A novel loss-of-function mutation in TTF-2 is associated with congenital hypothyroidism, thyroid agenesis and cleft palate

Mireille Castanet 2 Soo-Mi Park 1 Aaron Smith 1 Michel Bost 0 Juliane Le ger 2 Stanislas Lyonnet Anna Pelet Paul Czernichow Krishna Chatterjee 1 Michel Polak 2 0 Department ofPediatrics , CHU, Grenoble 1 Department ofMedicine, University ofCambridge, Addenbrooke's Hospital , Cambridge , UK 2 Paediatric Endocrinology Unit and INSERM U457 , Paris , France Thyroid dysgenesis is the most common cause of congenital hypothyroidism (CH) and its genetic basis is largely unknown. Here, we describe the second homozygous missense mutation in TTF-2 (or FOXE1), a transcription factor that has been implicated in thyroid development. Two male siblings, born to consanguineous parents, presented with CH, athyreosis and cleft palate and were found to be homozygous for a mutation corresponding to a serine to asparagine substitution at codon 57 (S57N) in the forkhead DNA binding domain of TTF-2. Their heterozygous parents were unaffected and this mutation was not found in 31 unrelated cases of athyreosis or normal controls. Consistent with its location, the S57N TTF-2 mutant protein showed impaired DNA binding and partial loss of transcriptional function. Such incomplete loss of TTF-2 function may account for the absence of choanal atresia and bifid epiglottis in our patients, anomalies which were present together with CH and cleft palate in two other individuals with the only other, more deleterious, TTF-2 mutation (A65V) described previously. Our observations support the role of TTF-2 in both thyroid and palate development but suggest phenotypic heterogeneity of this syndromic form of CH. France INTRODUCTION Congenital hypothyroidism (CH) is the most common neonatal endocrine disorder, being found at a rate of 1 in 30004000 live births (1) and results in severe neurodevelopmental impairment if treatment is delayed. Consequently, most countries operate a neonatal screening programme to enable early detection of cases and therapeutic intervention. CH is most commonly (85% of cases) due to defects in thyroid development leading to glandular dysgenesis. Thyroid dysgenesis comprises either complete agenesis (3540%), ectopic (5560%), or hypoplastic (5%) development of the gland. Remaining dyshormonogenetic causes of CH are associated either with a goiter or a normal-sized thyroid gland (24). The pathogenesis of thyroid dysgenesis is largely unknown. Whilst most cases are sporadic, up to 2% of patients with thyroid dysgenesis have a family history of the condition, suggesting the existence of genetic factors which could contribute to the disorder (5). Further evidence in support of a genetic basis for CH is provided by the higher incidence of non-thyroidal congenital anomalies (cardiac and renal malformations and hip dislocation) and chromosomal defects in infants with this disorder than in the general population (68). Thyroid transcription factor 2 (TTF-2) is a member of the forkhead/winged helix-domain protein family (9), many of which are key regulators of embryonic development. The mouse gene is located on chromosome 4 (9) and the human gene (known as TTF-2, FKHL15 or FOXE1) on chromosome 9q22 (10). Both the mouse and human gene contain a single coding exon (912). TTF-2 regulates the transcription of target genes such as thyroglobulin and thyroid peroxidase by binding to specific regulatory DNA sequences in their promoters via its forkhead DNA binding domain (1315). In the rat, TTF-2 is expressed in the thyroid primordium at the onset of its development and TTF-2 expression continues during migration of the thyroid diverticulum (9,10). Homozygous null mice with targeted disruption of TTF-2, exhibit cleft palate and thyroid malformation consisting of either thyroid agenesis or thyroid ectopy (16). We recently described the first human TTF-2 gene defect in two siblings born with congenital hypothyroidism due to thyroid agenesis together with other anomalies including cleft palate, choanal atresia, bifid epiglottis and spiky hair (12). Both individuals were homozygous for a missense mutation (A65V) in a highly conserved residue within the forkhead domain. The mutant TTF-2 protein exhibited complete loss of DNA binding and transcriptional function. Here, we describe a second family with two affected siblings, born to consanguineous parents, who presented with CH, thyroid dysgenesis (TD), cleft palate and spiky hair. They are homozygous for a novel missense mutation (S57N) in the forkhead domain of TTF-2. Functional studies in vitro indicate that the S57N mutant retains some specific DNA binding and transcriptional activating function. Unlike the cases described previously, our patients had an incomplete clinical phenotype, lacking choanal atresia and bifid epiglottis, which may indicate partial preservation of TTF-2 function in vivo. Clinical features Two male sibling probands (Fig. 1A, IV-5 and IV-11) were born at term after normal pregnancies to first cousin parents (Fig. 1A, III-1 and III-2) of Tunisian origin. They were both born with CH and extensive clefting of both soft and hard palate (Fig. 1B) in the absence of any other non-thyroidal congenital midline anomalies. Both are known to have normal karyotypes. Proband IV-5 was born before the establishment of routine neonatal screening for CH in France and therefore did not have the condition diagnosed until he was 4 months of age when he presented with features of frank hypothyroidism including developmental delay and poor growth (length 3 SD below mean). Measurement of circulating thyroid hormones showed a low total T4 of 9 nM/l (reference range 50137.5 nM/l). Knee radiographs showed severe delay in bone maturation with absent ossification centers in the superior tibial epiphyses and very short, dysplasic distal femoral epiphyses. 99Tc scanning and 123I scanning at 5 months (before treatment) and 11 y respectively, both showed no uptake of tracer in neck regions corresponding to the line of thyroid migration, suggesting thyroid agenesis. Thyroid autoantibodies are absent. Following his delayed diagnosis thyroxine replacement therapy was commenced but his compliance has been erratic. Consequently, he has mental retardation in addition to a significant psychiatric problem necessitating prolonged admissions to psychiatric institutions. Despite repeated operations on his palate, velopharyngeal incompetence remains. His brother, proband IV-11, was noted to have hypotonia, prolonged jaundice and poor weight gain in the neonatal period (birth weight 4.5 kg; birth length 55 cm) and CH was diagnosed shortly after birth by the neonatal screening programme. Additional dysmorphic clinical features noted at birth include hypertelorism, low-set posteriorly rotated ears, a low posterior hair line and spiky hair (Fig. 1C and D). His heel prick TSH level was 110 mU/ml (normal range <20 mU/ml) and his serum TSH and total T4 concentrations were 200 mU/ml (reference range 0.54.5 mU/ml) and 28 nM/l (reference range 50137.5 nM/l), respectively at 3 da (...truncated)