Total Iodide Organification Defect Research Articles

A truncating TPO mutation (Y55X) in patients with hypothyroidism and total iodide organification defect

AbsractPurpose: Mutations in the TPO gene have been reported to cause congenital hypothyroidism (CH), and our aim in this study was to determine the genetic basis of congenital hypothyroidism in two affected children coming from a consanguineous family.Methods: Since CH is usually inherited in autosomal recessive manner in consanguineous/multi case-families, we adopted a two-stage strategy of genetic linkage studies and targeted sequencing of the candidate genes. First we investigated the potential genetic linkage of the family to any known CH locus using microsatellite markers and then screened for mutations in linked-gene by Sanger sequencing. Results: The family showed potential linkage to the TPO gene and we detected a non-sense mutation (Y55X) in both cases that had total iodode organification defect (TIOD). The mutation segregated with disease status in the family. Y55X is the only truncating mutation in the exon 2 of the TPO gene reported in the literature and results in the earliest stop codon known in the gene to date. Conclusions: This study confirms the pathogenicity of Y55X mutation and demonstrates that a nonsense mutation in the amino-terminal coding region of the TPO gene could totally abolish the function of the TPO enzyme leading to TIOD. Thus it helps to establish a strong genotype/phenotype correlation associated with this mutation. It also highlights the importance of molecular genetic studies in the definitive diagnosis and accurate classification of CH.

Minimally Invasive Follicular Thyroid Carcinoma Developed in Dyshormonogenetic Multinodular Goiter Due to Thyroid Peroxidase Gene Mutation

The occurrence of thyroid carcinoma in patients with congenital hypothyroidism (CH) caused by dyshormonogenesis is very rare, and has only been reported in one patient harboring mutations in the thyroid peroxidase (TPO) gene. We report on a 29-year follow-up of two consanguineous siblings with CH due to total iodide organification defect who also had sensorineural hearing loss. Molecular analysis revealed a novel biallelic mutation of the TPO gene in which phenylalanine substitutes serine at codon 292 (c.875C>T, p.S292F) in exon 8. Despite early initiation, adequate doses of levothyroxine treatment and consequently normal thyrotropin (TSH) levels, the proposita developed a huge multinodular goiter (MNG) and underwent total thyroidectomy due to tracheal compression. Pathological examination revealed a unifocal follicular thyroid carcinoma without vascular invasion in the left lobe of the thyroid gland. Our finding of follicular thyroid carcinoma arising from dyshormonogenetic MNG in a patient without elevated serum TSH levels indicates that genetic and environmental factors other than TSH level might be involved in the development of thyroid carcinoma in dyshormonogenetic MNG. Despite the rare occurrence of thyroid carcinoma in dyshormonogenetic MNG, we recommend long-term follow-up and regular neck ultrasound imaging to prevent delayed diagnosis of thyroid carcinoma.

Minimal Invasive Follicular Thyroid Carcinoma Developed in Dyshormonogenetic Multinodular Goiter Due to Thyroid Peroxidase Gene Mutation.

Background: The occurrence of thyroid carcinoma in patients with congenital hypothyroidism (CH) caused by dyshormonogenesis is very rare, and has been reported in only one patient harboring mutations in the thyroid peroxidase (TPO) gene. Patient findings: We report on a 29-year follow-up of two consanguineous siblings with CH due to total iodide organification defect who also had sensorineural hearing loss. Molecular analysis revealed a novel biallelic mutation of the TPO gene in which phenylalanine substitutes serine at codon 292 (c.875C>T, p.S292F) in exon 8. Despite early initiation, adequate doses of L-thyroxine treatment and consequently normal TSH levels, the proposita developed a huge multinodular goiter (MNG) and underwent total thyroidectomy due to tracheal compression. Pathological examination revealed a unifocal follicular thyroid carcinoma without vascular invasion in the left lobe of the thyroid gland. Summary: Our finding of follicular thyroid carcinoma arising from dyshormonogenetic MNG in a patient without elevated serum TSH levels indicates that genetic and environmental factors other than TSH level might be involved in the development of thyroid carcinoma in dyshormonogenetic MNG. Conclusions: Despite the rare occurrence of thyroid carcinoma in dyshormonogenetic MNG, we recommend long-term follow-up and regular neck ultrasound imaging to prevent delayed diagnosis of thyroid carcinoma.

