Case report: Tissue positivity for SARS-CoV-2 in a preterm born infant death of thrombosis: possible intrauterine transmission.

Intrauterine transmission of SARS-CoV-2 (Severe Acute Respiratory Syndrome Corona Virus 2) is still matter of debate among scientists and there is limited information concerning this aspect of research. This could lead to severe complications of the growing fetus and, theoretically, of the newborn as well. We report the case of a male infant of 1,100 grams, born at 27th week of gestation to a SARS-CoV-2 mother, tested negative for viral detection at delivery. He was immediately admitted to neonatal Intensive Care Unit (ICU) for severe complications, where he died after 37 days by pulmonary embolism and thrombosis of the superior vena cava. After autopsy, SARS-CoV-2 N-protein and Spike RBD were detected in several tissues, particularly in the esophagus, stomach, spleen, and heart, with a significantly higher H-Score than the placenta. In conclusion, immunohistochemical analysis demonstrated SARS-CoV-2 NP and Spike RBD positivity in different tissues suggesting a possible intrauterine transmission. Newborn thrombo-embolism could be a complication of SARS-CoV-2 infection as observed in adult patients.

Counteracting the Common Shwachman-Diamond Syndrome-Causing SBDS c.258+2T>C Mutation by RNA Therapeutics and Base/Prime Editing.

Shwachman-Diamond syndrome (SDS) represents one of the most common inherited bone marrow failure syndromes and is mainly caused by SBDS gene mutations. Only supportive treatments are available, with hematopoietic cell transplantation required when marrow failure occurs. Among all causative mutations, the SBDS c.258+2T>C variant at the 5' splice site (ss) of exon 2 is one of the most frequent. Here, we investigated the molecular mechanisms underlying aberrant SBDS splicing and showed that SBDS exon 2 is dense in splicing regulatory elements and cryptic splice sites, complicating proper 5'ss selection. Studies ex vivo and in vitro demonstrated that the mutation alters splicing, but it is also compatible with tiny amounts of correct transcripts, which would explain the survival of SDS patients. Moreover, for the first time for SDS, we explored a panel of correction approaches at the RNA and DNA levels and provided experimental evidence that the mutation effect can be partially counteracted by engineered U1snRNA, trans-splicing, and base/prime editors, ultimately leading to correctly spliced transcripts (from barely detectable to 2.5-5.5%). Among them, we propose DNA editors that, by stably reverting the mutation and potentially conferring positive selection to bone-marrow cells, could lead to the development of an innovative SDS therapy.

Monitoring Somatic Genetic Alterations in Circulating Cell-Free DNA/RNA of Patients with "Oncogene-Addicted" Advanced Lung Adenocarcinoma: A Real-World Clinical Study.

Liquid biopsy has advantages over tissue biopsy, but also some technical limitations that hinder its wide use in clinical applications. In this study, we aimed to evaluate the usefulness of liquid biopsy for the clinical management of patients with advanced-stage oncogene-addicted non-small-cell lung adenocarcinomas. The investigation was conducted on a series of cases-641 plasma samples from 57 patients-collected in a prospective consecutive manner, which allowed us to assess the benefits and limitations of the approach in a real-world clinical context. Thirteen samples were collected at diagnosis, and the additional samples during the periodic follow-up visits. At diagnosis, we detected mutations in ctDNA in 10 of the 13 cases (77%). During follow-up, 36 patients progressed. In this subset of patients, molecular analyses of plasma DNA/RNA at progression revealed the appearance of mutations in 29 patients (80.6%). Mutations in ctDNA/RNA were typically detected an average of 80 days earlier than disease progression assessed by RECIST or clinical evaluations. Among the cases positive for mutations, we observed 13 de novo mutations, responsible for the development of resistance to therapy. This study allowed us to highlight the advantages and disadvantages of liquid biopsy, which led to suggesting algorithms for the use of liquid biopsy analyses at diagnosis and during monitoring of therapy response.

OTC intron 4 variations mediate pathogenic splicing patterns caused by the c.386G>A mutation in humans and spfash mice, and govern susceptibility to RNA-based therapies.

