RESUMEN
Cystic fibrosis (CF) is caused by defects of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR-modulating drugs may overcome specific defects, such as the case of Trikafta, which is a clinically approved triple combination of Elexacaftor, Tezacaftor and Ivacaftor (ETI) that exhibited a strong ability to rescue the function of the most frequent F508del pathogenic variant even in genotypes with the mutated allele in single copy. Nevertheless, most rare genotypes lacking the F508del allele are still not eligible for targeted therapies. Via the innovative approach of using nasal conditionally reprogrammed cell (CRC) cell-based models that mimic patient disease in vitro, which are obtainable from each patient due to the 100% efficiency of the cell culture establishment, we theratyped orphan CFTR mutation L1077P. Protein studies, Forskolin-induced organoid swelling, and Ussing chamber assays congruently proved the L1077P variant function rescue by ETI. Notably, this rescue takes place even in the context of a single-copy L1077P allele, which appears to enhance its expression. Thus, the possibility of single-allele treatment also arises for rare genotypes, with an allele-specific modulation as part of the mechanism. Of note, besides providing indication of drug efficacy with respect to specific CFTR pathogenic variants or genotypes, this approach allows the evaluation of the response of single-patient cells within their genetic background. In this view, our studies support in vitro guided personalized CF therapies also for rare patients who are nearly excluded from clinical trials.
Asunto(s)
Fibrosis Quística , Humanos , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genéticaRESUMEN
OBJECTIVES: Sars-CoV-2 acute infection is clinically heterogeneous, ranging from asymptomatic cases to patients with a severe, systemic clinical course. Among the involved factors age and preexisting morbidities play a major role; genetic host susceptibility contributes to modulating the clinical expression and outcome of the disease. Mannose-binding lectin is an acute-phase protein that activates the lectin-complement pathway, promotes opsonophagocytosis and modulates inflammation, and is involved in several bacterial and viral infections in humans. Understanding its role in Sars-CoV-2 infection could help select a better therapy. METHODS: We studied MBL2 haplotypes in 419 patients with acute COVID-19 in comparison to the general population and related the haplotypes to clinical and laboratory markers of severity. RESULTS: We recorded an enhanced frequency of MBL2 null alleles in patients with severe acute COVID-19. The homozygous null genotypes were significantly more frequent in patients with advanced WHO score 4-7 (OR of about 4) and related to more severe inflammation, neutrophilia, and lymphopenia. CONCLUSIONS: Subjects with a defective MBL2 genotype (i.e., 0/0) are predisposed to a more severe acute Sars-CoV-2 infection; they may benefit from early replacement therapy with recombinant MBL. Furthermore, a subset of subjects with the A/A MBL genotype develop a relevant increase of serum MBL during the early phases of the disease and develop a more severe pulmonary disease; in these patients, the targeting of the complement may help. Therefore, COVID-19 patients should be tested at hospitalization with serum MBL analysis and MBL2 genotype, to define the optimal therapy.
