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Next-generation sequencing analysis of DUOX2 in 192 Chinese subclinical congenital hypothyroidism (SCH) and CH patients
Next-generation sequencing analysis of DUOX2 in 192 Chinese subclinical congenital hypothyroidism (SCH) and CH patients Chunyun Fu, Shiyu Luo, Shujie Zhang, Jin Wang, Haiyang Zheng, Qi Yang, Bobo Xie, Xuyun Hu, Xin Fan, Jingsi Luo, Rongyu Chen, Jiasun Su, Yiping Shen, Xuefan Gu, Shaoke Chen PII: DOI: Reference:
S0009-8981(16)30144-9 doi: 10.1016/j.cca.2016.04.019 CCA 14339
To appear in:
Clinica Chimica Acta
Received date: Revised date: Accepted date:
25 February 2016 17 April 2016 17 April 2016
Please cite this article as: Fu Chunyun, Luo Shiyu, Zhang Shujie, Wang Jin, Zheng Haiyang, Yang Qi, Xie Bobo, Hu Xuyun, Fan Xin, Luo Jingsi, Chen Rongyu, Su Jiasun, Shen Yiping, Gu Xuefan, Chen Shaoke, Next-generation sequencing analysis of DUOX2 in 192 Chinese subclinical congenital hypothyroidism (SCH) and CH patients, Clinica Chimica Acta (2016), doi: 10.1016/j.cca.2016.04.019
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Next-generation sequencing analysis of DUOX2 in 192 Chinese Subclinical Congenital Hypothyroidism (CH) and CH patients
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Chunyun Fu1,2, Shiyu Luo1,2, Shujie Zhang1,2, Jin Wang1,2, Haiyang Zheng1,2, Qi Yang1,2, Bobo
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Xie1,2, Xuyun Hu1,2, Xin Fan1,2, Jingsi Luo1,2, Rongyu Chen1,2, Jiasun Su1,2, Yiping Shen1,3, Xuefan Gu4, Shaoke Chen1,2 1
Department of Genetic Metabolism, Children's Hospital, Maternal and Child Health Hospital
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of Guangxi Zhuang Autonomous Region, Nanning 530003, People’s Republic of China 2
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GuangXi Center for Birth Defects Research and Prevention, Nanning 530003, People’s
Republic of China
Boston Children’s Hospital, Harvard Medical School, Boston 02115, MA
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Endocrinology and Genetic Metabolism of Institute for Pediatric Research, Xinhua Hospital
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Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai 200092,China Chunyun Fu and Shiyu Luo contribute equally to this work.
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Address correspondences to: Shaoke Chen, E-mail: [email protected] Xuefan Gu, E-mail: [email protected]
ACCEPTED MANUSCRIPT ABSTRACT Background: Defects in the human dual oxidase 2 (DUOX2) gene are reported to be one of
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the major causes of congenital hypothyroidism (CH). This study was set to examine the
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DUOX2 mutation spectrum and prevalence among Chinese CH and subclinical congenital hypothyroidism (SCH) patients and to define the relationships between DUOX2 genotypes and clinical phenotypes.
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Methods: Peripheral venous blood samples were collected from 192 CH/SCH patients in
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Guangxi Zhuang Autonomous Region of China. All exons and their exon-intron boundary sequences of the 11 known CH associated genes including DUOX2 were screened by
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next-generation sequencing (NGS).
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Results: NGS analysis of DUOX2 revealed 18 rare non-polymorphic variants in 57 CH/SCH
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patients. Sequencing of other CH candidate genes in the 57 patients revealed 2 thyroglobulin (TG) variants. All variants included 11 known mutations, 8 novel variants in DUOX2 and one
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novel variant in TG, among which three variants p.K530X, p.L1343F and p.R683L are highly recurrent in our patient cohort. 35 (83%) of the 42 patients with one or two DUOX2 pathogenic variants turned out to be SCH or transient congenital hypothyroidism (TCH), whereas 13 (87%) of the 15 patients with three or more DUOX2 pathogenic variants are associated with permanent congenital hypothyroidism (PCH). The accumulation of defects in DUOX2 contribute to the more severe disease regarding thyroid stimulating hormone (TSH) levels, free thyroxine (FT4) levels and initial dose of L-thyroxine (L-T4). Conclusion: Our study expanded the mutational spectrum of the DUOX2 and TG genes and provided the best estimation of the DUOX2 mutation rate (29%) for CH/SCH patients in
ACCEPTED MANUSCRIPT Guangxi Zhuang Autonomous Region of China. Most one or two DUOX2 pathogenic variants turned out to be SCH or TCH, whereas patients with three or more DUOX2 pathogenic
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variants were mostly associated with PCH. The coexistence of multiple pathogenic variants
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may have contributed to the severity of the hypothyroid condition.
Keywords: subclinical congenital hypothyroidism; Dual oxidase 2 gene (DUOX2); gene
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mutations; next-generation sequencing; Chinese population
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Introduction
Congenital hypothyroidism (CH) is the most common neonatal endocrine disorder in infancy
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with prevalence ranging from 1:2000 to 1:4000 [1,2]. It’s reported that the most prevalent
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cause of CH worldwide is still iodine deficiency [3], however, considerable progress has been
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made to identify the genetic causes in CH patients. Based on genetic alterations, CH can be classified into two groups: 80%-85% of cases are caused by disorders of thyroid gland
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development (thyroid dysgenesis, comprising agenesis, ectopy, or hypoplasia) [4], which has been linked to gene mutations in thyroid-stimulating hormone receptor (TSHR), paired box gene 8 (PAX8), thyroid transcription factor 1 (TTF1/NKX2.1), thyroid transcription factor 2 (TTF2/FOXE1) and NK2 transcription factor related locus 5 (NKX2.5) [5]; the remaining 15%-20% of CH cases are caused by abnormalities in thyroid hormone synthesis (thyroid dyshormonogenesis), which is associated with the presence of goiter or with a eutopic gland of normal size [6]. Molecular studies have found that dyshormogenesis in CH is caused by defects in genes such as dual oxidase 2 (DUOX2), thyroglobulin (TG), thyroid peroxidase
ACCEPTED MANUSCRIPT (TPO), Pendrine (SLC26A4), dehalogenase 1 (DEHAL1) and sodium iodide symporter (NIS) [7].
