p.L571P in the linker domain of rat thyroglobulin causes intracellular retention

Citterio, Cintia E.; Siffo, Sofía; Moya, Christian M.; Pio, Mauricio Gomes; Molina, Maricel F.; Scheps, Karen G.; Rey, Osvaldo; Arvan, Peter; Rivolta, Carina M.; Targovnik, Héctor M.

doi:10.1016/j.mce.2020.110719

Cited by 7 publications

(5 citation statements)

References 55 publications

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“…The variant p.Q29* is adjacent to the type 1 TG-like repeat A, where the variant p.T59S fs *17 (protein product of c.177-2A>C, see below) is located, with the variant p.S523P being located in the linker domain spacing type 1 TG-like repeats D and F (domain E) within the amino-terminal domain (NTD). Recently, the functional characterization of the missense variant p.L571P revealed the relevance of the linker domain in the intracellular trafficking of TG [ 17 ]. The variant p.F1542V fs *20 is located in the type-1 repeat P within the arm domain, while the variant p.Y2563C is located in the homodimerization carboxy-terminal V domain, also known as the choline esterase-like (ChEL) domain.…”

Section: Resultsmentioning

confidence: 99%

“…HEK-293T cells were transiently transfected with an empty expression vector (Mock) or expression vectors encoding full-length mouse WT or p.Y2562C TG (equivalent to p.Y2563C in human TG). Western blot analysis revealed an expected specific band for TG with a molecular mass corresponding to ~330 kDa both in the cell lysate and in the supernatant, denoting TG expression and secretion, respectively ( Figure 3 A) [ 17 , 23 ]. In contrast, p.Y2562C TG protein expression in the cell lysate and its secretion to the supernatant was significantly reduced compared to WT TG ( Figure 3 B).…”

Section: Resultsmentioning

confidence: 99%

“…Then, after 24 h, the supernatants were collected and centrifuged to remove cellular debris. The cells were lysed in RIPA buffer containing protease inhibitor cocktail [ 17 ], and equal volumes of supernatants and cell lysates were resolved in SDS-PAGE gel under non-reducing conditions, followed by electrotransference to nitrocellulose membranes and immunoblotting, as previously described [ 23 ]. Membranes were blocked and incubated with rabbit monoclonal anti-TG primary antibody (ab156008, Abcam, Cambridge, UK) diluted 1:3000 in PBS containing 0.05% Tween-20.…”

Section: Methodsmentioning

confidence: 99%

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Targeted Next-Generation Sequencing of Congenital Hypothyroidism-Causative Genes Reveals Unexpected Thyroglobulin Gene Variants in Patients with Iodide Transport Defect

Barquero

Geysels

Jacques

et al. 2022

IJMS

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Congenital iodide transport defect is an uncommon autosomal recessive disorder caused by loss-of-function variants in the sodium iodide symporter (NIS)-coding SLC5A5 gene and leading to dyshormonogenic congenital hypothyroidism. Here, we conducted a targeted next-generation sequencing assessment of congenital hypothyroidism-causative genes in a cohort of nine unrelated pediatric patients suspected of having a congenital iodide transport defect based on the absence of 99mTc-pertechnetate accumulation in a eutopic thyroid gland. Although, unexpectedly, we could not detect pathogenic SLC5A5 gene variants, we identified two novel compound heterozygous TG gene variants (p.Q29* and c.177-2A>C), three novel heterozygous TG gene variants (p.F1542Vfs*20, p.Y2563C, and p.S523P), and a novel heterozygous DUOX2 gene variant (p.E1496Dfs*51). Splicing minigene reporter-based in vitro assays revealed that the variant c.177-2A>C affected normal TG pre-mRNA splicing, leading to the frameshift variant p.T59Sfs*17. The frameshift TG variants p.T59Sfs*17 and p.F1542Vfs*20, but not the DUOX2 variant p.E1496Dfs*51, were predicted to undergo nonsense-mediated decay. Moreover, functional in vitro expression assays revealed that the variant p.Y2563C reduced the secretion of the TG protein. Our investigation revealed unexpected findings regarding the genetics of congenital iodide transport defects, supporting the existence of yet to be discovered mechanisms involved in thyroid hormonogenesis.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Targeted Next-Generation Sequencing of Congenital Hypothyroidism-Causative Genes Reveals Unexpected Thyroglobulin Gene Variants in Patients with Iodide Transport Defect