BACKGROUND: Aberrant splicing is a common outcome in the presence of exonic or intronic variants that might hamper the intricate network of interactions defining an exon in a specific gene context. Therefore, the evaluation of the functional, and potentially pathological, role of nucleotide changes remains one of the major challenges in the modern genomic era. This aspect has also to be taken into account during the pre-clinical evaluation of innovative therapeutic approaches in animal models of human diseases. This is of particular relevance when developing therapeutics acting on splicing, an intriguing and expanding research area for several disorders. Here, we addressed species-specific splicing mechanisms triggered by the OTC c.386G>A mutation, relatively frequent in humans, leading to Ornithine TransCarbamylase Deficiency (OTCD) in patients and spfash mice, and its differential susceptibility to RNA therapeutics based on engineered U1snRNA. METHODS: Creation and co-expression of engineered U1snRNAs with human and mouse minigenes, either wild-type or harbouring different nucleotide changes, in human (HepG2) and mouse (Hepa1-6) hepatoma cells followed by analysis of splicing pattern. RNA pulldown studies to evaluate binding of specific splicing factors. RESULTS: Comparative nucleotide analysis suggested a role for the intronic +10-11 nucleotides, and pull-down assays showed that they confer preferential binding to the TIA1 splicing factor in the mouse context, where TIA1 overexpression further increases correct splicing. Consistently, the splicing profile of the human minigene with mouse +10-11 nucleotides overlapped that of mouse minigene, and restored responsiveness to TIA1 overexpression and to compensatory U1snRNA. Swapping the human +10-11 nucleotides into the mouse context had opposite effects. Moreover, the interplay between the authentic and the adjacent cryptic 5'ss in the human OTC dictates pathogenic mechanisms of several OTCD-causing 5'ss mutations, and only the c.386+5G>A change, abrogating the cryptic 5'ss, was rescuable by engineered U1snRNA. CONCLUSIONS: Subtle intronic variations explain species-specific OTC splicing patterns driven by the c.386G>A mutation, and the responsiveness to engineered U1snRNAs, which suggests careful elucidation of molecular mechanisms before proposing translation of tailored therapeutics from animal models to humans.

Cis-Segregation of c.1171C>T Stop Codon (p.R391*) in SERPINC1 Gene and c.1691G>A Transition (p.R506Q) in F5 Gene and Selected GWAS Multilocus Approach in Inherited Thrombophilia.

Inherited thrombophilia (e.g., venous thromboembolism, VTE) is due to rare loss-of-function mutations in anticoagulant factors genes (i.e., SERPINC1, PROC, PROS1), common gain-of-function mutations in procoagulant factors genes (i.e., F5, F2), and acquired risk conditions. Genome Wide Association Studies (GWAS) recently recognized several genes associated with VTE though gene defects may unpredictably remain asymptomatic, so calculating the individual genetic predisposition is a challenging task. We investigated a large family with severe, recurrent, early-onset VTE in which two sisters experienced VTE during pregnancies characterized by a perinatal in-utero thrombosis in the newborn and a life-saving pregnancy-interruption because of massive VTE, respectively. A nonsense mutation (CGA > TGA) generating a premature stop-codon (c.1171C>T; p.R391*) in the exon 6 of SERPINC1 gene (1q25.1) causing Antithrombin (AT) deficiency and the common missense mutation (c.1691G>A; p.R506Q) in the exon 10 of F5 gene (1q24.2) (i.e., FV Leiden; rs6025) were coinherited in all the symptomatic members investigated suspecting a cis-segregation further confirmed by STR-linkage-analyses [i.e., SERPINC1 IVS5 (ATT)5-18, F5 IVS2 (AT)6-33 and F5 IVS11 (GT)12-16] and SERPINC1 intragenic variants (i.e., rs5878 and rs677). A multilocus investigation of blood-coagulation balance genes detected the coexistence of FV Leiden (rs6025) in trans with FV HR2-haplotype (p.H1299R; rs1800595) in the aborted fetus, and F11 rs2289252, F12 rs1801020, F13A1 rs5985, and KNG1 rs710446 in the newborn and other members. Common selected gene variants may strongly synergize with less common mutations tuning potential life-threatening conditions when combined with rare severest mutations. Merging classic and newly GWAS-identified gene markers in at risk families is mandatory for VTE risk estimation in the clinical practice, avoiding partial risk score evaluation in unrecognized at risk patients.

Dissection of pleiotropic effects of variants in and adjacent to F8 exon 19 and rescue of mRNA splicing and protein function.

