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Clinical Chemistry 52: 182-191, 2006. First published December 1, 2005; 10.1373/clinchem.2005.058321
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(Clinical Chemistry. 2006;52:182-191.)
© 2006 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Three Mutations (p.Q36H, p.G418fsX482, and g.IVS19-2A>C) in the Dual Oxidase 2 Gene Responsible for Congenital Goiter and Iodide Organification Defect

Viviana Varela1, Carina M. Rivolta1, Sebastián A. Esperante1, Laura Gruñeiro-Papendieck2, Ana Chiesa2 and Héctor M. Targovnik1,a

1 Laboratorio de Biología Molecular, Cátedra de Genética y Biología Molecular, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires, Argentina.
2 Centro de Investigaciones Endocrinológicas, CEDIE-CONICET, División Endocrinología, Hospital de Niños "Ricardo Gutiérrez", Buenos Aires, Argentina.

aAddress correspondence to this author at: Laboratorio de Biología Molecular, Cátedra de Genética y Biología Molecular, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Av. Córdoba 2351, 4to piso-sala 5, 1120 Buenos Aires, Argentina. Fax 54-11-4508-3645; e-mail htargovn{at}huemul.ffyb.uba.ar.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Iodide organification defects are associated with mutations in the dual oxidase 2 (DUOX2) gene and are characterized by a positive perchlorate discharge test. These mutations produce a congenital goitrous hypothyroidism, usually transmitted in an autosomal recessive mode.

Methods: We studied the complete coding sequence of the human DUOX2 gene by single-strand conformational polymorphism (SSCP) analysis of DNA from 17 unrelated patients with iodide organification defects. Samples showing an aberrant pattern were directly sequenced. All mutations were validated by SSCP analysis. Finally, the effect of a splicing mutation was studied by construction of minigenes.

Results: Genomic DNA sequencing revealed 3 novel mutations [c.108G>C (p.Q36H), c.1253delG (p.G418fsX482), and g.IVS19-2A>C] and 1 previously reported mutation [c.2895-2898delGTTC (p.S965fsX994)] in 2 families with 1 (family 1) and 2 (family 2) affected members. This implies the inheritance of 2 compound heterozygous mutations, p.Q36H and p.S965fsX994 in family 1 and p.G418fsX482 and g.IVS19-2A>C in family 2. The c.1253delG mutation was associated with a c.1254C>A transversion. In vitro transcription analysis showed that exon 20 is skipped entirely when the g.IVS19-2A>C mutation is present. The wild-type glutamine residue at position 36 is strictly conserved.

Conclusions: Two previously unknown compound heterozygous mutations in the DUOX2 gene, p.Q36H/p.S965fsX994 and p.G418fsX482/g.IVS19-2A>C, are responsible for iodide organification defects in 2 unrelated families. Identification of the molecular basis of this disorder might be helpful for understanding the pathophysiology of this congenital hypothyroidism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Congenital hypothyroidism occurs with a prevalence of ~1 in 4000 newborns (1)(2). Patients with this syndrome can be divided into 2 groups: those with nongoitrous (dysembryogenesis) and those with goitrous (dyshormonogenesis) congenital hypothyroidism. Dysembryogenesis, or dysgenesis, congenital hypothyroidism, which accounts for 85% of cases, results from ectopic thyroid tissue at the base of the tongue or in any position along the thyroglossal tract, agenesis, and hypoplasia. In a minority of these patients, the congenital hypothyroidism is associated with mutations in genes responsible for the development or growth of thyroid follicular cells: thyroid transcription factor 1 (TTF-1; also known as TITF1, NKX2–1, or T/EBP) (3)(4), thyroid transcription factor 2 (TTF-2; also known as TITF2, FOXE1, or FKHL15) (5), paired box transcription factor 8 (PAX-8) (6)(7)(8)(9), thyrotropin (TSH) 1 (10)(11), and TSH receptor (12)(13)(14). The presence of congenital goiter (which accounts for the remaining 15% of cases) has been linked to mutations in the Na+/I– symporter (NIS) (15)(16)(17), thyroglobulin (TG) (18)(19)(20)(21), thyroperoxidase (TPO) (22)(23)(24), dual oxidase 2 (DUOX2; also known as ThOX2 and LNOX2) (25), and Pendrine (PDS; also known as SLC26A4) (26)(27) genes. These mutations produce a heterogeneous spectrum of congenital goitrous hypothyroidism, usually transmitted in an autosomal recessive mode.

