The present study reveals the clinical and molecular analysis of a cohort of sixteen children from unrelated families with CH and IOD in Argentinian population. Patients with IOD have a variable degree of primary hypothyroidism characterized by elevated TG serum levels, an increased and rapid uptake of iodine, a significant discharge of thyroidal radioiodine after perchlorate discharge test (PDT) (Supplementary Figure 1) and a thyroid gland enlargement depending on the severity of the defect. PDT allows to distinguish Total IOD (TIOD) from Partial IOD (PIOD, between 10 and 90%) . In untreated patients, a complete defect causes a severe phenotype resulting in mental retardation with a large goiter. Due to late initiation of treatment, many late-diagnosed hypothyroid patients suffer typical signs and symptoms of hypothyroidism even though they receive regular L-T4 treatment. Unfortunely this is the case of some of our patients who, together with others who interrupted their treatment for different reasons, experimented severe repercussions (patients 3:II-1, 4:II-1, 5:II-3, 6:II-1, 7:II-1, 8:II-1, 8:II-2, 12:II-1) (Supplementary Notes 1). So, they needed psycho-pedagogical or phonoaudiological assistance. The remaining 8 patients (1:II-1, 2:II-1, 9:II-3, 10:II-1, 11:II-1, 13:II-1, 14:II-1, 15:II-1 and 16:II-1) grew and developed normally attending normal schooling with good performance (Supplementary Notes 1). All the cases studied correspond to permanent CH (PCH) except the index patients of the families 10 and 11 whom transient CH (TCH) was diagnosed. It has been reported that TDH can cause PCH or TCH . Although variants in TPO and DUOX2 appear to be the most common cause of IOD, patients with hipoplasia and non-goitrous hypothyroidism have been reported recently harboring TPO  and DUOX2 mutations .
Previous reports indicate that CH caused by variants in the TPO gene is an autosomal recessive disorder, and most patients with biallelic TPO variants have PCH . In the present study, TPO variants have been associated to TCH as can been seen in the index patients of family 10 and 11 who presented severe hypothyroidism in the neonatal period.
Most cases of CH associated with alterations in the DUOX2 gene are caused by either biallelic or monoallelic variants which lead to extremely complex correlation between DUOX2 genotypes and clinical phenotypes [49, 54, 55]. Both biallelic and monoallelic DUOX2 variants could be associated with TCH or PCH [13, 46, 55-57]. DUOXA2 variants also are associated with TCH or PCH [1, 55]. Recently, Lui et al.  identified two monoallelic missense variants in DUOX1 and DUOXA1 genes causing TCH and PCH, respectively. Of note, biallelic inactivation of TG, SLC5A, SLC26A4 or SLC26A7 causes PCH too [59, 60].
Molecular diagnosis of CH has been traditionally established by PCR-based approaches followed by systematic Sanger sequencing analysis. The identification of simultaneous variants in the same gene or in different thyroid specific genes by introduction of NGS platforms contributes to the accurate diagnosis and classification of the defects CH. TPO and DUOXs variants were identified in a total of 205 (in 38 papers) and 663 (in 40 papers) patients with CH, respectively, using targeted NGS panel (TNGS) or whole-exome sequencing (WES). Patients and variants are summarized in Supplementaries Tables 1 and 2. These findings suggested that, not only monogenic inheritance, but also digenic or oligogenic inheritance are involved in the pathogenesis of IOD.
In this report, we have identified novel variants associated with CH and other variants previously described. Some of the latter were not associated with this pathology. So, ours results show that variants were most frequently found in TPO (68.2%), followed by DUOX2 (27.3%) and IYD (4.5%) genes. No definitively variants were found in SLC5A5, SLC26A4, DUOXA2 and TG by NGS technology. 10 missense mutations, 2 splice site variants, 2 duplications and 1 deletion were identified in TPO gene (Figure 4a), whereas, 6 variants were identified in DUOX2 gene: 3 missense and 3 deletions (Figure 4b). Only 1 missense variant was identified in IYD gene. Sixteen out of a total number of 22 variants was identified as likely pathogenic variants. We have identified 3 likely benign variants, two of them, in TPO gene: p.Met706Val and c.2007-9_-7delTCT and the other, in IYD gene: p.Arg292Cys as it was evidenced by Clinvar database and our studies using in silico predictive tools, whereas 3 variants, 1 in TPO (p.Ser398Thr) and two in DUOX2 gene (p.Pro138Leu and p.Pro142Arg) were classified as VUS. 13 variants are found in the extracellular portion of the TPO (Figure 4), in the myeloperoxidase (MPO)-like domain: p.Asn307Thr, p.Ala397Profs*76, p.Ser398Thr, p.Pro499Leu, p.Thr561 Met, p.Ala576Glu, p.Gln660Glu, p.Arg665Trp, c.2007-9_-7delTCT and p.Met706Val; in the Sushi domain: p.Val748Met and in the EGF domain: p.Glu799Lys and p.Cys808Leufs*72. Two variants are located in the intracellular part of TPO: c.2749-2A>C and p.Ser918Cysfs*62 (Figure 4).