Asunto(s)
COVID-19 , Lectina de Unión a Manosa , Humanos , COVID-19/genética , SARS-CoV-2 , Genotipo , Predisposición Genética a la Enfermedad , Lectina de Unión a Manosa/genética , InflamaciónRESUMEN
The recent pandemic years have prompted the scientific community to increasingly search for and adopt new and more efficient therapeutic and diagnostic approaches to deal with a new infection. In addition to the development of vaccines, which has played a leading role in fighting the pandemic, the development of monoclonal antibodies has also represented a valid approach in the prevention and treatment of many cases of CoronaVirus Disease 2019 (COVID-19). Recently, we reported the development of a human antibody, named D3, showing neutralizing activity against different SARS-CoV-2 variants, wild-type, UK, Delta and Gamma variants. Here, we have further characterized with different methods D3's ability to bind the Omicron-derived recombinant RBD by comparing it with the antibodies Cilgavimab and Tixagevimab, recently approved for prophylactic use of COVID-19. We demonstrate here that D3 binds to a distinct epitope from that recognized by Cilgavimab and shows a different binding kinetic behavior. Furthermore, we report that the ability of D3 to bind the recombinant Omicron RBD domain in vitro results in a good ability to also neutralize Omicron-pseudotyped virus infection in ACE2-expressing cell cultures. We point out here that D3 mAb maintains a good ability to recognize both the wild-type and Omicron Spike proteins, either when used as recombinant purified proteins or when expressed on pseudoviral particles despite the different variants, making it particularly useful both from a therapeutic and diagnostic point of view. On the basis of these results, we propose to exploit this mAb for combinatorial treatments with other neutralizing mAbs to increase their therapeutic efficacy and for diagnostic use to measure the viral load in biological samples in the current and future pandemic waves of coronaviruses.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes , Anticuerpos Antivirales/uso terapéuticoRESUMEN
Despite the promising results of new CFTR targeting drugs designed for the recovery of F508del- and class III variants activity, none of them have been approved for individuals with selected rare mutations, because uncharacterized CFTR variants lack information associated with the ability of these compounds in recovering their molecular defects. Here we used both rectal organoids (colonoids) and primary nasal brushed cells (hNEC) derived from a CF patient homozygous for A559T (c.1675G>A) variant to evaluate the responsiveness of this pathogenic variant to available CFTR targeted drugs that include VX-770, VX-809, VX-661 and VX-661 combined with VX-445. A559T is a rare mutation, found in African-Americans people with CF (PwCF) with only 85 patients registered in the CFTR2 database. At present, there is no treatment approved by FDA (U.S. Food and Drug Administration) for this genotype. Short-circuit current (Isc) measurements indicate that A559T-CFTR presents a minimal function. The acute addition of VX-770 following CFTR activation by forskolin had no significant increment of baseline level of anion transport in both colonoids and nasal cells. However, the combined treatment, VX-661-VX-445, significantly increases the chloride secretion in A559T-colonoids monolayers and hNEC, reaching approximately 10% of WT-CFTR function. These results were confirmed by forskolin-induced swelling assay and by western blotting in rectal organoids. Overall, our data show a relevant response to VX-661-VX-445 in rectal organoids and hNEC with CFTR genotype A559T/A559T. This could provide a strong rationale for treating patients carrying this variant with VX-661-VX-445-VX-770 combination.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Humanos , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , Colforsina/uso terapéutico , Benzodioxoles/farmacología , Mutación , Organoides , GenotipoRESUMEN
The frequent finding of thrombocytopenia in patients with severe SARS-CoV-2 infection (COVID-19) and previous evidence that several viruses enter platelets suggest that SARS-CoV-2 might be internalized by platelets of COVID-19. Aim of our study was to assess the presence of SARS-CoV-2 RNA in platelets from hospitalized patients with aconfirmed diagnosis of COVID-19. RNA was extracted from platelets, leukocytes and serum from 24 COVID-19 patients and 3 healthy controls, real-time PCR and ddPCR for viral genes were carried out. SARS-CoV-2 RNA was not detected in any of the samples analyzed nor in healthy controls, by either RT-PCR or ddPCR, while RNA samples from nasopharyngeal swabs of COVID-19 patients were correctly identified. Viral RNA was not detected independently of viral load, of positive nasopharyngeal swabs, or viremia, the last detected in only one patient (4.1%). SARS-CoV-2 entry in platelets is not acommon phenomenon in COVID-19 patients, differently from other viral infections.
Asunto(s)
Plaquetas/virología , COVID-19/sangre , COVID-19/virología , ARN Viral , SARS-CoV-2/fisiología , Anciano , COVID-19/diagnóstico , Prueba de COVID-19 , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena en Tiempo Real de la Polimerasa , SARS-CoV-2/aislamiento & purificación , Carga ViralRESUMEN
The rare but dangerous adverse events evidenced after massive vaccination against SARS-CoV-2 are represented by thrombosis and thrombocytopenia. The patients diagnosed with severe COVID-19 may develop a pro-thrombotic state with a much higher frequency, thus we decided to investigate the role of Spike protein (the only common product of the two conditions) or the anti-Spike antibodies in the etiopathogenesis of thrombosis. A pathogenic Platelet Factor 4 (PF4)-dependent syndrome, unrelated to the use of heparin therapy, has been reported after the administration of vaccines in the patients manifesting acute thrombocytopenia and thrombosis. Thus, we aimed at shedding light on the structural similarities of Spike of SARS-CoV-2 and PF4 on their eventual biochemical interactions and on the role of their specific antibodies. The similarities between PF4 and Spike-RBD proteins were evaluated by a comparison of the structures and by testing the cross-reactivity of their specific antibodies by ELISA assays. We found that the anti-Spike antibodies do not recognize PF4, on the contrary, the anti-PF4 antibodies show some cross-reactivity for Spike-RBD. More interestingly, we report for the first time that the PF4 and Spike-RBD proteins can bind each other. These data suggest that the interaction of the two proteins could be involved in the generation of anti-PF4 antibodies, their binding to Spike-RBD, which could lead to platelets aggregation due also to their high expression of ACE2.
