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Free access

Klemens Raile, Michele O'Connell, Angela Galler, George Werther, Peter Kühnen, Heiko Krude and Oliver Blankenstein

Background

Mutations of the preproinsulin gene (INS) account for both permanent neonatal diabetes (PND) and adult-onset diabetes. The molecular mechanism of complete INS deletion has recently been published and we now add clinical data of homozygous and heterozygous subjects as well as the detailed mapping of the 646 bp deletion of the INS gene.

Methods

Location and size of the INS deletion was mapped in one case with PND and INS genotype of the whole family was further characterized by breakpoint-spanning PCR. The phenotype of monoallelic loss of INS was studied in 33 adult family members of a large consanguineous kindred with INS deletion.

Results

The 646 bp deletion was found in two individuals with PND that included exons 1 and 2 of the INS gene (chr11: g.2138434_2139080del646) and results in loss of approximately half of the preproinsulin protein. The two boys with homozygous INS deletion (D/D) presented with reduced birth weight, PND within the first 24 h of life and complete absence of C-peptide. Adult family members with the N/D had diabetes onset with earliest 25 years, while the oldest subject without diabetes was 45 years. INS-deletion-diabetes was initially treated with oral antidiabetic drugs but then transferred to insulin within 5–16 years. Overall, N/D-subjects (n=11) had a higher risk to develop insulin-dependent diabetes up to the fifth decade, if compared with normal subjects (n=22).

Conclusion

Complete loss of the human INS gene results in neonatal diabetes, while heterozygous INS deletion is a strong risk factor for developing insulin-dependent diabetes at adult age.

Free access

Susanna Wiegand, Dagmar l'Allemand, Hanna Hübel, Heiko Krude, Mareike Bürmann, Peter Martus, Annette Grüters and Reinhard W Holl

Objective

To study whether metformin reduces obesity, homeostasis model assessment for insulin resistance index (HOMA-IR), and the metabolic syndrome (MtS) in obese European adolescents in addition to previous unsuccessful lifestyle intervention.

Design and methods

After 6 months of multiprofessional lifestyle intervention, 70 out of 86 adolescents without improvement in body mass index (BMI) and HOMA-IR were randomized into either the placebo (n=34) or the metformin group (2×500 mg/day, n=36) in addition to ongoing lifestyle intervention for another 6 months.

Results

Age was 13.8 years, BMI was 33.1 kg/m2, 65% were female, and 89% were Caucasians. During lifestyle intervention alone, BMI and HOMA-IR deteriorated significantly. In the subsequent medication period, HOMA-IR and fasting insulin improved similarly in the placebo and metformin groups (HOMA-IR decreased 73 vs 54% respectively in metformin versus placebo; P=0.048), but BMI remained unchanged. The insulin sensitivity index, however, only improved in the metformin group. High fasting insulin is correlated with a subsequent BMI increase irrespective of the medication. MtS remained unchanged.

Conclusions

Obese European adolescents' insulin sensitivity improved without weight change during placebo or metformin intervention in addition to lifestyle intervention. Most differences did not reach statistical significance, probably due to improved compliance with lifestyle intervention as a placebo effect. In addition, the metformin dose may be too low.

Free access

Eva Al Taji, Heike Biebermann, Zdeňka Límanová, Olga Hníková, Jaroslav Zikmund, Christof Dame, Annette Grüters, Jan Lebl and Heiko Krude

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.

Open access

Sally Tantawy, Lin Lin, Ilker Akkurt, Guntram Borck, Dietrich Klingmüller, Berthold P Hauffa, Heiko Krude, Heike Biebermann, John C Achermann and Birgit Köhler

Background

Steroidogenic factor 1 (SF-1, NR5A1) is a key transcriptional regulator of many genes involved in the hypothalamic–pituitary–gonadal axis and mutations in NR5A1 can result in 46,XY disorders of sex development (DSD). Patients with this condition typically present with ambiguous genitalia, partial gonadal dysgenesis, and absent/rudimentary Müllerian structures. In these cases, testosterone is usually low in early infancy, indicating significantly impaired androgen synthesis. Further, Sertoli cell dysfunction is seen (low inhibin B, anti-Müllerian hormone). However, gonadal function at puberty in patients with NR5A1 mutations is unknown.

Subjects and methods

Clinical assessment, endocrine evaluation, and genetic analysis were performed in one female and one male with 46,XY DSD who showed spontaneous virilization during puberty. The female patient presented at adolescence with clitoral hypertrophy, whereas the male patient presented at birth with severe hypospadias and entered puberty spontaneously. Molecular analysis of NR5A1 was performed followed by in vitro functional analysis of the two novel mutations detected.

