BUSCADOR RECOLECTA

Ground reaction force data from the recorded stance phases in the non-operated control sheep of the study (n = 3)., Peer reviewed

Complete set of all experimental data for all analyzed gait parameters (GRFpeak, GRFmean, t and Imp) in all animals in the study., Peer reviewed

Complete set of all data asymmetries in the analyzed gait parameters (GRFpeak, GRFmean, t and Imp) calculated between the hind- and forelimbs., Peer reviewed

Peer reviewed

Supplementary figure S1. Schematic representation of the hypothetical consequences of the THRB variant. In silico prediction of the effect of the spliceogenic variant identified in TRβ1 isoform (NM_001354712.2: c.283+1G>A) showed two scenarios. (A), The skipping of exon 5 disrupts the A/B domain, leaving a protein of 374 amino acids with a secondary structure different from the wild-type protein. The DNAbinding domain (DBD), hinge domain and ligand-binding domain (LBD) are not affected. (B), The skipping of exons 5 and 6 results in a premature codon stop that produces only a small peptide. The 3D models of the proteins were generated using the IntFOLD and PEP-FOLD4 online servers., Inherited retinal dystrophies (IRDs) are a clinically and genetically heterogeneous group of disorders that often severely impair vision. Some patients manifest poor central vision as the first symptom due to cone-dysfunction, which is consistent with cone dystrophy (COD), Stargardt disease (STGD), or macular dystrophy (MD) among others. Here, we aimed to identify the genetic cause of autosomal dominant COD in one family. WGS was performed in 3 affected and 1 unaffected individual using the TruSeq Nano DNA library kit and the NovaSeq 6,000 platform (Illumina). Data analysis identified a novel spliceogenic variant (c.283 + 1G>A) in the thyroid hormone receptor beta gene (THRB) as the candidate disease-associated variant. Further genetic analysis revealed the presence of the same heterozygous variant segregating in two additional unrelated dominant pedigrees including 9 affected individuals with a diagnosis of COD (1), STGD (4), MD (3) and unclear phenotype (1). THRB has been previously reported as a causal gene for autosomal dominant and recessive thyroid hormone resistance syndrome beta (RTHβ); however, none of the IRD patients exhibited RTHβ. Genotype-phenotype correlations showed that RTHβ can be caused by both truncating and missense variants, which are mainly located at the 3′ (C-terminal/ligand-binding) region, which is common to both THRB isoforms (TRβ1 and TRβ2). In contrast, the c.283 + 1G>A variant is predicted to disrupt a splice site in the 5′-region of the gene that encodes the N-terminal domain of the TRβ1 isoform protein, leaving the TRβ2 isoform intact, which would explain the phenotypic variability observed between RTHβ and IRD patients. Interestingly, although monochromacy or cone response alterations have already been described in a few RTHβ patients, herein we report the first genetic association between a pathogenic variant in THRB and non-syndromic IRDs. We thereby expand the phenotype of THRB pathogenic variants including COD, STGD, or MD as the main clinical manifestation, which also reflects the extraordinary complexity of retinal functions mediated by the different THRB isoforms., Peer reviewed

Supplementary Table 1. (A), Annotated rare SNVs from WGS data of family A. Dataset with the variants filtered by MAF<0.01 0.01 in gnomAD database from the four sequenced individuals of family A (FamA-II:1, II:2, III:1, and III:2). The prioritization was done using the settings and the cutoffs described elsewhere (Gonzalez-Del Pozo et al., 2022). The colum "Filtering step" shows the tag for the filter that allow priotitize the variant. (B), Structural variants (SVs) and copy number variants (CNVs) priorized for the manual curation from WGS data of family A. Manta (v. 1.5.0) was used for the variant calling of SVs and CNVs was identified by Control-FREEC (v.11.5) and PennCNV (v. 1.0.5). AnnotSV 2.2 online software was used for the annotation., Inherited retinal dystrophies (IRDs) are a clinically and genetically heterogeneous group of disorders that often severely impair vision. Some patients manifest poor central vision as the first symptom due to cone-dysfunction, which is consistent with cone dystrophy (COD), Stargardt disease (STGD), or macular dystrophy (MD) among others. Here, we aimed to identify the genetic cause of autosomal dominant COD in one family. WGS was performed in 3 affected and 1 unaffected individual using the TruSeq Nano DNA library kit and the NovaSeq 6,000 platform (Illumina). Data analysis identified a novel spliceogenic variant (c.283 + 1G>A) in the thyroid hormone receptor beta gene (THRB) as the candidate disease-associated variant. Further genetic analysis revealed the presence of the same heterozygous variant segregating in two additional unrelated dominant pedigrees including 9 affected individuals with a diagnosis of COD (1), STGD (4), MD (3) and unclear phenotype (1). THRB has been previously reported as a causal gene for autosomal dominant and recessive thyroid hormone resistance syndrome beta (RTHβ); however, none of the IRD patients exhibited RTHβ. Genotype-phenotype correlations showed that RTHβ can be caused by both truncating and missense variants, which are mainly located at the 3′ (C-terminal/ligand-binding) region, which is common to both THRB isoforms (TRβ1 and TRβ2). In contrast, the c.283 + 1G>A variant is predicted to disrupt a splice site in the 5′-region of the gene that encodes the N-terminal domain of the TRβ1 isoform protein, leaving the TRβ2 isoform intact, which would explain the phenotypic variability observed between RTHβ and IRD patients. Interestingly, although monochromacy or cone response alterations have already been described in a few RTHβ patients, herein we report the first genetic association between a pathogenic variant in THRB and non-syndromic IRDs. We thereby expand the phenotype of THRB pathogenic variants including COD, STGD, or MD as the main clinical manifestation, which also reflects the extraordinary complexity of retinal functions mediated by the different THRB isoforms., Peer reviewed

