Targeted next-generation sequencing of thirteen causative genes in Chinese patients with congenital hypothyroidism

Abstract

To identify the spectrum and prevalence of thirteen causative genes mutations in congenital hypothyroidism (CH) patients, we collected blood samples and extracted genomic DNA of 106 CH patients, and designed a customized targeted next-generation sequencing panel containing 13 CH-causing genes to detect mutations. A total of 132 mutations were identified in 65.09% of patients (69/106) on the following nine genes: DUOX2, TG, TPO, TSHR, TTF1, TTF2, NKX2-5, PAX8 and GNAS. 69.70% (92/132) mutations related to thyroid dyshormonogenesis genes, including DUOX2 (n = 49), TG (n = 35), and TPO (n = 8). 21.21% (28/132) mutations related to thyroid dysgenesis genes, including TSHR (n = 19), TTF1 (n = 5), TTF2 (n = 1), PAX8 (n = 2), and NKX2-5 (n = 1). 9.09% (12/132) mutations related to GNAS, which was associated with thyrotropin resistance. No mutation of THRA, TSHB, IYD or SLC5A5 was detected. Among 69 mutations detected patients, 41 (59.42%) patients were two or more mutations detected, and mutations of 30 (43.48%) patients related to two or three genes. According to the pathomechanism of the mutant genes, 57.97% CH patients were classified as thyroid dyshormonogenesis. Overall, DUOX2, TG and TSHR mutations were the most common genetic defects in Chinese CH patients, and thyroid dyshormonogenesis could be the first genetic etiology of CH in Chinese. Besides, multiple mutations accounts for a part of genetic pathogenesis.

THE CONDITION of thyroid hormone deficiency present at birth was defined as congenital hypothyroidism (CH), which is the most common neonatal endocrine disorder with an incidence of 1:2,000 to 1:4,000 live births [1]. Nowadays, methods of diagnosis and treatment for CH are well-developed, newborn screening tests allows for early diagnosis of CH, and L-T4 is widely used in the treatment of CH. However, the pathogenic mechanism of CH remains unclear.

Iodine deficiency is the most common cause of CH worldwide [2], however, genetic mutation have been considered as one cause of CH in numerous researches [3, 4], more than six hundred genomic variations have been recorded in the ClinVar database. Previous studies showed that 80%–85% of CH patients were caused by thyroid dysgenesis (including agenesis, ectopy and hypoplasia), which were related 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). However, only 10–15% of cases were caused by thyroid dyshormonogenesis, which were associated with mutations in thyroid oxidase 2 (DUOX2), dual-oxidase maturation factor 2 (DUOXA2), thyroglobulin (TG), thyroid peroxidase (TPO), solute carrier family 5 member 5 (SLC5A5), solute carrier family 26 member 4 (SLC26A4) and iodotyrosine deiodinase (IYD) genes [4]. Few studies on genetic causes of CH were available in Chinese population, more researches need to be conducted to clarify the causes and etiology of CH.

In this study, we performed mutations screening of CH-causing genes in the 106 CH Chinese by targeted next-generation sequencing (NGS). The current study aims to screen and characterize the mutations of thirteen causative genes, including DUOX2, TG, TPO, TSHR, TTF1, TTF2, PAX8, NKX2-5, GNAS, THRA, TSHB, IYD and SLC5A5.

Methods

Patients

Approval of this study was granted by the Ethics Committee of Changzhou Maternity and Child Health Care Hospital affiliated to Nanjing Medical University. One hundred and six non-consanguineous patients were recruited in ChangZhou City from January 2012 to September 2017 by newborn screening (NBS), patients with other congenital diseases were excluded. NBS of CH were done with filter paper between 72 h and 7 days after birth. Blood samples were collected from the heel and the TSH levels were measured by time-resolved fluorescence assay (Auto DELFIA 1235, Perkin Elmer). Subjects with increased TSH levels (TSH ≥9 mIU/L) were recalled for further evaluation. Serum TSH and free thyroxine (FT4) were determined by electrochemiluminescence assay (Cobas e601, Roche Diagnostics). Diagnosis of CH was based on elevated TSH levels (TSH ≥10 mIU/L) and decreased FT4 levels (FT4 <7.77 pmol/L). Blood samples for sequencing were collected from the heel or vein.

