Rifat Bircan

Genetics and phenomics of inherited and sporadic non-autoimmune hyperthyroidism

TSH receptor (TSHR) germline mutations occur as activating mutations in familial non-autoimmune hyperthyroidism (FNAH) or sporadic non-autoimmune hyperthyroidism (SNAH). Up to date 17 constitutively activating TSHR mutations have been reported in 24 families with FNAH. The diagnosis of FNAH should be considered in cases with a positive family history, early onset of hyperthyroidism, goiter, absence of clinical stigmata of autoimmunity and recurrent hyperthyroidism. Moreover, 14 subjects with sporadic non-autoimmune hyperthyroidism and 10 different TSH receptor germline mutations have been reported. The main characteristic of SNAH is a negative family history. Additional consequences of prolonged neonatal hyperthyroidism (mental retardation, speech disturbances and craniosynostosis) have often been reported in SNAH. No genotype-phenotype relationship has been reported in patients with germline TSHR mutations. There is no association of in vitro activities determined by linear regression analysis (LRA) and several clinical indicators of hyperthyroidism activity for SNAH. However, the comparison of the LRA values of sporadic TSHR mutations with LRA values of familial TSHR mutations does show a significantly higher median LRA value for sporadic as compared to familial autosomal dominant hyperthyroidism. This finding is in line with the clinical impression of a more active clinical course in patients with SNAH. However, additional genetic, constitutional or environmental factors are most likely responsible for the phenotypic variations of the disease and the lack of correlation between in vitro activities of the TSHR mutations and the severity of hyperthyroidism.

Cases of Borderline In Vitro Constitutive Thyrotropin Receptor Activity: How to Decide Whether a Thyrotropin Receptor Mutation Is Constitutively Active or Not?

BACKGROUND Previous in vitro data for several constitutively activating thyrotropin receptor (TSHR) mutations reported divergent results for the constitutive activity of the same mutations. Moreover, several case reports have highlighted the difficulties in determining whether a TSHR mutation is constitutively active or not. Retrospectively, this has repeatedly been the case for mutants with only a slight increase of basal cAMP activity. We re-examined 10 previously described TSHR germline mutations with minor increases of basal cAMP activity and analyzed the influences of the cell line and vector system on the basal receptor activity. METHODS TSHR mutations were characterized by determination of cell surface expression, cAMP accumulation, and linear regression analysis of constitutive activity. RESULTS Re-examination of the previously described constitutively active TSHR germline mutations did not show constitutive activity for R310C and N670S as tested in COS-7 cells and confirmed constitutive activity for the other eight mutations. However, mutant N670S showed a slight but significant increase of basal activity measured by linear regression analysis when analyzed in HEK(GT) cells transiently transfected with pcDNA but not with the pSVL vector. This was not the case for R310C. CONCLUSIONS Our findings indicate that current methods to precisely classify mutants with only a slight increase of the basal activity as constitutively active are limited. The results concerning the level of the basal activity can be influenced by the vector and/or the cell system. A comprehensive clinical characterization of the respective patients appears as a necessary and promising adjunct for the activity classification of these borderline mutations.

Recurrent EZH1 mutations are a second hit in autonomous thyroid adenomas.

Autonomous thyroid adenomas (ATAs) are a frequent cause of hyperthyroidism. Mutations in the genes encoding the TSH receptor (TSHR) or the Gs protein α subunit (GNAS) are found in approximately 70% of ATAs. The involvement of other genes and the pathogenesis of the remaining cases are presently unknown. Here, we performed whole-exome sequencing in 19 ATAs that were paired with normal DNA samples and identified a recurrent hot-spot mutation (c.1712A>G; p.Gln571Arg) in the enhancer of zeste homolog 1 (EZH1) gene, which codes for a catalytic subunit of the polycomb complex. Targeted screening in an independent cohort confirmed that this mutation occurs with high frequency (27%) in ATAs. EZH1 mutations were strongly associated with known (TSHR, GNAS) or presumed (adenylate cyclase 9 [ADCY9]) alterations in cAMP pathway genes. Furthermore, functional studies revealed that the p.Gln571Arg EZH1 mutation caused increased histone H3 trimethylation and increased proliferation of thyroid cells. In summary, this study revealed that a hot-spot mutation in EZH1 is the second most frequent genetic alteration in ATAs. The association between EZH1 and TSHR mutations suggests a 2-hit model for the pathogenesis of these tumors, whereby constitutive activation of the cAMP pathway and EZH1 mutations cooperate to induce the hyperproliferation of thyroid cells.

Angiotensin converting enzyme gene polymorphism in Turkish asthmatic patients.