Iodide organification defects are associated with mutations in the TPO or DUOX2 genes (22)(23)(24)(25) and are characterized by a positive perchlorate discharge test. TPO is the key enzyme in the formation of thyroid hormones; it catalyzes both the iodination and coupling of hormonogenic tyrosyl residues of TG with an absolute requirement of H2O2, which acts as an electron acceptor (28). H2O2 is generated on the apical plasma membrane of the thyroid follicular cell by a metabolic pathway involving 2 members of the NADPH oxidase family: Duox1 and -2 (29)(30)(31)(32)(33)(34). The DUOX2 gene (GenBank accession no. NT_010194) is located on chromosome 15q15.3 spanning 21.5 Kb of genomic DNA (31), which includes 33 coding exons. The mRNA (GenBank accession no. NM_014080) is 6376 nucleotides long, and the preprotein is composed of a putative 26-amino acid signal peptide followed by a 1522-amino acid polypeptide (31). The DUOX1 gene encodes a homologous 1551-amino acid protein displaying 83% sequence similarity. To date, only 4 inactivating mutations in the DUOX2 gene (p.R434X, p.Q686X, p.R701X, and p.S965fsX994) have been identified in patients with iodide organification defect (25).

In the present study, screening by single-strand conformational polymorphism (SSCP) and direct sequencing analysis of the DUOX2 gene revealed 3 previously unreported mutations and 1 previously reported mutation in 2 unrelated families with congenital goiter and iodide organification defect, constituting 2 compound heterozygous mutations (p.Q36H/p.S965fsX994 and p.G418fsX482/g.IVS19-2A>C).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
patient selection
We studied 17 selected unrelated patients with iodide organification defect (perchlorate discharge test ≥30%) from an original cohort of 40 patients with congenital goitrous hypothyroidism at screening. We previously reported that 7 of these patients harbored mutations in the TPO gene: 1 was compound heterozygous, and the other 6 were simply heterozygous for the mutations (24).

The project was approved by the Institutional Review Board, and written informed consent was obtained from all individuals involved in this study.

clinical report
Family 1: Index patient II-2.
The patient is the second child of a nonconsanguineous couple and was referred because a high serum TSH value on neonatal screening at day 3 of life. He was born at term after an uncomplicated pregnancy and delivery with a birth weight of 3.1 kg. At day 27 of life, he had persistent jaundice, large posterior fontanel, and umbilical hernia. Thyroid tissue was palpable. After confirmation of hypothyroidism, he was started with L-thyroxine supplementation. At age 5 years, his thyroid hormone supplementation was interrupted for 4 weeks to investigate the etiology. A technetium-99 scan showed a goiter, and hypothyroidism was confirmed. His perchlorate discharge test result (46%) indicated a iodide organification defect. Treatment was reinitiated. He had a very good compliance, and his growth and development are normal. His parents and sister have normal thyroid function. The results of thyroid function tests of the members of family 1 who were studied are shown in Table 1 .


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  		Table 1. Thyroid function test results for relatives and patients with congenital goitrous hypothyroidism and organification defect.1

Family 2: Patient II-2.
The patient is the second child of a nonconsanguineous couple referred at the age of 8 months with hypothyroidism. She had all of the signs of thyroid hormone deficiency with goiter, and after confirmation, treatment with L-thyroxine was started.

At the age of 4.5 years, the etiology of her condition was assessed after withdrawal of the thyroxine supplementation, and hypothyroidism attributable to iodide organification defect was suspected (perchlorate discharge test result, 68%). She grew up normally but is mildly mentally retarded.

At age 15 years, after a noncomplicated pregnancy and delivery, she gave birth to a 3.0-kg male infant (III-1) who had a normal serum TSH concentration at neonatal screening for congenital hypothyroidism. The boy grew and developed normally up to 3 years of age when, during a routine pediatric examination, a 30-g goiter was detected. Thyroid evaluation showed high serum TSH concentrations, a perchlorate discharge test result of 31%, and positive anti-thyroid antibodies [anti-TPO antibodies, 70 kIU/L (reference values <20 kIU/L]); anti-TG antibodies, 20 kIU/L (reference values <20 kIU/L)], indicating autoimmune thyroid disease.