Supplementary Table 2 Different THRB in vivo and in vitro knock-out and knockdown models and its phenotypic effect., Inherited retinal dystrophies (IRDs) are a clinically and genetically heterogeneous group of disorders that often severely impair vision. Some patients manifest poor central vision as the first symptom due to cone-dysfunction, which is consistent with cone dystrophy (COD), Stargardt disease (STGD), or macular dystrophy (MD) among others. Here, we aimed to identify the genetic cause of autosomal dominant COD in one family. WGS was performed in 3 affected and 1 unaffected individual using the TruSeq Nano DNA library kit and the NovaSeq 6,000 platform (Illumina). Data analysis identified a novel spliceogenic variant (c.283 + 1G>A) in the thyroid hormone receptor beta gene (THRB) as the candidate disease-associated variant. Further genetic analysis revealed the presence of the same heterozygous variant segregating in two additional unrelated dominant pedigrees including 9 affected individuals with a diagnosis of COD (1), STGD (4), MD (3) and unclear phenotype (1). THRB has been previously reported as a causal gene for autosomal dominant and recessive thyroid hormone resistance syndrome beta (RTHβ); however, none of the IRD patients exhibited RTHβ. Genotype-phenotype correlations showed that RTHβ can be caused by both truncating and missense variants, which are mainly located at the 3′ (C-terminal/ligand-binding) region, which is common to both THRB isoforms (TRβ1 and TRβ2). In contrast, the c.283 + 1G>A variant is predicted to disrupt a splice site in the 5′-region of the gene that encodes the N-terminal domain of the TRβ1 isoform protein, leaving the TRβ2 isoform intact, which would explain the phenotypic variability observed between RTHβ and IRD patients. Interestingly, although monochromacy or cone response alterations have already been described in a few RTHβ patients, herein we report the first genetic association between a pathogenic variant in THRB and non-syndromic IRDs. We thereby expand the phenotype of THRB pathogenic variants including COD, STGD, or MD as the main clinical manifestation, which also reflects the extraordinary complexity of retinal functions mediated by the different THRB isoforms., Peer reviewed

Supplementary Table 3 Clinical and biochemical parameters of available patients harboring the c.283+1G>A variant in THRB gene. Abnormal values are in bold numbers., Inherited retinal dystrophies (IRDs) are a clinically and genetically heterogeneous group of disorders that often severely impair vision. Some patients manifest poor central vision as the first symptom due to cone-dysfunction, which is consistent with cone dystrophy (COD), Stargardt disease (STGD), or macular dystrophy (MD) among others. Here, we aimed to identify the genetic cause of autosomal dominant COD in one family. WGS was performed in 3 affected and 1 unaffected individual using the TruSeq Nano DNA library kit and the NovaSeq 6,000 platform (Illumina). Data analysis identified a novel spliceogenic variant (c.283 + 1G>A) in the thyroid hormone receptor beta gene (THRB) as the candidate disease-associated variant. Further genetic analysis revealed the presence of the same heterozygous variant segregating in two additional unrelated dominant pedigrees including 9 affected individuals with a diagnosis of COD (1), STGD (4), MD (3) and unclear phenotype (1). THRB has been previously reported as a causal gene for autosomal dominant and recessive thyroid hormone resistance syndrome beta (RTHβ); however, none of the IRD patients exhibited RTHβ. Genotype-phenotype correlations showed that RTHβ can be caused by both truncating and missense variants, which are mainly located at the 3′ (C-terminal/ligand-binding) region, which is common to both THRB isoforms (TRβ1 and TRβ2). In contrast, the c.283 + 1G>A variant is predicted to disrupt a splice site in the 5′-region of the gene that encodes the N-terminal domain of the TRβ1 isoform protein, leaving the TRβ2 isoform intact, which would explain the phenotypic variability observed between RTHβ and IRD patients. Interestingly, although monochromacy or cone response alterations have already been described in a few RTHβ patients, herein we report the first genetic association between a pathogenic variant in THRB and non-syndromic IRDs. We thereby expand the phenotype of THRB pathogenic variants including COD, STGD, or MD as the main clinical manifestation, which also reflects the extraordinary complexity of retinal functions mediated by the different THRB isoforms., Peer reviewed

