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In the last decade, an incredible improvement has been made in elucidating the genetic bases of cardiomyopathies. Here we report the impact of either the European Society of Cardiology (ESC) guidelines or the use of whole exome sequencing (WES) in terms of a number of variants of uncertain significance (VUS) and missed diagnoses in a series of 260 patients affected by inherited cardiac disorders. Samples were analyzed using a targeted gene panel of 128 cardiac-related genes and/or WES in a subset of patients, with a three-tier approach. Analyzing (i) only a subset of genes related to the clinical presentation, strictly following the ESC guidelines, 20.77% positive test were assessed. The incremental diagnostic rate for (ii) the whole gene panel, and (iii) the WES was 4.71% and 11.67%, respectively. The diverse analytical approaches increased the number of VUSs and incidental findings. Indeed, the use of WES highlights that there is a small percentage of syndromic conditions that standard analysis would not have detected. Moreover, the use of targeted sequencing coupled with "narrow" analytical approach prevents the detection of variants in actionable genes that could allow for preventive treatment. Our data suggest that genetic testing might aid clinicians in the diagnosis of inheritable cardiac disorders.
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Forensic pathologists are frequently asked to investigate cases of sudden death (SD), and identifying the cause of death can be of particular importance, especially where it may be necessary to perform family screening among the relatives of the victim. A multidisciplinary approach inclusive of genetic analysis is therefore strongly recommended. According to forensic practice, arrhythmogenic cardiomyopathy (ACM) is a well-known cause of SD. However, cases of SD caused by a left ventricular pattern of ACM diagnosed at autopsy are rarely reported in the literature. We present the case of an apparently healthy, 37-year-old male found dead at his home. At autopsy, multiple foci of epicardial and mid-wall fibrous and fibro-adipose tissue were observed within the left ventricle and, to a lesser extent, within the interventricular septum. Toxicology was negative, whereas a filamin C truncating mutation was detected through genetic analysis. To our knowledge, this is the first instance of arrhythmogenic left ventricular cardiomyopathy being diagnosed at autopsy.
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Filaminas , Mutação , Humanos , Masculino , Adulto , Filaminas/genética , Autopsia , Displasia Arritmogênica Ventricular Direita/genética , Ventrículos do Coração/patologia , Patologia Legal/métodos , Morte Súbita Cardíaca/etiologiaRESUMO
Phenotypic and genotypic heterogeneity in congenital ocular diseases, especially in anterior segment dysgenesis (ASD), have created challenges for proper diagnosis and classification of diseases. Over the last decade, genomic research has indeed boosted our understanding in the molecular basis of ASD and genes associated with both autosomal dominant and recessive patterns of inheritance have been described with a wide range of expressivity. Here we describe the molecular characterization of a cohort of 162 patients displaying isolated or syndromic congenital ocular dysgenesis. Samples were analyzed with diverse techniques, such as direct sequencing, multiplex ligation-dependent probe amplification, and whole exome sequencing (WES), over 20 years. Our data reiterate the notion that PAX6 alterations are primarily associated with ASD, mostly aniridia, since the majority of the cohort (66.7%) has a pathogenic or likely pathogenic variant in the PAX6 locus. Unexpectedly, a high fraction of positive samples (20.3%) displayed deletions involving the 11p13 locus, either partially/totally involving PAX6 coding region or abolishing its critical regulatory region, underlying its significance. Most importantly, the use of WES has allowed us to both assess variants in known ASD genes (i.e., CYP1B1, ITPR1, MAB21L1, PXDN, and PITX2) and to identify rarer phenotypes (i.e., MIDAS, oculogastrointestinal-neurodevelopmental syndrome and Jacobsen syndrome). Our data clearly suggest that WES allows expanding the analytical portfolio of ocular dysgenesis, both isolated and syndromic, and that is pivotal for the differential diagnosis of those conditions in which there may be phenotypic overlaps and in general in ASD.
