TSHR Mutations as a Cause of Congenital Hypothyroidism in Japan: A ...

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TSHR Mutations as a Cause of Congenital Hypothyroidism in Japan: A Population-Based Genetic Epidemiology Study Satoshi Narumi, Koji Muroya, Yoichiro Abe, Masato Yasui, Yumi Asakura, Masanori Adachi, and Tomonobu Hasegawa Departments of Pediatrics (S.N., T.H.) and Pharmacology (Y.Ab., M.Y.), Keio University School of Medicine, Tokyo 160-8582, Japan; Department of Endocrinology and Metabolism (K.M., Y.As., M.A.), Kanagawa Children’s Medical Center, Yokohama 232-8555, Japan; and Neonatal Mass-Screening Committee (M.A.), Kanagawa Prefecture Medical Association, Yokohama 231-0037, Japan

Context: The prevalence of congenital hypothyroidism (CH) associated with mutations in the TSH receptor gene (TSHR) has not been established. Objective: We examined the frequency of TSHR mutations among patients with permanent primary CH and in the general population in Japan. Subjects and Methods: We enrolled 102 patients with permanent primary CH 关70 with “moderate to severe CH” (TSH, ⱖ10 mU/liter) and 32 with “mild CH” (TSH, 5–10 mU/liter)兴, who were identified through newborn screening among 353,000 newborns born in Kanagawa prefecture from October 1979 to June 2006. These subjects were tested for TSHR mutations by PCR-based direct sequencing. We further characterized molecular functions of identified mutant TSHRs in vitro. Results: We found three patients with moderate to severe CH who had biallelic mutations in TSHR and three patients with mild CH who had monoallelic mutations. Observed mutations included one previously characterized mutation (p.R450H) and three uncharacterized mutations (p.G132R, p.A204V, and p.D403N). In vitro experiments confirmed loss of functions of these four mutants. Among four mutations, p.R450H was particularly frequent: six of nine mutant alleles harbored p.R450H. All six alleles with p.R450H commonly carried a minor single nucleotide polymorphism, suggesting a founder effect. We estimated the prevalence of biallelic TSHR mutations to be 4.3% (three in 70) in Japanese patients with moderate to severe CH, and 1 in 118,000 (three in 353,000) in the general Japanese population. Conclusions: In Japan, TSHR mutations are relatively common among patients with CH, and a founder mutation (p.R450H) accounts for about 70% of mutants. (J Clin Endocrinol Metab 94: 1317–1323, 2009)

he most common congenital endocrine disorder is congenital hypothyroidism (CH). Permanent primary CH typically occurs in 1 in 3,000 to 4,000 births (1). Today, most of developed countries have introduced newborn screening for CH, and nearly all patients are diagnosed in their early infancy. These circumstances have been providing opportunities for various types of epidemiological investigations on CH.

T

Clinically, CH is classified into two types based on the presence of goiter: goitrous CH or nongoitrous CH. The etiology of goitrous CH is strongly associated with inborn errors of thyroid hormone synthesis; mutations in the gene involving thyroid hormone synthesis (e.g. TPO, TG etc.) are frequently observed among these patients (2– 4). In contrast, the etiology of nongoitrous CH remains obscure. Previous molecular genetic studies have identified seem-

ISSN Print 0021-972X ISSN Online 1945-7197 Printed in U.S.A. Copyright © 2009 by The Endocrine Society doi: 10.1210/jc.2008-1767 Received August 11, 2008. Accepted January 9, 2009. First Published Online January 21, 2009

Abbreviations: CH, Congenital hypothyroidism; FT4, free T4; SNP, single nucleotide polymorphism; TSHR, TSH receptor; WT, wild-type.

J Clin Endocrinol Metab, April 2009, 94(4):1317–1323

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Prevalence of TSHR Mutations in Japan

ingly rare cases with gene defects of TSH receptor (TSHR) (5) or thyroid-expressed transcription factors (e.g. PAX8, TITF1, FOXE1) (6 – 8). However, to what extent the etiology of nongoitrous CH can be explained by these mutations remains undetermined. One of the genes that, when mutated, can cause nongoitrous CH is TSHR (5). To date, more than 50 congenitally hypothyroid patients with biallelic inactivating TSHR mutations have been described. The prevalence of this gene defect has not been examined, although it is presumed to be rare (9). Here we report the first population-based investigation on the prevalence of inactivating TSHR mutations. We enrolled 102 patients with permanent primary CH who represented 353,000 regional general populations and screened for mutations in TSHR. The primary objective was to define the prevalence of biallelic inactivating TSHR mutations among patients with permanent primary CH and in the general population in Japan.