Monoallelic thyroid peroxidase gene mutation in a patient with congenital hypothyroidism with total iodide organification defect

The aim of this study was to identify the genetic defect of a patient with dyshormonogenetic congenital hypothyroidisms (CH) with total iodide organification defect (TIOD). A male child diagnosed with CH during neonatal screening. Laboratory tests confirmed the permanent and severe CH with TIOD (99% perchlorate release). The coding sequence of TPO, DUOX2, and DUOXA2 genes and 2957 base pairs (bp) of the TPO promoter were sequenced. Molecular analysis of patient's DNA identified the heterozygous duplication GGCC (c.1186_1187insGGCC) in exon 8 of the TPO gene. No additional mutation was detected either in the TPO gene, TPO promoter, DUOX2 or DUOXA2 genes. We have described a patient with a clear TIOD causing severe goitrous CH due to a monoallelic TPO mutation. A plausible explanation for the association between an autosomal recessive disorder with a single TPO-mutated allele is the presence of monoallelic TPO expression.

Analysis of TPO gene in Turkish children with iodide organification defect: identification of a novel mutation

The objective was to determine molecular genetic analysis of the TPO gene in Turkish children with iodide organification defect (IOD). Patients with a diagnosis of primary hypothyroidism were evaluated. Subjects having a definite diagnosis of autoimmune thyroiditis, thyroid gland dysplasia and, or iodine deficiency were excluded. A total of 10 patients from nine unrelated Turkish families, with an unknown etiology of hypothyroidism, and with a presumptive diagnosis of IOD were included in the study. A perchlorate discharge test (PDT) was performed to all subjects, and sequence analysis of TPO gene was applied in patients with a positive PDT. Five out of 10 patients have a total IOD, while the five remaining patients have a partial IOD according to PDT results. In two sisters, one has a partial and the other one has a total IOD a novel homozygous nonsense p.Q315X mutation was found in exon 8. Additionally, a previously known homozygous missense p.R314W mutation was detected in the same exon in another patient with a total IOD. No TPO gene mutation was detected in any of the seven remaining patients. Two different TPO gene mutations were found to be responsible for IOD in two unrelated Turkish families from the same ethnic background. More subjects should be screened for detecting the prevalence and spectrum profile of TPO mutations in our population that might be helpful for understanding the pathophysiology of congenital hypothyroidism.

Screening for mutations of the human thyroid peroxidase gene in patients with congenital hypothyroidism.

While congenital hypothyroidism in 80-90% of the affected individuals is caused by thyroid dysgenesis (athyrosis, ectopy or hypoplasia), hypothyroidism in patients with a thyroid gland of normal position and size can be due to regulatory or enzymatic defects of thyroid hormone biosynthesis. Beside defects of thyroglobulinsynthesis, defects of the sodium-iodide-transporter or the TSH-receptor, a defect of the thyroidperoxidase, the key-enzyme of thyroid hormone biosynthesis, can cause a total iodide organification defect and thereby congenital hypothyroidism. We screened 14 of 103 patients (13.6%) with non familial congenital hypothyroidism and a normally developed thyroid gland detected by the newborn screening program with the PCR-SSCP (single-stranded-conformational-polymorphism) technique for mutations in the exons 2, 8, 9, 10 and 14 of the human thyroperoxidase gene, and in which mutations had been described previously in Dutch and Brazilian families with total organification defects. Most of the previously reported mutations were found in exons 8, 9 and 10 which code for the caralytic part of the enzyme. In two patients a GGCC-duplication in exon 8 was detected leading to a premature stop codon in exon 9. While one patient without neonatal goiter was homozygous for this mutation, the second patient was only heterozygous thus demanding another mutation on the second TPO-allel to explain the phenotype. Since the GGCC duplication is easily demonstrable by a NaeI digestion, because it creates a restriction site for this enzyme, screening for this mutation is indicated since it is easy to perform. In contrast to the perchlorate discharge test molecular genetic studies are less invasive, but as useful in making a definitive diagnosis in the individual patient. Furthermore it is the first feasible step to study the etiology and epidemiology of the so far only putative defects of thyroid hormone biosynthesis leading to congenital hypothyroidism.