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Defects in the human DUOX2 gene are reported to be one of the major causes of CH. The
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DUOX2 gene is located on chromosome 15 and consists of 34 exons [8]. The DUOX2 protein is a 1548-amino-acid polypeptide located at the apical membrane of thyrocytes and generates the hydrogen peroxide (H2O2) needed by thyroid peroxidase for the incorporation of iodine
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into thyroglobulin, an essential step in thyroid hormone synthesis. Up to date, the mutational
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spectrum of the DUOX2 gene and the phenotype-genotype correlations have not yet fully been established. Both biallelic and monoallelic DUOX2 mutations lead to a wide spectrum of
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phenotypes, ranging from mild subclinical congenital hypothyroidism (SCH) with elevated
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thyroid stimulating hormone (TSH) levels but normal thyroid hormone levels, to overt CH
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with thyroid dyshormonogenesis. Also, little is known about DUOX2 mutation spectrum and its prevalence among Chinese CH/SCH patients. In our recent study, we identified DUOX2
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pathogenic variants in 13 of 45 cases (29%) and found that most monoallelic or biallelic DUOX2 pathogenic variants turned out to be TCH, while patients with triallelic DUOX2 pathogenic variants were associated with permanent congenital hypothyroidism (PCH) [9]. The aim of this study was to screen for the presence of mutations in DUOX2 gene among patients with CH/SCH in China, to define the relationships between DUOX2 genotypes and clinical phenotypes and to confirm our previous findings based on a larger sample size and comprehensive analysis of all known CH associated genes.
Methods
ACCEPTED MANUSCRIPT Patients A total of 192 newborns were enrolled in this study ( the previous 45 CH patients collected
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for DUOX2 mutation screening by Sanger sequencing were not included in this study [9]),
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including 140 patients with CH and 52 patients with SCH, who were identified through a large-scale newborn screening (NBS) covering 615,000 cases in Guangxi, China, from July 2009 to June 2013. CH NBS were done with filter paper between 72h and 7 days after birth.
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Blood samples were collected from the heel and the TSH level were measured by
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time-resolved fluorescence assay (Perkin Elmer, USA). Subjects with increased TSH levels (TSH ≥ 8 mIU/l) during NBS were followed-up for further evaluation. Serum TSH and free
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thyroxine (FT4) were determined by electrochemiluminescence assay (Cobas e601, Roche
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Diagnostics, USA). Diagnosis of CH was based on elevated TSH levels (TSH≥10 mIU/l) and
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decreased FT4 levels (FT4 < 12 pmol/l). Patients with elevated TSH levels and normal FT4 levels (normal range 12-22 pmol/l) were diagnosed as SCH. Permanent or transient CH was
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determined using results of thyroid function tests after temporary withdrawal of L-thyroxine (L-T4) therapy at approximately 2 years of age. Thyroid ultrasonography and
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scintigraphy were performed during the neonatal period before treatment. This study was approved by the local Medical Ethics Committee. Informed consent was obtained from the parents of the patients. Next generation sequencing and bioinformatics analysis Peripheral venous blood samples were collected from the patients. Genomic DNA was extracted from peripheral blood leukocytes using QIAamp DNA Blood Mini Kit (Qiagen, Germany) according to the manufacturer’s protocol. CH capture panel as Illumina Truseq
ACCEPTED MANUSCRIPT Custom Amplicon v1.5 kit was designed and 11 known CH associated genes (DUOX2, TSHR, PAX8, NKX2.1, NKX2.5, FOXE1,TG, TPO, NIS, SLC26A4 and DEHAL1) were included with
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the whole coding regions and flanking intronic sequences. The prepared sample library was
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sequenced by Illumina MiSeq instrument using MiSeq Reagent Kit v2, 500-cycles (Illumina Inc., San Diego, CA). Illumina Amplicon Viewer v1.3 and MiSeq Reporter v2.3 software were used for data analysis, and the SnpEff [10] was used for variant annotation. SIFT [11]
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and MutationTaster [12] were used to evaluate the pathogenicity of the novel variants.
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Multiple sequence alignment of the DUOX2 family protein among different species were carried out by DNAMAN software version 8 to analyze the amino acid conservation of the
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mutated sites. All variants were further validated by Sanger sequencing. In addition, a cohort
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of 400 ethnicity-matched healthy subjects with normal FT4 and TSH levels were sequenced
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Results
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to assess the variant frequencies in normal control population.
Next generation sequencing analysis of DUOX2 revealed 18 different variants in 57 individuals (47 CH and 10 SCH patients) including 31 with single heterozygous variation, 11 with two variants and 15 with three or more variants. Sequencing of other CH candidate genes in the 57 patients with DUOX2 mutations further revealed 2 TG variants. All variants were confirmed by Sanger sequencing (Fig. S1 , Fig. S2). The variants included 11 known mutations: c.3329G>A (p.R1110Q), c.1588A>T (p.K530X), c.2635G>A (p.E879K), c.3340delC (p.L1114SfsX56), c.903G>T (p.W301C), c.3413C>A (p.A1138D), c.1736T>C (p.L579P), c.2048G>T (p.R683L), c.2524C>T (p.R842X), c.4027G>T (p.L1343F) in DUOX2
ACCEPTED MANUSCRIPT and c.8119C>T (p.R2707X) in TG, as well as 9 novel variants: two in-frame deletions c.2102-2104delGAG (p.G702del) and c.3478-3480delCTG (p.L1160del), six missense
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variants c.3251G>A (p.R1084Q), c.4000C>T (p.R1334W), c.3967G>A (p.A1323T),
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c.4475G>A (p.R1492H), c.3391G>T (p.A1131S), c.244C>A (p.R82S) in DUOX2 and one nonsense mutation c.5766C>A (p.Y1922X) in TG (Fig. 1). The variants p.K530X, p.R683L, p.L1343F are highly recurrent in our patient cohort: p.K530X occuring in fourteen
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heterozygotes and one homozygote, p.R683L in six heterozygotes and ten homozygotes, and
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p.L1343F in nineteen heterozygotes.