Asunto(s)
Vacunas contra la COVID-19/efectos adversos , COVID-19/inmunología , Factor Plaquetario 4 , Glicoproteína de la Espiga del Coronavirus , Reacciones Cruzadas , Humanos , Factor Plaquetario 4/química , Factor Plaquetario 4/inmunología , Unión Proteica , Conformación Proteica , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunología , Trombocitopenia/patología , Trombosis/patologíaRESUMEN
Background and objectives: ischemic stroke (IS) is among the most frequent causes of death worldwide; thus, it is of paramount relevance to know predisposing factors that may help to identify and treat the high-risk subjects. Materials and Methods:we tested nine variants in genes involved in thrombotic pathway in 282 patients that experienced IS and 87 that had transient ischemic attacks (TIA) in comparison to 430 subjects from the general population (GP) of the same geographic area (southern Italy). We included cases of young and child IS to evaluate the eventual differences in the role of the analyzed variants. Results: we did not observe significant differences between TIA and the GP for any of the variants, while the allele frequencies of methylene-tetrahydrofolate reductase (MTHFR) C677T, beta-fibrinogen -455G>A and factor (FXIII) V34L were significantly higher in patients with IS than in the subjects from the GP. No significant interaction was observed with sex. Conclusions: the present data argue that some gene variants have a role in IS and this appears to be an interesting possibility to be pursued in large population studies to help design specific strategies for IS prevention.
Asunto(s)
Isquemia Encefálica , Factor XIII/genética , Ataque Isquémico Transitorio , Accidente Cerebrovascular Isquémico , Metilenotetrahidrofolato Reductasa (NADPH2) , Accidente Cerebrovascular , Isquemia Encefálica/genética , Niño , Predisposición Genética a la Enfermedad , Humanos , Ataque Isquémico Transitorio/genética , Accidente Cerebrovascular Isquémico/genética , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Polimorfismo Genético , Factores de Riesgo , Accidente Cerebrovascular/genéticaRESUMEN
Despite the unprecedented effort of the scientific community, the novel SARS-CoV-2 virus has infected more than 46 million people worldwide, killing over one million two hundred thousand. Understanding the mechanisms by which some individuals are more susceptible to SARS-CoV-2 infection and why a subgroup of them are prone to experience severe pneumonia, and death should lead to a better approach and more effective treatments for COVID-19. Here, we focus our attention on ACE2, a primary receptor of SARS-CoV-2. We will discuss its biology, tissue expression, and post-translational regulation that determine its potential to be employed by SARS-CoV-2 for cell entry. Particular attention will be given to how the ACE2 soluble form can have a great impact on disease progression and thus be used in a potential therapeutic strategy. Furthermore, we will discuss repercussions that SARS-CoV-2/ACE2 binding has on the renin-angiotensin system and beyond. Indeed, although mostly neglected, ACE2 can also act on [des-Arg 937]-bradykinin of the kinin-kallikrein system regulating coagulation and inflammation. Thorough comprehension of the role that ACE2 plays in different pathways will be the key to assess the impact that SARS-CoV-2/ACE2 binding has on organismal physiology and will help us to find better therapies and diagnostic tools.