The pathogenic significance of nucleotide variants commonly relies on nucleotide position within the gene, with exonic changes generally attributed to quantitative or qualitative alteration of protein biosynthesis, secretion, activity, or clearance. However, these changes may exert pleiotropic effects on both protein biology and mRNA splicing due to the overlapping of the amino acid and splicing codes, thus shaping the disease phenotypes. Here, we focused on hemophilia A, in which the definition of F8 variants' causative role and association to bleeding phenotypes is crucial for proper classification, genetic counseling, and management of affected individuals. We extensively characterized a large panel of hemophilia A-causing variants (n = 30) within F8 exon 19 by combining and comparing in silico and recombinant expression analyses. We identified exonic variants with pleiotropic effects and dissected the altered protein features of all missense changes. Importantly, results from multiple prediction algorithms provided qualitative results, while recombinant assays allowed us to correctly infer the likely phenotype severity for 90% of variants. Molecular characterization of pathogenic variants was also instrumental for the development of tailored correction approaches to rescue splicing affecting variants or missense changes impairing protein folding. A single engineered U1snRNA rescued mRNA splicing of nine different variants and the use of a chaperone-like drug resulted in improved factor VIII protein secretion for four missense variants. Overall, dissection of the molecular mechanisms of a large panel of HA variants allowed precise classification of HA-affected individuals and favored the development of personalized therapeutic approaches.

Next-generation sequencing and recombinant expression characterized aberrant splicing mechanisms and provided correction strategies in factor VII deficiency.

Despite the exhaustive screening of F7 gene exons and exon-intron boundaries and promoter region, a significant proportion of mutated alleles remains unidentified in patients with coagulation factor VII deficiency. Here, we applied next-generation sequencing to 13 FVII-deficient patients displaying genotype-phenotype discrepancies upon conventional sequencing, and identified six rare intronic variants. Computational analysis predicted splicing effects for three of them, which would strengthen (c.571+78G>A; c.806-329G>A) or create (c.572-392C>G) intronic 5' splice sites (5'ss). In F7 minigene assays, the c.806-329G>A was ineffective while the c.571+78G>A change led to usage of the +79 cryptic 5'ss with only trace levels of correct transcripts (3% of wild-type), in accordance with factor VII activity levels in homozygotes (1-3% of normal). The c.572-392C>G change led to pseudo-exonization and frame-shift, but also substantial levels of correct transcripts (approx. 70%). However, this variant was associated with the common F7 polymorphic haplotype, predicted to further decrease factor VII levels; this provided some kind of explanation for the 10% factor VII levels in the homozygous patient. Intriguingly, the effect of the c.571+78G>A and c.572-392C>G changes, and particularly of the former (the most severe and well-represented in our cohort), was counteracted by antisense U7snRNA variants targeting the intronic 5'ss, thus demonstrating their pathogenic role. In conclusion, the combination of next-generation sequencing of the entire F7 gene with the minigene expression studies elucidated the molecular bases of factor VII deficiency in 10 of 13 patients, thus improving diagnosis and genetic counseling. It also provided a potential therapeutic approach based on antisense molecules that has been successfully exploited in other disorders.

An Altered Splicing Registry Explains the Differential ExSpeU1-Mediated Rescue of Splicing Mutations Causing Haemophilia A.

The exon recognition and removal of introns (splicing) from pre-mRNA is a crucial step in the gene expression flow. The process is very complex and therefore susceptible to derangements. Not surprisingly, a significant and still underestimated proportion of disease-causing mutations affects splicing, with those occurring at the 5' splice site (5'ss) being the most severe ones. This led to the development of a correction approach based on variants of the spliceosomal U1snRNA, which has been proven on splicing mutations in several cellular and mouse models of human disease. Since the alternative splicing mechanisms are strictly related to the sequence context of the exon, we challenged the U1snRNA-mediated strategy in the singular model of the exon 5 of coagulation factor (F)VIII gene (F8) in which the authentic 5'ss is surrounded by various cryptic 5'ss. This scenario is further complicated in the presence of nucleotide changes associated with FVIII deficiency (Haemophilia A), which weaken the authentic 5'ss and create/strengthen cryptic 5'ss. We focused on the splicing mutations (c.602-32A > G, c.602-10T > G, c.602G > A, c.655G > A, c.667G > A, c.669A > G, c.669A > T, c.670G > T, c.670+1G > T, c.670+1G > A, c.670+2T > G, c.670+5G > A, and c.670+6T > C) found in patients with severe to mild Haemophilia A. Minigenes expression studies demonstrated that all mutations occurring within the 5'ss, both intronic or exonic, lead to aberrant transcripts arising from the usage of two cryptic intronic 5'ss at positions c.670+64 and c.670+176. For most of them, the observed proportion of correct transcripts is in accordance with the coagulation phenotype of patients. In co-transfection experiments, we identified a U1snRNA variant targeting an intronic region downstream of the defective exon (Exon Specific U1snRNA, U1sh7) capable to re-direct usage of the proper 5'ss (∼80%) for several mutations. However, deep investigation of rescued transcripts from +1 and +2 variants revealed only the usage of adjacent cryptic 5'ss, leading to frameshifted transcript forms. These data demonstrate that a single ExSpeU1 can efficiently rescue different mutations in the F8 exon 5, and provide the first evidence of the applicability of the U1snRNA-based approach to Haemophilia A.