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Sequenciamento do Exoma , Fator de Transcrição PAX6 , Humanos , Fator de Transcrição PAX6/genética , Masculino , Feminino , Anormalidades do Olho/genética , Anormalidades do Olho/diagnóstico , Anormalidades do Olho/patologia , Fenótipo , Segmento Anterior do Olho/anormalidades , Segmento Anterior do Olho/patologia , Mutação , Oftalmopatias/genética , Oftalmopatias/diagnóstico , Oftalmopatias/congênitoRESUMO
Tumor cells release exosomes, extracellular vesicle containing various bioactive molecules such as protein, DNA and RNA. The analysis of RNA molecules packaged in exosomes may provide new potential diagnostic or prognostic tumor biomarkers. The treatment of radioiodine-refractory aggressive thyroid cancer is still an unresolved clinical challenge, and the search for biomarkers that are detectable in early phase of the disease has become a fundamental goal for thyroid cancer research. By using transcriptome analysis, this study aimed to analyze the gene expression profiles of exosomes secreted by a non-tumorigenic thyroid cell line (Nthy-ori 3.1-exo) and a papillary thyroid cancer (TPC-1-exo) cell line, comparing them with those of cell bodies (Nthy-ori 3.1-cells and TPC-1-cells). A total of 9107 transcripts were identified as differentially expressed when comparing TPC-1-exo with TPC-1-cells and 5861 when comparing Nthy-ori 3.1-exo with Nthy-ori 3.1-cells. Among them, Sialic acid-binding immunoglobulin-like lectins 10 and 11 (SIGLEC10, SIGLEC11) and Keratin-associated protein 5 (KRTAP5-3) transcripts, genes known to be involved in cancer progression, turned out to be up-regulated only in TPC-1-exo. Gene ontology analysis revealed significantly enriched pathways, and only in TPC-1-exo were the differential expressed genes associated with an up-regulation in epigenetic processes. These findings provide a proof of concept that some mRNA species are specifically packaged in tumor-cell-derived exosomes and may constitute a starting point for the identification of new biomarkers for thyroid tumors.
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Exossomos , Neoplasias da Glândula Tireoide , Humanos , RNA/metabolismo , Exossomos/metabolismo , Corpo Celular/metabolismo , Corpo Celular/patologia , Radioisótopos do Iodo/metabolismo , Linhagem Celular Tumoral , Neoplasias da Glândula Tireoide/patologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proliferação de CélulasRESUMO
The pathogenesis of cerebral small vessel disease (CSVD) is largely unknown. Endothelial disfunction has been suggested as the turning point in CSVD development. In this study, we tested the effect of plasma from CSVD patients on human cerebral microvascular endothelial cells with the aim of describing the pattern of endothelial activation. Plasma samples from three groups of young subjects have been tested: PTs (subjects affected by early stage CSVD); CTRLs (control subjects without abnormalities at MRI scanning); BDs (blood donors). Human Brain Endothelial Cells 5i (HBEC5i) were treated with plasma and total RNA was extracted. RNAs were pooled to reduce gene expression-based variability and NGS analysis was performed. Differentially expressed genes were highlighted comparing PTs, CTRLs and BDs with HBEC5i untreated cells. No significantly altered pathway was evaluated in BD-related treatment. Regulation of p38 MAPK cascade (GO:1900744) was the only pathway altered in CTRL-related treatment. Indeed, 36 different biological processes turned out to be deregulated after PT treatment of HBEC5i, i.e., the cytokine-mediated signaling pathway (GO:0019221). Endothelial cells activate inflammatory pathways in response to stimuli from CSVD patients' plasma, suggesting the pathogenetic role of neuroinflammation from the early asymptomatic phases of cerebrovascular disease.
