Screening of frequent variants associated with congenital hypothyroidism: a comparison with next generation sequencing

Abstract

Congenital hypothyroidism (CH) is considered the most common congenital endocrine disorder of genetic origin. Next generation sequencing (NGS) is the standard method for identifying genetic mutations, but it is an expensive and complex technique. Therefore, we propose to use Sanger sequencing to identify selected variants of the four most common CH-causative genes: DUOX2, TG, TSHR, and PAX8. To analyze the performance of Sanger sequencing, we compared its variant detection ability with that of a CH NGS panel containing 53 genes. We performed Sanger sequencing of selected variants and panel NGS analysis of 25 Japanese patients with CH. Sanger sequencing identified nine variants in seven patients, while NGS identified 24 variants in 14 patients. Of these, eight, five, eight, two, and one were found to be potentially pathogenic in DUOX2, TSHR, TG, UBR1, and TPO genes, respectively. The percentage of detectable variants using Sanger sequencing compared with NGS was 37.5% (9/24 variants), whereas the percentage of detectable cases carrying variants using Sanger sequencing compared with NGS was 50% (7/14 patients). We proposed a system for screening commonly identified CH-related variants by Sanger sequencing. Sanger sequencing could therefore identify about a third of CH-causative variants, so is considered an effective and efficient form of pre-screening before NGS.

CONGENITAL HYPOTHYROIDISM (CH) is a congenital thyroid hormone deficiency caused by morphological abnormality or dysfunction of the thyroid gland that develops at the fetal or perinatal stage [1]. CH has been considered the most common congenital endocrine disorder, with a prevalence of approximately 1:2,000 to 1:4,000 live births [2]. While most cases are sporadic, about 15%–20% have a genetic origin and are associated with possible causative genes [3, 4]. Thyroid dysgenesis is mainly considered sporadic, but in some cases a genetic component has been proposed to be involved. Thyroid dyshormonogenesis shows autosomal recessive inheritance in some cases [1]. Clinical features differ depending on the type of mutation but can include thyroid dysgenesis and thyroid dyshormonogenesis. TSHR mutations are associated with a higher risk of mild and permanent CH (PCH) [5], while DUOX2 mutations can cause transient CH (TCH) [6]. TG and TPO mutations have been identified in patients with moderate or severe PCH and goiter [7]. Clinical phenotypes of PAX8 mutation carriers vary, ranging from overt CH with severe thyroid hypoplasia to subclinical hypothyroidism with a normal-sized thyroid [8].

The frequency of inherited CH is dependent upon regional biases. The reported genetic prevalence of mutations in DUOX2, TG, TSHR, PAX8, and TPO was 1/44,000 [6], 1/67,000 [6, 9], 1/117,000 [5], 1/180,000 [10], and 1/180,000 [6], respectively. Furthermore, each gene has mutation hotspots. For example, TSHR p.Arg450His (rs189261858) comprises 70% of the total TSHR variants in Japanese patients [5], DUOX2 p.His678Arg (rs57659670) is a functional single nucleotide polymorphism (SNP) with an allele frequency of 0.035 [6], TG p.Cys1264Arg (rs2076738) is a common variant [9], and PAX8 p.Arg31His (rs104893657) was identified as being located in a mutation hotspot [11, 12].

The genetic diagnosis of CH is helpful in understanding its genetic etiology. Recently, mutations associated with CH have been identified in several studies using next generation sequencing (NGS) [3, 4, 13-15]. Although NGS remains the most reliable technique in identifying sequence variants, it is inefficient as it requires a large number of samples for one measurement. Therefore, cheaper and simpler methods are required for clinical medicine.

Because many patients with CH are predicted to have SNPs and known mutations depending on their ethnicity and region, pre-analysis of common causative genes of CH using Sanger sequencing should enable the screening of variants in some patients before performing NGS. In this study, we compared the performance of Sanger sequencing to screen for common CH causative variants with NGS.

Materials and Methods

Patients

Twenty-five Japanese CH patients (12 males and 13 females) were recruited between April 2016 and July 2018 from the Department of Pediatric Endocrinology outpatients at University of Yamanashi Hospital. All patients had been diagnosed with CH by newborn screening (NBS). Patients with any chromosomal abnormalities, those with maternal or neonatal iodine exposure, and very preterm infants were excluded. Detailed clinical information of the patients is summarized in Table 1.

