Thyroid Peroxidase Mutations Research Articles

5143 Disulfide Bonds of Thyroid Peroxidase Are Critical Elements for Subcellular Localization, Proteasome-Dependent Degradation, and Enzyme Activity

Abstract Disclosure: H. Iwasaki: None. H. Suwanai: None. K. Kanekura: None. N. Satoshi: None. F. Yakou: None. H. Sakai: None. K. Ishii: None. N. hara: None. R. Suzuki: None. Background: Congenital hypothyroidism (CH) is caused by mutations in cysteine residues including Cys655 and Cys825 that form disulfide bonds in thyroid peroxidase (TPO). It is highly likely that disulfide bonds play an important role in TPO activity. However, no study has comprehensively analyzed cysteine mutations that form disulfide bonds in TPO. In this study, we induced mutations in cysteine residues involved in the formation of disulfide bonds and analyzed their effect on subcellular localization, degradation, and enzyme activities to evaluate the importance of disulfide bonds in TPO. Methods: Vector plasmid TPO mutants C655F and C825R in CH patients were transfected into HEK293 cells. TPO activity and protein expression levels were measured by Amplex red assay and western blotting. The same procedure was performed in the presence of proteasome inhibitor MG132. Subcellular localization was determined by immunocytochemistry and flow cytometry. The location of all disulfide bonds within TPO was predicted by in silico analysis. All TPO mutations associated with disulfide bonds were induced. TPO activity and protein expression levels were also measured in all TPO mutants associated with disulfide bonds using the Amplex red assay and western blotting. Results: C655F and C825R showed significantly decreased activity and protein expression compared to the wild-type (P < 0.05). In the presence of a proteasome inhibitor MG132, the protein expression level of TPO was increased to the level comparable with that of the wild-type but its activity did not. The degree of subcellular distribution of TPO to the cell surface in the mutant was lower than that in the wild-type TPO. Twenty-four cysteine residues were involved in the formation of 12 disulfide bonds in TPO, and all TPO mutants harboring an amino acid substitution in each cysteine showed significantly reduced TPO activity and protein expression levels. Furthermore, the differences in TPO activity depended on the position of the disulfide bond. Conclusions: All 12 disulfide bonds play an important role in the activity of TPO. Furthermore, the mutations lead to misfolding, degradation and membrane insertion. Presentation: 6/2/2024

Disulfide Bonds of Thyroid Peroxidase Are Critical Elements for Subcellular Localization, Proteasome-Dependent Degradation, and Enzyme Activity.

Background: Congenital hypothyroidism (CH) is caused by mutations in cysteine residues, including Cys655 and Cys825 that form disulfide bonds in thyroid peroxidase (TPO). It is highly likely that these disulfide bonds could play an important role in TPO activity. However, to date, no study has comprehensively analyzed cysteine mutations that form disulfide bonds in TPO. In this study, we induced mutations in cysteine residues involved in disulfide bonds formation and analyzed their effect on subcellular localization, degradation, and enzyme activities to evaluate the importance of disulfide bonds in TPO activity. Methods: Vector plasmid TPO mutants, C655F and C825R, known to occur in CH, were transfected into HEK293 cells. TPO activity and protein expression levels were measured by the Amplex red assay and Western blotting. The same procedure was performed in the presence of MG132 proteasome inhibitor. Subcellular localization was determined using immunocytochemistry and flow cytometry. The locations of all disulfide bonds within TPO were predicted using in silico analysis. All TPO mutations associated with disulfide bonds were induced. TPO activity and protein expression levels were also measured in all TPO mutants associated with disulfide bonds using the Amplex red assay and Western blotting. Results: C655F and C825R showed significantly decreased activity and protein expression compared with the wild type (WT) (p < 0.05). In the presence of the MG132 proteasome inhibitor, the protein expression level of TPO increased to a level comparable with that of the WT without increases in its activity. The degree of subcellular distribution of TPO to the cell surface in the mutants was lower compared with the WT TPO. Twenty-four cysteine residues were involved in the formation of 12 disulfide bonds in TPO. All TPO mutants harboring an amino acid substitution in each cysteine showed significantly reduced TPO activity and protein expression levels. Furthermore, the differences in TPO activity depended on the position of the disulfide bond. Conclusions: All 12 disulfide bonds play an important role in the activity of TPO. Furthermore, the mutations lead to misfolding, degradation, and membrane insertion.