Family 2: Index patient II-4.
The patient was the fourth child of this family. He was referred at 1 month of age with severe hypothyroidism (persistent jaundice, myxedematous face, large posterior fontanel, and umbilical hernia) and goiter. Hypothyroidism was confirmed, and treatment with L-thyroxine was started. At age 4 years, the etiology of his condition was evaluated after withdrawal of thyroxine treatment. His findings resembled those of his sister.

The parents and the 2 healthy siblings of this family were evaluated. Thyroid function was normal in patient II-1, whereas the mother (I-2) and a sister (II-3) showed high serum TSH concentrations and positive anti-thyroid antibodies: for the mother (I-2), anti-TPO antibodies were 279 kIU/L and the anti-TG antibodies were <20 kIU/L; for the sister (II-3), the anti-TPO antibodies were 70 kIU/L and the anti-TG antibodies were 40 kIU/L. The perchlorate discharge test in I-2 was 42%. The father (I-1) had TSH concentrations within reference values and also positive anti-thyroid antibodies (anti-TPO antibodies, 64 kIU/L; anti-TG antibodies, 33 kIU/L. These findings indicate an autoimmune thyroid disease in I-1, I-2, and II-3.

The results of thyroid function tests of the family 2 members studied are shown in Table 1Up .

thyroid function tests
The perchlorate discharge test was performed according to standard procedures. Briefly, 25 µCi of radioiodine (I-131) was administered orally, and thyroid uptake was measured at 15-min intervals for 1 h. At this point, 500 mg of potassium perchlorate was given orally, and sequential uptakes were measured at 15-min intervals for an additional period of 2 h. Discharge was calculated as the difference between the uptake at the time of perchlorate administration and the uptake 120 min after perchlorate administration, expressed as a percentage. Values >10% were considered as a failure to retain the administered radioiodine, probably because of a congenital organification defect or an autoimmune thyroid disease.

Serum total thyroxine (TT4) and serum TSH were determined by electrochemiluminescence immunoassay (ELECSYSTM system; Roche). Serum TG was measured by immunofluorometric assay (Delfia; Perkin-Elmer). Anti-TPO and anti-TG antibodies were measured by immunochemiluminometric assay (Immulite; Diagnostic Products Corporation).

sscp analysis
SSCP analysis was used to screen for the presence of mutations in each exon of the DUOX2 gene. Genomic DNA was isolated from leukocytes by the sodium dodecyl sulfate–proteinase K method. The complete coding sequence of the human DUOX2 gene, along with the flanking intronic regions of each intron, were amplified from the affected patients. PCR was performed in 50-µL reactions containing a standard PCR buffer (Invitrogen, Life Technologies), 250 ng of genomic DNA, 1.5 or 2.5 mM MgCl2, 200 µM each of dATP, dCTP, dTTP, and dGTP; with or without 80 nL/µL dimethyl sulfoxide; 1 U of Taq polymerase (Invitrogen, Life Technologies); and 40 pmol each of the forward and reverse primers. Intronic primers were specially designed or were as reported previously (25) for each of the 33 DUOX2 coding exons. Their oligonucleotide sequences and the positions of their 5' coding sequence ends are shown in Table 2 . Samples were denatured at 95 °C for 3 min followed by 35 cycles of amplification, each consisting of denaturation at 95 °C for 30 s, primer annealing at 55–66 °C for 30 s, and primer extension at 72 °C for 1 min. After the last cycle, the samples were incubated for an additional 10 min at 72 °C to ensure that the final extension step was complete. The amplified products were analyzed in 2% agarose gels. The gel matrix for SSCP analysis contained 8% or 10% polyacrylamide (29:1; Invitrogen, Life Technologies), with or without 100 mL/L glycerol. Samples were electrophoresed for 18 h at a constant temperature (4 °C). DNA was visualized by silver staining.


