Original Paper
HOR MON E RE SE ARCH I N PÆDIATRIC S
Received: November 11, 2013 Accepted: March 11, 2014 Published online: September 20, 2014
Horm Res Paediatr 2014;82:252–260 DOI: 10.1159/000362235
High Frequency of DUOX2 Mutations in Transient or Permanent Congenital Hypothyroidism with Eutopic Thyroid Glands Hye Young Jin a Sun-Hee Heo b Yoo-Mi Kim a Gu-Hwan Kim c Jin-Ho Choi a Beom-Hee Lee a Han-Wook Yoo a
a
Division of Pediatric Endocrinology and Metabolism, Department of Pediatrics, b Genome Research Center for Birth Defects and Genetic Disorders, and c Medical Genetics Center, Asan Medical Center Children’s Hospital, University of Ulsan College of Medicine, Seoul, Korea
Abstract Background/Aims: This study aimed to clarify the frequency, phenotypes, and molecular spectrum of DUOX2, TPO, TSHR, and TG mutations in patients with congenital hypothyroidism (CH) with enlarged or normal-sized eutopic thyroid glands. Methods: The study cohort included 43 subjects from 41 unrelated families who had CH with eutopic thyroid glands. Mutation analyses of DUOX2, TPO, and TSHR were performed. The functional capacities of novel missense variants of DUOX2 were verified by measuring H2O2 generation in vitro. Results: Of the 43 subjects, 23 (53.5%) had sequence variants in at least one gene. Twelve different DUOX2 variants, including seven novel variants, were identified in 20 subjects. A functional analysis of the DUOX2 variants revealed that most variants, other than p.G206V and p.H678R, caused a significant reduction in H2O2 generation. Therefore, 15 subjects harbored functionally deleterious DUOX2 variants. Of these, 5 subjects had transient CH, and 10 were found to have permanent CH. Sequence variants in TSHR were identified in 5 subjects. One of the 43 subjects (2.3%) had sequence variants in two different genes. Conclusions: DUOX2 variants are a relatively common cause of CH with
© 2014 S. Karger AG, Basel 1663–2818/14/0824–0252$39.50/0 E-Mail [email protected] www.karger.com/hrp
normal-sized or enlarged eutopic thyroid glands. Variable phenotypes were associated with partial loss of the functional activity of DUOX2 variants. © 2014 S. Karger AG, Basel
Introduction
Patients with congenital hypothyroidism (CH) with a eutopic thyroid can have various etiologies: resistance to thyroid-stimulating hormone (TSH), thyroid dyshormonogenesis, and transient hypothyroidism [1]. Dyshormonogenic CH is caused by several genetic defects, including defects in thyroid peroxidase (TPO), thyroglobulin (TG), dual oxidase 2 (DUOX2), the dual oxidase maturation factor (DUOXA2), the sodium-iodide symporter (SLC5A5), pendrin (SLC26A4), and iodotyrosine deiodinase (IYD) [2, 3]. Most genetic defects responsible for dyshormonogenesis are inherited in an autosomal recessive manner and manifest as goitrous CH. The prevalence of thyroid dyshormonogenesis due to TPO and TG was reported to be 1: 66,000 in the Netherlands and 1:67,000 in Japan [4, 5]. Dyshormonogenesis with a eutopic thyroid appears to be the
H.Y.J. and S.-H.H. contributed equally to this work.
Han-Wook Yoo, MD, PhD Division of Pediatric Endocrinology and Metabolism, Department of Pediatrics Asan Medical Center Children’s Hospital, University of Ulsan College of Medicine 388-1, Pungnap-Dong, Songpa-Gu, Seoul 138-736 (Korea) E-Mail hwyoo @ amc.seoul.kr
Downloaded by: Univ. of California San Diego 132.239.1.231 - 10/2/2014 4:08:55 PM
Key Words Congenital hypothyroidism · DUOX2 · Dyshormonogenesis
TSH, free T4, and thyroglobulin (Tg). In case of hypothyroidism in siblings, thyroid functions were followed up, even though their screening TSH levels were lower than 10 mU/l. Antibodies (Ab) against thyroperoxidase (TPO) (anti-TPO Ab), Tg (anti-Tg Ab), and the TSH receptor (anti-TSH Ab) were also measured. At confirmatory diagnosis, subjects with the TSH levels higher than 10 mU/l were considered to have CH [6, 11]. A 99mTc scintigraphy was performed to assess the location and size of the thyroid gland. Thyroid size was measured using ultrasonography according to a Japanese study [17]; thyroid widths equal to or >+2 SD were defined as goiter, whereas ones equal to or +2 SD.
1 (3/F)b 2 (4/M) 3 (4/M) 4 (7/M)c 5 (5/F)c 6 (5/M)
Subject No. (current age, years/gender)
Table 1. Hormonal and mutational data for patients with transient CH in the study cohort harboring sequence variants
none none none none TSHR: p.G132R/ p.G132R
none none none TSHR: p.G132R TSHR: p.G132R TSHR: p.R450H none none none TSHR: p.R450H none none
Second gene (TPO, TSHR, TG)
none none none none none none
Second gene (TPO, TSHR, TG)
in A549 cells transfected with the indicated expression vectors to measure H2O2-producing capacity. The activity of each mutant is expressed as a percentage of wildtype activity. Each bar represents the mean, and the error bars represent SDS. The p. G206V and p.H678R mutants showed relatively high residual function (more than 75% of the wild-type activity). The significant inhibition of H2O2-generating activity was observed when the remaining mutant vectors were transfected into the A549 cells (* p < 0.05).