Clinical Description of Infants with Congenital Hypothyroidism and Iodide Organification Defects

Aims: To describe the phenotype of a large group of children with congenital hypothyroidism (CH) and iodide organification defect (IOD), suspected based on normal thyroid position and abnormal perchlorate discharge test, as first step of a project evaluating correlations between phenotypes and genotypes. Methods: 71 children born in Paris between 1980 and 2006 were included. Two groups were defined according to perchlorate discharge: total IOD (TIOD) when the release was above 90% and partial IOD (PIOD) between 10 and 90%. Comparisons between groups were performed using SPSS 14.0 for Windows. Results: The incidence of IOD over the 2003–2006 period was 1:20,660. Of the 71 children, 61 had PIOD and 10 TIOD. Compared to PIOD, TIOD was characterized by greater clinical severity. A wide spectrum of clinical features was seen in the PIOD group. Evolution showed transient hypothyroidism in 10/61 patients with PIOD and 1/10 TIOD patients. Conclusions: Severe presentation in the majority of TIOD patients suggests dysfunction of a key iodide-organification enzyme. In contrast, the variety of clinical features in PIOD group suggests that diverse mechanisms may lead to PIOD, such as delayed or reduced activity of enzymes involved in hormonogenesis or defects in iodine storage and release.

Thyroid scintigraphy and perchlorate test after recombinant human TSH: a new tool for the differential diagnosis of congenital hypothyroidism during infancy

Prompt initiation of L-thyroxine therapy in neonates with congenital hypothyroidism (CH) often prevents the performance of functional studies. Aetiological diagnosis is thus postponed until after infancy, when the required investigations are performed after L-thyroxine withdrawal. The aim of this study was to verify the efficacy and safety of new protocols for rhTSH (Thyrogen) testing during L-thyroxine replacement in the differential diagnosis of CH. Ten CH patients (15-144 months old) were studied. Seven had neonatal evidence of gland in situ at the ultrasound examination performed at enrolment and received two rhTSH injections (4 microg/kg daily, i.m.) with 123I scintigraphy and perchlorate test on day 3. Three patients with an ultrasound diagnosis of thyroid dysgenesis received three rhTSH injections with 123I scintigraphy on days 3 and 4. TSH and thyroglobulin (Tg) determinations were performed on days 1, 3 and 4, and neck ultrasound on day 1. rhTSH stimulation caused Tg levels to increase in eight cases. Blunted Tg responses were seen in two patients with ectopia and hypoplasia. Interestingly, in two cases the association of different developmental defects was demonstrated. Perchlorate test revealed a total iodide organification defect in two patients, including one with a neonatal diagnosis of Pendred's syndrome, who were subsequently found to harbour TPO mutations. rhTSH did not cause notable side-effects. These new rhTSH protocols always resulted in accurate disease characterisation, allowing specific management and targeted genetic analyses. Thus, rhTSH represents a valid and safe alternative to L: -thyroxine withdrawal in the differential diagnosis of CH in paediatric patients.

