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Thyroid dysgenesis caused by PAX8 mutation: The hypermutability with CpG dinucleotides at codon 31
Thyroid dysgenesis caused by PAX8 mutation: The hypermutability with CpG dinucleotides at codon 31 Masaki Komatsu, MD, Tsutomu Takahashi, MD, Ikuko Takahashi, MD, Masaaki Nakamura, MD, Ikuo Takahashi, MD, and Goro Takada, MD We identified a novel mutation (CGC to TGC) at codon 31 of the Paired box 8 gene, an important transcription factor in the development of the thyroid gland. Mutations at this codon have been independently reported in 2 cases (CGC to CAC). These transitions are considered typical CpG-consequence mutations and account for hypermutability at this position. (J Pediatr 2001;139:597-9.)
Congenital hypothyroidism is a very common disorder among congenital endocrine diseases, being found in 1 in 3000 to 4000 infants. The major cause of congenital hypothyroidism is dysgenesis of the thyroid gland (80%-85%). One third of patients with thyroid dysgenesis have thyroid aplasia, and in the remainder, the gland is located ectopically. Recent investigations have shown the role of thyroid-specific transcription factors including thyroid transcription factor-1 (TTF-1), thyroid transcription factor-2 (TTF-2), and paired box 8 (PAX8) in thyroid gland development. The transcription of the thyroglobulin and thyroperoxidase genes was also found to be regulated by these factors.1 Mutations of these
transcription factors have been found in about 2% of patients with congenital hypothyroidism. A heterozygous TTF-1 mutation was found in an infant with mild hypothyroidism and respiratory distress syndrome.2 A homozygous missense mutation (A65V) in TTF-2 was found in 2 siblings with cleft palate, choanal atresia, and congenital hypothyroidism.1 Recently, autosomal dominant mutations of the PAX8 gene were described in patients with different forms of thyroid dysgenesis.3,4 In this report, we describe a Japanese family with thyroid dysgenesis caused by a novel missense mutation at codon 31 of the PAX8 gene.
METHODS Patients
From the Department of Pediatrics, Akita University School of Medicine, Akita, Japan; and the Divisions of Surgery and Pediatrics, Ogachi Chuo General Hospital, Akita, Japan.
Submitted for publication Dec 12, 2000; revision received Mar 26, 2001; accepted Apr 30, 2001. Reprint requests: Tsutomu Takahashi, Department of Pediatrics, Akita University School of Medicine, Hondo 1-1-1, Akita-shi, Akita, 010-8543, Japan. Copyright © 2001 by Mosby, Inc. 0022-3476/2001/$35.00 + 0 9/22/117071 doi:10.1067/mpd.2001.117071
The propositus (P1), an 8-year-old girl, was born to nonconsanguineous Japanese parents after an uneventful pregnancy. Her mother had hypothyroidism and was treated with thyroid hormone through her pregnancy. The propositus was believed to have congenital hypothyroidism because hyperthyrotropinemia was detected by neonatal mass screening (thyrotropin level, 40 mU/L; normal <5.0 mU/L). Because serum triiodothyronine and
thyroxine levels were normal, she was carefully observed without treatment. Thyroxine treatment was started when she was 3 months old, when her serum thyrotropin level was 109 mU/L and she had failure to thrive. At the age of 8 years, after discontinuation of thyroxine treatment for 3 weeks, her thyroid function and structure were examined. The results were as follows: thyrotropin 176 mU/L (normal 0.4-4.7 mU/L), free thyroxine 9.0 pmol/L (12.9-41.7 pmol/L), and thyroglobulin 8.1 µg/mL (normal PAX8 TTF-1 TTF-2 TRH FT4
Paired box 8 Thyroid transcription factor-1 Thyroid transcription factor-2 Thyrotropin-releasing hormone Free thyroxine
<30 µg/mL). Anti-microsomal and antithyroglobulin antibodies were negative. A thyrotropin-releasing hormone (TRH) stimulation test induced a hyperreactive thyrotropin response (0 minutes, 176 mU/L; 30 minutes, 474 mU/L; 60 minutes, 319 mU/L; 90 minutes, 279 mU/L; and 120 minutes, 216 mU/L). Iodine 123 uptake (3 hours) was 0.8% (normal, 5.4%-12.0%) in the normal position. Cervical ultrasonography demonstrated an extremely small thyroid gland. Her mother (P2), a 39-year-old woman, was given a diagnosis of hypothyroidism when she was 14 years old and treated with thyroid hormone. She does not show signs of severe mental retardation, despite lack of thyroid replacement treatment from birth to age 14, but her final height is extremely short, 132 cm. No thyroid gland tissue was detected by ultrasonography. The 597
KOMATSU ET AL
THE JOURNAL OF PEDIATRICS OCTOBER 2001
Figure. Direct sequence of exon 2 of PAX8 gene. Asterisk indicates mutated nucleotide.
