Clinical, Biochemical, and Molecular Genetic Characterization of Two Patients with Congenital Hypothyroidism Harboring Novel Compound Heterozygous Variants in the Thyroglobulin Gene

Clinical, Biochemical, and Molecular Genetic Characterization of Two Patients with Congenital Hypothyroidism Harboring Novel Compound Heterozygous Variants in the Thyroglobulin Gene | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Clinical, Biochemical, and Molecular Genetic Characterization of Two Patients with Congenital Hypothyroidism Harboring Novel Compound Heterozygous Variants in the T hyroglobulin Gene Héctor M. Targovnik, Valentina Ricci, Valeria F. Garzón, Mauricio Gomes Pio, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9117442/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Context Thyroglobulin (TG) is the most abundant glycoprotein secreted by thyrocytes into the follicular lumen, serving as the essential substrate for thyroid hormone biosynthesis. The clinical spectrum of TG defects is highly heterogeneous, ranging from euthyroid states to mild or severe permanent hypothyroidism. The aim of this study was to identify and characterize novel TG variants to advance our understanding of the molecular mechanisms underlying thyroid dyshormonogenesis. Methods Two patients from unrelated, non-consanguineous families with impaired TG synthesis were investigated, both undergoing comprehensive clinical, biochemical, and imaging evaluations. Genetic testing included DNA sequencing, genotyping, and bioinformatics analyses. Main results Molecular analysis revealed two previously unreported TG variants—NM_003235.5:c.1375C>T (NP_003226.4:p.(Gln459Ter)) and NM_003235.5:c.5509_5518delAAAGACACAG (NP_003226.4:p.(Lys1837CysfsTer12))—alongside two known variants, NM_003235.5:c.378C>A (NP_003226.4:p.(Tyr126Ter)) and NM_003235.5:c.7880A>G (NP_003226.4:p.(Asp2627Gly)). A frameshift variant, combined with a clinically relevant missense variant in the ChEL domain (NP_003226.4:p.(Lys1837CysfsTer12)/ NP_003226.4:p.(Asp2627Gly)), was identified in one patient with congenital hypothyroidism (CH) who presented with normal serum TG levels. In contrast, the second patient, with classic goitrous CH and reduced serum TG levels, carried two nonsense variants introducing premature stop codons (NP_003226.4:p.(Tyr126Ter)/NP_003226.4(p.Gln459Ter)), resulting in truncated proteins and severely impaired hormone synthesis. The deleterious impact predicted by amino acid analysis software, together with strict evolutionary conservation and three-dimensional modeling of NP_003226.4:p.(Asp2627Gly) in the ChEL domain strongly supports a pivotal role of this residue in maintaining TG structural integrity. Conclusion The systematic exhaustive analysis of two novel variants, together with two previously reported TG variants, using molecular and bioinformatics tools, broadens the spectrum of deleterious TG variants and provides deeper insights into the etiology of CH. Molecular Genetics Thyroglobulin Gene Variant Congenital Hypothyroidism Thyroid Dyshormonogenesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Congenital hypothyroidism (CH) is the most frequent thyroid disorder in childhood, with an incidence of approximately 1 in 2,000–4,000 live births and a female-to-male ratio of 2:1. It is typically characterized by elevated thyroid-stimulating hormone (TSH) levels and reduced circulating thyroid hormone (TH) concentrations [1-4]. Affected children may exhibit a wide spectrum of clinical manifestations. The advent of high-throughput sequencing technologies has enabled large-scale detection of genetic variants, leading to the discovery of novel inactivating variants in candidate genes associated with CH. Importantly, the pathogenesis of CH is not limited to monogenic inheritance; digenic and oligogenic mechanisms have also been implicated [4,5]. CH can be classified into three major categories: environmental causes (such as iodine deficiency), thyroid dysgenesis, and thyroid dyshormonogenesis (TDH) [1,4,6-8]. Thyroid dysgenesis encompasses structural abnormalities of the gland, including complete absence (agenesis or athyreosis), reduced size (hypoplasia), or ectopic localization (thyroid ectopia) [1,4,6-8]. Genetic variants in several transcription factors and signaling genes—such as NKX2.1 , NKX2.5 , FOXE1 , PAX8, TSHR, CDCA8, ELN, GLIS3, HOXD3 , HOXB3 , JAG1 , KMT2D , TBX1 , TUBB 1, and URBI —have been reported in patients with thyroid dysgenesis phenotypes [1,4,9,10]. In contrast, TDH represents a group of genetic defects that impair TH synthesis due to pathogenic variants in genes encoding proteins essential for multiple steps of TH biosynthesis. These include Solute Carrier Family 5 Member 5 ( SLC5A5 , encoding the sodium/iodide symporter, NIS), Solute Carrier Family 26 Member 4 ( SLC26A4 , encoding pendrin), Solute Carrier Family 26 Member 7 ( SLC26A7 ), Thyroid Peroxidase ( TPO ), Dual Oxidase 1 ( DUOX1 ), DUOX Maturation Factor 1 ( DUOXA1 ), Dual Oxidase 2 (DUOX2 ), DUOX Maturation Factor 2 ( DUOXA2 ), Iodotyrosine Deiodinase ( IYD ), and Thyroglobulin ( TG ) [1,4,6-8]. Moreover, pathogenic variants in SLC26A4 , SLC5A5 , TPO , DUOX1 , DUOX2 , and TG have also been implicated in thyroid dysgenesis [9,11-15]. TDH caused by TG gene variants (TDH3) is most often inherited in an autosomal recessive pattern, with an estimated incidence ranging from 1 in 67,000 to 1 in 100,000 live births [16,17]. The clinical spectrum is broad, spanning from euthyroid states to mild or severe permanent hypothyroidism. To date, more than 300 pathogenic TG variants have been identified, encompassing missense mutations that affect conserved cysteine residues and the ChEL domain, as well as duplications, deletions, insertions, partial inversions, splice-site defects, and nonsense variants [5,18-20]. In this study, we report the clinical, biochemical, and molecular features of two patients from unrelated families diagnosed with CH. Molecular analysis identified two TG variants not previously published—NM_003235.5:c.1375C>T (NP_003226.4:p.(Gln459Ter)) and NM_003235.5:c.5509-5518delAAAGACACAG (NP_003226.4:p.(Lys1837CysfsTer12)) and—together with two published variants, NM_003235.5:c.378C>A (NP_003226.4:p.(Tyr126Ter)) and NM_003235.5:c.7880A>G (NP_003226.4:p.(Asp2627Gly)). The identification and comprehensive molecular characterization of TG variants in patients with TDH3 provide valuable insights that enhance diagnostic accuracy and genetic counseling. Methods Subjects Two patients with suspected CH diagnosis were referred for clinical and biochemical evaluation and confirmation of diagnosis to the Endocrinology Division of the Hospital de Niños “Ricardo Gutiérrez” (HNRG). Serum TSH, total T 4 (T 4 ), total T 3 (T 3 ), and free T 4 (FT 4 ) levels were measured using electrochemiluminescence immunoassay (Elecsys 2010, Roche Diagnostics, Indianapolis, IN, USA). Anti-thyroid peroxidase (TPO) antibodies, anti-thyroglobulin (TG) antibodies, and serum TG levels were assessed by chemiluminescence immunoassay (Immulite 2000, Siemens Healthcare Diagnostics, New York, NY, USA). Antibodies against the TSH receptor (TRAb) were determined using electrochemiluminescence immunoassay (Elecsys Anti-TSHR, Roche Diagnostics). DNA isolation The genomic DNA was extracted from the peripheral venous blood cells as previously described [21]. The DNA was quantified using a high-performance microvolume spectrophotometer NanoPhotometer® NP60 (Implen Inc., München, Germany). DNA purity was assessed by measuring the absorbance ratio 260/280 nm; further DNA sample processing was performed only if the ratio was between 1.8 and 2.1. Custom-panel sequencing For the index patient II-2 of Family SRA, DNA library preparation and hybridization were performed using a Twist Custom Panel (Twist Bioscience, South San Francisco, CA, USA). The quality of genomic DNA fragmentation was verified using an Agilent capillary system Fragment Analyzer™ (Agilent Technologies, Santa Clara, CA, USA). Next-generation Sequencing (NGS) by synthesis with fluorescent reversible terminator deoxyribonucleotides was performed using a NextSeq 500 system (Illumina, San Diego, CA, USA) at the Translational Medicine Unit of the HNRG. Whole-exome sequencing Whole-exome sequencing (WES) was performed for the index patient II-2 of Family BAS at Macrogen Inc. (Seoul, Republic of Korea). Genomic DNA extracted from Family BAS, index patient II-2 was physically fragmented to a target peak size of 150–200 bp with the Covaris LE220 focused-ultrasonicator (Covaris, Woburn, MA, USA), and the exome capture library was enriched using an Agilent Sure select XT V6 kit (Agilent) for the entire coding sequence and intron–exon boundaries of 20,000 human genes. The postcapture library was sequenced using the NovaSeq 6000 System (Illumina) producing 150 bp paired-end reads. The total number of bases sequenced was 6,760,596,672 bp, and the total number of reads was 45,265,924. Genome analysis FASTQ files were processed using a Genome Analysis Toolkit (GATK, https://www.broadinstitute.org/gatk/) v4.0.5.1-based pipeline. Sequence data were aligned with GRCh38/hg38 human reference genome using the BWA-MEM algorithm of Burrows–Wheeler Aligner software to perform variant calls and annotations. Duplicates were removed using Picard (Broad Institute). Data was analyzed for single-nucleotide variants (SNVs) and insertions/deletions (indels). The target region included the coding exons, consensus splice sites (± 2 bases from exon boundaries), and the extended splice region (±3 to ±10 bases). Coverage depth and read quality were evaluated with the Integrative Genomics Viewer (https://www.broadinstitute.org/scientific-community/software/integrative-genomics-viewer) v2.16.0. TheAmerican College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) guidelines [22], and the ClinGen Sequence Variant Interpretation Working Group recommendations were followed to determine the pathogenicity of a variant. The description of the variants' position in the DNA (genomic and coding) and in the protein is according to the HGVS nomenclature, based on the genome build GRCh38/hg38.(https://hgvs-nomenclature.org/stable/). Sanger sequencing Putative disease-causing variants were confirmed in patients and parents using Sanger sequencing. Target exons were amplified by polymerase chain reaction (PCR) with specific primers, reported previously [23] and sequenced with the same sense and antisense specific primers or M13 universal primers with the Big Dye deoxyterminator Cycle Sequencing Kit (Applied Biosystems, Weiterstadt, Germany). The samples were analyzed on the 3500XL Genetic Analyzer (Applied Biosystems) Genomics and Bioinformatics Unit of the Instituto Nacional de Tecnología Agropecuaria (INTA) or at the Translational Medicine Unit of the HNRG. The sequences were compared to the reference sequence and analyzed using ChromasPro (Technelysium Pty Ltd., Queensland, Australia). Detection of exon copy number variants Copy Number Variants (CNVs) were predicted using the coverage based DECoN (Detection of Exon Copy Number) algorithm (https://github.com/RahmanTeam/DECoN) [24,25]. Protein homology analysis Amino acid sequence homology within the ChEL domain was evaluated through multiple sequence alignment using CLUSTAL W (version 1.83) (http://www.ch.embnet.org/software/ClustalW.html). Protein sequences were retrieved from the NCBI database (https://www.ncbi.nlm.nih.gov) for the following species: Alligator mississippiensis (XP_059579449.1), Aquarana catesbeiana (XP_073488945.1), Astyanax mexicanus (XP_022529282.2), Bos Taurus (NP_776308.1), Canis lupus familiaris (NP_001041569.1), Carassius auratus (XP_026120244.1), Cavia porcellus (XP_003467392.1), Chelonia mydas (XP_043394997.1), Clupea harengus (XP_031429786.1), Columba livia (XP_021154537.2), Crotalus tigris (XP_039206379.1), Cynoglossus semilaevis (XP_008321228.1), Cyprinus carpio (XP_042628676.1), Danio rerio (NP_001316794.1), Eublepharis macularius ( XP_054841882.1), Gallus gallus (NP_001376406.2), Homo sapiens (NP_003226.4), Lampetra fluviatilis (CAL5909345.1), Lampetra planeri (CAL5920418.1), Larus michahellis (XP_074433977.1), Lepisosteus oculatus (XP_015212882.2), Macaca fascicularis (XP_045254657.2), Macaca mulatta (XP_028708128.1), Mus musculus (NP_033401.2), Oryzias latipes (XP_011484169.1), Pan troglodytes (XP_016815373.4), Panthera leo (XP_042780307.1), Pongo pygmaeus (XP_054355108.2), Python bivittatus (XP_025021113.1), Rattus norvegicus (NP_112250.2), Stegastes partitus (XP_008304814.1), Struthio camelus (XP_068789869.1), Taeniopygia guttata (XP_072781126.1), Trichechus manatus latirostris (XP_004373071.1), Xenopus tropicalis (NP_001316486.1) and Xiphophorus maculatus (XP_023185937.1). The TG sequence for Gorilla gorilla was obtained from UniProt [https://www.uniprot.org; accession G3QS68]. Amino acid prediction analysis SNVs were analyzed with the sequence-based predictors included in the dbNSFP v4 (https://www.dbnsfp.org/): SIFT, SIFT4G, Polyphen2-HDIV, Polyphen2-HVAR, LRT, MutationTaster, MutationAssessor, FATHMM, PROVEAN, VEST4, MetaSVM, MetaLR, MetaRNN, M-CAP, REVEL, MutPred2, PrimateAI, DEOGEN2, BayesDel_addAF, BayesDel_noAF, ClinPred, LIST-S2 VARITY_R, VARITY_ER, EVE, AlphaMissense, CADD, DANN, FATHMM-MKL, FATHMM-XF [26-35]. 3D modeling analysis The UCSF Chimera program (UCSF Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, https://www.cgl.ucsf.edu/chimera/) [36-37] was used to obtain the 3D model of the human TG (PDB 6SCJ, resolution: 3.60 Å) [38] . Results Clinical and biochemical description Family SRA The index patient, SRA:II-2, was born in 2010 at 37 weeks of gestation (birth weight: 2910 g) by cesarean section to an unrelated couple. Her neonatal course was complicated by a four-day hospitalization in the Neonatal Intensive Care Unit for suspected sepsis. Neonatal screening on postnatal day 3 identified primary hypothyroidism with a TSH of 65.05 µUI/ml (cut off: 10 µUI/ml). The patient was referred to the Endocrinology Department of the HNRG at 15 days of life. Initial physical examination revealed an infant who was clinically euthyroid but presented with a palpable, enlarged thyroid gland. Confirmatory serum biochemistry at day 15 showed TSH: 62.47 µUI/ml (reference range between 0 and 60 days: 1.3–10 µUI/ml), T 4 : 8.3 µg/dl (reference range between 0 and 30 days: 6-8 µg/dl), FT 4 : 1.14 ng/dl (reference range between 0 and 30 days: 1.0-2.6 ng/dl) and T 3 : 202 ng/dl (reference range between 0 and 30 days: 80-260 ng/dl). Serum TG was 61.7 ng/ml (reference range between 0-2 months: 10-100 ng/ml). To rule out immune-mediated or transplacental etiology, thyroid autoantibodies (anti-TPO/anti-TG) and TRABs were measured and found to be negative in both the infant and the mother, who was herself healthy with normal thyroid function. Replacement therapy with L-T 4 was initiated at 50 µg/day. Imaging studies included a 99m Tc thyroid scintigraphy, which demonstrated an eutopic, enlarged gland consistent with diffuse goiter. A knee radiograph confirmed the presence of distal femoral and proximal tibial epiphyses. After 20 days of treatment, biochemical control showed TSH: 19.11 µUI/ml and FT4: 1.36 ng/dl. The patient maintained a steady growth and developmental trajectory through childhood with regular L-T 4 adjustments. At age 5, the L-T 4 withdrawal trial confirmed permanent CH. Laboratory results were as follows, TSH: >100 µUI/ml (reference range: 0.5-6.5 µUI/ml), T 4 of 3.7 µg/dl (reference range: 6-14 µg/dl), FT 4 of 0.41 ng/dl (reference range: 0.8-2.2 ng/dl) and T 3 of 119 ng/dl (reference range: 80-220 ng/dl), while TG remained at 13.9 ng/ml (reference range: 6-40 ng/ml). The perchlorate discharge test was negative. The thyroid ultrasound showed a regular, eutopic gland with a homogeneous structure and normal vasculature, right lobe size: 2.36 x 0.86 x 1.05 cm, left lobe size: 1.94 x 0.88 x 1.02 cm, and total volume: 2.01 cm 3 (mean to 5 years: 4.10 cm 3 )[39]. Thyroid volume was calculated by multiplying of length, breadth and depth by a corrective factor (0.52) for each lobe [40]. Audiometry and ophthalmological evaluation were normal. Cognitive assessment performed at age 6 years showed scores within the average range, though processing speed was just below the average range. Pubertal progression was normal, with menarche occurring at age 12. As the patient progressed into adolescence, the thyroid gland remained consistently palpable. The most recent thyroid ultrasound at age 13 demonstrated a diffusely heterogeneous echo-structure and lobulated contours, but without focal nodules or cystic degeneration, right lobe size: 1.2 x 1.4 x 3.4 cm, left lobe size: 1.1 x 1.1 x 2.9 cm, and total volume: 4.79 cm 3 (mean to 13 years: 9.66 cm 3 ) [39]. At age 14, the patient’s thyroid function remained well-controlled (TSH: 3.51 µUI/ml, FT4: 1.35 ng/dl) with a final adult height of 163 cm. Family BAS Index patient BAS:II-2, born in 2018, is the first and only child of nonconsanguineous healthy parents. Born from an uneventful pregnancy and delivery, was identified as CH in the neonatal screening with TSH >100 µUI/ml (cut off: 10 µUI/ml). Confirmation serum studies showed TSH: >100 µUI/ml, T 4 : 4 ug/dl, FT 4 : 0.48 ng/dl, T 3 : 124 ng/dl, anti-TPO, anti-TG negatives and TG: < 0.9 ng/ml at 10 days of life. He presented jaundice, puffy face, and palpable goiter, as evidenced by a 99m Tc thyroid scan. He started L-T 4 treatment with good adherence and began follow-up. He grew and developed normally (90 th percentile of height and weight) till age 3, when treatment was withdrawn for a month, and reevaluation of thyroid function confirmed hypothyroidism with TSH: >95 µUI/ml, FT 4 : 0.40 ng/dl and TG: <0.9 ng/ml. Thyroid ultrasound showed an eutopic enlarged thyroid gland without nodules or cysts, right lobe size: 3.4 x 1.0 x 1.3 cm, left lobe size: 3.3 x 1.1 x 1.4 cm, and total volume: 4.94 cm 3 (mean to 3 years: 2.9 cm 3 ) [39]. He restarted treatment. At age 4, he grows normally. His neurocognitive outcome is normal. Molecular Genetic Analysis Family SRA A compound of heterozygous for NM_003235.5:c.5509_5518delAAAGACACAG (NC_000008.11:g.132963035_132963044delAAAGACACAG) and NM_003235.5:c.7880A>G (NC_000008.11:g.133131829A>G) in the TG gene was identified by custom-panel sequencing in index patient SRA:II-1. The c.5509_5518del variant in exon 29 causes a frameshift starting with codon lysine 1837 , changes this amino acid to a cysteine residue, and creates a premature stop codon at position 12 of the new reading frame (NP_003226.4:p.