Physiology and Pathophysiology of the DUOXes

The dual oxidases (DUOXes) 1 and 2 are named based on their having both a domain homologous to the NADPH-oxidase of the phagocyte NADPH-oxidase gp91( phox )/NOX2 and a domain homologous to thyroid peroxidase. The DUOX1 and DUOX2 mRNAs were originally cloned from thyroid tissue, and the corresponding proteins were recognized as intricate components of the thyroid hormone synthesis process, providing hydrogen peroxide essential for the organification of iodide. The function of DUOX2 in thyroid hormonogenesis has been firmly established by linking the congenital hypothyroid phenotype "total iodide organification defect" to biallelic inactivating DUOX2 mutations. Based on the expression of both DUOXes in combination with a peroxidase in a range of different tissues and functional studies; the concept evolves that DUOX is important not only for thyroid hormonogenesis but also as an integral part of the host defense system of mucosal surfaces, participates in the control of epithelial infection, augments surface B-cell receptor signaling in lymphocytes, and is involved in generating a respiratory burst at fertilization.

Total Iodide Organification Defect: Clinical and Molecular Characterization of an Italian Family

Thyroid peroxidase (TPO) deficiency is frequently involved in total iodide organification defects (TIOD). According to the recessive mode of inheritance, mutations are found in homozygous or in compound heterozygous states. However, a single heterozygous TPO mutation is reported in a high percentage (approximately 20%) of patients with typical TIOD phenotype. In the present study, the genetic and clinical evaluation of a TIOD family is reported. The propositus is an Italian girl with congenital hypothyroidism and positive perchlorate discharge test. Two TPO frameshift mutations were documented: a C deletion at 477 in exon 5, and a GGCC duplication at 1277 in exon 8. Unaffected family members, heterozygous for one of the two TPO mutations, were also studied in order to evaluate in vivo the functional activity of a single TPO allele. They have been found to have normal thyroid morphology and function with normal perchlorate test. In conclusion, the present study reports the clinical and molecular investigations in an Italian TIOD family. The results show that the TIOD phenotype in the propositus is associated to a compound heterozygous pattern, while a single TPO mutation does not significantly affect in vivo the efficiency of iodide organification. Therefore, extensive analyses of TPO gene and 2p25 locus are needed in the frequent TIOD cases in whom conventional investigations disclosed only one mutant allele.

Mutation screening of the thyroid peroxidase gene in a cohort of 55 Portuguese patients with congenital hypothyroidism

Defects in the human thyroid peroxidase (TPO) gene are reported to be one of the causes of congenital hypothyroidism (CH) due to a total iodide organification defect. The aim of the present study was to determine the nature and frequency of TPO gene mutations in patients with CH, characterised by elevated TSH levels and orthotopic thyroid gland, identified in the Portuguese National Neonatal Screening Programme. The sample comprised 55 patients, from 53 unrelated families, with follow-up in the endocrinology clinics of the treatment centres of Porto and Lisbon. Mutation screening in the TPO gene (exons 1-17) was performed by single-strand conformational analysis followed by sequencing of fragments with abnormal migration patterns. Eight different mutations were detected in 13 patients (seven homozygotes and six compound heterozygotes). Novel mutations included three missense mutations, namely 391T > C (S131P), 1274A > G (N425S) and 2512T > A (C838S), as well as the predictable splice mutation 2748G > A (Q916Q/spl?). The undocumented polymorphism 180-47A > C was also detected. The results are in accordance with previous observations confirming the genetic heterogeneity of TPO defects. The proportion of patients in which the aetiology was determined justifies the implementation of this molecular testing in our CH patients with dyshormonogenesis.

Persistent mild hypothyroidism associated with novel sequence variants of theDUOX2 gene in two siblings

