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Background: Friedreich ataxia is the most common inherited ataxia in Europe and is mainly caused by biallelic pathogenic expansions of the GAA trinucleotide repeat in intron 1 of the FXN gene that lead to a decrease in frataxin protein levels. Rarely, affected individuals carry either a large intragenic deletion or whole-gene deletion of FXN on one allele and a full-penetrance expanded GAA repeat on the other allele., Case Presentation: We report here a patient that presented the typical clinical features of FRDA and genetic analysis of FXN intron 1 led to the assumption that the patient carried the common biallelic expansion. Subsequently, parental sample testing led to the identification of a novel intragenic deletion involving the 5'UTR upstream region and exons 1 and 2 of the FXN gene by MLPA., Conclusions: With this case, we want to raise awareness about the potentially higher prevalence of intragenic deletions and underline the essential role of parental sample testing in providing accurate genetic counselling., (© 2023. The Author(s).)

Background: Hereditary hemorrhagic telangiectasia (HHT) is a rare vascular disease inherited in an autosomal dominant manner. Disease-causing variants in endoglin ( ENG ) and activin A receptor type II-like 1 ( ACVRL1 ) genes are detected in more than 90% of the patients undergoing molecular testing. The identification of variants of unknown significance is often seen as a challenge in clinical practice that makes family screening and genetic counseling difficult. Here, we show that the implementation of cDNA analysis to assess the effect of splice site variants on mRNA splicing is a powerful tool., Methods: Gene panel sequencing of genes associated with HHT and other arteriovenous malformation-related syndromes was performed. To evaluate the effect of the splice site variants, cDNA analysis of ENG and ACVRL1 genes was carried out., Results: three novel splice site variants were identified in ENG (c.68-2A > T and c.1311+4_1311+8del) and ACVLR1 (c.526-6C > G) genes correspondingly in three individuals with HHT that met ≥ 3 Curaçao criteria. All three variants led to an aberrant splicing inducing exon skipping ( ENG :c.68-2A > T and ACVRL1 :c.526-6C > G) or intron retention ( ENG :c.1311+4_1311+8del) allowing the confirmation of the predicted effect on splicing and the reclassification from unknown significance to pathogenic/likely pathogenic of two of them., Conclusions: RNA analysis should be performed to assess and/or confirm the impact of variants on splicing. The molecular diagnosis of HHT patients is crucial to allow family screening and accurate genetic counseling. A multidisciplinary approach including clinicians and geneticists is crucial when dealing with patients with rare diseases.