Asunto(s)
Enzima Convertidora de Angiotensina 2/fisiología , COVID-19/etiología , SARS-CoV-2/fisiología , COVID-19/diagnóstico , COVID-19/terapia , Humanos , Receptores de Coronavirus/fisiología , Sistema Renina-Angiotensina/fisiología , Internalización del VirusRESUMEN
Pharmacological rescue of mutant cystic fibrosis transmembrane conductance regulator (CFTR) in cystic fibrosis (CF) depends on the specific defect caused by different mutation classes. We asked whether a patient with the rare p.Gly970Asp (c.2909G>A) mutation could benefit from CFTR pharmacotherapy since a similar missense mutant p.Gly970Arg (c.2908G>C) was previously found to be sensitive to potentiators in vitro but not in vivo. By complementary DNA transfection, we found that both mutations are associated with defective CFTR function amenable to pharmacological treatment. However, analysis of messenger RNA (mRNA) from patient's cells revealed that c.2908G>C impairs RNA splicing whereas c.2909G>A does not perturb splicing and leads to the expected p.Gly970Asp mutation. In agreement with these results, nasal epithelial cells from the p.Gly970Asp patient showed significant improvement of CFTR function upon pharmacological treatment. Our results underline the importance of controlling the effect of CF mutation at the mRNA level to determine if the pharmacotherapy of CFTR basic defect is appropriate.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/genética , Mutación Puntual , Codón , Fibrosis Quística/metabolismo , Células HEK293 , Humanos , Fenotipo , Empalme del ARN , TransfecciónRESUMEN
In cystic fibrosis, deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel causes misfolding and premature degradation. One possible approach to reducing the detrimental health effects of cystic fibrosis could be the identification of proteins whose suppression rescues F508del-CFTR function in bronchial epithelial cells. However, searches for these potential targets have not yet been conducted, particularly in a relevant airway background using a functional readout. To identify proteins associated with F508del-CFTR processing, we used a high-throughput functional assay to screen an siRNA library targeting 6,650 different cellular proteins. We identified 37 proteins whose silencing significantly rescued F508del-CFTR activity, as indicated by enhanced anion transport through the plasma membrane. These proteins included FAU, UBE2I, UBA52, MLLT6, UBA2, CHD4, PLXNA1, and TRIM24, among others. We focused our attention on FAU, a poorly characterized protein with unknown function. FAU knockdown increased the plasma membrane targeting and function of F508del-CFTR, but not of wild-type CFTR. Investigation into the mechanism of action revealed a preferential physical interaction of FAU with mutant CFTR, leading to its degradation. FAU and other proteins identified in our screening may offer a therapeutically relevant panel of drug targets to correct basic defects in F508del-CFTR processing.
Asunto(s)
Bronquios/metabolismo , Membrana Celular/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células Epiteliales/metabolismo , Mutación , Proteínas Ribosómicas/metabolismo , Bronquios/patología , Membrana Celular/patología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Células Epiteliales/patología , Humanos , Proteolisis , Proteínas Ribosómicas/genéticaRESUMEN
BACKGROUND: The effect of complex alleles in cystic fibrosis (CF) is poorly defined for the lack of functional studies. OBJECTIVES: To describe the genotype-phenotype correlation and the results of either in vitro and ex vivo studies performed on nasal epithelial cells (NEC) in a cohort of patients with CF carrying cystic fibrosis transmembrane conductance regulator (CFTR) complex alleles. METHODS: We studied 70 homozygous, compound heterozygous or heterozygous for CFTR mutations: p.[Arg74Trp;Val201Met;Asp1270Asn], n=8; p.[Ile148Thr;Ile1023_Val1024del], n=5; p.[Arg117Leu;Leu997Phe], n=6; c.[1210-34TG[12];1210-12T[5];2930C>T], n=3; p.[Arg74Trp;Asp1270Asn], n=4; p.Asp1270Asn, n=2; p.Ile148Thr, n=6; p.Leu997Phe, n=36. In 39 patients, we analysed the CFTR gating activity on NEC in comparison with patients with CF (n=8) and carriers (n=4). Finally, we analysed in vitro the p.