(Lys1837CysfsTer12)), while the NM_003235.5:c.7880A>G variant in exon 46 produces the substitution of a aspartic acid for glycine at codon 2627 (NP_003226.4:p.(Asp2627Gly)) (shown in Fig. 1). Direct sequencing of exon 29 and 46 of both parents' genomic DNA indicated that the patient inherited NM_003235.5:c.5509_5518del variant from the healthy father and NM_003235.5:c.7880A>G variant from the healthy mother (shown in Fig. 1). The NM_003235.5:c.5509_5518del variant is reported in the gnomAD v4.1 database with a frequency of 0.0001% (1/1614046, European non-Finnish), the NM_003235.5:c.7880A>G variant is also indexed with a frequency of 0.0001% (1/1614092, European non-Finnish). The NM_003235.5:c.5509_5518del variant has not previously been reported in association with CH, whereas the NM_003235.5:c.7880A>G variant was recently described by us in association with hyperthyrotropinemia [41]. Considering the structural relevance of analyzing polymorphic coding variants across TG to confirm heterozygosity and to facilitate future comparative allele analyses with newly reported cases of similar genotype, eight heterozygous TG variants were identified in the index patient SAR:II-1. These included five non-synonymous substitutions— (NM_003235.5:c.2200T>G (NP_003226.4:p.(Ser734Ala)), NM_003235.5:c.3082A>G (NP_003226.4:p.(Met1028Val)), NM_003235.5:c.5921T>C (NP_003226.4:p.(Met1974Thr)) NM_003235.5:c.7501T>C (NP_003226.4:p.(rp2501Arg)), and NM_003235.5:c.7589G>A (NP_003226.4:p.(Arg2530Gln))— as well as three synonymous SNVs: (NM_003235.5:c.2334T>C (NP_003226.4(p.Pro778=)), NM_003235.5:c.7408C>T (NP_003226.4:p.(Leu2470=)) and NM_003235.5:c.7920C>T [NP_003226.4:p.(Tyr2640=)). No CNVs were detected in the coding sequence of the TG gene, indicating the absence of exon-level deletions or duplications. Other candidate genes implicated in the development of congenital hypothyroidism— DUOX1 , DUOXA1 , DUOX2 , DUOXA2 , FOXE1 , GLIS3 , GNAS , IYD , NKX2-1 , NKX2-5 , PAX8 , SLC26A7 , SLC5A5 , TBX1 , TPO , TSHR , JAG1 , TUBB1 , CDCA8 , THRB , TBL1X , IGSF1 , TSHB , FGFR1 , FOXA2 , GLI2 , IRS4 , PROP1 , SECISBP2 , SLC16A2 , SOX3 , POU1F1 , LHX3 , LHX4 , OTX2 —were also examined. No pathogenic variants were detected in any of these genes. All exons were covered at 90-100% (with a depth of at least 20x). Family BAS A compound of heterozygous for NM_003235.5:c.378C>A (NC_000008.11:g.132871451C>A) and NM_003235.5:c.1375C>T (NC_000008.11:g.132886747C>T) in the TG gene was identified by WES in index patient BAS:II-1. The previously documented variant NM_003235.5:c.378C>A in exon 4 replaces a tyrosine residue at position 126 with a premature stop codon (NP_003226.4:p.(Y126Ter)) [42,43], while the novel variant NM_003235.5:c.1375C>T in exon 9 replaces a glutamine residue at position 459 with a premature stop codon (NP_003226.4:p.(Gln459Ter)) (shown in Fig. 2). Direct sequencing of exons 4 and 9 of both parents' genomic DNA indicated that the patient inherited NM_003235.5:c.378C>A variant from the healthy father and NM_003235.5:c.1375C>T variant from the healthy mother (shown in Fig. 2). Neither variant was indexed in the gnomAD v4.1database. Seven coding polymorphic TG variants were identified in the index patient BAS:II-1. These including four non-synonymoussubstitutions—(NM_003235.5:c.3935A>G (NP_003226.4:p.(Asp1312Gly)), NM_003235.5:c.5512G>A (NP_003226.4:p.(Asp1838Asn)), NM_003235.5:c.7501T>C (NP_003226.4:p.(Trp2501Arg)) and NM_003235.5:c.7589G>A (NP_003226.4:p.(Arg2530Gln))— as well as three synonymous SNVs: (NM_003235.5:c.4506T>C (NP_003226.4:p.(Ala1502=)), NM_003235.5:c.7408C>T (NP_003226.4:p.(Leu2470=)) and NM_003235.5:c.7920C>T (NP_003226.4:p.(Tyr2640=)). All variants were in the heterozygous state, except for NM_003235.5:c.7501T>C, which was homozygous. Furthermore, no CNVs were identified in the TG gene and acceptor or donor splicing consensus sequences were rigorously respected in all introns of the TG gene. The other main genes involved in the development of congenital goiter and hypothyroidism were also analyzed: DUOX1 , DUOXA1 , DUOX2 , DUOXA2 , FOXE1 , GLIS3 , GNAS , IYD , NKX2-1 , NKX2-5 , PAX8 , SLC26A4 , SLC26A7 , SLC5A5 , TBX1 , TPO , TSHR , DIO1, DIO2 , DIO3 , DNAJC17 , JAG1 , TUBB1 , CDCA8 , THRA , THRB , TBL1X , SERPINA7 , IGSF1 , TRH , TRHR , TSHB , HHEX , KCNJ16 , HOXA3 , ANO1 , FGF8 , FGFR1 , FOXA2 , GLI2 , IRS4 , PROP1 , SECISBP2 , SLC16A2 , SOX2 , SOX3 , URB1 , RGS12 , GRPEL1 , CLIC6 , WFS1 , TREX1 , POU1F1 , LHX3 , LHX4 , HESX1 , KAT6B , PTPN22 , TSBP1 , B3GNT2 , FGF1 , FGF2 , B3GLCT , KCNJ10 , OTX2 , ALB , LRP2, ELN, HOXD3, HOXB3, and KMT2D. No deleterious variants were identified in any of these genes. All exons were covered at 100%. The GT-AG splicing consensus sequences were rigorously respected in all introns of all genes studied. Amino acid prediction analysis of the NP_003226.4:p.(Asp2627Gly) variant A comprehensive set of in silico algorithms was applied to evaluate the pathogenic potential of the NP_003226.4:p.(Asp2627Gly) substitution (shown in Table 1). Of the 30 prediction tools, 27 indicated a deleterious impact, whereas only LRT, FATHMM, and PrimateAI classified the variant as neutral or tolerated. Conservation analysis of the aspartic acid residue 2627 Comparative evaluation of the ChEL domain across 37 vertebrate species—including mammals, birds, reptiles, amphibians, ray-finned fishes, and jawless vertebrates—demonstrates complete conservation of aspartic acid at position 2627 (shown in Fig. 3). This strict evolutionary preservation strongly supports a pivotal role for this residue in maintaining the structural integrity of TG within the ChEL domain. 3D modeling analysis of the NP_003226.4:p.(Asp2627Gly) mutant Structural modeling localized aspartic acid 2627 to the central region of the ChEL domain (Region IV) of the TG monomer. In the wild-type model, aspartic acid 2627 formed hydrogen bonds with tyrosine 2611 and arginine 2386 , contributing to the intramolecular hydrogen bond network within the ChEL domain (shown in Fig. 4a). Substitution of aspartic acid 2627 by glycine (NP_003226.4:p.(Asp2627Gly)) resulted in the complete loss of these hydrogen bonds, with no additional hydrogen bond disruptions detected (shown in Fig. 4b). Structural inspection of the mutant model revealed no clashes and no detectable changes in the overall tertiary conformation or hydrophobicity profile of the TG monomer. In addition, aspartic acid 2627 is not part of a canonical N-glycosylation consensus sequence (N-X-S/T) and is not spatially associated with modeled N-glycan structures, indicating that the NP_003226.4:p.(Asp2627Gly) variant does not directly involve N-glycan attachment sites. Fig. 4c shows aspartic acid 2627 located within the central region of the ChEL domain. Discussion We report two previously unpublished TG variants—NM_003235.5:c.1375C>T (NP_003226.4:p.(Gln459Ter)) and NM_003235.5:c.5509_5518del (NP_003226.4:p.(Lys1837CysfsTer12))—together with two previously documented variants, NM_003235.5:c.378C>A (NP_003226.4:p.(Tyr126Ter)) [42,43] and NM_003235.5:c.7880A>G (NP_003226.4:p.(Asp2627Gly)) [41]. The pathogenicity of the variants was manually assessed according to ACMG/AMP recommendations [22], as follows. The c.1375C>T variant, classified as pathogenic, is predicted to result in loss of normal protein function through either protein truncation or nonsense-mediated mRNA decay (NMD) (PVS1). This variant is absent from population databases (PM2_Supp), confirmed in trans with a pathogenic variant in our patient (PM3), and the patient’s phenotype is highly specific for TDH3 (PP4) (shown in Table 2). The NM_003235.5:c.378C>A and NM_003235.5:c.5509_5518del variants, both classified as pathogenic, meet the criteria PM2_Supp, PVS1, PM3_strong, and PP4; and PM2_Supp, PVS1, and PP4, respectively (shown in Table 2). In contrast, the c.7880A>G variant, classified as of uncertain significance but with a tendency toward pathogenicity (Hot VUS, shown in Table 2), fulfills PM2_Supp (absent from controls or at extremely low frequency), PM3 (in trans with a pathogenic variant in an affected individual), and PP4 (highly specific phenotype), together with PP3 (computational tools, shown in Table 1). The clinical and biochemical criteria indicative of CH due to TG defects comprise low serum TG levels, elevated serum TSH, and a concomitant reduction in total serum T 4 with low or normal T 3 concentrations. A markedly reduced serum TG level is considered a key diagnostic hallmark of TG defects [5]. The index patient BAS:II-1 meets this criterion, whereas in the SRA:II-1 case, the TG value may fall within the normal range despite the presence of biallelic variants in the TG gene; nevertheless, it remains consistently higher than the values usually described in individuals with TG gene variants. Other cases of TG variants associated with normal or elevated serum TG levels have been well documented, including homozygous variants [43-46] and compound heterozygotes [43,47]. TG is a 660 kDa homodimeric glycoprotein that functions as the indispensable precursor for TH biosynthesis. Within its structure, specific tyrosyl residues undergo iodination and subsequent coupling reactions catalyzed by TPO, in concert with the DUOX system, ultimately yielding T 4 and T 3 [5,18-20]. The human TG gene , located on chromosome 8 (chr8:132,866,958–133,134,903; GRCh38/hg38), spans approximately 268 kb and contains a coding sequence of 8,453 nucleotides distributed across 48 exons (NCBI RefSeq: NM_003235.5) [48-52]. Translation produces a 19-amino acid signal peptide followed by a 2,748-residue polypeptide that assembles into the mature TG monomer (NCBI: NP_003226.4; UniProt: P01266) [48,52,53]. The canonical primary structure of TG is organized into four regions (I–IV), comprising TG type 1, 2, and 3 repeats, as well as a cholinesterase-like (ChEL) domain at the C-terminus (shown in Fig. 5) [5,48,50-55]. This domain is essential for TG dimerization and acts both as an intramolecular chaperone and as a molecular escort for TG regions I, II, and III. [56,57]. All truncated forms detected in this study eliminate the ChEL domain in its entirety. NP_003226.4:p.(Gln459Ter) and NP_003226.4:p.(Tyr126Ter) mutants comprise only a part of region I (shown in Fig. 5), while NP_003226.4:p.(Lys1837CysfsTer12) includes regions I, II, and only a part of region III (shown in Fig. 5). The functional consequences of the nonsense variants identified in this study may involve structural alterations in the protein that impair TH biosynthesis, primarily through the loss of the C-terminal hormonogenic site at position 2,766. This region harbors the principal coupling site for T 3 formation, mediated by the MIT 2766 –DIT 2766 interaction between opposing monomers [58,59]. Nevertheless, the NP_003226.4:p.(Gln459Ter) and NP_003226.4:p.(Lys1837CysfsTer12) mutants retain partial capacity for T 4 synthesis, as they preserve both the acceptor DIT 24 (exon 2) and the donor DIT 149 (exon 4) within the N-terminal hormonogenic site [60,61]. In contrast, NP_003226.4:p.(Tyr126Ter) mutant additionally loses the principal coupling site required for T 4 formation. It can be hypothesized that a short amino-terminal portion of TG containing a single hormonogenic site, even if producing only low physiological levels of TH, was sufficient to sustain vertebrate complexification from its earliest emergence until the fusion of the ChEL domain. This hypothesis is supported by the observation that a milder hypothyroidism phenotype has been reported in some homozygous patients carrying the NP_003226.4:p.Arg296Ter variant [10,42,43,45,62-64]. However, transient expression studies revealed that secretion of the NP_003226.4:p.Arg296Ter mutant was essentially undetectable [65]. In addition to the limited ability to generate active TH as a pathophysiological mechanism underlying CH, RNA surveillance pathways also operate in the presence of truncated proteins. Among these, NMD ensures the rapid cytoplasmic degradation of transcripts containing premature stop codons, thereby preventing the accumulation of truncated and potentially deleterious proteins [66]. In the present study, we extended the analysis of the NP_003226.4:p.(Asp2627Gly) variant, recently reported by us, by performing amino acid substitution prediction, protein homology assessment, and 3D structural modeling. Twenty-seven of the thirty predictors indicated a deleterious effect of the NP_003226.4:p.(Asp2627Gly) variant (shown in Table 1), and the wild-type aspartic acid at position 2,627 is strictly conserved in TG across thirty-seven vertebrate species analyzed (shown in Fig. 3), supporting an essential role of this residue in TG structure and/or proper functionality. Aspartic acid 2627 is located within the core of the ChEL domain (shown in Fig. 4), a region characterized by a dense and highly interconnected network of intramolecular interactions. Its substitution by glycine disrupts hydrogen bonds with tyrosine 2611 and arginine 2386 , weakening this tightly organized interaction network. Importantly, although aspartic acid 2627 is not itself part of a canonical N-glycosylation consensus sequence and is not directly associated with N-glycan attachment sites, its central positioning within the ChEL domain suggests that local destabilization may propagate through the domain and induce subtle long-range structural rearrangements. Such secondary effects could alter the spatial organization or accessibility of distal glycosylated residues, potentially compromising proper glycan presentation or recognition by the endoplasmic reticulum (ER) quality-control machinery. In this context, even modest perturbations in ChEL domain folding may contribute to impaired protein maturation. Misfolded TG molecules may accumulate within ER as a consequence of missense variants in the ChEL domain—such as NP_003226.4:p.(Asp2627Gly)—or of partial or complete deletions—such as NP_003226.4:p.(Tyr126Ter), NP_003226.4:p.(Gln459Ter) and NP_003226.4:p.