other 3 members of this family, the father and 2 sisters, are healthy and have no sign of hypothyroidism.
Molecular Studies Genomic DNA was extracted from peripheral blood lymphocytes. Polymerase chain reaction amplification from exon 2 to exon 9 of the PAX8 gene was performed with the primer pairs described by Macchia et al.3 The cycling conditions were 94°C for 1 minute, 57°C for 1 minute, and 72°C for 1 minute for 30 cycles, followed by a final extension step at 72°C for 7 minutes. Both forward and reverse strands were directly sequenced by using an automatic DNA sequencer (ABI PRISM 310 DNA sequencer; PE Applied Biosystems, Foster City, Calif).
RESULTS AND DISCUSSION P1 and P2 showed an arginine (CGC) to cysteine (TGC) substitution at codon 31 of the PAX8 gene (designated as R31C) (Figure). In addition, we analyzed 50 volunteers without features of hypothyroidism after informed 598
consent had been obtained. R31C was not identified in 100 alleles from healthy persons, demonstrating that it was not a polymorphism (data not shown). Codon 31 is located in the coding region of the paired domain in the PAX8 gene.5 The mutations in this codon must be critical, because the DNA-binding ability is reduced and results in abnormal development of the thyroid gland.3 The mutation at codon 31 has been independently reported in 2 other cases, an Italian patient3 and one Japanese family including 3 patients.4 These patients had an arginine (CGC) to histidine (CAC) substitution at this codon (designated as R31H). The missense mutations of codon 31, R31C (CGC to TGC) and R31H (CGC to CAC), may be ascribed to the hypermutability at CpG dinucleotides. It is well known that CpG dinucleotides can be a mutational hot spot in the human genome. In eukaryotic genomes, 5methylcytosine occurs predominantly in CpG dinucleotides, the majority of which appear to be methylated. Spontaneous deamination of 5-methylcytosine to thymine leads to TpG or CpA mutations.6-8 This mechanism may be the cause of the missense mutations in the 3 cases.
To date, only 3 mutations of the PAX8 gene, R108X, R31H, and L62R, have been identified by screening the genomic DNA of 145 Italian patients with thyroid dysgenesis.3 In those mutations, R108X (CGA to TGA) and R31H (CGC to CAC) are de novo mutations, which can be considered typical CpG-consequence mutations. Recently, the same R31H mutation was identified in a Japanese family with thyroid dysgenesis,4 suggesting that the R31H mutation is a recurrent mutation occurring in a mutational hot spot of the PAX8 gene. We identified a novel mutation, R31C (CGC to TGC), in the same codon as the R31H mutation, and R31C is also thought to be caused by the transformation of cytosine to thymine at the CpG nucleotide through the methylation-deamination mechanism. Among the 5 mutations identified from the PAX8 gene, 3 were located at the CpG nucleotide of codon 31. Thus, we propose that codon 31 is a hot spot for recurrent mutations in the human PAX8 gene.