The most frequent variants are, the reported previously p.Val748Met identified in 4 patients, the p.Thr561Met presents in 3 patients, the p.Ala397Profs*76 found in 2 patients and the p.Asn307Thr identified en 2 patients too. The pathogenic p.Val748Met variant produces several changes in molecular surface. Significant structural alterations have been detected in the p.Thr561Met variant, which would alter the secundary structure and affect the function of TPO protein. Our results agree with the investigations of Fu et al. . On the other hand additional prediction studies would indicate the pathogenity of the p.Thr561Met variant. The p.Ala397Profs*76, described for the first time by Abramowicz et al.  is a common alteration of the TPO gene in Caucasian population.
The p.Asn307Thr identified previously in our laboratory  has been classified in the present study as likely pathogenic variant. The change origines a new hydrogen bond, a local increase in electropositive charges. On the other hand, the position 307 is a known glycoslylation site for which we hyphotesize that the alteration of the normal protein glycosylation pattern could affect its transport and correct localization in the thyrocyte membrane.
Three variants identified here have been previously functionally characterized, p.Ser398Thr [9, 10], p.Pro499Leu  and p.Glu799Lys . The variant p.Ser398Thr has been classified by us as VUS, which would be consistent with the following explanations: (a) the 3D modelling analysis of the p.Ser398Thr showed the breaking of a hydrogen bond between an asparagine at position 557 and an isoleucine at position 497 and several changes in the molecular surface of the mutated protein; (b) Guria et al. , demostrated that p.Ser398Thr had and slightly decreased enzyme activity determinated by the I− and guaiacol assays, this findings are consistent with the molecular docking-based study carried out by Begum et al.  in which p.Ser398Thr showed a moderate influence on the interaction between the TPO protein and the heme prosthetic group. If we establish a genotype-phenotype relationship, we could hypothesize that this variant would not be the cause of the CH, since the mother and one of the index patient's siblings are euthyroid and homozygous for it (Figure 1a). In a previous work we have functionally analyzed the p.Pro499Leu variant, described here as likely pathogenic variant using the Baculovirus System as a model of enzyme expression of human TPO. Significantly lower activity was recorded . Bikker et al.  expressed the p.Glu799Lys in CHO-K1 cells. The protein appeared to insert normally in the cell membrane, since it showed distribution similar to that of wild-type TPO. The position of the variant is far from the active site, but still the mutant protein is devoid of enzymatic activity, suggesting that the exchange of an acidic for a basic amino acid in exon 14 may lead to improper folding of the molecule .
Most cases of CH associated with alterations in the TPO gene are caused by either homozygous or compound heterozygous genotypes. However, two or even more variations in one or more CH associated genes can be found in a single patient, and the coexistence of multiple variants may affect the severity of the hypothyroid condition. In the present study, a variant heterozygous for c.2749-2A>C in TPO combined with the heterozygous IYD variant for p.Arg292Cys were identified in patient 6: II-1 and three variants have been identified in patient 9:II-3, two in TPO gene: the homozygous p.Met706Val and the heterozygous p.Val748M and one homozygous variant in DUOX2 gene: p.Pro138Leu.
Aditionally we have identified 6 DUOX2 variants. p.Pro138Leu, p.Pro142Arg and p.Leu264Cysfs*57 are located in the peroxidase-like domain, whereas p.Gln899Serfs*21, p.Thr920Ile and p.Phe966Serfs*29 are present in the transmembrane domain. Only two variants were described previously associated with congenital hypothyroidism, the p.Pro138Leu [46, 47] and the p.Phe966Serfs*29 variant [6, 11, 24, 40, 41, 46, 48-51]. The frameshift p.Phe966Serfs*29, commonly reported as p.S965fsX994 is one of the most frequent mutations in DUOX2 gene. Muzza et al.  showed that the transcripts carrying this deletion were submitted to nonsense mRNA decay, indicating the existence of mRNA quality control active on the DUOX2 gene. Despite p.Pro138Leu appears functionally silent, with limited damaging impact, has been significantly associated with CH in Japanese and Italian patients and has been classified in this study as VUS . Several molecular surface changes can be seen in the affected area and in the whole protein in our 3D modeling analysis.
In this study, eight patients were monogenic and compound heterozygous for variants in TPO and DUOX2 genes (Patients 1:II-1, 2:II-1, 3:II-1, 4:II-1, 5:II-3, 7:II-1, 14:II-1, 15:II-1). All of them harbored variants that were possibly contributing to CH. Five likely pathogenic and monoallelic variants have been identified here, all of them were absent in the healthy control (Patients 8:II-1 and 8:II-2, 11:II-1, 12:II-1, 13:II-1, 16:II-1). These variants are usually assumed to coexist with an additional undetected CNV, intronic or regulatory mutation on the other chromosome . This may occur in our patients since the sequencing techniques used here would not detect mutations in noncoding regions of the genome and although CNVs were not identified. On the other hand, monoallelic variants could coexist with other genes not incorporated in the design of our panel and that also contribute to the hypothyroid phenotype. Only two patients were heterozygous for digenic variants in TPO/IYD (6:II-1) and in TPO/DUOX2 (9:II-3) genes. In the first case only the TPO variant would be the cause of the pathology due to the IYD variant is likely benign according to bioinformatics predictors.
In summary, five novel variants have been identified, two en TPO: c.2749-2A>C and c.2752_2753delAG, [p.Ser918Cysfs*62] and three variants in DUOX2 gene: c.425C>G [p.Pro142Arg]; c.790delC [p.Leu264Cysfs*57] and c.2695delC [p.Gln899Serfs*21] expanding so the causative mutation spectrum of TDH in Argentinian patients.