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NK2 genes (NKX2 gene cluster in humans) encode for homeodomain-containing transcription factors that are conserved along the phylogeny. According to the most detailed classifications, vertebrate NKX2 genes are classified into two distinct families, NK2.1 and NK2.2. The former is constituted by NKX2-1 and NKX2-4 genes, which are homologous to the Drosophila scro gene; the latter includes NKX2-2 and NKX2-8 genes, which are homologous to the Drosophila vnd gene. Conservation of these genes is not only related to molecular structure and expression, but also to biological functions. In Drosophila and vertebrates, NK2 genes share roles in the development of ventral regions of the central nervous system. In vertebrates, NKX2 genes have a relevant role in the development of several other organs such as the thyroid, lung, and pancreas. Loss-of-function mutations in NKX2-1 and NKX2-2 are the monogenic cause of the brain-lung-thyroid syndrome and neonatal diabetes, respectively. Alterations in NKX2-4 and NKX2-8 genes may play a role in multifactorial diseases, autism spectrum disorder, and neural tube defects, respectively. NKX2-1, NKX2-2, and NKX2-8 are expressed in various cancer types as either oncogenes or tumor suppressor genes. Several data indicate that evaluation of their expression in tumors has diagnostic and/or prognostic value.
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PURPOSE: According to the American College of Medical Genetics (ACMG) classification, variants of uncertain significance (VUS) are gene variations whose impact on the disease risk is not yet known. VUS, therefore, represent an unmet need for genetic counselling. Aim of the study is the use the AlphaFold artificial intelligence algorithm to predict the impact of novel mutations of the IGFALS gene, detected in a subject with short stature and initially classified as VUS according to the ACMG classification. METHODS: A short-stature girl and her parents have been investigated. IGFALS mutations have been detected through clinical exome and confirmed by Sanger sequencing. The potential presence of co-occurring gene alterations was investigated in the proband by whole exome and CGH array. Structure of the ALS protein (encoded by the IGFALS gene) was evaluated through the AlphaFold artificial intelligence algorithm. RESULTS: Two IGFALS variants were found in the proband: c.1349T > C (p.Leu450Pro) and c.1363_1365delCTC (p.Leu455del), both classified as VUS, according to ACMG. Parents' analysis highlighted the in trans position of the two variants. AlphaFold showed that the mutated positions were found the concave side a horseshoe structure of the ALS protein, likely interfering with protein-protein interactions. According to a loss of function (LoF) effect of the two variants, reduced levels of the IGF1 and IGFBP-3 proteins, as well as a growth hormone (GH) excess were detected in the proband's serum. CONCLUSIONS: By using the AlphaFold structure we were able to predict two IGFALS gene mutations initially classified as VUS, as potentially pathogenetic. Our proof-of-concept showed a potential application of AlphaFold as tool to a better inform VUS interpretation of genetic tests.
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Esclerose Lateral Amiotrófica , Humanos , Feminino , Inteligência Artificial , Proteínas de Transporte , Glicoproteínas/genética , MutaçãoRESUMO
Classical aniridia is a congenital and progressive panocular disorder almost exclusively caused by heterozygous loss-of-function variants at the PAX6 locus. We report nine individuals from five families with severe aniridia and/or microphthalmia (with no detectable PAX6 mutation) with ultrarare monoallelic missense variants altering the Arg51 codon of MAB21L1. These mutations occurred de novo in 3/5 families, with the remaining families being compatible with autosomal dominant inheritance. Mice engineered to carry the p.Arg51Leu change showed a highly-penetrant optic disc anomaly in heterozygous animals with severe microphthalmia in homozygotes. Substitutions of the same codon (Arg51) in MAB21L2, a close homolog of MAB21L1, cause severe ocular and skeletal malformations in humans and mice. The predicted nucleotidyltransferase function of MAB21L1 could not be demonstrated using purified protein with a variety of nucleotide substrates and oligonucleotide activators. Induced expression of GFP-tagged wildtype and mutant MAB21L1 in human cells caused only modest transcriptional changes. Mass spectrometry of immunoprecipitated protein revealed that both mutant and wildtype MAB21L1 associate with transcription factors that are known regulators of PAX6 (MEIS1, MEIS2 and PBX1) and with poly(A) RNA binding proteins. Arg51 substitutions reduce the association of wild-type MAB21L1 with TBL1XR1, a component of the NCoR complex. We found limited evidence for mutation-specific interactions with MSI2/Musashi-2, an RNA-binding proteins with effects on many different developmental pathways. Given that biallelic loss-of-function variants in MAB21L1 result in a milder eye phenotype we suggest that Arg51-altering monoallelic variants most plausibly perturb eye development via a gain-of-function mechanism.