(Lys1837CysfsTer12)—ultimately resulting in premature degradation [67]. This retention induces ER distention, a pathological condition termed ER storage disease (ERSD) [68]. Misfolded proteins are subsequently eliminated through the ER-associated degradation (ERAD) pathway. On the other hand, Zhang et al. [69] demonstrated that, in cases of TG defects caused by deleterious variants, T 4 production can originate from mutant TG retained intracellularly and subsequently released into the follicular lumen by dead thyrocytes. Once released, these mutant TG molecules are reutilized—or “cannibalized”—by the TH biosynthetic machinery of neighboring viable thyrocytes, which iodinate them and synthesize T 4 . Based on this mechanism, we hypothesize that in patient SRA:II-1, who carries the missense TG variant NP_003226.4:p.(Asp2627Gly) affecting the ChEL domain in combination with the frameshift variant NP_003226.4:p.(Lys1837CysfsTer12) on the opposite allele, this cannibalization process may partially compensate for impaired TG secretion at the apical membrane–colloid interface. This mechanism could account for the normal serum levels of TG, attributable to extensive thyrocyte loss. In conclusion, we report novel TG variants that expand the spectrum of TG defects. A clinically relevant missense variant in the ChEL domain, in combination with a frameshift, was identified in a patient with CH and normal serum TG levels. In contrast, a second patient with classic goitrous CH and low serum TG levels harbored two nonsense variants introducing premature stop codons within the TG coding sequence, resulting in truncated proteins with severely impaired TH synthesis. Collectively, our findings underscore the phenotypic diversity of CH attributable to TG variants. Declarations Data availability Data and material are available from the authors upon request. Acknowledgement We thank the study participants and their family members. V.R. and M.E.M. are research fellows of the Fondo para la Investigación Científica y Tecnológica (FONCyT-Agencia I+D+I). M.G.P. is a research fellow of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). H.M.T., M.G.R., A.E.C., C.M.R., and M.L.T. are established investigators of the CONICET. We thank Dr. Gabriela Sansó, Mrs. MG Gutiérrez Moyano, and Mr. Rodolfo De Bellis for their kind and skillful technical assistance. We thank Mrs. Rosenbrock Lambois for her assistance with study coordination. Author Contributions H.M.T. was involved in WES analysis, bioinformatic predictions, funding acquisition, study conception and design, and manuscript writing. V.R. contributed to formal analysis, data interpretation, validation, and variant curation. V.F.G. performed WES analysis, applied bioinformatic prediction tools, and carried out sample preparation for Sanger sequencing. M.G.P. conducted the structural modeling analyses. P.S. and M.E.A. performed custom-panel sequencing and contributed to variant curation. M.E.M. and A.E.C. participated in patient recruitment and the collection of clinical data and blood samples. A.I. was responsible for the bioinformatic analysis of custom-panel sequencing. M.G.R. provided essential resources and financial support for the study. C.M.R. contributed to funding acquisition and study conception and design. M.L.T. performed validation and variant curation, acquired funding, conceived and designed the study, and contributed to manuscript writing. All authors critically reviewed the manuscript, contributed to its revision, and approved the final version. Funding This study was funded by grants from the Fondo para la Investigación Científica y Tecnológica (FONCyT-ANPCyT-MINCyT, PICT-2018-02146 to H.M.T. and PIDC-2019-0007 to A.E.C. and M.L.T.), CONICET (PIP 2021-11220200102976CO to C.M.R.), and Universidad de Buenos Aires (UBACyT 2020-20020190100050BA to C.M.R.). The funders had no role in the design, data collection, data analysis, and reporting of this study. Conflict of Interest On behalf of all authors, the corresponding author states that there is no conflict of interest to declare. Ethical approval The studies involving human participants were reviewed and approved by the Ethical Committee of the Faculty of Pharmacy and Biochemistry of the University of Buenos Aires (CEICFFyB, No. 1094) and the Research Ethics Committee of the HNRG, Buenos Aires, Argentina (C.E.I. 21.33). 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J Endocrinol 170(2):307–321. https://doi.org/10.1677/joe.0.1700307 Holzer G, Morishita Y, Fini JB, Lorin T, Gillet B, Hughes S, Tohmé M, Deléage G, Demeneix B, Arvan P, Laudet V (2016) Thyroglobulin represents a novel molecular architecture of vertebrates. J Biol Chem 291(32):16553–16566. https://doi.org/10.1074/jbc.M116.719047 Swillens S, Ludgate M, Mercken L, Dumont JE, Vassart G (1986) Analysis of sequence and structure homologies between thyroglobulin and acetylcholinesterase: possible functional and clinical significance. Biochem Biophys Res Commun 137(1):142-148. https://doi.org/10.1016/0006-291x(86)91187-3 Molina F, Bouanani M, Pau B, Granier C (1996) Characterization of the type-1 repeat from thyroglobulin, a cysteine-rich module found in proteins from different families. Eur J Biochem. 240(1):125–133. https://doi.org/10.1111/j.1432-1033.1996.0125h.x Lee J, Di Jeso B, Arvan P (2008) The cholinesterase-like domain of thyroglobulin functions as an intramolecular chaperone. J Clin Invest 118(8):2950–2958. https://doi.org/10.1172/JCI35164 Lee J, Wang X, Di Jeso B, Arvan P (2009) The cholinesterase-like domain, essential in thyroglobulin trafficking for thyroid hormone synthesis, is required for protein dimerization. J Biol Chem. 284(19):12752–12761 https://doi.org/10.1074/jbc.M806898200 Citterio CE, Veluswamy B, Morgan SJ, Galton VA, Banga JP, Atkins S, Morishita Y, Neumann S, Latif R, Gershengorn MC, Smith TJ, Arvan P (2017) De novo triiodothyronine formation from thyrocytes activated by thyroid-stimulating hormone. J Biol Chem 292(37):15434–15444. https://doi.org/10.1074/jbc.M117.784447 Citterio CE, Morishita Y, Dakka N, Veluswamy B, Arvan P (2018) Relationship between the dimerization of thyroglobulin and its ability to form triiodothyronine. J Biol Chem 293(13):4860–4869. https://doi.org/10.1074/jbc.RA118.001786 Dunn AD, Corsi CM, Myers HE, Dunn JT (1998) Tyrosine 130 is an important outer ring donor for thyroxine formation in thyroglobulin. J Biol Chem 273(39):25223–25229. https://doi.org/10.1074/jbc.273.39.25223 Lamas L, Anderson PC, Fox JW, Dunn JT (1989) Consensus sequences for early iodination and hormonogenesis in human thyroglobulin. J Biol Chem. 264(23):13541–13545. Caputo M, Rivolta CM, Esperante SA, Gruñeiro-Papendieck L, Chiesa A, Pellizas CG, González-Sarmiento R, Targovnik HM (2007) Congenital hypothyroidism with goitre caused by new mutations in the thyroglobulin gene. Clin Endocrinol. 67(3):351–357. https://doi.org/10.1111/j.1365-2265.2007.02889.x Rivolta CM, Moya CM, Gutnisky VJ, Varela V, Miralles-García JM, González-Sarmiento R, Targovnik HM (2005) A new case of congenital goiter with hypothyroidism due to a homozygous p.R277X mutation in the exon 7 of the thyroglobulin gene: a mutational hot spot could explain the recurrence of this mutation. J Clin Endocrinol Metab. 90(6):3766–3770. https://doi.org/10.1210/jc.2005-0278 van de Graaf SAR, Ris-Stalpers C, Veenboer GJM, Cammenga M, Santos C, Targovnik HM, de Vijlder JJM, Medeiros-Neto G (1999) A premature stopcodon in thyroglobulin mRNA results in familial goiter and moderate hypothyroidism. J Clin Endocrinol Metab 84(7):2537–2542. https://doi.org/10.1210/jcem.84.7.5862 Lee J, Di Jeso B, Arvan P (2011) Maturation of thyroglobulin protein region I. J Biol Chem 286(38):33045–33052. https://doi.org/10.1074/jbc.M111.281337 Behm-Ansmant I, Kashima I, Rehwinkel J, Saulière J, Wittkopp N, Izaurralde E (2007) mRNA quality control: an ancient machinery recognizes and degrades mRNAs with nonsense codons. FEBS Lett. 581(15):2845–2853. https://doi.org/10.1016/j.febslet.2007.05.027 Siffo S, Gomes Pio M, Martínez EB, Lachlan K, Walker J, Weill J, González-Sarmiento R, Rivolta CM, Targovnik HM (2023) The p.Pro2232Leu variant in the ChEL domain of thyroglobulin gene causes intracellular transport disorder and congenital hypothyroidism. Endocrine 80(1):47–53. https://doi.org/10.1007/s12020-022-03284-5 Kim PS, Arvan P (1998) Endocrinopathies in the family of endoplasmic reticulum (ER) storage diseases: disorders of protein trafficking and the role of ER molecular chaperones. Endocr Rev 19(2):173–202. https://doi.org/10.1210/edrv.19.2.0327 Zhang X, Kellogg AP, Citterio CE, Zhang H, Larkin D, Morishita Y, Targovnik HM, Balbi VA, Arvan P (2021) Thyroid hormone synthesis continues despite biallelic thyroglobulin mutation with cell death. JCI Insight 6(11):e148496. https://doi.org/10.1172/jci.insight.148496 Tables Table 1. Bioinformatic prediction of functional effects of the variant NP_003226.4:p.(Asp2627Gly) in the ChEL domain of the thyroglobulin. Predictor Score Prediction Rankscore Reference SIFT 0.0 Damaging >0.05 Tolerated, 0.05 Tolerated, <=0.05 Damaging [32]; dbNSFP v4 Polyphen2_HDIV 1.0 Probably damaging <=0.452 Benign, =0.453 Possibly Damaging, >=0.957 Probably Damaging [27] Polyphen2_HVAR 0.999 Probably damaging <=0.452 Benign, =0.453 Possibly Damaging, >=0.957 Probably Damaging [27] LRT 0.012546 Neutral >0.001 Neutral or Unknown, <=0.001 Deleterious [31-32]; dbNSFP v4 MutationTaster 0.987212 Disease causing 0.5 Diseases Causing Automatic or Diseases Causing [27-32]; dbNSFP v4 MutationAssessor 3.27 Medium impact <=0.8 Neutral impact (NI), 0.8 Low impact (LI), 1.935 Medium impact (MI), >3.5 High impact (HI). Benign = NI and LI, Deleterious = MI and HI [27-32]; dbNSFP v4 FATHMM -0.43 Tolerated >-1.5 Tolerated, -2.5 Neutral, <=-2.5 Deleterious [32]; dbNSFP v4 VEST4 0.937 Pathogenic =0.75 Pathogenic [35] MetaSVM 0.2184 Damaging 0 Damaging [31] MetaLR 0.5323 Damaging 0.5 Damaging [31] MetaRNN 0.95961165 Damaging 0.5 Damaging [32]; dbNSFP v4 M-CAP 0.059366 Possibly Pathogenic 0.025 Possibly Pathogenic [29] http://bejerano.stanford.edu/mcap REVEL 0.682 Pathogenic =0.5 Pathogenic [34-35] MutPred 0.753 Pathogenic =0.5 Pathogenic [35] PrimateAI 0.470505654812 Tolerated 0.803 Damaging [32]; dbNSFP v4 DEOGEN2 0.612807 Damaging 0.5 Damaging [32-33]; dbNSFP v4 BayesDel_addAF 0.326526 Damaging 0.0692655 Damaging [32]; dbNSFP v4 BayesDel_noAF 0.231256 Damaging -0.0570105 Damaging [32]; dbNSFP v4 ClinPred 0.998569965362549 Damaging 0.5 Damaging [32]; dbNSFP v4 LIST-S2 0.866213 Damaging 0.85 Damaging [32]; dbNSFP v4 VARITY_R 0.9111106 Damaging 0.5 Damaging [30] VARITY_ER 0.895924 Damaging 0.5 Damaging [30] EVE _Class90 0.8051757528573578 Pathogenic =0.6 Pathogenic, >0.4 - <0.6 Uncertain [28-32]; dbNSFP v4 AlphaMissense 0.8056 Likely Pathogenic 0.564 Likely Pathogenic [26] CADD 29.7 Damaging 24 Damaging [31] DANN 0.99813861375246238 Damaging =0.99 Damaging [31] Fathmm-MKL_coding 0.93153 Damaging 0.5 Damaging [31] Fathmm-XF_coding 0.715970 Pathogenic 0.5 Damaging [32]; dbNSFP v4 Table 2: Information on the Single Nucleotide Variants identified in the thyroglobulin gene and genetic diagnosis of the patients. Patient Single Nucleotide Variant Segregation ACMG/AMP Criteria Classification* Conclusion SRA:II-1 NM_003235.5:c.5509_5518del ( p.(Lys1837CysfsTer12)) Father PM2_supp, PVS1, PP4 Pathogenic Possibly Solved NM_003235.5:c.7880A>G (p.(Asp2627Gly)) Mother PM2_supp, PP3, PM3, PP4 Hot VUS BAS:II-1 NM_003235.5:c.378C>A (p.(Tyr126Ter)) Father PM2_supp, PVS1, PM3_strong, PP4 Pathogenic Solved NM_003235.5:c.1375C>T (p.(Gln459Ter) Mother PM2_supp, PVS1, PM3, PP4 Pathogenic *A Bayesian scoring algorithm was used. Scores were binned into the following categories: 0= Benign; 0.001-0.051= Likely Benign; 0.100-0.188= VUS (variant of uncertain significance) leaning to Benign; 0.325-0.500= VUS; 0.675-0.812= VUS leaning to Pathogenic (Hot VUS); 0.900-0.988= Likely Pathogenic; 0.994-0.999= Pathogenic. Additional Declarations The authors declare no competing interests. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9117442","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":605839671,"identity":"fe9fd741-bf67-4ab5-ab8c-382faab3ddb3","order_by":0,"name":"Héctor M. 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Buenos Aires, Argentina.","correspondingAuthor":true,"prefix":"","firstName":"Héctor","middleName":"M.","lastName":"Targovnik","suffix":""},{"id":605840268,"identity":"ed020216-e653-4373-ad60-57160c928955","order_by":1,"name":"Valentina Ricci","email":"","orcid":"","institution":"Centro de Investigaciones Endocrinológicas “Dr. César Bergadá” (CEDIE) CONICET – FEI – División de Endocrinología, Hospital de Niños Ricardo Gutiérrez, Buenos Aires.","correspondingAuthor":false,"prefix":"","firstName":"Valentina","middleName":"","lastName":"Ricci","suffix":""},{"id":605840269,"identity":"69670b99-1ede-4356-a154-8ece2cc4910d","order_by":2,"name":"Valeria F. Garzón","email":"","orcid":"","institution":"Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología, Biotecnología y Genética/Cátedra de Genética. 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Departamento de Microbiología, Inmunología, Biotecnología y Genética/Cátedra de Genética. Buenos Aires, Argentina.","correspondingAuthor":false,"prefix":"","firstName":"Carina","middleName":"M.","lastName":"Rivolta","suffix":""},{"id":605843296,"identity":"c2c6cafb-341e-4689-a9c9-1e34df2c0528","order_by":11,"name":"Mariana L. Tellechea","email":"","orcid":"","institution":"Centro de Investigaciones Endocrinológicas “Dr. César Bergadá” (CEDIE) CONICET – FEI – División de Endocrinología, Hospital de Niños Ricardo Gutiérrez, Buenos Aires.","correspondingAuthor":false,"prefix":"","firstName":"Mariana","middleName":"L.","lastName":"Tellechea","suffix":""}],"badges":[],"createdAt":"2026-03-13 18:37:49","currentVersionCode":1,"declarations":{"humanSubjects":true,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":true,"humanSubjectConsent":true,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-9117442/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9117442/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104876973,"identity":"e825d992-a115-4b53-bca6-3e9a930d22ea","added_by":"auto","created_at":"2026-03-18 08:44:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":386545,"visible":true,"origin":"","legend":"\u003cp\u003eFamily SRA, pedigree and genotyping results. Partial sequencing chromatograms of genomic DNA are shown (Sense strand). Squares represent males and circles females. Filled symbols denote affected individuals and half-filled symbols, unaffected heterozygote individuals carrying the variant and not diseased. Single peaks in the chromatogram denote homozygosity while two overlapping peaks at the same locus indicate heterozygosity. The arrows indicate the position of the identified variants. Solid symbol represent allele with variant NM_003235.5:c.5509-5518del (NP_003226.4:p.(Lys1837CysfsTer12)) and hatched symbol allele with variant NM_003235.5:c.7880A\u0026gt;G (NP_003226.4:p.(Asp2627Gly)). WT: wild-type.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-9117442/v1/d5c04ef355e393497b1fa562.png"},{"id":104876987,"identity":"596c527e-540c-47ff-8472-aab34911abc5","added_by":"auto","created_at":"2026-03-18 08:44:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":337558,"visible":true,"origin":"","legend":"\u003cp\u003eFamily BSA, pedigree and genotyping results. Partial sequencing chromatograms of genomic DNA are shown (Sense strand). Squares represent males and circles females. Filled symbols denote affected individuals and half-filled symbols, unaffected heterozygote individuals carrying the variant and not diseased. Single peaks in the chromatogram denote homozygosity while two overlapping peaks at the same locus indicate heterozygosity. The arrows indicate the position of the identified variants. Solid symbol represent allele with variant NM_003235.5:c.378C\u0026gt;A (NP_003226.4:p.(Tyr126Ter)) and hatched symbol allele with variant NM_003235.5: c.1375C\u0026gt;T (NP_003226.4:p.(Gln459Ter)). WT: wild-type.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-9117442/v1/24b333a64ffe78c86a92514a.png"},{"id":104876964,"identity":"b13dfc46-d567-454d-b2b4-f4c407721380","added_by":"auto","created_at":"2026-03-18 08:44:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1211437,"visible":true,"origin":"","legend":"\u003cp\u003eEvolutionary conservation of the NP_003226.4:p.