[Arg74Trp;Val201Met;Asp1270Asn] complex allele. RESULTS: The p.[Ile148Thr;Ile1023_Val1024del] caused severe CF in five compound heterozygous with a class I-II mutation. Their CFTR activity on NEC was comparable with patients with two class I-II mutations (mean 7.3% vs 6.9%). The p.[Arg74Trp;Asp1270Asn] and the p.Asp1270Asn have scarce functional effects, while p.[Arg74Trp;Val201Met;Asp1270Asn] caused mild CF in four of five subjects carrying a class I-II mutation in trans, or CFTR-related disorders (CFTR-RD) in three having in trans a class IV-V mutation. The p.[Arg74Trp;Val201Met;Asp1270Asn] causes significantly (p<0.001) higher CFTR activity compared with compound heterozygous for class I-II mutations. Furthermore, five of six compounds heterozygous with the p.[Arg117Leu;Leu997Phe] had mild CF, whereas the p.Leu997Phe, in trans with a class I-II CFTR mutation, caused CFTR-RD or a healthy status (CFTR activity: 21.3-36.9%). Finally, compounds heterozygous for the c.[1210-34TG[12];1210-12T[5];2930C>T] and a class I-II mutation had mild CF or CFTR-RD (gating activity: 18.5-19.0%). CONCLUSIONS: The effect of complex alleles partially depends on the mutation in trans. Although larger studies are necessary, the CFTR activity on NEC is a rapid contributory tool to classify patients with CFTR dysfunction.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Adolescente , Adulto , Alelos , Niño , Preescolar , Fibrosis Quística/patología , Femenino , Genotipo , Heterocigoto , Homocigoto , Humanos , Masculino , Persona de Mediana Edad , Mutación , Mucosa Nasal/metabolismo , Mucosa Nasal/patología , Fenotipo , Adulto JovenRESUMEN
OBJECTIVES: We aimed to improve the knowledge of pathogenic mutations in sporadic cases of congenital chloride diarrhea (CCD) and emphasize the importance of functional studies to define the effect of novel mutations. METHODS: All member 3 of solute carrier family 26 (SLC26A3) coding regions were sequenced in 17 sporadic patients with CCD. Moreover, the minigene system was used to analyze the effect of 2 novel splicing mutations. RESULTS: We defined the SLC26A3 genotype of all 17 patients with CCD and identified 12 novel mutations. Using the minigene system, we confirmed the in silico prediction of a complete disruption of splicing pattern caused by 2 of these novel mutations: the c.971+3_971+4delAA and c.735+4_c.735+7delAGTA. Moreover, several prediction tools and a structure-function prediction defined the pathogenic role of 6 novel missense mutations. CONCLUSIONS: We confirm the molecular heterogeneity of sporadic CCD adding 12 novel mutations to the list of known pathogenic mutations. Moreover, we underline the importance, for laboratories that offer molecular diagnosis and genetic counseling, to perform fast functional analysis of novel mutations.
Asunto(s)
Antiportadores de Cloruro-Bicarbonato/genética , Diarrea/congénito , Errores Innatos del Metabolismo/genética , Mutación , Estudios de Casos y Controles , Diarrea/diagnóstico , Diarrea/genética , Marcadores Genéticos , Pruebas Genéticas , Genotipo , Técnicas de Genotipaje , Humanos , Errores Innatos del Metabolismo/diagnóstico , Transportadores de SulfatoRESUMEN
Cystic Fibrosis (CF) is one of the most common life shortening conditions in Caucasians. CF is caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene which result in reduced or altered CFTR functionality. Several microRNAs (miRNAs) downregulate the expression of CFTR, thus causing or exacerbating the symptoms of CF. In this context, the design of anti-miRNA agents represents a valid functional tool, but its translation to the clinic might lead to unpredictable side effects because of the interference with the expression of other genes regulated by the same miRNAs. Herein, for the first time, is proposed the use of peptide nucleic acids (PNAs) to protect specific sequences in the 3'UTR (untranslated region) of the CFTR messenger RNA (mRNA) by action of miRNAs. Two PNAs (7 and 13 bases long) carrying the tetrapeptide Gly-SerP-SerP-Gly at their C-end, fully complementary to the 3'UTR sequence recognized by miR-509-3p, have been synthesized and the structural features of target PNA/RNA heteroduplexes have been investigated by spectroscopic and molecular dynamics studies. The co-transfection of the pLuc-CFTR-3´UTR vector with different combinations of PNAs, miR-509-3p, and controls in A549 cells demonstrated the ability of the longer PNA to rescue the luciferase activity by up to 70% of the control, thus supporting the use of suitable PNAs to counteract the reduction in the CFTR expression.