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Monoallelic Expression of Mutant Thyroid Peroxidase Allele Causing Total Iodide Organification Defect

Institute of Endocrine Sciences (L.F., N.C., D.M., G.V., C.F., L.P., P.B.-P.), School of Medicine, University of Milan, Milan, Italy; Ospedale Maggiore Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) (L.F., P.B.-P.), 20122 Milan, Italy; and Istituto Auxologico Italiano IRCCS (L.P.), 20145 Milan, Italy

Address all correspondence and requests for reprints to: Paolo Beck-Peccoz, M.D., Institute of Endocrine Sciences (Pad. Granelli), Ospedale Maggiore Istituto di Ricovero e Cura a Carattere Scientifico, Via F. Sforza, 35, 20122 Milan, Italy. E-mail: paolo.beckpeccoz{at}unimi.it.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
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.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
HEREDITARY INBORN ERRORS in thyroid hormone synthesis are found in 10–20% of congenital hypothyroid patients and are frequently attributable to thyroid peroxidase (TPO) deficiency.

The human TPO (hTPO) gene, which localizes on chromosome 2p25, is divided into 17 exons and covers approximately 150 kb of DNA (1, 2). TPO is a membrane-bound hemoprotein located at the apical membrane of the thyroid cell that catalyzes both iodination and coupling of iodotyrosine residues into the thyroglobulin (Tg), thus leading to the synthesis of thyroid hormones.

Impaired TPO function results in total iodide organification defects (TIOD). Patients present with congenital hypothyroidism, generally of a severe degree with high plasma TSH and Tg concentrations. A rapid and elevated radioiodine uptake is observed, with a complete release after perchlorate (KClO₄) administration, consistent with the defect in iodide organification. To date, 26 different TPO mutations, mostly located in exons 8, 9, and 14, have been described in patients with TIOD (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15). According to the recessive mode of inheritance, the affected subjects are homozygous or compound heterozygous for gene mutations. Recently, two patients have been described in which the phenotype was associated with a single TPO mutation, in one case due to a maternal isodisomy for chromosome 2p (16) and in the other case due to the deletion of the paternal TPO gene at chromosome 2p25 (17). However, beyond these two cases, the literature reports other families from different origin with TIOD phenotype and a single TPO-mutated allele (7, 9, 13, 15). The explanation for TIOD in these single heterozygous cases is presently unknown.

In the present study, we describe an intriguing family affected with TIOD in which a single paternal mutation is responsible for the phenotype due to lack of maternal allele transcripts at the thyroid tissue level.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The family studied is composed of the unaffected mother, father, and one daughter (subject 1) and by three affected siblings (patient 2, a male; and patients 3 and 4, both females). The parents denied consanguinity, but descended from a small village in the central part of Italy. The mother had six pregnancies; the second ended with a spontaneous abortion at the third month, whereas the fourth pregnancy led to a normal delivery but the newborn died soon after birth. The three affected siblings (third, fifth, and sixth pregnancies, respectively) were born at term, before the introduction of the neonatal screening in Italy. Early diagnosis was missed, despite signs and symptoms of severe hypothyroidism in the first weeks after birth, and the start of L-T₄ therapy was delayed in all three cases (5, 3, and 4 months, respectively). Because of the presence of goiter (small in patient 3 and large in patients 2 and 4) and sensorineural hearing loss (mild in patient 3 and moderate in patients 2 and 4), they were diagnosed as affected with Pendred’s syndrome.