(Asp2627Gly) variant in thyroglobulin. a Comparative alignment of partial ChEL domain sequences from 37 vertebrate species, generated using the Clustal Omega program. Amino acids are shown using single-letter codes. Conserved cysteine residues are highlighted in yellow, and the conserved aspartic acid2627 residue is boxed.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-9117442/v1/cb8697330c3953a297bfb4d5.png"},{"id":104876962,"identity":"82d21c70-dd5f-4d33-8b0b-a5bdd072e94f","added_by":"auto","created_at":"2026-03-18 08:44:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2228423,"visible":true,"origin":"","legend":"\u003cp\u003eThree-dimensional structure of the thyroglobulin NP_003226.4:p.(Asp2627Gly) variant. The “wild-type” thyroglobulin monomer is displayed at the center (surface view) according to the classical model, divided into four regions: Region I (yellow), Region II (violet), Region III (green), and Region IV (red), which contains the ChEL domain. A) Tertiary structure analysis: The black arrow highlights the wild-type aspartic acid2627 residue, while the blue dotted lines indicate the 22 hydrogen bonds identified. Hydrogen bonds #8 and #9 (underlined) are directly associated with the wild-type aspartic acid2627 residue, interacting with tyrosine2611 and arginine2386. B) Mutant conformation: The open structure shows the substitution of aspartic acid2627for glycine, resulting in the loss of hydrogen bonds #8 and #9. No steric clashes were detected in the mutated model. No conformational or hydrophobicity profile changes were observed. C) The thyroglobulin monomer, with the dark pink region indicating the location of aspartic acid2627 within the central region of the ChEL domain.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-9117442/v1/2491eb1646a6cc75ce978894.png"},{"id":104876892,"identity":"52be5a95-e273-44ed-b545-61e0dc976e08","added_by":"auto","created_at":"2026-03-18 08:44:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":166980,"visible":true,"origin":"","legend":"\u003cp\u003eStructural organization of wild-type thyroglobulin and variants NP_003226.4:p.(Tyr126Ter), NP_003226.4:p.(Gln459Ter), NP_003226.4:p.(Lys1837CysfsTer12), and NP_003226.4:p.(Asp2627Gly). Classical model of the primary structure adapted from Malthiéry \u0026amp; Lissitzky [48], Mercken et al. [50], Parma et al. [51], van de Graaf et al. [52], Holzer et al. [53], Swillens et al. [54] and Molina et al. [55]. Thyroglobulin signal peptide (SP), TG type 1, TG type 2, and TG type 3 repeating units, linker and hinge modules, spacers 1, 2 and 3, and acetylcholinesterase (ChEL) homology domain are drawn to scale and represented by boxes. The thyroglobulin monomer is organized into four framework regions (I, II, III, and IV). Shown are the sites of formation of N-terminal T4 (coupling of a DIT149 donor with the DIT24 acceptor) and C-terminal T3 (coupling of an MIT2766 on the antepenultimate residue of a TG monomer with the antepenultimate DIT2766 on the opposite monomer). Amino acids are numbered, including the 19 amino acids of the signal peptide, following NCBI numbering: NP_003226.4 and UniProt P01266.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-9117442/v1/8dc5ace6196f0461740ced00.png"},{"id":105033750,"identity":"f3215d0d-199d-416c-91c2-140b2ababfef","added_by":"auto","created_at":"2026-03-20 07:21:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5837220,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9117442/v1/8ca52a5d-8bfa-4a93-9d0e-593301b0d4b8.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eClinical, Biochemical, and Molecular Genetic Characterization of Two Patients with Congenital Hypothyroidism Harboring Novel Compound Heterozygous Variants in the T\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ehyroglobulin \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eGene\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCongenital hypothyroidism (CH) is the most frequent thyroid disorder in childhood, with an incidence of approximately 1 in 2,000\u0026ndash;4,000 live births and a female-to-male ratio of 2:1. It is typically characterized by elevated thyroid-stimulating hormone (TSH) levels and reduced circulating thyroid hormone (TH) concentrations [1-4]. Affected children may exhibit a wide spectrum of clinical manifestations. The advent of high-throughput sequencing technologies has enabled large-scale detection of genetic variants, leading to the discovery of novel inactivating variants in candidate genes associated with CH. Importantly, the pathogenesis of CH is not limited to monogenic inheritance; digenic and oligogenic mechanisms have also been implicated [4,5].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;CH can be classified into three major categories: environmental causes (such as iodine deficiency), thyroid dysgenesis, and thyroid dyshormonogenesis (TDH) [1,4,6-8]. Thyroid dysgenesis encompasses structural abnormalities of the gland, including complete absence (agenesis or athyreosis), reduced size (hypoplasia), or ectopic localization (thyroid ectopia) [1,4,6-8]. Genetic variants in several transcription factors and signaling genes\u0026mdash;such as \u003cem\u003eNKX2.1\u003c/em\u003e, \u003cem\u003eNKX2.5\u003c/em\u003e, \u003cem\u003eFOXE1\u003c/em\u003e, PAX8, TSHR, CDCA8, ELN, GLIS3, \u003cem\u003eHOXD3\u003c/em\u003e, \u003cem\u003eHOXB3\u003c/em\u003e, \u003cem\u003eJAG1\u003c/em\u003e, \u003cem\u003eKMT2D\u003c/em\u003e, \u003cem\u003eTBX1\u003c/em\u003e, \u003cem\u003eTUBB\u003c/em\u003e1, and \u003cem\u003eURBI\u003c/em\u003e\u0026mdash;have been reported in patients with thyroid dysgenesis phenotypes [1,4,9,10]. In contrast, TDH represents a group of genetic defects that impair TH synthesis due to pathogenic variants in genes encoding proteins essential for multiple steps of TH biosynthesis. These include \u003cem\u003eSolute Carrier Family 5 Member 5\u003c/em\u003e (\u003cem\u003eSLC5A5\u003c/em\u003e, encoding the sodium/iodide symporter, NIS), \u003cem\u003eSolute Carrier Family 26 Member 4\u003c/em\u003e (\u003cem\u003eSLC26A4\u003c/em\u003e, encoding pendrin), \u003cem\u003eSolute Carrier Family 26 Member 7\u003c/em\u003e (\u003cem\u003eSLC26A7\u003c/em\u003e), \u003cem\u003eThyroid Peroxidase\u0026nbsp;\u003c/em\u003e(\u003cem\u003eTPO\u003c/em\u003e), \u003cem\u003eDual Oxidase 1\u003c/em\u003e (\u003cem\u003eDUOX1\u003c/em\u003e), \u003cem\u003eDUOX Maturation Factor 1\u003c/em\u003e (\u003cem\u003eDUOXA1\u003c/em\u003e), \u003cem\u003eDual Oxidase\u003c/em\u003e 2 \u003cem\u003e(DUOX2\u003c/em\u003e), \u003cem\u003eDUOX Maturation Factor 2\u003c/em\u003e (\u003cem\u003eDUOXA2\u003c/em\u003e), \u003cem\u003eIodotyrosine Deiodinase\u0026nbsp;\u003c/em\u003e(\u003cem\u003eIYD\u003c/em\u003e), and \u003cem\u003eThyroglobulin\u0026nbsp;\u003c/em\u003e(\u003cem\u003eTG\u003c/em\u003e) [1,4,6-8]. Moreover, pathogenic variants in \u003cstrong\u003e\u003cem\u003eSLC26A4\u003c/em\u003e, \u003cem\u003eSLC5A5\u003c/em\u003e, \u003cem\u003eTPO\u003c/em\u003e, \u003cem\u003eDUOX1\u003c/em\u003e, \u003cem\u003eDUOX2\u003c/em\u003e,\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;\u003cstrong\u003e\u003cem\u003eTG\u003c/em\u003e\u003c/strong\u003e\u003c/strong\u003e have also been implicated in thyroid dysgenesis [9,11-15].\u003c/p\u003e\n\u003cp\u003eTDH caused by \u003cstrong\u003e\u003cem\u003eTG\u0026nbsp;\u003c/em\u003egene variants (TDH3)\u003c/strong\u003e is most often inherited in an autosomal recessive pattern, with an estimated incidence ranging from 1 in 67,000 to 1 in 100,000 live births [16,17]. The clinical spectrum is broad, spanning from euthyroid states to mild or severe permanent hypothyroidism. To date, more than 300 pathogenic \u003cem\u003eTG\u003c/em\u003e variants have been identified, encompassing missense mutations that affect conserved cysteine residues and the ChEL domain, as well as duplications, deletions, insertions, partial inversions, splice-site defects, and nonsense variants [5,18-20].\u003c/p\u003e\n\u003cp\u003eIn this study, we report the clinical, biochemical, and molecular features of two patients from unrelated families diagnosed with CH. Molecular analysis identified two TG variants not previously published\u0026mdash;NM_003235.5:c.1375C\u0026gt;T (NP_003226.4:p.(Gln459Ter)) and NM_003235.5:c.5509-5518delAAAGACACAG (NP_003226.4:p.(Lys1837CysfsTer12)) and\u0026mdash;together with two published variants, NM_003235.5:c.378C\u0026gt;A (NP_003226.4:p.(Tyr126Ter)) and NM_003235.5:c.7880A\u0026gt;G (NP_003226.4:p.(Asp2627Gly)). The identification and comprehensive molecular characterization of TG variants in patients with TDH3 provide valuable insights that enhance diagnostic accuracy and genetic counseling.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eSubjects\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwo patients with suspected CH diagnosis were referred for clinical and biochemical evaluation and confirmation of diagnosis to the Endocrinology Division of the Hospital de Ni\u0026ntilde;os \u0026ldquo;Ricardo Guti\u0026eacute;rrez\u0026rdquo; (HNRG).\u003c/p\u003e\n\u003cp\u003eSerum TSH, total T\u003csub\u003e4\u003c/sub\u003e (T\u003csub\u003e4\u003c/sub\u003e), total T\u003csub\u003e3\u003c/sub\u003e (T\u003csub\u003e3\u003c/sub\u003e), and free T\u003csub\u003e4\u003c/sub\u003e (FT\u003csub\u003e4\u003c/sub\u003e) levels were measured using electrochemiluminescence immunoassay (Elecsys 2010, Roche Diagnostics, Indianapolis, IN, USA). Anti-thyroid peroxidase (TPO) antibodies, anti-thyroglobulin (TG) antibodies, and serum TG levels were assessed by chemiluminescence immunoassay (Immulite 2000, Siemens Healthcare Diagnostics, New York, NY, USA). Antibodies against the TSH receptor (TRAb) were determined using electrochemiluminescence immunoassay (Elecsys Anti-TSHR, Roche Diagnostics).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA isolation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe genomic DNA was extracted from the peripheral venous blood cells as previously described [21]. The DNA was quantified using a high-performance microvolume spectrophotometer NanoPhotometer\u0026reg; NP60 (Implen Inc.,\u0026nbsp;M\u0026uuml;nchen, Germany). DNA purity was assessed by measuring the absorbance ratio 260/280 nm; further DNA sample processing was performed only if the ratio was between 1.8 and 2.1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCustom-panel\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003esequencing\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the index patient II-2 of Family SRA, DNA library preparation and hybridization were performed using a Twist Custom Panel (Twist Bioscience, South San Francisco, CA, USA). The quality of genomic DNA fragmentation was verified using an Agilent capillary system Fragment Analyzer\u0026trade; (Agilent Technologies, Santa Clara, CA, USA). Next-generation Sequencing (NGS) by synthesis with fluorescent reversible terminator deoxyribonucleotides was performed using a NextSeq 500 system (Illumina, San Diego, CA, USA) at the Translational Medicine Unit of the HNRG.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eWhole-exome sequencing\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhole-exome sequencing (WES) was performed for the index patient II-2 of Family BAS at Macrogen Inc. (Seoul, Republic of Korea). Genomic DNA extracted from Family BAS, index patient II-2 was physically fragmented to a target peak size of 150\u0026ndash;200 bp with the Covaris LE220 focused-ultrasonicator (Covaris, Woburn, MA, USA), and the exome capture library was enriched using an Agilent Sure select XT V6 kit (Agilent) for the entire coding sequence and intron\u0026ndash;exon boundaries of 20,000 human genes. The postcapture library was sequenced using the NovaSeq 6000 System\u0026nbsp;(Illumina) producing 150 bp paired-end reads.\u0026nbsp;The total number of bases sequenced was\u0026nbsp;6,760,596,672\u0026nbsp;bp, and the total number of reads was\u0026nbsp;45,265,924.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eGenome analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFASTQ files were processed using a Genome Analysis Toolkit (GATK, https://www.broadinstitute.org/gatk/) v4.0.5.1-based pipeline. Sequence data were aligned with GRCh38/hg38 human reference genome using the BWA-MEM algorithm of Burrows\u0026ndash;Wheeler Aligner software to perform variant calls and annotations. Duplicates were removed using Picard (Broad Institute). Data was analyzed for single-nucleotide variants (SNVs) and insertions/deletions (indels). The target region included the coding exons, consensus splice sites (\u0026plusmn; 2 bases from exon boundaries), and the extended splice region (\u0026plusmn;3 to \u0026plusmn;10 bases). Coverage depth and read quality were evaluated with the Integrative Genomics Viewer (https://www.broadinstitute.org/scientific-community/software/integrative-genomics-viewer) v2.16.0.\u0026nbsp;TheAmerican College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) guidelines [22], and the ClinGen Sequence Variant Interpretation Working Group recommendations were followed to determine the pathogenicity of a variant.\u0026nbsp;The description of the variants\u0026apos; position in the DNA (genomic and coding) and in the protein is according to the\u0026nbsp;HGVS\u0026nbsp;nomenclature, based\u0026nbsp;on the genome build GRCh38/hg38.(https://hgvs-nomenclature.org/stable/).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSanger sequencing\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePutative disease-causing variants were confirmed in patients and parents using Sanger sequencing. Target exons were amplified by polymerase chain reaction (PCR) with specific primers,\u0026nbsp;reported previously [23]\u0026nbsp;and sequenced with the same sense and antisense specific primers or M13 universal primers with the Big Dye deoxyterminator Cycle Sequencing Kit (Applied Biosystems, Weiterstadt, Germany). The samples were analyzed on the 3500XL Genetic Analyzer (Applied Biosystems)\u0026nbsp;Genomics and Bioinformatics Unit of the Instituto Nacional de Tecnolog\u0026iacute;a Agropecuaria (INTA) or\u0026nbsp;at the Translational Medicine Unit of the HNRG. The sequences were compared to the reference sequence and analyzed using ChromasPro (Technelysium Pty Ltd.,\u0026nbsp;Queensland, Australia).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDetection of exon copy number variants\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCopy Number Variants (CNVs) were predicted using the coverage based DECoN (Detection of Exon Copy Number) algorithm (https://github.com/RahmanTeam/DECoN) [24,25].