Table 1 Clinical characteristics and thyroid function test of CH patients

	n, median	(range)
Case	25
Sex (M:F)	12:13
Age (years)	6	(1–19)
Gestational age (weeks)	39	(32–40)
Birth weight (g)	2,886	(1,732–3,759)
Family history	2
Struma	4
Dysgenesis	3
Serum TSH (μIU/mL)	12.0	(1.59–1,180)
Serum free thyroxine (ng/dL)	1.46	(0.18–2.15)
Thyroglobulin (ng/mL)	106	(18.2–15,588)

M, male; F, female; TSH, thyroid-stimulating hormone

Neonate NBS followed the Guidelines for Mass Screening of Congenital Hypothyroidism in Japan. NBS samples were taken four to six days after birth to measure thyroid-stimulating hormone (TSH) levels. A neonate with TSH exceeding 15–30 μIU/mL in the first blood sample was immediately referred to the hospital for a detailed examination. Neonates with TSH 7.5–15 μIU/mL were recalled for a second blood sample at the facility that performed the first blood test. If TSH in the second blood test was higher than 5 μIU/mL, the neonate underwent a detailed examination.

At the first evaluation, serum levels of TSH, triiodothyronine, free thyroxine (fT4), and thyroglobulin were measured. Thyroid ultrasonography was performed to determine the position and size of the thyroid gland. Additional information about the possible existence of thyroid disease in family members was also collected from all patients. CH was diagnosed from the results of NBS, clinical symptoms, imaging, and thyroid function tests.

Levothyroxine (l-T4) replacement was initiated according to previously published guidelines [1]. Treatment was started immediately in the following cases: i) serum TSH ≥30 μIU/mL or TSH 15–30 μIU/mL with low fT4 (≤1.5 ng/dL), ii) no evidence of distal femoral nucleus and/or the inability of ultrasonography to identify the thyroid gland, or iii) goiter. The initiation of treatment was considered if TSH was >10 μIU/mL at 3–4 weeks after birth. l-T4 administration was discontinued at 3 years of age in some patients, and the thyroid function test was performed again. If a patient was untreated, the thyroid function test was performed again 1–2 weeks later and the patient was followed-up.

The classification of CH was based on the condition of withdrawing treatment according to previously published guidelines [1]. CH was classified as CH requiring continuous treatment (PCH), TCH, and subclinical CH (SCH). PCH was defined as a condition requiring lifelong hormone therapy because of insufficient thyroid hormone production and included patients who required l-T4 treatment after 3 years of age. TCH was defined as a condition requiring transient hormone therapy during the early stages of life and included patients who discontinued l-T4 therapy at 3 years of age and did not require l-T4 re-administration for more than 1 year after l-T4 discontinuation [1, 16, 17]. Patients with TSH ≥10 μIU/mL at <6 months after birth or TSH ≥5 μIU/mL at 12 months after birth were categorized as having SCH [1, 18]. Other patients were categorized as undecided.

The parents or guardians of all patients provided their written informed consent for study participation, and the study was approved by the institutional review board at University of Yamanashi (no.1780).

Sanger sequencing

Common causative CH genes in the Japanese population have been reported previously [5, 6, 9, 11]. We selected the high-frequency variants DUOX2 p.His678Arg, TG p.Cys1264Arg, TSHR p.Arg450His, and PAX8 p.Arg31His for analysis by Sanger sequencing.

Genomic DNA was extracted from patient peripheral blood by a standard method and amplified by PCR using primers specific to the mutation sites. The forward and reverse primers for exon 17 of DUOX2 were 5'-GGACATCTGCTGAACTACCC-3' and 5'-TGATAATGGAGTCGTGTGAGG-3', respectively; the forward and reverse primers for exon 17 of TG were 5'-CACCACGGCTCCACTTTC-3' and 5'-GGGGTGCAGGATAGATGCTC-3', respectively; the forward and reverse primers for exon 11 of TSHR were 5'-CTGGGTGACAGCATTGTTGG-3' and 5'-GCGAGAAGGAAGCAGCAAAC-3', respectively; and the forward and reverse primers of exon 3 for PAX8 were 5'-ATCCCCACCCAAACTCCTAC-3' and 5'-CCCTGAGATCAGCTGGAGAA-3', respectively. PCR was performed using the following thermocycling conditions: initial denaturation at 95°C for 10 min, followed by 35 cycles of denaturation at 95°C for 15 s, annealing at 58°C for 30 s, and extension at 72°C for 1 min, with a final extension at 72°C for 7 min. PCR products were analyzed using direct sequencing.

