Molecular insights into the possible role of Kir4.1 and Kir5.1 in thyroid hormone biosynthesis.

INTRODUCTION: Thyroid morphogenesis is a complex process. Inwardly rectifying potassium (Kir) genes play a role in hormone release, cell excitability, pH and K(+) homeostasis in many tissues. OBJECTIVES: To investigate the thyroid developmental expression of three members, Kir4.1, Kir4.2 and Kir5.1, in mice. To postulate the K(+) channel role in thyroid hormone secretion. MATERIAL AND METHODS: Quantitative RT-PCR analysis of Kir4.1, Kir4.2 and Kir5.1 in mice of different stages (E13.5-E18.5). RESULTS: mRNA for Kir4.1, Kir4.2 and Kir5.1 were identified and increased with age in mice. Both Kir4.1 and Kir4.2 genes are better expressed after E16.5. Kir4.2 greatly increases from E13.5 to E16.5 (p ≤ 0.05). CONCLUSION: Quantitative PCR shows that the mouse thyroid presents increased expression for Kir channels during development. The role of Kir in thyroid morphogenesis and differentiation might be understood in future studies. We speculate that thyroglobulin trafficking might be modulated by Kir4.1/5.1.

Functional characterization of the novel sequence variant p.S304R in the hinge region of TSHR in a congenital hypothyroidism patients and analogy with other formerly known mutations of this gene portion.

CONTEXT: Thyroid dysgenesis may be associated with loss-of-function mutations in the thyrotropin receptor (TSHR) gene. OBJECTIVES: The aim of this study was to characterize a novel TSHR gene variant found in one patient harboring congenital hypothyroidism (CH) from a cohort of patients with various types of thyroid defects. MATERIALS AND METHODS: This cross-sectional cohort study involved 118 patients with CH and their family members, including 45 with familial and 73 with sporadic diseases. The thyroid gland was normal in 23 patients, 25 patients had hypoplasia, 25 hemithyroid agenesis, 21 had athyreosis, and 21 had ectopy. Genomic DNA was extracted, and 10 exons of the TSHR gene were amplified and sequenced. Mutations in other candidate genes were investigated. Ortholog alignment was performed, and TSHR functional assays were evaluated. RESULTS: We identified one previously unknown missense variation in the hinge region (HinR) of the TSHR gene (p.S304R) in one patient with thyroid hypoplasia. This variant is conserved in our ortholog alignment. However, the p.S304R TSHR variant presented a normal glycosylation pattern and signal transduction activity in functional analysis. CONCLUSION: We report the ocurrence of a novel nonsynonymous substitution in the HinR of the large N-terminal extracellular domain of the TSHR gene in a patient with thyroid hypoplasia. In contrast with four others in whom TSHR mutations of the hinge portion were previously identified, the p.S304R TSHR variation neither affected TSH binding nor cAMP pathway activation. This TSHR gene variant was documented in a CH patient, but the current data do not support its role in the clinical phenotype.

A novel FOXE1 mutation (R73S) in Bamforth-Lazarus syndrome causing increased thyroidal gene expression.