100
*
* 80
40
*
0
Em
*
*
20
pty
*
*
60
ve
*
*
*
*
r L e T S Y K R R V Q Q c to t y p N 4 3 A 7 2 P 9 6 2 0 6 4 8 8 6 7 8 E 8 7 9 8 8 5 1 1 0 1 1 2 3 3 3 4 W G 1 G H R ld i A R R1 W
allelic variants in DUOX2 (subjects 1–3, 7–9, 13–17, 19), 8 (subjects 8, 9, 13–17, 19) turned out to have permanent CH. Of these, 1 patient (subject 16) was associated with a second gene mutation (TSHR p.R450H) (table 2). Six patients (subjects 7–12) showed persistent TSH elevation after levothyroxine withdrawal at age 3. Their TSH levels were followed up until 3–6 months after age 3, and eventually levothyroxine treatment was resumed. Among the 4 familial cases from two independent pedigrees, subjects 1 and 17 were compound heterozygous for p.G206V and p.E879K, and subjects 4 and 5, siblings born from non-consanguineous parents, were homozygous for p.G488R in DUOX2. The parents of subjects 1 and 17, heterozygous for either substitution (p.G206V or p.E879K), were thought to be in a clinically euthyroid state without a goiter based on medical history, although their thyroid function tests were not available. The parents of subjects 4 and 5, who were heterozygous for p. G488R, had normal TSH levels, suggesting autosomal recessive inheritance (fig. 3).
The findings in this study demonstrate that DUOX2 mutations are one of the most common causes of dyshormonogenic CH. A significant portion of the cases appear to be due to the impairment of H2O2 generation caused by DUOX2 mutations. A recent Japanese study demonstrated that DUOX2 mutations are one of the leading causes of permanent CH, comprising 41% of dyshor-
monogenesis cases [7, 10, 24]. Our study included transient and permanent CH cases with normal-sized to enlarged eutopic thyroids, which we found appeared at a comparable frequency (43.9%). Unlike other dyshormonogenic defects, such as those caused by mutations of TPO, TG, NIS, and PDS, monoallelic DUOX2 mutations cause milder, transient hypothyroidism, whereas biallelic mutations result in severe, permanent hypothyroidism [14, 25]. In the present study, subjects 1, 4, and 5 with biallelic DUOX2 variants were confirmed as having transient CH, although they presented with overt hypothyroidism at the initial diagnosis, and subjects 13 and 19 with monoallelic DUOX2 variants were considered to have permanent CH. Our finding that phenotypic variability is not related to the number of mutant alleles is consistent with previous reports on transient, mild phenotypes in CH patients with biallelic DUOX2 mutations [8, 15]. Variable phenotypes are presumed to be caused by (1) another H2O2-generating oxidase in thyrocytes, such as DUOX1; (2) other genetic factors such as DUOXA2; (3) ethnic differences, and (4) environmental factors such as iodine intake [8, 15]. However, possibilities of monoallelic mutation in one allele and deletion of DUOX2 in the other allele cannot be ruled out unless array comparative genomic hybridization is performed [13]. Both transient and permanent CH were found to be associated with DUOX2 mutations in our present analyses. On re-evaluation of the patients at 3 years of age, serum TSH levels were not so high in the permanent CH group, whereas mild hyperthyrotropinemia (TSH levels
Molecular Basis of Congenital Hypothyroidism
Horm Res Paediatr 2014;82:252–260 DOI: 10.1159/000362235
Discussion
257
Downloaded by: Univ. of California San Diego 132.239.1.231 - 10/2/2014 4:08:55 PM
Fig. 2. DUOX2-mediated H2O2 generation
Relative H2O2 production (%)
120
DUOX2
G206V/H678R
E879K/WT
G488R/WT
WT/G488R
DUOX2
Subject 17 G206V/WT WT/E879K H678R/WT
Subject 1 G206V/WT WT/E879K H678R/WT
Subject 4 G488R/G488R
Subject 5 G488R/G488R
At screening TSH (mU/l) Free T4 (ng/dl)
76.8 0.11
26.9 1.0
>200 0.25
77.0 0.24
At re-evaluation TSH (mU/l) Free T4 (ng/dl)
13.4 1.3
3.8 1.2
2.3 1.4
2.8 1.5
Permanent CH
Transient CH
Transient CH
Transient CH
Fig. 3. Pedigrees of familial CH.