Next generation sequencing

We developed a custom Ion Ampliseq panel (Ion Torrent, Thermo Fisher Scientific, Waltham, MA, USA) targeting 53 genes associated with CH (TRHR, TSHB, IGSF1, NKX2-1/TTF1, FOXE1/TTF2, NKX2-5, PAX8, TSHR, SLC5A5/NIS, SLC26A4, TG, TPO, DUOX2, DUOXA2, IYD, THRB, THRA, SLC16A2, and SECISBP2) [1] or relevant to thyroid diseases such as hypothyroidism, goiter, and Graves’ disease (POU1F1, PROP1, LHX3, LHX4, OTX2, HESX1, GSTO2, TBL1X, GLIS3, EZH2, NSD1, KATB6, UBR1, SH2B3, BCHE, NEFL, NEFM, CTLA4, FCRL3, PTGS2, PTPN22, ITPR3, IFIH1, GC, ABO, RNASET2, C6orf10, KCNJ10, ARID5B, B3GNT2, PHTF1, IRS4, JAG1, and NEFH). The library was prepared using an Ion Ampliseq Library Kit Plus and Ion Library Quantitation Kit (Ion Torrent, Thermo Fisher Scientific). Emulsion PCR and enrichment steps were performed using an Ion Personal Genome Machine (PGM) Hi-Q View Chef Kit (Ion Torrent, Thermo Fisher Scientific). Prepared amplicon libraries were sequenced using an Ion Chef system, an Ion PGM Hi-Q View Chef Kit, and Ion 318 Chip Kit v2 (Ion Torrent, Thermo Fisher Scientific). Sequencing was performed using an Ion Torrent PGM system (Ion Torrent, Thermo Fisher Scientific). All steps were performed according to the manufacturer’s instructions.

Bioinformatics analysis was conducted using Torrent Suite 5.10 software (Thermo Fisher Scientific). The read was aligned to hg19, and variant calling was performed using the Germ Line-PGM-High Stringency setting. Called variants were functionally annotated using the ANNOVAR tool. All variants were screened according to the location, frequency, and type of mutation.

We applied the following criteria to select the variants: a minor allele frequency (MAF) <0.2 in the 1000 Genome Project (https://www.internationalgenome.org/1000-genomes-browsers) and the 4.7 K Japan (https://jmorp.megabank.tohoku.ac.jp/202001/); located within exons or splicing regions; and missense, nonsense, and frameshift mutations, but not synonymous variants. The public version of the Human Genome Mutation Database (http://www.hgmd.cf.ac.uk/ac/index.php), ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/), and PubMed databases (https://pubmed.ncbi.nlm.nih.gov/) were searched to evaluate whether the variants were known or unknown.

We aimed to identify novel variants, including unknown variants or those with a MAF <0.01. In the in silico analysis, variants were included that were predicted to be “probably damaging; >0.908”, “deleterious; <0.05”, and “possibly pathogenic; >0.025” by the Polyphen-2 (http://genetics.bwh.harvard.edu/pph2/index.shtml), SIFT (https://sift.bii.a-star.edu.sg/), and M-CAP (http://bejerano.stanford.edu/MCAP/) prediction tools, respectively. Finally, we used the American College of Medical Genetics and Genomics (ACMG) guidelines to classify variants [19].

Comparing the performance of analyses using Sanger sequencing with NGS

We identified the frequencies of variants identified by Sanger sequencing and NGS and confirmed that variants identified by NGS were also identified by Sanger sequencing. We also directly compared results from the two forms of sequencing in terms of number of cases, coverage of variants, cost and time, and the prediction of patient clinical features.