BACKGROUND: Homozygous loss-of-function mutations in the FOXE1 gene have been reported in several patients with partial or complete Bamforth-Lazarus syndrome: congenital hypothyroidism (CH) with thyroid dysgenesis (usually athyreosis), cleft palate, spiky hair, with or without choanal atresia, and bifid epiglottis. Here, our objective was to evaluate potential functional consequences of a FOXE1 mutation in a patient with a similar clinical phenotype. METHODS: FOXE1 was sequenced in eight patients with thyroid dysgenesis and cleft palate. Transient transfection was performed in HEK293 cells using the thyroglobulin (TG) and thyroid peroxidase (TPO) promoters in luciferase reporter plasmids to assess the functional impact of the FOXE1 mutations. Primary human thyrocytes transfected with wild type and mutant FOXE1 served to assess the impact of the mutation on endogenous TG and TPO expression. RESULTS: We identified and characterized the function of a new homozygous FOXE1 missense mutation (p.R73S) in a boy with a typical phenotype (athyreosis, cleft palate, and partial choanal atresia). This new mutation located within the forkhead domain was inherited from the heterozygous healthy consanguineous parents. In vitro functional studies in HEK293 cells showed that this mutant gene enhanced the activity of the TG and TPO gene promoters (1.5-fold and 1.7-fold respectively vs. wild type FOXE1; p<0.05), unlike the five mutations previously reported in Bamforth-Lazarus syndrome. The gain-of-function effect of the FOXE1-p.R73S mutant gene was confirmed by an increase in endogenous TG production in primary human thyrocytes. CONCLUSION: We identified a new homozygous FOXE1 mutation responsible for enhanced expression of the TG and TPO genes in a boy whose phenotype is similar to that reported previously in patients with loss-of-function FOXE1 mutations. This finding further delineates the role for FOXE1 in both thyroid and palate development, and shows that enhanced gene activity should be considered among the mechanisms underlying Bamforth-Lazarus syndrome.

Inflammatory peeling skin syndrome caused by homozygous genomic deletion in the PSORS1 region encompassing the CDSN gene.

Peeling skin syndrome (PSS) type B is a rare recessive genodermatosis characterized by lifelong widespread, reddish peeling of the skin with pruritus. The disease is caused by small-scale mutations in the Corneodesmosin gene (CDSN) leading to premature termination codons. We report for the first time a Japanese case resulting from complete deletion of CDSN. Corneodesmosin was undetectable in the epidermis, and CDSN was unamplifiable by PCR. QMPSF analysis demonstrated deletion of CDSN exons inherited from each parent. Deletion mapping using microsatellite haplotyping, CGH array and PCR analysis established that the genomic deletion spanned 49-72 kb between HCG22 and TCF19, removing CDSN as well as five other genes within the psoriasis susceptibility region 1 (PSORS1) on 6p21.33. This observation widens the spectrum of molecular defects underlying PSS type B and shows that loss of these five genes from the PSORS1 region does not result in an additional cutaneous phenotype.

Clinical expression and new SPINK5 splicing defects in Netherton syndrome: unmasking a frequent founder synonymous mutation and unconventional intronic mutations.

Netherton syndrome (NS) is a severe skin disease caused by loss-of-function mutations in SPINK5 (serine protease inhibitor Kazal-type 5) encoding the serine protease inhibitor LEKTI (lympho-epithelial Kazal type-related inhibitor). Here, we disclose new SPINK5 defects in 12 patients, who presented a clinical triad suggestive of NS with variations in inter- and intra-familial disease expression. We identified a new and frequent synonymous mutation c.891C>T (p.Cys297Cys) in exon 11 of the 12 NS patients. This mutation disrupts an exonic splicing enhancer sequence and causes out-of-frame skipping of exon 11. Haplotype analysis indicates that this mutation is a founder mutation in Greece. Two other new deep intronic mutations, c.283-12T>A in intron 4 and c.1820+53G>A in intron 19, induced partial intronic sequence retention. A new nonsense c.2557C>T (p.Arg853X) mutation was also identified. All mutations led to a premature termination codon resulting in no detectable LEKTI on skin sections. Two patients with deep intronic mutations showed residual LEKTI fragments in cultured keratinocytes. These fragments retained some functional activity, and could therefore, together with other determinants, contribute to modulate the disease phenotype. This new founder mutation, the most frequent mutation described in European populations so far, and these unusual intronic mutations, widen the clinical and molecular spectrum of NS and offer new diagnostic perspectives for NS patients.

Hes1 is required for appropriate morphogenesis and differentiation during mouse thyroid gland development.