258
Horm Res Paediatr 2014;82:252–260 DOI: 10.1159/000362235
erozygous mutation in TSHR manifest overt hypothyroidism [7]. Functional analysis of missense DUOX2 variants in vitro showed wide variability in relative H2O2 production. Interestingly, p.H678R and p.G206V yielded a high H2O2 production of more than 80%, although the difference with wild-type alleles was statistically significant. The p.H678R variant was present in 3 subjects with transient hypothyroidism and in 7 subjects with permanent hypothyroidism. In most cases except 4 (subjects 6, 20, 21, 22), the variant was associated with other mutations in DUOX2 in the other allele or a second gene mutation. In 3 subjects with permanent hypothyroidism (subjects 20, 21 and 22), the p.H678R variant was the only alteration found. Considering that p.H678R is a benign variant, it is noticeable that subjects with p.H678R have hypothyroidism. However, subjects 6 and 22 with heterozygous p.H678R have transient and permanent hypothyroidism, respectively. Also, subjects 20 and 21 with homozygous p.H678R have permanent hypothyroidism. Therefore, it is presumed that CH is not solely caused by p. H678R and would be pathogenic only when a second gene mutation or as-yet unidentified genetic defects coexists, necessitating further analysis on epigenetic, environmental, or other genetic modifiers, as suggested previously [7]. Jin/Heo/Kim/Kim/Choi/Lee/Yoo
Downloaded by: Univ. of California San Diego 132.239.1.231 - 10/2/2014 4:08:55 PM
ranging from 5 to 10 mU/l) was observed in the mild dysfunction group. In silico studies have suggested that one variant (p.P96L) is benign in all six prediction programs. However, the absence of the p.P96L variant in healthy controls and in the 1000 Genomes database suggests the variant is unique to our cohort. The p.P96L variant might contribute to partial defects in the organification process and thus partially impair H2O2 generation during in vitro functional analyses. As described earlier, the broad phenotypic spectrum of CH with a eutopic thyroid is presumed to be the result of other genetic modifiers, epigenetic changes, second gene mutations, or differences related to ethnicity, nutrition, or environmental factors. For example, the p. H678R variant has been regarded as an SNP, based on previous findings where it was observed in 8 out of 60 controls (13.3%) and where it showed a similar amount of H2O2 release in functional analysis compared to what was seen in a normal control [11]. In addition, minor allele frequency of p.H678R in Japanese control was 3.5%, which is similar to our study, indicating a polymorphism [7]. However, in our current study, p.H678R showed a partial loss of functional activity, similar to an earlier Japanese study, indicating functional SNP [7]. Furthermore, homozygous DUOX2 p.H678R cases with a het-
On the other hand, the p.G206V variant was found in 1 subject with transient hypothyroidism (subject 1) and in another with permanent hypothyroidism (subject 17). In both cases, p.G206V was associated with other mutations in DUOX2 in the other allele. These data could suggest that p.H678R and p.G206V are benign variants or functional SNPs, and that subjects 21 and 22 may harbor another mutation in an as-yet unidentified gene. However, the luminescence-based method to determine H2O2 generation in vitro have shown unexplained inconsistencies in the capacity to detect WT DUOX2 and DUOXA2 activity, resulting in an eightfold variation in H2O2 generation [9, 11]. Therefore, it cannot exclude possibility that p.H678R and p.G206V are pathogenic mutations. Subject 10, who had monoallelic DUOX2 variant (p. H678R), was heterozygous for the TSHR variant p.G132R. Subjects 11 and 12 were heterozygous for TSHR, p.G132R and p.R450H, respectively. At re-evaluation, subjects 10– 12 showed persistent hyperthyrotropinemia (TSH 5–10 mU/l) until 6 months after discontinuation of levothyroxine, resulting in resuming of treatment. Considering the benign nature of the p.H678R variant and autosomal recessive pattern of thyroid dyshormonogenesis, it is possible that these 3 subjects have genetic defects in other genes that were not screened in this study. Subject 16, who had monoallelic DUOX2 variant (p.A1123T), was
heterozygous for the TSHR variant p.R450H. These findings suggest digenic causes are the one of the feature in CH with a eutopic thyroid and the necessity to screen for other genes, such as TPO, TG, NIS, PDS, and TSHR, in patients with dyshormonogenic CH. The limitation of this study is that perchlorate discharge tests were not performed, so it remains speculative whether the subjects have dyshomonogenesis caused by complete or partial iodide organification defects. In conclusion, DUOX2 variants frequently caused transient or permanent CH in subjects with normal-sized or enlarged eutopic thyroid glands. Moreover, screening of DUOX2 is helpful in patients with a non-syndromic partial iodide organification defect to verify the underlying etiology of CH. Some functional SNPs and monoallelic DUOX2 mutations have been identified in many cases of transient hypothyroidism. The phenotypic variability of patients with DUOX2 mutations could be explained by digenicity, epigenetic changes, and ethnic or environmental differences. Acknowledgement This study was supported by a grant from the Korean Center for Disease Control and Prevention, The Ministry for Health, Welfare and Family Affairs, Republic of Korea (Grant No. 2013E7400400).
References
Molecular Basis of Congenital Hypothyroidism
6 Corbetta C, Weber G, Cortinovis F, Calebiro D, Passoni A, Vigone MC, Beck-Peccoz P, Chiumello G, Persani L: A 7-year experience with low blood TSH cutoff levels for neonatal screening reveals an unsuspected frequency of congenital hypothyroidism. Clin Endocrinol (Oxf) 2009;71:739–745. 7 Narumi S, Muroya K, Asakura Y, Aachi M, Hasegawa T: Molecular basis of thyroid dyshormonogenesis: genetic screening in population-based Japanese patients. J Clin Endocrinol Metab 2011;96:E1838–E1842. 8 Maruo Y, Takahashi H, Soeda I, Nishikura N, Matsui K, Ota Y, Mimura Y, Mori A, Sato H, Takeuchi Y: Transient congenital hypothyroidism caused by biallelic mutations of the dual oxidase 2 gene in Japanese patients detected by a neonatal screening program. J Clin Endocrinol Metab 2008; 93: 4261– 4267. 9 Tonacchera M, De Marco G, Agretti P, Montanelli L, Di Cosmo C, Freitas Ferreira AC, Dimida A, Ferrarini E, Ramos HE, Ceccarelli C, Brozzi F, Pinchera A, Vitti P: Identification and functional studies of two new dual-oxi-
dase 2 (DUOX2) mutations in a child with congenital hypothyroidism and a eutopic normal-size thyroid gland. J Clin Endocrinol Metab 2009;94:4309–4314. 10 Narumi S, Muroya K, Abe Y, Yasui M, Asakura Y, Adachi M, Hasegawa T: TSHR mutations as a cause of congenital hypothyroidism in Japan: a population-based genetic epidemiology study. J Clin Endocrinol Metab 2009;94: 1317–1323. 11 De Marco G, Agretti P, Montanelli L, Di Cosmo C, Bagattini B, De Servi M, Ferrarini E, Dimida A, Freitas Ferreira AC, Molinaro A, Ceccarelli C, Brozzi F, Pinchera A, Vitti P, Tonacchera M: 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–E1339. 12 Grasberger H, De Deken X, Miot F, Pohlenz J, Refetoff S: Missense mutations of dual oxidase 2 (DUOX2) implicated in congenital hypothyroidism have impaired trafficking in cells reconstituted with DUOX2 maturation factor. Mol Endocrinol 2007;21:1408–1421.