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  		Table 2. DUOX2 oligonucleotides used as primers in PCR amplification and sequence reactions.1

dna sequencing
Samples showing an aberrant pattern in SSCP analysis were directly sequenced with the Taq polymerase-based chain terminator method (fmol; Promega); the DUOX2 forward and reverse primers were the same as those used in the SSCP screening (Table 2Up ). The results were analyzed by use of the PC gene (Intelligenetics), DNASTAR (DNASTAR Inc.), and Nucleotide BLAST (http://www.ncbi.nlm.nih.gov/BLAST) software programs.

cloning of wild-type and mutated exon 11 pcr fragments
The amplified fragment corresponding to exon 11 from index patient II-4 (family 2) was T-A cloned into pGEM-T Easy vector (Promega). DH5{alpha}-competent cells were used for transformations. After transformation, the recombinant clones were identified by color screening on indicator plates containing isopropyl-thio-ß-galactoside and 5-bromo-4-chloro-3-indolyl-ß-D-galactoside (X-Gal), and plasmid DNA was isolated by standard procedures. DNA sequencing was performed as described above from wild-type and mutant allele clones.

validation of DUOX2 mutations by sscp analysis
We validated all mutations by studying healthy unrelated individuals by SSCP analysis under conditions identical to those described above, using the same intronic primers to amplify exons 2, 11, 20, and 21 (Table 2Up ).

ACII restriction analysis
The mutant cytosine, detected by nucleotide sequencing in position 108 (p.Q36H), creates a new AciI restriction site that is absent in the wild-type allele, which has guanine at position 108. The presence of the mutation was therefore independently confirmed by restriction analysis with AciI (New England BioLabs Inc.) in index patient II-2 (family 1) and his sister and parents. A 226-bp fragment containing exon 2 was generated by PCR under conditions identical to those described above with the same exon 2 intronic primers (Table 2Up ). The PCR product (1 µg) was digested overnight at 37 °C with 10 U of AciI. After digestion, the DNA fragments were separated on a 2% agarose gel and visualized with ethidium bromide. Digestion of the mutant allele produced 2 fragments of 142 and 84 bp.

construction and expression of the minigenes
To study the effect of the g.IVS19-2A>C mutation, we constructed wild-type and mutated minigenes, using the exon trapping vector pSPL3 (Life Technologies). The genomic DNA region from index patient II-4 (family 2) between exons 18 and 22 and the intronic flanking sequences (158 bp upstream from the 5' exon 18 end and 156 bp downstream from the 3' exon 22 end) were amplified by long PCR, as described previously (35). The forward primer (pSPL3-In17F; 5'-CACCGAATGCGGCCGCAACAGAGGCCCCAGTCAGTA-3') contained the NotI site, and the reverse primer (pSPL3-In22R; 5'-GCGGATCCGAGACTCAGGATGGTCGCTT-3') contained the BamHI site. The 3900-bp (3876 of which were DUOX2 sequences) PCR products were purified from the agarose gel by use if the GFX PCR DNA and Gel Band Purification Kit (Amersham Biosciences) and were sequentially digested with NotI and BamHI. The insert was directionally cloned into the NotI and BamHI sites of the pSPL3 vector. Because index patient II-4 (family 2) is heterozygous for the IVS19-2A>C mutation, the wild-type and mutated alleles were thus cloned. The recombinant plasmids were amplified in DH5{alpha}-competent cells and purified by use of the Concert High Purity Plasmid Miniprep System (Life Technologies). The correct sequence was confirmed by sequencing with the intronic forward primer used for PCR amplification of exon 20 (Table 2Up ).

CV-1 cells were grown in 3.8-cm dishes in DMEM supplemented with 50 mL/L bovine calf serum and 100 kIU/L penicillin–streptomycin in a 5% CO2 atmosphere at 37 °C. When the cells reached ~90% confluence, they were transfected with 1 µg of plasmid DNA (wild-type, mutant, and control pSPL3) per 3.8-cm dish with Lipofectamine 2000 (Invitrogen, Life Technologies). After incubation for 48 h, the cells were harvested and total RNA was extracted with Trizol (Invitrogen, Life Technologies). Reverse transcription-PCR (RT-PCR) was performed in the SuperScript One-Step RT-PCR System (Invitrogen, Life Technologies) with the exon 18 forward primer (e18F; 5'-TGACAAGGATGGCAATGGCT-3') and the exon 22 reverse primer (e22R; 5'-GCCAAACCTCTTCTTCAGTCCA-3'). Samples were incubated at 45 °C for 50 min, then at 94 °C for 10 min, followed by 40 cycles of DNA denaturation (94 °C for 45 s), annealing (55 °C for 1 min), and polymerization (72 °C for 1 min). After the last cycle, the samples were incubated for an additional 10 min at 72 °C. The RT-PCR products were purified from the agarose gel by use of the GFX PCR DNA and Gel Band Purification Kit (Amersham Biosciences) and then direct sequenced with the e18F and e22R primers.