The novel truncating variant p.Y1922X was not detected in 400 control individuals and also
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absent from public population databases such as 1000 Genomes Project. It was classified as
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pathogenic variants (PVS1+PS4) according to our assessment using the ACMG/AMP
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guideline [13].
The other novel variants were not detected in our 400 control individuals. Both the identified
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novel missense variants and in-frame deletions were found to be located in the highly conserved regions of DUOX2 (Fig. 2). SIFT and MutationTaster predicted that the novel missense variants likely had deleterious effects by damaging DUOX2 function. The two novel deletions (c.2102-2104delGAG and c.3478-3480delCTG) both removed 3 nucleotides, resulting in an in-frame deletion of Glycine 702 in the Topological domain and an in-frame deletion of Leucine 1160 in the Transmembrane area, respectively. Those all suggested that the amino acid substitutions/deletions might be pathologic. Because of a lack of data, we were unable to perform mutation segregation with phenotype within the family.
ACCEPTED MANUSCRIPT The clinical features and laboratory test results were summarized in Table S1. All patients were born at full-term to unrelated parents and diagnosed with CH/SCH by NBS. L-T4
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replacement therapy was started immediately after clinical diagnosis and the dose was
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adjusted according to the serum TSH and FT4 levels. After temporary withdrawal of L-T4 therapy at approximately 2 years of age, 35 (83%) of the 42 patients with one or two DUOX2 pathogenic variants turned out to be SCH or transient congenital hypothyroidism (TCH), four
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of them turned out to be permanent congenital hypothyroidism (PCH), and the other three
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patients were currently under two years of age thus still remained to be determined. By contrast, 13 (87%) of the 15 patients with three or more DUOX2 pathogenic variants are
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associated with PCH and the other two of them turned out to be TCH. Most patients with one
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or two DUOX2 pathogenic variants turned out to be SCH or TCH, whereas patients with three
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or more DUOX2 pathogenic variants were mostly associated with PCH. The accumulation of defects in DUOX2 contribute to the more severe condition regarding TSH levels, FT4 levels
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and initial dose of L-T4 (Fig. 3). Of the 57 CH/SCH patients, 37 patients showed thyroid gland of normal size and location, 17 subjects had increased size, ultrasonography showed Patient 7 (right lobe: 1.1× 0.7 × 0.5 cm; left: 1.1 × 0.6 × 0.5 cm), Patent 4 (right lobe: 1.1× 0.7 × 0.5 cm; left: 1.0 × 0.5 × 0.4 cm) and Patient 23 (right lobe: 1.2× 0.8 × 0.7 cm; left: 1.1 × 0.7 × 0.6 cm) with decreased size of thyroid gland which was confirmed by thyroid scintigraphy. Upon the completion of L-T4 replacement therapy, the physical and intellectual development of 55 in 57 patients are in line with their contemporaries, except for patient 3 and 4 who were short stature due to poor compliance to therapy.
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Discussion
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We conducted the largest DUOX2 gene mutation screening so far in CH/SCH patients. The
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result of our study revealed a 29% of DUOX2 mutation rate among 192 patients with
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CH/SCH in Guangxi Zhuang Autonomous Region, China. This rate is the same with our previous study [9] and similar to the published studies in Dutch (44.4%) [14], Japanese (43%) [15] and Italian (45.5%) [16], but higher than that of 14.9% reported in a smaller cohort of 67
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Han Chinese patients with CH and goitre based on Sanger sequencing. The difference may be explained by the target population and methods involved, although some genetic diversity in
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different ethnic groups may also be implicated.
DUOX2 is a common gene responsible for thyroid dyshormonogenesis when mutated, most
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patients with DUOX2 pathogenic variants have a increased or normal size of thyroid gland.
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Of the 57 CH/SCH patients with DUOX2 variants in our study, 37 patients showed normal size and location of the thyroid gland, 17 subjects had increased size. Interestingly, the
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ultrasound showed patients 4, 9 and 23 had a decreased thyroid gland. No mutation was found in the three patients in any other known CH associated genes. The different phenotype provide an excellent model for studying novel candidate genes with modifying effect on
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thyroid gland.
DUOX2 alterations were first described in 2002 [14] and more mutations have since been identified.
However,
the
mutational
spectrum
of
the
DUOX2
gene
and
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phenotype-genotype correlations has not yet been fully established. Most cases of CH associated with alterations in the DUOX2 gene are caused by either biallelic or monoallelic mutations, and both biallelic or monoallelic DUOX2 mutations lead to a wide spectrum of phenotypes, ranging from mild SCH to severe PCH [16-18]. In the present study, we performed the 11 known CH associated genes screening for a large CH/SCH cohort from Guangxi, China. We found most one or two DUOX2 pathogenic variants turned out to be SCH or TCH, whereas patients with three or more DUOX2 pathogenic variants were mostly
ACCEPTED MANUSCRIPT associated with PCH. The results was in accordance with our previous observations [9]. We also analysed and compared the TSH levels, FT4 levels and initial dose of L-T4 between patients with one, two and multiple DUOX2 pathogenic variants and found that the
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accumulation of mutations contribute to the more severe disease regarding above indexes. Our present study identified nine novel pathogenic variants and eleven previously reported
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mutations over 57 different patients. The variants p.K530X, p.R683L and p.L1343F are highly recurrent in our patient cohort, 7.7%, 8.3% and 9.8% of the CH patients in this study
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carried the three variants, respectively. Once again, our study showed the phenotypic heterogeneity of DUOX2. The nine patients (patient 9-17) carrying a same heterozygous
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mutation p.K530X, but the clinical phenotypes of TSH levels and thyroid morphology varied greatly. Moreover, the clinical classifying of PCH or TCH and the thyroid morphology also
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varied in eight patients (patient 46-53) with same genotype p.R683L/p.R683L/p.L1343F. It’s
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presumed the phenotypic variability are caused by (1) environmental factors; (2) individual differences or stochastic phenomena, and (3) other variants of novel genes associated with
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CH.
