The genetic characteristics of congenital hypothyroidism in China by comprehensive screening of 21 candidate genes

Abstract

Objective: Congenital hypothyroidism (CH), the most common neonatal metabolic disorder, is characterized by impaired neurodevelopment. Although several candidate genes have been associated with CH, comprehensive screening of causative genes has been limited.

Design and methods: One hundred ten patients with primary CH were recruited in this study. All exons and exon-intron boundaries of 21 candidate genes for CH were analyzed by next-generation sequencing. And the inheritance pattern of causative genes was analyzed by the study of family pedigrees.

Results: Our results showed that 57 patients (51.82%) carried biallelic mutations (containing compound heterozygous mutations and homozygous mutations) in six genes (DUOX2, DUOXA2, DUOXA1, TG, TPO and TSHR) involved in thyroid hormone synthesis. Autosomal recessive inheritance of CH caused by mutations in DUOX2, DUOXA2, TG and TPO was confirmed by analysis of 22 family pedigrees. Notably, eight mutations in four genes (FOXE1, NKX2-1, PAX8 and HHEX) that lead to thyroid dysgenesis were identified in eight probands. These mutations were heterozygous in all cases and hypothyroidism was not observed in parents of these probands.

Conclusions: Most cases of congenital hypothyroidism in China were caused by thyroid dyshormonogenesis rather than thyroid dysgenesis. This study identified previously reported causative genes for 57/110 Chinese patients and revealed DUOX2 was the most frequently mutated gene in these patients. Our study expanded the mutation spectrum of CH in Chinese patients, which was significantly different from Western countries.

Figure 1

The flow chart of data analysis. The raw NGS data of 110 CH patients were generated by GATK. After quality control of raw data as described in main text, 823 relative credible mutations were selected (including 679 SNVs and 144 Indels), then intergenic and 3′/5′ UTR variants, nonsplice-related intronic variants, synonymous variants were excluded and the remaining 241 mutations (including 219 SNVs and 22 Indels) were chose to be verified by Sanger sequencing. Finally, a total of 218 mutations were verified by Sanger sequencing (including 199 SNVs and 19 Indels). SNVs, single nucleotide variants.

Figure 2

Mutations detected in 89 patients with congenital hypothyroidism. The right side were the 15 mutated genes, the bottom were the patient ID. Each column represents one patient and each row represents one gene. Blue blocks represent biallelic mutations (containing compound heterozygous mutations and homozygous mutations) and green blocks represent monoallelic mutations (heterozygous mutation). For example, patient 4 carries mutations in two genes, biallelic mutations in the DUOX2 gene in addition to a monoallelic TG mutation. Patient 5 carries mutations in three genes, biallelic mutations in the DUOX2 gene and a monoallelic mutation in DUOXA1 in addition to a monoallelic TSHR mutation. A total of 57 patients carried biallelic mutations in DUOX2, TG, TPO, TSHR, DUOXA2 or DUOXA1. A total of 66 patients harbored mutations in DUOX2, which was the most frequently mutated gene.

Figure 3

Mutation sites in the secondary structure of DUOX2, TG, TSHR and TPO proteins. (A) Fifty-one mutation sites distributed in the secondary structure of DUOX2 protein, which contains 4 domains, peroxidase-like domain, EF-hand domain, ferric oxidoreductase domain and FAD-binding FR-type domain. A total of 41 mutations were located in these domains region. (B) Twenty-one mutation sites located in the secondary structure of TG protein, which include 3 domains, type 1 domain, type 2 domain, type 3 domain and carboxylesterase family domain. Fifteen mutation sites were in domain region. (C) Nine mutation sites were in TPO protein with CCP/SCR domain and calcium domain, only two mutations (L764P, C756fs) were in domain region. (D) Ten mutation sites were located in TSHR protein, of which 3 were in LRR domain and one was in PDZ-binding domain. FAD, flavin adenine dinucleotide; CCP, the complement control protein; SCR, short consensus repeat; LRR, leucine-rich repeats. Missense mutations, stopgain mutations, splicing mutations, frameshift deletion mutations, frameshift insertion mutations and nonframeshift deletion mutations are indicated with black, brown, red, green, purple and blue font, respectively. *Denotes the novel mutations and ^#denotes the recurrent mutations.

Figure 4

Twenty-two pedigrees with probands carried biallelic mutation. Seventeen pedigrees carried DUOX2 mutations, 2 harbored TPO mutations, two had DUOXA2 mutation, and one had TG mutation. For example, the mothers of proband 1 and 2 from family 1 were sisters who shared the same DUOX2 mutation, which was transmitted to their children respectively. Another DUOX2 mutation carried by proband 1 and 2 was inherited from their fathers respectively. The finding that individuals with biallelic mutations have congenital hypothyroidism (CH), and parents with heterozygous mutations are euthyroid indicates the CH caused by DUOX2, DUOXA2, TG and TPO is inherited in an autosomal recessive manner.