Targeted next generation sequencing

Genomic DNA was extracted from peripheral blood leukocytes using the QIAamp DNA blood kit according to the manufacturer’s protocol. A total of 10 ng DNA per sample was used for sequencing using the CH capture panel, which was designed base on the Illumina Truseq Custom Amplicon v1.5 kit. Thirteen pathogenic genes were screened in all patients, including the entire coding regions and exon-intron boundaries. The genetic fragments were between 250–280 bp and were prepared using the Covaris and Agencourt AMPure XP kits, which include purified and captured gene fragments. Adaptor-ligated amplicons were prepared using the Illumina Paired-End Sample Preparation kit. Illumina multi-PE-adaptors were bound to terminal genes and target enrichment was performed by multiplex PCR. After 12 PCR cycles, amplicons were purified using Agencourt AMpurc SPRI XP beads and captured on an Illumina MiSeq 2000 instrument.

Mutations analysis

Illumina Amplicon Viewer v1.3 and MiSeq Reporter v2.3 software were used for data analysis, and the SnpEff was used for mutation annotation. We also used automatic tools (including SIFT, polyphen-2 and MutationTaster) to predict the impact of mutations on the function and structure of their respective proteins, synonymous mutations were not evaluated.

Results

A total of 132 potentially functional mutations were detected after removing polymorphic or low-impact mutations in 69 patients, whereas 37 patients showed no potentially functional mutations, the overview of mutations was showed in Fig. 1. Seventeen mutations were homozygous, including ten DUOX2 mutations, three TG mutations, three TSHR mutations and one TPO mutation, other mutations were heterozygous. The distribution of 132 mutations in 69 patients was showed in Fig. 2. 69.70% (92/132) mutations related to thyroid dyshormonogenesis genes, including DUOX2 (n = 49), TG (n = 35), and TPO (n = 8). 21.21% (28/132) mutations related to thyroid dysgenesis genes, including TSHR (n = 19), TTF1 (n = 5), TTF2 (n = 1), PAX8 (n = 2), and NKX2-5 (n = 1). 9.09% (12/132) mutations related to GNAS, which was associated with thyrotropin resistance. No mutation of THRA, TSHB, IYD or SLC5A5 was detected.

Fig. 1

Overview of mutations in 69 patients with congenital hypothyroidism

Blue blocks represent homozygous mutations, green blocks represent heterozygous mutation, and purple blocks represent compound mutations (containing compound homozygous mutations and heterozygous mutations).

Fig. 2

Distribution of 132 mutations on nine causative genes

After data preparation, 83 different mutations were identified in 69 CH patients. Mutations related to thyroid dyshormonogenesis genes were showed in Table 1. Twenty-four different DUOX2 mutations were detected, including twenty reported mutations and four novel mutations (c.3721A>T, c.3321delC, c.1007_1009del and c.1300_1320del). Twenty TG mutations included nine reported mutations, six novel stopgain mutations (c.3457A>T, c.3538C>T, c.7799G>A, c.6185G>A, c.976C>T and c.3994C>T), two novel missense mutations (c.5020C>A and c.2593C>A), and three novel frameshift mutations (c.1000delG, c.5018_5019insAAATA and c.1712delT). One novel missense mutation (c.2080T>C) and six reported mutations were detected in TPO gene, and most of these mutations were missense.