A Computational Approach: The Functional Effects of Thyroid Peroxidase Variants in Thyroid Cancer and Genetic Disorders.

Thyroid peroxidase (TPO) is essential for the synthesis of thyroid hormones. However, specific mutations render TPO antigenic and prone to autoimmune attacks leading to thyroid cancer, TPO deficiency, and congenital hypothyroidism (CH). Despite technological advancement, most experimental procedures cannot quickly identify the genetic causes of CH nor detect thyroid cancer in the early stages. We performed saturated computational mutagenesis to calculate the folding energy changes (∆∆G) caused by missense mutations and analyzed the mutations involved in post-translational modifications (PTMs). Our results showed that the functional important missense mutations occurred in the heme peroxidase domain. Through computational saturation mutagenesis, we identified the TPO mutations in G393 and G348 affecting protein stability and PTMs. Our folding energy calculations revealed that seven of nine somatic thyroid cancer mutations destabilized TPO. These findings highlight the impact of these specific mutations on TPO stability, linking them to thyroid cancer and other genetic thyroid-related disorders. Our results show that computational mutagenesis of proteins provides a quick insight into rare mutations causing Mendelian disorders and cancers in humans.

Tpo knockout in zebrafish partially recapitulates clinical manifestations of congenital hypothyroidism and reveals the involvement of TH in proper development of glucose homeostasis

Congenital hypothyroidism (CH) is a highly prevalent but treatable neonatal endocrine disorder. Thyroid peroxidase (TPO) catalyzes key reactions in thyroid hormone (TH) synthesis. TPO mutations have been found to underlie approximately 5% of congenital hypothyroidism in Chinese patients with more severe phenotypes, the treatment of whom usually requires a higher dose of L-thyroxine. The Tpo gene of zebrafish has 66% homology with the human TPO gene, and synteny analysis has indicated that it is likely a human TPO ortholog. In this study, we generated a tpo-/- mutant zebrafish line through knockout of tpo with CRISPR/Cas9 and investigated the associated phenotypes. Tpo-/- mutant zebrafish displayed growth retardation; an increased number of thyroid follicular cells; and abnormal extrathyroidal phenotypes including pigmentation defects, erythema in the thoracic region, delayed scale development and failure of swim bladder secondary lobe formation. All these abnormal phenotypes were reversed by 30 nM thyroxine (T4) treatment starting at 1 month of age. Tpo-/- mutants also showed increased glucose levels during larval stages, and the increases were induced at least in part by increasing glucagon and decreasing insulin expression. Our work indicates that tpo-mutant zebrafish may serve as a human congenital hypothyroidism model for studying TPO- and TH-related disease mechanisms.

Screening and Functional Analysis of TPO Gene Mutations in a Cohort of Chinese Patients With Congenital Hypothyroidism.

BackgroundsAs a crucial enzyme in thyroid hormone synthesis, the genetic defective thyroid peroxidase (TPO) was one of the main genetic factors leading to congenital hypothyroidism (CH).MethodsMutations in the TPO gene were screened and identified in 219 patients with CH from northwest China by using high-throughput sequencing and bioinformatics analysis. The biological function of detected variants was studied by in vitro experiments and homology modeling.ResultsNineteen rare variants, including seven novel ones, were detected in 17 of 219 patients (7.8%). Most cases were detected with one single heterozygous variant, and only two patients were detected with multiple variants, i.e., compounds for (1) IVS7-1G>A, p.Ala443Val, and p.Arg769Trp and (2) p.Asn592Ser and p.Asn798Lys. The biological function of the four missense mutations (i.e., p.Ala443Val, p.Arg769Trp, p.Asn592Ser, and p.Asn798Lys) they carried were further studied. Experimental data showed that these four mutations did not affect the protein expression level of the TPO gene but remarkably reduced the peroxidase activity toward guaiacol oxidation, retaining 8–32% of activity of the wild-type protein. The comparison of the predicted 3-D structures of wild-type and mutant TPO proteins showed that these four amino acid substitutions changed the non-covalent interactions of studied residues that might alter the structure and function of the TPO protein.ConclusionThis study was the first to analyze the TPO mutation spectrum of patients with CH in northwest China. Our data indicated that the TPO mutation was not a common reason to cause CH in China. The functional data may help to clarify the structure-function relationship of the TPO protein and provide further evidence for the elucidation of the genetic etiology of CH.