Notch signalling plays an important role in endocrine development, through its target gene Hes1. Hes1, a bHLH transcriptional repressor, influences progenitor cell proliferation and differentiation. Recently, Hes1 was shown to be expressed in the thyroid and regulate expression of the sodium iodide symporter (Nis). To investigate the role of Hes1 for thyroid development, we studied thyroid morphology and function in mice lacking Hes1. During normal mouse thyroid development, Hes1 was detected from E9.5 onwards in the median anlage, and at E11.5 in the ultimobranchial bodies. Hes1(-/-) mouse embryos had a significantly lower number of Nkx2-1-positive progenitor cells (p<0.05) at E9.5 and at E11.5. Moreover, Hes1(-/-) mouse embryos showed a significantly smaller total thyroid surface area (-40 to -60%) compared to wild type mice at all study time points (E9.5-E16.5). In both Hes1(-/-) and wild type mouse embryos, most Nkx2-1-positive thyroid cells expressed the cell cycle inhibitor p57 at E9.5 in correlation with low proliferation index. In Hes1(-/-) mouse embryos, fusion of the median anlage with the ultimobranchial bodies was delayed by 3 days (E16.5 vs. E13.5 in wild type mice). After fusion of thyroid anlages, hypoplastic Hes1(-/-) thyroids revealed a significantly decreased labelling area for T4 (-78%) and calcitonin (-65%) normalized to Nkx2-1 positive cells. Decreased T4-synthesis might be due to reduced Nis labelling area (-69%). These findings suggest a dual role of Hes1 during thyroid development: first, control of the number of both thyrocyte and C-cell progenitors, via a p57-independent mechanism; second, adequate differentiation and endocrine function of thyrocytes and C-cells.

Pregnancy in women heterozygous for MCT8 mutations: risk of maternal hypothyroxinemia and fetal care.

CONTEXT: Monocarboxylate transporter 8 (MCT8 or SLC16A2) mutations cause X-linked Allan-Herndon-Dudley syndrome. Heterozygous females are usually asymptomatic, but pregnancy may modify thyroid function and MCT8 is expressed in the placenta, suggesting that maternal and fetal abnormalities might develop even in the absence of MCT8 fetal mutation. Genetic counseling is so far based on X-linked transmission, and prenatal diagnosis is rarely performed. OBJECTIVE: To describe thyroid function and the prenatal diagnosis in pregnant mothers harboring heterozygous MCT8 mutations and management of the persistent maternal hypothyroxinemia. Patients Two women heterozygous for MCT8 mutations (c.1690G>A and c.1393-1G>C) were monitored throughout pregnancy. METHODS: Prenatal diagnosis included sex determination, direct MCT8 sequencing, and familial linkage analysis. Ultrasonography and hormonal assays for maternal thyroid function evaluation were performed serially during pregnancy. Neonatal thyroid hormonal status was assessed. RESULTS: None of the three fetuses (two males and one female) carried MCT8 mutations. One of the two heterozygous mothers revealed gestational hypothyroxinemia, prompting early levothyroxine (l-T4) therapy until delivery. The second heterozygous mother showed normal thyroid function but was preventively traited by l-T4 and all of the three neonates had normal thyroid hormone levels and thyroid gland at birth, suggesting advantages of prenatal care and/or compensatory mechanisms. CONCLUSION: Heterozygous MCT8 women should be monitored for requirement of l-T4 therapy to prevent fetal and neonatal hypothyroidism and to avoid risk of potential cognitive delay due to gestational hypothyroxinemia. Moreover, when the disease-causing mutation is known and/or the first child is affected, prenatal diagnosis for male fetuses should be assessed early for MCT8 mutations by direct sequencing.

New cases of isolated congenital central hypothyroidism due to homozygous thyrotropin beta gene mutations: a pitfall to neonatal screening.