In summary, we conducted the largest DUOX2 mutation screening and identified eight novel DUOX2 pathogenic variants and one novel TG pathogenic variants in a cohort of 192 patients
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in Guangxi Zhuang Autonomous Region of China. Our study expands the DUOX2/TG mutation spectrum and provides the best estimation of the DUOX2 mutation rate (29%) for Chinese patients in this region of China with CH/SCH. Most one or two DUOX2 pathogenic variants turned out to be SCH or TCH, whereas patients with three or more DUOX2 pathogenic variants were mostly associated with PCH. The coexistence of multiple pathogenic variants may have contributed to the severity of the hypothyroid condition.
ACCEPTED MANUSCRIPT Figure legends Figure 1. Novel variants in DUOX2 and TG in the 57 patients with CH/SCH.
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Figure 2. Multiple sequence alignment of DUOX2 from different species. A) The Arginine
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82 residue is located within a highly conserved region. B) The Glycine 702 residue is located within a highly conserved region. C) The Arginine 1084 residue is located within a highly conserved region. D) The Alanine 1131 residue is located within a highly conserved region. E)
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The Leucine 1160 residue is located within a highly conserved region. F) The Alanine 1323
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residue is located within a highly conserved region. G) The Arginine 1334 residue is located within a highly conserved region. H) The Arginine 1492 residue is located within a highly
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conserved region.
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Figure 3. Analysis and compare of TSH levels, FT4 levels and initial dose of L-T4
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between patients with one, two and multiple DUOX2 pathogenic variants. A) Analysis and compare of TSH levels between patients with one, two and multiple DUOX2 pathogenic
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variants. B) Analysis and compare of FT4 levels between patients with one, two and multiple DUOX2 pathogenic variants. C) Analysis and compare of initial dose of L-T4 between patients with one, two and multiple DUOX2 pathogenic variants.
ACCEPTED MANUSCRIPT Declaration of interest The authors declare that there is no conflict of interest that could be perceived as prejudicing
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the impartiality of the research reported.
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Funding
This work was supported in part by grants from the National Natural Science Foundation of
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China (81260126), Key Projects of Guangxi Health Department (2012025) and Guangxi Natural Science Foundation Program (2012GXNSFAA053174).
Acknowledgments
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We thank all subjects for their collaborative participation in our study. We thank all members
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of the research group for their contributions to this investigation.
ACCEPTED MANUSCRIPT REFERENCES [1] Szinnai G. Clinical genetics of congenital hypothyroidism. Endocr Dev 2014; 26:60-78. [2] Fu C, Chen R, Zhang S, et al. PAX8 pathogenic variants in Chinese patients with congenital hypothyroidism. Clin Chim Acta 2015; 450: 322-326.
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[3] Kibirige MS, Hutchison S, Owen CJ, et al. Prevalence of maternal dietary iodine insufficiency in
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the north east of England: implications for the fetus. Arch Dis Child Fetal Neonatal Ed 2004; 89: F436-439.
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[4] Park SM, Chatterjee VK. Genetics of congenital hypothyroidism. J Med Genet 2005; 42: 379-389. [5] Hermanns P, Grasberger H, Cohen R, et al. Two cases of thyroid dysgenesis caused by different novel PAX8 mutations in the DNA-binding region: in vitro studies reveal different pathogenic mechanisms. Thyroid 2013; 23: 791-796.
[6]Jain V, Agarwal R, Deorari AK, et al. Congenital hypothyroidism. Indian J Pediatr 2008; 75:
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363-367.
[7] Targovnik HM, Esperante SA, Rivolta CM. Genetics and phenomics of hypothyroidism and goiter due to thyroglobulin mutations. Mol Cell Endocrinol 2010; 322: 44-55.
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[8] Varela V, Rivolta CM, Esperante SA, et al. Three mutations (p.Q36H, p.G418fsX482, and g.IVS19-2A>C) in the dual oxidase 2 gene responsible for congenital goiter and iodide organification defect. Clin Chem 2006; 52: 182-191.
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[9] Fu C, Zhang S, Su J, et al. Mutation screening of DUOX2 in Chinese patients with congenital hypothyroidism. J Endocrinol Invest 2015; 38: 1219-1224.
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[10] Cingolani P, Platts A, Wang le L, et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly (Austin) 2012; 6: 80-92.
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[11] Sim NL, Kumar P, Hu J, et al. SIFT web server: predicting effects of amino acid substitutions on proteins. Nucleic Acids Res 2012; 40: W452-457. [12] Schwarz JM, Rodelsperger C, Schuelke M, et al. MutationTaster evaluates disease-causing potential of sequence alterations. Nat Methods 2010; 7: 575-576.
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[13] Richards S, Aziz N, Bale S, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 2015; 17: 405-424. [14] Moreno JC, Bikker H, Kempers MJ, et al. Inactivating mutations in the gene for thyroid oxidase 2 (THOX2) and congenital hypothyroidism. N Engl J Med 2002; 347: 95-102. [15] Narumi S, Muroya K, Asakura Y, et al. Molecular basis of thyroid dyshormonogenesis: genetic screening in population-based Japanese patients. J Clin Endocrinol Metab 2011; 96: E1838-1842. [16] De Marco G, Agretti P, Montanelli L, et al. Identification and functional analysis of novel dual oxidase 2 (DUOX2) mutations in children with congenital or subclinical hypothyroidism. J Clin Endocrinol Metab 2011; 96: E1335-1339. [17] Wang F, Lu K, Yang Z, et al. Genotypes and phenotypes of congenital goitre and hypothyroidism caused by mutations in dual oxidase 2 genes. Clin Endocrinol (Oxf) 2014; 81: 452-457. [18] Moreno JC, Visser TJ. New phenotypes in thyroid dyshormonogenesis: hypothyroidism due to DUOX2 mutations. Endocr Dev 2007; 10: 99-117.