Barquero

Geysels

Jacques

et al. 2022

IJMS

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“…5'-gggtggggcagcatatAgaactccaaggaattg-3'. The nal construct was veri ed by sequencing with the primers listed in Citterio et al [33].…”

Section: Site-directed Mutagenesismentioning

confidence: 99%

The p.Pro2232Leu variant in the ChEL domain of thyroglobulin gene causes intracellular transport disorder and congenital hypothyroidism

et al. 2022

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Thyroglobulin (TG), the predominant glycoprotein of the thyroid gland, functions as matrix protein in thyroid hormonegenesis. TG de ciency results in thyroid dyshormonogenesis. These variants produce a heterogeneous spectrum of congenital goitre, with an autosomal recessive mode of inheritance. The purpose of this study was to identify and functionally characterize new variants in the TG gene in order to increase the understanding of the molecular mechanisms responsible for thyroid dyshormonogenesis. A total of four patients from two non-consanguineous families with marked alteration of TG synthesis were studied.The two families were previously analysed in our laboratory, only one deleterious allele, in each one, was detected after sequencing the TG gene (c.2359C > T [p.Arg787*], c.5560G > T [p.Glu1854*]). These ndings were con rmed in the present studies by Next-Generation Sequencing. The single nucleotide coding variants of the TG gene were then analyzed to predict the possible variant causing the disease. The p.Pro2232Leu (c.6695C > T), identi ed in both families, showing a low frequency population in gnomAD v2.1.1 database and protein homology, amino acid prediction, and 3D modeling analysis predict a potential pathogenic effect of this variant. We also transiently express p.Pro2232Leu in a full-length rat TG cDNA clone and con rmed that this point variant was su cient to cause intracellular retention of mutant TG in HEK293T cells. Consequently, each family carried a compound heterozygous for p.Arg787*/p.Pro2232Leu or p.Glu1854*/p.Pro2232Leu variants.In conclusion, our results con rm the pathophysiological importance of altered TG folding as a consequence of missense variants located in the ChEL domain of TG.

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“…A general investigation on genetic changes found in cases of congenital goiter was presented by Nicholas et al [128] in 2016. A specific mutation affecting the linker domain of thyroglobulin, i.e., p.L571P, has been recently described in relation to the conformational maturation and intracellular handling of rat thyroglobulin leading to intracellular retention [129] . An extensive description of the genetics and biochemistry of thyroglobulin was recently published by Citterio et al [130] .…”

Section: Morphologymentioning

confidence: 99%

Global view on the pathogenesis of benign thyroid disease based on historical, experimental, biochemical and genetic data, identifying the role of magnesium, selenium, coenzyme Q10 and iron in the context of the unfolded protein response and protein quality control of thyroglobulin

Moncayo¹,

Moncayo²,

J³

2020

jtgg

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p.L571P in the linker domain of rat thyroglobulin causes intracellular retention

Cited by 7 publications

References 55 publications

Targeted Next-Generation Sequencing of Congenital Hypothyroidism-Causative Genes Reveals Unexpected Thyroglobulin Gene Variants in Patients with Iodide Transport Defect

Targeted Next-Generation Sequencing of Congenital Hypothyroidism-Causative Genes Reveals Unexpected Thyroglobulin Gene Variants in Patients with Iodide Transport Defect

The p.Pro2232Leu variant in the ChEL domain of thyroglobulin gene causes intracellular transport disorder and congenital hypothyroidism

Global view on the pathogenesis of benign thyroid disease based on historical, experimental, biochemical and genetic data, identifying the role of magnesium, selenium, coenzyme Q10 and iron in the context of the unfolded protein response and protein quality control of thyroglobulin

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