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Aniridia , Microftalmia , Humanos , Animais , Camundongos , Microftalmia/genética , Fator de Transcrição PAX6/genética , Aniridia/genética , Mutação de Sentido Incorreto , Heterozigoto , Fatores de Transcrição/genética , Proteínas de Homeodomínio/genética , Proteínas de Ligação a RNA/genética , Proteínas do Olho/genética , Peptídeos e Proteínas de Sinalização Intracelular/genéticaRESUMO
N6-methyladenosine (m6A) is the most abundant internal modification of RNA in eukaryotic cells, and, in recent years, it has gained increasing attention. A good amount of data support the involvement of m6A modification in tumorigenesis, tumor progression, and metastatic dissemination. However, the role of this RNA modification in thyroid cancer still remains poorly investigated. In this study, m6A-related RNA methylation profiles are compared between a normal thyroid cell line and different thyroid cancer cell lines. With this approach, it was possible to identify the different patterns of m6A modification in different thyroid cancer models. Furthermore, by silencing METTL3, which is the main player in the RNA methylation machinery, it was possible to evaluate the impact of m6A modification on gene expression in an anaplastic thyroid cancer model. This experimental approach allowed us to identify DDI2 as a gene specifically controlled by the m6A modification in anaplastic thyroid cancer cell lines. Altogether, these data are a proof of concept that RNA methylation widely occurs in thyroid cancer cell models and open a way forward in the search for new molecular patterns for diagnostic discrimination between benign and malignant lesions.
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Carcinoma Anaplásico da Tireoide , Neoplasias da Glândula Tireoide , Linhagem Celular , Humanos , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , RNA/metabolismo , Neoplasias da Glândula Tireoide/genéticaRESUMO
The release of molecules in exosomal cargoes is involved in tumor development and progression. We compared the profiles of exosomal microRNAs released by two thyroid cancer cell lines (TPC-1 and K1) with that of non-tumorigenic thyroid cells (Nthy-ori-3-1), and we explored the network of miRNA-target interaction. After extraction and characterization of exosomes, expression levels of microRNAs were investigated using custom TaqMan Advanced array cards, and compared with those expressed in the total cell extracts. The functional enrichment and network-based analysis of the miRNAs' targets was also performed. Five microRNAs (miR-21-5p, miR-31-5p, miR-221-3p, miR-222-3p, and let-7i-3p) were significantly deregulated in the exosomes of tumor cells vs. non-tumorigenic cells, and three of them (miR-31-5p, miR-222-3p, and let-7i-3p) in the more aggressive K1 compared to TPC-1 cells. The network analysis of the five miRNAs identified some genes as targets of more than one miRNAs. These findings permitted the identification of exosomal microRNAs secreted by aggressive PTC cells, and indicated that their main targets are regulators of the tumor microenvironment. A deeper analysis of the functional role of the targets of exosomal miRNAs will provide further information on novel targets of molecular treatments for these neoplasms.
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Toward the discovery of novel reliable biomarkers, epigenetic alterations have been repeatedly proposed for the diagnosis and the development of therapeutic strategies against cancer. Indeed, for promoter methylation to actively become a tumor marker for clinical use, it must be combined with a highly informative technology evaluated in an appropriate biospecimen. Methodological standardization related to epigenetic research is, in fact, one of the most challenging tasks. Moreover, tissue-based biopsy is being complemented and, in some cases, replaced by liquid biopsy. This review will highlight the advancements made for both pre-analytical and analytical implementation for the prospective use of methylation biomarkers in clinical settings, with particular emphasis on liquid biopsy.