Background: A higher incidence of thrombotic events, mainly pulmonary embolism (PE), has been reported in hospitalized patients with COVID-19. The main objective was to assess clinical and laboratory differences in hospitalized COVID-19 patients according to occurrence of PE., Methods: This retrospective study included all consecutive patients hospitalized with COVID-19 who underwent a computed tomography (CT) angiography for PE clinical suspicion. Clinical data and median blood test results distributed into weekly periods from COVID-19 symptoms onset, were compared between PE and non-PE patients., Results: Ninety-two patients were included, 29 (32%) had PE. PE patients were younger (63.9 (SD 13.7) vs 69.9 (SD 12.5) years). Clinical symptoms and COVID-19 CT features were similar in both groups. PE was diagnosed after a mean of 20.0 (SD 8.6) days from the onset of COVID-19 symptoms. Corticosteroid boluses were more frequently used in PE patients (62% vs. 43%). No patients met ISTH DIC criteria. Any parameter was statistically significant or clinically relevant except for D-Dimer when comparing both groups. Median values [IQR] of D-dimer in PE vs non-PE patients were: week 2 (2010.7 [770.1-11208.9] vs 626.0 [374.0-2382.2]; p = 0.004); week 3 (3893.1 [1388.2-6694.0] vs 1184.4 [461.8-2447.8]; p = 0.003); and week 4 (2736.3 [1202.1-8514.1] vs 1129.1 [542.5-2834.6]; p = 0.01). Median fold-increase of D-dimer between week 1 and 2 differed between groups (6.64 [3.02-23.05] vs 1.57 [0.64-2.71], p = 0.003); ROC curve AUC was 0.879 (p = 0.003) with a sensitivity and specificity for PE of 86% and 80%, respectively., Conclusions: Among hospitalized COVID-19 patients, D-dimer levels are higher at weeks 2, 3 and 4 after COVID-19 symptom onset in patients who develop PE. This difference is more pronounced when the fold increase between weeks 1 and 2 is compared., Competing Interests: The authors have declared that no competing interests exist.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family, which mediates apoptosis by the extrinsic pathway. Up-regulation of decoy receptors, DcR1 and DcR2, may result in diminished binding of TRAIL to their functional receptors. DcR1 expression was assessed in normal endometrial tissue (NE) and endometrial carcinoma (EC) samples by immunohistochemistry (IHC) and quantitative real-time polymerase chain reaction (PCR). IHC was performed in two tissue microarrays; one composed of 80 samples of NE and a second one constructed from paraffin-embedded blocks of 62 EC. For quantitative real-time RT-PCR analysis, RNA was obtained from 19 NE and 28 EC samples using Trizol. mRNA expression of DcR1 was assessed with Taqman-based assays in an Abi-Prism 700 SDS. Results were correlated with stage, histological type, and grade. By IHC, cytoplasmic expression of DcR1 was frequently seen in NE (79.6%) and varied according to the menstrual cycle. Positive DcR1 immunostaining was also detected in EC (98.1% of the cases) without any specific statistical association with histological type, grade, and stage. By quantitative real-time PCR, all NE had similar levels of DcR1expression (0.8-1.7 RQ), which were considered the basal levels of DcR1 expression in NE. Increased DcR1 expression (> or =5-fold higher than the basal levels) was detected in 13 of 28 EC (46.4%). High DcR1 expression levels were found in ECs of different stages: IA, four of 12 (33%); IB, two of four (50%); IC, four of six (66%); and IIA and IIB three of six (50%). Results suggest that DcR1 expression occurs in a subset of EC and may contribute to resistance to TRAIL-induced apoptosis.

TNF-alpha has been reported to be relevant in stroke-induced neuronal death. However the precise function of TNF-alpha in brain ischemia remains controversial since there are data supporting either a detrimental or a protective effect. Here we show that TNF-alpha is released after oxygen-glucose deprivation (OGD) of cortical cultures and is a major contributor to the apoptotic death observed without affecting the OGD-mediated necrotic cell death. In this paradigm, apoptosis depends on TNF-alpha-induced activation of caspase-8 and -3 without affecting the activation of caspase-9. By using knock-out mice for TNF-alpha receptor 1, we show that the activation of both caspase-3 and -8 by TNF-alpha is mediated by TNF-alpha receptor 1. The pro-apoptotic role of TNF-alpha in OGD is restricted to neurons and microglia, since astrocytes do not express either TNF-alpha or TNF-alpha receptor 1. Altogether, these results show that apoptosis of cortical neurons after OGD is mediated by TNF-alpha/TNF-alpha receptor 1.