Subjects and Methods Study site Clinical study sites were all in Kanagawa prefecture, which is located in the central part of Japan and had about 8.9 million inhabitants by the end of 2006 (approximately 7% of the total Japanese population). Average amount of consumption of seaweed (a major source of dietary iodine) was 15.8 g/d in the prefecture and 14.3 g/d in Japan, according to the National Health and Nutrition Survey conducted in 2005.

Newborn screening The newborn screening procedure of Kanagawa prefecture has been based on determination of TSH and T4 or free T4 (FT4) in a filter-paper blood spot obtained at 4 –7 d after birth. Children with high TSH levels (ⱖ30 mU/liter) were immediately referred to pediatric endocrinologists. Children with borderline TSH levels (15–30 mU/liter) and/or low T4 levels (T4 ⬍4.0 ␮g/dl or FT4 ⬍0.7 ng/dl) were reevaluated. When the result was abnormal or again borderline, the child was referred to a pediatric endocrinologist.

Evaluation and selection of patients All study subjects have been followed at Kanagawa Children’s Medical Center. They were routinely evaluated for serum levels of TSH, free T3, and FT4 at the time of first visit. Thyroid morphology was evaluated by ultrasonography and/or scintigraphy in almost all patients with severe CH and in a part of patients with mild to moderate CH. When a patient was suspected of having transient CH or transient hyperthyrotropinemia, we discontinued the treatment and reassessed the thyroid function. We enrolled patients with permanent primary CH who met the criteria as follows: 1) having TSH levels of at least 5 mU/liter under discontinuation of treatment; or 2) having radiological evidence of thyroid dysgenesis (i.e. thyroid aplasia, ectopia, or severe hypoplasia). We excluded patients with any chromosomal abnormalities or central CH. We also excluded patients who had neither discontinuation data nor thyroid dysgenesis because they could have transient CH or hyperthyrotropinemia. We classified study subjects into two subgroups according to disease severity. Patients of which serum TSH levels were at least 10 mU/liter (under discontinuation) were classified as “moderate to severe CH,” whereas patients with TSH levels of 5–10 mU/liter (under discontinuation) were classified as “mild CH.” Patients who lacked discontinuation data but had radiological evidence of thyroid dysgenesis were classified as moderate to severe CH. We obtained written informed consent to participate in the study from the patients or parents. Institutional review boards of Kanagawa


Children’s Medical Center and Keio University School of Medicine approved the study.

Mutational analysis Genomic DNA was extracted from peripheral blood by a standard technique. All 10 coding exons and flanking introns of TSHR were analyzed by PCR-direct sequencing (for details, see the supplemental data published on The Endocrine Society’s Journals Online web site at http:// jcem.endojournals.org). Detected sequence variations were tested in 100 unrelated healthy Japanese individuals.

Allele-specific sequencing In the patients that were heterozygous for both p.D403N and p.R450H, PCR from genomic DNA was performed to amplify fragments spanning codons 403 and 450. In four patients that were heterozygous for p.R450H, PCR from genomic DNA was performed to amplify fragments spanning codon 450 and rs1991517 关a single nucleotide polymorphism (SNP) located 832-bp downstream of codon 450兴. Those fragments were subcloned with a TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA), and individual alleles were sequenced separately.

Construction of expression vectors cDNA encoding human TSHR was generated by PCR from human thyroid cDNA (Gene Pool Human cDNA; Invitrogen) with primers 5⬘-GAA TTC ACC ATG AGG CCG GCG GAC TTG CTG-3⬘ and 5⬘-CCG CGG TTA CAA AAC CGT TTG CAT ATA CTC-3⬘ and was subcloned into pCR2.1-TOPO. The expression vector was constructed as follows: 1) insert the SV40 polyadenylation signal (derived from pEGFP-N1) into SacII-SacI site of pBluescript II SK(⫹); 2) insert the SV40 promoter sequence (derived from pSV-␤-Galactosidase) into XhoI-HindIII site; and 3) insert the human TSHR cDNA via the introduced EcoRI and SacII restriction sites. The mutants for G132R, A204V, D403N, and R450H were introduced by site-directed mutagenesis (supplemental data). All final constructs were verified by direct DNA sequencing.