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eProtein homology analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmino acid sequence homology within the ChEL domain was evaluated through multiple sequence alignment using CLUSTAL W (version 1.83) (http://www.ch.embnet.org/software/ClustalW.html). Protein sequences were retrieved from the NCBI database (https://www.ncbi.nlm.nih.gov) for the following species: \u003cem\u003eAlligator mississippiensis\u0026nbsp;\u003c/em\u003e(XP_059579449.1),\u003cem\u003e\u0026nbsp;Aquarana catesbeiana\u003c/em\u003e (XP_073488945.1), \u003cem\u003eAstyanax mexicanus\u003c/em\u003e (XP_022529282.2), \u003cem\u003eBos Taurus\u003c/em\u003e (NP_776308.1), \u003cem\u003eCanis lupus familiaris\u003c/em\u003e (NP_001041569.1),\u003cem\u003eCarassius\u0026nbsp;auratus\u003c/em\u003e (XP_026120244.1),\u003cem\u003eCavia porcellus\u003c/em\u003e (XP_003467392.1),\u0026nbsp;\u003cem\u003eChelonia mydas\u003c/em\u003e (XP_043394997.1),\u003cem\u003eClupea harengus\u003c/em\u003e (XP_031429786.1),\u0026nbsp;\u003cem\u003eColumba livia\u003c/em\u003e (XP_021154537.2),\u0026nbsp;\u003cem\u003eCrotalus tigris\u003c/em\u003e (XP_039206379.1),\u0026nbsp;\u003cem\u003eCynoglossus semilaevis\u003c/em\u003e (XP_008321228.1),\u0026nbsp;\u003cem\u003eCyprinus carpio\u003c/em\u003e (XP_042628676.1),\u0026nbsp;\u003cem\u003eDanio rerio\u003c/em\u003e (NP_001316794.1),\u0026nbsp;\u003cem\u003eEublepharis macularius\u003c/em\u003e\u003cem\u003e\u0026nbsp;(\u003c/em\u003eXP_054841882.1),\u0026nbsp;\u003cem\u003eGallus gallus\u003c/em\u003e (NP_001376406.2),\u0026nbsp;\u003cem\u003eHomo sapiens\u003c/em\u003e (NP_003226.4),\u0026nbsp;\u003cem\u003eLampetra fluviatilis\u003c/em\u003e (CAL5909345.1),\u0026nbsp;\u003cem\u003eLampetra planeri\u0026nbsp;\u003c/em\u003e(CAL5920418.1),\u0026nbsp;\u003cem\u003eLarus michahellis\u003c/em\u003e (XP_074433977.1),\u0026nbsp;\u003cem\u003eLepisosteus oculatus\u003c/em\u003e (XP_015212882.2),\u0026nbsp;\u003cem\u003eMacaca fascicularis\u003c/em\u003e (XP_045254657.2),\u0026nbsp;\u003cem\u003eMacaca mulatta\u003c/em\u003e (XP_028708128.1),\u0026nbsp;\u003cem\u003eMus musculus\u003c/em\u003e (NP_033401.2),\u0026nbsp;\u003cem\u003eOryzias latipes\u003c/em\u003e (XP_011484169.1),\u0026nbsp;\u003cem\u003ePan troglodytes\u003c/em\u003e (XP_016815373.4),\u0026nbsp;\u003cem\u003ePanthera leo\u003c/em\u003e (XP_042780307.1),\u0026nbsp;\u003cem\u003ePongo pygmaeus\u003c/em\u003e (XP_054355108.2),\u0026nbsp;\u003cem\u003ePython bivittatus\u003c/em\u003e (XP_025021113.1),\u003cem\u003e\u0026nbsp;Rattus norvegicus\u003c/em\u003e (NP_112250.2),\u0026nbsp;\u003cem\u003eStegastes partitus\u003c/em\u003e (XP_008304814.1),\u0026nbsp;\u003cem\u003eStruthio camelus\u003c/em\u003e (XP_068789869.1),\u0026nbsp;\u003cem\u003eTaeniopygia guttata\u003c/em\u003e (XP_072781126.1),\u0026nbsp;\u003cem\u003eTrichechus manatus latirostris\u003c/em\u003e (XP_004373071.1),\u0026nbsp;\u003cem\u003eXenopus tropicalis\u003c/em\u003e (NP_001316486.1) and\u0026nbsp;\u003cem\u003eXiphophorus maculatus\u003c/em\u003e (XP_023185937.1).\u0026nbsp;The TG sequence for \u003cem\u003eGorilla gorilla\u003c/em\u003e was obtained from UniProt [https://www.uniprot.org; accession G3QS68].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAmino acid prediction analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSNVs were analyzed with the sequence-based predictors included in the dbNSFP v4 (https://www.dbnsfp.org/): \u0026nbsp;SIFT, SIFT4G, Polyphen2-HDIV, Polyphen2-HVAR, LRT, MutationTaster, MutationAssessor, FATHMM, PROVEAN, VEST4, MetaSVM, MetaLR, MetaRNN, M-CAP, REVEL, MutPred2, PrimateAI, DEOGEN2, BayesDel_addAF, BayesDel_noAF, ClinPred, LIST-S2 VARITY_R, VARITY_ER, EVE, AlphaMissense, CADD, DANN, FATHMM-MKL, FATHMM-XF [26-35].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3D modeling analysis\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe UCSF Chimera program (UCSF Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, https://www.cgl.ucsf.edu/chimera/) [36-37] was used to obtain the 3D model of the human TG (PDB 6SCJ, resolution: 3.60 \u0026Aring;) [38]\u003cstrong\u003e\u003cem\u003e.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eClinical and biochemical description\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFamily SRA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe index patient, SRA:II-2, was born in 2010 at 37 weeks of gestation (birth weight: 2910 g) by cesarean section to an unrelated couple.\u0026nbsp;Her neonatal course was complicated by a four-day hospitalization in the Neonatal Intensive Care Unit for suspected sepsis. Neonatal screening on postnatal day 3 identified primary hypothyroidism with a TSH of 65.05 µUI/ml (cut off: 10 µUI/ml). The patient was referred to the Endocrinology Department of the HNRG at 15 days of life. Initial physical examination revealed an infant who was clinically euthyroid but presented with a palpable, enlarged thyroid gland. Confirmatory serum biochemistry at day 15 showed TSH: 62.47 µUI/ml (reference range between 0 and 60 days: 1.3–10 µUI/ml), T\u003csub\u003e4\u003c/sub\u003e: 8.3 µg/dl (reference range between 0 and 30 days: 6-8 µg/dl), FT\u003csub\u003e4\u003c/sub\u003e: 1.14 ng/dl (reference range between 0 and 30 days: 1.0-2.6 ng/dl) and T\u003csub\u003e3\u003c/sub\u003e: 202 ng/dl (reference range between 0 and 30 days: 80-260 ng/dl). Serum TG was 61.7 ng/ml (reference range between 0-2 months: 10-100 ng/ml). To rule out immune-mediated or transplacental etiology, thyroid autoantibodies (anti-TPO/anti-TG) and TRABs were measured and found to be negative in both the infant and the mother, who was herself healthy with normal thyroid function. Replacement therapy with L-T\u003csub\u003e4\u003c/sub\u003e was initiated at 50 µg/day. Imaging studies included a \u003csup\u003e99m\u003c/sup\u003eTc thyroid scintigraphy, which demonstrated an eutopic, enlarged gland consistent with diffuse goiter. A knee radiograph confirmed the presence of distal femoral and proximal tibial epiphyses. After 20 days of treatment, biochemical control showed TSH: 19.11 µUI/ml and FT4: 1.36 ng/dl. The patient maintained a steady growth and developmental trajectory through childhood with regular L-T\u003csub\u003e4\u003c/sub\u003e adjustments. At age 5, the L-T\u003csub\u003e4\u003c/sub\u003e withdrawal trial confirmed permanent CH. Laboratory results were as follows, TSH: \u0026gt;100 µUI/ml (reference range: 0.5-6.5 µUI/ml), T\u003csub\u003e4\u003c/sub\u003e of 3.7 µg/dl (reference range: \u0026nbsp;6-14 µg/dl), FT\u003csub\u003e4\u003c/sub\u003e of 0.41 ng/dl (reference range: 0.8-2.2 ng/dl) and T\u003csub\u003e3\u003c/sub\u003e of 119 ng/dl (reference range: 80-220 ng/dl), while TG remained at 13.9 ng/ml (reference range: \u0026nbsp;6-40 ng/ml). The perchlorate discharge test was negative. The thyroid ultrasound showed a regular, eutopic gland with a homogeneous structure and normal vasculature, right lobe size: 2.36 x 0.86 x 1.05 cm, left lobe size: 1.94 x 0.88 x 1.02 cm, and total volume: 2.01 cm\u003csup\u003e3\u003c/sup\u003e (mean to 5 years: 4.10 cm\u003csup\u003e3\u003c/sup\u003e)[39]. Thyroid volume was calculated by multiplying of length, breadth and depth by a corrective factor (0.52) for each lobe [40].\u003c/p\u003e\n\u003cp\u003eAudiometry and ophthalmological evaluation were normal. Cognitive assessment performed at age 6 years showed scores within the average range, though processing speed was just below the average range. Pubertal progression was normal, with menarche occurring at age 12. As the patient progressed into adolescence, the thyroid gland remained consistently palpable. The most recent thyroid ultrasound at age 13 demonstrated a diffusely heterogeneous echo-structure and lobulated contours, but without focal nodules or cystic degeneration, right lobe size: 1.2 x 1.4 x 3.4 cm, left lobe size: 1.1 x 1.1 x 2.9 cm, and total volume: 4.79 cm\u003csup\u003e3\u003c/sup\u003e (mean to 13 years: 9.66 cm\u003csup\u003e3\u003c/sup\u003e) [39]. At age 14, the patient’s thyroid function remained well-controlled (TSH: 3.51 µUI/ml, FT4: 1.35 ng/dl) with a final adult height of 163 cm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFamily BAS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIndex patient BAS:II-2, born in 2018, is the first and only child of nonconsanguineous healthy parents. Born from an uneventful pregnancy and delivery, was identified as CH in the neonatal screening with TSH \u0026gt;100 µUI/ml (cut off: 10 µUI/ml). Confirmation serum studies showed TSH: \u0026gt;100 µUI/ml, T\u003csub\u003e4\u003c/sub\u003e: 4 ug/dl, FT\u003csub\u003e4\u003c/sub\u003e: 0.48 ng/dl, T\u003csub\u003e3\u003c/sub\u003e: 124 ng/dl, anti-TPO, anti-TG negatives and TG: \u0026lt; 0.9 ng/ml at 10 days of life. He presented jaundice, puffy face, and palpable goiter, as evidenced by a \u003csup\u003e99m\u003c/sup\u003eTc thyroid scan. He started L-T\u003csub\u003e4\u003c/sub\u003e treatment with good adherence and began follow-up.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHe grew and developed normally (90\u003csup\u003eth\u003c/sup\u003e percentile of height and weight) till age 3, when treatment was withdrawn for a month, and reevaluation of thyroid function confirmed hypothyroidism with TSH: \u0026gt;95 µUI/ml, FT\u003csub\u003e4\u003c/sub\u003e: 0.40 ng/dl and TG: \u0026lt;0.9 ng/ml. Thyroid ultrasound showed an eutopic enlarged thyroid gland without nodules or cysts, right lobe size: 3.4 x 1.0 x 1.3 cm, left lobe size: 3.3 x 1.1 x 1.4 cm, and total volume: 4.94 cm\u003csup\u003e3\u003c/sup\u003e (mean to 3 years: 2.9 cm\u003csup\u003e3\u003c/sup\u003e) [39]. He restarted treatment. At age 4, he grows normally. His neurocognitive outcome is normal.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMolecular Genetic Analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFamily SRA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA compound of heterozygous for NM_003235.5:c.5509_5518delAAAGACACAG\u0026nbsp;(NC_000008.11:g.132963035_132963044delAAAGACACAG)\u0026nbsp;\u0026nbsp;and NM_003235.5:c.7880A\u0026gt;G (NC_000008.11:g.133131829A\u0026gt;G) in the \u003cem\u003eTG\u003c/em\u003e gene was identified by custom-panel\u0026nbsp;sequencing\u0026nbsp;in index patient SRA:II-1.\u0026nbsp;The c.5509_5518del variant in exon 29 causes a frameshift starting with codon lysine\u003csup\u003e1837\u003c/sup\u003e, changes this amino acid to a cysteine residue, and creates a premature stop codon at position 12 of the new reading frame (NP_003226.4:p.(Lys1837CysfsTer12)), while the NM_003235.5:c.7880A\u0026gt;G variant\u0026nbsp;in exon 46 produces the substitution of a aspartic acid for\u0026nbsp;glycine\u0026nbsp;at codon 2627 (NP_003226.4:p.(Asp2627Gly)) (shown in Fig. 1).\u0026nbsp;Direct sequencing of exon 29 and 46 of both parents' genomic DNA indicated that the patient inherited\u0026nbsp;NM_003235.5:c.5509_5518del\u0026nbsp;variant from the healthy father and\u0026nbsp;NM_003235.5:c.7880A\u0026gt;G\u0026nbsp;variant from the healthy mother (shown in Fig. 1). The NM_003235.5:c.5509_5518del variant is reported in the gnomAD v4.1 database with a frequency of 0.0001% (1/1614046, European non-Finnish), the NM_003235.5:c.7880A\u0026gt;G variant is also indexed with a frequency of 0.0001% (1/1614092, European non-Finnish). The NM_003235.5:c.5509_5518del variant has not previously been reported in association with CH, whereas the NM_003235.5:c.7880A\u0026gt;G variant was recently described by us in association with hyperthyrotropinemia [41].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConsidering the structural relevance of analyzing polymorphic coding variants across TG to confirm heterozygosity and to facilitate future comparative allele analyses with newly reported cases of similar genotype, eight heterozygous TG variants were identified in the index patient SAR:II-1. These included five non-synonymous substitutions—\u0026nbsp;(NM_003235.5:c.2200T\u0026gt;G (NP_003226.4:p.(Ser734Ala)), NM_003235.5:c.3082A\u0026gt;G (NP_003226.4:p.(Met1028Val)), NM_003235.5:c.5921T\u0026gt;C (NP_003226.4:p.(Met1974Thr)) NM_003235.5:c.7501T\u0026gt;C (NP_003226.4:p.(rp2501Arg)), and \u0026nbsp;NM_003235.5:c.7589G\u0026gt;A (NP_003226.4:p.(Arg2530Gln))— as well as\u0026nbsp;three synonymous SNVs:\u0026nbsp;(NM_003235.5:c.2334T\u0026gt;C (NP_003226.4(p.Pro778=)), NM_003235.5:c.7408C\u0026gt;T (NP_003226.4:p.(Leu2470=)) and NM_003235.5:c.7920C\u0026gt;T [NP_003226.4:p.(Tyr2640=)).\u0026nbsp;No CNVs were detected in the coding sequence of the \u003cem\u003eTG\u003c/em\u003e gene, indicating the absence of exon-level deletions or duplications.\u003c/p\u003e\n\u003cp\u003eOther candidate genes implicated in the development of congenital hypothyroidism—\u0026nbsp;\u003cem\u003eDUOX1\u003c/em\u003e, \u003cem\u003eDUOXA1\u003c/em\u003e, \u003cem\u003eDUOX2\u003c/em\u003e, \u003cem\u003eDUOXA2\u003c/em\u003e, \u003cem\u003eFOXE1\u003c/em\u003e, \u003cem\u003eGLIS3\u003c/em\u003e, \u003cem\u003eGNAS\u003c/em\u003e, \u003cem\u003eIYD\u003c/em\u003e, \u003cem\u003eNKX2-1\u003c/em\u003e, \u003cem\u003eNKX2-5\u003c/em\u003e, \u003cem\u003ePAX8\u003c/em\u003e, \u003cem\u003eSLC26A7\u003c/em\u003e, \u003cem\u003eSLC5A5\u003c/em\u003e, \u003cem\u003eTBX1\u003c/em\u003e, \u003cem\u003eTPO\u003c/em\u003e, \u003cem\u003eTSHR\u003c/em\u003e, \u003cem\u003eJAG1\u003c/em\u003e, \u003cem\u003eTUBB1\u003c/em\u003e, \u003cem\u003eCDCA8\u003c/em\u003e, \u003cem\u003eTHRB\u003c/em\u003e, \u003cem\u003eTBL1X\u003c/em\u003e, \u003cem\u003eIGSF1\u003c/em\u003e, \u003cem\u003eTSHB\u003c/em\u003e, \u003cem\u003eFGFR1\u003c/em\u003e, \u003cem\u003eFOXA2\u003c/em\u003e, \u003cem\u003eGLI2\u003c/em\u003e, \u003cem\u003eIRS4\u003c/em\u003e, \u003cem\u003ePROP1\u003c/em\u003e, \u003cem\u003eSECISBP2\u003c/em\u003e, \u003cem\u003eSLC16A2\u003c/em\u003e, \u003cem\u003eSOX3\u003c/em\u003e, \u003cem\u003ePOU1F1\u003c/em\u003e, \u003cem\u003eLHX3\u003c/em\u003e, \u003cem\u003eLHX4\u003c/em\u003e, \u003cem\u003eOTX2\u003c/em\u003e—were also examined. No pathogenic variants were detected in any of these genes.\u0026nbsp;All exons were covered at 90-100% (with a depth of at least 20x).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFamily BAS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA compound of heterozygous for NM_003235.5:c.378C\u0026gt;A (NC_000008.11:g.132871451C\u0026gt;A) \u0026nbsp;and NM_003235.5:c.1375C\u0026gt;T (NC_000008.11:g.132886747C\u0026gt;T) in the \u003cem\u003eTG\u003c/em\u003e gene was identified by\u0026nbsp;WES\u0026nbsp;in index patient BAS:II-1.\u0026nbsp;The\u0026nbsp;previously documented\u0026nbsp;variant\u0026nbsp;NM_003235.5:c.378C\u0026gt;A in exon 4\u0026nbsp;replaces a tyrosine residue at position 126 with a premature stop codon (NP_003226.4:p.(Y126Ter)) [42,43], while the novel variant NM_003235.5:c.1375C\u0026gt;T in exon 9 replaces a glutamine residue at position 459 with a premature stop codon (NP_003226.4:p.(Gln459Ter)) (shown in Fig. 2).\u0026nbsp;Direct sequencing of exons 4 and 9 of both parents' genomic DNA indicated that the patient inherited NM_003235.5:c.378C\u0026gt;A variant from the healthy father and NM_003235.5:c.1375C\u0026gt;T variant from the healthy mother (shown in Fig. 2). Neither variant was indexed in the gnomAD v4.1database.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSeven coding polymorphic TG variants were identified in the index patient BAS:II-1. These including four non-synonymoussubstitutions—(NM_003235.5:c.3935A\u0026gt;G (NP_003226.4:p.(Asp1312Gly)), NM_003235.5:c.5512G\u0026gt;A (NP_003226.4:p.(Asp1838Asn)), NM_003235.5:c.7501T\u0026gt;C (NP_003226.4:p.(Trp2501Arg)) and NM_003235.5:c.7589G\u0026gt;A (NP_003226.4:p.(Arg2530Gln))— as well as three synonymous SNVs:\u0026nbsp;(NM_003235.5:c.4506T\u0026gt;C (NP_003226.4:p.(Ala1502=)), NM_003235.5:c.7408C\u0026gt;T (NP_003226.4:p.(Leu2470=)) and NM_003235.5:c.7920C\u0026gt;T (NP_003226.4:p.(Tyr2640=)).\u0026nbsp;All variants were in the heterozygous state, except for NM_003235.5:c.7501T\u0026gt;C, which was homozygous.\u0026nbsp;Furthermore, no CNVs were identified in the \u003cem\u003eTG\u003c/em\u003e gene and acceptor or donor splicing consensus sequences were rigorously respected in all introns of the \u003cem\u003eTG\u0026nbsp;\u003c/em\u003egene.