One of the steps in thyroid hormone biosynthesis is the generation of hydrogen peroxide by dual oxidases (DUOX). Only one study reported mutations in DUOX2 gene in congenital hypothyroidism (CH) associated with total iodide organification defect (TIOD) in homozygosity or with partial iodide organification defect (PIOD) in heterozygous patients. We report genetic and phenotypic characterization of a family affected with isolated CH. The proband was positive at neonatal TSH screening. High serum TSH with low FT4 confirmed the diagnosis. At 4 years, TSH was high after L-T(4) withdrawal and (123)I scintigraphy with perchlorate discharge test revealed a PIOD. His brother was negative at TSH screening, but perinatal iodine overload was documented by urinary test. Serum TSH was elevated at postnatal day 11 and progressively increased together with a decline in urinary iodine. Reevaluation at 4 years confirmed a persistent hyperthyrotropinemia associated with PIOD. Both siblings resulted compound heterozygotes for two novel DUOX2 variants, a nonsense mutation (c.2524C>T, p.Arg842X) and a missense substitution (c.1126C>T, p.Arg376Trp), undetected in 140 control alleles. The parents had normal thyroid function and were heterozygous carriers of mutant alleles. In conclusion, we report two novel sequence variants in DUOX2 gene that are associated with persistent mild hypothyroidism and PIOD in two siblings. Different neonatal iodine supply apparently acted as disease modifier, justifying the discrepant results at TSH screening in the two siblings with same DUOX2 genotype and suggesting that mild dyshormonogenic defects may remain undisclosed in areas characterized by elevated iodine intake.

Monoallelic expression of mutant thyroid peroxidase allele causing total iodide organification defect.

Mutations in the thyroid peroxidase (TPO) gene lead to severe congenital hypothyroidism due to total iodide organification defect (TIOD). According to the recessive mode of inheritance, patients are homozygous or compound heterozygous for gene mutations. However, about 17% of cases with typical phenotype harbor a single TPO-mutated allele. We present a TIOD family in which the three affected siblings had a single genomic TPO mutation (R693W) inherited from the unaffected father. Other mutations were not found, although all TPO coding exons and exon/intron boundaries were sequenced. Eleven different polymorphisms were found in hetero- or homozygosity in all family members. On the contrary, using retrotranscribed thyroid tissue RNA, all heterozygous polymorphisms and the mutation were homozygous. The distribution of the polymorphisms indicated that only the mutant paternal allele is transcribed at the thyroid tissue level. We excluded the presence of major deletions involving the maternal chromosome at 2p25 and of maternal imprinting or mutations in part of the regulatory regions of the gene. In summary, we report one family with TIOD due to monoallelic expression of a mutant TPO allele in the thyroid. This mechanism might be generally involved in TIOD cases with a single TPO-mutated allele.

High prevalence of a novel mutation (2268 insT) of the thyroid peroxidase gene in Taiwanese patients with total iodide organification defect, and evidence for a founder effect.

The mutation of the thyroid peroxidase (TPO) gene that causes the total iodide organification defect (TIOD) is a common and severe condition leading to dyshormonogenesis of the thyroid gland in Caucasians. However, the role of TIOD in Chinese patients with thyroid dyshormonogenesis is unknown. In this study we followed 16 patients from 16 unrelated families in Taiwan and performed perchlorate discharge examination. Seven patients had TIOD and 2 had the partial iodine organification defect (PIOD) among the 16 families. These 9 patients underwent screening in search of TPO gene mutations. Three new mutations (2268 insT, 2243 delT, and G157C) were detected in the 7 patients with TIOD, whereas no mutation in the TPO gene was found in the 2 patients with PIOD. The 2268 insT mutation was noted to be the most common among these TIOD patients (12 of 14 studied alleles, 86%). With 3 intragenic polymorphic markers, we found that the alleles carrying the 2268 insT mutation in Taiwan Chinese TIOD patients were tightly linked to a specific haplotype. The allele frequencies of this haplotype in the 8 patients with homozygous 2268 insT (5 unrelated families, 10 studied alleles) and in 49 normal individuals (98 studied alleles) were 1.00 and 0.02, respectively (P < 0.0001). This indicates that this common novel mutation among Taiwanese patients with TIOD is due to a founder effect.

Mutation analysis of thyroid peroxidase gene in Chinese patients with total iodide organification defect: identification of five novel mutations.