Cell culture and in vitro functional analysis COS-7 cells were maintained in DMEM supplemented with 50 U/ml penicillin, 50 ␮g/ml streptomycin, and 10% fetal bovine serum. Cells were transfected with each expression construct by Lipofectamine reagent enhanced with Plus Reagent (Invitrogen) according to the manufacturer’s instructions. For immunoblot analysis, crude cell extracts were prepared from the cells transfected for 48 h and were separated by 10% SDS-PAGE. Western blotting was performed with a monoclonal anti-TSHR antibody clone 2C11 (10) (Serotec Ltd., Oxford, UK) used at a 1:200 dilution. Horseradish peroxidase-labeled rat antimouse antibody IgG was used as a second antibody. Bound antibody was revealed with a chemiluminescence kit (GE Healthcare, Buckinghamshire, UK). For analysis of competitive TSH binding, 500,000 cells were transfected with each plasmid and were reseeded onto 24-well plates (100,000 cells/well) at 24 h after transfection. Forty-eight hours after transfection, the medium was removed, and the cells were incubated with 0.03 U/liter of 125I-TSH (60 ␮Ci/␮g, 80 U/mg; Cosmic Corporation, Tokyo, Japan) alone or in the presence of various concentrations of unlabeled bovine TSH (Sigma-Aldrich, St. Louis, MO) in the binding buffer 关10 mM HEPES/NaOH (pH 7.4), 140 mM NaCl, 4 mM KCl, 1 mM MgCl2, 1.25 mM CaCl2, 1 mM NaH2PO4, 11 mM D-glucose, 0.2% BSA; final volume, 0.2 ml兴 for 2 h at 37 C. Then, the buffer was removed, the cells were lysed with 0.2 ml of 1 M NaOH, and the radioactivity was counted by a gamma-counter. For analysis of TSH-stimulated cAMP production, 200,000 cells were transfected and were cultured for 24 h. Then, the cells were reseeded onto 96-well plates (10,000 cells/well). Forty-eight hours after transfection, the medium was removed, and the cells were incubated with various concentrations of bovine TSH in the binding buffer (final volume, 0.1 ml) for 30 min at 37 C. Subsequently, intracellular cAMP was measured by


an enzyme immunoassay kit (GE Healthcare) according to the manufacturer’s protocol.

Haplotype analysis Two polymorphic microsatellite markers, D14S0274i and D14S0071i (resides 134-kb upstream and 65-kb downstream of TSHR, respectively; data from http://www.h-invitational.jp/gdbs/, accessed on January 1, 2008) were used for haplotype analysis. PCR products were synthesized with fluorescently labeled primer pairs and were analyzed by an ABI310 (Applied Biosystems, Foster City, CA) (supplemental data). Haplotypes were established for each individual.

Statistics Between the participants and the patients who were eligible but did not participate, the age and the blood-spot TSH levels were compared by the Mann-Whitney U test, and the categorical variables were compared by the Fisher’s exact test. The results of the cAMP production studies were compared by the Welch’s t test.

Results Subjects Between October 1979 and June 2006, a total of 1,922,592 newborns were screened for CH in Kanagawa prefecture (Fig. 1). As a result, 3268 newborns were suspected of CH and were referred to pediatric endocrinologists. Among them, 1001 individuals (30.6%) visited to Kanagawa Children’s Medical Center. Of these individuals, 170 patients were eligible for enrollment. Fifty-two patients were not followed at the start of the study (January 2007), chiefly due to moving to other regions and transition to adult health care. The remaining 118 patients were being followed, and all of them (or their parents) were asked to

FIG. 1. Enrollment of the study subjects. Between October 1979 and June 2006, a total of 1,922,592 newborns were screened for CH in Kanagawa prefecture, and 3,268 were referred to pediatric endocrinologists. We evaluated 30.6% (1001 of 3268) of them. A total of 170 patients met the criteria for enrollment (described in Subjects and Methods), and 60.0% (102 of 170) of them participated in the study. Thus, the participants were thought to represent 353,000 (⫽ 1.92 ⫻ 106 ⫻ 30.6% ⫻ 60.0%) regional general populations. TBG, T4-binding globulin.