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Biomarcadores Tumorais , Oncologia , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Metilação de DNA , Biópsia Líquida , Estudos ProspectivosRESUMO
In-depth study of the entire SARS-CoV-2 genome has uncovered many mutations, which have replaced the lineage that characterized the first wave of infections all around the world. In December 2020, the outbreak of variant of concern (VOC) 202012/01 (lineage B.1.1.7) in the United Kingdom defined a turning point during the pandemic, immediately posing a worldwide threat on the Covid-19 vaccination campaign. Here, we reported the evolution of B.1.1.7 lineage-related infections, analyzing samples collected from January 1st 2021, until April 15th 2021, in Friuli Venezia Giulia, a northeastern region of Italy. A cohort of 1508 nasopharyngeal swabs was analyzed by High Resolution Melting (HRM) and 479 randomly selected samples underwent Next Generation Sequencing analysis (NGS), uncovering a steady and continuous accumulation of B.1.1.7 lineage-related specimens, joined by sporadic cases of other known lineages (i.e. harboring the Spike glycoprotein p.E484K mutation). All the SARS-CoV-2 genome has been analyzed in order to highlight all the rare mutations that may eventually result in a new variant of interest. This work suggests that a thorough monitoring of the SARS-CoV-2 genome by NGS is essential to contain any new variant that could jeopardize all the efforts that have been made so far to resolve the emergence of the pandemic.
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COVID-19/diagnóstico , Nasofaringe/virologia , SARS-CoV-2/classificação , Análise de Sequência de RNA/métodos , COVID-19/epidemiologia , Surtos de Doenças , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Itália/epidemiologia , Filogenia , Filogeografia , RNA Viral/genética , SARS-CoV-2/genética , Reino Unido/epidemiologiaRESUMO
BACKGROUND: Cornelia de Lange syndrome (CdLS) is a rare and clinically variable syndrome characterized by growth impairment, multi-organ anomalies, and a typical set of facial dysmorphisms. Here we describe a 2-year-old female child harboring a novel de novo missense variant in HDAC8, whose phenotypical score, according to the recent consensus on CdLS clinical diagnostic criteria, allowed the diagnosis of a non-classic CdLS. METHODS: Clinical exome sequencing was performed on the trio, identifying a de novo heterozygous variant in HDAC8 (NM_018486; c. 356C>G p.Thr119Arg). Molecular modeling was performed to evaluate putative functional consequence of the HDAC8 protein. RESULTS: The variant HDAC8 c.356C>G is classified as pathogenic following the ACMG (American College of Medical Genetics and Genomics)/AMP (Association for Molecular Pathology) guidelines. By molecular modeling, we confirmed the deleterious effect of this variant, since the amino acid change compromises the conformational flexibility of the HDAC8 loop required for optimal catalytic function. CONCLUSION: We described a novel Thr119Arg mutation in HDAC8 in a patient displaying the major phenotypic traits of the CdLS. Our results suggest that a modest change outside an active site is capable of triggering global structural changes that propagate through the protein scaffold to the catalytic site, creating de facto haploinsufficiency.
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Síndrome de Cornélia de Lange/genética , Histona Desacetilases/genética , Mutação de Sentido Incorreto , Proteínas Repressoras/genética , Pré-Escolar , Síndrome de Cornélia de Lange/patologia , Feminino , Histona Desacetilases/química , Humanos , Conformação Proteica , Proteínas Repressoras/químicaRESUMO
Exosomes released from tumor cells are instrumental in shaping the local tumor microenvironment to allow cancer progression. Recently, it has been shown that tumor exosomes carry large fragments of dsDNA, which may reflect the mutational status of parental cells. Although it has been described that a stressful microenvironment can influence exosomal cargo, the effects on DNA packing and its transfer into recipient cells have yet to be investigated. Here, we report that exosomes derived from SW480 (human colorectal adenocarcinoma cell line) cells can carry dsDNA fragments containing the entire coding sequence of both TP53 and KRAS genes, harboring the SW480-related TP53 c.818G > A and KRAS c.35G > T typical mutations. We also report the following: that cell stimulation with lipopolysaccharides (LPS) promotes the selective packaging of the TP53 gene, but not the KRAS gene; that exosomes secreted by SW480 cells efficiently transfer the mutated sequences into normal CCD841-CoN colon epithelial and THLE-2 hepatic cells; that this mechanism is more efficient when the cells had been previously incubated with pro-inflammatory cytokines; that the TP53 gene appears actively transcribed in both recipient cells; and that mutated mRNA levels are not influenced by cytokine treatment. Our data strongly suggest that pro-inflammatory stimulation promotes the horizontal transfer of an oncogene by exosomes, although this remains a rare event. Further studies are needed to assess the impact of the oncogenic transfer by exosomes in malignant transformation and its role in tumor progression.
