Lateralized overgrowth with vascular malformation caused by a somatic PTPN11 pathogenic variant: Another piece added to the puzzle of mosaic RASopathies.

Lateralized/segmental overgrowth disorders (LOs) encompass a heterogeneous group of congenital conditions with excessive body tissue growth. Documented molecular alterations in LOs mostly consist of somatic variants in genes of the PI3KCA/AKT/mTOR pathway or of chromosome band 11p15.5 imprinted region anomalies. In some cases, somatic pathogenic variants in genes of the RAS/MAPK pathway have been reported. We present the first case of a somatic pathogenic variant (T507K) in PTPN11 causing a LO phenotype characterized by severe lateralized overgrowth, vascular proliferation, and cerebral astrocytoma. The T507K variant was detected in DNA from overgrown tissue in a leg with capillary malformation. The astrocytoma tissue showed a higher PTPN11 variant allele frequency. A pathogenic variant in FGFR1 was also found in tumor tissue, representing a second hit on the RAS/MAPK pathway. These findings indicate that RAS/MAPK cascade overactivation can cause mosaic overgrowth phenotypes resembling PIK3CA-related overgrowth disorders (PROS) with cancer predisposition and are consistent with the hypothesis that RAS/MAPK hyperactivation can be involved in the pathogenesis of astrocytoma. This observation raises the issue of cancer predisposition in patients with RAS/MAPK pathway gene variants and expands genotype spectrum of LOs and the treatment options for similar cases through inhibition of the RAS/MAPK oversignaling.

Clinical presentation and genetic analyses of neurofibromatosis type 1 in independent patients with monoallelic double de novo closely spaced mutations in the NF1 gene.

Neurofibromatosis type 1 (NF1) belongs to RASopathies, a group of syndromes caused by germline mutations in Ras/MAPK pathway genes. Most NF1 patients exhibit single inactivating pathogenic variants within the NF1 gene. We performed extensive genetic analyses in two NF1 families disclosing the first two cases of double de novo monoallelic NF1 variants. Both index patients described in this study had classical NF1. Probands were born from fathers in their late 30s and presented closely spaced double mutations (<100 bp) in NF1 regions showing an excess of somatic mutations. Closely spaced multiple mutations have been reported in RAS/MAPK signaling genes but never in NF1. Mutagenesis is a quasi-random process in humans, therefore two causative variants in the same gene, moreover in the same allele are exceptional. Here, we discuss possible mechanisms for this ultrarare event. Our findings confirm the possibility of a higher risk of concurrent de novo variants in NF1.

Metabolomic Approaches for Detection and Identification of Biomarkers and Altered Pathways in Bladder Cancer.

Metabolomic analysis has proven to be a useful tool in biomarker discovery and the molecular classification of cancers. In order to find new biomarkers, and to better understand its pathological behavior, bladder cancer also has been studied using a metabolomics approach. In this article, we review the literature on metabolomic studies of bladder cancer, focusing on the different available samples (urine, blood, tissue samples) used to perform the studies and their relative findings. Moreover, the multi-omic approach in bladder cancer research has found novel insights into its metabolic behavior, providing excellent start-points for new diagnostic and therapeutic strategies. Metabolomics data analysis can lead to the discovery of a "signature pathway" associated with the progression of bladder cancer; this aspect could be potentially valuable in predictions of clinical outcomes and the introduction of new treatments. However, further studies are needed to give stronger evidence and to make these tools feasible for use in clinical practice.

Novel Insights into Autophagy and Prostate Cancer: A Comprehensive Review.

Autophagy is a complex process involved in several cell activities, including tissue growth, differentiation, metabolic modulation, and cancer development. In prostate cancer, autophagy has a pivotal role in the regulation of apoptosis and disease progression. Several molecular pathways are involved, including PI3K/AKT/mTOR. However, depending on the cellular context, autophagy may play either a detrimental or a protective role in prostate cancer. For this purpose, current evidence has investigated how autophagy interacts within these complex interactions. In this article, we discuss novel findings about autophagic machinery in order to better understand the therapeutic response and the chemotherapy resistance of prostate cancer. Autophagic-modulation drugs have been employed in clinical trials to regulate autophagy, aiming to improve the response to chemotherapy or to anti-cancer treatments. Furthermore, the genetic signature of autophagy has been found to have a potential means to stratify prostate cancer aggressiveness. Unfortunately, stronger evidence is needed to better understand this field, and the application of these findings in clinical practice still remains poorly feasible.