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ACCEPTED MANUSCRIPT Highlights We provided the best estimation of the DUOX2 mutation rate (29%) for CH/SCH patients in Guangxi Zhuang Autonomous Region of China. Most one or two DUOX2 pathogenic variants turned out to be SCH or TCH. Patients with three or more DUOX2 pathogenic variants were mostly associated with PCH.
The coexistence of multiple pathogenic variants may have contributed to the severity of the hypothyroid condition.
We reported 8 novel variants in DUOX2 and one novel variant in TG.
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S0009-8981(16)30144-9 doi: 10.1016/j.cca.2016.04.019 CCA 14339
To appear in:
Clinica Chimica Acta
Received date: Revised date: Accepted date:
25 February 2016 17 April 2016 17 April 2016
Please cite this article as: Fu Chunyun, Luo Shiyu, Zhang Shujie, Wang Jin, Zheng Haiyang, Yang Qi, Xie Bobo, Hu Xuyun, Fan Xin, Luo Jingsi, Chen Rongyu, Su Jiasun, Shen Yiping, Gu Xuefan, Chen Shaoke, Next-generation sequencing analysis of DUOX2 in 192 Chinese subclinical congenital hypothyroidism (SCH) and CH patients, Clinica Chimica Acta (2016), doi: 10.1016/j.cca.2016.04.019
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Next-generation sequencing analysis of DUOX2 in 192 Chinese Subclinical Congenital Hypothyroidism (CH) and CH patients
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Chunyun Fu1,2, Shiyu Luo1,2, Shujie Zhang1,2, Jin Wang1,2, Haiyang Zheng1,2, Qi Yang1,2, Bobo
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Xie1,2, Xuyun Hu1,2, Xin Fan1,2, Jingsi Luo1,2, Rongyu Chen1,2, Jiasun Su1,2, Yiping Shen1,3, Xuefan Gu4, Shaoke Chen1,2 1
Department of Genetic Metabolism, Children's Hospital, Maternal and Child Health Hospital
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of Guangxi Zhuang Autonomous Region, Nanning 530003, People’s Republic of China 2
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GuangXi Center for Birth Defects Research and Prevention, Nanning 530003, People’s
Republic of China
Boston Children’s Hospital, Harvard Medical School, Boston 02115, MA
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4
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Endocrinology and Genetic Metabolism of Institute for Pediatric Research, Xinhua Hospital
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Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai 200092,China Chunyun Fu and Shiyu Luo contribute equally to this work.
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Address correspondences to: Shaoke Chen, E-mail: [email protected] Xuefan Gu, E-mail: [email protected]
ACCEPTED MANUSCRIPT ABSTRACT Background: Defects in the human dual oxidase 2 (DUOX2) gene are reported to be one of
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the major causes of congenital hypothyroidism (CH). This study was set to examine the
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DUOX2 mutation spectrum and prevalence among Chinese CH and subclinical congenital hypothyroidism (SCH) patients and to define the relationships between DUOX2 genotypes and clinical phenotypes.
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Methods: Peripheral venous blood samples were collected from 192 CH/SCH patients in
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Guangxi Zhuang Autonomous Region of China. All exons and their exon-intron boundary sequences of the 11 known CH associated genes including DUOX2 were screened by
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next-generation sequencing (NGS).
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Results: NGS analysis of DUOX2 revealed 18 rare non-polymorphic variants in 57 CH/SCH
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patients. Sequencing of other CH candidate genes in the 57 patients revealed 2 thyroglobulin (TG) variants. All variants included 11 known mutations, 8 novel variants in DUOX2 and one
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novel variant in TG, among which three variants p.K530X, p.L1343F and p.R683L are highly recurrent in our patient cohort. 35 (83%) of the 42 patients with one or two DUOX2 pathogenic variants turned out to be SCH or transient congenital hypothyroidism (TCH), whereas 13 (87%) of the 15 patients with three or more DUOX2 pathogenic variants are associated with permanent congenital hypothyroidism (PCH). The accumulation of defects in DUOX2 contribute to the more severe disease regarding thyroid stimulating hormone (TSH) levels, free thyroxine (FT4) levels and initial dose of L-thyroxine (L-T4). Conclusion: Our study expanded the mutational spectrum of the DUOX2 and TG genes and provided the best estimation of the DUOX2 mutation rate (29%) for CH/SCH patients in
ACCEPTED MANUSCRIPT Guangxi Zhuang Autonomous Region of China. Most one or two DUOX2 pathogenic variants turned out to be SCH or TCH, whereas patients with three or more DUOX2 pathogenic
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variants were mostly associated with PCH. The coexistence of multiple pathogenic variants
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may have contributed to the severity of the hypothyroid condition.
Keywords: subclinical congenital hypothyroidism; Dual oxidase 2 gene (DUOX2); gene
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mutations; next-generation sequencing; Chinese population
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Introduction
Congenital hypothyroidism (CH) is the most common neonatal endocrine disorder in infancy
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with prevalence ranging from 1:2000 to 1:4000 [1,2]. It’s reported that the most prevalent
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cause of CH worldwide is still iodine deficiency [3], however, considerable progress has been
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made to identify the genetic causes in CH patients. Based on genetic alterations, CH can be classified into two groups: 80%-85% of cases are caused by disorders of thyroid gland
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development (thyroid dysgenesis, comprising agenesis, ectopy, or hypoplasia) [4], which has been linked to gene mutations in thyroid-stimulating hormone receptor (TSHR), paired box gene 8 (PAX8), thyroid transcription factor 1 (TTF1/NKX2.1), thyroid transcription factor 2 (TTF2/FOXE1) and NK2 transcription factor related locus 5 (NKX2.5) [5]; the remaining 15%-20% of CH cases are caused by abnormalities in thyroid hormone synthesis (thyroid dyshormonogenesis), which is associated with the presence of goiter or with a eutopic gland of normal size [6]. Molecular studies have found that dyshormogenesis in CH is caused by defects in genes such as dual oxidase 2 (DUOX2), thyroglobulin (TG), thyroid peroxidase
ACCEPTED MANUSCRIPT (TPO), Pendrine (SLC26A4), dehalogenase 1 (DEHAL1) and sodium iodide symporter (NIS) [7].