Note added in proof During the course of our study, the novel mutation, C57Y, was reported by Vilain et al.9
REFERENCES 1. Clifton-Bligh RJ, Wentworth JM, Heinz P, Crisp MS, John R, Lazarus JH, et al. Mutation of the gene encoding human TTF-2 associated with thyroid agenesis, cleft palate and choanal atresia. Nat Genet 1998; 19:399-401. 2. Devriendt K, Vanhole C, Matthijs G, de Zegher F. Deletion of thyroid transcription factor-1 gene in an infant with neonatal thyroid dysfunction and respiratory failure. N Engl J Med 1998;338:1317-8. 3. Macchia PE, Lapi P, Krude H, Pirro MT, Missero C, Chiovato L, et al. PAX8 mutations associated with congenital hypothyroidism caused by thyroid dysgenesis. Nat Genet 1998; 19:83-6. 4. Gouji K, Matsuo M. Folica Endocrinologica Japonica 2000;76:153.
KOMATSU ET AL
THE JOURNAL OF PEDIATRICS
VOLUME 139, NUMBER 4 5. Xu W, Rould MA, Jun S, Desplan C, Pabo CO. Crystal structure of paired domain-DNA complex at 2.5Å resolution reveals structural basis for Pax development mutation. Cell 1995; 80:639-50. 6. Cooper DN, Krawczak M, Antonarakis SE. The nature and mechanisms of human gene mutation. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors.
The metabolic and molecular bases of inherited disease. 7th ed. New York: McGraw-Hill; 1995. p. 259-91. 7. Ollila J, Lappalainen I, Vihinen M. Sequence specificity in CpG mutation hotspots. FEBS Lett 1996; 396:119-22. 8. El Antri S, Mauffret O, Monnot M, Lescot E, Convert O, Fermandjian S. Structural deviation at CpG provide a plausible explanation for the high frequency of mu-
tation at this site. Phosphorus nuclear magnetic resonance and circular dichroism studies. J Mol Biol 1993;230:373-8. 9. Vilain C, Rydlewski C, Duprez L, Heinrichs C, Abramowicz M, Malvaux P, et al. Autosomal dominant transmission of congenital thyroid hypoplasia due to loss-of-function mutation of PAX8. J Clin Endocrinol Metab 2001;86:234-8.
599
Congenital hypothyroidism is a very common disorder among congenital endocrine diseases, being found in 1 in 3000 to 4000 infants. The major cause of congenital hypothyroidism is dysgenesis of the thyroid gland (80%-85%). One third of patients with thyroid dysgenesis have thyroid aplasia, and in the remainder, the gland is located ectopically. Recent investigations have shown the role of thyroid-specific transcription factors including thyroid transcription factor-1 (TTF-1), thyroid transcription factor-2 (TTF-2), and paired box 8 (PAX8) in thyroid gland development. The transcription of the thyroglobulin and thyroperoxidase genes was also found to be regulated by these factors.1 Mutations of these
transcription factors have been found in about 2% of patients with congenital hypothyroidism. A heterozygous TTF-1 mutation was found in an infant with mild hypothyroidism and respiratory distress syndrome.2 A homozygous missense mutation (A65V) in TTF-2 was found in 2 siblings with cleft palate, choanal atresia, and congenital hypothyroidism.1 Recently, autosomal dominant mutations of the PAX8 gene were described in patients with different forms of thyroid dysgenesis.3,4 In this report, we describe a Japanese family with thyroid dysgenesis caused by a novel missense mutation at codon 31 of the PAX8 gene.
METHODS Patients
From the Department of Pediatrics, Akita University School of Medicine, Akita, Japan; and the Divisions of Surgery and Pediatrics, Ogachi Chuo General Hospital, Akita, Japan.