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TABLE 1. Characteristics of the participants Variable n (males/females) Age, median (range) (yr) Family history of CH, n (%) TSH at screening, median (range) (mU/liter) Thyroid morphology, n (%) Ectopia Aplasia Hypoplasia Goiter Normal Not evaluated

Moderate to severe CH 70 (31/39) 12.2 (3.1 to 25.2) 7 (10)

Mild CH 32 (16/16) 9.3 (3.8 to 21.2) 1 (3)

130.5 (16.9 to ⬎200) 34.9 (18.1 to ⬎200)

36 (51) 6 (9) 6 (9) 7 (10) 9 (13) 6 (9)

1 (3) 0 (0) 2 (6) 4 (13) 20 (63) 5 (16)

participate in the study. Of these patients, 102 (60.0% of the eligible patients) gave informed consent to participation. The characteristics of the participants are summarized in Table 1. We calculated the number of regional populations (n) that our participants represented as follows: n ⫽ 1.92 ⫻ 106 ⫻ 30.6% ⫻ 60.0% ⫽ 3.53 ⫻ 105. The participants (n ⫽ 102) were significantly younger than the patients who were eligible but did not participate (n ⫽ 68) (median age, 11.0 yr and 19.8 yr, respectively; P ⬍ 0.001). There were no significant differences between the two groups with respect to the proportion of male sex (46 and 43%, respectively), patients with thyroid dysgenesis (52 and 60%, respectively), and blood-spot TSH levels at screening (median, 70.5 and 87.5 mU/ liter, respectively). Detection and characterization of TSHR mutations We identified four nonsynonymous sequence variations in six patients. Observed variations included one previously reported and characterized mutation (c.1349G⬎A, p.R450H) (11), one previously reported but not characterized sequence variation (c.1207G⬎A, p.D403N) (12), and two novel sequence variations (c.394G⬎C, p.G132R; c.611C⬎T, p.A204V). Representative chromatograms are shown in Fig. 2A. None of them were found in 100 control individuals. Three patients had biallelic sequence variations (Table 2, patients 1–3): patient 1 was homozygous for p.R450H, patient 2 was heterozygous for p.G132R and p.R450H, and patient 3 was heterozygous for p.D403N and p.R450H. Three other patients had monoallelic sequence variations (Table 2, patients 4 – 6): patients 4 and 5 had heterozygous p.R450H, and patient 6 had heterozygous p.A204V. For patient 3, we confirmed the compound heterozygosity using allele-specific sequencing (data not shown). For patient 2, we could not confirm the compound heterozygosity, although the genotype of the mother (heterozygous for p.R450H) was consistent with it. To assess molecular functions of four putative mutant TSHRs, we examined the signaling properties of them using in vitro expression experiments. The expression levels of each mutant TSHR protein were comparable to that of wild-type (WT)

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(data not shown), indicating that these mutations do not cause destabilization of the TSHR protein. Competitive TSH binding studies showed reduced binding activities of four mutant receptors (Fig. 2B): G132R-TSHR showed an abrogated binding activity; A204V-TSHR and R450H-TSHR had moderately reduced binding activities; and D403N-TSHR exhibited a mild reduction of binding activity. Abilities of the mutant TSHRs to produce cAMP in response to TSH were roughly parallel to their TSH binding activities (Fig. 2C): G132R-TSHR showed the lowest cAMP response, which was 26% of WT (P ⬍ 0.001); A204V-TSHR showed 50% response compared with WT (P ⬍ 0.001); and D403N-TSHR and the R450HTSHR exhibited comparable cAMP production activities, which were 80% (P ⬍ 0.05) and 78% (P ⬍ 0.001) of WT, respectively. Prevalence of TSHR mutations The percentage prevalence of biallelic TSHR mutation carriers was 4.3% (three in 70) among patients with moderate to severe CH, and that of monoallelic mutations was 9.4% (three in 32) among patients with mild CH. We estimated the prevalence of biallelic TSHR mutation carriers in the general Japanese population to be one in 118,000 (three in 353,000). Hypothesizing that the frequency of TSHR mutations follows a HardyWeinberg distribution, we calculated the prevalence of heterozygous TSHR mutation carriers to be 1 in 172.