Long-Term Outcome of Patients with TPO Mutations.

Introduction: Thyroid peroxidase (TPO) deficiency is the most common enzymatic defect causing congenital hypothyroidism (CH). We aimed to characterize the long-term outcome of patients with TPO deficiency. Methods: Clinical and genetic data were collected retrospectively. Results: Thirty-three patients with primary CH caused by TPO deficiency were enrolled. The follow-up period was up to 43 years. Over time, 20 patients (61%) developed MNG. Eight patients (24%) underwent thyroidectomy: one of them had minimal invasive follicular thyroid carcinoma. No association was found between elevated lifetime TSH levels and the development of goiter over the years. Conclusions: This cohort represents the largest long-term follow up of patients with TPO deficiency. Our results indicate that elevated TSH alone cannot explain the high rate of goiter occurrence in patients with TPO deficiency, suggesting additional factors in goiter development. The high rate of MNG development and the risk for thyroid carcinoma indicate a need for long-term follow up with annual ultrasound scans.

Functional analysis of thyroid peroxidase gene mutations resulting in congenital hypothyroidism.

Thyroid peroxidase (TPO) is essential for thyroid hormone biosynthesis. TPO mutations might lead to congenital hypothyroidism. In the present study, we analysed the function of a compound heterozygous TPO mutation in a Chinese family. We studied a 23-year-old Chinese girl with a history of growth retardation and severe constipation from the age of 3months, who was diagnosed as having congenital hypothyroidism. Genomic DNA was extracted from peripheral blood samples obtained from the patient's family members. The genomic DNA was sequenced to detect mutations in a panel of genes associated with congenital hypothyroidism. Bioinformatic analysis and structural modelling predicted the potential disease-causing potential mutant genes and the microstructure of the mutant protein, respectively. Western blotting and ELISA were used to measure protein expression, and guaiacol oxidation assay measured the TPO activity of the mutant protein. We identified a compound heterozygous mutation (c.C1993T, c.T2473C) in the TPO gene. Bioinformatic analysis predicted that the TPO mutations were potentially disease causing. Structural modelling predicted damage to the microstructure of the mutant TPO protein. Western blotting and ELISA showed reduced protein levels of the mutant TPO protein compared with that of the wild-type protein. The mutant TPO protein showed weaker activity compared with that of the wild-type protein. A novel compound heterozygous mutation of TPO gene was identified in a Chinese family. This mutation might alter the extracellular microstructure of TPO, and decrease its expression and the activity, resulting in congenital hypothyroidism.

The TPO mutation screening and genotype-phenotype analysis in 230 Chinese patients with congenital hypothyroidism

Inborn defects in thyroid hormone biosynthesis contribute to nearly half of congenital hypothyroidism (CH) cases in China. The thyroid peroxidase (TPO) mutation is one of the most frequent mutations that results in thyroid dyshormonogenesis. In this study, 35 non-synonymous mutations in 15 TPO sites, including 6 novel mutations, were identified in 230 Chinese patients with CH. The enzyme activity of the mutations in TPO was investigated in vitro, and patients with less than 15% residual enzyme activity showed severe CH, such as markedly increased thyroid-stimulating hormone (TSH) at diagnosis (>100 μIU/mL) and pronounced goiter, and required a higher dose of L-thyroxine to maintain the euthyroid. However, CH patients with greater than 16% TPO activity showed mild CH, a typical childhood socially without L-thyroxine treatment before 3 years of age, and the appearance of a macroscopic goiter at childhood. The findings indicated that the residual enzymatic activity of TPO was correlated with clinical phenotypes of CH patients with TPO biallelic mutations.

Increased Prevalence of TG and TPO Mutations in Sudanese Children With Congenital Hypothyroidism.