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Defects in the human DUOX2 gene are reported to be one of the major causes of CH. The
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DUOX2 gene is located on chromosome 15 and consists of 34 exons [8]. The DUOX2 protein is a 1548-amino-acid polypeptide located at the apical membrane of thyrocytes and generates the hydrogen peroxide (H2O2) needed by thyroid peroxidase for the incorporation of iodine
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into thyroglobulin, an essential step in thyroid hormone synthesis. Up to date, the mutational
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spectrum of the DUOX2 gene and the phenotype-genotype correlations have not yet fully been established. Both biallelic and monoallelic DUOX2 mutations lead to a wide spectrum of
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phenotypes, ranging from mild subclinical congenital hypothyroidism (SCH) with elevated
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thyroid stimulating hormone (TSH) levels but normal thyroid hormone levels, to overt CH
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with thyroid dyshormonogenesis. Also, little is known about DUOX2 mutation spectrum and its prevalence among Chinese CH/SCH patients. In our recent study, we identified DUOX2
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pathogenic variants in 13 of 45 cases (29%) and found that most monoallelic or biallelic DUOX2 pathogenic variants turned out to be TCH, while patients with triallelic DUOX2 pathogenic variants were associated with permanent congenital hypothyroidism (PCH) [9]. The aim of this study was to screen for the presence of mutations in DUOX2 gene among patients with CH/SCH in China, to define the relationships between DUOX2 genotypes and clinical phenotypes and to confirm our previous findings based on a larger sample size and comprehensive analysis of all known CH associated genes.
Methods
ACCEPTED MANUSCRIPT Patients A total of 192 newborns were enrolled in this study ( the previous 45 CH patients collected
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for DUOX2 mutation screening by Sanger sequencing were not included in this study [9]),
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including 140 patients with CH and 52 patients with SCH, who were identified through a large-scale newborn screening (NBS) covering 615,000 cases in Guangxi, China, from July 2009 to June 2013. CH NBS were done with filter paper between 72h and 7 days after birth.
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Blood samples were collected from the heel and the TSH level were measured by
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time-resolved fluorescence assay (Perkin Elmer, USA). Subjects with increased TSH levels (TSH ≥ 8 mIU/l) during NBS were followed-up for further evaluation. Serum TSH and free
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thyroxine (FT4) were determined by electrochemiluminescence assay (Cobas e601, Roche
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Diagnostics, USA). Diagnosis of CH was based on elevated TSH levels (TSH≥10 mIU/l) and
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decreased FT4 levels (FT4 < 12 pmol/l). Patients with elevated TSH levels and normal FT4 levels (normal range 12-22 pmol/l) were diagnosed as SCH. Permanent or transient CH was
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determined using results of thyroid function tests after temporary withdrawal of L-thyroxine (L-T4) therapy at approximately 2 years of age. Thyroid ultrasonography and
99m
Tc
scintigraphy were performed during the neonatal period before treatment. This study was approved by the local Medical Ethics Committee. Informed consent was obtained from the parents of the patients. Next generation sequencing and bioinformatics analysis Peripheral venous blood samples were collected from the patients. Genomic DNA was extracted from peripheral blood leukocytes using QIAamp DNA Blood Mini Kit (Qiagen, Germany) according to the manufacturer’s protocol. CH capture panel as Illumina Truseq
ACCEPTED MANUSCRIPT Custom Amplicon v1.5 kit was designed and 11 known CH associated genes (DUOX2, TSHR, PAX8, NKX2.1, NKX2.5, FOXE1,TG, TPO, NIS, SLC26A4 and DEHAL1) were included with
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the whole coding regions and flanking intronic sequences. The prepared sample library was
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sequenced by Illumina MiSeq instrument using MiSeq Reagent Kit v2, 500-cycles (Illumina Inc., San Diego, CA). Illumina Amplicon Viewer v1.3 and MiSeq Reporter v2.3 software were used for data analysis, and the SnpEff [10] was used for variant annotation. SIFT [11]
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and MutationTaster [12] were used to evaluate the pathogenicity of the novel variants.
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Multiple sequence alignment of the DUOX2 family protein among different species were carried out by DNAMAN software version 8 to analyze the amino acid conservation of the
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mutated sites. All variants were further validated by Sanger sequencing. In addition, a cohort
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of 400 ethnicity-matched healthy subjects with normal FT4 and TSH levels were sequenced
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Results
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to assess the variant frequencies in normal control population.
Next generation sequencing analysis of DUOX2 revealed 18 different variants in 57 individuals (47 CH and 10 SCH patients) including 31 with single heterozygous variation, 11 with two variants and 15 with three or more variants. Sequencing of other CH candidate genes in the 57 patients with DUOX2 mutations further revealed 2 TG variants. All variants were confirmed by Sanger sequencing (Fig. S1 , Fig. S2). The variants included 11 known mutations: c.3329G>A (p.R1110Q), c.1588A>T (p.K530X), c.2635G>A (p.E879K), c.3340delC (p.L1114SfsX56), c.903G>T (p.W301C), c.3413C>A (p.A1138D), c.1736T>C (p.L579P), c.2048G>T (p.R683L), c.2524C>T (p.R842X), c.4027G>T (p.L1343F) in DUOX2
ACCEPTED MANUSCRIPT and c.8119C>T (p.R2707X) in TG, as well as 9 novel variants: two in-frame deletions c.2102-2104delGAG (p.G702del) and c.3478-3480delCTG (p.L1160del), six missense
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variants c.3251G>A (p.R1084Q), c.4000C>T (p.R1334W), c.3967G>A (p.A1323T),
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c.4475G>A (p.R1492H), c.3391G>T (p.A1131S), c.244C>A (p.R82S) in DUOX2 and one nonsense mutation c.5766C>A (p.Y1922X) in TG (Fig. 1). The variants p.K530X, p.R683L, p.L1343F are highly recurrent in our patient cohort: p.K530X occuring in fourteen
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heterozygotes and one homozygote, p.R683L in six heterozygotes and ten homozygotes, and
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p.L1343F in nineteen heterozygotes.