FIG. 2. Identification and functional characterization of the inactivating TSHR mutations. A, Partial sequences of PCR products of patients are shown. The heterozygous substitutions of arginine (CGC) in place of glycine (GGC) at codon 132, valine (GTT) in place of alanine (GCT) at codon 204, asparagine (AAT) in place of aspartate (GAT) at codon 403, and histidine (CAC) in place of arginine (CGC) at codon 450 are indicated by the arrows. The homozygous substitution of codon 450 is also shown. B, Competitive TSH binding assay. We conducted displacement experiments using 125I-TSH and unlabeled bovine TSH (bTSH). Values are means ⫾ SEM of four independent transfections. Four mutants exhibited impaired TSH binding of various degrees. Mutations within the ligand binding domain (G132R-TSHR and A204V-TSHR) tend to have more profound loss of binding activities. C, TSH-stimulated cAMP production assay. bTSH stimulated the production of cAMP in a concentration-dependent manner in COS-7 cells transfected with either WT or the mutant TSHRs. Data are corrected for the amount of cAMP produced by transfected cells with WT TSHR and stimulated by 1 U/liter of bTSH (expressed in arbitrary units). Values are means ⫾ SEM of four independent transfections. cAMP production activities of the mutant receptors were roughly parallel to their binding activities. G132R-TSHR, which exhibited abrogated binding activity, could produce a reduced amount of cAMP. This is probably due to the enhancement of intracellular signal transduction by the G protein-adenylate cyclase system.

Founder effect In the present and previous studies (11, 13–15), p.R450H has been frequently found among Japanese patients with CH. To distinguish whether those p.R450H alleles were derived from a common ancestral allele or were produced by independent recurrent mutations, we attempted to characterize each allele harboring p.R450H. During the mutational screening, we noticed that patients who were heterozygous for p.R450H (patients 2–5) commonly had a minor SNP 关the G genotype of rs1991517; allele frequency among general Japanese population is 0.122 according to HapMap data (http://www.hapmap.org/, accessed on May 1, 2008)兴. The patient with homozygous p.R450H (patient 1) was homozygous for the minor SNP. These observations indicated a linkage between p.R450H and the SNP, and we confirmed it using allele-specific sequencing (data not shown). When we hypothesize that six p.R450H alleles were produced by independent recurrent mutations, its occurrence would be as low as 3.3 ⫻ 10⫺6 关⫽ (0.122)6兴, suggesting a founder effect. We also analyzed haplotypes of two microsatellite markers spanning the TSHR locus and found no common pattern of polymorphisms among five patients having p.R450H (data not shown). Clinical phenotypes of TSHR mutations All six patients carrying TSHR mutations had elevated serum TSH levels accompanied by normal serum T4 levels, namely compensated hypothyroidism. Three patients with biallelic mutations (Table 2, patients 1–3) had moderately elevated TSH levels (13.5–31.2 mU/liter; institutional reference, 0.5–5.0), and three patients with monoallelic mutations (Table 2, patients 4 – 6) had only slightly increased TSH levels (5.3–9.6 mU/liter). As for thyroid morphology, patients 1, 4, and 6 had normal-sized and placed gland, whereas patient 2 had slightly hypoplastic and



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TABLE 2. Clinical phenotypes of the patients with TSHR mutations Variable Age (yr), sex TSHR genotype Screening Age (d) Blood-spot TSH (mU/liter) Blood-spot FT4 (ng/dl) Blood-spot T4 (␮g/dl) Initial evaluation Age (d) Serum TSH (mU/liter) Serum FT4 (ng/dl) Serum Tg (ng/ml) L-T4 discontinuation Age (yr) Serum TSH (mU/liter) Serum FT4 (ng/dl) Serum Tg (ng/ml) Thyroid morphology L-T4 L-T4, a

dose (␮g/kg 䡠 d)

Patient 1

Patient 2

Patient 3

Patient 4

Patient 5

Patient 6

17, M p.关R450H兴 ⫹ 关R450H兴

17, F p.关G132R (⫹) R450H兴

12, M p.关D403N兴 ⫹ 关R450H兴

5, M p.R450H (1 allele)

13, M p.R450H (1 allele)

12, F p.A204V (1 allele)

5 22.9 2.1

6 44.3

5 19.8 2.3

4 18.4 1.9

5 39.6 1.9

5 36.5 0.6

9.2 54 21.2 1.6 18

14 18.0 1.6 NA

23 55.0 1.0 NA

19 66.1 1.1 710

15 6.0 1.3 NA

15 3.7 2.1 NA

2 27.8 1.4 NA Normal

12 31.2 1.1 NA Mild hypoplasia

3 13.5 1.2 41 NA

5 8.2 1.4 24 Normal

1 9.6 1.1 NA NA

3 5.3 1.4 56 Normal

2.7

2.1

2.3

1.4

Never treated

2.6

Referencea

⬍15.0b ⬎0.7b ⬎4.0b

1.7–9.1c 0.9 –2.3c 3.8 –56.3d

0.5–5.0 0.9 –1.6 ⬍30

Levothyroxine; NA, not available; Tg, thyroglobulin; M, male; F, female.