Congenital hypothyroidism (CH) is due to dyshormonogenesis in 10% to 15% of subjects worldwide but accounts for 60% of CH cases in the Sudan. To investigate the molecular basis of CH in Sudanese families. Clinical phenotype reporting and serum thyroid hormone measurements. Deoxyribonucelic acid extraction for whole-exome sequencing and Sanger sequencing. University research center. Twenty-six Sudanese families with CH. Clinical evaluation, thyroid function tests, genetic sequencing, and analysis. Our samples and information regarding samples from the literature were used to compare TG (thyroglobulin) and TPO (thyroid peroxidase) mutation rates in the Sudanese population with all populations. Mutations were found in dual-oxidase 1 (DUOX1), dual-oxidase 2 (DUOX2), iodotyrosine deiodinase (IYD), solute-carrier (SLC) 26A4, SLC26A7, SLC5A5, TG, and TPO genes. The molecular basis of the CH in 7 families remains unknown. TG mutations were significantly higher on average in the Sudanese population compared with the average number of TG mutations in other populations (P < 0.05). All described mutations occur in domains important for protein structure and function, predicting the CH phenotype. Genotype prediction based on phenotype includes low or undetectable thyroglobulin levels for TG gene mutations and markedly higher thyroglobulin levels for TPO mutations. The reasons for higher incidence of TG gene mutations include gene length and possible positive genetic selection due to endemic iodine deficiency.

Mutation Spectrum in TPO Gene of Bangladeshi Patients with Thyroid Dyshormonogenesis and Analysis of the Effects of Different Mutations on the Structural Features and Functions of TPO Protein through In Silico Approach.

Although thyroid dyshormonogenesis (TDH) accounts for 10-20% of congenital hypothyroidism (CH), the molecular etiology of TDH is unknown in Bangladesh. Thyroid peroxidase (TPO) is most frequently associated with TDH and the present study investigated the spectrum of TPO mutations in Bangladeshi patients and analyzed the effects of mutations on TPO protein structure through in silico approach. Sequencing-based analysis of TPO gene revealed four mutations in 36 diagnosed patients with TDH including three nonsynonymous mutations, namely, p.Ala373Ser, p.Ser398Thr, and p.Thr725Pro, and one synonymous mutation p.Pro715Pro. Homology modelling-based analysis of predicted structures of MPO-like domain (TPO142-738) and the full-length TPO protein (TPO1-933) revealed differences between mutant and wild type structures. Molecular docking studies were performed between predicted structures and heme. TPO1-933 predicted structure showed more reliable results in terms of interactions with the heme prosthetic group as the binding energies were -11.5 kcal/mol, -3.2 kcal/mol, -11.5 kcal/mol, and -7.9 kcal/mol for WT, p.Ala373Ser, p.Ser398Thr, and p.Thr725Pro, respectively, implying that p.Ala373Ser and p.Thr725Pro mutations were more damaging than p.Ser398Thr. However, for the TPO142-738 predicted structures, the binding energies were -11.9 kcal/mol, -10.8 kcal/mol, -2.5 kcal/mol, and -5.3 kcal/mol for the wild type protein, mutant proteins with p.Ala373Ser, p.Ser398Thr, and p.Thr725Pro substitutions, respectively. However, when the interactions between the crucial residues including residues His239, Arg396, Glu399, and His494 of TPO protein and heme were taken into consideration using both TPO1-933 and TPO142-738 predicted structures, it appeared that p.Ala373Ser and p.Thr725Pro could affect the interactions more severely than the p.Ser398Thr. Validation of the molecular docking results was performed by computer simulation in terms of quantum mechanics/molecular mechanics (QM/MM) and molecular dynamics (MD) simulation. In conclusion, the substitutions mutations, namely, p.Ala373Ser, p.Ser398Thr, and p.Thr725Pro, had been involved in Bangladeshi patients with TDH and molecular docking-based study revealed that these mutations had damaging effect on the TPO protein activity.

Mutation screening of the TPO gene in a cohort of 192 Chinese patients with congenital hypothyroidism

ObjectivesDefects in the human thyroid peroxidase (TPO) gene are reported to be one of the causes of congenital hypothyroidism (CH) due to dyshormonogenesis. The aim of this study was to...

