| Congenital hypothyroidism is a frequent endocrine metabolic disease and one of the most common causes of mental retardation in the childhood,85%of which was caused by thyroid agenesis (TD) approximately. A number of genes have been identified to be associated with the CH, but it cannot explain the presence of such a large quantity of CH cases with a disproportionate low mutation rate, suggesting that there must be other undiscovered CH candidate gene. This study aimed to enrich the NKX2.5gene mutation library and explore more candidate gene mutations of CH.The study is divided into two parts. In part I, a screening of NKX2.5mutations in90patients associated with congenital hypothyroidism with ectopy was performed to research the gene NKX2.5mutation and genetic characteristics of patients with TD ectopic in Shandong Province, and it has never been reported to screen the volunteer genes for CH in such a mass of patients with ectopy in China, and the quantity is also at the forefront in the world. Blood samples were collected from90TD patients with ectopy who has been proved to exclude other congenital diseases, especially Congenital Heart Disease. Genomic DNA was extracted and two exons of gene NKX2.5were amplified using PCR and direct sequencing to find new mutations types of gene NKX2.5. Analysis of NKX2.5in90TD patients with ectoy revealed no mutations in the area of both exons and joint area between exons and introns. Nevertheless, an SNP (rs2277923, c.63T>C) which was demonstrated to be a synonymous mutation(Glu→Glu) was found in exon1, and the allele frequency of this SNP was T=0.4167. It’s demonstrated to have no significant difference with the allele frequency of MAF minor allele count (P>0.05), suggesting that there may be not a correlation between this rs2277923and TD. The data we gathered suggest that NKX2.5mutations are highly rare in TD patients with ectopy and not serve as the main cause of congenital hypothyroidism in Shandong province,In the second part of this study, we use whole exome sequencing (WES) to screen gene mutations in patients with congenital hypothyroidism. Firstly, selecting10patients associated with congenital hypothyroidism with Thyroid athyreosis excluded known gene mutations [NKX2.1(TTF1), FOXE1(TTF2), PAX8, NKX2.5, and TSHR]. Agilent Human All Exon50M Kit chip was used to capture all the exons in genome and the WES was performed on Illumina MiSeq2000high-throughput sequencing platforms. By the analysis of the WES results, we got10candidate gene mutations. Sanger sequencing was performed on the samples in which mutations was found in the WES to exclude the possibility of false positive and all mutations which had been found in the WES was detected again. Through literature review, function prediction and the close relationship between these mutations and reported mutations, we inferred that FOXC2gene may be a causative gene of CH. However, in the latter screening (90athyreosis&100ectopy), no mutations were found in this gene, suggesting that the rate FOXC2gene mutation was very low. Intraspecific comparative genomics suggested the close relationship between FOXC2and FOXE2which had been proved to a causative gene of CH, and by extracting mRNA from mature thyroid tissue and RT-PCR, we validated the existence of FOXC2in the mature thyroid tissue in this study, which can illustrated an association between FOXC2and CH in some extent. The study initially considered FOXC2a causative gene of congenital hypothyroidism, proving a new direction to the study of genetic mechanism of CH, besides it confirmed that the WES could be used to search rare gene mutations that cause monogenic diseases.
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