Second primary malignancies in patients with non-melanoma skin cancer: Results from a cancer registry-based study in Emilia Romagna, north-east Italy.

BACKGROUND: previous research on the risk of subsequent, primary non-cutaneous malignancies among patients with non-melanoma skin cancers (NMSCs) led to conflicting results. We aimed to investigate a possible link between NMSC and second primary malignancies by using the population-based data available in cancer registries. METHODS: this observational study retrospectively assessed the risk of occurrence of both synchronous and methachronous second primary tumours in a cohort of cancer patients whose first diagnosis was NMSC. The cohort came from the network of general cancer registries of the Emilia-Romagna Region, northeast Italy, in the period between 1978 and 2012, and was compared with the general population living in the same area. Two main indexes were used: i) Standardized Incidence Ratio (SIR), calculated as the ratio between the observed and the expected number of second cancers and ii) Excess Absolute Risk (EAR), expressing the absolute excess or deficit of second cancer incidence. RESULTS: in the period analysed (1978-2012, 72,503,157 person/years, PYs), 89,912 primary NMSC were found in 76,414 patients. Among them, 14,195 developed a second primary cancer in the subsequent 501,763 follow-up PYs. NMSC patients showed an overall SIR of 1.22 (CI 95% 1.20-1,24) and an EAR of 5.11 cases/1000 PYs (CI 95% 4.48-5.74). CONCLUSIONS: the study results showed that NMSC patients had an increase in relative risk and, at least for some tumours, in absolute risk of developing a second cancer when compared with the general population. Genetic, environmental and personal risk factors may influence this finding.

Disease-causing variants of the conserved +2T of 5' splice sites can be rescued by engineered U1snRNAs.

The ability of variants of the spliceosomal U1snRNA to rescue splicing has been proven in several human disease models, but not for nucleotide changes at the conserved GT nucleotide of 5' splice sites (5'ss), frequent and associated with severe phenotypes. Here, we focused on variants at the 5'ss of F9 intron 3, leading to factor IX (FIX) deficiency (hemophilia B). Through minigene expression, we demonstrated that all changes induce complete exon 3 skipping, which explains the associated hemophilia B phenotype. Interestingly, engineered U1snRNAs remarkably increased the proportion of correct transcripts in the presence of the c.277+4A>G (∼60%) and also c.277+2T>C mutation (∼20%). Expression of splicing-competent cDNA constructs indicated that the splicing rescue produces an appreciable increase of secreted FIX protein levels. These data provide the first experimental evidence that even part of variants at the conserved 5'ss +2T nucleotide can be rescued, thus expanding the applicability of this U1snRNA-based approach.

Antibody response to polyomavirus primary infection: high seroprevalence of Merkel cell polyomavirus and lymphoid tissue involvement.