Hearing Impairment in Hypothyroid Dwarf Mice Caused by Mutations of the Thyroid Peroxidase Gene

Thyroid hormone (TH) is essential for proper cochlear development and function, and TH deficiencies cause variable hearing impairment in humans and mice. Thyroid peroxidase (TPO) catalyzes key reactions in TH synthesis, and TPO mutations have been found to underlie many cases of congenital hypothyroidism in human patients. In contrast, only a single mutation of the mouse TPO gene has been reported previously (Tpo(R479C)) but was not evaluated for auditory function. Here, we describe and characterize two new mouse mutations of Tpo with an emphasis on their associated auditory deficits. Mice homozygous for these recessive mutations have dysplastic thyroid glands and lack detectable levels of TH. Because of the small size of mutant mice, the mutations were named teeny (symbol Tpo(tee)) and teeny-2 Jackson (Tpo(tee-2J)). Tpo(tee) is a single base-pair missense mutation that was induced by ENU, and Tpo(tee-2J) is a 64 bp intragenic deletion that arose spontaneously. The Tpo(tee) mutation changes the codon for a highly conserved tyrosine to asparagine (p.Y614N), and the Tpo(tee-2J) mutation deletes a splice donor site, which results in exon skipping and aberrant transcripts. Mutant mice are profoundly hearing impaired with auditory brainstem response (ABR) thresholds about 60 dB above those of non-mutant controls. The maturation of cochlear structures is delayed in mutant mice and tectorial membranes are abnormally thick. To evaluate the effect of genetic background on auditory phenotype, we produced a C3.B6-Tpo(tee-2J) congenic strain and found that ABR thresholds of mutant mice on the C3H/HeJ strain background are 10-12 dB lower than those of mutant mice on the C57BL/6 J background. The Tpo mutant strains described here provide new heritable mouse models of congenital hypothyroidism that will be valuable for future studies of thyroid hormones' role in auditory development and function.

A common thyroid peroxidase gene mutation (G319R) in Turkish patients with congenital hypothyroidism could be due to a founder effect

The most common congenital endocrine disorder is congenital hypothyroidism (CH), which can lead to mental retardation if untreated. Majority of the patients have been found to have defects in thyroid development and migration disorders (dysgenesis), and the remaining ones have thyroid hormone synthesis defects (dyshormonogenesis). One of the most common mechanisms to cause dyshormonogenesis is a defect in the thyroid peroxidase (TPO) enzyme. In familial cases, mutations in the TPO gene are fairly prevalent. To date, more than 80 mutations have been identified, which result in variably decreasing TPO bioactivities. Clinical manifestations of TPO defects are typically permanent CH and with or without goiter. In this report, we presented two children with CH who were born to consanguineous parents and were homozygous carriers of a missense (G319R) TPO mutation, the mutation segregated with the disease status in the families confirming its pathogenicity. G319R mutation seemed to be a common cause of CH in Turkish population, which could originate from a common founder ancestor. Moreover, our results also confirmed the phenotypic variability associated with different TPO mutations.

Congenital Goitrous Primary Hypothyroidism in Two German Families Caused by Novel Thyroid Peroxidase (TPO) Gene Mutations

Congenital hypothyroidism occurs with a prevalence of approximately 1:3 500. Defects in thyroid hormone synthesis which lead to goitrous hypothyroidism account for 10-15% of these cases. Several genetic defects have been characterized and mutations in the thyroid peroxidase (TPO) gene are the most common cause for dyshormonogenesis.So far, more than 80 mutations in the TPO gene have been described, resulting in a variable decrease in TPO bioactivity. Clinically TPO defects manifest with congenital primary goitrous hypothyroidism.We here present 2 children with congenital primary hypothyroidism, who were identified to have compound heterozygous TPO mutations. They both shared the same novel mutation in the TPO gene (C756R) in exon 13. One case presented with an apparently dominant inheritance of thyroid dyshormonogenesis.