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Transformação Celular Neoplásica/patologia , Neoplasias Colorretais/patologia , Exossomos/genética , Mediadores da Inflamação/imunologia , Mutação , Microambiente Tumoral/imunologia , Proteína Supressora de Tumor p53/genética , Transformação Celular Neoplásica/genética , Neoplasias Colorretais/genética , Humanos , Células Tumorais CultivadasRESUMO
BACKGROUND: Colorectal cancer (CRC) represents the fourth leading cause of cancer-related deaths. The heterogeneity of CRC identity limits the usage of cell lines to study this type of tumor because of the limited representation of multiple features of the original malignancy. Patient-derived colon organoids (PDCOs) are a promising 3D-cell model to study tumor identity for personalized medicine, although this approach still lacks detailed characterization regarding molecular stability during culturing conditions. Correlation analysis that considers genomic, transcriptomic, and proteomic data, as well as thawing, timing, and culturing conditions, is missing. METHODS: Through integrated multi-omics strategies, we characterized PDCOs under different growing and timing conditions, to define their ability to recapitulate the original tumor. RESULTS: Whole Exome Sequencing allowed detecting temporal acquisition of somatic variants, in a patient-specific manner, having deleterious effects on driver genes CRC-associated. Moreover, the targeted NGS approach confirmed that organoids faithfully recapitulated patients' tumor tissue. Using RNA-seq experiments, we identified 5125 differentially expressed transcripts in tumor versus normal organoids at different time points, in which the PTEN pathway resulted of particular interest, as also confirmed by further phospho-proteomics analysis. Interestingly, we identified the PTEN c.806_817dup (NM_000314) mutation, which has never been reported previously and is predicted to be deleterious according to the American College of Medical Genetics and Genomics (ACMG) classification. CONCLUSION: The crosstalk of genomic, transcriptomic and phosphoproteomic data allowed to observe that PDCOs recapitulate, at the molecular level, the tumor of origin, accumulating mutations over time that potentially mimic the evolution of the patient's tumor, underlining relevant potentialities of this 3D model.
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Neoplasias Colorretais/enzimologia , Organoides/enzimologia , PTEN Fosfo-Hidrolase/metabolismo , Neoplasias Colorretais/genética , Progressão da Doença , Humanos , Proteômica/métodos , Sequenciamento do Exoma/métodosRESUMO
SPG6 accounts for 1% of autosomal dominant Hereditary Spastic Paraplegia (HSP) and is caused by pathogenic variants in NIPA1, which encodes a magnesium transporter located in plasma membrane and early endosomes, implicated in neuronal development and maintenance. Here we report a 39-year-old woman affected by progressive gait disturbance associated to absence seizures episodes within childhood. Clinical exome sequencing identified a likely pathogenic de novo heterozygous variant in NIPA1 (NM_144599.5 c.249 C > G; p.Asn83Lys). Molecular modelling was performed to evaluate putative functional consequence of the NIPA1 protein. Indeed, the Asn83Lys modification is predicted to induce a significant perturbation of the protein structure, altering signal transduction or small-molecule transport by modulating the length of the second transmembrane domain. This is the first study reporting a SPG6-affected patient harbouring the NIPA1 p.Asn83Lys mutation.
