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Arquivos Brasileiros de Endocrinologia & Metabologia

On-line version ISSN 1677-9487

Arq Bras Endocrinol Metab vol.56 no.3 São Paulo Apr. 2012 



Absence of mutations in PAX8, NKX2.5, and TSH receptor genes in patients with thyroid dysgenesis


Ausência de mutações nos genes PAX-8, NKX2.5 e receptor de TSH em pacientes com disgenesia tireoidiana



Ester S. BrustI: Cristine B. BeltraoII; Maria C. ChammasIII; Tomoco WatanabeIII; Marcelo T. SapienzaIII; Suemi MaruiI

IUnidade de Tireoide, Laboratório de Endocrinologia Celular e Molecular (LIM-25), Disciplina de Endocrinologia, Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo (HCFMUSP), São Paulo, SP, Brazil
IIAssociação de Pais e Amigos dos Excepcionais (APAE) de São Caetano do Sul; Unidade de Tireoide, Laboratório de Endocrinologia Celular e Molecular (LIM-25), Disciplina de Endocrinologia, HCFMUSP, São Paulo, SP, Brazil
IIIInstituto de Radiologia (InRad), FMUSP, São Paulo, SP, Brazil

Correspondence to




OBJECTIVES: To precisely classify the various forms of TD, and then to screen for mutations in transcription factor genes active in thyroid development.
SUBJECTS AND METHODS: Patients underwent ultrasound, thyroid scan, and serum thyroglobulin measurement to accurately diagnose the form of TD. DNA was extracted from peripheral leukocytes. The PAX8, and NKX2.5 genes were evaluated in all patients, and TSH receptor (TSHR) gene in those with hypoplasia.
RESULTS: In 27 nonconsanguineous patients with TD, 13 were diagnosed with ectopia, 11 with hypoplasia, and 3 with athyreosis. No mutations were detected in any of the genes studied.
CONCLUSION: Sporadic cases of TD are likely to be caused by epigenetic factors, rather than mutations in thyroid transcription factors or genes involved in thyroid development.

Keywords: Thyroid dysgenesis; PAX8; NKX2.5; TSHR; congenital hypothyroidism; mutation


OBJETIVOS: Classificar corretamente as várias formas de DT e depois rastrear por mutações em genes que participam no desenvolvimento da tireoide.
Os pacientes realizaram ultrassonografia, cintilografia e tireoglobulina sérica para o diagnóstico preciso de DT. DNA foi extraído de leucócitos periféricos. Os genes PAX8 e NKX2.5 foram estudados em todos os pacientes e o gene do receptor do TSH (TSHR) foi estudado na hipoplasia.
RESULTADOS: Avaliaram-se 27 pacientes sem consanguinidade com DT, dos quais 13 foram diagnosticados com ectopia, 11 com hipoplasia e 3 com atireose. Nenhuma mutação foi detectada nos genes estudados.
CONCLUSÃO: Casos esporádicos de DT são provavelmente causados mais por fatores epigenéticos do que por mutações em fatores de transcrição ou genes envolvidos no desenvolvimento tireoidiano.

Descritores: Disgenesia tireoidiana; PAX-8; NKX2.5; TSHR; hipotireoidismo congênito; mutação




Thyroid dysgenesis (TD) is the major cause of congenital and permanent hypothyroidism. Most cases are sporadic, affecting more females, and frequently associated to heart defects (1). Clinical presentation of TD includes athyreosis, the absence of thyroid tissue, hemiagenesis, the presence of only one thyroid lobe, hypoplasia, and ectopia (2,3). Transcription factors PAX8 and NKX2.5 are active in thyroid development (4), and are possible candidates for TD. Mutations in the TSH receptor (TSHR) cause hypothyroidism of variable severity via thyroid hypoplasia (5).

PAX8 is a member of the PAX family of transcription factors, active in several germline tissues in the human embryo (6). PAX8 is crucial to follicular development and thyroid hormone production induced by many genes. Few patients with congenital hypothyroidism caused by ectopia or athyreosis have PAX8 mutations inherited in an autosomal dominant fashion (7-9).

NKX2.5 is a homeobox-containing transcription factor essential to heart morphogenesis. In mouse embryos, Nkx2.5 transcripts were observed in thyroid precursor cells in the pharyngeal floor. In later stages of development, Nkx2.5 expression is limited to the thyroid primordium area. Mutations in NKX2.5 have been described not only in patients with heart defects, but also in patients with thyroid ectopia or athyreosis without heart involvement (1,10).