A thyroid peroxidase (TPO) mutation in dogs reveals a canid-specific gene structure

Congenital hypothyroidism with goiter (CHG) occurring as an autosomal recessive disorder is typically due to a defect of thyroid hormone synthesis (aka dyshormonogenesis). Thyroid peroxidase (TPO) is a multifunctional, heme-containing enzyme whose activity is required, and several inactivating TPO mutations causing CHG in humans and dogs have been described. Recently, two half-sib Spanish water dog (SWD) pups were diagnosed with CHG based on clinical signs, endocrine testing, and thyroid histology. TPO enzyme activity was absent, and immuno-cross-reactive TPO was undetectable in affected-dog thyroid tissue. A single guanosine insertion was observed in the first exon of the affected-dog TPO cDNA at a site not previously thought to be within the coding sequence. The insertion allele segregated with the deduced disease allele in the SWD breed and was not observed in unrelated dogs of various breeds. Comparison of the insertion site (an 8-nt poly-G tract) with the orthologous sequences of other mammalian reference genomes revealed that the octa-G tract obliterated the intron 1 splice acceptor site and the exon 2 translation initiation codon found at that position in other species. An in-frame ATG in strong Kozak consensus context was observed in the normal dog sequence 12 codons 5' of the usual mammalian start site, suggesting that dogs have lost the noncoding exon 1 demonstrated in human and mouse. A survey of TPO sequences in other carnivore species indicates that the poly-G tract necessitating an alternative translation initiation site is a canid-specific feature.

Detection of heterozygous c.1708C&gt;T and c.1978C&gt;G thyroid peroxidase (TPO) mutations in Iraqi patients with toxic and nontoxic goiter

Sixty-three Arabic patients (16 males and 47 females) with thyroid toxic and nontoxic goiter who attended the endocrinologist in Nuclear Medicine Hospital and Al Yarmok Nuclear Medicine Department in Baghdad, Iraq were examined for thyroid peroxidase (TPO) gene mutations. A total of ten heterozygous mutations have been identified in the human TPO gene associated with thyroid toxic and nontoxic goiter. These mutations involved transition or transversion of cysteine either by thymine or guanine at the position 1708 of the exon 10 (c.1708C>T) and the position 1978 of the exon 11 (c.1978C>G). From a total of ten detected mutations, two c.1978C>G mutations were detected in nontoxic goiter patients and eight (two c.1708C>T and six c.1978C>G mutations) were detected in toxic goiter. In conclusion, this study identified ten TPO mutations associated with toxic and nontoxic goiter that have not been yet reported in Iraq, and most of them are detected among females (90 %) and adults age between 30 and 50 years old (80 %).

The role of Na/I symporter, thyroid peroxidase and type 1 iodothyronine deiodinase gene in the pathogenesis of hashimoto's thyroiditis coexistent with papillary thyroid carcinoma

Objective To investigate the role of Na/I symporter (NIS),thyroid peroxidase (TPO) and type 1 iodothyronine deiodinase (DIOI) mutation in the pathogenesis of Hashimoto' s thyroiditis (HT) coexistent with papillary thyroid carcinoma (PTC).Methods Thirty-four HT patients with PTC were selected as subjects and 26 cases of HT as controls.The genomic DNA of the specimens was extracted.By using polymerase chain reaction (PCR),interesting fragments were obtained and sequenced.Results Among 34 patients of HT with PTC,no PCR products were found with the mutation in the NIS gene.Ten out of 34 patients and 2 out of 26 controls had 6 TPO point mutations and 1 insertion mutation,and 5 of out of 34 patients and 1 out of 26 controls had 3 DIOI point mutations.Statistical analysis showed there was significance in mutation positivity between HT with PTC and HT.In addition,mutation positivity had significant difference between patients with different thyroid function in HT and PTC group ( P ＜ 0.05 ).Conclusion It is possible that TPO and DIOI gene mutation had a potential molecular biological link with the two diseases. Key words: Na/I symporter; Thyroid peroxidase; Hashimoto's thyroiditis; Papillary thyroid carcinoma

Molecular screening of the TSH receptor (TSHR) and thyroid peroxidase (TPO) genes in Korean patients with nonsyndromic congenital hypothyroidism