The affected patients came to our attention at the ages of 30, 31, and 25 yr, respectively. L-T₄ therapy was withdrawn for 4 wk to perform a complete clinical evaluation. The biochemical parameters showed very low levels of free T₃ and free T₄, very high levels of TSH and Tg, negative anti-Tg and anti-TPO antibodies, goiter of different sizes, high initial radioiodine uptake with spontaneous decrease and complete release after potassium perchlorate oral administration, consistent with a total organification defect (Table 1). The affected patients are also affected with mental retardation, language disturbances, minor skeletal dysmorphisms, and abnormalities in coordination and movement (particularly patient 2). The karyotype of patient 2 was normal, thus excluding major structural chromosomal abnormalities. The clinical data argued against a PDS gene involvement, because in Pendred’s syndrome, hypothyroidism is usually absent or subclinical, the organification defect is partial, and the degree of sensorineural hearing loss is severe (18). Indeed, the sequence of all PDS coding exons was normal in the three patients (19), and the data obtained at reevaluation were strongly indicative of a TPO involvement. At the age of 33 yr, patient 2 underwent total thyroidectomy due to the enlarged volume of the multinodular goiter with symptoms and signs of tracheal compression and deviation, despite TSH suppressive therapy with L-T₄. The histological examination showed no malignancy. Two nodules from the right lobe, one nodule from the left lobe, and extranodular thyroid tissue from both lobes were stored separately at -80 C.

The experimental studies have been approved by the Ethics Committee of the Institution. Informed consent for both clinical and genetic studies was obtained from all of the family members.

Genetic analysis of the TPO gene using leukocytes DNA

DNA was extracted by standard methods from whole blood of all family members. Exons 1–17 of the TPO gene were amplified by means of newly designed intronic primers (Table 2). Samples were subjected to 10 min denaturation at 98 C, followed by 35 three-step cycles (appropriate annealing temperature for 1 min, 72 C for 2 min, 94 C for 1 min), 72 C for 10 min in a TouchDown Thermal Cycler (Hybaid, Middlesex, UK). PCR products were directly sequenced after removal of unincorporated deoxynucleoside triphosphates and primers by GFX PCR DNA purification kit (Amersham Pharmacia Biotech, Uppsala, Sweden). An aliquot of 3–10 ng/100 bp of purified DNA and 3.2 pmol of either the forward or reverse primer was used in standard cycle sequencing reactions with ABI PRISM Big Dye terminators and run on an ABI PRISM 310 Genetic Analyzer (PE Applied Biosystems, Foster City, CA). The cycle-sequencing conditions consisted of 25 cycles of 96 C for 30 sec, 50 C for 15 sec, and 60 C for 4 min. One sequence read from each direction across the entire coding region and including intron-exon boundaries was obtained for each subject. The entire sequence of exon 8 was read using one set of internal primers (8R and 8F3 in Ref. 5).

The central part of a right thyroid nodule, previously stored at -80 C, was excised and divided into two specimens for the extraction of DNA and total RNA, respectively. The same procedure was repeated for the left nodule and for extranodular thyroid tissue. Total RNA (500 ng) was retrotranscribed (Ready-to-Go You-Prime First-Strand Beads, Amersham Pharmacia Biotech) in a total reaction volume of 33 µl containing 2 µl of random hexamers (Promega Corp., Madison, WI). After the first strand of cDNA synthesis, forward and reverse primers (Table 4, T1–T8) were added, and exons 1, 2, 7, 11, 12, and 15 were PCR amplified and directly sequenced individually. The remaining exons were amplified by means of primers encompassing exons 2–7, exons 7–9, exons 8–11, and exons 12–17 (Table 4, D1–D8), and these RT-PCR fragments were compared on an agarose gel with those obtained from a normal thyroid cDNA. The identity of these fragments was confirmed by direct sequencing.

Genetic analysis of TPO using cultured skin fibroblast

Skin fibroblast obtained from cutaneous punch biopsy in the three affected patients were grown, and the nucleic acids were extracted at confluence. Retrotranscription, amplification, and direct sequencing of exons 1–17 were performed as described above.