Horm Res Paediatr 2014;82:252–260 DOI: 10.1159/000362235
259
Downloaded by: Univ. of California San Diego 132.239.1.231 - 10/2/2014 4:08:55 PM
1 Rastogi MV, LaFranchi SH: Congenital hypothyroidism. Orphanet J Rare Dis 2010;5:17. 2 Park SM, Chatterjee VK: Genetics of congenital hypothyroidism. J Med Genet 2005; 42: 379–389. 3 Grasberger H, Refetoff S: Genetic causes of congenital hypothyroidism due to dyshormonogenesis. Curr Opin Pediatr 2011; 23: 421–428. 4 Bakker B, Bikker H, Vulsma T, de Randamie JS, Wiedijk BM, De Vijlder JJ: Two decades of screening for congenital hypothyroidism in the Netherlands: TPO gene mutations in total iodide organification defects (an update). J Clin Endocrinol Metab 2000; 85: 3708–3712. 5 Hishinuma A, Fukata S, Nishiyama S, Nishi Y, Oh-Ishi M, Murata Y, Ohyama Y, Matsuura N, Kasai K, Harada S, Kitanaka S, Takamatsu J, Kiwaki K, Ohye H, Uruno T, Tomoda C, Tajima T, Kuma K, Miyauchi A, Ieiri T: Haplotype analysis reveals founder effects of thyroglobulin gene mutations C1058R and C1977S in Japan. J Clin Endocrinol Metab 2006;91:3100–3104.
260
17 Yasumoto M, Inoue H, Ohashi I, Shibuya H, Onishi T: Simple new technique for sonographic measurement of the thyroid in neonates and small children. J Clin Ultrasound 2004;32:82–85. 18 Takashima S, Nomura N, Tanaka H, Itoh Y, Miki K, Harada T: Congenital hypothyroidism: assessment with ultrasound. AJNR Am J Neuroradiol 1995;16:1117–1123. 19 Srinivasan R, Harigopal S, Turner S, Cheetham T: Permanent and transient congenital hypothyroidism in preterm infants. Acta Paediatr 2012;101:e179–e182. 20 Surks MI, Ortiz E, Daniels GH, Sawin CT, Col NF, Cobin RH, Franklyn JA, Hershman JM, Burman KD, Denke MA, Gorman C, Cooper RS, Weissman NJ: Subclinical thyroid disease: scientific review and guidelines for diagnosis and management. JAMA 2004;291:228–238.
Horm Res Paediatr 2014;82:252–260 DOI: 10.1159/000362235
21 Camilot M, Teofoli F, Gandini A, Franceschi R, Rapa A, Corrias A, Bona G, Radetti G, Tato L: Thyrotropin receptor gene mutations and TSH resistance: variable expressivity in the heterozygotes. Clin Endocrinol (Oxf) 2005; 63:146–151. 22 Grasberger H, Refetoff S: Identification of the maturation factor for dual oxidase. Evolution of a eukaryotic operon equivalent. J Biol Chem 2006;281:18269–18272. 23 Lee ST, Lee DH, Kim JY, Kwon MJ, Kim JW, Hong YH, Lee YW, Ki CS: Molecular screening of the TSH receptor and thyroid peroxidase genes in Korean patients with non-syndromic congenital hypothyroidism. Clin Endocrinol (Oxf) 2011; 75: 715– 721. 24 Narumi S, Muroya K, Asakura Y, Adachi M, Hasegawa T: Transcription factor mutations and congenital hypothyroidism: systematic genetic screening of a population-based cohort of Japanese patients. J Clin Endocrinol Metab 2010;95:1981–1985. 25 Grasberger H: Defects of thyroidal hydrogen peroxide generation in congenital hypothyroidism. Mol Cell Endocrinol 2010; 322: 99– 106.
Jin/Heo/Kim/Kim/Choi/Lee/Yoo
Downloaded by: Univ. of California San Diego 132.239.1.231 - 10/2/2014 4:08:55 PM
13 Hoste C, Rigutto S, Van Vliet G, Miot F, De Deken X: Compound heterozygosity for a novel hemizygous missense mutation and a partial deletion affecting the catalytic core of the H2O2-generating enzyme DUOX2 associated with transient congenital hypothyroidism. Hum Mutat 2010;31:E1304–E1319. 14 Moreno JC, Bikker H, Kempers MJ, van Trotsenburg AS, Baas F, de Vijlder JJ, Vulsma T, Ris-Stalpers C: Inactivating mutations in the gene for thyroid oxidase 2 (THOX2) and congenital hypothyroidism. N Engl J Med 2002; 347:95–102. 15 Vigone MC, Fugazzola L, Zamproni I, Passoni A, Di Candia S, Chiumello G, Persani L, Weber G: Persistent mild hypothyroidism associated with novel sequence variants of the DUOX2 gene in two siblings. Hum Mutat 2005;26:395. 16 Sriphrapradang C, Tenenbaum-Rakover Y, Weiss M, Barkoff MS, Admoni O, Kawthar D, Caltabiano G, Pardo L, Dumitrescu AM, Refetoff S: The coexistence of a novel inactivating mutant thyrotropin receptor allele with two thyroid peroxidase mutations: a genotype-phenotype correlation. J Clin Endocrinol Metab 2011;96:E1001–E1006.
