The TPO mutation screening and genotype-phenotype analysis in 230 Chinese patients with congenital hypothyroidism
Introduction
Congenital hypothyroidism (CH) is the most common congenital endocrine disorder, affecting approximately 1 in 3000 to 4000 newborns in Caucasians (Peters C et al., 2018) and approximately 1 in 2000 to 2200 live births in China (Yu B et al., 2018). Eighty-five percent of CH cases are caused by thyroid dysgenesis in Caucasians, while nearly 50% of CH in China is caused by thyroid dyshormonogenesis (DH), which is a result of defects in thyroid hormone biosynthesis. The thyroperoxidase (TPO) mutation, which causes a defect of thyroid hormone biosynthesis, is one of the most common mutations (Mio and Grani, 2020; Ris-SC et al., 2010; Stoupa A et al., 2018; Kotani T et al., 2003; Targovnik HM, 2017). TPO mutations were identified in 46% patients with thyroid dyshormonogenesis in Europe (Avbelj M et al., 2007) but only in approximately 1–10% of patients with thyroid dyshormonogenesis in China (Fu C et al., 2016; Yu B et al., 2018), indicating that the frequency of TPO mutations in patients with thyroid dyshormonogenesis was higher in Caucasians than in the Chinese Han population.
TPO is located at the apical membrane of thyrocytes and plays a key role in thyroid hormone biosynthesis. TPO oxidizes iodide ions to form iodine atoms, which activate the iodide ions to iodize tyrosine residues on thyroglobulin (TG), leading to the generation of mono- or di-iodotyrosine residues. TPO also catalyzes the coupling reaction of two iodotyrosine residues, ultimately forming triiodothyroxine (T3) or tetraiodothyronine (T4) residues in TG (Cangul H et al., 2013; Nilsson M, 2017). TPO is specifically expressed in thyrocytes, including 17 exons, and encodes 933 amino acid proteins (107 kDa) in three primary domains: the myeloperoxidase-like domain (MPO), the complement control protein-like domain (Sushi domain/CCP), and the epidermal growth factor-like domain (EGF) (Williams DE et al., 2018). The MPO domain includes α-helical and little β-sheet, which is significant for the heme group being covalently bound to TPO. CCP is crucial for hydrogen bonding that contains three β-strands. EGF in TPO's ectodomain forms three disulfide bonds. MPO and CCP are more important for TPO protein function, and all three domains are involved in composing epitopes (Targovnik HM et al., 2019; Umeki et al., 2004a).
TPO mutation was first reported in 1992 by Abramowicz et al. (Abramowicz MJ et al., 1992) as an autosomal-recessive pathogenic pattern of CH characterized by inability to utilize iodine in the thyroid. Currently, 151 mutations in TPO defined as ‘disease causing’ are reported in the Human Gene Mutation Database (HGMD) (HGMD Professional, 2019.4). The effect of most of the mutations on TPO enzyme activity remains unknown. The mutation p.C756fs was found in Malaysian-Chinese patients with goitrous CH (Lee CC et al., 2015).
We expanded samples based on our previous work for screening the TPO mutations and characterized the phenotypes of patients with CH who carried TPO mutations. We further evaluated the functions of 14 TPO mutants (including 5 novel mutations) and one insertion in the 3′untranslated region in human embryonic kidney 293 cell line that contains the SV40 T-antigen (293 T cells).
Section snippets
Recruiting patients with congenital hypothyroidism
Newborn screening for CH is based on the TSH level using filter-paper blood spots acquired 3–5 days after birth (Bauer AJ et al., 2019; Kaplowitz PB et al., 2019). Infants with elevated TSH levels (≥10 mU/L) during the initial screening went through a second screening: serum TSH, free triiodothyronine (FT3), and free thyroxin (FT4) were determined by performing an immunochemiluminometric assay (UniCelDxI 800, Beckman, USA). The details of the diagnostic criteria to establish permanent CH in
Mutation of TPO screening in 230 patients with congenital hypothyroidism
All the exons and exon-intron boundaries were amplified by multiplex PCR using customized primers designed to generate approximately 225 bp amplicons. After the quality control, 95.64% of base pairs in the exon of TPO were covered to depths of at least 30 × . Among 230 Chinese patients with CH, 35 non-synonymous mutations in 15 sites of TPO were identified from 23 patients. Out of the 23 patients with mutation of TPO, 13 patients carried biallelic mutations in TPO. All mutations included six
Discussion
In the present study, the TPO mutation was screened in 230 Chinese patients with congenital hypothyroidism (CH). Thirty-five non-synonymous mutations in 15 sites of TPO were identified in 23 CH patients (23/230, 10%), including six novel mutations (p.S37P, p.C269S, p.A430E, p.V748M, p.E799D, and p.R846W). Of these, 13 CH patients carried biallelic mutations in TPO (13/230, 5.65%). Based on our family analysis, we have proved that congenital hypothyroidism is caused by TPO mutations in
Conclusion
We found that 5.65% of congenital hypothyroidism in Chinese patients was caused by a TPO biallelic mutation. The residual enzymatic activity of TPO was correlated with clinical phenotypes of CH patients with TPO biallelic mutations. Three patients with biallelic mutations of TPO, whose residual enzymatic activity of TPO was greater than 16%, showed mild CH and had a normal social life without L-T4 treatment before 3 years of age, but they appeared to have macroscopic goiter that appeared during
Funding
This work was supported by Chinese National Key Research Program (2017YFC1001801) and the National Natural Science Foundation of China (81770786, 81661168016, 81870537, 81670717, 31501015 and 81870540).
CRediT authorship contribution statement
Rui-Jia Zhang: Writing - original draft, Data curation. Feng Sun: Methodology. Feng Chen: Supervision. Ya Fang: Resources. Chen-Yan Yan: Formal analysis. Chang-Run Zhang: Project administration. Ying-Xia Ying: Resources. Zheng Wang: Software. Cao-Xu Zhang: Validation. Feng-Yao Wu: Visualization. Bing Han: Funding acquisition. Jun Liang: Funding acquisition. Shuang-Xia Zhao: Funding acquisition, Formal analysis, Writing - review & editing. Huai-Dong Song: Funding acquisition, Conceptualization,
Declaration of competing interest
None.
Acknowledgments
The authors are grateful to all patients and their parents who participated in this study.
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