protein analysis
We compared amino acid sequence homologies between several species of Duox2 and Duox1 families, using the Protein Blast and Search for Conserved Domains (http://www.ncbi.nlm.nih.gov/BLAST) software programs.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
screening of mutations in the DUOX2 gene by sscp and sequence analysis
All 33 coding exons of the DUOX2 gene, along with the flanking intronic sequences, from 17 unrelated patients with iodide organification defect and healthy controls were screened by SSCP analysis. Analysis of PCR products showed 4 different patterns of migration in 2 patients (II-2 in family 1 and II-4 in family 2) that were not detected in the healthy controls. The remaining 15 unrelated patients did not show aberrant bands, suggesting the absence of DUOX2 gene mutations. Sequence analysis of the samples showing the abnormal SSCP patterns revealed 3 previously unreported mutations and 1 previously identified mutation (c.2895-2898delGTTC) (25). One was a missense mutation involving a guanine-to-cytosine transition at nucleotide 108 in exon 2 (c.108G>C), which replaces the wild-type glutamine at codon 36 with a histidine (p.Q36H; see Fig. 1 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol52/issue2/ ). The second was a guanine deletion at nucleotide 1253 in exon 11 (c.1253delG), which produced a frameshift at amino acid 418 in exon 11 with a putative premature stop codon at amino acid position 482 in exon 12 (p.G418fsX482; see Fig. 2 in the online Data Supplement). The third was a adenine-to-cytosine transversion at position –2 of the splice acceptor site in intron 19 (g.IVS19-2A>C; see Fig. 3 in the online Data Supplement). This infers the possibility that the splice-site mutation might lead to total elimination of exon 20 of the DUOX2 gene. Translation of the resulting mutated transcript showed that the reading frame was not maintained by the junction of exons 19 and 21 and that, consequently, a frameshift was generated with a putative premature stop codon at amino acid position 886 in exon 21.

The last mutation detected by SSCP analysis was a previously reported GTTC deletion in exon 21 (c.2895-2898delGTTC) (25), which causes a frameshift at amino acid 965 in exon 21 with a putative premature stop codon at amino acid position 994 in exon 22 (p.S965fsX994; see Fig. 4 in the online Data Supplement).

These findings indicate that index patient II-2 (family 1) is compound heterozygous for p.Q36H/p.S965fsX994, whereas index patient II-4 (family 2) is compound heterozygous for p.G418fsX482/g.IVS19-2A>C.

The c.1253delG heterozygous state of index patient II-4 was confirmed by cloning and sequencing of both wild-type and mutant alleles. Another difference identified in the mutant allele was a cytosine-to-adenine transversion in position 1254 (c.1254C>A; see Fig. 2 in the online Data Supplement).

Previous SSCP analysis did not show evidence of TPO gene mutations in either index patient (II-2 in family 1 and II-4 in family 2) (24).

validation of DUOX2 mutations by sscp analysis
We ruled out the possibility that the p.Q36H mutation could be a polymorphism because it was not detected in 134 chromosomes from the general population by SSCP analysis (data not shown). The p.G418fsX482 and g.IVS19-2A>C mutations were not detected in 100 alleles from healthy individuals screened by SSCP analysis. In contrast, 1 of the 200 chromosomes also analyzed by SSCP had the p.S965fsX994 mutation (data not shown).

segregation analysis of DUOX2 mutations in family 1
SSCP analysis of PCR products of exons 2 and 21 from index patient II-2, his father (I-1), his mother (I-2), and his unaffected sister (II-1; Fig. 1 ) showed that II-2 inherited 1 copy of the p.Q36H mutation from his mother and 1 copy of the p.S965fsX994 mutation from his father. The healthy sister (II-1) was heterozygous for the p.Q36H mutation and did not carry the p.S965fsX994 mutation (Fig. 1 ).