Table 1 Overview of the mutations relevant to thyroid dyshormonogenesis

Gene	Exon position	Nucleotide position	Amino acid position	RS ID	n.
DUOX2 (NM_014080)
	exon14	c.1588A>T	p.K530X	rs180671269	6
	exon30	c.4027C>T	p.L1343F	rs147945181	5
	exon25	c.3329G>A	p.R1110Q	rs368488511	5
	exon28	c.3632G>A	p.R1211H	rs141763307	4
	exon4	c.227C>T	p.P76L	rs767705906	3
	exon19	c.2335G>A	p.V779M	rs145061993	2
	exon20	c.2654G>A	p.R885Q	rs181461079	2
	exon17	c.2048G>T	p.R683L	rs8028305	2
	exon22	c.2894C>T	p.S965L	rs144153950	1
	exon25	c.3391G>T	p.A1131S	rs147540920	1
	exon12	c.1265G>A	p.R422H	rs201135069	1
	exon33	c.4405G>A	p.E1469K	rs376623263	1
	exon19	c.2413G>A	p.E805K	rs756995727	1
	exon15	c.1717C>T	p.Q573X	rs769605933	1
	exon18	c.2202G>A	p.W734X	rs769789467	1
	exon16	c.1873C>T	p.R625X	rs770083296	1
	exon29	c.3721A>T	p.I1241F*	—	1
	exon16	c.1883delA	p.K628fs	rs200592893	1
	exon17	c.2104_2106del	p.702_702del	rs779340990	1
	exon26	c.3478_3480del	p.1160_1160del	rs758318135	3
	exon6	c.605_621del	p.Q202fs	rs769318570	3
	exon25	c.3321delC	p.T1107fs*	—	1
	exon12	c.1300_1320del	p.434_440del*	—	1
	exon9	c.1007_1009del	p.336_337del*	—	1
TG (NM_003235)
	exon45	c.7847A>T	p.N2616I	rs10091530	11
	exon42	c.7364G>A	p.R2455H	rs2272707	4
	exon12	c.3040G>C	p.D1014H	rs114772213	2
	exon34	c.6185G>A	p.W2062X*	—	2
	exon9	c.1900G>A	p.G634R	rs116417639	1
	exon41	c.7123G>A	p.G2375R	rs137854434	1
	exon4	c.301C>G	p.Q101E	rs531772882	1
	exon7	c.866C>A	p.S289X	rs746023979	1
	exon47	c.8137G>A	p.A2713T	rs749517425	1
	exon28	c.5447A>G	p.Q1816R	rs761047115	1
	exon8	c.976C>T	p.Q326X*	—	1
	exon10	c.2593C>A	p.P865T*	—	1
	exon16	c.3457A>T	p.K1153X*	—	1
	exon16	c.3538C>T	p.Q1180X*	—	1
	exon18	c.3994C>T	p.Q1332X*	—	1
	exon25	c.5020C>A	p.P1674T*	—	1
	exon45	c.7799G>A	p.W2600X*	—	1
	exon8	c.1000delG	p.G334fs*	—	1
	exon9	c.1712delT	p.L571fs*	—	1
	exon25	c.5018_5019insAAATA	p.S1673fs*	—	1
TPO
NM_000547	exon8	c.1082G>T	p.R361L	rs201781919	2
NM_175722	exon13	c.2080T>C	p.S694P*	—	1
NM_175722	exon11	c.1786C>T	p.R596W	rs114406277	1
NM_175722	exon5	c.502G>A	p.A168T	rs150812908	1
NM_175722	exon11	c.1723G>A	p.V575M	rs28991292	1
NM_175722	exon14	c.2146G>A	p.G716R	rs546683738	1
NM_000547	exon14	c.2422delT	p.C808fs	rs763662774	1

*: Novel mutations.

Mutations related to thyroid dysgenesis genes were showed in Table 2. Fifteen missense TSHR mutations were detected, including eleven reported mutations and four novel mutations (c.700T>C, c.501C>G, c.152C>A and c.1384T>C). Four missense TTF1 mutations were detected, including one reported mutations and three novel mutations (c.269G>A, c.1598C>T, and c.515A>G). Two novel mutations (c.398G>A and c.275T>C) were detected in PAX8 gene. One reported missense TTF2 mutation (c.2423G>C) and one novel missense NKX2-5 mutation (c.416G>A) were detected. Nine GNAS mutations related to thyrotropin resistance were detected, including five reported mutations and four novel mutations (c.700T>C, c.501C>G, c.152C>A, and c.1384T>C), see Table 3.

Table 2 Overview of the mutations relevant to thyroid dysgenesis

Gene	Exon position	Nucleotide position	Amino acid position	RS ID	n.
TSHR (NM_000369)
	exon10	c.1349G>A	p.R450H	rs189261858	3
	exon10	c.1574T>C	p.F525S	rs200138601	2
	exon5	c.394G>C	p.G132R	rs760874290	2
	exon10	c.1591C>T	p.R531W	rs139892516	1
	exon10	c.915T>A	p.S305R	rs142122217	1
	exon9	c.823G>A	p.A275T	rs180762551	1
	exon9	c.733G>A	p.G245S	rs189506473	1
	exon10	c.1222T>C	p.C408R	rs199702292	1
	exon10	c.2098A>G	p.K700E	rs201306565	1
	exon10	c.1838A>G	p.Y613C	rs540799629	1
	exon10	c.1270G>T	p.V424F	rs587778742	1
	exon1	c.152C>A	p.P51Q*	—	1
	exon6	c.501C>G	p.I167M*	—	1
	exon9	c.700T>C	p.S234P*	—	1
	exon10	c.1384T>C	p.C462R*	—	1
TTF1 (NM_007344)
	exon4	c.1598C>T	p.A533V*	—	1
	exon2	c.515A>G	p.Q172R*	—	1
	exon2	c.269G>A	p.R90K*	—	2
	exon2	c.1348C>T	p.H450Y	rs765553668	1
TTF2 (NM_003594)
	exon14	c.2423G>C	p.R808P	rs770497978	1
PAX8 (NM_013953)
	exon5	c.398G>A*	p.R133Q*	—	1
	exon4	c.275T>C*	p.I92T*	—	1
NKX2-5 (NM_001166176)
	exon2	c.416G>A*	p.S139N*	—	1

*: Novel mutations.