Human polyomaviruses (HPyVs) asymptomatically infect the human population establishing latency in the host, and their seroprevalence can reach 90% in healthy adults. Few studies have focused on the pediatric population, and there are no reports regarding the seroprevalence of all the newly isolated HPyVs among Italian children. Therefore, we investigated the frequency of serum antibodies against 12 PyVs in 182 immunocompetent children from Northeast Italy, by means of a multiplex antibody detection system. Additionally, secondary lymphoid tissues were collected to analyze the presence of HPyV DNA sequences using a specific real-time PCRs or PCRs. Almost 100% of subjects were seropositive for at least one PyV. Seropositivity ranged from 3% for antibodies against simian virus 40 (SV40) in children from 0 to 3 years, to 91% for antibodies against WU polyomavirus (WUPyV) and HPyV10 in children from 8 to 17 years. The mean number of PyV for which children were seropositive increased with the increasing of age: 4 standard deviations (SD) 1.8 in the 0-3-year group, 5 (SD 1.9) in the 4-7-year group, and 6 (SD 2.2) in the 8-17-year group. JC polyomavirus (JCPyV) DNA was detected in 1% of the adenoids, WUPyV in 12% of the tonsils, and 28% of the adenoids, and Merkel cell polyomavirus (MCPyV) was present in 6 and 2% of the tonsils and adenoids, respectively. Our study gives new insights on the serological evidence of exposure to PyVs during childhood, and on their possible respiratory route of transmission.

MBL2 polymorphisms in women with atypical squamous cells of undetermined significance.

Infection with high risk Human papillomavirus (HPV) is the main known cause of cervical cancer. HPV induces different grades of lesions: among them, Atypical squamous cells of undetermined significance are abnormal lesions that could evolve in pre-cancer lesions or spontaneously regress. The mannose binding lectin (MBL) is an innate immunity serum protein also found in cervico-vaginal mucosa, whose expression is known to be affected by polymorphisms in exon 1 and promoter of the MBL2 gene. In the present study the possible association between MBL2 functional polymorphisms and susceptibility to develop atypical squamous cells of undetermined significance was investigated in a group of women from North-East of Italy, stratified for HPV infection status. The MBL2 D and O alleles and the deficient producer combined genotypes, responsible for low MBL production, were more represented among atypical squamous cells of undetermined significance positive women than healthy controls and the results were confirmed when only HPV negative samples were considered. These results suggest a possible involvement of MBL2 functional polymorphisms in atypical squamous cells of undetermined significance susceptibility.

Beta defensin-1 gene polymorphisms and susceptibility to atypical squamous cells of undetermined significance lesions in Italian gynecological patients.

The role of the human beta-defensin 1 (hBD-1) in the susceptibility to the onset of the Atypical Squamous Cells of Undetermined Significance (ASCUS) lesion, in the presence or not of HPV infection, is still unknown. In the current study, the three functional single nucleotide polymorphisms (SNPs) -52G > A, -44C > G, and -20G > A at the 5' un-translated region (UTR) of DEFB1 gene, encoding hBD-1, were analyzed in ASCUS lesion gynecological patients and healthy women from the north-east of Italy (Trieste). Cervical samples from 249 European-Caucasian women were collected, screened for HPV and cytologically evaluated; DEFB1 genotyping has been performed by direct sequencing. No significant differences were found for -52G > A, -44C > G, and -20G > A SNPs allele and genotype frequencies between women with and without ASCUS lesions. DEFB1 minor haplotypes were significantly more frequent in ASCUS lesion positive than negative women, associating with an increased risk of this type of lesion. When women were stratified according to HPV infection status, significant differences in the distribution of -52G > A SNP genotype frequencies were found: the presence of the A allele in the homozygous genotype A/A associated with a lower risk of developing ASCUS lesions in HPV negative women. DEFB1 minor haplotypes were also associated with an increased risk of developing ASCUS lesions, being significantly more frequent in HPV negative women with lesions, than without lesions. Although these results highlight the possible involvement of DEFB1, further studies are needed to support the role of DEFB1 in the modulation of the susceptibility to ASCUS lesions.

NLRP1 polymorphisms in patients with asbestos-associated mesothelioma.