Inactivating mutations in TSHR have been described in patients with congenital hypothyroidism and thyroid hypoplasia. Some of these patients had been diagnosed with athyreosis, but serum thyroglobulin was detectable, denoting thyroid tissue that was not visible by conventional imaging methods (11). The definition of hypoplasia is extremely difficult due to many variables that determine thyroid size in childhood, such as gender, age, height, body surface area, puberty, and iodine sufficiency. In prior studies, we used ultrasound in combination with thyroid scan and serum thyroglobulin levels to more precisely define primary congenital hypothyroidism (12).

In addition to defining the conditions of ectopia, athyreosis, and thyroid hypoplasia in patients with permanent and primary hypothyroidism using a combination of ultrasound, thyroid scan, and serum thyroglobulin levels, the aim of this study was to define candidate genes and search for mutations related to various clinical presentations of TD. Since PAX8 and NKX2.5 genes are involved in all steps of thyroid development (formation, migration, differentiation, and proliferation), these transcription factors were studied in all patients with TD. The TSHR gene was studied only in patients with thyroid hypoplasia, since this gene is involved only in the proliferation of thyroid cells.



Twenty-seven patients aged 3-19 years, diagnosed with primary congenital hypothyroidism were recruited in the outpatient clinic of the Association for Parents and Friends of Disabled Individuals (APAE), São Caetano, and referred to the Governmental Neonatal Screening Service to be studied at the Hospital das Clinicas – FMUSP (12). Informed consent was obtained from all parents, and the protocol was approved by the Ethics Committee of the Institution. Patients underwent color Doppler ultrasound (CD-US), combined serum thyroglobulin (TG) measurement, and thyroid scan with uptake of 99Tc Pertechnetate (99mTc) and radioactive iodine (131I).

CD-US was performed using a Phillips scanner with a 7.5-12 MHZ transducer focusing on the thyroid gland and cervical region, from the mandible bone to the manubrium. Total thyroid volume was calculated as described elsewhere (13,14), and compared according to height, sex, age, and body surface area.

After a four-week washout with no levothyroxine treatment, total T3 and T4, free T4 (FT4), TSH, thyroglobulin (TG) and anti-TG antibody were measured in all patients by immunofluorometric assays (Autodelfia®, Wallac Oy, Turku, Finland). Patients with anti-TG antibodies were excluded from the study. A radionuclide scan was performed after the four- week levothyroxine washout and two weeks on a low iodine diet. Uptake of 131I (5 µCi) was measured at 2 and 24 h after oral administration. Similar uptake measurements were carried out on the following day after intravenous injection of 99Tc Pertechnetate (10 mCi).

Athyreosis diagnosis was determined when no thyroid was visualized by any of the imaging techniques used (CD-US and scan). Thyroid hypoplasia was diagnosed when total thyroid volume measured by CD-US was calculated to be less than 2 SD from the normal value for height, gender, chronological age, and body surface area. Ectopia was diagnosed when thyroid tissue was observed outside the normal bed. After patients were diagnosed and classified according to the various clinical presentations of TD, the TSHR gene was studied only in patients with thyroid hypoplasia. The PAX8 and NKX2.5 genes were studied in all patients.

Mutation screening by DNA sequencing

DNA was extracted from peripheral leukocytes (15). Coding regions and exon-intron boundaries of the candidate genes were amplified by PCR and sequenced using the ABI Prism 3130 xl (Applied Biosystems, Foster City, CA, USA), as previously described (1,7). DNA from 50 adults with normal thyroid function was used for comparison in gene sequencing.



We studied 27 nonconsanguineous patients (20 female) aged 3 to 19 years (median 4.6 years) with TD (Table 1). All patients presented high TSH levels after 4 weeks of levothyroxine suspension, confirming the diagnosis of primary congenital hypothyroidism. No patients had heart defects detected by echocardiogram. All had normal kidney function. None had respiratory insufficiency.



Twelve patients were diagnosed with ectopia, having thyroid tissue in the submandibular region observed on thyroid scan. One additional patient presented ectopia associated with left lobe hemiagenesia. Thyroglobulin levels of the 13 patients with ectopia ranged from 4.5 to 123 ng/mL (mean and SD = 46.2 ± 37.9 ng/mL, median 28.4 ng/mL) (Figure 1).



Eight patients were diagnosed with thyroid hypoplasia, having less than 2 SD of the normal thyroid volume, as assessed by CD-US. Thyroglobulin levels of these patients ranged from 6.8 to 65.2 ng/mL (mean and SD = 34.7 ± 18.6 ng/mL, median 35.2 ng/mL) (Figure 1).