Table 3 Overview of the mutations relevant to thyrotropin resistance

Gene	Exon position	Nucleotide position	Amino acid position	Mutation types	n.
GNAS
NM_016592	exon1	c.571G>A	p.G191R	rs762734492	1
NM_016592	exon1	c.334G>A	p.E112K	rs201307445	1
NM_001309840	exon6	c.301C>T	p.R101C*	—	1
NM_001077490	exon1	c.1384G>A	p.A462T	rs745875776	1
NM_001077490	exon1	c.1225C>G	p.L409V	rs56102209	1
NM_001077490	exon1	c.1189C>G	p.L397V	rs148033592	1
NM_001077490	exon1	c.1211T>C	p.M404T*	—	3
NM_000516	exon4	c.308T>C	p.I103T*	—	2
NM_000516	exon12	c.1018T>C	p.F340L*	—	1

*: Novel mutations.

Distribution of potentially functional mutations or causative genes were investigated in 69 mutated patients. 28 (40.58%) patients had one potentially functional mutation, 26 (37.68%) patients had two mutations, and 15 (21.74%) patients had three or more mutations (Fig. 3A). Similarly, 39 (56.52%) patients had mutations in one gene and 30 (43.483%) patients in two or three genes (Fig. 3B). According to the pathomechanism of the mutant genes, we classified the type of the 69 CH patients, 40 (57.97%) CH patients were classified as thyroid dyshormonogenesis (Table 4).

Fig. 3

The distribution of 69 CH patients with different mutations

A: Frequency distribution of the patients with different number of potentially functional mutations; B: Frequency distribution of the patients with different number of mutation-detected causative genes.

Table 4 Classification of CH according to the pathomechanism of mutant genes

Type	NO.	Related genes (number)
TDH	n = 40 (57.97%)	DUOX2 (12), TG (13), TPO (4), DUOX2/TG (9), DUOX2/TPO (2)
TD	n = 9 (13.04%)	TSHR (5), TTF2 (1), PAX8 (1), TSHR/TTF1 (2)
TR	n = 3 (4.35%)	GNAS (3)
TDH/TD	n = 10 (14.49%)	DUOX2/TSHR (3), DUOX2/TTF1 (1), TG/TSHR (1), TG/PAX8 (1), DUOX2/TPO/TSHR (2), DUOX2/TG/TSHR (1), DUOX2/TG/TTF1 (1)
TDH/TR	n = 4 (5.80%)	DUOX2/GNAS (2), TG/GNAS (2)
TD/TR	n = 2 (2.90%)	TTF1/GNAS (1), NKX2-5/GNAS (1)
TDH/TD/TR	n = 1 (1.45%)	DUOX2/TSHR/GNAS (1)

TDH, thyroid dyshormonogenesis; TD, thyroid dysgenesis; TR, thyrotropin resistance

Discussion

In the present study, we studied the mutations status of thirteen causative genes by NGS in a cohort of 106 CH patients. Eighty-three different mutations were identified in 69 patients in the following nine genes: DUOX2, TG, TPO, TSHR, TTF1, TTF2, NKX2-5, PAX8 and GNAS, and most of the mutations were heterozygous.

Mutations in DUOX2 were responsible for thyroid dyshormonogenesis in numerous researches [5]. Most patients with DUOX2 pathogenic mutations have an ectopic thyroid gland with an increased or normal size [6]. However, the mutational spectrum of the DUOX2 gene and the correlations between phenotype and genotype has not yet been fully established. The mutation of c.1588A>T in DUOX2 was highly recurrent with an approximately prevalence of 1/40,000 in our cohort, which was responsible for thyroid dyshormonogenesis. c.1588A>T had population-specificity and mainly reported in Asia population, including China [7-9], Japan [10, 11] and Malaysia [12]. Mutations of c.4027C>T [13], c.3329G>A [7, 14], c.3632G>A [15], c.2335G>A [11, 16] and c.2654G>A [17] were also mainly reported in Asians, and most of them were identified in Chinese Han population. c.1883delA [9-11, 14], c.3478_3480del [8, 14, 18] and c.605_621del [9, 19, 20] were scattered distribution in Asia, including China, Japan and Korea. Six other mutations, including c.2048G>T [8], c.227C>T [21], c.2894C>T [16], c.3391G>T [8, 22], c.2202G>A [22] and c.2104_2106del [8] were reported only in Chinese, and the missense mutation of c.4405G>A was first identified among Chinese population in our study, which was previous reported in Korean [14]. The mutations of c.1873C>T was identified as a novel pathogenic mutation by the qCarrier test in a reproductive carrier-testing program [23], but no direct evidence showed that the mutation was related with CH. In addition, c.1265G>A, c.2413G>A, c.1717C>T, c.3721A>T, c.3321delC, c.1300_1320del and c.1007_1009del mutations were first identified in our study, which may be related to CH.