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/terapia , MicroARNs/genética , Ácidos Nucleicos de Péptidos/uso terapéutico , Regiones no Traducidas 3'/genética , Células A549 , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/antagonistas & inhibidores , Humanos , MicroARNs/antagonistas & inhibidores , Mutación , Ácidos Nucleicos de Péptidos/genética , ARN Mensajero/genética , TransfecciónAsunto(s)
Cloroquina/efectos adversos , Cloroquina/uso terapéutico , Infecciones por Coronavirus/tratamiento farmacológico , Glucosafosfato Deshidrogenasa/efectos de los fármacos , Neumonía Viral/tratamiento farmacológico , Betacoronavirus/genética , Betacoronavirus/patogenicidad , COVID-19 , Femenino , Glucosafosfato Deshidrogenasa/genética , Deficiencia de Glucosafosfato Deshidrogenasa/tratamiento farmacológico , Heterocigoto , Humanos , Masculino , Pandemias , SARS-CoV-2Asunto(s)
Síndrome Coronario Agudo/genética , Trastornos de la Coagulación Sanguínea Heredados/genética , Coagulación Sanguínea/genética , Variación Genética , Infarto del Miocardio/genética , Síndrome Coronario Agudo/sangre , Síndrome Coronario Agudo/epidemiología , Síndrome Coronario Agudo/prevención & control , Adolescente , Adulto , Edad de Inicio , Anciano , Anciano de 80 o más Años , Trastornos de la Coagulación Sanguínea Heredados/sangre , Trastornos de la Coagulación Sanguínea Heredados/tratamiento farmacológico , Trastornos de la Coagulación Sanguínea Heredados/epidemiología , Niño , Preescolar , Femenino , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Humanos , Masculino , Persona de Mediana Edad , Infarto del Miocardio/sangre , Infarto del Miocardio/epidemiología , Infarto del Miocardio/prevención & control , Fenotipo , Estudios Retrospectivos , Medición de Riesgo , Factores de Riesgo , Adulto JovenRESUMEN
OBJECTIVE: The aim of our study was that to assess the allelic and genotype frequencies of nine prothrombotic gene variants in patients with a history of pregnancy loss and recurrent pregnancy loss (RPL). Women who underwent assisted reproductive technology (ART) with ongoing pregnancy and those with recurrent implantation failure (RIF) were also included. METHODS: Nine prothrombotic gene variants were evaluated: factor V Leiden (FVL), factor V, H1299R variant (FVR2), factor II (FII) G20210A, methylene-tetrahydrofolate reductase (MTHFR) C677T and A1298C, beta-fibrinogen -455G>A, factor XIII (FXIII) V34L, human platelet antigen-1 (HPA-1) L33P variants, and plasminogen activator inhibitor-1 (PAI-1) 4G/5G. The following study groups were assessed: (1) women who experienced one (n = 334) or two (n = 264) episodes of pregnancy loss; (2) 468 women who experienced RPL; (3) 214 women who underwent ART followed by ongoing pregnancies; and (4) 282 women who experienced RIF after ART, that is, three or more consecutive implantation failures following high-quality embryo transfers to the uterus with an appropriate endometrium. As control group, 430 subjects from the general population were enrolled. RESULTS: FVL, the -455G>A variant of beta-fibrinogen, and PAI-1 4G were associated with a higher risk of developing RPL compared with the general population. Furthermore, FVL, FVR2, FII G20210A and MTHFR C677T conferred a significantly higher risk of RIF in women who performed ART compared with the general population. No statistical differences between the general population and other study groups were observed. CONCLUSIONS: Specific prothrombotic genetic variants are more frequently expressed in women with RPL and RIF, supporting their role in the development of polimicrothrombosis and impairing the invasion during embryo implantation.