Renal Cell Carcinoma as a Metabolic Disease: An Update on Main Pathways, Potential Biomarkers, and Therapeutic Targets.

Clear cell renal cell carcinoma (ccRCC) is the most frequent histological kidney cancer subtype. Over the last decade, significant progress has been made in identifying the genetic and metabolic alterations driving ccRCC development. In particular, an integrated approach using transcriptomics, metabolomics, and lipidomics has led to a better understanding of ccRCC as a metabolic disease. The metabolic profiling of this cancer could help define and predict its behavior in terms of aggressiveness, prognosis, and therapeutic responsiveness, and would be an innovative strategy for choosing the optimal therapy for a specific patient. This review article describes the current state-of-the-art in research on ccRCC metabolic pathways and potential therapeutic applications. In addition, the clinical implication of pharmacometabolomic intervention is analyzed, which represents a new field for novel stage-related and patient-tailored strategies according to the specific susceptibility to new classes of drugs.

Gastric polyposis and desmoid tumours as a new familial adenomatous polyposis clinical variant associated with APC mutation at the extreme 3'-end.

Germline mutations of the APC gene, which encodes a multidomain protein of 2843 amino acid residues, cause familial adenomatous polyposis (FAP). Three FAP clinical variants are correlated with the location of APC mutations: (1) classic FAP with profuse polyposis (>1000 adenomas), associated with mutations from codon 1250 to 1424; (2) attenuated FAP (<100 adenomas), associated with mutations at APC extremities (before codon 157 and after codon 1595); (3) classic FAP with intermediate colonic polyposis (100-1000 adenomas), associated with mutations located in the remaining part of APC In an effort to decipher the clinical phenotype associated with APC C-terminal germline truncating mutations in patients with FAP, after screening APC mutations in one family whose members (n=4) developed gastric polyposis, colon oligo-polyposis and desmoid tumours, we performed a literature meta-analysis of clinically characterised patients (n=97) harbouring truncating mutations in APC C-terminus. The APC distal mutations identified in this study cluster with a phenotype characterised by colon oligo-polyposis, diffuse gastric polyposis and desmoid tumours. In conclusion, we describe a novel FAP clinical variant, which we propose to refer to as Gastric Polyposis and Desmoid FAP, that may require tailored management.

Blood-based test for diagnosis and functional subtyping of familial Mediterranean fever.

BACKGROUND AND OBJECTIVE: Familial Mediterranean fever (FMF) is the most common monogenic autoinflammatory disease (AID) worldwide. The disease is caused by mutations in the MEFV gene encoding the inflammasome sensor Pyrin. Clinical diagnosis of FMF is complicated by overlap in symptoms with other diseases, and interpretation of genetic testing is confounded by the lack of a clear genotype-phenotype association for most of the 340 reported MEFV variants. In this study, the authors designed a functional assay and evaluated its potential in supporting FMF diagnosis. METHODS: Peripheral blood mononuclear cells (PBMCs) were obtained from patients with Pyrin-associated autoinflammation with an FMF phenotype (n=43) or with autoinflammatory features not compatible with FMF (n=8), 10 asymptomatic carriers and 48 healthy donors. Sera were obtained from patients with distinct AIDs (n=10), and whole blood from a subset of patients and controls. The clinical, demographic, molecular genetic factors and other characteristics of the patient population were assessed for their impact on the diagnostic test read-out. Interleukin (IL)-1ß and IL-18 levels were measured by Luminex assay. RESULTS: The ex vivo colchicine assay may be performed on whole blood or PBMC. The functional assay robustly segregated patients with FMF from healthy controls and patients with related clinical disorders. The diagnostic test distinguished patients with classical FMF mutations (M694V, M694I, M680I, R761H) from patients with other MEFV mutations and variants (K695R, P369S, R202Q, E148Q) that are considered benign or of uncertain clinical significance. CONCLUSION: The ex vivo colchicine assay may support diagnosis of FMF and functional subtyping of Pyrin-associated autoinflammation.

