Stepanka Pruhova

Molecular genetics and phenotypic characteristics of MODY caused by hepatocyte nuclear factor 4α mutations in a large European collection

Aims/hypothesisHeterozygous mutations in the gene of the transcription factor hepatocyte nuclear factor 4α (HNF-4α) are considered a rare cause of MODY with only 14 mutations reported to date. The description of the phenotype is limited to single families. We investigated the genetics and phenotype of HNF-4α mutations in a large European Caucasian collection.MethodsHNF-4α was sequenced in 48 MODY probands, selected for a phenotype of HNF-1α MODY but negative for HNF-1α mutations. Clinical characteristics and biochemistry were compared between 54 HNF-4α mutation carriers and 32 familial controls from ten newly detected or previously described families.ResultsMutations in HNF-4α were found in 14/48 (29%) probands negative for HNF-1α mutations. The mutations found included seven novel mutations: S34X, D206Y, E276D, L332P, I314F, L332insCTG and IVS5nt+1G>A. I314F is the first reported de novo HNF-4α mutation. The average age of diagnosis was 22.9 years with frequent clinical evidence of sensitivity to sulphonylureas. Beta cell function, but not insulin sensitivity, was reduced in diabetic mutation carriers compared to control subjects (homeostasis model assessment of beta cell function 29% p<0.001 vs controls). HNF-4α mutations were associated with lower apolipoprotein A2 (p=0.001), A1 (p=0.04) and total HDL-cholesterol (p=0.02) than in control subjects. However, in contrast to some previous reports, levels of triglycerides and apolipoprotein C3 were normal.Conclusions/interpretationHNF-4α mutations are common when no HNF-1α mutation is found in strictly defined MODY families. The HNF-4α clinical phenotype and beta cell dysfunction are similar to HNF-1α MODY and are associated with reduced apolipoprotein A2 levels. We suggest that sequencing of HNF-4α should be performed in patients with clinical characteristics of HNF-1α MODY in whom mutations in HNF-1α are not found.

Genetic evidence that HNF-1α–dependent transcriptional control of HNF-4α is essential for human pancreatic β cell function

Mutations in the genes encoding hepatocyte nuclear factor 4α (HNF-4α) and HNF-1α impair insulin secretion and cause maturity onset diabetes of the young (MODY). HNF-4α is known to be an essential positive regulator of HNF-1α. More recent data demonstrates that HNF-4α expression is dependent on HNF-1α in mouse pancreatic islets and exocrine cells. This effect is mediated by binding of HNF-1α to a tissue-specific promoter (P2) located 45.6 kb upstream from the previously characterized Hnf4α promoter (P1). Here we report that the expression of HNF-4α in human islets and exocrine cells is primarily mediated by the P2 promoter. Furthermore, we describe a G → A mutation in a conserved nucleotide position of the HNF-1α binding site of the P2 promoter, which cosegregates with MODY. The mutation results in decreased affinity for HNF-1α, and consequently in reduced HNF-1α–dependent activation. These findings provide genetic evidence that HNF-1α serves as an upstream regulator of HNF-4α and interacts directly with the P2 promoter in human pancreatic cells. Furthermore, they indicate that this regulation is essential to maintain normal pancreatic function.

Genetic epidemiology of MODY in the Czech republic: new mutations in the MODY genes HNF-4α, GCK and HNF-1α

Aims/hypothesisThe aim of this study was to examine the prevalence and nature of mutations in HNF4α/MODY1, GCK/MODY2 and HNF-1α/MODY3 genes in Czech subjects with clinical diagnosis of MODY.MethodsWe studied 61 unrelated index probands of Czech origin (28 males, 33 females) with a clinical diagnosis of MODY and 202 family members. The mean age of probands was 22.7±12.0 years (range, 6–62) and the mean age at the first recognition of hyperglycaemia was 14.7±6.0 years (range, 1–25). The promotor and coding regions inclusive intron exon boundaries of the HNF-4α, GCK and HNF-1α genes were examined by PCR-dHPLC (HNF-1α and GCK) and direct sequencing.ResultsWe identified 20 different mutations in the HNF-4α, GCK and HNF-1α in 29 families (48% of all families studied), giving a relative prevalence of 5% of MODY1, 31% of MODY2 and 11.5% of MODY3 among the Czech kindred with MODY. Three of 3, 10 of 11 and 1 of 6 of the mutations identified in HNF-4α, GCK and HNF-1α respectively, were new.Conclusion/interpretationOf the families 48% carried mutations in the MODY1–3 genes and of the identified mutations 70% were new. In 52% of Czech families with clinical characteristics of MODY, no mutations were found in the analysed genes. This finding shows that the majority of MODY mutations in a central European population are local and that other MODY genes could be responsible for autosomal dominant transmission of diabetes mellitus.