Methylation assay

Eventual methylation at one CpG-rich island (GGCCCGGCGC) located at -132 bp in the TPO gene promoter region was tested by the bisulfite genomic sequencing protocol, as previously reported (21). Genomic DNA from the mother and from one normal control and DNA obtained from thyroid tissue of patient 2 were used as template. At the end of the reaction, free bisulfite was removed by means of a purification kit (Wizard DNA Clean-up, Promega Corp.). Recovered DNA was amplified using primers designed to favor the amplification of fully bisulfite-converted DNA (Table 3, DEM1 and DEM2). Ten microliters of the PCR product (523 bp) were added to 5 U HpaII in a total reaction volume of 50 µl; the digestion mixture was incubated overnight and run on a 2% agarose gel. Non-bisulfite-converted maternal DNA was used as negative control, whereas HpaII digestion was monitored by using another nonconverted fragment from the same CpG-rich region of maternal TPO gene that was amplified by means of appropriate primers (Table 3, NC1S and NC1AS).

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Genetic analysis of TPO gene using leukocyte DNA

The sequencing of the TPO gene, including the noncoding exon 1 and 870 bp of the promoter, showed 12 sequence variants corresponding to 11 different polymorphisms and one missense mutation. Polymorphisms were located in the promoter (-799G>A, -706G>A, -35A>G); in exon 1 (11G>A); in exon 2 (102C>G, Leu>Leu); in exon 7 (859G>T, Ala>Ser); in exon 11 (2088C>T, Asp>Asp); in intron 11 (IVS11–33C>T); in exon 12 (2235C>T, Pro>Pro, 2263A>C, Thr>Pro); and in exon 15 (2630T>C, Val>Ala). The mutation is located in exon 12 (2167 C>T), it leads to the substitution of Arg to Trp at codon 693, and it was recently described in homozygosity in a Dutch family from Afghan descent (13). In the present family, R693W has been found in simple heterozygosity in the unaffected father and in the three affected siblings, but not in the mother or the unaffected daughter. No other mutations were found, although all TPO exons and exon/intron boundaries were sequenced. In particular, a range of 23–192 bp of intronic sequences at 5' and a range of 44–295 bp of intronic sequences at 3' of each exon have been analyzed and found normal. No variations were found in the TATAA box, in the CpG-rich region, or in the CRE-like element.

All polymorphisms have been reported already in the literature (5, 22), with the exception of that lying in exon 2, which is a silent single nucleotide substitution, and of two promoter variations (-799 G>A and -706 G>A), which were found only in the mother and the unaffected daughter (patient 1).

Polymorphisms have been found in heterozygosity or homozygosity in the different members of the family. It is worth noting that the same pattern was found in the father and in the three affected siblings.

Genetic analysis of TPO gene on thyroid tissue DNA and RNA

In patient 2, although the sequence of TPO gene using somatic DNA was identical with that observed with leukocyte DNA, a different pattern was seen using retrotranscribed thyroid tissue RNA. In this case, all heterozygous exonic polymorphisms and the mutation were homozygous (Fig. 1).

View larger version (51K): [in this window] [in a new window]   FIG. 1. Direct sequencing of the TPO gene in genomic DNA from leukocytes and in DNA and cDNA (obtained from retrotranscribed RNA) from thyroid tissue. The mutation in exon 12 and all informative polymorphisms (exon 11, exon 12, and exon 15) are shown. In all cases, the sequence variation is present in heterozygosity in the blood and tissue DNA and in homozygosity in the tissue cDNA. Polymorphisms in intron 11, exon 1, and exon 2 were not informative and are not shown in the figure.

View larger version (42K): [in this window] [in a new window]   FIG. 2. Allelic distribution in the family studied. Black circles and square indicate affected members. Polymorphisms and the mutation have been named by base substitution and nucleotide position in accordance with recent nomenclature recommendations (33 ). The mutation in exon 12 (2167C>T, Arg693Trp) is underlined. The affected siblings inherited the paternal allele carrying the mutation, and the unaffected daughter inherited the other paternal allele. A different maternal allele has been inherited by the three affected siblings and the unaffected daughter. At the thyroid tissue level, the lack of transcripts corresponding to the maternal allele inherited by the affected siblings leads to the homozygous expression of Arg693Trp mutation. A recombination event has been recorded for the unaffected daughter. The asterisk indicates intron 11 polymorphism (IVS11–33C>T).