Values listed are those used in Kanagawa Children’s Medical Center if not otherwise stated.

b

Cutoff levels were obtained from the Kanagawa prefecture screening program for congenital hypothyroidism.

c

Values are from Nelson et al. (22).

d

Values are from Sobrero et al. (23).

normal-placed gland (Table 2). In the remaining two patients, thyroid morphology was not evaluated. Family studies revealed four additional individuals that were heterozygous for p.R450H (Fig. 3). The serum TSH levels of those four individuals (3.4 – 4.1 mU/liter) were all within our institutional reference range, but were comparable to the upper limit of the age-specific reference range that was determined in National Health and Nutrition Examination Survey III population without known risk factors for thyroid diseases (16).

is the first population-based investigation on prevalence of a genetic defect that causes nongoitrous CH. In general, pathogenesis of nongoitrous CH cases is seldom determined. This fact highlights the significance of our study that found about 4% of Japanese patients with moderate to severe CH had biallelic mutations in TSHR. Estimated prevalence of biallelic TSHR mutations in the general Japanese population (one in 118,000) seems lower than that of TG mutation carriers in Japan 关one in 67,000 (4)兴 and that of TPO mutation carriers in The Netherlands 关one in 66,000 (3)兴. Estimated prevalence of monoallelic mutation carriers (one in 172) is comparable to the data reported from Wales 关W546X, one in 180 (17)兴, although they are not Discussion population-based. We conducted a population-based genetic epidemiology study To date, inactivating TSHR mutations have been found in 15 regarding inactivating TSHR mutations. To our knowledge, this Japanese patients (belonging to nine families) with CH (11, 13–15). Among these mutations, p.R450H accounts for 75% (12 of 16) of unrelated mutant alleles. This proportion is similar to our population-based data, in which p.R450H accounted for 67% (six of nine) of unrelated mutant alleles. In the present report, we demonstrated the founder effect of p.R450H, and this seems to explain why the mutation is common in Japan but has so far not been detected among Caucasians. As for the geographical distribution of p.R450H, it was found in several distant regions in Japan (our unpublished FIG. 3. Family studies. We genotyped six family members belonging to three families and found four observation), and furthermore, was also additional heterozygous TSHR mutation carriers. TSH levels are expressed in milliunits per liter. Rx, found in a patient who immigrated from Levothyroxine treatment.

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China (our unpublished observation). These findings imply a relatively older evolutionary origin of p.R450H. The result of microsatellite haplotype analysis, which showed no common combination of polymorphisms, supports this speculation because it means that there was sufficient time for recombination events between p.R450H and adjacent microsatellites. The phenotypes of Japanese patients with biallelic inactivating TSHR mutations are relatively mild compared with previously reported cases: none of 15 previously described patients (including three patients that appeared in this report) had overt hypothyroidism or severe thyroid hypoplasia (11, 13–15). Of note, all 15 patients had at least one allele harboring p.R450H. Therefore, these milder phenotypes are presumably due to high residual function of R450H-TSHR as shown in functional analyses of the present and previous studies (11, 13). These findings are consistent with the concept of genotype-phenotype correlation suggested by Tiosano et al. (18), who stated that subjects having at least one allele with a missense mutation exhibiting reduced but not absent TSH-mediated signal transduction would have compensated hypothyroidism. Several previous reports have detected monoallelic TSHR mutations in individuals with subclinical hypothyroidism and have argued the significance of monoallelic mutations (19, 20). Calebiro et al. (21) observed that some missense mutations had dominant negative effect in vitro and proposed the idea of “dominant TSH resistance.” On the contrary, more than half of the monoallelic mutation carriers documented in the previous reports (mostly detected through family studies) had normal thyroid functions (data not shown). It is worth noting that those individuals do not represent the entire population of monoallelic mutation carriers owing to bias associated with subject enrollment. Our mutational screening showed that three patients with mild CH had monoallelic TSHR mutations. Considering the high estimated prevalence (one in 172) of monoallelic mutation carriers (based on Hardy-Weinberg equilibrium equation), the vast majority of monoallelic mutation carriers might have normal or only minimally impaired thyroid function and thus be negative for newborn screening. Moreover, in the pedigree of patient 4, intrafamilial variability of serum TSH levels among heterozygous R450H carriers was observed. These lines of evidence imply that additional factor(s) (genetic and/or nongenetic) may influence the individual thyroid function of monoallelic mutation carriers. Further studies are required to elucidate completely the pathological significance of monoallelic inactivating TSHR mutations. Our study has several limitations. First, study subjects were collected in only one study site, and the sample size was relatively small. This is because we prioritized obtaining uniform quality data considering that accurate enrollment and clinical evaluation are essential for this genetic epidemiology study. Nonetheless, we believe that the conclusions of the study would be valid throughout Japan, because Kanagawa prefecture is not isolated geographically or demographically. Second, we cannot exclude selection bias associated with subject enrollment. The participants were significantly younger than the patients who were eligible but did not participate. This difference could be a potential source of bias, although we confirmed that there were no sig-