Figure 1
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  		Figure 1. Segregation analysis of the DUOX2 mutated alleles in members of families 1 and 2.

(A), pedigrees showing the pattern of inheritance of the DUOX2 mutated alleles. Squares and circles indicate male and female members, respectively. Filled symbols denote affected patients; half-filled symbols, unaffected heterozygote individuals. The open arrows indicate the index patients (II-2 in family 1 and II-4 in family 2). In family 1, the striped symbols indicate the p.Q36H allele, and the filled symbols the p.S965fsX994 allele. In family 2, the striped symbols indicate the p.G418fsX482 allele, and the filled symbols the g.IVS19-2A>C allele. (B), SSCP analysis of exons 2, 11, 20, and 21. The PCR products were subjected to electrophoresis in 8%–10% nondenaturing acrylamide gels without glycerol. The filled arrows indicate the aberrant migration bands.

The guanine-to-cytosine transversion at position 108 creates a restriction site for AciI. The index patient II-2, his mother, and his unaffected sister (II-1) have a wild-type allele (226 bp) containing guanine at position 108, which is resistant to digestion with AciI, and a mutant allele containing cytosine at position 108, which is digested with AciI, producing 2 fragments of 142 and 84 bp. In contrast, the father has only the wild-type sequence at this position (data not shown).

segregation analysis of DUOX2 mutations in family 2
SSCP analysis of PCR products of exons 11 and 20 from index patient II-4, his father (I-1), his mother (I-2), his unaffected sister (II-1), his unaffected brother (II-3), his affected sister (II-2), and the unaffected nephew (III-1; Fig. 1Up ) showed that both affected siblings II-2 and II-4 inherited 1 copy of the p.G418fsX482 mutation from their mother and 1 copy of the g.IVS19-2A>C mutation from their father. The healthy sister and brother (II-1 and II-3) and the unaffected nephew (III-1) were heterozygous for the g.IVS19-2A>C mutation and did not carry the p.G418fsX482 mutation (Fig. 1Up ).

minigene analysis
Using the exon trapping system, we tested a minigene containing the g.IVS19-2A>C mutation for abnormal splicing. In vitro transcription showed that exon 20, which consists of 197 bp, is skipped entirely when the g.IVS19-2A>C mutation is present, whereas the minigene that contains the wild-type allele is correctly spliced (Fig. 2 ). Interestingly, RT-PCR amplification products of both minigenes lacked exon 21. Two nonspecific PCR bands were detected in the pSPL3 vector control and in both wild-type and mutant minigenes (Fig. 2B ).


Figure 2
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  		Figure 2. In vitro expression of the wild-type and mutant g.IVS19-2A>C minigenes.

(A), schematic representation of the genomic organization of the wild-type and mutant minigenes and their RT-PCR products. The 3876-bp PCR amplified fragments from index patient II-4 (family 2) were directionally cloned into the NotI and BamHI sites of the exon-trapping pSPL3 vector, which was produced in CV-1 cells. Vector and genomic DNA splice donor (gt) and acceptor (ag) sites are shown. cDNA was synthesized from transcribed mRNA and amplified with primers e18F and e22R, located in exons 18 and 22 of the DUOX2 gene, respectively. Processing of the wild-type transfected sequences produced a majority PCR product of 438 bp. In contrast, the IVS19-2A>C mutation leads to the skipping of exon 20, producing a 241-bp fragment. (B), agarose electrophoresis of the RT-PCR products. The RT-PCR fragments from wild-type (WT) and mutant (Mut) minigenes, pSPL3 vector control (C) and the negative PCR control (–) were electrophoresed on a 2% agarose gel, stained with ethidium bromide, and visualized under ultraviolet light. The nonspecific PCR bands are indicated by filled arrows. The DNA size marker (MW) was a 100-bp DNA ladder (Invitrogen, Life Technologies).

protein analysis
Comparison of the human Duox2 with sequences found in the GenBank database, using the Blast network service, revealed that the wild-type glutamine residue at position 36 was strictly conserved in all Duox1 and Duox2 species analyzed (Fig. 3 ).