Six patients were diagnosed with athyreosis using CD-US and thyroid scan. However, 3 of these 6 patients had measurable thyroglobulin levels of 4.0, 5.4, and 9.1 ng/mL. Therefore they were reclassified as having thyroid hypoplasia.

The coding regions and exon-intron boundaries of the PAX8 and NKX2.5 genes were fully sequenced in all 27 patients with TD. We identified no mutations. The single nucleotide polymorphism (SNP) rs2277923 ( within the NKX2.5 gene was detected in 93% of patients, but the differences in the allele and genotype frequencies between patients and controls were not statistically significant (p > 0.05).

The coding regions and exon-intron boundaries of the TSHR gene were fully sequenced in the 8 patients with thyroid hypoplasia and in the 3 patients with athyreosis who were reclassified as having thyroid hypoplasia due to detectable serum thyroglobulin. Similarly, no mutations were identified.



Thyroid dysgenesis (TD) comprises a broad spectrum of clinical presentations, including athyreosis, hemiagenesia, hypoplasia, and ectopia. All cause definitive hypothyroidism. It is possible that each subgroup of TD could be caused by a specific genetic modification (1). We therefore decided to better define each clinical presentation in order to more efficiently search for the involvement of candidate genes.

Kreisner and cols. suggested an initial approach with ultrasound to define congenital hypothyroidism (16). If the thyroid gland was absent, TD is the most probable diagnosis, with athyreosis or ectopia. If the thyroid gland was present at ultrasound, more exams are necessary to establish the etiology. Combined use of ultrasound and serum thyroglobulin measurement allows the differentiation between patients with ectopia, athyreosis, and thyroid hypoplasia. For example, we were able to reclassify three patients who had been misdiagnosed with athyreosis using only imaging methods. The presence of serum thyroglobulin in these patients revealed that their correct diagnosis was actually thyroid hypoplasia. Since thyroid scan is more difficult to perform, serum thyroglobulin measurement is an important tool in the differential diagnosis of TD, and should be routinely performed in cases of primary congenital hypothyroidism. Patients with undetectable serum thyroglobulin, in the absence of anti-thyroglobulin antibodies, certainly present athyreosis or thyroglobulin deficiency (dyshormonogenesis). In thyroglobulin deficiency, the presence of the thyroid, palpable or visible on the ultrasound, enables dyshormonogenesis diagnosis (12).

In this study, all 27 patients with TD were screened for mutations in the PAX8 and NKX2.5 genes, since these are transcription factors involved in thyroid embryogenesis. Mutations in these genes have been previously described in cases of athyreosis, ectopia, and hypoplasia (7,8,10,17-20). We found no mutations in the PAX8 or NKX2.5 genes in our cohort. The only variation we observed was SNP rs2277923 within the NKX2.5 gene, which is a synonymous variation, and does not cause an amino acid change. However, we do not believe this SNP to be related to TD since the frequency of the SNP within patients and controls was not significantly different. In the patients with thyroid hypoplasia, we looked for mutations in the TSHR gene, but found none.

Our negative findings demonstrate the rare genetic etiology in sporadic cases of TD, at least in genes known to be involved in the formation and migration of follicular cells. Several previously studies in different ethnic populations with TD did not find mutations, either, particularly in sporadic cases (1,7,9,21-27) (Table 2). It is likely that other epigenetic factors determine TD, such as differential gene expressions or methylation (28,29). Considering the candidate genes for TD described above in a large number of negative patients with different genetic backgrounds studied so far, including those from our group, we can conclude that TD should be weakly correlated with inherited genetic defects. This finding make it necessary to carry out further molecular analyses, as the genes that are known to cause the disorder account for only very few cases.



Acknowledgments: the authors would like to thank the patients and their parents, as well as the staff at Associação de Pais e Amigos do Excepcional (APAE), São Caetano do Sul.

Financial support: Fundação de Amparo à Pesquisa do Estado de São Paulo (Fapesp) 06/05800-1 and 08/04786-0.

Disclosure: no potential conflict of interest relevant to this article was reported.



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Correspondence to:
Suemi Marui
Faculdade de Medicina, Universidade de São Paulo
Av. Dr. Arnaldo, 455, sala 4305, 4º andar
01243-903 – São Paulo, SP, Brazil

Received on 3/Aug/2011
Accepted on 13/Mar/2012

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