Results

Table 2 shows the clinical characteristics at the time of close inspection and genotypes of the 25 patients. Sanger sequencing detected variants in a total of seven patients, including one with a compound heterozygous TSHR variant, four with monoallelic DUOX2 variants, one with a monoallelic TSHR variant, and one with monoallelic variants in different combinations of DUOX2 and TSHR (Fig. 1). Sanger sequencing also identified the following monogenic variants: DUOX2 p.His678Arg (n = 4), TG p.Cys1264Arg (n = 0), TSHR p.Arg450His (n = 1), and PAX8 p.Arg31His (n = 0). It also identified two heterozygous variants (DUOX2 p.His678Arg and TSHR p.Arg450His) in one patient. Direct sequencing of TSHR revealed p.Val473Ile and p.Gly464fs compound heterozygous variants in one patient, giving a total of nine variants identified in seven patients. TSHR variants (n = 2) were seen in patients with PCH (n = 2), while DUOX2 variants (n = 4) were seen in patients with TCH or SCH (n = 2), PCH (n = 1), and undecided (n = 1). Combination variants in TSHR and DUOX2 were seen in one patient with PCH.

Table 2 Patient phenotypic and genetic characteristics

No.	Sex	Age (years)	GA	BW	FH	Struma	Dysgenesis	Serum TSH (μIU/mL)	Serum fT4 (ng/dL)	Tg (ng/mL)	Oral administration of levothyroxine	Clinical phenotype	Variant	ACMG
1	F	6	35	1,778				108	0.58	NA	Yes	PCH
2	F	3	38	3,278				10.7	1.81	83.0	Yes	PCH	TSHR p.Arg450His heterozygote TPO p.Pro883Ser heterozygote	P LP
3	F	9	37	2,704	Yes			17.1	1.46	97.5	Yes	PCH	TSHR p.Val473Ile/p.Gly464fs compound heterozygote	P/LP
4	F	17	40	2,716		Yes		1,180	0.18	NA	Yes	PCH
5	F	13	37	2,628				1.59	1.50	94.6	Yes	PCH	DUOX2 p.His678Arg/p.Ser1322Tyr compound heterozygote	B/VUS
6	M	19	39	3,436			Yes	252	0.73	1,114	Yes	PCH
7	F	19	39	2,354			Yes	66.2	1.19	865	Yes	PCH	TG p.Lys2720Thr Heterozygote	VUS
8	M	16	39	3,068				8.05	1.36	105	Yes	PCH
9	F	6	39	2,890			Yes	35.7	0.91	24.0	Yes	PCH	TG p.Thr1498Met heterozygote UBR1 p.Asp1619Val heterozygote	VUS VUS
10	M	19	40	2,818				41.0	1.59	160	Yes	PCH	TSHR p.Arg450His/p.Arg109Gln compound heterozygote DUOX2 p.His678Arg heterozygote	P/P B
11	M	2	39	2,922				6.34	1.67	221	Yes	PCH
12	M	2	38	3,452				6.82	1.41	106	Yes	PCH
13	M	15	NA	3,190				10.2	1.48	NA	Finished	TCH	TG p.Thr1498Met Heterozygote	VUS
14	M	6	38	2,808				11.2	1.53	93.9	Finished	TCH	TG p.Thr1498Met heterozygote UBR1 p.Arg1380Cys heterozygote	VUS VUS
15	M	12	39	2,786		Yes		19.7	1.07	NA	Finished	TCH	TG p.Arg1066Cys/p.Thr1498Met compound heterozygote	VUS/VUS
16	M	12	40	3,759		Yes		5.78	1.91	160	Finished	TCH	DUOX2 p.His678Arg heterozygote	B
17	M	2	35	2,476		Yes		43.2	0.54	15,588	Finished	TCH
18	F	17	NA	2,946		Yes		7.56	2.15	404	No	SCH
19	F	3	38	2,692				7.95	1.75	62.5	No	SCH	TG p.Thr1498Met heterozygote	VUS
20	F	2	38	2,886	Yes			14.4	1.94	102	No	SCH	DUOX2 p.His678Arg heterozygote TG p.Gln830Glu heterozygote	B VUS
21	F	1	39	3,118				8.30	1.79	229	No	SCH
22	F	2	40	2,932				7.16	1.43	98.7	No	SCH	DUOX2 p.Leu1160del Heterozygote	P
23	M	1	39	2,902				87.6	1.12	852	Yes	undecided	DUOX2 p.His678Arg/p.Arg885Gln compound heterozygote	B/P
24	M	2	39	2,712				12.8	1.39	276	Yes	undecided
25	F	1	32	1,732				12.0	1.65	18.2	Yes	undecided