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe other main genes involved in the development of congenital goiter and hypothyroidism were also analyzed:\u0026nbsp;\u003cem\u003eDUOX1\u003c/em\u003e,\u003cem\u003e\u0026nbsp;DUOXA1\u003c/em\u003e,\u003cem\u003e\u0026nbsp;DUOX2\u003c/em\u003e,\u003cem\u003e\u0026nbsp;DUOXA2\u003c/em\u003e,\u003cem\u003e\u0026nbsp;FOXE1\u003c/em\u003e,\u003cem\u003e\u0026nbsp;GLIS3\u003c/em\u003e,\u003cem\u003e\u0026nbsp;GNAS\u003c/em\u003e,\u003cem\u003e\u0026nbsp;IYD\u003c/em\u003e,\u003cem\u003e\u0026nbsp;NKX2-1\u003c/em\u003e,\u003cem\u003eNKX2-5\u003c/em\u003e,\u003cem\u003e\u0026nbsp;PAX8\u003c/em\u003e,\u003cem\u003e\u0026nbsp;SLC26A4\u003c/em\u003e,\u003cem\u003e\u0026nbsp;SLC26A7\u003c/em\u003e,\u003cem\u003eSLC5A5\u003c/em\u003e,\u003cem\u003e\u0026nbsp;TBX1\u003c/em\u003e,\u003cem\u003e\u0026nbsp; TPO\u003c/em\u003e,\u003cem\u003e\u0026nbsp;TSHR\u003c/em\u003e,\u003cem\u003e\u0026nbsp;DIO1, DIO2\u003c/em\u003e,\u003cem\u003e\u0026nbsp;DIO3\u003c/em\u003e,\u003cem\u003e\u0026nbsp;DNAJC17\u003c/em\u003e,\u003cem\u003e\u0026nbsp;JAG1\u003c/em\u003e,\u003cem\u003e\u0026nbsp;TUBB1\u003c/em\u003e,\u003cem\u003e\u0026nbsp;CDCA8\u003c/em\u003e,\u003cem\u003eTHRA\u003c/em\u003e,\u003cem\u003e\u0026nbsp;THRB\u003c/em\u003e,\u003cem\u003e\u0026nbsp;TBL1X\u003c/em\u003e, \u003cem\u003eSERPINA7\u003c/em\u003e,\u003cem\u003e\u0026nbsp;IGSF1\u003c/em\u003e,\u003cem\u003e\u0026nbsp;TRH\u003c/em\u003e,\u003cem\u003e\u0026nbsp;TRHR\u003c/em\u003e,\u003cem\u003e\u0026nbsp;TSHB\u003c/em\u003e,\u003cem\u003e\u0026nbsp;HHEX\u003c/em\u003e, \u003cem\u003eKCNJ16\u003c/em\u003e,\u003cem\u003e\u0026nbsp;HOXA3\u003c/em\u003e,\u003cem\u003e\u0026nbsp;ANO1\u003c/em\u003e,\u003cem\u003e\u0026nbsp;FGF8\u003c/em\u003e,\u003cem\u003e\u0026nbsp;FGFR1\u003c/em\u003e,\u003cem\u003e\u0026nbsp;FOXA2\u003c/em\u003e,\u003cem\u003e\u0026nbsp;GLI2\u003c/em\u003e,\u003cem\u003e\u0026nbsp;IRS4\u003c/em\u003e,\u003cem\u003e\u0026nbsp;PROP1\u003c/em\u003e, \u003cem\u003eSECISBP2\u003c/em\u003e, \u003cem\u003eSLC16A2\u003c/em\u003e,\u003cem\u003e\u0026nbsp;SOX2\u003c/em\u003e,\u003cem\u003e\u0026nbsp;SOX3\u003c/em\u003e,\u003cem\u003e\u0026nbsp;URB1\u003c/em\u003e,\u003cem\u003e\u0026nbsp;RGS12\u003c/em\u003e,\u003cem\u003e\u0026nbsp;GRPEL1\u003c/em\u003e,\u003cem\u003e\u0026nbsp;CLIC6\u003c/em\u003e, \u003cem\u003eWFS1\u003c/em\u003e,\u003cem\u003e\u0026nbsp;TREX1\u003c/em\u003e,\u003cem\u003e\u0026nbsp;POU1F1\u003c/em\u003e,\u003cem\u003e\u0026nbsp;LHX3\u003c/em\u003e,\u003cem\u003e\u0026nbsp;LHX4\u003c/em\u003e,\u003cem\u003e\u0026nbsp;HESX1\u003c/em\u003e,\u003cem\u003e\u0026nbsp;KAT6B\u003c/em\u003e, \u003cem\u003ePTPN22\u003c/em\u003e,\u003cem\u003e\u0026nbsp;TSBP1\u003c/em\u003e,\u003cem\u003e\u0026nbsp;B3GNT2\u003c/em\u003e, \u003cem\u003eFGF1\u003c/em\u003e,\u003cem\u003e\u0026nbsp;FGF2\u003c/em\u003e,\u003cem\u003e\u0026nbsp;B3GLCT\u003c/em\u003e, \u003cem\u003eKCNJ10\u003c/em\u003e,\u003cem\u003e\u0026nbsp;OTX2\u003c/em\u003e,\u003cem\u003e\u0026nbsp;ALB\u003c/em\u003e,\u003cem\u003e\u0026nbsp;LRP2, ELN, HOXD3, HOXB3,\u0026nbsp;\u003c/em\u003eand \u003cem\u003eKMT2D.\u0026nbsp;\u003c/em\u003eNo deleterious variants were identified in any of these genes.\u0026nbsp;All exons were covered at 100%.\u0026nbsp;The GT-AG splicing consensus sequences were rigorously respected in all introns of all genes studied.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAmino acid prediction analysis of the\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eNP_003226.4:p.(Asp2627Gly) variant\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA comprehensive set of \u003cem\u003ein silico\u003c/em\u003e algorithms was applied to evaluate the pathogenic potential of the NP_003226.4:p.(Asp2627Gly)\u0026nbsp;substitution (shown in Table 1). Of the 30 prediction tools, 27 indicated a deleterious impact, whereas only LRT, FATHMM, and PrimateAI classified the variant as neutral or tolerated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConservation analysis of the \u003cstrong\u003easpartic acid residue 2627\u003c/strong\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eComparative evaluation of the ChEL domain across 37 vertebrate species—including mammals, birds, reptiles, amphibians, ray-finned fishes, and jawless vertebrates—demonstrates complete conservation of aspartic acid at position 2627 (shown in Fig. 3). This strict evolutionary preservation strongly supports a pivotal role for this residue in maintaining the structural integrity of TG within the ChEL domain.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3D modeling analysis of the\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;NP_003226.4:p.(Asp2627Gly) mutant\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Structural modeling localized\u0026nbsp;aspartic acid\u003csup\u003e2627\u003c/sup\u003eto the central region of the ChEL domain (Region IV) of the TG monomer. In the wild-type model,\u0026nbsp;aspartic acid\u003csup\u003e2627\u003c/sup\u003eformed hydrogen bonds with tyrosine\u003csup\u003e2611\u003c/sup\u003eand arginine\u003csup\u003e2386\u003c/sup\u003e, contributing to the intramolecular hydrogen bond network within the ChEL domain (shown in Fig. 4a). Substitution of\u0026nbsp;aspartic acid\u003csup\u003e2627\u003c/sup\u003eby glycine (NP_003226.4:p.(Asp2627Gly))\u0026nbsp;resulted in the complete loss of these hydrogen bonds, with no additional hydrogen bond disruptions detected (shown in Fig. 4b). Structural inspection of the mutant model revealed no clashes and no detectable changes in the overall tertiary conformation or hydrophobicity profile of the TG monomer. In addition,\u0026nbsp;aspartic acid\u003csup\u003e2627\u003c/sup\u003eis not part of a canonical N-glycosylation consensus sequence (N-X-S/T) and is not spatially associated with modeled N-glycan structures, indicating that the\u0026nbsp;NP_003226.4:p.(Asp2627Gly) variant does not directly involve N-glycan attachment sites.\u0026nbsp;Fig. 4c\u0026nbsp;shows aspartic acid\u003csup\u003e2627\u003c/sup\u003e located within the central region of the ChEL domain.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe report two previously unpublished TG variants\u0026mdash;NM_003235.5:c.1375C\u0026gt;T (NP_003226.4:p.(Gln459Ter)) and NM_003235.5:c.5509_5518del (NP_003226.4:p.(Lys1837CysfsTer12))\u0026mdash;together with two previously documented variants, NM_003235.5:c.378C\u0026gt;A (NP_003226.4:p.(Tyr126Ter)) [42,43] and NM_003235.5:c.7880A\u0026gt;G (NP_003226.4:p.(Asp2627Gly)) [41]. The pathogenicity of the variants was manually assessed according to ACMG/AMP recommendations [22], as follows. The c.1375C\u0026gt;T variant, classified as pathogenic, is predicted to result in loss of normal protein function through either protein truncation or nonsense-mediated mRNA decay (NMD) (PVS1). This variant is absent from population databases (PM2_Supp), confirmed in trans with a pathogenic variant in our patient (PM3), and the patient\u0026rsquo;s phenotype is highly specific for TDH3 (PP4) (shown in Table 2). The NM_003235.5:c.378C\u0026gt;A and NM_003235.5:c.5509_5518del variants, both classified as pathogenic, meet the criteria PM2_Supp, PVS1, PM3_strong, and PP4; and PM2_Supp, PVS1, and PP4, respectively (shown in Table 2). In contrast, the c.7880A\u0026gt;G variant, classified as of uncertain significance but with a tendency toward pathogenicity (Hot VUS, shown\u0026nbsp;in Table 2), fulfills PM2_Supp\u0026nbsp;(absent from controls or at extremely low frequency), PM3\u0026nbsp;(in trans with a pathogenic variant in an affected individual), and PP4\u0026nbsp;(highly specific phenotype), together with PP3\u0026nbsp;(computational tools, shown in Table 1).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The clinical and biochemical criteria indicative of CH due to TG defects comprise low serum TG levels, elevated serum TSH, and a concomitant reduction in total serum T\u003csub\u003e4\u003c/sub\u003e with low or normal T\u003csub\u003e3\u003c/sub\u003e concentrations. A markedly reduced serum TG level is considered a key diagnostic hallmark of TG defects [5]. The index patient BAS:II-1 meets this criterion, whereas in the SRA:II-1 case, the TG value may fall within the normal range despite the presence of biallelic variants in the \u003cem\u003eTG\u003c/em\u003e gene; nevertheless, it remains consistently higher than the values usually described in individuals with \u003cem\u003eTG\u003c/em\u003e gene variants. Other cases of \u003cem\u003eTG\u0026nbsp;\u003c/em\u003evariants associated with normal or elevated serum TG levels have been well documented, including homozygous variants [43-46] and compound heterozygotes [43,47].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTG is a 660 kDa homodimeric glycoprotein that functions as the indispensable precursor for TH biosynthesis. Within its structure, specific tyrosyl residues undergo iodination and subsequent coupling reactions catalyzed by TPO, in concert with the DUOX system, ultimately yielding T\u003csub\u003e4\u003c/sub\u003e and T\u003csub\u003e3\u003c/sub\u003e [5,18-20]. The human \u003cstrong\u003e\u003cem\u003eTG\u003c/em\u003e gene\u003c/strong\u003e, located on chromosome 8 (chr8:132,866,958\u0026ndash;133,134,903; GRCh38/hg38), spans approximately 268 kb and contains a coding sequence of 8,453 nucleotides distributed across 48 exons (NCBI RefSeq: NM_003235.5) [48-52]. Translation produces a 19-amino acid signal peptide followed by a 2,748-residue polypeptide that assembles into the mature TG monomer (NCBI: NP_003226.4; UniProt: P01266) [48,52,53]. The canonical primary structure of TG is organized into four regions (I\u0026ndash;IV), comprising TG type 1, 2, and 3 repeats, as well as a cholinesterase-like (ChEL) domain at the C-terminus (shown in Fig. 5) [5,48,50-55]. This domain is essential for TG dimerization and acts both as an intramolecular chaperone and as a molecular escort for TG regions I, II, and III. [56,57]. All truncated forms detected in this study eliminate the ChEL domain in its entirety. NP_003226.4:p.(Gln459Ter)\u0026nbsp;and\u0026nbsp;NP_003226.4:p.(Tyr126Ter)\u0026nbsp;mutants comprise only a part of region I (shown in Fig. 5), while\u0026nbsp;NP_003226.4:p.(Lys1837CysfsTer12) includes regions I, II, and only a part of region III (shown in Fig. 5).\u0026nbsp;The functional consequences of the nonsense variants identified in this study may involve structural alterations in the protein that impair TH biosynthesis, primarily through the loss of the C-terminal hormonogenic site at position 2,766. This region harbors the principal coupling site for T\u003csub\u003e3\u003c/sub\u003e formation, mediated by the MIT\u003csup\u003e2766\u003c/sup\u003e\u0026ndash;DIT\u003csup\u003e2766\u003c/sup\u003e interaction between opposing monomers [58,59]. Nevertheless, the NP_003226.4:p.(Gln459Ter) and NP_003226.4:p.(Lys1837CysfsTer12) mutants retain partial capacity for T\u003csub\u003e4\u0026nbsp;\u003c/sub\u003esynthesis, as they preserve both the acceptor DIT\u003csup\u003e24\u003c/sup\u003e (exon 2) and the donor DIT\u003csup\u003e149\u003c/sup\u003e (exon 4) within the N-terminal hormonogenic site [60,61]. In contrast, NP_003226.4:p.(Tyr126Ter) mutant\u0026nbsp;additionally\u0026nbsp;loses the principal coupling site required for T\u003csub\u003e4\u003c/sub\u003e formation. It can be hypothesized that a short amino-terminal portion of TG containing a single hormonogenic site, even if producing only low physiological levels of TH, was sufficient to sustain vertebrate complexification from its earliest emergence until the fusion of the ChEL domain. This hypothesis is supported by the observation that a milder hypothyroidism phenotype has been reported in some homozygous patients carrying the\u0026nbsp;NP_003226.4:p.Arg296Ter\u0026nbsp;variant [10,42,43,45,62-64]. However,\u0026nbsp;transient expression studies\u0026nbsp;revealed that secretion of the\u0026nbsp;NP_003226.4:p.Arg296Ter mutant was essentially undetectable [65]. In addition to the limited ability to generate active TH as a pathophysiological mechanism underlying CH, RNA surveillance pathways also operate in the presence of truncated proteins. Among these, NMD ensures the rapid cytoplasmic degradation of transcripts containing premature stop codons, thereby preventing the accumulation of truncated and potentially deleterious proteins [66].\u003c/p\u003e\n\u003cp\u003eIn the present study, we extended the analysis of the NP_003226.4:p.(Asp2627Gly) variant, recently reported by us, by performing amino acid substitution prediction, protein homology assessment, and 3D structural modeling. Twenty-seven of the thirty predictors indicated a deleterious effect of the NP_003226.4:p.(Asp2627Gly) variant (shown in Table 1), and the wild-type aspartic acid at position 2,627 is strictly conserved in TG across thirty-seven vertebrate species analyzed (shown in Fig. 3), supporting an essential role of this residue in TG structure and/or proper functionality. Aspartic acid\u003csup\u003e2627\u003c/sup\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eis located within the core of the ChEL domain (shown in Fig. 4), a region characterized by a dense and highly interconnected network of intramolecular interactions. Its substitution by glycine disrupts hydrogen bonds with tyrosine\u003csup\u003e2611\u003c/sup\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eand arginine\u003csup\u003e2386\u003c/sup\u003e, weakening this tightly organized interaction network. Importantly, although\u0026nbsp;aspartic acid\u003csup\u003e2627\u003c/sup\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eis not itself part of a canonical N-glycosylation consensus sequence and is not directly associated with N-glycan attachment sites, its central positioning within the ChEL domain suggests that local destabilization may propagate through the domain and induce subtle long-range structural rearrangements. Such secondary effects could alter the spatial organization or accessibility of distal glycosylated residues, potentially compromising proper glycan presentation or recognition by the endoplasmic reticulum (ER) quality-control machinery. In this context, even modest perturbations in ChEL domain folding may contribute to impaired protein maturation.\u0026nbsp;Misfolded TG molecules may accumulate within ER as a consequence of missense variants in the ChEL domain\u0026mdash;such as NP_003226.4:p.(Asp2627Gly)\u0026mdash;or of partial or complete deletions\u0026mdash;such as NP_003226.4:p.(Tyr126Ter), NP_003226.4:p.(Gln459Ter) and NP_003226.4:p.(Lys1837CysfsTer12)\u0026mdash;ultimately resulting in premature degradation [67]. This retention induces ER distention, a pathological condition termed ER storage disease (ERSD) [68]. Misfolded proteins are subsequently eliminated through the ER-associated degradation (ERAD) pathway. On the other hand, Zhang et al. [69] demonstrated that, in cases of TG defects caused by deleterious variants, T\u003csub\u003e4\u003c/sub\u003e production can originate from mutant TG retained intracellularly and subsequently released into the follicular lumen by dead thyrocytes. Once released, these mutant TG molecules are reutilized\u0026mdash;or \u0026ldquo;cannibalized\u0026rdquo;\u0026mdash;by the TH biosynthetic machinery of neighboring viable thyrocytes, which iodinate them and synthesize T\u003csub\u003e4\u003c/sub\u003e. Based on this mechanism, we hypothesize that in patient SRA:II-1, who carries the missense TG variant NP_003226.4:p.(Asp2627Gly) affecting the ChEL domain in combination with the frameshift variant NP_003226.4:p.(Lys1837CysfsTer12) on the opposite allele, this cannibalization process may partially compensate for impaired TG secretion at the apical membrane\u0026ndash;colloid interface. This mechanism could account for the normal serum levels of TG, attributable to extensive thyrocyte loss.\u003c/p\u003e\n\u003cp\u003eIn conclusion, we report novel TG variants that expand the spectrum of TG defects. A clinically relevant missense variant in the ChEL domain, in combination with a frameshift, was identified in a patient with CH and normal serum TG levels. In contrast, a second patient with classic goitrous CH and low serum TG levels harbored two nonsense variants introducing premature stop codons within the TG coding sequence, resulting in truncated proteins with severely impaired TH synthesis. Collectively, our findings underscore the phenotypic diversity of CH attributable to TG variants.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eData availability\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData and material are available from the authors upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAcknowledgement\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the study participants and their family members. V.R. and M.E.M. are research fellows of the Fondo para la Investigaci\u0026oacute;n Cient\u0026iacute;fica y Tecnol\u0026oacute;gica (FONCyT-Agencia I+D+I). M.G.P. is a research fellow of the Consejo Nacional de Investigaciones Cient\u0026iacute;ficas y T\u0026eacute;cnicas (CONICET). \u0026nbsp;H.M.T., M.G.R., A.E.C., C.M.R., and M.L.T. are established investigators of the CONICET. We thank Dr. Gabriela Sans\u0026oacute;, Mrs. MG Gutiérrez Moyano, and Mr. Rodolfo De Bellis for their kind and skillful technical assistance. We thank Mrs. Rosenbrock Lambois for her assistance with study coordination.