Novel Non-Synonymous Mutations of PAX8 in a Cohort of Chinese with Congenital Hypothyroidism
Congenital hypothyroidism (CH) is the most common endocrine disorder present at birth, characterized by thyroid hormone deficiency that can lead to severe mental and growth retardation if left untreated. The incidence of CH has been progressively increasing, with reported rates ranging from 1:1400 to 1:2800 live births. CH can be broadly classified into two major types: congenital non-goitrous hypothyroidism (CHNG) and thyroid dyshormonogenesis (TDH). CHNG, which accounts for 75% to 85% of CH cases, is caused by defects in the development and migration of the thyroid gland, leading to conditions such as athyreosis, hypoplasia, or ectopy. TDH, on the other hand, is caused by genetic defects in proteins involved in thyroid hormone synthesis and is associated with goitrous enlargement of the thyroid gland.
PAX8, a transcription factor belonging to the PAX family, plays a crucial role in thyroid organogenesis and the expression of thyroid-specific genes. Mutations in PAX8 have been associated with various thyroid disorders, including thyroid follicular carcinomas, atypical follicular thyroid adenomas, and thyroid dysgenesis. The PAX8 gene spans 63 kb and contains 12 exons, encoding a protein with a PAX domain responsible for DNA binding. Most pathogenic mutations in PAX8 are located within the PAX domain, impairing its DNA binding ability and leading to thyroid dysgenesis.
The prevalence of PAX8 mutations varies significantly among different ethnic populations. For instance, mutations in PAX8 have been detected in 8.4% of French patients with CH, while only 0.5% of Czech patients with CH carry such mutations. In China, the prevalence of PAX8 mutations also varies across different regions, with reported rates of 2.38% in Guangxi, 2.73% in Shanghai, and 1.14% in Shandong province. This study aimed to identify and characterize PAX8 mutations in a cohort of Chinese patients with CH and explore the prevalence of these mutations.
The study enrolled 105 unrelated Chinese patients with CH, including 40 females and 65 males, with a mean age of 1.8 years. These patients were identified through newborn screening and followed up in pediatric clinics across four major hospitals in China. Genomic DNA was extracted from peripheral blood samples, and exome sequencing was performed using the Hiseq 2000 platform. Variants were identified and annotated using BWA, SAM tools, and ANNOVAR. Functional predictions for the identified mutations were made using Polyphen-2 and SIFT. The effects of the mutations on the transcription of thyroid peroxidase (TPO) were assessed using luciferase reporter assays.
Three PAX8 mutations were identified in four subjects among the 105 samples. One variant, rs189229644, was detected in two subjects and categorized as uncertain significance. The other two missense mutations, c.275T>C (p.Ile92Thr) and c.398G>A (p.Arg133Gln), were novel and not detected in three large-scale genotyping projects: the 1000 Genome Project, Exome Aggregation Consortium, and GO Exome Sequencing Project. These mutations were also absent in 347 ethnically matched healthy controls, indicating their potential pathogenicity. Functional studies revealed that both mutations impaired the transcription ability of PAX8 on its target gene, TPO, confirming their role in the pathogenesis of CHNG2.
The patient with the c.275T>C mutation was a male born at 41 weeks of gestation with a birth weight of 3200 g and length of 52 cm. He was diagnosed with CH through neonatal screening, which revealed a high TSH level (>100 mU/mL) and low FT4 level (6.2 pmol/L). The patient with the c.398G>A mutation was a female born at 38 weeks of gestation with a birth weight of 12,000 g and length of 85 cm. Her neonatal screening also showed a high TSH level (97.1 mU/mL) and low FT4 level (3.1 pmol/L).
Multiple sequence alignment of PAX8 protein sequences from 12 different species revealed that the Ile92 and Arg133 residues are highly conserved, underscoring their functional importance. Luciferase reporter assays demonstrated that both mutations significantly reduced the transcriptional activity of PAX8 on the TPO promoter, further supporting their pathogenic role.
The study also reviewed the prevalence of PAX8 mutations in Chinese patients with CH, combining data from previous studies. An average frequency of 1.74% (21/1209) was observed, consistent with the low prevalence reported in other ethnic populations. For example, in 17 different ethnic cohorts of European patients with CH, the average frequency of PAX8 mutations was 1.0%, ranging from 0 to 3.4%. The variability in PAX8 mutation prevalence across different regions in China highlights the need for comprehensive genetic screening in diverse populations.
In conclusion, this study identified two novel PAX8 mutations, c.275T>C and c.398G>A, in a cohort of Chinese patients with CH. These mutations were shown to impair the transcription ability of PAX8, leading to thyroid dysgenesis and CH. The prevalence of PAX8 mutations in this cohort was 1.90%, similar to other studies involving Chinese patients from different regions. The findings underscore the importance of genetic screening and functional characterization of PAX8 mutations in understanding the molecular pathogenesis of CH.
doi.org/10.1097/CM9.0000000000000213
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