Upon activation, tumor necrosis factor alpha (TNF-alpha) receptor can engage apoptotic or survival pathways. Inhibition of macromolecular synthesis is known to sensitize cells to TNF-alpha-induced cell death. It is believed that this sensitization is due to the transcriptional blockade of genes regulated by NF-kappaB. Nevertheless, such evidence has remained elusive in the nervous system. Here, we show that TNF-alpha cannot normally induce apoptosis in PC12 cells or cortical neurons. However, cells treated with Actinomycin D (ActD) become susceptible to TNF-alpha-induced cell death through the activation of caspase-8, generation of tBid and activation of caspase-9 and -3. Analysis of several proteins involved in TNF-alpha receptor signaling showed no significant downregulation of NF-kappaB target genes, such as IAPs or FLIP, under such conditions. However, Bcl-x(L) protein levels, but not those of Bcl-2, Bax and Bak, are reduced by ActD or TNF-alpha/ActD treatments. Moreover, Bcl-x(L) overexpression fully protects cells against TNF-alpha/ActD-induced cell death. When endogenous levels of Bcl-x(L) are specifically downregulated by lentiviral-based RNAi, cells no longer require ActD to be sensitive to TNF-alpha-triggered apoptosis. Furthermore, Bcl-x(L) downregulation does not affect TNF-alpha-mediated NF-kappaB activation. Altogether, our results demonstrate that Bcl-x(L), and not Bcl-2, FLIP or IAPs, acts as the endogenous regulator of neuronal resistance/sensitivity to TNF-alpha-induced apoptosis in an NF-kappaB-independent manner.

Inactivation of a tumor suppressor gene typically occurs in two steps, thus fulfilling Knudson hypothesis. One "hit" is frequently a point mutation or a small deletion. The other alteration is usually a large genomic loss of part of a gene, or even part of a chromosome, or the whole chromosome. However, it is not clear which of these two events occurs first. Loss of heterozygosity (LOH) analysis allows the identification of one of the 2 hits. Although microsatellite polymerase chain reaction is the technique most frequently used to assess LOH, other different approaches can also be used. The LOH can also be assessed by restriction fragment length polymorphism analysis, single strand conformation polymorphism analysis, oligonucleotide microarrays capable to simultaneously determine the genotype of thousands of single-nucleotide polymorphism (single-nucleotide polymorphism arrays), comparative genomic hybridization, multiplex amplification and probe hybridization, and multiplex ligation-dependent probe amplification. In this article, the authors review the results obtained with molecular analysis of LOH in the understanding of development and progression of endometrial carcinoma. Particular attention is given to: (1) the presence of widespread LOH in nonendometrioid carcinoma, probably reflecting the existence of chromosomal instability; and (2) specific LOH patterns associated with some clinicopathologic features.

Mitochondrial alterations have been associated with the cytotoxic effect of 6-hydroxydopamine (6-OHDA), a widely used toxin to study Parkinson's disease. In previous work, we have demonstrated that 6-OHDA increases mitochondrial membrane permeability leading to cytochrome c release, but the precise mechanisms involved in this process remain unknown. Herein we studied the mechanism of increased mitochondrial permeability of SH-SY5Y neuroblastoma cells in response to 6-OHDA. Cytochrome c release induced by 6-OHDA occurred, in both SH-SY5Y cells and primary cultures, in the absence of mitochondrial swelling or a decrease in mitochondrial calcein fluorescence, suggesting little involvement of the mitochondrial permeability transition pore in this process. In contrast, 6-OHDA-induced cell death was associated with a significant translocation of the pro-apoptotic Bax protein from the cytosol to mitochondria and with a significant induction of the BH3-only protein PUMA. Experiments in mouse embryonic fibroblasts deficient in Bax or PUMA demonstrated a role for both proteins in 6-OHDA-induced apoptosis. Although 6-OHDA elevated both total and nuclear p53 protein levels, activation of p53 was not essential for subsequent cell death. In contrast, we found that p38 mitogen-activated protein kinase (MAPK) was activated early during 6-OHDA-induced apoptosis, and that treatment with the p38 MAPK inhibitor SKF86002 potently inhibited PUMA induction, green fluorescent protein-Bax redistribution and apoptosis in response to 6-OHDA. These data demonstrate a critical involvement of p38 MAPK, PUMA, and Bax in 6-OHDA-induced apoptosis.