Results

Testosterone levels were normal during puberty in both patients. Analysis of NR5A1 revealed two novel heterozygous missense mutations in the ligand-binding domain of SF-1 (patient 1: p.L376F; patient 2: p.G328V). The mutant proteins showed reduced transactivation of the CYP11A promoter in vitro.

Conclusion

Patients with 46,XY DSD and NR5A1 mutations can produce sufficient testosterone for spontaneous virilization during puberty. Phenotypic females (46,XY) with NR5A1 mutations can present with clitoromegaly at puberty, a phenotype similar to other partial defects of androgen synthesis or action. Testosterone production in 46,XY males with NR5A1 mutations can be sufficient for virilization at puberty. As progressive gonadal dysgenesis is likely, gonadal function should be monitored in adolescence and adulthood, and early sperm cryopreservation considered in male patients if possible.

Free access

Jeannine Sauber, Jessica Grothe, Maria Behm, André Scherag, Harald Grallert, Thomas Illig, Anke Hinney, Johannes Hebebrand, Susanna Wiegand, Annette Grüters, Heiko Krude and Heike Biebermann

Objective

In the past 20 years, obesity has become a major health problem due to associated diseases like type 2 diabetes mellitus. The gastric inhibitory polypeptide receptor (GIPR) modulates body weight and glucose homeostasis and, therefore, represents an interesting candidate gene for obesity and the comorbidity impaired glucose homeostasis. Recently, a GIPR variation was found to be associated with impaired insulin response in humans.

In this study, we screened the GIPR gene for mutations and examined the association between three single-nucleotide polymorphisms (SNPs; rs8111428, rs2302382, rs1800437) and childhood obesity, as well as impaired glucose homeostasis.

Methods

The coding region of the GIPR was screened for mutations by direct sequencing. We genotyped three known SNPs in 2280 healthy normal weight (1696) and obese (584) children and adolescents. Genotyping was performed using the SNaPshot protocol, the iplex, and matrix-assisted laser desorption ionization time-of-flight spectrometry technique. Obesity was defined by a body mass index SDS above 2; homeostatic model assessment was calculated.

Results

No evidence for an association was found between the SNPs and the obesity phenotype. Significant association was found between the minor allele C of the SNP rs1800437 and elevated homeostasis model of insulin resistance values (P=0.001). No further sequence variations in the GIPR were found to be associated with childhood obesity.

Conclusion

Variations of the GIPR sequence are not associated with childhood obesity. This study points to a potential role for rs1800437 in glucose homeostasis. Further studies are necessary to confirm these results.

Free access

Eva K Wirth, Sien-Yi Sheu, Jazmin Chiu-Ugalde, Remy Sapin, Marc O Klein, Ilona Mossbrugger, Leticia Quintanilla-Martinez, Martin Hrabě de Angelis, Heiko Krude, Thomas Riebel, Karin Rothe, Josef Köhrle, Kurt W Schmid, Ulrich Schweizer and Annette Grüters

Context

Thyroid hormone transport across the plasma membrane depends on transmembrane transport proteins, including monocarboxylate transporter 8 (MCT8). Mutations in MCT8 (or SLC16A2) lead to a severe form of X-linked psychomotor retardation, which is characterised by elevated plasma triiodothyronine (T3) and low/normal thyroxine (T4). MCT8 contributes to hormone release from the thyroid gland.

Objective

To characterise the potential impact of MCT8-deficiency on thyroid morphology in a patient and in Mct8-deficient mice.

Design

Thyroid morphology in a patient carrying the A224V mutation was followed by ultrasound imaging for over 10 years. After thyroidectomy, a histopathological analysis was carried out. The findings were compared with histological analyses of mouse thyroids from the Mct8 −/y model.

Results

We show that an inactivating mutation in MCT8 leads to a unique, progressive thyroid follicular pathology in a patient. After thyroidectomy, histological analysis revealed gross morphological changes, including several hyperplastic nodules, microfollicular areas with stromal fibrosis and a small focus of microfollicular structures with nuclear features reminiscent of papillary thyroid carcinoma (PTC). These findings are supported by an Mct8-null mouse model in which we found massive papillary hyperplasia in 6- to 12-month-old mice and nuclear features consistent with PTC in almost 2-year-old animals. After complete thyroidectomy and substitution with levothyroxine (l-T4), the preoperative, inadequately low T4 and free T4 remained, while increasing the l-T4 dosage led to T3 serum concentrations above the normal range.

Conclusions

Our results implicate peripheral deiodination in the peculiar hormonal constellation of MCT8-deficient patients. Other MCT8-deficient patients should be closely monitored for potential thyroid abnormalities.