View larger version (59K): [in this window] [in a new window]   FIG. 3. A, Methylation assay. After bisulfite treatment (leading to the conversion of nonmethylated cytosine to uracil), genomic maternal DNA (M), tissue DNA of patient 2 (T), and genomic DNA from a control (N) yielded a PCR product of the expected size and were not cut by HpaII enzyme, thus excluding the possible presence of methylation at this CpG-rich island of the TPO promoter. No PCR products were obtained using untreated maternal DNA and primers specific for converted DNA (C1), demonstrating the specificity of these primers. Untreated maternal DNA was amplified by means of primers for nonconverted DNA and the two expected bands of 322 and 156 bp were obtained after incubation with HpaII, thus indicating the efficiency of the restriction reaction (C2). B, The TPO gene regions not including heterozygous polymorphisms were amplified by means of primers encompassing exons 2–7 (fragment A), exons 7–9 (fragment B), exons 8–11 (fragment C), and exon 12-3' UTR (fragment D). RT-PCR fragments generated from the total RNA of the thyroid tissue of patient 2 were compared on an agarose gel with those obtained from a normal thyroid total RNA (N). The molecular weight of these fragments was identical and corresponding to the expected transcript for a normal exonic/intronic sequence of the TPO gene. Alternative transcripts corresponding to hTPO-2 (23 ), in which exon 10 is deleted, and to hTPO-Zanelli (24 ), in which exon 16 is deleted, were identified in both samples. A blank (b) sample was included in each experiment. MW, Molecular weight.

The fibroblast DNA was found to harbor the same pattern of leukocyte DNA and somatic DNA from thyroid tissue. No expression of the TPO gene using fibroblast retrotranscribed RNA was detected.

Methylation assay

After bisulfite treatment (leading to the conversion of nonmethylated cytosine to uracil), genomic maternal DNA, tissue DNA of patient 2, and genomic DNA from a normal control yielded a PCR product of the expected size and were not cut by HpaII enzyme, thus excluding the possible presence of methylation at this CpG-rich island of the TPO promoter. The efficiency of the restriction reaction was demonstrated by the presence of the two expected bands of 322 and 156 bp using a nonconverted control DNA fragment (Fig. 3B).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
In the present study, we report a family in which the three siblings affected with TIOD show a severe congenital hypothyroidism. The haplotyping of the TPO alleles, performed thanks to the presence of numerous informative polymorphisms both in the coding and uncoding regions, proved that the TIOD phenotype in this family is the result of the expression of a mutant paternal allele associated to the lack of maternal allele transcripts at the thyroid tissue level. Possible underlying mechanisms for maternal TPO allele defect include major genomic deletions, accelerated mRNA decay, maternal imprinting, or alterations in the regulatory regions of the gene.

Major deletions involving the maternal chromosome at 2p25 can be discarded due to the evidence of heterozygosity in the DNA derived from different tissues, such as leukocytes, skin fibroblast, and thyroid. To verify the existence of TPO transcripts of a low molecular weight, generated in the thyroid tissue from the maternal allele as a consequence of exon skipping or premature stop codons, we set up the amplification of four TPO fragments encompassing exons 2–7, exons 7–9, exons 8–11, and exon 12-3' UTR. These fragments were found to be identical, comparing the cDNA generated from the thyroid tissue of patient 2 to a normal control and corresponding to the expected transcript for a conserved exonic/intronic sequence of the TPO gene. By means of extensive analyses of retrotranscribed materials from thyroid tissues, we could confirm the existence of alternative splicing products, such as hTPO-2 and hTPO-Zanelli, but no extra bands were detected in the case of the affected goiter. The alternatively spliced hTPO-Zanelli had been previously described only in Graves’ disease (24); here we show its existence also in normal and dyshormonogenic thyroid tissues.