Total iodide organification defect (TIOD), where the iodide in the thyroid gland cannot be oxidized and/or bound to the protein, is caused by a defect in the thyroid peroxidase (TPO) gene. Single strand conformation polymorphism analysis was used to screen for mutations in the TPO gene from five unrelated TIOD patients in Taiwan, and five novel mutations were detected. Three of these were frameshift mutations: a single T insertion between nucleotide position 2268 and 2269 (c.2268-2269 insT) in exon 13 and two single C deletions at nucleotide positions 843 (c.843 delC) and 2413 (c.2413 delC) in exon 8 and 14 respectively. The other two were single nucleotide substitutions (c.G1477>A and c.G2386>T) located in exons 9 and 13 respectively. While the former would result in amino acid substitution (Gly493Ser) in the highly conserved region of the TPO polypeptide, the latter would result in either amino acid substitution (Asp796Tyr) or alternative splicing. Of those identified TPO mutations, c.2268-2269 insT was most prevalent and was detected as heterozygous in all but one TIOD patients. All five TIOD patients investigated in this study were compound heterozygous. The method presented in this study could be used for carrier assessment and mutation analysis of newly identified TIOD patients.

Loss of heterozygocity at the thyroid peroxidase gene locus in solitary cold thyroid nodules.

Germline mutations in both alleles of the thyroid peroxidase (TPO) gene have been reported as a frequent cause of congenital hypothyroidism resulting from a total iodide organification defect (TIOD). Because TPO mutations have a prevalence of 1 in 66,000 newborns and is inherited in an autosomal recessive mode the frequency of a heterozygous germline mutation in the TPO gene should reach about 1 in 260 in the population. A somatic TPO mutation coinciding with a somatic loss of one of the TPO alleles or a TPO germline mutation could lead to somatic loss of TPO activity with impairment of thyroid hormone synthesis and decrease of growth control. The latter would lead to increased thyroid epithelial cell proliferation and the subsequent development of a scintigraphically cold thyroid nodule (CTN). To test this hypothesis we studied 40 CTN for the presence of mutations or loss of heterozygosity (LOH) in the TPO gene. For comparisons we also studied LOH in 17 autonomously functioning thyroid nodules (AFTN). Genomic DNA was extracted from nodular and surrounding tissue, polymerase chain reaction (PCR) amplified, sequenced, and analyzed for LOH. In 6 CTNs of 37 informative cases we detected LOH using the genomic markers sRA, D2S2268, and D2S319 within or near the TPO gene locus (2p24-25). In contrast, a genomic marker closer to the centromer (D2S144, 2p24-21) shows LOH in only 1 CTN. We did not detect LOH in AFTN. In none of the cases a germline or somatic mutation in the TPO gene was detectable in the TPO gene. LOH in 6 of 37 CTNs suggests that genetic defects at the TPO or the chromosomal locus 2p24-25 might play a role in the etiology of CTNs. However, we did not find the combination of LOH with a somatic mutation in the TPO gene. It is therefore likely that a gene defect near the TPO locus is part of the neoplastic process in a subgroup of CTNs.

Maternal Isodisomy for Chromosome 2p Causing Severe Congenital Hypothyroidism

Severe congenital hypothyroidism (CH) due to a total iodide organification defect (TIOD) is usually due to mutations in the thyroid peroxidase (TPO) gene located at chromosome 2p25. A homozygous deletion[Δ T2512 (codon 808)] in exon 14 was identified in a patient with classical TIOD. The transmission pattern of the TPO gene in this family was anomalous; the mother was heterozygous for the deletion; and the mutation was absent in the father. Polymorphic short tandem repeat (STR) markers confirmed paternity and demonstrated on chromosome 2 that the propositus was homozygous for most markers on chromosome 2p and that these were identical to one of the maternal 2p homologs. A normal karyotype was found in the propositus, his parents and sister. We conclude that the homozygosity in the patient is due to partial maternal isodisomy of the short arm of chromosome 2, carrying a defective TPO gene. The patient, born small for gestational age, develops and grows well and appears healthy (while being treated with thyroxine) and has a normal phenotype except for a unilateral preauricular skin tag. This shows that partial maternal isodisomy for chromosome 2p (2pter - 2p12) is compatible with a minimal influence on normal development.