Since the first TG mutation was reported [24], over 150 mutations were reported to be related with iodotyrosyl coupling defect or thyroid dyshormonogenesis in the ClinVar, we also detected a high prevalence of TG mutations in this study. According to previous researches [25-27], c.7847A>T mutation in TG was population-specificity with an allele frequency of 0.02062 in East Asian, and this mutation was also the most common mutation with an approximately prevalence of 1/22,000 in our cohort. c.1712delT and c.7123G>A were first identified in Chinese, which were previous identified in Brazil, Japan [28], UK [29] Argentina [30], and Taiwanese [27]. Other mutations, including 3040G>C, c.6185G>A, c.301C>G, c.866C>A, c.8137G>A, c.976C>T, c.2593C>A, c.3457A>T, c.3538C>T, c.5020C>A, c.7799G>A, c.1000delG and c.5018_5019insAAATA were first identified in our study.

Mutations of TSHR were third prevalence in our cohort. TSHR, which locates on the surface of thyroid follicular cells and mediates the effects of TSH, is important for the development and function of the thyroid gland. TSHR gene mutations were reported to be one of the causes of thyroid dysgenesis. Fifteen different TSHR mutations were identified in our study, eight of them, including c.1349G>A [31], c.1574T>C [32], c.394G>C [33], c.1591C>T [34], c.915T>A [35], c.733G>A [32], c.1838A>G [36] and c.1270G>T [37] were reported related to CH.

In addition, a total of 24 mutations of TPO, TTF1, TTF2, NKX2-5, PAX8 and GNAS were identified, which were infrequent in Chinese and most of them were unreported. No pathogenic mutation of THRA, TSHB, IYD and SLC5A5 was identified in our cohort, which was similar to relevant report [38].

We found almost 70% mutations related to thyroid dyshormonogenesis in our cohort, and most of mutations were present as heterozygous, which was different from the reports in other regions [39]. We classified the type of mutant CH patients according to the pathomechanism of the genes, 57.97% CH patients were classified as thyroid dyshormonogenesis. Previous research showed that 80%–85% of CH cases were caused by thyroid dysgenesis in the Caucasian areas [40], but our results indicated that thyroid dyshormonogenesis could be the first etiology of CH in Chinese. We assume the difference of mutation may be a result of difference in study populations, races, living environment and geographical locations.

Previous study showed that multiple mutations were detected in CH patients [33, 41], our study also indicated that mutation of multiple site or multi-gene take a considerable proportion in Chinese CH patients. Multiple mutations may cause a more serious phenotype in the CH patients. Patients with one or two DUOX2 pathogenic mutations turned out to be subclinical or transient CH, while patients with three or more DUOX2 pathogenic mutations were mostly associated with permanent CH [8]. The coexistence of multiple pathogenic mutations may have contributed to the severity of the hypothyroid condition and mutations of multiple gene may led a great genotype-phenotype variability [17, 42, 43]. Therefore, further studies were still needed to enlarge the mutation spectrum of CH and to verify the function of mutations, which may give an more profound insight to the etiology of CH. In summary, DUOX2, TG and TSHR were the top three mutant genes in Chinese CH patients, while the frequencies of TTF1, TTF2, NKX2-5 and PAX8 mutations were few. This study could help to further understand the gene mutations spectrum and genetic pathogenesis of CH.

Acknowledgments

This work was supported by Jiangsu provincial maternal and child health research project (F201671), Key research and development plan project of Jiangsu Province (BE2017650) and Changzhou science and technology support project (Social Development CE20175021). The study design and protocol were reviewed and approved by the ethics committee of Changzhou Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University. We thank Lihua Jiang and Yan Li for assistance of samples, Fang Guo and Rui Yang for writing and revise, and Dr. Jianbin Liu for the bioinformatics analysis.

Disclosure of Conflicts of Interest

The authors declare that they have no competing interests.

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