nificant differences in the thyroid phenotypes. Third, we could not detect complex abnormalities such as exon-level deletion, duplication, or inversion in the locus because we performed the mutational screening using direct sequencing. In conclusion, we conducted mutation screening for TSHR using population-based genome samples and found that TSHR mutations are relatively common among Japanese patients with permanent primary CH. A common founder mutation p.R450H, which accounted for as high as 67% of the total mutations, was hypomorphic, and it seems to explain the milder phenotypes of Japanese patients with biallelic inactivating TSHR mutations. Further genetic epidemiology studies dealing with not only other populations but also other causative genes will be necessary to reach for the total genetic landscape of CH. Such attempts will provide valuable insights into the etiology of CH and fundamental resources for more appropriate genetic counseling.

Acknowledgments We thank Fumiko Kato for technical assistance. We also thank Prof. Takao Takahashi (Department of Pediatrics, Keio University School of Medicine) for fruitful discussion and Prof. Pinchas Cohen (Pediatric Endocrinology, University of California at Los Angeles) for critical comments. Address all correspondence and requests for reprints to: Tomonobu Hasegawa, M.D., Ph.D., Department of Pediatrics, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail: [email protected]. This work was supported in part by a grant for Child Health and Development from the Ministry of Health, Labor and Welfare of Japan (05-045-0375). S.N. is a research associate of Global Center-of-Excellence for Human Metabolomics Systems Biology from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Disclosure Summary: The authors have nothing to disclose.

References 1. Toublanc JE 1992 Comparison of epidemiological data on congenital hypothyroidism in Europe with those of other parts in the world. Horm Res 38: 230 –235 2. Ambrugger P, Stoeva I, Biebermann H, Torresani T, Leitner C, Gru¨ters A 2001 Novel mutations of the thyroid peroxidase gene in patients with permanent congenital hypothyroidism. Eur J Endocrinol 145:19 –24 3. Bakker B, Bikker H, Vulsma T, de Randamie JS, Wiedijk BM, De Vijlder JJ 2000 Two decades of screening for congenital hypothyroidism in The Netherlands: TPO gene mutations in total iodide organification defects (an update). J Clin Endocrinol Metab 85:3708 –3712 4. 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 2006 Haplotype analysis reveals founder effects of thyroglobulin gene mutations C1058R and C1977S in Japan. J Clin Endocrinol Metab 91:3100 –3104 5. Sunthornthepvarakui T, Gottschalk ME, Hayashi Y, Refetoff S 1995 Brief report: resistance to thyrotropin caused by mutations in the thyrotropin-receptor gene. N Engl J Med 332:155–160 6. Macchia PE, Lapi P, Krude H, Pirro MT, Missero C, Chiovato L, Souabni A, Baserga M, Tassi V, Pinchera A, Fenzi G, Gruters A, Busslinger M, Di Lauro R 1998 PAX8 mutations associated with congenital hypothyroidism caused by thyroid dysgenesis. Nat Genet 19:83– 86 7. Devriendt K, Vanhole C, Matthijs G, de Zegher F 1998 Deletion of thyroid transcription factor-1 gene in an infant with neonatal thyroid dysfunction and respiratory failure. N Engl J Med 338:1317–1318 8. Clifton-Bligh RJ, Wentworth JM, Heinz P, Crisp MS, John R, Lazarus JH, Ludgate M, Chatterjee VK 1998 Mutation of the gene encoding human TTF-2


9. 10.

11.

12.

13.

14.

15.

