Comprehensive genotyping and clinical characterisation reveal 27 novel NKX2-1 mutations and expand the phenotypic spectrum.

BACKGROUND: NKX2-1 encodes a transcription factor with large impact on the development of brain, lung and thyroid. Germline mutations of NKX2-1 can lead to dysfunction and malformations of these organs. Starting from the largest coherent collection of patients with a suspected phenotype to date, we systematically evaluated frequency, quality and spectrum of phenotypic consequences of NKX2-1 mutations. METHODS: After identifying mutations by Sanger sequencing and array CGH, we comprehensively reanalysed the phenotype of affected patients and their relatives. We employed electrophoretic mobility shift assay (EMSA) to detect alterations of NKX2-1 DNA binding. Gene expression was monitored by means of in situ hybridisation and compared with the expression level of MBIP, a candidate gene presumably involved in the disorders and closely located in close genomic proximity to NKX2-1. RESULTS: Within 101 index patients, we detected 17 point mutations and 10 deletions. Neurological symptoms were the most consistent finding (100%), followed by lung affection (78%) and thyroidal dysfunction (75%). Novel symptoms associated with NKX2-1 mutations comprise abnormal height, bouts of fever and cardiac septum defects. In contrast to previous reports, our data suggest that missense mutations in the homeodomain of NKX2-1 not necessarily modify its DNA binding capacity and that this specific type of mutations may be associated with mild pulmonary phenotypes such as asthma. Two deletions did not include NKX2-1, but MBIP, whose expression spatially and temporarily coincides with NKX2-1 in early murine development. CONCLUSIONS: The high incidence of NKX2-1 mutations strongly recommends the routine screen for mutations in patients with corresponding symptoms. However, this analysis should not be confined to the exonic sequence alone, but should take advantage of affordable NGS technology to expand the target to adjacent regulatory sequences and the NKX2-1 interactome in order to maximise the yield of this diagnostic effort.

Analysis of the gene coding for steroidogenic factor 1 (SF1, NR5A1) in a cohort of 50 Egyptian patients with 46,XY disorders of sex development.

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.

Arteries define the position of the thyroid gland during its developmental relocalisation.

During vertebrate development, the thyroid gland undergoes a unique relocalisation from its site of induction to a distant species-specific position in the cervical mesenchyme. We have analysed thyroid morphogenesis in wild-type and mutant zebrafish and mice, and find that localisation of growing thyroid tissue along the anteroposterior axis in zebrafish is linked to the development of the ventral aorta. In grafting experiments, ectopic vascular cells influence the localisation of thyroid tissue cell non-autonomously, showing that vessels provide guidance cues in zebrafish thyroid morphogenesis. In mouse thyroid development, the midline primordium bifurcates and two lobes relocalise cranially along the bilateral pair of carotid arteries. In hedgehog-deficient mice, thyroid tissue always develops along the ectopically and asymmetrically positioned carotid arteries, suggesting that, in mice (as in zebrafish), co-developing major arteries define the position of the thyroid. The similarity between zebrafish and mouse mutant phenotypes further indicates that thyroid relocalisation involves two morphogenetic phases, and that variation in the second phase accounts for species-specific differences in thyroid morphology. Moreover, the involvement of vessels in thyroid relocalisation sheds new light on the interpretation of congenital thyroid defects in humans.