Jan Lebl

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.

Tumor risk in disorders of sex development

Certain patients with disorders of sex development (DSD), who bear Y chromosome material in their karyotype, are at increased risk for the development of type II germ cell tumors (GCT), which arise from early fetal germ cells. DSD gonads frequently harbor immature germ cells which express early fetal germ cell markers. Some of them (e.g. OCT3/4 and NANOG) seem to be of pathogenetic relevance in GCT development providing cells with the ability of pluripotency, proliferation and apoptosis suppression. Also TSPY (testis-specific protein Y-encoded), the main candidate for the so-called gonadoblastoma locus on Y chromosome, is overexpressed in germ cells of DSD patients and possibly contributes to their survival and proliferation. Nowadays, the use of immunohistochemical methods is highly relevant in identifying DSD gonads at risk. The risk for GCT development varies. While the prevalence of GCT is 15% in patients with partial androgen insensitivity, it may reach more than 30% in patients with gonadal dysgenesis. Patients with complete androgen insensitivity and ovotesticular DSD develop malignancies in 0.8% and 2.6% of cases, respectively. However, these data may be biased for various reasons. To better estimate the risk in individual groups of DSD, further investigations on large patient series are needed.

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.

Screening for mutations in transcription factors in a Czech cohort of 170 patients with congenital and early-onset hypothyroidism: identification of a novel PAX8 mutation in dominantly inherited early-onset non-autoimmune hypothyroidism.

OBJECTIVE Mutations in NKX2.1, NKX2.5, FOXE1 and PAX8 genes, encoding for transcription factors involved in the development of the thyroid gland, have been identified in a minority of patients with syndromic and non-syndromic congenital hypothyroidism (CH). DESIGN In a phenotype-selected cohort of 170 Czech paediatric and adolescent patients with non-goitre CH, including thyroid dysgenesis, or non-goitre early-onset hypothyroidism, PAX8, NKX2.1, NKX2.5, FOXE1 and HHEX genes were analysed for mutations. METHODS NKX2.1, NKX2.5, FOXE1 and HHEX genes were directly sequenced in patients with syndromic CH. PAX8 mutational screening was performed in all 170 patients by single-stranded conformation polymorphism, followed by direct sequencing of samples with abnormal findings. The R52P PAX8 mutation was functionally characterized by DNA binding studies. RESULTS We identified a novel PAX8 mutation R52P, dominantly inherited in a three-generation pedigree and leading to non-congenital, early-onset, non-goitre, non-autoimmune hypothyroidism with gradual postnatal regression of the thyroid size and function. The R52P PAX8 mutation results in the substitution of a highly conserved residue of the DNA-binding domain with a loss-of-function effect. CONCLUSIONS The very low frequency of genetic defects in a population-based cohort of children affected by non-goitre congenital and early-onset hypothyroidism, even in a phenotype-focussed screening study, suggests the pathogenetic role of either non-classic genetic mechanisms or the involvement of genes unknown so far. Identification of a novel PAX8 mutation in a particular variant of non-congenital early-onset hypothyroidism indicates a key function of PAX8 in the postnatal growth and functional maintenance of the thyroid gland.

Gonadal pathology and tumor risk in relation to clinical characteristics in patients with 45,X/46,XY mosaicism

CONTEXT Gonadectomy is avoided whenever possible in boys with 45,X/46,XY. However, no clinical markers are currently available to guide clinicians in predicting gonadal tumor risk or hormone production. OBJECTIVE The objective of the study was to test the hypothesis that gonadal histology and risk for development of a malignant germ cell tumor are reflected by the clinical presentation of a 45,X/46,XY individual. DESIGN The design of the study was the correlation of clinical data [external masculinization score (EMS), pubertal outcome] with pathology data (gonadal phenotype, tumor risk). SETTING This was a multicenter study involving two multidisciplinary disorder of sex development teams. PATIENTS Patients included genetically proven 45,X/46,XY (and variants) cases, of whom at least one gonadal biopsy or gonadectomy specimen was available, together with clinical details. INTERVENTIONS Patients (n = 48) were divided into three groups, based on the EMS. Gonadal histology and tumor risk were assessed on paraffin-embedded samples (n = 87) by morphology and immunohistochemistry on the basis of established criteria. MAIN OUTCOME MEASURES Gonadal differentiation and tumor risk in the three clinical groups were measured. Clinical outcome in patients with at least one preserved gonad was also measured. RESULTS Tumor risk in the three groups was significantly related to the gonadal differentiation pattern (P < 0.001). In boys, hormone production was sufficient and was not predicted by the EMS. CONCLUSIONS The EMS reflects gonadal differentiation and tumor risk in patients with 45,X/46,XY. In boys, testosterone production is often sufficient, but strict follow-up is warranted because of malignancy risk, which appears inversely related to EMS. In girls, tumor risk is limited but gonads are not functional, making gonadectomy the most reasonable option.