HOR MON E RE SE ARCH I N PÆDIATRIC S
Received: November 11, 2013 Accepted: March 11, 2014 Published online: September 20, 2014
Horm Res Paediatr 2014;82:252–260 DOI: 10.1159/000362235
High Frequency of DUOX2 Mutations in Transient or Permanent Congenital Hypothyroidism with Eutopic Thyroid Glands Hye Young Jin a Sun-Hee Heo b Yoo-Mi Kim a Gu-Hwan Kim c Jin-Ho Choi a Beom-Hee Lee a Han-Wook Yoo a
a
Division of Pediatric Endocrinology and Metabolism, Department of Pediatrics, b Genome Research Center for Birth Defects and Genetic Disorders, and c Medical Genetics Center, Asan Medical Center Children’s Hospital, University of Ulsan College of Medicine, Seoul, Korea
Abstract Background/Aims: This study aimed to clarify the frequency, phenotypes, and molecular spectrum of DUOX2, TPO, TSHR, and TG mutations in patients with congenital hypothyroidism (CH) with enlarged or normal-sized eutopic thyroid glands. Methods: The study cohort included 43 subjects from 41 unrelated families who had CH with eutopic thyroid glands. Mutation analyses of DUOX2, TPO, and TSHR were performed. The functional capacities of novel missense variants of DUOX2 were verified by measuring H2O2 generation in vitro. Results: Of the 43 subjects, 23 (53.5%) had sequence variants in at least one gene. Twelve different DUOX2 variants, including seven novel variants, were identified in 20 subjects. A functional analysis of the DUOX2 variants revealed that most variants, other than p.G206V and p.H678R, caused a significant reduction in H2O2 generation. Therefore, 15 subjects harbored functionally deleterious DUOX2 variants. Of these, 5 subjects had transient CH, and 10 were found to have permanent CH. Sequence variants in TSHR were identified in 5 subjects. One of the 43 subjects (2.3%) had sequence variants in two different genes. Conclusions: DUOX2 variants are a relatively common cause of CH with
© 2014 S. Karger AG, Basel 1663–2818/14/0824–0252$39.50/0 E-Mail [email protected] www.karger.com/hrp
normal-sized or enlarged eutopic thyroid glands. Variable phenotypes were associated with partial loss of the functional activity of DUOX2 variants. © 2014 S. Karger AG, Basel
Introduction
Patients with congenital hypothyroidism (CH) with a eutopic thyroid can have various etiologies: resistance to thyroid-stimulating hormone (TSH), thyroid dyshormonogenesis, and transient hypothyroidism [1]. Dyshormonogenic CH is caused by several genetic defects, including defects in thyroid peroxidase (TPO), thyroglobulin (TG), dual oxidase 2 (DUOX2), the dual oxidase maturation factor (DUOXA2), the sodium-iodide symporter (SLC5A5), pendrin (SLC26A4), and iodotyrosine deiodinase (IYD) [2, 3]. Most genetic defects responsible for dyshormonogenesis are inherited in an autosomal recessive manner and manifest as goitrous CH. The prevalence of thyroid dyshormonogenesis due to TPO and TG was reported to be 1: 66,000 in the Netherlands and 1:67,000 in Japan [4, 5]. Dyshormonogenesis with a eutopic thyroid appears to be the
H.Y.J. and S.-H.H. contributed equally to this work.
Han-Wook Yoo, MD, PhD Division of Pediatric Endocrinology and Metabolism, Department of Pediatrics Asan Medical Center Children’s Hospital, University of Ulsan College of Medicine 388-1, Pungnap-Dong, Songpa-Gu, Seoul 138-736 (Korea) E-Mail hwyoo @ amc.seoul.kr
Downloaded by: Univ. of California San Diego 132.239.1.231 - 10/2/2014 4:08:55 PM
Key Words Congenital hypothyroidism · DUOX2 · Dyshormonogenesis
TSH, free T4, and thyroglobulin (Tg). In case of hypothyroidism in siblings, thyroid functions were followed up, even though their screening TSH levels were lower than 10 mU/l. Antibodies (Ab) against thyroperoxidase (TPO) (anti-TPO Ab), Tg (anti-Tg Ab), and the TSH receptor (anti-TSH Ab) were also measured. At confirmatory diagnosis, subjects with the TSH levels higher than 10 mU/l were considered to have CH [6, 11]. A 99mTc scintigraphy was performed to assess the location and size of the thyroid gland. Thyroid size was measured using ultrasonography according to a Japanese study [17]; thyroid widths equal to or >+2 SD were defined as goiter, whereas ones equal to or +2 SD.
1 (3/F)b 2 (4/M) 3 (4/M) 4 (7/M)c 5 (5/F)c 6 (5/M)
Subject No. (current age, years/gender)
Table 1. Hormonal and mutational data for patients with transient CH in the study cohort harboring sequence variants
none none none none TSHR: p.G132R/ p.G132R
none none none TSHR: p.G132R TSHR: p.G132R TSHR: p.R450H none none none TSHR: p.R450H none none
Second gene (TPO, TSHR, TG)
none none none none none none
Second gene (TPO, TSHR, TG)
in A549 cells transfected with the indicated expression vectors to measure H2O2-producing capacity. The activity of each mutant is expressed as a percentage of wildtype activity. Each bar represents the mean, and the error bars represent SDS. The p. G206V and p.H678R mutants showed relatively high residual function (more than 75% of the wild-type activity). The significant inhibition of H2O2-generating activity was observed when the remaining mutant vectors were transfected into the A549 cells (* p < 0.05).