Further evidence that mutations in INS can be a rare cause of Maturity-Onset Diabetes of the Young (MODY)

BackgroundInsulin gene (INS) mutations have recently been described as a common cause of permanent neonatal diabetes (PNDM) and a rare cause of diabetes diagnosed in childhood or adulthood.MethodsINS was sequenced in 116 maturity-onset diabetes of the young (MODYX) patients (n = 48 Danish and n = 68 Czech), 83 patients with gestational diabetes mellitus (GDM), 34 type 1 diabetic patients screened negative for glutamic acid decarboxylase (GAD), and 96 glucose tolerant individuals. The control group was randomly selected from the population-based sampled Inter99 study.ResultsOne novel heterozygous mutation c.17G>A, R6H, was identified in the pre-proinsulin gene (INS) in a Danish MODYX family. The proband was diagnosed at 20 years of age with mild diabetes and treated with diet and oral hypoglycaemic agent. Two other family members who carried the INS R6H were diagnosed with diabetes when 51 years old and with GDM when 27 years old, respectively. A fourth mutation carrier had normal glucose tolerance when 20 years old. Two carriers of INS R6H were also examined twice with an oral glucose tolerance test (OGTT) with 5 years interval. They both had a ~30% reduction in beta-cell function measured as insulinogenic index. In a Czech MODYX family a previously described R46Q mutation was found. The proband was diagnosed at 13 years of age and had been treated with insulin since onset of diabetes. Her mother and grandmother were diagnosed at 14 and 35 years of age, respectively, and were treated with oral hypoglycaemic agents and/or insulin.ConclusionMutations in INS can be a rare cause of MODY and we conclude that screening for mutations in INS should be recommended in MODYX patients.

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.

Glucokinase diabetes in 103 families from a country‐based study in the Czech Republic: geographically restricted distribution of two prevalent GCK mutations

Pruhova S, Dusatkova P, Sumnik Z, Kolouskova S, Pedersen O, Hansen T, Cinek O, Lebl J. Glucokinase diabetes in 103 families from a country‐based study in the Czech Republic: geographically restricted distribution of two prevalent GCK mutations.

Hepatic phenotypes of HNF1B gene mutations: A case of neonatal cholestasis requiring portoenterostomy and literature review

Hepatocyte nuclear factor 1-β (HNF1B) defects cause renal cysts and diabetes syndrome (RCAD), or HNF1B-maturity-onset diabetes of the young. However, the hepatic phenotype of HNF1B variants is not well studied. We present a female neonate born small for her gestational age [birth weight 2360 g; -2.02 standard deviations (SD) and birth length 45 cm; -2.40 SD at the 38(th) gestational week]. She developed neonatal cholestasis due to biliary atresia and required surgical intervention (portoenterostomy) when 32-d old. Following the operation, icterus resolved, but laboratory signs of liver dysfunction persisted. She had hyperechogenic kidneys prenatally with bilateral renal cysts and pancreatic hypoplasia postnatally that led to the diagnosis of an HNF1B deletion. This represents the most severe hepatic phenotype of an HNF1B variant recognized thus far. A review of 12 published cases with hepatic phenotypes of HNF1B defects allowed us to distinguish three severity levels, ranging from neonatal cholestasis through adult-onset cholestasis to non-cholestatic liver impairment, all of these are associated with congenital renal cysts and mostly with diabetes later in life. We conclude that to detect HNF1B variants, neonates with cholestasis should be checked for the presence of renal cysts, with special focus on those who are born small for their gestational age. Additionally, patients with diabetes and renal cysts at any age who develop cholestasis and/or exocrine pancreatic insufficiency should be tested for HNF1B variants as the true etiological factor of all disease components. Further observations are needed to confirm the potential reversibility of cholestasis in infancy in HNF1B mutation/deletion carriers.

Six novel mutations in the GCK gene in MODY patients.