The novel truncating variant p.Y1922X was not detected in 400 control individuals and also
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absent from public population databases such as 1000 Genomes Project. It was classified as
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pathogenic variants (PVS1+PS4) according to our assessment using the ACMG/AMP
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guideline [13].
The other novel variants were not detected in our 400 control individuals. Both the identified
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novel missense variants and in-frame deletions were found to be located in the highly conserved regions of DUOX2 (Fig. 2). SIFT and MutationTaster predicted that the novel missense variants likely had deleterious effects by damaging DUOX2 function. The two novel deletions (c.2102-2104delGAG and c.3478-3480delCTG) both removed 3 nucleotides, resulting in an in-frame deletion of Glycine 702 in the Topological domain and an in-frame deletion of Leucine 1160 in the Transmembrane area, respectively. Those all suggested that the amino acid substitutions/deletions might be pathologic. Because of a lack of data, we were unable to perform mutation segregation with phenotype within the family.
ACCEPTED MANUSCRIPT The clinical features and laboratory test results were summarized in Table S1. All patients were born at full-term to unrelated parents and diagnosed with CH/SCH by NBS. L-T4
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replacement therapy was started immediately after clinical diagnosis and the dose was
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adjusted according to the serum TSH and FT4 levels. After temporary withdrawal of L-T4 therapy at approximately 2 years of age, 35 (83%) of the 42 patients with one or two DUOX2 pathogenic variants turned out to be SCH or transient congenital hypothyroidism (TCH), four
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of them turned out to be permanent congenital hypothyroidism (PCH), and the other three
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patients were currently under two years of age thus still remained to be determined. By contrast, 13 (87%) of the 15 patients with three or more DUOX2 pathogenic variants are
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associated with PCH and the other two of them turned out to be TCH. Most patients with one
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or two DUOX2 pathogenic variants turned out to be SCH or TCH, whereas patients with three
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or more DUOX2 pathogenic variants were mostly associated with PCH. The accumulation of defects in DUOX2 contribute to the more severe condition regarding TSH levels, FT4 levels
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and initial dose of L-T4 (Fig. 3). Of the 57 CH/SCH patients, 37 patients showed thyroid gland of normal size and location, 17 subjects had increased size, ultrasonography showed Patient 7 (right lobe: 1.1× 0.7 × 0.5 cm; left: 1.1 × 0.6 × 0.5 cm), Patent 4 (right lobe: 1.1× 0.7 × 0.5 cm; left: 1.0 × 0.5 × 0.4 cm) and Patient 23 (right lobe: 1.2× 0.8 × 0.7 cm; left: 1.1 × 0.7 × 0.6 cm) with decreased size of thyroid gland which was confirmed by thyroid scintigraphy. Upon the completion of L-T4 replacement therapy, the physical and intellectual development of 55 in 57 patients are in line with their contemporaries, except for patient 3 and 4 who were short stature due to poor compliance to therapy.
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Discussion
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We conducted the largest DUOX2 gene mutation screening so far in CH/SCH patients. The
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result of our study revealed a 29% of DUOX2 mutation rate among 192 patients with
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CH/SCH in Guangxi Zhuang Autonomous Region, China. This rate is the same with our previous study [9] and similar to the published studies in Dutch (44.4%) [14], Japanese (43%) [15] and Italian (45.5%) [16], but higher than that of 14.9% reported in a smaller cohort of 67
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Han Chinese patients with CH and goitre based on Sanger sequencing. The difference may be explained by the target population and methods involved, although some genetic diversity in
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different ethnic groups may also be implicated.
DUOX2 is a common gene responsible for thyroid dyshormonogenesis when mutated, most
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patients with DUOX2 pathogenic variants have a increased or normal size of thyroid gland.
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Of the 57 CH/SCH patients with DUOX2 variants in our study, 37 patients showed normal size and location of the thyroid gland, 17 subjects had increased size. Interestingly, the
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ultrasound showed patients 4, 9 and 23 had a decreased thyroid gland. No mutation was found in the three patients in any other known CH associated genes. The different phenotype provide an excellent model for studying novel candidate genes with modifying effect on
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thyroid gland.
DUOX2 alterations were first described in 2002 [14] and more mutations have since been identified.
However,
the
mutational
spectrum
of
the
DUOX2
gene
and
the
phenotype-genotype correlations has not yet been fully established. Most cases of CH associated with alterations in the DUOX2 gene are caused by either biallelic or monoallelic mutations, and both biallelic or monoallelic DUOX2 mutations lead to a wide spectrum of phenotypes, ranging from mild SCH to severe PCH [16-18]. In the present study, we performed the 11 known CH associated genes screening for a large CH/SCH cohort from Guangxi, China. We found most one or two DUOX2 pathogenic variants turned out to be SCH or TCH, whereas patients with three or more DUOX2 pathogenic variants were mostly
ACCEPTED MANUSCRIPT associated with PCH. The results was in accordance with our previous observations [9]. We also analysed and compared the TSH levels, FT4 levels and initial dose of L-T4 between patients with one, two and multiple DUOX2 pathogenic variants and found that the
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accumulation of mutations contribute to the more severe disease regarding above indexes. Our present study identified nine novel pathogenic variants and eleven previously reported
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mutations over 57 different patients. The variants p.K530X, p.R683L and p.L1343F are highly recurrent in our patient cohort, 7.7%, 8.3% and 9.8% of the CH patients in this study
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carried the three variants, respectively. Once again, our study showed the phenotypic heterogeneity of DUOX2. The nine patients (patient 9-17) carrying a same heterozygous
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mutation p.K530X, but the clinical phenotypes of TSH levels and thyroid morphology varied greatly. Moreover, the clinical classifying of PCH or TCH and the thyroid morphology also
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varied in eight patients (patient 46-53) with same genotype p.R683L/p.R683L/p.L1343F. It’s
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presumed the phenotypic variability are caused by (1) environmental factors; (2) individual differences or stochastic phenomena, and (3) other variants of novel genes associated with
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CH.