Free access

Sally Tantawy, Inas Mazen, Hala Soliman, Ghada Anwar, Abeer Atef, Mona El-Gammal, Ahmed El-Kotoury, Mona Mekkawy, Ahmad Torky, Agnes Rudolf, Pamela Schrumpf, Annette Grüters, Heiko Krude, Marie-Charlotte Dumargne, Rebekka Astudillo, Anu Bashamboo, Heike Biebermann and Birgit Köhler

Objective

Steroidogenic factor 1 (SF1, NR5A1) is a key transcriptional regulator of genes involved in the hypothalamic–pituitary–gonadal axis. Recently, SF1 mutations were found to be a frequent cause of 46,XY disorders of sex development (DSD) in humans. We investigate the frequency of NR5A1 mutations in an Egyptian cohort of XY DSD.

Design

Clinical assessment, endocrine evaluation and genetic analysis of 50 Egyptian XY DSD patients (without adrenal insufficiency) with a wide phenotypic spectrum.

Methods

Molecular analysis of NR5A1 gene by direct sequencing followed by in vitro functional analysis of the two novel missense mutations detected.

Results

Three novel heterozygous mutations of the coding region in patients with hypospadias were detected. p.Glu121AlafsX25 results in severely truncated protein, p.Arg62Cys lies in DNA-binding zinc finger, whereas p.Ala154Thr lies in the hinge region of SF1 protein. Transactivation assays using reporter constructs carrying promoters of anti-Müllerian hormone (AMH), CYP11A1 and TESCO core enhancer of Sox9 showed that p.Ala154Thr and p.Arg62Cys mutations result in aberrant biological activity of NR5A1. A total of 17 patients (34%) harboured the p.Gly146Ala polymorphism.

Conclusion

We identified two novel NR5A1 mutations showing impaired function in 23 Egyptian XY DSD patients with hypospadias (8.5%). This is the first study searching for NR5A1 mutations in oriental patients from the Middle East and Arab region with XY DSD and no adrenal insufficiency, revealing a frequency similar to that in European patients (6.5–15%). We recommend screening of NR5A1 in patients with hypospadias and gonadal dysgenesis. Yearly follow-ups of gonadal function and early cryoconservation of sperms should be performed in XY DSD patients with NR5A1 mutations given the risk of future fertility problems due to early gonadal failure.

Free access

Anne-Kathrin Wermter, Kathrin Reichwald, Thomas Büch, Frank Geller, Cornelia Platzer, Klaus Huse, Claudia Hess, Helmut Remschmidt, Thomas Gudermann, Gerald Preibisch, Wolfgang Siegfried, Hans-Peter Goldschmidt, Wei-Dong Li, R Arlen Price, Heike Biebermann, Heiko Krude, Caren Vollmert, H-Erich Wichmann, Thomas Illig, Thorkild I A Sørensen, Arne Astrup, Lesli Hingstrup Larsen, Oluf Pedersen, Delphine Eberlé, Karine Clément, John Blundell, Martin Wabitsch, Helmut Schäfer, Matthias Platzer, Anke Hinney and Johannes Hebebrand

Objective: The importance of the melanin-concentrating hormone (MCH) system for regulation of energy homeostasis and body weight has been demonstrated in rodents. We analysed the human MCH receptor 1 gene (MCHR1) with respect to human obesity.

Design: This consisted of genomic screening of 13.4 kb encompassing the MCHR1 in extremely obese German children and adolescents and association analyses for two coding single nucleotide polymorphisms (SNPs). To confirm initial positive association results, additional association studies and transmission disequilibrium tests in further German, Danish, French and American samples were conducted. Selected SNPs were investigated using functional in vitro studies and reporter gene assays.

Methods: Single-stranded conformation polymorphism analysis, re-sequencing, PCR-restriction fragment length polymorphism analyses, tetra-primer amplification refractory mutation systems, matrix-assisted laser desorption/ionization time of flight mass spectrometry and reporter gene assays were carried out as well as measuring inositol phosphate formation, inhibition of cAMP formation and activation of p42/44 MAP kinase.

Results: We identified 11 infrequent variations and two SNPs in the MCHR1 coding sequence and 18 SNPs (eight novel) in the flanking sequence. Association and transmission disequilibrium with obesity were detected for several SNPs in independent study groups of German obese children and adolescents and controls. In two German samples, encompassing 4056 and 295 individuals, trends towards association with obesity were detected. Findings in a second epidemiological German sample and in Danish, French and American samples were negative. Functional in vitro studies as well as reporter gene assays revealed no significant results.

Conclusion: Our initial association of MCHR1 alleles/haplotype detected might be related to juvenile-onset obesity, conditional on a particular genetic and/or environmental background. Alternatively, we could not exclude the possibility that the initially detected association represented a false positive finding.