To the Editor: Maturity-onset diabetes of the young (MODY; MIM# 606391) is a genetically and clinically heterogeneous form of diabetes mellitus, characterized by an autosomal dominant inheritance, early-onset non-insulin-dependent diabetes mellitus and by a primary defect in the pancreatic beta-cell function (1). Until now, six types of MODY diabetes have been identified, depending on the gene causing the disease (2). Screening for glucokinase (GCK) mutations in subjects with clinical characteristics of MODY allows distinguishing between patients with a benign metabolic condition (GCK mutation positive, clinical diagnosis MODY2) and those with a higher risk of progressive hyperglycemia associated with more prevalent and severe diabetic complications (GCK mutation negative). The first mutation in the GCK gene was reported in 1992 (3). Up to now, 195 mutations in GCK have been described, in 285 families (4). Diabetic complications are rare in GCK–MODY, thus GCK–MODY patients only need to be followed by annual HbA1c examination. Also, screening of GCK for heterozygous inactivating mutations allows to determine the subtype of MODY diabetes and to predict the lifelong prognosis. All 12 exons (exons 1a, 1b, 1c and 2–10), the intron–exon boundaries and promotor region of GCK (GenBank accession number, AF04101222) were screened; in 92 Czech probands fulfilling classical MODY criteria, using denaturing highperformance liquid chromatography as previously described (5). The nature of identified mutations was established by direct nucleotide sequencing using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, USA) according to manufacture’s instructions. Mutations were confirmed using a second, independent amplification of the affected part of GCK and re-sequenced the following day. The probands were recruited from pediatricians and endocrinologists from the entire Czech Republic. Fifteen different missense mutations were identified in 27 patients. Of these, six were novel missense mutations R250C (exon 7, c.748C.T), L315H (exon 8, c.944T.A), F316V (exon 8, c.946T.G), F419L (exon 10, c.1255T.C), I436N (exon 10, c.1307T.A) and A454E (exon 10, c.1361C.A). Some of the identified GCK missense mutations are located near putative functional domains: R250C was found in the close vicinity of a putative glucose binding site, while F419L was detected near a putative MgATP binding site and could thus affect binding kinetics (6). Five of these mutations co-segregated with hyperglycemia in the family, suggesting that the variants are new diseasecausing mutations. For the novel R250C variant, family members were not available for cosegregation studies. All codons, which are changed by the six novel mutations, are conserved in the human, mouse, rat and chimpanzee genomes and we found none of these mutations in 50 unrelated healthy Czech Caucasian subjects. Therefore, we assume that the mutations are probably novel disease-causing mutations. We also compared the clinical characteristics of patients with GCK mutations and those without mutation in GCK (data not shown in details). In short – the treatment of hyperglycemia with diet was more frequent (p , 0.001) in the group of probands with mutations in GCK and they had a significantly lower frequency of diabetic complications (p 1⁄4 0.02). None of the patients with mutations in GCK was treated with insulin (p , 0.001). Moreover, GCK mutation carriers had a lower level of glycosylated hemoglobin (p1⁄4 0.02). ThemeanHbA1c (%) inGCK-positive probands vs negative was 5.7 0.2 vs 6.5 0.2. In conclusion, we identified 29% of GCK mutation carriers among Czech MODY probands, confirming that mutations in GCK are a common cause of MODY in the Czech population. The present high relative prevalence of GCK–MODY, compared with some other European studies, might reflect not only a specific genetic background, but also the mode of recruitment, because most of the probands in the present investigation were recruited by

The common p.R114W HNF4A mutation causes a distinct clinical subtype of monogenic diabetes

HNF4A mutations cause increased birth weight, transient neonatal hypoglycemia, and maturity onset diabetes of the young (MODY). The most frequently reported HNF4A mutation is p.R114W (previously p.R127W), but functional studies have shown inconsistent results; there is a lack of cosegregation in some pedigrees and an unexpectedly high frequency in public variant databases. We confirm that p.R114W is a pathogenic mutation with an odds ratio of 30.4 (95% CI 9.79–125, P = 2 × 10−21) for diabetes in our MODY cohort compared with control subjects. p.R114W heterozygotes did not have the increased birth weight of patients with other HNF4A mutations (3,476 g vs. 4,147 g, P = 0.0004), and fewer patients responded to sulfonylurea treatment (48% vs. 73%, P = 0.038). p.R114W has reduced penetrance; only 54% of heterozygotes developed diabetes by age 30 years compared with 71% for other HNF4A mutations. We redefine p.R114W as a pathogenic mutation that causes a distinct clinical subtype of HNF4A MODY with reduced penetrance, reduced sensitivity to sulfonylurea treatment, and no effect on birth weight. This has implications for diabetes treatment, management of pregnancy, and predictive testing of at-risk relatives. The increasing availability of large-scale sequence data is likely to reveal similar examples of rare, low-penetrance MODY mutations.

Two Cases of Diabetic Ketoacidosis in HNF1A-MODY Linked to Severe Dehydration: Is it time to change the diagnostic criteria for MODY?

OBJECTIVE Hepatocyte nuclear factor-1A maturity-onset diabetes of the young (HNF1A-MODY) is a monogenic form of diabetes caused by heterozygous mutations in HNF1A. Currently, a history of diabetic ketoacidosis (DKA) is an exclusion criterion for genetic testing for MODY. HISTORY AND EXAMINATION In this article, we describe two unrelated patients aged 17 and 24 years with severe DKA developed several years after the diagnosis of HNF1A-MODY. INVESTIGATION Both patients were treated with insulin, but their metabolic control was poor (HbA1c 15%, 140 mmol/mol and 13%, 119 mmol/mol, respectively) due to noncompliance and missed insulin injections. In both patients, DKA followed a course of recurrent vomiting with dehydration and prerenal acute kidney injury. Their glycemia, blood pH, and base excess at admission were 97 mmol/L [1,748 mg/dL], 6.80, and −33 mmol/L (patient 1) and 34 mmol/L [613 mg/dL], 7.03, and −14 mmol/L (patient 2). CONCLUSIONS This anecdotal observation supports the notion that a history of DKA does not exclude MODY.