100
*
* 80
40
*
0
Em
*
*
20
pty
*
*
60
ve
*
*
*
*
r L e T S Y K R R V Q Q c to t y p N 4 3 A 7 2 P 9 6 2 0 6 4 8 8 6 7 8 E 8 7 9 8 8 5 1 1 0 1 1 2 3 3 3 4 W G 1 G H R ld i A R R1 W
allelic variants in DUOX2 (subjects 1–3, 7–9, 13–17, 19), 8 (subjects 8, 9, 13–17, 19) turned out to have permanent CH. Of these, 1 patient (subject 16) was associated with a second gene mutation (TSHR p.R450H) (table 2). Six patients (subjects 7–12) showed persistent TSH elevation after levothyroxine withdrawal at age 3. Their TSH levels were followed up until 3–6 months after age 3, and eventually levothyroxine treatment was resumed. Among the 4 familial cases from two independent pedigrees, subjects 1 and 17 were compound heterozygous for p.G206V and p.E879K, and subjects 4 and 5, siblings born from non-consanguineous parents, were homozygous for p.G488R in DUOX2. The parents of subjects 1 and 17, heterozygous for either substitution (p.G206V or p.E879K), were thought to be in a clinically euthyroid state without a goiter based on medical history, although their thyroid function tests were not available. The parents of subjects 4 and 5, who were heterozygous for p. G488R, had normal TSH levels, suggesting autosomal recessive inheritance (fig. 3).
The findings in this study demonstrate that DUOX2 mutations are one of the most common causes of dyshormonogenic CH. A significant portion of the cases appear to be due to the impairment of H2O2 generation caused by DUOX2 mutations. A recent Japanese study demonstrated that DUOX2 mutations are one of the leading causes of permanent CH, comprising 41% of dyshor-
monogenesis cases [7, 10, 24]. Our study included transient and permanent CH cases with normal-sized to enlarged eutopic thyroids, which we found appeared at a comparable frequency (43.9%). Unlike other dyshormonogenic defects, such as those caused by mutations of TPO, TG, NIS, and PDS, monoallelic DUOX2 mutations cause milder, transient hypothyroidism, whereas biallelic mutations result in severe, permanent hypothyroidism [14, 25]. In the present study, subjects 1, 4, and 5 with biallelic DUOX2 variants were confirmed as having transient CH, although they presented with overt hypothyroidism at the initial diagnosis, and subjects 13 and 19 with monoallelic DUOX2 variants were considered to have permanent CH. Our finding that phenotypic variability is not related to the number of mutant alleles is consistent with previous reports on transient, mild phenotypes in CH patients with biallelic DUOX2 mutations [8, 15]. Variable phenotypes are presumed to be caused by (1) another H2O2-generating oxidase in thyrocytes, such as DUOX1; (2) other genetic factors such as DUOXA2; (3) ethnic differences, and (4) environmental factors such as iodine intake [8, 15]. However, possibilities of monoallelic mutation in one allele and deletion of DUOX2 in the other allele cannot be ruled out unless array comparative genomic hybridization is performed [13]. Both transient and permanent CH were found to be associated with DUOX2 mutations in our present analyses. On re-evaluation of the patients at 3 years of age, serum TSH levels were not so high in the permanent CH group, whereas mild hyperthyrotropinemia (TSH levels
Molecular Basis of Congenital Hypothyroidism
Horm Res Paediatr 2014;82:252–260 DOI: 10.1159/000362235
Discussion
257
Downloaded by: Univ. of California San Diego 132.239.1.231 - 10/2/2014 4:08:55 PM
Fig. 2. DUOX2-mediated H2O2 generation
Relative H2O2 production (%)
120
DUOX2
G206V/H678R
E879K/WT
G488R/WT
WT/G488R
DUOX2
Subject 17 G206V/WT WT/E879K H678R/WT
Subject 1 G206V/WT WT/E879K H678R/WT
Subject 4 G488R/G488R
Subject 5 G488R/G488R
At screening TSH (mU/l) Free T4 (ng/dl)
76.8 0.11
26.9 1.0
>200 0.25
77.0 0.24
At re-evaluation TSH (mU/l) Free T4 (ng/dl)
13.4 1.3
3.8 1.2
2.3 1.4
2.8 1.5
Permanent CH
Transient CH
Transient CH
Transient CH
Fig. 3. Pedigrees of familial CH.