16.

associated with thyroid agenesis, cleft palate and choanal atresia. Nat Genet 19:399 – 401 Fisher DA 2002 Disorders of the thyroid in the newborn and infant. In: Sperling MA, ed. Pediatric endocrinology. 2nd ed. Philadelphia: Saunders; 161–185 Johnstone AP, Cridland JC, DaCosta CR, Harfst E, Shepherd PS 1994 Monoclonal antibodies that recognize the native human thyrotropin receptor. Mol Cell Endocrinol 105:R1–R9 Nagashima T, Murakami M, Onigata K, Morimura T, Nagashima K, Mori M, Morikawa A 2001 Novel inactivating missense mutations in the thyrotropin receptor gene in Japanese children with resistance to thyrotropin. Thyroid 11:551–559 Camilot M, Teofoli F, Gandini A, Franceschi R, Rapa A, Corrias A, Bona G, Radetti G, Tato L 2005 Thyrotropin receptor gene mutations and TSH resistance: variable expressivity in the heterozygotes. Clin Endocrinol (Oxf) 63: 146 –151 Tsunekawa K, Onigata K, Morimura T, Kasahara T, Nishiyama S, Kamoda T, Mori M, Morikawa A, Murakami M 2006 Identification and functional analysis of novel inactivating thyrotropin receptor mutations in patients with thyrotropin resistance. Thyroid 16:471– 479 Kanda K, Mizuno H, Sugiyama Y, Imamine H, Togari H, Onigata K 2006 Clinical significance of heterozygous carriers associated with compensated hypothyroidism in R450H, a common inactivating mutation of the thyrotropin receptor gene in Japanese. Endocrine 30:383–388 Shibayama K, Ohyama Y, Hishinuma A, Yokota Y, Kazahari K, Kazahari M, Ieiri T, Matsuura N 2005 Subclinical hypothyroidism caused by a mutation of the thyrotropin receptor gene. Pediatr Int 47:105–108 Hollowell JG, Staehling NW, Flanders WD, Hannon WH, Gunter EW, Spencer CA, Braverman LE 2002 Serum TSH, T(4), and thyroid antibodies in the United States population (1988 to 1994): National Health and Nutrition Examination Survey (NHANES III). J Clin Endocrinol Metab 87:489 – 499


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17. Jordan N, Williams N, Gregory J, Evans C, Owen M, Ludgate M 2003 The W546X mutation of the thyrotropin receptor gene: potential major contributor to thyroid dysfunction in a Caucasian population. J Clin Endocrinol Metab 88:1002–1005 18. Tiosano D, Pannain S, Vassart G, Parma J, Gershoni-Baruch R, Mandel H, Lotan R, Zaharan Y, Pery M, Weiss RE, Refetoff S, Hochberg Z 1999 The hypothyroidism in an inbred kindred with congenital thyroid hormone and glucocorticoid deficiency is due to a mutation producing a truncated thyrotropin receptor. Thyroid 9:887– 894 19. Alberti L, Proverbio MC, Costagliola S, Romoli R, Boldrighini B, Vigone MC, Weber G, Chiumello G, Beck-Peccoz P, Persani L 2002 Germline mutations of TSH receptor gene as cause of nonautoimmune subclinical hypothyroidism. J Clin Endocrinol Metab 87:2549 –2555 20. Tonacchera M, Perri A, De Marco G, Agretti P, Banco ME, Di Cosmo C, Grasso L, Vitti P, Chiovato L, Pinchera A 2004 Low prevalence of thyrotropin receptor mutations in a large series of subjects with sporadic and familial nonautoimmune subclinical hypothyroidism. J Clin Endocrinol Metab 89: 5787–5793 21. Calebiro D, De Filippis T, Lucchi S, Covino C, Panigone S, Beck-Peccoz P, Dunlap D, Persani L 2005 Intracellular entrapment of wild-type TSH receptor by oligomerization with mutants linked to dominant TSH resistance. Hum Mol Genet 14:2991–3002 22. Nelson JC, Clark SJ, Borut DL, Tomei RT, Carlton EI 1993 Age-related changes in serum free thyroxine during childhood and adolescence. J Pediatr 123:899 –905 23. Sobrero G, Munoz L, Bazzara L, Martin S, Silvano L, Iorkansky S, Bergoglio L, Spencer C, Miras M 2007 Thyroglobulin reference values in a pediatric infant population. Thyroid 17:1049 –1054