Supplementary Figure 2 Generation of a HVEM-deficient A20-GFP tumor cell line using a CRISPR-Cas9 approach. (A) Mouse HVEM gene is encoded by eight exons. Deletion of exon 1 encoding the first part of the signal peptide led to abrogation of HVEM expression on the cell membrane of tumor cells. Different domains of HVEM are abbreviated as follows: TM, transmembrane region; CRD, Cystein-Rich Domain; IC, intracellular domain. (B) Cleavage efficiency of different sgRNA guides designed over exon 1: control HVEM DNA target sequence, HVEM DNA target sequence plus Cas9 or HVEM DNA plus Cas9 incubated with different sgRNA guides were loaded and run in a 1% agarose gel. (C) PCR amplification of HVEM mutation of exon 1 from A20-GFP HVEM WT (LCL-7) tumor cell line and A20-GFP HVEM KO deficient tumor cell line (LCL-25). The expected band for the amplicon obtained with the flanking primers designed on exon 1 was 1090 bp (A20 HVEM WT), whereas in HVEM deficient tumor cell line was 618 bp. The indel mutation introduced into exon 1 encompassed 472 bp affecting the 5´UTR region, exon 1, exon 1 and part of intron 1-2 sequences. (D) Amino acid sequence alignment showing the complete deletion of the first part of the signal peptide encoded by exon 1 of mouse HVEM gene. The alignment of the amino acid sequence of the HVEM WT protein versus HVEM KO protein was performed with Clustal Omega (http://www.ebi.ac.uk/tools/clustalo/. An asterisk displays identical amino acids indicating perfect alignment (*). (E) Flow cytometry histograms of A20 HVEM WT, A20-GFP HVEM WT (LCL-7) and A20-GFP HVEM KO (LCL-25) cell lines stained with biotinylated isotype matched control (rat IgG2a, AFRC MAC 157) antibody or biotinylated rat anti-mouse HVEM monoclonal antibody (clone 6C9) and biotinylated rat anti-mouse BTLA (clon 4G12b) mAbs. The binding reactions were developed using streptavidin (SA)-Alexa Fluor 647 or SA-BV421. (F) In vitro rate of proliferation of HVEM WT and HVEM KO cell lines. Four replicates of A20 HVEM WT or A20 HVEM KO tumor cells were seeded in a 24 well plate at the rate of 5000 cells per well and cell counting was performed every day over a period of 6 days. Total number of cells (×106) in culture is plotted at different time points., Introduction: A high frequency of mutations affecting the gene encoding Herpes Virus Entry Mediator (HVEM, TNFRSF14) is a common clinical finding in a wide variety of human tumors, including those of hematological origin., Methods: We have addressed how HVEM expression on A20 leukemia cells influences tumor survival and its involvement in the modulation of the anti-tumor immune responses in a parental into F1 mouse tumor model of hybrid resistance by knocking-out HVEM expression. HVEM WT or HVEM KO leukemia cells were then injected intravenously into semiallogeneic F1 recipients and the extent of tumor dissemination was evaluated., Results: The loss of HVEM expression on A20 leukemia cells led to a significant increase of lymphoid and myeloid tumor cell infiltration curbing tumor progression. NK cells and to a lesser extent NKT cells and monocytes were the predominant innate populations contributing to the global increase of immune infiltrates in HVEM KO tumors compared to that present in HVEM KO tumors. In the overall increase of the adaptive T cell immune infiltrates, the stem cell-like PD-1- T cells progenitors and the effector T cell populations derived from them were more prominently present than terminally differentiated PD-1+ T cells., Conclusions: These results suggest that the PD-1- T cell subpopulation is likely to be a more relevant contributor to tumor rejection than the PD-1+ T cell subpopulation. These findings highlight the role of co-inhibitory signals delivered by HVEM upon engagement of BTLA on T cells and NK cells, placing HVEM/BTLA interaction in the spotlight as a novel immune checkpoint for the reinforcement of the anti-tumor responses in malignancies of hematopoietic origin., Peer reviewed

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/w15203679/s1, Figure S1: Chemical structure of pollutants, Figure S2: Pollutants structures as a function of the pH; Table S1: Characteristics of the pharmaceuticals; Supplementary references: [93,94,95,96,97,98]., Peer reviewed