heterozygous INSR mutations are the most common cause of monogenic insulin resistance. However, growth retardation is only reported with homozygous or compound heterozygous mutations. We describe a novel translocation [t(7,19)(p15.2;p13.2)] cosegregating with insulin resistance and pre- and postnatal growth deficiency. Chromosome translocations present a unique opportunity to identify modifying loci; therefore, our objective was to determine the mutational mechanism resulting in this complex phenotype. RESEARCH DESIGN AND METHODS Breakpoint mapping was performed by fluorescence in situ hybridization (FISH) on patient chromosomes. Sequencing and gene expression studies of disrupted and adjacent genes were performed on patient-derived tissues. RESULTS Affected individuals had increased insulin, C-peptide, insulin–to–C-peptide ratio, and adiponectin levels consistent with an insulin receptoropathy. FISH mapping established that the translocation breakpoints disrupt INSR on chromosome 19p15.2 and CHN2 on chromosome 7p13.2. Sequencing demonstrated INSR haploinsufficiency accounting for elevated insulin levels and dysglycemia. CHN2 encoding β-2 chimerin was shown to be expressed in insulin-sensitive tissues, and its disruption was shown to result in decreased gene expression in patient-derived adipose tissue. CONCLUSIONS We present a likely digenic cause of insulin resistance and growth deficiency resulting from the combined heterozygous disruption of INSR and CHN2, implicating CHN2 for the first time as a key element of proximal insulin signaling in vivo.

Identification and functional characterisation of novel glucokinase mutations causing maturity-onset diabetes of the young in Slovakia.

Heterozygous glucokinase (GCK) mutations cause a subtype of maturity-onset diabetes of the young (GCK-MODY). Over 600 GCK mutations have been reported of which ∼65% are missense. In many cases co-segregation has not been established and despite the importance of functional studies in ascribing pathogenicity for missense variants these have only been performed for <10% of mutations. The aim of this study was to determine the minimum prevalence of GCK-MODY amongst diabetic subjects in Slovakia by sequencing GCK in 100 Slovakian probands with a phenotype consistent with GCK-MODY and to explore the pathogenicity of identified variants through family and functional studies. Twenty-two mutations were identified in 36 families (17 missense) of which 7 (I110N, V200A, N204D, G258R, F419S, c.580-2A>C, c.1113–1114delGC) were novel. Parental DNA was available for 22 probands (covering 14/22 mutations) and co-segregation established in all cases. Bioinformatic analysis predicted all missense mutations to be damaging. Nine (I110N, V200A, N204D, G223S, G258R, F419S, V244G, L315H, I436N) mutations were functionally evaluated. Basic kinetic analysis explained pathogenicity for 7 mutants which showed reduced glucokinase activity with relative activity indices (RAI) between 0.6 to <0.001 compared to wild-type GCK (1.0). For the remaining 2 mutants additional molecular mechanisms were investigated. Differences in glucokinase regulatory protein (GKRP) –mediated-inhibition of GCK were observed for both L315H & I436N when compared to wild type (IC50 14.6±0.1 mM & 20.3±1.6 mM vs.13.3±0.1 mM respectively [p<0.03]). Protein instability as assessed by thermal lability studies demonstrated that both L315H and I436N show marked thermal instability compared to wild-type GCK (RAI at 55°C 8.8±0.8% & 3.1±0.4% vs. 42.5±3.9% respectively [p<0.001]). The minimum prevalence of GCK-MODY amongst Slovakian patients with diabetes was 0.03%. In conclusion, we have identified 22 GCK mutations in 36 Slovakian probands and demonstrate that combining family, bioinformatic and functional studies can aid the interpretation of variants identified by molecular diagnostic screening.

Novel monogenic diabetes mutations in the P2 promoter of the HNF4A gene are associated with impaired function in vitro

Diabet. Med. 27, 631–635 (2010)

Lack of Association Between Peripheral Activity of Thyroid Hormones and Elevated TSH Levels in Childhood Obesity

Objective: An elevated thyroid stimulating hormone (TSH) level is a frequent finding in obese children, but its association with peripheral hormone metabolism is not fully understood. We hypothesized that in obesity, the changes in thyroid hormone metabolism in peripheral tissues might lead to dysregulation in the thyroid axis. The purpose of this study was to investigate the association of TSH with thyroid hormones in a group of obese children as compared to normal-weight controls. Methods: Serum TSH, free thyroxine (fT4) and free triiodothyronine (fT3) levels were measured in 101 obese children and in 40 controls. Serum reverse T3 (rT3) levels were also measured in a subgroup of 51 obese children and in 15 controls. Results: Serum TSH level was significantly higher in obese children compared to controls (2.78 vs. 1.99 mIU/L, p<0.001), while no difference was found in fT4, fT3, rT3 levels and in fT3/rT3 ratio. In the obese group, fT3 level positively correlated with fT4 (r=0.217, p=0.033) and inversely with rT3 (r=-0.288, p=0.045). However, thyroid hormone levels and TSH levels were not correlated. Conclusion: In obese children, normal fT4, fT3 and rT3 levels suggest an undisturbed peripheral hormone metabolism. These levels show no correlation with elevated TSH levels.