NA, not available; F, female; M, male; GA, gestational age; BW, birth weight; FH, family history; TSH, thyroid-stimulating hormone; fT4, free thyroxine; Tg, thyroglobulin; CH, congenital hypothyroidism; PCH, permanent CH; TCH, transient CH; SCH, subclinical CH; ACMG, American College of Medical Genetics and Genomics; P, pathogenic; LP, likely pathogenic; VUS, variants of uncertain significance; B, benign; Bold, variants detected by Sanger sequencing

Fig. 1

Variant analysis in CH patients by Sanger sequencing (left) and NGS (right). Sanger sequencing showed that one, four, and one patient had TSHR p.Val473Ile/p.Gly464fs compound heterozygous, DUOX2 p.His678Arg heterozygous, and TSHR p.Arg450His heterozygous variants, respectively. Combination variants TSHR p.Arg450His and DUOX2 p.His678Arg were seen in one patient. Variants were detected in 14 of 25 patients by NGS. Five of these patients (20%) carried biallelic variants. Five patients (20%) and four patients (16%) carried monoallelic variants and combinations of a heterozygous variant in two genes. The percentage of detectable patients determined using Sanger sequencing compared with NGS was 50% (7/14 patients). The percentage of detectable variants determined using Sanger sequencing compared with NGS was 37.5% (9/24 variants).

The overview of variant identification by NGS is shown in Fig. 2. NGS data of 25 CH patients were generated, and 578 variants were selected for analysis. After quality control and the exclusion of synonymous variants, the remaining 205 variants were verified. NGS identified a total of 24 variants in 14 patients after removing unselected variants. As shown in Table 3, the variants were distributed in 15 sites (six novel sites and nine previously reported sites) of five genes [6, 20-26]. Eight, five, eight, two, and one variants were found to be potentially pathogenic in DUOX2, TSHR, TG, UBR1, and TPO genes, respectively. Variants of DUOX2 p.His678Arg were seen in patients with compound heterozygous variants or combinations of two genes (n = 4), and monogenic variants (n = 1). Biallelic variants comprised 20% (5/25) of the variants identified by NGS. One patient had a combined THSR biallelic variant and DUOX2 monoallelic variant. Monoallelic heterozygous variants comprised 20% (5/25) of variants identified by NGS. Monoallelic variants in different combinations of two genes represented 16% of the variants identified (4/25): DUOX2 and TG (n = 1), TSHR and TPO (n = 1), and TG and UBR1 (n = 2). A total of 11 patients had no potentially functional variants.

Fig. 2

Flow chart of NGS data analysis in 25 CH patients with 578 variants. After quality control and the exclusion of synonymous variants, the remaining 205 variants were verified. Finally, 24 variants were identified as pathogenic of CH.

Table 3 Overview of genetic variants identified in CH patients

n	Gene	Exon	Location	rs number	Amino acid change	MAF	Polyphen	SIFT	M-CAP	ACMG	Reference
5	DUOX2	17	chr15:45398438	rs57659670	p.His678Arg	0.165	0	1	NA	B	Maruo (2008) [20]
1	DUOX2	20	chr15:45396158	rs181461079	p.Arg885Gln	0.001	0.998	0	0.108	P	Maruo (2008) [20]
1	DUOX2	26	chr15:45391615	rs758318135	p.Leu1160del	0	NA	NA	NA	P	Narumi (2011) [6]
1	DUOX2	30	chr15:45388141	NA	p.Ser1322Tyr	0	1	0	0.122	VUS	NA
1	TG	10	chr8:133900540	rs2076737	p.Gln830Glu	0.001	0.988	0	0.039	VUS	NA
1	TG	13	chr8:133910470	rs200122163	p.Arg1066Cys	0.001	1	0	0.214	VUS	NA
5	TG	21	chr8:133931735	rs74590117	p.Thr1498Met	0.013	0.991	0.15	NA	VUS	Jiang (2016) [21]
1	TG	47	chr8:134145875	NA	p.Lys2720Thr	0	0.984	0.03	0.029	VUS	NA
1	TPO	12	chr2:1544394	rs190968346	p.Pro883Ser	0.001	0.01	0.02	0.013	LP	Umeki (2004) [22]
1	TSHR	4	chr14:81554306	rs121908865	p.Arg109Gln	0	1	0.53	0.106	P	Clifton-Bligh (1997) [23]
2	TSHR	10	chr14:81609751	rs189261858	p.Arg450His	0	1	0	0.554	P	Nagashima (2001) [24]
1	TSHR	10	chr14:81609793	NA	p.Gly464fs	0	NA	NA	NA	LP	Watanabe (2020) [25]
1	TSHR	10	chr14:81609819	NA	p.Val473Ile	0	1	0	0.098	P	Tsunekawa (2006) [26]
1	UBR1	37	chr15:43276107	rs372200839	p.Arg1380Cys	0	1	0	0.060	VUS	NA
1	UBR1	45	chr15:43244626	rs753088815	p.Asp1619Val	0	0.995	0	0.131	VUS	NA