To investigate thyroid-stimulating hormone receptor (TSHR) and thyroid peroxidase (TPO) mutations in Korean patients with primary congenital hypothyroidism (CH). Congenital hypothyroidism is a common genetic disorder in which the majority of mutations occur in the TSHR and TPO genes. We examined the frequencies of TSHR and TPO mutations among Korean patients with primary CH. Furthermore, we explored the relationships between imaging findings and mutation status. A total of 193 paediatric patients with nonsyndromic CH were enrolled in the present study. Patients with decreased (99m) Tc uptake were screened for TSHR mutations using Sanger sequencing, and those with increased uptake were screened for TPO mutations. The relationships between scintigraphic and ultrasonographic findings and mutation status were analysed. Thirteen (16·5%) of 79 patients with decreased (99m) Tc uptake were found to harbour TSHR mutations including G132R, G245S, R450H, R519C and F525S. The R450H mutation was present in 13 (72·2%) of 18 disease alleles. Seven (10·3%) of 68 patients with increased (99m) Tc uptake harboured TPO mutations including R189Q, K439E, G493S, C808LfsX72, A863T, R875Hfs and P883S. The TSHR and TPO mutations were observed only in patients with normal to slightly enlarged thyroid glands. This study identified underlying TSHR and TPO mutations in Korean patients with CH and revealed a possible relationship between imaging findings and mutation status. In addition, the low rate of mutation positivity suggests significant genetic heterogeneity of CH in the Korean population.

The Coexistence of a Novel Inactivating Mutant Thyrotropin Receptor Allele with Two Thyroid Peroxidase Mutations: A Genotype-Phenotype Correlation

TSH receptor (TSHR) and thyroid peroxidase (TPO) gene mutations occur independently. This is the first report of their coexistence in the same individuals. The objective of the study was to evaluate the genotype-phenotype correlations when mutations in both genes are present alone or together in the same individual. Thirty subjects from an extended Arab kindred underwent clinical investigation and molecular studies of the mutant TSHRs. A novel mutant TSHR was identified, involving four nucleotides at three sites on the same allele, c.267G>T (L89L), c.269/270AG>CT (Q90P), and c.790C>T (P264S). In addition, two known TPO gene mutations, G493S and R540X, were identified. Thirteen heterozygotes for the mutant TSHR allele had mild hyperthyrotropinemia. In nine of theses, the coexistence of a TPO mutation in one allele did not magnify the hyperthyrotropinemia. Homozygotes for the mutant TSHR and a compound heterozygote for the TPO mutations presented frank hypothyroidism. In vitro studies showed increasing loss of function for Q90P less than P264S less than Q90P/P264S TSHR mutants, the latter being that expressed in the subjects under investigation. The two interchangeably used WT TSHR vectors, L87 and V87, although functionally identical, differed in structure and function in the presence of the Q90P mutation. TSHR and TPO gene mutations were identified alone and together in individuals of a consanguineous kindred. Homozygotes for the TSHR and a compound heterozygote for the TPO mutations were hypothyroid. The mild hyperthyrotropinemia of heterozygotes for the mutant TSHR allele was not aggravated by the coexistence of a TPO defect in one allele.

Two compound heterozygous mutations (c.215delA/c.2422T→C and c.387delC/c.1159G→A) in the thyroid peroxidase gene responsible for congenital goitre and iodide organification defect

Iodide organification defects are frequently but not always associated with mutations in the thyroid peroxidase (TPO) gene and characterized by a positive perchlorate discharge test. These mutations phenotypically produce a congenital goitrous hypothyroidism, with an autosomal recessive mode of inheritance. In the present study we extended our initial molecular studies in six unrelated patients heterozygous for the TPO mutations, in order to identify the second mutation in this autosomal recessive disease. The promoter and the complete coding regions of the human TPO and DUOXA2 genes, along with the flanking regions of each intron were analysed by direct DNA sequencing. Four different inactivating TPO mutations were identified in two patients: two novel mutations (c.215delA [p.Q72fsX86] and c.1159G-->A [p.G387R]) and two previously reported (c.387delC [p.N129fsX208] and c.2422T-->C [p.C808R]), confirming the inheritance of two different compound heterozygous mutations, c.215delA/c.2422T-->C and c.387delC/c.1159G-->A. The remaining four patients did not show additional inactivating mutations in the TPO gene and all had only the wild type sequencing in the DUOXA2 gene. We have reported two patients with iodide organification defect caused by two compound heterozygous mutations, c.215delA/c.2422T-->C [p.Q72fsX86/p.C808R] and c.387delC/c.1159G-->A [p.N129fsX208/p.G387R], in the TPO gene and four patients with monoallelic TPO defect. Identification of the molecular basis of this disorder might be helpful for understanding the pathophysiology of congenital hypothyroidism.