Asunto(s)
Aborto Habitual , Trombofilia , Embarazo , Humanos , Femenino , Inhibidor 1 de Activador Plasminogénico/genética , Estudios Retrospectivos , Aborto Habitual/genética , Factor V/genética , Implantación del Embrión/genética , Protrombina/genética , Fibrinógeno/genética , Trombofilia/genéticaRESUMEN
Cystic fibrosis (CF) is a life-limiting genetic disorder characterized by defective chloride ion transport due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Early detection through newborn screening programs significantly improves outcomes for individuals with CF by enabling timely intervention. Here, we report the identification of an Alu element insertion within the exon 15 of CFTR gene, initially overlooked in standard next-generation sequencing analyses. However, using traditional molecular techniques, based on polymerase chain reaction and Sanger sequencing, allowed the identification of the Alu element and the reporting of a correct diagnosis. Our analysis, based on bioinformatics tools and molecular techniques, revealed that the Alu element insertion severely affects the gene expression, splicing patterns, and structure of CFTR protein. In conclusion, this study emphasizes the importance of how the integration of human expertise and modern technologies represents a pivotal step forward in genomic medicine, ensuring the delivery of precision healthcare to individuals affected by genetic diseases.
Asunto(s)
Elementos Alu , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Pruebas Genéticas , Humanos , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Elementos Alu/genética , Fibrosis Quística/genética , Fibrosis Quística/diagnóstico , Pruebas Genéticas/métodos , Recién Nacido , Masculino , FemeninoRESUMEN
We have recently witnessed that considerable progresses have been made in the rapid detection and appropriate treatments of COVID-19, but still this virus remains one of the main targets of world research. Based on the knowledge of the complex mechanism of viral infection we designed peptide-dendrimer inhibitors of SARS-CoV-2with the aim to block cell infection through interfering with the host-pathogen interactions. We used two different strategies: i) the first one aims at hindering the virus anchorage to the human cell; ii) the second -strategy points to interfere with the mechanism of virus-cell membrane fusion. We propose the use of different nanosized carriers, formed by several carbosilane dendritic wedges to deliver two different peptides designed to inhibit host interaction or virus entry. The antiviral activity of the peptide-dendrimers, as well as of free peptides and free dendrimers was evaluated through the use of SARS-CoV-2 pseudotyped lentivirus. The results obtained show that peptides designed to block host-pathogen interaction represent a valuable strategy for viral inhibition.
Asunto(s)
Antivirales , Tratamiento Farmacológico de COVID-19 , Dendrímeros , Péptidos , SARS-CoV-2 , Dendrímeros/química , Dendrímeros/farmacología , Humanos , SARS-CoV-2/efectos de los fármacos , Antivirales/farmacología , Antivirales/química , Péptidos/química , Péptidos/farmacología , Internalización del Virus/efectos de los fármacos , Diseño de Fármacos , COVID-19/virología , Silanos/química , Silanos/farmacología , Interacciones Huésped-PatógenoRESUMEN
Inherited ichthyoses are a group of clinically and genetically heterogeneous rare disorders of skin keratinization with overlapping phenotypes. The clinical picture and family history are crucial to formulating the diagnostic hypothesis, but only the identification of the genetic defect allows the correct classification. In the attempt to molecularly classify 17 unrelated Italian patients referred with congenital nonsyndromic ichthyosis, we performed massively parallel sequencing of over 50 ichthyosis-related genes. Genetic data of 300 Italian unaffected subjects were also analyzed to evaluate frequencies of putative disease-causing alleles in our population. For all patients, we identified the molecular cause of the disease. Eight patients were affected by autosomal recessive congenital ichthyosis associated with ALOX12B, NIPAL4, and TGM1 mutations. Three patients had biallelic loss-of-function variants in FLG, whereas 6/11 males were affected by X-linked ichthyosis. Among the 24 different disease-causing alleles we identified, 8 carried novel variants, including a synonymous TGM1 variant that resulted in a splicing defect. Moreover, we generated a priority list of the ichthyosis-related genes that showed a significant number of rare and novel variants in our population. In conclusion, our comprehensive molecular analysis resulted in an effective first-tier test for the early classification of ichthyosis patients. It also expands the genetic, mutational, and phenotypic spectra of inherited ichthyosis and provides new insight into the current understanding of etiologies and epidemiology of this group of rare disorders.