NA, Not available; MAF, Minor allele frequency; Polyphen, Polymorphism Phenotyping; SIFT, Sorting Intolerant From Tolerant; M-CAP, Mendelian Clinically Applicable Pathogenicity; ACMG, American College of Medical Genetics and Genomics; P, pathogenic; LP, likely pathogenic; VUS, variants of uncertain significance; B, benign

NGS showed that the TSHR variant (n = 1) was seen in a patient with PCH (n = 1), while DUOX2 variants (n = 4) were seen in patients with TCH or SCH (n = 2), PCH (n = 1), and undecided (n = 1). TG variants (n = 5) were seen in patients with PCH (n = 2) and TCH or SCH (n = 3). Combination variants in TSHR and TPO (n = 1), and DUOX2 and TG (n = 1) were seen in a patient with PCH (n = 1) and one with SCH (n = 1), respectively. Combination variants in TG and UBR1 (n = 2) were seen in a patient with PCT (n = 1) and one with TCH (n = 1), respectively.

Comparing the performance of Sanger sequencing with NGS

CH-related variants were identified by Sanger sequencing in seven patients, and by NGS in 14 patients. The percentage of detectable variants determined using Sanger sequencing compared with NGS was 37.5% (9/24 variants; Fig. 1). All single gene variants in one patient were identified using Sanger sequencing, while Sanger sequencing identified no variants in 18 patients; NGS identified variants in seven of these 18 patients.

The results of Sanger sequencing can be given to the patient about 1 week after recruitment. However, NGS findings cannot be revealed for up to 2 years. In our study, Sanger sequencing costs were approximately $50 per person compared with NGS costs of approximately $520 per person.

TSHR and DUOX2 variants were more commonly identified in patients with PCH and TCH or SCH, respectively, by Sanger sequencing and NGS. Moreover, combination variants in two genes, including TSHR, were more common in patients with PCH. The prediction of patient clinical features, such as TSHR variants being more common in patients with PCH and DUOX2 variants being more common in patients with TCH or SCH, were almost comparable between Sanger sequencing and NGS.

Discussion

The findings of this study provide information about the use of Sanger sequencing for identifying genetic variants associated with CH. Sanger sequencing identified CH-related variants in seven patients, while NGS identified them in 14 patients. The percentage of detectable variants determined using Sanger sequencing compared with NGS was 37.5% (9/24 variants). This indicates the ability of Sanger sequencing to detect CH in advance of NGS in certain situations.

Several studies have screened for CH genetic mutations by NGS, with differences in populations, subjects, study methods, genes, and pathogenicity criteria [3, 4, 13-15]. We included CH patients of various classifications, such as PCH, TCH, and SCH. Our cohort is similar to a regional-based cohort of CH. We analyzed 53 genes, including various thyroid disease-related genes and previously reported CH causative genes. We identified 20% of single-gene variants by NGS, which is similar to the ratio reported in other studies. CH causative genes are constant, therefore we think it be useful to select the genes to be analyzed, as performed in Sanger sequencing.