The above reported results exclude the presence of even small in frame deletions/insertions in the maternal allele, but not the occurrence of accelerated mRNA decay resulting in total absence of maternal TPO transcript. We consider it very unlikely that this could be due to unrecognized mutations in the sequenced regions, because high-quality electropherograms were obtained for all TPO exons and were carefully read in both directions, starting from different PCR products. Nevertheless, accelerated mRNA decay could also be the consequence of deep intronic alterations or partial gene deletion. In particular, a deletion of exon(s) not harboring one of the numerous informative polymorphisms could be missed at genomic DNA sequencing.

Another possible underlying mechanism could be maternal imprinting of chromosome 2 or TPO gene. Indeed, it has been hypothesized that chromosome 2 might be imprinted, based on its homology with mouse chromosome 2, which is known to be imprinted (25); moreover six different families presenting with various degrees of abnormal development have been described as associated with a maternal disomy of chromosome 2 (for review, see Ref. 16). However, another patient with a complete chromosome 2 maternal isodisomy was described to be completely normal (26), thus excluding possible maternal imprinting of chromosome 2. Furthermore, the revision of the literature allowed us to exclude the possibility of parental imprinting of the TPO gene. Indeed, among the nine unrelated TIOD families described with a single TPO mutation (Refs. 7, 9, 13, 15 , and the present study), in three the genetic analysis of the parents is available and shows that in one family the mutation is inherited from the mother (15) and in two families from the father (Ref. 9 and the present study). Finally, because DNA methylation in CpG islands may suppress gene transcription (27), the methylation at one CpG-rich site in the regulatory region of the TPO gene has been excluded in the present family by using an appropriate test. On these bases, genomic imprinting of TPO gene does not appear to be involved in the pathogenesis of TIOD.

The promoter region of the TPO gene has been previously characterized up to 900 bp from the transcription start site (20). Mutations in this regulatory region have been excluded by direct sequencing. The two novel variations found in the promoter sequence are located in the maternal allele that was not inherited by the affected patients.

Functional analyses have not been performed for the mutation in exon 12, but it is worth noting that this mutant cosegregates with TIOD in two unrelated families (Ref. 13 and the present study). Furthermore, there is evidence strongly indicating that Arg693 is an essential residue for peroxidase activity. Firstly, Arg693 is highly conserved among human peroxidases, a family of structurally and functionally related proteins such as myeloperoxidase, eosinophil peroxidase, and lactoperoxidase (28); it is also found in all known TPO polypeptides (rat, mouse, and pig; Refs. 29, 30, 31 ; Fig. 4). According to the predicted structure of hTPO reported by Banga et al. (32), this residue lies in a highly conserved region of the gene. Finally, because residues from 684–697 are predicted to constitute one of the two most hydrophilic regions of the protein, the substitution at codon 693 of an Arg with a hydrophobic Trp is very likely to induce major defects in TPO activity. No clinical data have been reported on the patient of the other family harboring this mutation in compound heterozygosity (13), but in the present patients it is associated with a severe phenotype and likely leads to complete inactivation of the function of the enzyme. Because TIOD is not associated with sensorineural hearing loss, we speculated that this defect in the three affected patients could be due to an alteration in one of the numerous genes involved in deafness.

View larger version (25K): [in this window] [in a new window]   FIG. 4. Amino acid sequence of the mutant TPO as compared with other human peroxidases (28 ) and with several animal TPO (29 30 31 ). Conserved amino acids are boxed. Arrow marks the missense mutation Arg693Trp. Arginine (R) is a basic, positively charged and polar amino acid, whereas tryptophan (W) is a neutral, noncharged and aromatic residue.

	Footnotes

 
This work was partially supported by Research Funds of Ricerca Corrente of Istituto Auxologico Italiano IRCCS (to L.P.).

Results from this work were presented in part at the 28th Annual Meeting of the European Thyroid Association, Göteborg, Sweden, September 2002.

Abbreviations: CRE, cAMP response element; hTPO, Human TPO; Tg, thyroglobulin; TIOD, total iodide organification defect; TPO, thyroid peroxidase; UTR, untranslated region.

Received August 29, 2002.

Accepted March 25, 2003.

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