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Estudos de Associação Genética , Predisposição Genética para Doença , Proteínas de Membrana/genética , Mutação de Sentido Incorreto , Paraplegia Espástica Hereditária/diagnóstico , Paraplegia Espástica Hereditária/genética , Adulto , Alelos , Substituição de Aminoácidos , Feminino , Estudos de Associação Genética/métodos , Genótipo , Humanos , FenótipoRESUMO
BACKGROUND: Understanding the molecular mechanisms underlying papillary thyroid cancer (PTC) proved to be vital not only for diagnostic purposes but also for tailored treatments. Despite the strong evidence of heritability, only a small subset of alterations has been implicated in PTC pathogenesis. To this reason, we used targeted next-generation sequencing (NGS) to identify candidate variants implicated in PTC pathogenesis, progression, and invasiveness. METHODS: A total of 42 primary PTC tissues were investigated using a targeted next-generation sequencing (NGS) panel enlisting 47 genes involved in DNA repair and tumor progression. RESULTS: We identified 57 point mutations in 78.5% of samples (n = 32). Thirty-two somatic mutations were identified exclusively in known thyroid cancer genes (BRAF, KRAS, NRAS, and TERT). Unpredictably, 45% of the all identified mutations (n = 25) resulted to be germline, most affecting DNA repair genes. Interestingly, none of the latter variants was in the main population databases. Following ACMG classification, 20% of pathogenic/likely pathogenic and 68% of variant of unknown significance were identified. CONCLUSIONS: Overall, our results support the hypothesis that rare germline variants in DNA repair genes are accountable for PTC susceptibility. More data, including the segregation analysis in affected families, should be collected before definitely annotate these alterations and to establish their potential prognostic and treatment implications.
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Neoplasias da Glândula Tireoide , Reparo do DNA/genética , Células Germinativas , Mutação em Linhagem Germinativa , Humanos , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Câncer Papilífero da Tireoide/genética , Neoplasias da Glândula Tireoide/genéticaRESUMO
BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has rapidly spread worldwide from the beginning of 2020. Quantitative reverse transcription-PCR (RT-qPCR) is, to this day, the preferred methodology for viral RNA detection, even if not without problems. To overcome some of the limitations still existing for the detection and quantification of nucleic acids in various applications, the use of one-step reverse transcription-droplet digital PCR (RT-ddPCR) has been established. The purpose of this study was, then, to evaluate the efficacy of ddPCR for the detection of SARS-CoV-2 RNA in nasopharyngeal swabs, optimizing the detection of low-viral load-burdened samples. METHODS: The RT-ddPCR workflow was validated for sensitivity, specificity, linearity, reproducibility, and precision using samples from 90 COVID-19-infected patients referred to the Department of Laboratory Medicine of the University Hospital of Udine (Italy). RESULTS: The present study shows that RT-ddPCR allows the detection of as low as 10.3 copies of a SARS-COV-2 E-gene per sample with a higher level of accuracy and precision, especially at low concentration. CONCLUSION: During the postpeak phase of the SARS-CoV-2 pandemic, it is essential to rely on a highly robust molecular biology method to identify infected subjects, whether they have symptoms or not, in order to prepare appropriate containment measures.
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Teste de Ácido Nucleico para COVID-19/métodos , Nasofaringe/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , SARS-CoV-2/genética , Portador Sadio/virologia , Humanos , Limite de Detecção , RNA Viral/genética , Carga Viral , Fluxo de TrabalhoRESUMO
The Silver-Russell syndrome (SRS) is a rare disorder characterized by heterogeneous clinical features, including growth retardation, typical facial dysmorphisms, and body asymmetry. Genetic alterations causative of SRS mostly affect imprinted genes located on chromosomes 7 or 11. Hypomethylation of the Imprinting Center 1 (IC1) of the chromosome 11p15.5 is the most common cause of SRS, while the Imprinting Center 2 (IC2) has been more rarely involved. Specifically, maternally inherited 11p15.5 deletions including the IC2 have been associated with the Beckwith-Wiedemann Syndrome (BWS), while paternal deletions with a variable spectrum of phenotypes. Here, we describe the case of a girl with a mild SRS phenotype associated with a paternally inherited 1.4 kb deletion of IC2. The father of the proband inherited the deletion from his mother and showed normal growth, while the paternal grandmother had the deletion on her paternal chromosome and exhibited short stature. Together with previous findings obtained in mouse and humans, our data support the notion that deletion of the paternal copy of IC2 can cause SRS.