BACKGROUND: An increasing incidence of malignant mesothelioma (MM) cases in patients with low levels of asbestos exposure suggests the interference of alternative cofactors. SV40 infection was detected, as co-morbidity factor, only in 22% of asbestos-MM patients from a North-Eastern Italy area. An additional mechanism of injury related to asbestos exposure in MM development has been recently associated to inflammatory responses, principally driven by interleukin (IL)-1 beta (ß) activated within the inflammasome complex.NLRP3 inflammosome has been described as the intracellular sensor for asbestos able to induce inflammasome activation and IL-1ß secretion while NLRP1 is expressed in lung epithelial cells and alveolar macrophages and contributes to the immune response and to survival/apoptosis balance. This study proposes to evaluate the impact of known NLRP3 and NLRP1 polymorphisms in the individual susceptibility to asbestos-induced mesothelioma in subjects from a hyperendemic area for MM. METHODS: 134 Italian patients with diagnosis of mesothelioma due (MMAE, n=69) or not (MMAF, n=65) to asbestos, 256 healthy Italian blood donors and 101 Italian healthy subjects exposed to asbestos (HCAE) were genotyped for NLRP1 (rs2670660 and rs12150220) and NLRP3 (rs35829419 and rs10754558) polymorphisms. RESULTS: While NLRP3 SNPs were not associated to mesothelioma, the NLRP1 rs12150220 allele T was significantly more frequent in MMAE (0.55) than in HCAE (0.41) (p=0.011; OR=1.79) suggesting a predisponent effect of this allele on the development of mesothelioma. This effect was amplified when the NLRP1 rs2670660 allele was combined with the NLRP1 rs12150220 allele (p=0.004; OR=0.52). CONCLUSION: Although NLRP3 SNPs was not involved in mesothelioma predisposition, these data proposed NLRP1 as a novel factor possibly involved in the development of mesothelioma.

Merkel-cell polyomavirus (MCPyV) is rarely associated to B-chronic lymphocytic leukemia (1 out of 50) samples and occurs late in the natural history of the disease.

BACKGROUND: Previous studies have reported conflicting results on the frequency and potential pathogenetic role of Merkel-cell polyomavirus (MCPyV) in B-chronic lymphocytic leukemia (B-CLL). OBJECTIVES: To evaluate the association of MCPyV to B-CLL and to investigate the occurrence of MCPyV infection in relationship to the natural history of B-CLL. STUDY DESIGN: Samples of primary B-CLL peripheral blood mononuclear cells were obtained from two distinct University Hospitals of Italy from January 2010. For one B-CLL patient, it was possible to retrospectively examine the blood sample at diagnosis of B-CLL (March 2004) and several pathological tissues of cutaneous tumors occurring during the course of the disease. RESULTS: Only one out of 50 B-CLL blood samples examined was positive for MCPyV DNA. Retrospective analysis revealed that MCPyV DNA was absent in peripheral blood sample at diagnosis, becoming present only in advanced disease stages also in tonsil tissue as well as in a biopsy of differentiated squamous cell carcinoma. CONCLUSIONS: The association with MCPyV seems to represent a rare and late event during the natural history of B-CLL.

Asbestos and SV40 in malignant pleural mesothelioma from a hyperendemic area of north-eastern Italy.

AIMS AND BACKGROUND: Malignant mesothelioma is a fatal cancer of increasing incidence in north-eastern Italy. Together with asbestos, the polyomavirus SV40 was hypothesized to contribute to the onset of malignant mesothelioma. To investigate the putative role of SV40 in the individual susceptibility to asbestos-induced malignant mesothelioma, we conducted a molecular epidemiological study on a series of malignant mesothelioma patients from an area in north-eastern Italy hyperendemic for malignant pleural mesothelioma. METHODS AND STUDY DESIGN: We collected 63 mesothelioma samples from incidence cases of patients diagnosed with malignant pleural mesothelioma in the period 2009-2010. DNA was extracted from patients' tissue biopsies using the BioRobot EZ1 Qiagen workstation. SV40 sequence detection and quantification was performed by specific real time PCR. The 74.6% of the 63 enrolled patients had a history of asbestos exposure. The epithelioid histotype was more prevalent in males (64.0%) and the mixed in females (61.5%) who showed significantly higher cancer co-morbidity (46.1% vs 12%, P = 0.005). SV40 was detected in 22% of MM tumors, with a low viral load. In SV40-positive patients, a threefold increased risk of asbestos exposure was observed, more evident in females (OR 4.32) than in males (OR 1.20). CONCLUSIONS: Our findings indicate that a high prevalence of SV40 was present in malignant mesothelioma incident cases from an area hyperendemic for malignant mesothelioma in north-eastern Italy. Although asbestos is considered the main risk factor in malignant mesothelioma onset, a role for SV40 could be hypothesized.

Activation of a cryptic splice site in a potentially lethal coagulation defect accounts for a functional protein variant.