Figure 3
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  		Figure 3. Protein homology analysis.

Shown is the partial protein alignment of the different species of the Duox2 and Duox1 families. Conserved residues are indicated by gray shading, and the glutamine residue at position 36, involved in the missense mutation (p.Q36H), is boxed.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
An organification defect attributable to DUOX2 gene mutations is one of the causes for thyroid dyshormonogenesis (25). This type of primary congenital hypothyroidism is characterized by intact iodide trapping, normal TG protein, and aberrant organification of iodide (1)(2). In this study, all 3 affected individuals had clinical and biochemical criteria suggestive of congenital goiter associated with deficiency in the organification of iodide: presence of goiter, hypothyroidism, positive perchlorate discharge test, and high serum TG and TSH concentrations with simultaneous low serum TT4 concentrations (1)(2). Molecular analyses indicated that the affected individuals carry either compound heterozygous mutations for p.Q36H/p.S965fsX994 or p.G418fsX482/g.IVS19-2A>C.

Duox2 contains 7 transmembrane domains (TMDs), a domain of homology with peroxidase (TPO-like), 5 potential sites for N-glycosylation, 1 conserved arginine and 4 specific histidines involved in the heme prosthetic group binding, 1 FAD- and 4 NADPH-binding sites, and 2 EF-hand domains (31)(32)(33)(34) (see Fig. 5A in the online Data Supplement). The p.Q36H missense mutation is located in the TPO-like domain of Duox2 (see Fig. 5B in the online Data Supplement). The putative function of the peroxidase homology domain is not yet clear. Edens et al. (36) reported that the TPO-like domain of Duox1 catalyzes protein cross-linking by forming dityrosine bonds. The p.Q36H mutation was not detected in the general population, suggesting that this change is not a polymorphism. More importantly, the wild-type glutamine residue at position 36 is strictly conserved in all species for which suitable Duox1 and Duox2 sequences have been reported. On the basis of such observations, we hypothesize that the glutamine residue in this position plays a critical structural role in the Duox2 protein. Consequently, the p.Q36H mutation may cause structural instability leading to deficient Duox2 function.

The putative truncated protein of 481 amino acids generated by the c.1253delG mutation (p.G418fsX482) eliminates the last 2 potential sites for N-glycosylation, both EF-hand domains, all histidine and arginine sites, all TMDs, and the FAD- and NADPH-binding sites (see Fig. 5C in the online Data Supplement), whereas the putative truncated protein of 993 amino acids generated by the c.2895–2898delGTTC mutation (p.S965fsX994) eliminates all histidine and arginine sites, the last 6 TMDs, and the FAD- and NADPH-binding sites (see Fig. 5E in the online Data Supplement). We found the c.1253delG mutation associated with the c.1254C>A transversion in the same allele. This substitution was not detected in any of the 100 alleles from healthy individuals screened by SSCP analysis, suggesting that it is not a polymorphic nucleotide position. However, a hypothetical allele with the c.1254C>A transversion and without the c.1253delG mutation would conserve glycine-418 (GGC>GGA). The functional consequences of both truncated proteins, on the one hand, is insufficient thyroidal production of H2O2, which prevents the synthesis of sufficient quantities of thyroid hormones. On the other hand, deletion of the middle region and/or C-terminal of Duox2 could alter the protein folding and assembly, leading to a marked reduction in the ability to export the protein from the endoplasmic reticulum (37)(38). An interesting but unsolved question, which could not be investigated because the thyroid tissue from patients was unavailable and we thus could not perform mRNA analysis, would have been to estimate the DUOX2 transcript concentration in goiters. In general, mRNA containing nonsense mutations is found in low concentrations. This decrease is attributable to an accelerated degradation rate of the mutated mRNA because the untranslated part of the messenger is not protected by ribosomes. To complement this scenario, it is not possible to exclude that an alternative splicing mechanism, by activation of cryptic splice sites, might restore the normal reading frame disrupted by the mutation and eliminate the stop codon that would truncate the protein. As is reported for the human TPO gene, a GGCC insertion-duplication at position 1186 in the eighth exon (p.R396fsX472) would lead to a frameshift at amino acid 396 with a premature stop at 472 in exon 9 (22). Alternative splicing by a cryptic acceptor splice site in exon 9 restores the normal reading frame disrupted by the mutation and eliminates the stop codon.