Absence of breast-feeding is associated with the risk of type 1 diabetes: a case–control study in a population with rapidly increasing incidence

There are indications that the effect of environmental factors on the risk of type 1 diabetes mellitus (T1DM) is increasing over time. This can be documented by the rapid increase of T1DM incidence in genetically stable populations. Our aim was to study an association of T1DM with the variable factors of the perinatal period and of early infancy, using data from children born over a period of changing exposure to some of the studied factors. A case–control dataset was analysed, consisting of 868 diabetic children and 1,466 anonymous controls, mostly schoolmates of the children with T1DM. The data were collected using structured questionnaires completed by parents. After performing univariate analyses, the associations were analysed using multiple logistic regression adjusted for potential confounders, including the year of birth. The risk of T1DM decreased with increasing duration of breast-feeding, while no breast-feeding was associated with an increased T1DM risk, OR=1.93 [95% CI: 1.33–2.80], breast-feeding for more than 12 months was protective, OR=0.42 [95% CI: 0.22–0.81], both being relative to the reference category of breast-feeding for 1–3 months. A short duration of day-care attendance (none or less than 1 year) was weakly associated with the risk of T1DM, OR=1.65 [95% CI: 1.05–2.62]. No association was detected between T1DM and signs of prenatal infections, perinatal stress factors, birth size and weight, indicators of crowding or the presence of a domestic pet in the household. Short breast-feeding period and short attendance to day care is associated with the risk of T1DM in Czech children.

Growth Patterns and Final Height in Congenital Adrenal Hyperplasia due to Classical 21-Hydroxylase Deficiency

Background: Longitudinal growth and bone age (BA) development are the most important clinical parameters for monitoring adequate glucocorticoid replacement in children with congenital adrenal hyperplasia (CAH). Aim of the Study: To analyze the growth pattern of patients treated for CAH of the salt wasting (SW) and simple virilizing (SV) clinical forms; to evaluate final height as compared to reference data and individual target height; to evaluate the course of BA development. Patients and Methods: A large database of 598 patients with CAH was created in 5 Central European countries and growth data of 341 treated patients with 21-hydroxylase deficiency were analyzed retrospectively. The patients were of Caucasian origin. Centiles were constructed in a cross-sectional manner and an additional longitudinal analysis was performed in order to evaluate the pubertal growth spurt by applying particular statistical methods (Preece-Baines model). Results: The growth of SW CAH patients was impaired in infancy and early childhood (0–3 years of age), but followed normal patterns in childhood until puberty. In contrast, children with SV CAH had normal patterns of growth in infancy and early childhood and were considerably taller than healthy references during childhood. In the longitudinal study, peak height velocity in both boys and girls was normal, but it occurred at an earlier age than in the standard population. The final height of patients with CAH was reduced in comparison to both the reference and the individual target height. No correlations were found between final height and age at the start of the therapy in SV patients or between final height and year of birth. BA was advanced in both types of CAH, but more accelerated in SV patients. Conclusion: Characteristic growth patterns for treated SV and SW CAH children were identified, with a normal pubertal growth spurt and reduced final height being observed.

Screening detected celiac disease in children with type 1 diabetes mellitus : Effect on the clinical course - (A case control study)

Objective: To investigate clinical and metabolic characteristics of diabetic children with screening detected celiac disease in a multicenter case-control study. Methods: Cases: 98 diabetic patients were diagnosed as having silent celiac disease by screening with endomysial antibodies and subsequent biopsy. Controls: two controls in the same center were chosen, (stratified by age and age-at-diabetes onset) who were negative for endomysial antibodies (n = 195). Height, weight, HbA1c, insulin dosage and acute complications were documented for at least 1 year of follow up. Results: Mean age of diabetes manifestation was 6.5 ± 4.1 years and diagnosis of celiac disease was made at 10.0 ± 5.4 years. Biopsy showed total or subtotal mucosal atrophy in 74 patients. The mean observation period after the diagnosis of celiac disease was 3.3 ± 1.9 years. Mean HbA1c levels were similar between cases and controls (8.63% ± 1.45% versus 8.50% ± 1.39%; P = 0.35). There was also no difference in the frequency of severe hypoglycemia, ketoacidosis and the applied insulin dosage (P = 0.45). Body mass index-standard deviation score at celiac disease diagnosis (0.57 ± 1.24 versus 0.52 ± 1.07) and height-standard deviation score (0.14 ± 1.13 versus 0.30 ± 0.95) did not differ between cases and controls. After diagnosis of celiac disease, weight gain was diminished in boys with celiac disease compared with their controls (P < 0.05). Female cases also had a lower body mass index than female controls (P = 0.067). Conclusion: In a cohort of diabetic children, silent celiac disease had no obvious effect on metabolic control but negatively influenced weight gain.

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.