258
Horm Res Paediatr 2014;82:252–260 DOI: 10.1159/000362235
erozygous mutation in TSHR manifest overt hypothyroidism [7]. Functional analysis of missense DUOX2 variants in vitro showed wide variability in relative H2O2 production. Interestingly, p.H678R and p.G206V yielded a high H2O2 production of more than 80%, although the difference with wild-type alleles was statistically significant. The p.H678R variant was present in 3 subjects with transient hypothyroidism and in 7 subjects with permanent hypothyroidism. In most cases except 4 (subjects 6, 20, 21, 22), the variant was associated with other mutations in DUOX2 in the other allele or a second gene mutation. In 3 subjects with permanent hypothyroidism (subjects 20, 21 and 22), the p.H678R variant was the only alteration found. Considering that p.H678R is a benign variant, it is noticeable that subjects with p.H678R have hypothyroidism. However, subjects 6 and 22 with heterozygous p.H678R have transient and permanent hypothyroidism, respectively. Also, subjects 20 and 21 with homozygous p.H678R have permanent hypothyroidism. Therefore, it is presumed that CH is not solely caused by p. H678R and would be pathogenic only when a second gene mutation or as-yet unidentified genetic defects coexists, necessitating further analysis on epigenetic, environmental, or other genetic modifiers, as suggested previously [7]. Jin/Heo/Kim/Kim/Choi/Lee/Yoo
Downloaded by: Univ. of California San Diego 132.239.1.231 - 10/2/2014 4:08:55 PM
ranging from 5 to 10 mU/l) was observed in the mild dysfunction group. In silico studies have suggested that one variant (p.P96L) is benign in all six prediction programs. However, the absence of the p.P96L variant in healthy controls and in the 1000 Genomes database suggests the variant is unique to our cohort. The p.P96L variant might contribute to partial defects in the organification process and thus partially impair H2O2 generation during in vitro functional analyses. As described earlier, the broad phenotypic spectrum of CH with a eutopic thyroid is presumed to be the result of other genetic modifiers, epigenetic changes, second gene mutations, or differences related to ethnicity, nutrition, or environmental factors. For example, the p. H678R variant has been regarded as an SNP, based on previous findings where it was observed in 8 out of 60 controls (13.3%) and where it showed a similar amount of H2O2 release in functional analysis compared to what was seen in a normal control [11]. In addition, minor allele frequency of p.H678R in Japanese control was 3.5%, which is similar to our study, indicating a polymorphism [7]. However, in our current study, p.H678R showed a partial loss of functional activity, similar to an earlier Japanese study, indicating functional SNP [7]. Furthermore, homozygous DUOX2 p.H678R cases with a het-
On the other hand, the p.G206V variant was found in 1 subject with transient hypothyroidism (subject 1) and in another with permanent hypothyroidism (subject 17). In both cases, p.G206V was associated with other mutations in DUOX2 in the other allele. These data could suggest that p.H678R and p.G206V are benign variants or functional SNPs, and that subjects 21 and 22 may harbor another mutation in an as-yet unidentified gene. However, the luminescence-based method to determine H2O2 generation in vitro have shown unexplained inconsistencies in the capacity to detect WT DUOX2 and DUOXA2 activity, resulting in an eightfold variation in H2O2 generation [9, 11]. Therefore, it cannot exclude possibility that p.H678R and p.G206V are pathogenic mutations. Subject 10, who had monoallelic DUOX2 variant (p. H678R), was heterozygous for the TSHR variant p.G132R. Subjects 11 and 12 were heterozygous for TSHR, p.G132R and p.R450H, respectively. At re-evaluation, subjects 10– 12 showed persistent hyperthyrotropinemia (TSH 5–10 mU/l) until 6 months after discontinuation of levothyroxine, resulting in resuming of treatment. Considering the benign nature of the p.H678R variant and autosomal recessive pattern of thyroid dyshormonogenesis, it is possible that these 3 subjects have genetic defects in other genes that were not screened in this study. Subject 16, who had monoallelic DUOX2 variant (p.A1123T), was
heterozygous for the TSHR variant p.R450H. These findings suggest digenic causes are the one of the feature in CH with a eutopic thyroid and the necessity to screen for other genes, such as TPO, TG, NIS, PDS, and TSHR, in patients with dyshormonogenic CH. The limitation of this study is that perchlorate discharge tests were not performed, so it remains speculative whether the subjects have dyshomonogenesis caused by complete or partial iodide organification defects. In conclusion, DUOX2 variants frequently caused transient or permanent CH in subjects with normal-sized or enlarged eutopic thyroid glands. Moreover, screening of DUOX2 is helpful in patients with a non-syndromic partial iodide organification defect to verify the underlying etiology of CH. Some functional SNPs and monoallelic DUOX2 mutations have been identified in many cases of transient hypothyroidism. The phenotypic variability of patients with DUOX2 mutations could be explained by digenicity, epigenetic changes, and ethnic or environmental differences. Acknowledgement This study was supported by a grant from the Korean Center for Disease Control and Prevention, The Ministry for Health, Welfare and Family Affairs, Republic of Korea (Grant No. 2013E7400400).
References
Molecular Basis of Congenital Hypothyroidism
6 Corbetta C, Weber G, Cortinovis F, Calebiro D, Passoni A, Vigone MC, Beck-Peccoz P, Chiumello G, Persani L: A 7-year experience with low blood TSH cutoff levels for neonatal screening reveals an unsuspected frequency of congenital hypothyroidism. Clin Endocrinol (Oxf) 2009;71:739–745. 7 Narumi S, Muroya K, Asakura Y, Aachi M, Hasegawa T: Molecular basis of thyroid dyshormonogenesis: genetic screening in population-based Japanese patients. J Clin Endocrinol Metab 2011;96:E1838–E1842. 8 Maruo Y, Takahashi H, Soeda I, Nishikura N, Matsui K, Ota Y, Mimura Y, Mori A, Sato H, Takeuchi Y: Transient congenital hypothyroidism caused by biallelic mutations of the dual oxidase 2 gene in Japanese patients detected by a neonatal screening program. J Clin Endocrinol Metab 2008; 93: 4261– 4267. 9 Tonacchera M, De Marco G, Agretti P, Montanelli L, Di Cosmo C, Freitas Ferreira AC, Dimida A, Ferrarini E, Ramos HE, Ceccarelli C, Brozzi F, Pinchera A, Vitti P: Identification and functional studies of two new dual-oxi-
dase 2 (DUOX2) mutations in a child with congenital hypothyroidism and a eutopic normal-size thyroid gland. J Clin Endocrinol Metab 2009;94:4309–4314. 10 Narumi S, Muroya K, Abe Y, Yasui M, Asakura Y, Adachi M, Hasegawa T: TSHR mutations as a cause of congenital hypothyroidism in Japan: a population-based genetic epidemiology study. J Clin Endocrinol Metab 2009;94: 1317–1323. 11 De Marco G, Agretti P, Montanelli L, Di Cosmo C, Bagattini B, De Servi M, Ferrarini E, Dimida A, Freitas Ferreira AC, Molinaro A, Ceccarelli C, Brozzi F, Pinchera A, Vitti P, Tonacchera M: 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–E1339. 12 Grasberger H, De Deken X, Miot F, Pohlenz J, Refetoff S: Missense mutations of dual oxidase 2 (DUOX2) implicated in congenital hypothyroidism have impaired trafficking in cells reconstituted with DUOX2 maturation factor. Mol Endocrinol 2007;21:1408–1421.