Submitted for publication Dec 12, 2000; revision received Mar 26, 2001; accepted Apr 30, 2001. Reprint requests: Tsutomu Takahashi, Department of Pediatrics, Akita University School of Medicine, Hondo 1-1-1, Akita-shi, Akita, 010-8543, Japan. Copyright © 2001 by Mosby, Inc. 0022-3476/2001/$35.00 + 0 9/22/117071 doi:10.1067/mpd.2001.117071
The propositus (P1), an 8-year-old girl, was born to nonconsanguineous Japanese parents after an uneventful pregnancy. Her mother had hypothyroidism and was treated with thyroid hormone through her pregnancy. The propositus was believed to have congenital hypothyroidism because hyperthyrotropinemia was detected by neonatal mass screening (thyrotropin level, 40 mU/L; normal <5.0 mU/L). Because serum triiodothyronine and
thyroxine levels were normal, she was carefully observed without treatment. Thyroxine treatment was started when she was 3 months old, when her serum thyrotropin level was 109 mU/L and she had failure to thrive. At the age of 8 years, after discontinuation of thyroxine treatment for 3 weeks, her thyroid function and structure were examined. The results were as follows: thyrotropin 176 mU/L (normal 0.4-4.7 mU/L), free thyroxine 9.0 pmol/L (12.9-41.7 pmol/L), and thyroglobulin 8.1 µg/mL (normal PAX8 TTF-1 TTF-2 TRH FT4
Paired box 8 Thyroid transcription factor-1 Thyroid transcription factor-2 Thyrotropin-releasing hormone Free thyroxine
<30 µg/mL). Anti-microsomal and antithyroglobulin antibodies were negative. A thyrotropin-releasing hormone (TRH) stimulation test induced a hyperreactive thyrotropin response (0 minutes, 176 mU/L; 30 minutes, 474 mU/L; 60 minutes, 319 mU/L; 90 minutes, 279 mU/L; and 120 minutes, 216 mU/L). Iodine 123 uptake (3 hours) was 0.8% (normal, 5.4%-12.0%) in the normal position. Cervical ultrasonography demonstrated an extremely small thyroid gland. Her mother (P2), a 39-year-old woman, was given a diagnosis of hypothyroidism when she was 14 years old and treated with thyroid hormone. She does not show signs of severe mental retardation, despite lack of thyroid replacement treatment from birth to age 14, but her final height is extremely short, 132 cm. No thyroid gland tissue was detected by ultrasonography. The 597
KOMATSU ET AL
THE JOURNAL OF PEDIATRICS OCTOBER 2001
Figure. Direct sequence of exon 2 of PAX8 gene. Asterisk indicates mutated nucleotide.
other 3 members of this family, the father and 2 sisters, are healthy and have no sign of hypothyroidism.
Molecular Studies Genomic DNA was extracted from peripheral blood lymphocytes. Polymerase chain reaction amplification from exon 2 to exon 9 of the PAX8 gene was performed with the primer pairs described by Macchia et al.3 The cycling conditions were 94°C for 1 minute, 57°C for 1 minute, and 72°C for 1 minute for 30 cycles, followed by a final extension step at 72°C for 7 minutes. Both forward and reverse strands were directly sequenced by using an automatic DNA sequencer (ABI PRISM 310 DNA sequencer; PE Applied Biosystems, Foster City, Calif).
RESULTS AND DISCUSSION P1 and P2 showed an arginine (CGC) to cysteine (TGC) substitution at codon 31 of the PAX8 gene (designated as R31C) (Figure). In addition, we analyzed 50 volunteers without features of hypothyroidism after informed 598
consent had been obtained. R31C was not identified in 100 alleles from healthy persons, demonstrating that it was not a polymorphism (data not shown). Codon 31 is located in the coding region of the paired domain in the PAX8 gene.5 The mutations in this codon must be critical, because the DNA-binding ability is reduced and results in abnormal development of the thyroid gland.3 The mutation at codon 31 has been independently reported in 2 other cases, an Italian patient3 and one Japanese family including 3 patients.4 These patients had an arginine (CGC) to histidine (CAC) substitution at this codon (designated as R31H). The missense mutations of codon 31, R31C (CGC to TGC) and R31H (CGC to CAC), may be ascribed to the hypermutability at CpG dinucleotides. It is well known that CpG dinucleotides can be a mutational hot spot in the human genome. In eukaryotic genomes, 5methylcytosine occurs predominantly in CpG dinucleotides, the majority of which appear to be methylated. Spontaneous deamination of 5-methylcytosine to thymine leads to TpG or CpA mutations.6-8 This mechanism may be the cause of the missense mutations in the 3 cases.