Two decades of screening for congenital hypothyroidism in The Netherlands: TPO gene mutations in total iodide organification defects (an update).

Presented is a cohort study to assess the nature and frequency of thyroid peroxidase (TPO) mutations in 45 patients (35 families) with congenital hypothyroidism due to a total iodide organification defect; incidence is 1:66,000 in The Netherlands. The presentation is consistently similar with a severe form of congenital hypothyroidism and also characterized by a complete and immediate release of accumulated radioiodide from the thyroid after sodium perchlorate administration. Sixteen different mutations were found, including eight novel mutations; the majority occurs in exons 8, 9, or 10. The GGCC insertion in exon 8 at nucleotide 1277, leading to an early termination signal in exon 9, is the most frequently occurring mutation. These mutations were detected in 29 families in both TPO alleles (13 homozygous and 16 compound heterozygous). In one family, partial maternal isodisomy of 2p was detected, in four families only one mutated TPO allele could be detected, and in one family no inactivating TPO mutation could be found. Because all patients clearly had the clinicopathologic features of a total iodide organification defect, we conclude that in these five families the mutations in the (other) alleles could be either located in the intronic sequences or in the promoter region. Mutations in the TPO gene result in total iodide organification defects.

Two Decades of Screening for Congenital Hypothyroidism in the Netherlands: TPO Gene Mutations in Total Iodide Organification Defects (an Update)

Presented is a cohort study to assess the nature and frequency of thyroid peroxidase (TPO) mutations in 45 patients (35 families) with congenital hypothyroidism due to a total iodide organification defect; incidence is 1:66,000 in The Netherlands. The presentation is consistently similar with a severe form of congenital hypothyroidism and also characterized by a complete and immediate release of accumulated radioiodide from the thyroid after sodium perchlorate administration. Sixteen different mutations were found, including eight novel mutations; the majority occurs in exons 8, 9, or 10. The GGCC insertion in exon 8 at nucleotide 1277, leading to an early termination signal in exon 9, is the most frequently occurring mutation. These mutations were detected in 29 families in both TPO alleles (13 homozygous and 16 compound heterozygous). In one family, partial maternal isodisomy of 2p was detected, in four families only one mutated TPO allele could be detected, and in one family no inactivating TPO mutation could be found. Because all patients clearly had the clinicopathologic features of a total iodide organification defect, we conclude that in these five families the mutations in the (other) alleles could be either located in the intronic sequences or in the promoter region. Mutations in the TPO gene result in total iodide organification defects.

Identification of five novel inactivating mutations in the human thyroid peroxidase gene by denaturing gradient gel electrophoresis.

Thyroid peroxidase (TPO) is the key enzyme in the synthesis of thyroid hormones. Defects in the TPO gene are reported to be the cause of congenital hypothyroidism due to a Total Iodide Organification Defect (TIOD). This type of defect, where iodide taken up by the thyroid gland cannot be oxidized and bound to protein, is the most common hereditary inborn error causing congenital hypothyroidism in the Netherlands. Denaturing Gradient Gel Electrophoresis (DGGE) of PCR amplified genomic DNA was used to screen for mutations in the TPO gene of TIOD patients from nine apparently unrelated families, and seven different mutations were detected. Three frameshift mutations were found: a 20 bp duplication in exon 2, a 4 bp duplication in exon 8, and an insertion of a single nucleotide (C) at pos. 2505 in exon 14. In addition, four single nucleotide substitutions were identified: one single-base mutation resulted in a premature termination codon (C-->T at pos. 1708 in exon 10), two single-base substitutions changed an amino acid in highly conserved regions of the gene (Tyr-->Asp in exon 9 and Glu-->Lys in exon 14). The fourth single-base mutation located at the exon 10/intron 10 border altered a conserved Gly into Ser and could also affect splicing. Nine TIOD patients from five families were compound heterozygotes and six patients from four families were homozygous for one of the mentioned mutations in the TPO gene.