Horm Res Paediatr 2014;82:252–260 DOI: 10.1159/000362235
259
Downloaded by: Univ. of California San Diego 132.239.1.231 - 10/2/2014 4:08:55 PM
1 Rastogi MV, LaFranchi SH: Congenital hypothyroidism. Orphanet J Rare Dis 2010;5:17. 2 Park SM, Chatterjee VK: Genetics of congenital hypothyroidism. J Med Genet 2005; 42: 379–389. 3 Grasberger H, Refetoff S: Genetic causes of congenital hypothyroidism due to dyshormonogenesis. Curr Opin Pediatr 2011; 23: 421–428. 4 Bakker B, Bikker H, Vulsma T, de Randamie JS, Wiedijk BM, De Vijlder JJ: Two decades of screening for congenital hypothyroidism in the Netherlands: TPO gene mutations in total iodide organification defects (an update). J Clin Endocrinol Metab 2000; 85: 3708–3712. 5 Hishinuma A, Fukata S, Nishiyama S, Nishi Y, Oh-Ishi M, Murata Y, Ohyama Y, Matsuura N, Kasai K, Harada S, Kitanaka S, Takamatsu J, Kiwaki K, Ohye H, Uruno T, Tomoda C, Tajima T, Kuma K, Miyauchi A, Ieiri T: Haplotype analysis reveals founder effects of thyroglobulin gene mutations C1058R and C1977S in Japan. J Clin Endocrinol Metab 2006;91:3100–3104.
260
17 Yasumoto M, Inoue H, Ohashi I, Shibuya H, Onishi T: Simple new technique for sonographic measurement of the thyroid in neonates and small children. J Clin Ultrasound 2004;32:82–85. 18 Takashima S, Nomura N, Tanaka H, Itoh Y, Miki K, Harada T: Congenital hypothyroidism: assessment with ultrasound. AJNR Am J Neuroradiol 1995;16:1117–1123. 19 Srinivasan R, Harigopal S, Turner S, Cheetham T: Permanent and transient congenital hypothyroidism in preterm infants. Acta Paediatr 2012;101:e179–e182. 20 Surks MI, Ortiz E, Daniels GH, Sawin CT, Col NF, Cobin RH, Franklyn JA, Hershman JM, Burman KD, Denke MA, Gorman C, Cooper RS, Weissman NJ: Subclinical thyroid disease: scientific review and guidelines for diagnosis and management. JAMA 2004;291:228–238.
Horm Res Paediatr 2014;82:252–260 DOI: 10.1159/000362235
21 Camilot M, Teofoli F, Gandini A, Franceschi R, Rapa A, Corrias A, Bona G, Radetti G, Tato L: Thyrotropin receptor gene mutations and TSH resistance: variable expressivity in the heterozygotes. Clin Endocrinol (Oxf) 2005; 63:146–151. 22 Grasberger H, Refetoff S: Identification of the maturation factor for dual oxidase. Evolution of a eukaryotic operon equivalent. J Biol Chem 2006;281:18269–18272. 23 Lee ST, Lee DH, Kim JY, Kwon MJ, Kim JW, Hong YH, Lee YW, Ki CS: Molecular screening of the TSH receptor and thyroid peroxidase genes in Korean patients with non-syndromic congenital hypothyroidism. Clin Endocrinol (Oxf) 2011; 75: 715– 721. 24 Narumi S, Muroya K, Asakura Y, Adachi M, Hasegawa T: Transcription factor mutations and congenital hypothyroidism: systematic genetic screening of a population-based cohort of Japanese patients. J Clin Endocrinol Metab 2010;95:1981–1985. 25 Grasberger H: Defects of thyroidal hydrogen peroxide generation in congenital hypothyroidism. Mol Cell Endocrinol 2010; 322: 99– 106.
Jin/Heo/Kim/Kim/Choi/Lee/Yoo
Downloaded by: Univ. of California San Diego 132.239.1.231 - 10/2/2014 4:08:55 PM
13 Hoste C, Rigutto S, Van Vliet G, Miot F, De Deken X: Compound heterozygosity for a novel hemizygous missense mutation and a partial deletion affecting the catalytic core of the H2O2-generating enzyme DUOX2 associated with transient congenital hypothyroidism. Hum Mutat 2010;31:E1304–E1319. 14 Moreno JC, Bikker H, Kempers MJ, van Trotsenburg AS, Baas F, de Vijlder JJ, Vulsma T, Ris-Stalpers C: Inactivating mutations in the gene for thyroid oxidase 2 (THOX2) and congenital hypothyroidism. N Engl J Med 2002; 347:95–102. 15 Vigone MC, Fugazzola L, Zamproni I, Passoni A, Di Candia S, Chiumello G, Persani L, Weber G: Persistent mild hypothyroidism associated with novel sequence variants of the DUOX2 gene in two siblings. Hum Mutat 2005;26:395. 16 Sriphrapradang C, Tenenbaum-Rakover Y, Weiss M, Barkoff MS, Admoni O, Kawthar D, Caltabiano G, Pardo L, Dumitrescu AM, Refetoff S: The coexistence of a novel inactivating mutant thyrotropin receptor allele with two thyroid peroxidase mutations: a genotype-phenotype correlation. J Clin Endocrinol Metab 2011;96:E1001–E1006.