To date, only 3 mutations of the PAX8 gene, R108X, R31H, and L62R, have been identified by screening the genomic DNA of 145 Italian patients with thyroid dysgenesis.3 In those mutations, R108X (CGA to TGA) and R31H (CGC to CAC) are de novo mutations, which can be considered typical CpG-consequence mutations. Recently, the same R31H mutation was identified in a Japanese family with thyroid dysgenesis,4 suggesting that the R31H mutation is a recurrent mutation occurring in a mutational hot spot of the PAX8 gene. We identified a novel mutation, R31C (CGC to TGC), in the same codon as the R31H mutation, and R31C is also thought to be caused by the transformation of cytosine to thymine at the CpG nucleotide through the methylation-deamination mechanism. Among the 5 mutations identified from the PAX8 gene, 3 were located at the CpG nucleotide of codon 31. Thus, we propose that codon 31 is a hot spot for recurrent mutations in the human PAX8 gene.
Note added in proof During the course of our study, the novel mutation, C57Y, was reported by Vilain et al.9
REFERENCES 1. Clifton-Bligh RJ, Wentworth JM, Heinz P, Crisp MS, John R, Lazarus JH, et al. Mutation of the gene encoding human TTF-2 associated with thyroid agenesis, cleft palate and choanal atresia. Nat Genet 1998; 19:399-401. 2. Devriendt K, Vanhole C, Matthijs G, de Zegher F. Deletion of thyroid transcription factor-1 gene in an infant with neonatal thyroid dysfunction and respiratory failure. N Engl J Med 1998;338:1317-8. 3. Macchia PE, Lapi P, Krude H, Pirro MT, Missero C, Chiovato L, et al. PAX8 mutations associated with congenital hypothyroidism caused by thyroid dysgenesis. Nat Genet 1998; 19:83-6. 4. Gouji K, Matsuo M. Folica Endocrinologica Japonica 2000;76:153.
KOMATSU ET AL
THE JOURNAL OF PEDIATRICS
VOLUME 139, NUMBER 4 5. Xu W, Rould MA, Jun S, Desplan C, Pabo CO. Crystal structure of paired domain-DNA complex at 2.5Å resolution reveals structural basis for Pax development mutation. Cell 1995; 80:639-50. 6. Cooper DN, Krawczak M, Antonarakis SE. The nature and mechanisms of human gene mutation. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors.
The metabolic and molecular bases of inherited disease. 7th ed. New York: McGraw-Hill; 1995. p. 259-91. 7. Ollila J, Lappalainen I, Vihinen M. Sequence specificity in CpG mutation hotspots. FEBS Lett 1996; 396:119-22. 8. El Antri S, Mauffret O, Monnot M, Lescot E, Convert O, Fermandjian S. Structural deviation at CpG provide a plausible explanation for the high frequency of mu-
tation at this site. Phosphorus nuclear magnetic resonance and circular dichroism studies. J Mol Biol 1993;230:373-8. 9. Vilain C, Rydlewski C, Duprez L, Heinrichs C, Abramowicz M, Malvaux P, et al. Autosomal dominant transmission of congenital thyroid hypoplasia due to loss-of-function mutation of PAX8. J Clin Endocrinol Metab 2001;86:234-8.
599