Changes at the invariable donor splice site +1 guanine, relatively frequent in human genetic disease, are predicted to abrogate correct splicing, and thus are classified as null mutations. However, their ability to direct residual expression, which might have pathophysiological implications in several diseases, has been poorly investigated. As a model to address this issue, we studied the IVS6+1G>T mutation found in patients with severe deficiency of the protease triggering coagulation, factor VII (FVII), whose absence is considered lethal. In expression studies, the IVS6+1G>T induced exon 6 skipping and frame-shift, and prevented synthesis of correct FVII transcripts detectable by radioactive/fluorescent labelling or real-time RT-PCR. Intriguingly, the mutation induced the activation of a cryptic donor splice site in exon 6 and production of an in-frame 30bp deleted transcript (8 ± 2%). Expression of this cDNA variant, lacking 10 residues in the activation domain, resulted in secretion of trace amounts (0.2 ± 0.04%) of protein with appreciable specific activity (48 ± 16% of wt-FVII). Altogether these data indicate that the IVS6+1G>T mutation is compatible with the synthesis of functional FVII molecules (~0.01% of normal, 1pM), which could trigger coagulation. The low but detectable thrombin generation (352 ± 55nM) measured in plasma from an IVS6+1G>T homozygote was consistent with a minimal initiation of the enzymatic cascade. In conclusion, we provide experimental clues for traces of FVII expression, which might have reverted an otherwise perinatally lethal genetic condition.

MicroRNA profiling for the identification of cancers with unknown primary tissue-of-origin.

Cancer of unknown primary (CUP) represents a common and important clinical problem. There is evidence that most CUPs are metastases of carcinomas whose primary site cannot be recognized. Driven by the hypothesis that the knowledge of primary cancer could improve patient's prognosis, we investigated microRNA expression profiling as a tool for identifying the tissue of origin of metastases. We assessed microRNA expression from 101 formalin-fixed, paraffin-embedded (FFPE) samples from primary cancers and metastasis samples by using a microarray platform. Forty samples representing ten different cancer types were used for defining a cancer-type-specific microRNA signature, which was used for predicting primary sites of metastatic cancers. A 47-miRNA signature was identified and used to estimate tissue-of-origin probabilities for each sample. Overall, accuracy reached 100% for primary cancers and 78% for metastases in our cohort of samples. When the signature was applied to an independent published dataset of 170 samples, accuracy remained high: correct prediction was found within the first two options in 86% of the metastasis cases (first prediction was correct in 68% of cases). This signature was also applied to predict 16 CUPs. In this group, first predictions exhibited probabilities higher than 90% in most of the cases. These results establish that FFPE samples can be used to reveal the tissue of origin of metastatic cancers by using microRNA expression profiling and suggest that the approach, if applied, could provide strong indications for CUPs, whose correct diagnosis is presently undefined.

Rescue of coagulation factor VII function by the U1+5A snRNA.

Our previous studies with genomic minigenes have demonstrated that an engineered small nuclear RNA-U1 (U1+5a) partially rescued coagulation factor VII (FVII) mRNA processing impaired by the 9726+5G>A mutation. Here, to evaluate the U1+5a effects on FVII function, we devised a full-length FVII splicing-competent construct (pSCFVII-wt). This construct drove in COS-1 cells the synthesis of properly processed FVII transcripts and of secreted functional FVII (23 +/- 4 ng/mL), which were virtually undetectable upon introduction of the 9726+5G>A mutation (pSCFVII-9726+5a). Cotransfection of pSCFVII-9726+5a with pU1+5a resulted in a partial rescue of FVII splicing and protein biosynthesis. The level increase in medium was dose dependent and, with a molar excess (1.5x) of pU1+5a, reached 9.5% plus or minus 3.2% (5.0 +/- 2.8 ng/mL) of FVII-wt coagulant activity. These data provide the first insights into the U1-snRNA-mediated rescue of donor splice sites at protein level, thus further highlighting its therapeutic implications in bleeding disorders, which would benefit even from tiny increase of functional levels.

Evaluation of factor V mRNA to define the residual factor V expression levels in severe factor V deficiency.

We evaluated FV mRNA in severe factor V deficiency caused by the -12T/A IVS18 mutation, activating a cryptic splice site and leading to premature translation termination. Quantitative evaluation of factor V cDNA from homozygous and heterozygous subjects, and correction for nonsense mediated decay, suggested the presence of 0.1% of normal factor V mRNA.