Sanger sequencing is considered as effective and efficient for pre-screening before NGS by several reasons. First, it can be used to help predict the clinical course which aids the treatment decision process. Recently, Long et al. [7] estimated the relationship between mutations and phenotypic characteristics in pooled patients with CH. Mutations prevailed in PAX8, TSHR, FOXE1, and NKX2-5, and patients with these mutations had a higher risk of PCH [7]. DUOX2 mutations have been shown to cause higher prevalence of TCH than PCH [27]. In the present study, Sanger sequencing showed that TSHR and DUOX2 variants were more common in patients with PCH (n = 2) and TCH or SCH (n = 2), respectively, while combination variants in TSHR and DUOX2 were seen in one patient with PCH. We were able to clinically predict five out of 25 cases, suggesting that Sanger sequencing could be used as a tool for clinical prediction in the future.

Second, it is possible to efficiently analyze in small number of patients using Sanger sequencing, unlike NGS necessary to accumulate patients because the unit cost per person is higher. Additionally, results can be obtained and communicated quickly. The cost per person for Sanger sequencing is about 1/10 that of NGS, suggesting that it might be more effective for smaller populations and facilities not equipped for NGS.

Third, Sanger sequencing can be efficient in certain situations. The percentage of detectable genetic variation determined using Sanger sequencing was 37.5% compared with NGS (9/24 mutations). Although many genetic variants can be identified using NGS, the presence or absence of biallelic variants is useful for conducting simple etiological estimations using Sanger sequencing. In the present study, variants were detected by Sanger sequencing in seven patients, including one in whom all single-gene variants were identified using Sanger sequencing. NGS analysis is more efficient to identify patients with unknown causes, Sanger sequencing is also effective to select patients with common variants.

Target genes for Sanger sequencing should be carefully considered to improve its performance. TSHR p.Arg450His is frequent in East Asia [18, 28-30] and comprises 70% of all Japanese TSHR variants [5]. Patients with TSHR p.Arg450His showed moderately decreased TSH binding and cAMP response [24, 26], and presented with thyroid morphology indicating mild CH. Analysis of TSHR gene is the most effective in such Asian small cohort. While TPO mutations appear to be the most common cause of CH in Western countries [7]. TG p.Cys1264Arg and PAX8 p.Arg31His were not found in our cohort, but TG p.Thr1498Met was commonly seen despite being a rare variant in the general population. TG variants are known to show regional variation in Japan, so the cohort region may affect its prevalence [9]. In recent reports of Japanese patients, DUOX2 p.Gly488Arg (rs191759494), p.Leu1160del (rs758318135), and p.Arg1110Gln (rs368488511) are identified as common variants [27, 31]. On the other hand, DUOX2 p.His678Arg is reported as functional SNP, which is responsible for partial hydrogen peroxide production intolerance and partial transfer efficiency reduction [6]. In our study, most of compound heterozygous and mild CH patients contained this variant. Biallelic inactivation of the DUOX2 is reported as a common cause of PCH or TCH, so DUOX2 mutations are described as genetic etiology of oligogenic involvement in latest Consensus Guidelines of CH [32, 33].

Our study has several limitations. The population was selected and the sample size was relatively small, so we cannot exclude selection bias associated with subject enrollment. Therefore, studies based on a larger cohort should be conducted to confirm our findings. Moreover, interpretation of the genetic analysis of CH is quite difficult. Some of the variants were classified as benign and variants of uncertain significance (VUS) by the ACMG guidelines. We did not conduct functional studies of the analyzed variants and assumed that they were VUS based on in silico analysis and frequency. This may have identified SNPs such as DUOX2 p.His678Arg with limited effects as potentially causative, so relevant functional studies are needed to verify variant functions. Additionally, our study focused on known CH genes, so it was not possible to rule out the action of variants outside the target of our analysis. Moreover, both NGS and Sanger sequencing fail to detect large deletions. Finally, future studies should include statistical power analysis and consider the cost-effectiveness of mutation detection methods.

In conclusion, we proposed a system for screening commonly identified CH-related variants by Sanger sequencing. We identified 37.5% of CH-causing genetic variants detected by NGS in half of the patients using Sanger sequencing. Pre-analysis of common causative genes of CH using Sanger sequencing would enable the screening of variants in a select group of patients before performing NGS. Thus, it would be possible to identify causative CH genes more simply and cheaply than using NGS.

Acknowledgement

We thank Sarah Williams, PhD, from Edanz Group (https://en-author-services.edanz.com/ac) for editing a draft of this manuscript.

Disclosure

None of the authors have any potential conflicts of interest associated with this research.

Conflict of Interest

None declared.

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