Death receptors (DRs) and their ligands are expressed in developing nervous system. However, neurons are generally resistant to death induction through DRs and rather their activation promotes neuronal outgrowth and branching. These results suppose the existence of DRs antagonists expressed in the nervous system. Fas apoptosis inhibitory molecule (FAIM(S)) was first identified as a Fas antagonist in B-cells. Soon after, a longer alternative spliced isoform with unknown function was identified and named FAIM(L). FAIM(S) is widely expressed, including the nervous system, and we have shown previously that it promotes neuronal differentiation but it is not an anti-apoptotic molecule in this system. Here, we demonstrate that FAIM(L) is expressed specifically in neurons, and its expression is regulated during the development. Expression could be induced by NGF through the extracellular regulated kinase pathway in PC12 (pheochromocytoma cell line) cells. Contrary to FAIM(S), FAIM(L) does not increase the neurite outgrowth induced by neurotrophins and does not interfere with nuclear factor kappaB pathway activation as FAIM(S) does. Cells overexpressing FAIM(L) are resistant to apoptotic cell death induced by DRs such as Fas or tumor necrosis factor R1. Reduction of endogenous expression by small interfering RNA shows that endogenous FAIM(L) protects primary neurons from DR-induced cell death. The detailed analysis of this antagonism shows that FAIM(L) can bind to Fas receptor and prevent the activation of the initiator caspase-8 induced by Fas. In conclusion, our results indicate that FAIM(L) could be responsible for maintaining initiator caspases inactive after receptor engagement protecting neurons from the cytotoxic action of death ligands.

The familial paraganglioma syndrome is an autosomal dominant disorder characterized by the presence of carotid body paragangliomas and, less frequently, paragangliomas of the glomus jugulare, glomus vagale, and adrenal pheochromocytomas. Germline mutations of the genes for succinate dehydrogenase subunits D, B, or C (SDHD, SDHB, SDHC) have been identified in some kindreds with familial paraganglioma. In this study, we report the clinicopathologic features of four different kindreds with familial paraganglioma, which were screened for germline mutations in the SDHD gene. DNA was obtained from tumor and normal tissue, as well as from peripheral blood. Mutation analysis was performed by single-strand conformation polymorphism analysis and DNA sequencing. SDHD germline mutations were detected in the affected family members of the four families, as well as in several asymptomatic carriers. An identical mutation in exon 4 of SDHD (334-337delACTG) was identified in two apparently unrelated kindreds. The third family showed a germline mutation in exon 2 (W43X). The mutations present in these three families had been previously described in Spanish families, suggesting a founder effect. The fourth family exhibited a mutation in exon 2 of SDHD (170-171delTT), which had not been previously identified. The affected family members of the four kindreds showed paragangliomas, located in the head and neck region, and all of them were benign. These results confirm that genetic testing of SDHD may be a powerful tool for the identification of the syndrome in patients with multiple or bilateral paragangliomas.

Caspase-activated DNase is responsible for the oligonucleosomal DNA degradation during apoptosis. DNA degradation is thought to be important for multicellular organisms to prevent oncogenic transformation or as a mechanism of viral defense. It has been reported that certain cells, including some neuroblastoma cell lines such as IMR-5, enter apoptosis without digesting DNA in such a way. We have analyzed the causes for the absence of DNA laddering in staurosporine-treated IMR-5 cells, and we have found that most of the molecular mechanisms controlling apoptosis are well preserved in this cell line. These include degradation of substrates for caspases, blockade of cell death by antiapoptotic genes such as Bcl-2 or Bcl-X(L), or normal levels and adequate activation of caspase-3. Moreover, these cells display normal levels of caspase-activated DNase and its inhibitory protein, inhibitor of caspase-activated DNase, and their cDNA sequences are identical to those reported previously. Nevertheless, IMR-5 cells lose caspase-activated DNase during apoptosis and recover their ability to degrade DNA when human recombinant caspase-activated DNase is overexpressed. Our results lead to the conclusion that caspase-activated DNase is processed during apoptosis of IMR-5 cells, making these cells a good model to study the relevance of this endonuclease in physiological or pathological conditions.