BACKGROUND: Congenital central hypothyroidism (CCH) is a rare condition that is often diagnosed in late childhood in countries where neonatal screening programs rely solely on detecting thyrotropin (TSH) elevation. TSHbeta gene mutation is one of the causes of CCH. We describe two cases of c.Q49X mutation and three cases of c.C105Vfs114X mutation in exon 3 of the TSH beta-subunit gene. SUMMARY: We found two different TSHbeta gene mutations in two families. In one family, we identified a missense mutation in exon 3 leading to a premature stop at position 49 (c.Q49X) in the two affected twins. In the other family, the three affected siblings had a 313delT nucleotide deletion leading to a frame shift responsible for premature termination at codon 114 (c.C105Vfs114X); neonatal screening showed very low TSH levels in all three patients. The presence of inappropriately low TSH levels at birth in the three affected members of the second family raises questions about the value of the TSH level for CCH screening. CONCLUSIONS: The marked phenotypic variability in patients with the c.Q49X mutation suggests modulation by interacting genes and/or differences in the genetic background. TSHbeta gene mutations should be suspected in neonates with inappropriately low TSH levels.

NKX2-1 mutations leading to surfactant protein promoter dysregulation cause interstitial lung disease in "Brain-Lung-Thyroid Syndrome".

NKX2-1 (NK2 homeobox 1) is a critical regulator of transcription for the surfactant protein (SP)-B and -C genes (SFTPB and SFTPC, respectively). We identified and functionally characterized two new de novo NKX2-1 mutations c.493C>T (p.R165W) and c.786_787del2 (p.L263fs) in infants with closely similar severe interstitial lung disease (ILD), hypotonia, and congenital hypothyroidism. Functional analyses using A549 and HeLa cells revealed that NKX2-1-p.L263fs induced neither SFTPB nor SFTPC promoter activation and had a dominant negative effect on wild-type (WT) NKX2-1. In contrast,NKX2-1-p.R165W activated SFTPC, to a significantly greater extent than did WTNKX2-1, while SFTPB activation was only significantly reduced in HeLa cells. In accordance with our in vitro data, we found decreased amounts of SP-B and SP-C by western blot in bronchoalveolar lavage fluid (patient with p.L263fs) and features of altered surfactant protein metabolism on lung histology (patient with NKX2-1-p.R165W). In conclusion, ILD in patients with NKX2-1 mutations was associated with altered surfactant protein metabolism, and both gain and loss of function of the mutated NKX2-1 genes on surfactant protein promoters were associated with ILD in "Brain-Lung-Thyroid syndrome".

Five new TTF1/NKX2.1 mutations in brain-lung-thyroid syndrome: rescue by PAX8 synergism in one case.

Thyroid transcription factor 1 (NKX2-1/TITF1) mutations cause brain-lung-thyroid syndrome, characterized by congenital hypothyroidism (CH), infant respiratory distress syndrome (IRDS) and benign hereditary chorea (BHC). The objectives of the present study were (i) detection of NKX2-1 mutations in patients with CH associated with pneumopathy and/or BHC, (ii) functional analysis of new mutations in vitro and (iii) description of the phenotypic spectrum of brain-lung-thyroid syndrome. We identified three new heterozygous missense mutations (L176V, P202L, Q210P), a splice site mutation (376-2A-->G), and one deletion of NKX2-1 at 14q13. Functional analysis of the three missense mutations revealed loss of transactivation capacity on the human thyroglobulin enhancer/promoter. Interestingly, we showed that deficient transcriptional activity of NKX2-1-P202L was completely rescued by cotransfected PAX8-WT, whereas the synergistic effect was abolished by L176V and Q210P. The clinical spectrum of 6 own and 40 published patients with NKX2-1 mutations ranged from the complete triad of brain-lung-thyroid syndrome (50%), brain and thyroid disease (30%), to isolated BHC (13%). Thyroid morphology was normal (55%) and compensated hypothyroidism occurred in 61%. Lung disease occurred in 54% of patients (IRDS at term 76%; recurrent pulmonary infections 24%). On follow-up, 20% developed severe chronic interstitial lung disease, and 16% died. In conclusion, we describe five new NKX2.1 mutations with, for the first time, complete rescue by PAX8 of the deficient transactivating capacity in one case. Additionally, our review shows that the majority of affected patients display neurological and/or thyroidal problems and that, although less frequent, lung disease is responsible for a considerable mortality.