Congenital hyperinsulinism and glycogenosis-like phenotype due to a novel HNF4A mutation

AIM Congenital hyperinsulinism (CHI) and glycogen storage disease (glycogenosis) are both causing hypoglycemia during infancy, but with different additional clinical features and therapeutic approach. We aimed to identify a genetic cause in a child with an ambiguous phenotype. METHODS AND RESULTS We present a child with hyperinsulinemic hypoglycemia, physiological 3-OH butyrate, increased triglyceride serum levels, increased level of glycogen in erythrocytes, increased liver transaminases, and increased echogenicity on liver ultrasonography. As both parents of the proband were referred as healthy, we raised a clinical suspicion on glycogenosis with recessive inheritance. However, whole exome sequencing revealed no mutation in genes causing glycogenosis, but a novel heterozygous variant LRG_483t1: c.427-1G>A in the HNF4A gene was identified. Aberrant splicing resulting in in-frame deletion c.429_476del, p.(T144_I159del) was confirmed by sequencing of HNF4A transcripts reverse-transcribed from whole blood RNA. The same variant was found in five of eight tested family relatives (one of them already had diabetes, two had prediabetes). With regard to the results of DNA analysis, we added diazoxide to the therapy. Consequently, the frequency and severity of hypoglycemia in the proband decreased. We have also recommended sulfonylurea treatment after diabetes onset in adult mutation carriers. CONCLUSIONS We have identified a novel HNF4A gene mutation in our patient with CHI and glycogenosis-like phenotype. The proband and her family members benefited from the genetic testing by WES method and consequently personalized therapy. Nevertheless, the HNF4A gene testing may be considered in selected CHI cases with glycogenosis-like phenotype prior WES analysis.

Coincidence of a novel KCNJ11 missense variant R365H with a paternally inherited 6q24 duplication in a patient with transient neonatal diabetes

OBJECTIVE—Neonatal diabetes is a heterogeneous group of disorders with diabetes manifestation in the first 6 months of life. The most common etiology in permanent neonatal diabetes is mutations of the ATP-sensitive K+ channel subunits; in transient neonatal diabetes, chromosome 6q24 abnormalities are the most common cause. RESEARCH DESIGN AND METHODS—We report a sporadic case of diabetes without ketoacidosis diagnosed on the fourth day of life. RESULTS—Analysis of the KCNJ11 gene found a novel R365H mutation in the proband and her unaffected father. The functional analysis did not support pathogenicity of this variant. When the patients diabetes remitted in the seventh month of life, the 6q24 region was analyzed and a paternally inherited duplication was identified. CONCLUSIONS—Our case reports a coincidental novel KCNJ11 variant in a patient with transient neonatal diabetes due to a 6q24 duplication, illustrating the difficulty in testing neonates before the clinical course of neonatal diabetes is known.

Mutations in SURF1 are important genetic causes of Leigh syndrome in Slovak patients

Abstract Objectives. Leigh syndrome is a progressive early onset neurodegenerative disease typically presenting with psychomotor regression, signs of brainstem and/or basal ganglia disease, lactic acidosis, and characteristic magnetic resonance imaging findings. At molecular level, deficiency of respiratory complexes and/or pyruvate dehydrogenase complex is usually observed. Nuclear gene SURF1 encodes an assembly factor for cytochrome c-oxidase complex of the respiratory chain and autosomal recessive mutations in SURF1 are one of the most frequent causes of cytochrome c-oxidase-related Leigh syndrome cases. Here, we aimed to elucidate the genetic basis of Leigh syndrome in three Slovak families. Methods and results. Three probands presenting with Leigh syndrome were selected for DNA analysis. The first proband, presenting with atypical LS onset without abnormal basal ganglia magnetic resonance imaging findings, was analyzed with whole exome sequencing. In the two remaining probands, SURF1 was screened by Sanger sequencing. Four different heterozygous mutations were identified in SURF1: c.312_321delinsAT:p.(Pro104Profs*1), c.588+1G>A, c.823_833+7del:p. (?) and c.845_846del:p.(Ser282Cysfs*9). All the mutations are predicted to have a loss-of-function effect. Conclusions. We identified disease-causing mutations in all three probands, which points to the important role of SURF1 gene in etiology of Leigh syndrome in Slovakia. Our data showed that patients with atypical Leigh syndrome phenotype without lesions in basal ganglia may benefit from the whole exome sequencing method. In the case of probands presenting the typical phenotype, Sanger sequencing of the SURF1 gene seems to be an effective method of DNA analysis.