In summary, we conducted the largest DUOX2 mutation screening and identified eight novel DUOX2 pathogenic variants and one novel TG pathogenic variants in a cohort of 192 patients
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in Guangxi Zhuang Autonomous Region of China. Our study expands the DUOX2/TG mutation spectrum and provides the best estimation of the DUOX2 mutation rate (29%) for Chinese patients in this region of China with CH/SCH. Most one or two DUOX2 pathogenic variants turned out to be SCH or TCH, whereas patients with three or more DUOX2 pathogenic variants were mostly associated with PCH. The coexistence of multiple pathogenic variants may have contributed to the severity of the hypothyroid condition.
ACCEPTED MANUSCRIPT Figure legends Figure 1. Novel variants in DUOX2 and TG in the 57 patients with CH/SCH.
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Figure 2. Multiple sequence alignment of DUOX2 from different species. A) The Arginine
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82 residue is located within a highly conserved region. B) The Glycine 702 residue is located within a highly conserved region. C) The Arginine 1084 residue is located within a highly conserved region. D) The Alanine 1131 residue is located within a highly conserved region. E)
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The Leucine 1160 residue is located within a highly conserved region. F) The Alanine 1323
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residue is located within a highly conserved region. G) The Arginine 1334 residue is located within a highly conserved region. H) The Arginine 1492 residue is located within a highly
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conserved region.
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Figure 3. Analysis and compare of TSH levels, FT4 levels and initial dose of L-T4
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between patients with one, two and multiple DUOX2 pathogenic variants. A) Analysis and compare of TSH levels between patients with one, two and multiple DUOX2 pathogenic
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variants. B) Analysis and compare of FT4 levels between patients with one, two and multiple DUOX2 pathogenic variants. C) Analysis and compare of initial dose of L-T4 between patients with one, two and multiple DUOX2 pathogenic variants.
ACCEPTED MANUSCRIPT Declaration of interest The authors declare that there is no conflict of interest that could be perceived as prejudicing
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the impartiality of the research reported.
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Funding
This work was supported in part by grants from the National Natural Science Foundation of
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China (81260126), Key Projects of Guangxi Health Department (2012025) and Guangxi Natural Science Foundation Program (2012GXNSFAA053174).
Acknowledgments
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We thank all subjects for their collaborative participation in our study. We thank all members
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of the research group for their contributions to this investigation.
ACCEPTED MANUSCRIPT REFERENCES [1] Szinnai G. Clinical genetics of congenital hypothyroidism. Endocr Dev 2014; 26:60-78. [2] Fu C, Chen R, Zhang S, et al. PAX8 pathogenic variants in Chinese patients with congenital hypothyroidism. Clin Chim Acta 2015; 450: 322-326.
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[3] Kibirige MS, Hutchison S, Owen CJ, et al. Prevalence of maternal dietary iodine insufficiency in
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[4] Park SM, Chatterjee VK. Genetics of congenital hypothyroidism. J Med Genet 2005; 42: 379-389. [5] Hermanns P, Grasberger H, Cohen R, et al. Two cases of thyroid dysgenesis caused by different novel PAX8 mutations in the DNA-binding region: in vitro studies reveal different pathogenic mechanisms. Thyroid 2013; 23: 791-796.
[6]Jain V, Agarwal R, Deorari AK, et al. Congenital hypothyroidism. Indian J Pediatr 2008; 75:
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[7] Targovnik HM, Esperante SA, Rivolta CM. Genetics and phenomics of hypothyroidism and goiter due to thyroglobulin mutations. Mol Cell Endocrinol 2010; 322: 44-55.
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[8] Varela V, Rivolta CM, Esperante SA, et al. Three mutations (p.Q36H, p.G418fsX482, and g.IVS19-2A>C) in the dual oxidase 2 gene responsible for congenital goiter and iodide organification defect. Clin Chem 2006; 52: 182-191.
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[9] Fu C, Zhang S, Su J, et al. Mutation screening of DUOX2 in Chinese patients with congenital hypothyroidism. J Endocrinol Invest 2015; 38: 1219-1224.
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[11] Sim NL, Kumar P, Hu J, et al. SIFT web server: predicting effects of amino acid substitutions on proteins. Nucleic Acids Res 2012; 40: W452-457. [12] Schwarz JM, Rodelsperger C, Schuelke M, et al. MutationTaster evaluates disease-causing potential of sequence alterations. Nat Methods 2010; 7: 575-576.
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[13] Richards S, Aziz N, Bale S, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 2015; 17: 405-424. [14] Moreno JC, Bikker H, Kempers MJ, et al. Inactivating mutations in the gene for thyroid oxidase 2 (THOX2) and congenital hypothyroidism. N Engl J Med 2002; 347: 95-102. [15] Narumi S, Muroya K, Asakura Y, et al. Molecular basis of thyroid dyshormonogenesis: genetic screening in population-based Japanese patients. J Clin Endocrinol Metab 2011; 96: E1838-1842. [16] De Marco G, Agretti P, Montanelli L, et al. Identification and functional analysis of novel dual oxidase 2 (DUOX2) mutations in children with congenital or subclinical hypothyroidism. J Clin Endocrinol Metab 2011; 96: E1335-1339. [17] Wang F, Lu K, Yang Z, et al. Genotypes and phenotypes of congenital goitre and hypothyroidism caused by mutations in dual oxidase 2 genes. Clin Endocrinol (Oxf) 2014; 81: 452-457. [18] Moreno JC, Visser TJ. New phenotypes in thyroid dyshormonogenesis: hypothyroidism due to DUOX2 mutations. Endocr Dev 2007; 10: 99-117.
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Figure 1
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Figure 2
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Figure 3
ACCEPTED MANUSCRIPT Highlights We provided the best estimation of the DUOX2 mutation rate (29%) for CH/SCH patients in Guangxi Zhuang Autonomous Region of China. Most one or two DUOX2 pathogenic variants turned out to be SCH or TCH. Patients with three or more DUOX2 pathogenic variants were mostly associated with PCH.
The coexistence of multiple pathogenic variants may have contributed to the severity of the hypothyroid condition.
We reported 8 novel variants in DUOX2 and one novel variant in TG.
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