Asthma is a chronic inflammatory disease of the airways. Several candidate genes have been identified with a potential role in the pathogenesis of asthma, including the angiotensin converting enzyme (ACE) gene. We aimed to investigate the frequency of an ACE gene polymorphism in Turkish asthmatic patients and to determine its impact on clinical parameters and disease severity. Ninety-seven asthmatic patients (M/F 25/72, mean age 39 ± 13 years) and 96 healthy subjects (M/F 26/70, mean age 38 ± 12 years) were included. At baseline, all participants completed a questionnaire on demographics, symptoms, triggering factors, severity of asthma, and the presence of atopism. Blood samples were obtained from all patients and genomic DNA was isolated. The frequency of the ACE genotypes (I = insertion and D = deletion) among asthmatics and controls were compared: asthmatics showed a 40.2% prevalence of the DD genotype (n = 39), ID was 45.4% (n = 44), and II was 14.4% (n = 14.4). In the control subjects, the frequency of DD was18.8% (n = 18), ID was 50% (n = 48) and II was 31.3% (n = 30). The DD ACEgenotype was significantly more frequent in asthmatics compared with controls (p < 0.001). Asthmatics with the ID ACE genotype showed a higher frequency of drug allergies, although this was not statistically significant (p = 0.08). Asthmatics with the DD genotype appeared to have a higher incidence of asthmatic episode exacerbations due to viral infections, but again this was not statistically significant (p = 0.08). Patients with mild or moderate-severe asthma had similar frequencies of these mutations. We found a higher frequency of the ACE DD gene mutation in Turkish asthmatic patients compared with non-asthmatics, suggesting that this ACE gene polymorphism may be a risk factor for asthma but does not increase the severity of the disease.

Shared Sporadic and Somatic Thyrotropin Receptor Mutations Display More Active In Vitro Activities than Familial Thyrotropin Receptor Mutations

BACKGROUND Germline thyrotropin receptor (TSHR) mutations are associated with sporadic congenital nonautoimmune hyperthyroidism and familial nonautoimmune hyperthyroidism. Somatic TSHR mutations are associated with toxic thyroid nodules (TTNs). The objective of the study was to define a relation of the clinical appearance and the in vitro activity (IVA) of the TSHR mutations described by several authors for these thyroid disorders. METHODS We analyzed the IVAs published as linear regression analysis (LRA) of the constitutive activity as a function of the TSHR expression and the basal cyclic adenosine monophosphate (cAMP) values to determine differences between exclusively somatic, exclusively familial, and shared sporadic and somatic TSHR-mutations. Further, we investigated correlations of the LRAs/basal cAMP values with clinical activity characteristics (CACs) of TTNs, such as largest diameter of the TTN and the age of the patient at thyroid surgery. RESULTS Shared sporadic and somatic mutations showed higher median LRA (14.5) and higher median basal cAMP values (fivefold) than exclusively familial mutations (6.1, p = 0.0002; 2.9-fold, p < 0.0001, respectively). Moreover, mutations shared between sporadic congenital nonautoimmune hyperthyroidism and toxic thyroid nodules (TTNs) showed higher median LRA/basal cAMP values (p < 0.0001) than exclusively somatic mutations in TTNs (5.1; 3.89-fold, respectively). Exclusively somatic mutations and exclusively familial mutations showed no significant difference in their median LRA values (p = 0.786) but a significant difference for basal cAMP values (p = 0.0006). The two examined CACs showed no correlation with the IVA characterized by LRA/basal cAMP values or with the presence or absence of a TSHR-mutation. CONCLUSIONS This systematic analysis of published constitutively activating TSHR-mutations, their CACs, and their IVA provides evidence for higher IVA of shared sporadic and somatic TSHR mutations as compared with familial TSHR mutations. CACs of somatic TSHR mutations in TTNs did not have a clear association with the IVA as characterized by LRA or basal cAMP values.

A novel mutation in the MC2R gene causing familial glucocorticoid deficiency type 1.

Familial glucocorticoid deficiency (FGD) or hereditary unresponsiveness to adrenocorticotropin (ACTH) is an autosomal recessive disorder characterized by isolated glucocorticoid deficiency associated with normal mineralocorticoid secretion. Mutations in genes encoding either ACTH receptor or melanocortin 2 receptor accessory protein are responsible for the disease in about 50% of cases, named FGD type 1 and type 2, respectively. Patients may present with hyperpigmentation, recurrent infections, failure to thrive, hypoglycemic seizures, and coma in infancy or early childhood. Here we report the case of a 17-day-old newborn diagnosed with FGD type 1 who presented with hyperbilirubinemia and hyperpigmentation, a sign which was erroneously assumed to be due to prolonged phototherapy by the referring physician. Hormone analysis showed low cortisol and high ACTH levels with normal serum electrolytes and renin-aldosterone axis. Genetic analysis revealed a novel homozygous melanocortin 2 receptor mutation p.Leu225Arg in the patient. The healthy parents were heterozygous for the mutation.