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthor Contributions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH.M.T. was involved in WES analysis, bioinformatic predictions, funding acquisition, study conception and design, and manuscript writing. V.R. contributed to formal analysis, data interpretation, validation, and variant curation. V.F.G. performed WES analysis, applied bioinformatic prediction tools, and carried out sample preparation for Sanger sequencing. M.G.P. conducted the structural modeling analyses. P.S. and M.E.A. performed custom-panel sequencing and contributed to variant curation. M.E.M. and A.E.C. participated in patient recruitment and the collection of clinical data and blood samples. A.I. was responsible for the bioinformatic analysis of custom-panel sequencing. M.G.R. provided essential resources and financial support for the study.\u0026nbsp;C.M.R. contributed to funding acquisition and study conception and design.\u0026nbsp;M.L.T. performed validation and variant curation, acquired funding, conceived and designed the study, and contributed to manuscript writing.\u0026nbsp;All authors critically reviewed the manuscript, contributed to its revision, and approved the final version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding \u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by grants from the Fondo para la Investigaci\u0026oacute;n Cient\u0026iacute;fica y Tecnol\u0026oacute;gica (FONCyT-ANPCyT-MINCyT, PICT-2018-02146 to H.M.T. and PIDC-2019-0007 to A.E.C. and M.L.T.), CONICET (PIP 2021-11220200102976CO to C.M.R.), and Universidad de Buenos Aires (UBACyT 2020-20020190100050BA to C.M.R.). The funders had no role in the design, data collection, data analysis, and reporting of this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConflict of Interest\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOn behalf of all authors, the corresponding author states that there is no conflict of interest to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthical approval\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe studies involving human participants were reviewed and approved by the Ethical Committee of the Faculty of Pharmacy and Biochemistry of the University of Buenos Aires (CEICFFyB, No. 1094) and the Research Ethics Committee of the HNRG, Buenos Aires, Argentina (C.E.I. 21.33).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eInformed consent\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe patient\u0026rsquo;s parents, acting as legal guardians, provided informed consent for participation in this study and agreed to the publication of the clinical case.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKwak MJ (2018) Clinical genetics of defects in thyroid hormone synthesis. 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Arch Dis Child Fetal Neonatal Ed 87(3):F209\u0026ndash;F211. https://doi.org/10.1136/fn.87.3.f209\u003c/li\u003e\n\u003cli\u003eRicci V, Masnata ME, Villanueva Gonzalez MD, Enac\u0026aacute;n RE, Izquierdo A, Adrover E, Esnaola Azcoiti M, Sans\u0026oacute; G, Scaglia PA, Rivolta CM, Targovnik HM, Rey RA, Ropelato MG, Chiesa AE, Nicola JP, Tellechea ML (2025) Variation spectra in mild isolated hyperthyrotropinemia: pilot cohort and systematic review. Front Endocrinol (Lausanne). 16:1612450. https://doi.org/10.3389/fendo.2025.1612450\u003c/li\u003e\n\u003cli\u003eCitterio CE, Machiavelli GA, Miras MB, Gru\u0026ntilde;eiro-Papendieck L, Lachlan K, Sobrero G, Chiesa A, Walker J, Mu\u0026ntilde;oz L, Testa G, Belforte FS, Gonz\u0026aacute;lez-Sarmiento R, Rivolta CM, Targovnik HM (2013) New insights into thyroglobulin gene: molecular analysis of seven novel mutations associated with goiter and hypothyroidism. 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J Clin Endocrinol Metab 84(4):1438\u0026ndash;1444. https://doi.org/10.1210/jcem.84.4.5633\u003c/li\u003e\n\u003cli\u003ePardo V, Vono-Toniolo J, Rubio IGS, Knobel M, Possato RF, Targovnik HM, Kopp P, Medeiros-Neto G (2009) The p.A2215D thyroglobulin gene mutation leads to deficient synthesis and secretion of the mutated protein and congenital hypothyroidism with wide phenotype variation. J Clin Endocrinol Metab 94(8):2938\u0026ndash;2944. https://doi.org/10.1210/jc.2009-0150\u003c/li\u003e\n\u003cli\u003eWatanabe Y, Sharwood E, Goodwin B, Creech MK, Hassan HY, Netea MG, Jaeger M, Dumitrescu A, Refetoff S, Huynh T, Weiss RE (2018) A novel mutation in the TG gene (G2322S) causing congenital hypothyroidism in a Sudanese family: a case report. BMC Med Genet 19(1):69. https://doi.org/10.1186/s12881-018-0588-7\u003c/li\u003e\n\u003cli\u003eFern\u0026aacute;ndez-Cancio M, Antol\u0026iacute;n M, Clemente M, Campos-Martorell A, Mogas E, Baz-Red\u0026oacute;n N, Leno-Colorado J, Comas-Armangu\u0026eacute; G, Garc\u0026iacute;a-Arum\u0026iacute; E, Soler-Colomer L, Gonz\u0026aacute;lez-Llorens N, Camats-Tarruella N, Yeste D (2024) Clinical and molecular study of patients with thyroid dyshormogenesis and variants in the thyroglobulin gene. Front Endocrinol (Lausanne) 15:1367808. https://doi.org/10.3389/fendo.2024.1367808\u003c/li\u003e\n\u003cli\u003eMalthi\u0026eacute;ry Y, Lissitzky S (1987) Primary structure of human thyroglobulin deduced from the sequence of its 8448-base complementary DNA. Eur J Biochem 165(3):491\u0026ndash;498. https://doi.org/10.1111/j.1432-1033.1987.tb11466.x\u003c/li\u003e\n\u003cli\u003eMendive FM, Rivolta CM, Moya CM, Vassart G, Targovnik HM (2001) Genomic organization of the human thyroglobulin gene: the complete intron-exon structure. Eur J Endocrinol 145(4):485\u0026ndash;496. https://doi.org/10.1530/eje.0.1450485\u003c/li\u003e\n\u003cli\u003eMercken L, Simons MJ, Swillens S, Massaer M, Vassart G. (1985) Primary structure of bovine thyroglobulin deduced from the sequence of its 8,431-base complementary DNA. Nature 316(6029):647\u0026ndash;651. https://doi.org/10.1038/316647a0\u003c/li\u003e\n\u003cli\u003eParma J, Christophe D, Pohl V, Vassart G (1987) Structural organization of the 5\u0026apos; region of the thyroglobulin gene. Evidence for intron loss and \u0026quot;exonization\u0026quot; during evolution. J Mol Biol 196(4):769\u0026ndash;779. https://doi.org/10.1016/0022-2836(87)90403-7\u003c/li\u003e\n\u003cli\u003evan de Graaf SAR, Ris-Stalpers C, Pauws E, Mendive FM, Targovnik HM, de Vijlder JJM (2001) Up to date with human thyroglobulin. J Endocrinol 170(2):307\u0026ndash;321. https://doi.org/10.1677/joe.0.1700307 \u003c/li\u003e\n\u003cli\u003eHolzer G, Morishita Y, Fini JB, Lorin T, Gillet B, Hughes S, Tohm\u0026eacute; M, Del\u0026eacute;age G, Demeneix B, Arvan P, Laudet V (2016) Thyroglobulin represents a novel molecular architecture of vertebrates. J Biol Chem 291(32):16553\u0026ndash;16566. https://doi.org/10.1074/jbc.M116.719047\u003c/li\u003e\n\u003cli\u003eSwillens S, Ludgate M, Mercken L, Dumont JE, Vassart G (1986) Analysis of sequence and structure homologies between thyroglobulin and acetylcholinesterase: possible functional and clinical significance. Biochem Biophys Res Commun 137(1):142-148. https://doi.org/10.1016/0006-291x(86)91187-3\u003c/li\u003e\n\u003cli\u003eMolina F, Bouanani M, Pau B, Granier C (1996) Characterization of the type-1 repeat from thyroglobulin, a cysteine-rich module found in proteins from different families. Eur J Biochem. 240(1):125\u0026ndash;133. https://doi.org/10.1111/j.1432-1033.1996.0125h.x\u003c/li\u003e\n\u003cli\u003eLee J, Di Jeso B, Arvan P (2008) The cholinesterase-like domain of thyroglobulin functions as an intramolecular chaperone. J Clin Invest 118(8):2950\u0026ndash;2958. https://doi.org/10.1172/JCI35164\u003c/li\u003e\n\u003cli\u003eLee J, Wang X, Di Jeso B, Arvan P (2009) The cholinesterase-like domain, essential in thyroglobulin trafficking for thyroid hormone synthesis, is required for protein dimerization. J Biol Chem. 284(19):12752\u0026ndash;12761 https://doi.org/10.1074/jbc.M806898200\u003c/li\u003e\n\u003cli\u003eCitterio CE, Veluswamy B, Morgan SJ, Galton VA, Banga JP, Atkins S, Morishita Y, Neumann S, Latif R, Gershengorn MC, Smith TJ, Arvan P (2017) De novo triiodothyronine formation from thyrocytes activated by thyroid-stimulating hormone. J Biol Chem 292(37):15434\u0026ndash;15444. https://doi.org/10.1074/jbc.M117.784447\u003c/li\u003e\n\u003cli\u003eCitterio CE, Morishita Y, Dakka N, Veluswamy B, Arvan P (2018) Relationship between the dimerization of thyroglobulin and its ability to form triiodothyronine. J Biol Chem 293(13):4860\u0026ndash;4869. https://doi.org/10.1074/jbc.RA118.001786\u003c/li\u003e\n\u003cli\u003eDunn AD, Corsi CM, Myers HE, Dunn JT (1998) Tyrosine 130 is an important outer ring donor for thyroxine formation in thyroglobulin. J Biol Chem 273(39):25223\u0026ndash;25229. https://doi.org/10.1074/jbc.273.39.25223\u003c/li\u003e\n\u003cli\u003eLamas L, Anderson PC, Fox JW, Dunn JT (1989) Consensus sequences for early iodination and hormonogenesis in human thyroglobulin. J Biol Chem. 264(23):13541\u0026ndash;13545.\u003c/li\u003e\n\u003cli\u003eCaputo M, Rivolta CM, Esperante SA, Gru\u0026ntilde;eiro-Papendieck L, Chiesa A, Pellizas CG, Gonz\u0026aacute;lez-Sarmiento R, Targovnik HM (2007) Congenital hypothyroidism with goitre caused by new mutations in the thyroglobulin gene. Clin Endocrinol. 67(3):351\u0026ndash;357. https://doi.org/10.1111/j.1365-2265.2007.02889.x\u003c/li\u003e\n\u003cli\u003eRivolta CM, Moya CM, Gutnisky VJ, Varela V, Miralles-Garc\u0026iacute;a JM, Gonz\u0026aacute;lez-Sarmiento R, Targovnik HM (2005) A new case of congenital goiter with hypothyroidism due to a homozygous p.R277X mutation in the exon 7 of the thyroglobulin gene: a mutational hot spot could explain the recurrence of this mutation. J Clin Endocrinol Metab. 90(6):3766\u0026ndash;3770. https://doi.org/10.1210/jc.2005-0278\u003c/li\u003e\n\u003cli\u003evan de Graaf SAR, Ris-Stalpers C, Veenboer GJM, Cammenga M, Santos C, Targovnik HM, de Vijlder JJM, Medeiros-Neto G (1999) A premature stopcodon in thyroglobulin mRNA results in familial goiter and moderate hypothyroidism. J Clin Endocrinol Metab 84(7):2537\u0026ndash;2542. https://doi.org/10.1210/jcem.84.7.5862\u003c/li\u003e\n\u003cli\u003eLee J, Di Jeso B, Arvan P (2011) Maturation of thyroglobulin protein region I. J Biol Chem 286(38):33045\u0026ndash;33052. https://doi.org/10.1074/jbc.M111.281337 \u003c/li\u003e\n\u003cli\u003eBehm-Ansmant I, Kashima I, Rehwinkel J, Sauli\u0026egrave;re J, Wittkopp N, Izaurralde E (2007) mRNA quality control: an ancient machinery recognizes and degrades mRNAs with nonsense codons. FEBS Lett. 581(15):2845\u0026ndash;2853. https://doi.org/10.1016/j.febslet.2007.05.027\u003c/li\u003e\n\u003cli\u003eSiffo S, Gomes Pio M, Mart\u0026iacute;nez EB, Lachlan K, Walker J, Weill J, Gonz\u0026aacute;lez-Sarmiento R, Rivolta CM, Targovnik HM (2023) The p.Pro2232Leu variant in the ChEL domain of thyroglobulin gene causes intracellular transport disorder and congenital hypothyroidism. Endocrine 80(1):47\u0026ndash;53. https://doi.org/10.1007/s12020-022-03284-5\u003c/li\u003e\n\u003cli\u003eKim PS, Arvan P (1998) Endocrinopathies in the family of endoplasmic reticulum (ER) storage diseases: disorders of protein trafficking and the role of ER molecular chaperones. Endocr Rev 19(2):173\u0026ndash;202. https://doi.org/10.1210/edrv.19.2.0327\u003c/li\u003e\n\u003cli\u003eZhang X, Kellogg AP, Citterio CE, Zhang H, Larkin D, Morishita Y, Targovnik HM, Balbi VA, Arvan P (2021) Thyroid hormone synthesis continues despite biallelic thyroglobulin mutation with cell death. JCI Insight 6(11):e148496. https://doi.org/10.1172/jci.insight.148496\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eBioinformatic prediction of functional effects of the variant NP_003226.4:p.(Asp2627Gly) in the ChEL domain of the thyroglobulin.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"955\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePredictor\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eScore\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePrediction\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 344px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRankscore\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eReference\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSIFT\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 138px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003eDamaging\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 344px;\"\u003e\n \u003cp\u003e\u0026gt;0.05 Tolerated, \u0026lt;=0.05 Damaging\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e\u0026nbsp;[31]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSIFT4G\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 138px;\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003eDamaging\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 344px;\"\u003e\n \u003cp\u003e\u0026gt;0.05 Tolerated, \u0026lt;=0.05 Damaging\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e[32];\u0026nbsp;dbNSFP v4\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePolyphen2_HDIV\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 138px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003eProbably damaging\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 344px;\"\u003e\n \u003cp\u003e\u0026lt;=0.452 Benign, \u0026lt;=0.956-\u0026gt;=0.453 Possibly Damaging, \u0026gt;=0.957 Probably Damaging\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e\u0026nbsp;[27]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePolyphen2_HVAR\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 138px;\"\u003e\n \u003cp\u003e0.999\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003eProbably damaging\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 344px;\"\u003e\n \u003cp\u003e\u0026lt;=0.452 Benign, \u0026lt;=0.956-\u0026gt;=0.453 Possibly Damaging, \u0026gt;=0.957 Probably Damaging\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e\u0026nbsp;[27]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLRT\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 138px;\"\u003e\n \u003cp\u003e0.012546\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003eNeutral\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 344px;\"\u003e\n \u003cp\u003e\u0026gt;0.001 Neutral or Unknown, \u0026lt;=0.001 Deleterious\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e[31-32];\u0026nbsp;dbNSFP v4\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMutationTaster\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 138px;\"\u003e\n \u003cp\u003e0.987212\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003eDisease causing\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 344px;\"\u003e\n \u003cp\u003e\u0026lt;=0.5 Polymorphism or Polymorphism Automatic, \u0026gt;0.5 Diseases Causing Automatic or Diseases Causing\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e[27-32];\u0026nbsp;dbNSFP v4\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMutationAssessor\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 138px;\"\u003e\n \u003cp\u003e3.27\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003eMedium impact\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 344px;\"\u003e\n \u003cp\u003e\u0026lt;=0.8 Neutral impact (NI), \u0026lt;=1.935 - \u0026gt;0.8 Low impact (LI), \u0026lt;=3.5 - \u0026gt;1.935 Medium impact (MI), \u0026gt;3.5 High impact (HI). Benign = NI and LI, Deleterious = MI and HI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e\u0026nbsp;[27-32]; dbNSFP v4\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFATHMM\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 138px;\"\u003e\n \u003cp\u003e-0.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003eTolerated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 344px;\"\u003e\n \u003cp\u003e\u0026gt;-1.5 Tolerated, \u0026lt;=-1.5 Damaging\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e[32];\u0026nbsp;dbNSFP v4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePROVEAN\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 138px;\"\u003e\n \u003cp\u003e-5.