Another mutation found in this study is a previously unreported A-to-C transversion at position –2 in the acceptor site of intron 19. This splice site mutation might lead to total elimination of exon 20 of the DUOX2 gene. It is known from other genes that mutations in this site that alter the consensus acceptor site can lead to exon skipping (18). Because of the thyroid tissue from II-4 is unavailable, we used an in vitro exon-trapping system to evaluate whether the g.IVS19-2A>C mutation produces an abnormal transcript by a defect in exon splicing. We found that during in vitro transcription, the mutation in the acceptor splice site causes skipping of exon 20. The excision of exon 20 in the DUOX2 mRNA might generate a frameshift with a putative premature stop codon in exon 21, which encodes a grossly truncated protein of 885 amino acids. The premature stop eliminates all histidine and arginine sites, the last 6 TMDs, and the FAD- and NADPH-binding sites (see Fig. 5D in the online Data Supplement). Unexpectedly, the RT-PCR products of both wild-type and mutant minigenes lacked the sequence corresponding to exon 21. At present, there are no published reports referring to the existence of alternatively spliced mRNAs lacking exon 21 of the DUOX2 gene. However, the excision of exons 20 and 21 would restore the normal reading frame in the mutated allele. Further experiments are required to confirm excision of exon 21 in the mRNA and to determine their functional consequences.

Finally, it is interesting to note that patient II-2 (family 1), who carries a missense mutation in one allele and a putative premature stop codon in the other allele (p.Q36H/p.S965fsX994), had a perchlorate discharge test result of 46%, whereas patients II-2 and II-4 (family 2), who carry 2 putative premature stop codons, one in each allele (p.G418fsX482/g.IVS19-2A>C), have major iodide discharges: 68% and 60%, respectively. Subsequently, we could hypothesize that the p.Q36H mutation causes a slight reduction in Duox2 function. However, the positive perchlorate discharge test observed in members I-2 (42%) and III-1 (31%) of family 2, both heterozygous for the p.G418fsX482 and g.IVS19-2A>C mutations, respectively, could be caused for their autoimmune thyroid diseases.

From our study, we can draw 5 conclusions: (a) SSCP analysis of the entire coding sequence of the human DUOX2 gene, including the flanking intronic regions, and sequencing of the fragments presenting aberrant migration patterns revealed 3 previously unreported mutations (p.Q36H, p.G418fsX482, and g.IVS19-2A>C) and 1 previously reported mutation (p.S965fsX994). (b) These finding established the inheritance in 2 unrelated family of 2 compound heterozygous mutations (p.Q36H/p.S965fsX994 and p.G418fsX482/g.IVS19-2A>C). (c) Analysis of the GenBank database revealed that the wild-type glutamine residue at position 36 is strictly conserved. (d) In vitro transcription analysis confirmed that exon 20 is skipped entirely when the g.IVS19-2A>C mutation is present. (e) The p.S965fsX994 mutation is the most frequent mutation in the DUOX2 gene; consequently, it would be helpful to investigate further cases with congenital hypothyroidism regarding this mutation. The identification and characterization of an increasing number of natural mutations have provided important insights into the structure–function relationship of Duox2.


   Acknowledgments
 
C.M. Rivolta and H.M. Targovnik are established investigators of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). S.A. Esperante is a research fellow of the Agencia Nacional de Promoción Científica y Tecnológica-Fondo para la Investigación Científica y Tecnológica (ANPCyT-FONCyT). This work was supported by grants from Universidad de Buenos Aires (B 087/2001 and B 057/2004) and ANPCyT-FONCyT (05-08838/PICT 2000/2001)


   Footnotes
 
1 Nonstandard abbreviations: TSH, thyrotropin; NIS, Na+/I– symporter; TG, thyroglobulin; TPO, thyroperoxidase; DUOX and Doux, dual oxidase gene (according to the recommendations of the Human Gene Nomenclature Committee) and protein; PDS, Pendrine; SSCP, single-strand conformational polymorphism; TT4, total thyroxine; RT-PCR, reverse transcription-PCR; and TMD, transmembrane domain.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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