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003eDeleterious\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 344px;\"\u003e\n \u003cp\u003e\u0026gt;-2.5 Neutral, \u0026lt;=-2.5 Deleterious\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e[32];\u0026nbsp;dbNSFP v4\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVEST4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 138px;\"\u003e\n \u003cp\u003e0.937\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003ePathogenic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 344px;\"\u003e\n \u003cp\u003e\u0026lt;0.75 Benign, \u0026gt;=0.75 Pathogenic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e\u0026nbsp;[35]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMetaSVM\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 138px;\"\u003e\n \u003cp\u003e0.2184\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003eDamaging\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 344px;\"\u003e\n \u003cp\u003e\u0026lt;=0 Tolerated, \u0026gt;0 Damaging\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e\u0026nbsp;[31]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMetaLR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 138px;\"\u003e\n \u003cp\u003e0.5323\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003eDamaging\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 344px;\"\u003e\n \u003cp\u003e\u0026lt;=0.5 Tolerated, \u0026gt;0.5 Damaging\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e\u0026nbsp;[31]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMetaRNN\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 138px;\"\u003e\n \u003cp\u003e0.95961165\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003eDamaging\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 344px;\"\u003e\n \u003cp\u003e\u0026lt;=0.5 Tolerated, \u0026gt;0.5 Damaging\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e[32];\u0026nbsp;dbNSFP v4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eM-CAP\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 138px;\"\u003e\n \u003cp\u003e0.059366\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003ePossibly Pathogenic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 344px;\"\u003e\n \u003cp\u003e\u0026lt;=0.025 Likely Benign, \u0026gt;0.025 Possibly Pathogenic \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e\u0026nbsp;[29]\u0026nbsp;http://bejerano.stanford.edu/mcap\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eREVEL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 138px;\"\u003e\n \u003cp\u003e0.682\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003ePathogenic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 344px;\"\u003e\n \u003cp\u003e\u0026lt;0.5 Neutral, \u0026gt;=0.5 Pathogenic\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e\u0026nbsp;[34-35]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMutPred\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 138px;\"\u003e\n \u003cp\u003e0.753\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003ePathogenic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 344px;\"\u003e\n \u003cp\u003e\u0026lt;0.5 Benign, \u0026gt;=0.5 Pathogenic\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e\u0026nbsp;[35]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePrimateAI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 138px;\"\u003e\n \u003cp\u003e0.470505654812\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003eTolerated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 344px;\"\u003e\n \u003cp\u003e\u0026lt;=0.803 Tolerated, \u0026gt;0.803 Damaging\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e[32]; dbNSFP v4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDEOGEN2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 138px;\"\u003e\n \u003cp\u003e0.612807\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003eDamaging\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 344px;\"\u003e\n \u003cp\u003e\u0026lt;=0.5 Tolerated, \u0026gt;0.5 Damaging\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e[32-33]; dbNSFP v4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBayesDel_addAF\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 138px;\"\u003e\n \u003cp\u003e0.326526\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003eDamaging\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 344px;\"\u003e\n \u003cp\u003e\u0026lt;=0.0692655 Tolerated, \u0026gt;0.0692655 Damaging\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e[32]; dbNSFP v4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBayesDel_noAF\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 138px;\"\u003e\n \u003cp\u003e0.231256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003eDamaging\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 344px;\"\u003e\n \u003cp\u003e\u0026lt;=-0.0570105 Tolerated, \u0026gt;-0.0570105 Damaging\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e[32];\u0026nbsp;dbNSFP v4\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eClinPred\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 138px;\"\u003e\n \u003cp\u003e0.998569965362549\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003eDamaging\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 344px;\"\u003e\n \u003cp\u003e\u0026lt;=0.5 Tolerated, \u0026gt;0.5 Damaging\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e[32];\u0026nbsp;dbNSFP v4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLIST-S2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 138px;\"\u003e\n \u003cp\u003e0.866213\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003eDamaging\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 344px;\"\u003e\n \u003cp\u003e\u0026lt;=0.85 Tolerated, \u0026gt;0.85 Damaging\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e[32]; dbNSFP v4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVARITY_R\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 138px;\"\u003e\n \u003cp\u003e0.9111106\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003eDamaging\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 344px;\"\u003e\n \u003cp\u003e\u0026lt;=0.5 Tolerated, \u0026gt;0.5 Damaging\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e\u0026nbsp;[30]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVARITY_ER\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 138px;\"\u003e\n \u003cp\u003e0.895924\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003eDamaging\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 344px;\"\u003e\n \u003cp\u003e\u0026lt;=0.5 Tolerated, \u0026gt;0.5 Damaging\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e\u0026nbsp;[30]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eEVE\u003c/strong\u003e\u003cstrong\u003e_Class90\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 138px;\"\u003e\n \u003cp\u003e0.8051757528573578\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003ePathogenic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 344px;\"\u003e\n \u003cp\u003e\u0026lt;=0.4 Benign, \u0026gt;=0.6 Pathogenic, \u0026gt;0.4 - \u0026lt;0.6 Uncertain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e\u0026nbsp;[28-32]; dbNSFP v4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAlphaMissense\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 138px;\"\u003e\n \u003cp\u003e0.8056\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003eLikely Pathogenic\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 344px;\"\u003e\n \u003cp\u003e\u0026lt;0.34 Likely Benign, 0.34 - 0.564 Ambiguous, \u0026gt;0.564 Likely Pathogenic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e\u0026nbsp;[26]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCADD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 138px;\"\u003e\n \u003cp\u003e29.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003eDamaging\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 344px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u0026lt;=24 Tolerated, \u0026gt;24 Damaging\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e\u0026nbsp;[31]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDANN\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 138px;\"\u003e\n \u003cp\u003e0.99813861375246238\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003eDamaging\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 344px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u0026lt;0.99 Tolerated, \u0026gt;=0.99 Damaging\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e\u0026nbsp;[31]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFathmm-MKL_coding\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 138px;\"\u003e\n \u003cp\u003e0.93153\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003eDamaging\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 344px;\"\u003e\n \u003cp\u003e\u0026lt;=0.5 Neutral, \u0026gt;0.5 Damaging\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e\u0026nbsp;[31]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFathmm-XF_coding\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 138px;\"\u003e\n \u003cp\u003e0.715970\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003ePathogenic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 344px;\"\u003e\n \u003cp\u003e\u0026lt;=0.5 Neutral, \u0026gt;0.5 Damaging\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e[32]; dbNSFP v4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2: Information on the Single Nucleotide Variants identified in the thyroglobulin\u0026nbsp;gene and genetic diagnosis of the patients.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"983\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePatient\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 270px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSingle Nucleotide Variant\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSegregation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 255px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eACMG/AMP Criteria\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eClassification*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSRA:II-1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 270px;\"\u003e\n \u003cp\u003eNM_003235.5:c.5509_5518del\u003cstrong\u003e\u0026nbsp;(\u003c/strong\u003e\u003cstrong\u003ep.(Lys1837CysfsTer12))\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eFather\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 255px;\"\u003e\n \u003cp\u003ePM2_supp, PVS1, PP4\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003ePathogenic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003ePossibly Solved\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 270px;\"\u003e\n \u003cp\u003eNM_003235.5:c.7880A\u0026gt;G (p.(Asp2627Gly))\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eMother\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 255px;\"\u003e\n \u003cp\u003ePM2_supp, PP3, PM3, PP4\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003eHot VUS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\" valign=\"top\" style=\"width: 983px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBAS:II-1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 270px;\"\u003e\n \u003cp\u003eNM_003235.5:c.378C\u0026gt;A\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(p.(Tyr126Ter))\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eFather\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 255px;\"\u003e\n \u003cp\u003ePM2_supp, PVS1, PM3_strong, PP4\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003ePathogenic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eSolved\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 270px;\"\u003e\n \u003cp\u003eNM_003235.5:c.1375C\u0026gt;T\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(p.(Gln459Ter)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eMother\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 255px;\"\u003e\n \u003cp\u003ePM2_supp, PVS1, PM3, PP4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003ePathogenic\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*A Bayesian scoring algorithm was used. Scores were binned into the following categories: 0= Benign; 0.001-0.051= Likely Benign; 0.100-0.188= VUS (variant of uncertain significance) leaning to Benign; 0.325-0.500= VUS; 0.675-0.812= VUS leaning to Pathogenic (Hot VUS); 0.900-0.988= Likely Pathogenic; 0.994-0.999= Pathogenic.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"a12d3b04-762d-4bf8-a64f-452d6603bab6","identifier":"10.13039/501100006668","name":"Fondo para la Investigación Científica y Tecnológica","awardNumber":"PICT-2018-02146 ","order_by":0},{"identity":"ad3d0153-71a0-4fdb-9757-24870d01e924","identifier":"10.13039/501100006668","name":"Fondo para la Investigación Científica y Tecnológica","awardNumber":"PIDC-2019-0007 ","order_by":1},{"identity":"31145af8-ca97-47b3-8531-09dee6d6e3ff","identifier":"10.13039/501100002923","name":"Consejo Nacional de Investigaciones Científicas y Técnicas","awardNumber":"PIP 2021-11220200102976CO ","order_by":2},{"identity":"04e3297a-fa07-44ff-8433-c1410ff78c22","identifier":"10.13039/501100005363","name":"Universidad de Buenos Aires","awardNumber":"UBACyT 2020-20020190100050BA ","order_by":3}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"University of Buenos Aires","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Thyroglobulin, Gene, Variant, Congenital Hypothyroidism, Thyroid Dyshormonogenesis","lastPublishedDoi":"10.21203/rs.3.rs-9117442/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9117442/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eContext\u003c/strong\u003e Thyroglobulin (TG) is the most abundant glycoprotein secreted by thyrocytes into the follicular lumen, serving as the essential substrate for thyroid hormone biosynthesis. The clinical spectrum of TG defects is highly heterogeneous, ranging from euthyroid states to mild or severe permanent hypothyroidism. The aim of this study was to identify and characterize novel TG variants to advance our understanding of the molecular mechanisms underlying thyroid dyshormonogenesis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e Two patients from unrelated, non-consanguineous families with impaired TG synthesis were investigated, both undergoing comprehensive clinical, biochemical, and imaging evaluations. Genetic testing included DNA sequencing, genotyping, and bioinformatics analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMain results\u003c/strong\u003e Molecular analysis revealed two previously unreported TG variants—NM_003235.5:c.1375C\u0026gt;T (NP_003226.4:p.(Gln459Ter)) and NM_003235.5:c.5509_5518delAAAGACACAG (NP_003226.4:p.(Lys1837CysfsTer12))—alongside two known variants, NM_003235.5:c.378C\u0026gt;A (NP_003226.4:p.(Tyr126Ter)) and NM_003235.5:c.7880A\u0026gt;G (NP_003226.4:p.(Asp2627Gly)). A frameshift variant, combined with a clinically relevant missense variant in the ChEL domain (NP_003226.4:p.(Lys1837CysfsTer12)/ NP_003226.4:p.(Asp2627Gly)), was identified in one patient with congenital hypothyroidism (CH) who presented with normal serum TG levels. In contrast, the second patient, with classic goitrous CH and reduced serum TG levels, carried two nonsense variants introducing premature stop codons (NP_003226.4:p.(Tyr126Ter)/NP_003226.4(p.Gln459Ter)), resulting in truncated proteins and severely impaired hormone synthesis. The deleterious impact predicted by amino acid analysis software, together with strict evolutionary conservation and three-dimensional modeling of NP_003226.4:p.(Asp2627Gly) in the ChEL domain strongly supports a pivotal role of this residue in maintaining TG structural integrity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e The systematic exhaustive analysis of two novel variants, together with two previously reported TG variants, using molecular and bioinformatics tools, broadens the spectrum of deleterious TG variants and provides deeper insights into the etiology of CH.\u003c/p\u003e","manuscriptTitle":"Clinical, Biochemical, and Molecular Genetic Characterization of Two Patients with Congenital Hypothyroidism Harboring Novel Compound Heterozygous Variants in the Thyroglobulin Gene","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-18 08:43:04","doi":"10.21203/rs.3.rs-9117442/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7b100416-079d-459a-afda-3f676101a014","owner":[],"postedDate":"March 18th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":64623973,"name":"Molecular Genetics"}],"tags":[],"updatedAt":"2026-03-18T08:43:05+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-18 08:43:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9117442","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9117442","identity":"rs-9117442","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}