Improvement of MEFV gene variants classification to aid treatment decision making in familial Mediterranean fever.

OBJECTIVE: FMF is an inherited autoinflammatory syndrome caused by mutations in the MEFV gene. MEFV variants are still largely classified as acvariant of uncertain significance, or with unresolved classification, posing significant challenges in FMF diagnosis. Rare Exome Variant Ensemble Learner (REVEL) is a recently developed variant metapredictor tool. To reduce the number of MEFV variants with ambiguous classification, we extracted REVEL scores for all missense variants present in the INFEVERS database, and analysed its correlation with expert-based classification and localization in the MEFV-encoded pyrin functional domains. METHODS: The data set of 216 MEFV missense variants was divided into four categories (likely benign, variant of uncertain significance, likely pathogenic and unresolved). Variants were plotted onto the pyrin protein, the distribution of REVEL scores in each category was computed and means, confidence intervals, and area under the receiver operating curve were calculated. RESULTS: We observed a non-random distribution of pathogenic variants along the pyrin functional domains. The REVEL scores demonstrated a good correlation with the consensus classification of the International Study Group for Systemic Autoinflammatory Diseases. Sensitivity, specificity and accuracy were calculated for different cut-off values of REVEL scores and a gene-specific-threshold of 0.298 was computed with confidence boundary limits. This cut-off value allowed us to propose a reclassification of 96 MEFV gene variants, thus reducing the variant of uncertain significance proportion from 61.6% to 17.6%. CONCLUSION: The combination of available expert information with sensitive predictor tools could result in a more accurate interpretation of clinical consequences of MEFV gene variants, and to a better genetic counselling and patient management.

In-silico Analysis of NF1 Missense Variants in ClinVar: Translating Variant Predictions into Variant Interpretation and Classification.

Background: With the advent of next-generation sequencing in genetic testing, predicting the pathogenicity of missense variants represents a major challenge potentially leading to misdiagnoses in the clinical setting. In neurofibromatosis type 1 (NF1), where clinical criteria for diagnosis may not be fully present until late infancy, correct assessment of variant pathogenicity is fundamental for appropriate patients' management. Methods: Here, we analyzed three different computational methods, VEST3, REVEL and ClinPred, and after extracting predictions scores for 1585 NF1 missense variants listed in ClinVar, evaluated their performances and the score distribution throughout the neurofibromin protein. Results: For all the three methods, no significant differences were present between the scores of "likely benign", "benign", and "likely pathogenic", "pathogenic" variants that were consequently collapsed into a single category. The cutoff values for pathogenicity were significantly different for the three methods and among benign and pathogenic variants for all methods. After training five different models with a subset of benign and pathogenic variants, we could reclassify variants in three sharply separated categories. Conclusions: The recently developed metapredictors, which integrate information from multiple components, after gene-specific fine-tuning, could represent useful tools for variant interpretation, particularly in genetic diseases where a clinical diagnosis can be difficult.

Familial Mediterranean fever: breaking all the (genetic) rules.

OBJECTIVE: FMF is an inherited autoinflammatory syndrome, characterized by attacks of painful periodic fever caused by diffuse serositis and risk of secondary amyloidosis due to IL-1ß-mediated inflammation. The disease appears to be transmitted through autosomal recessive mutations in the MEFV gene encoding the pyrin protein Although more than 300 variants have been reported worldwide so far, their association with symptom severity, the relative frequencies in different populations and the disease penetrance are far from being completely understood. We investigated genotype-phenotype correlations in two large nuclear families and verified whether commonly used web-based tools can usefully predict variant pathogenicity in FMF. METHODS: Peripheral blood samples were obtained from 15 patients of two families who had been diagnosed with FMF according to international criteria. The entire MEFV coding region was sequenced in all subjects, and 179 MEFV variants were surveyed with five different pathogenicity predictors. RESULTS: The inheritance of FMF could not be explained by traditional autosomal recessivity in both families. In silico tools demonstrated a significant association of variants' pathogenicity with their position along the coding sequence but not with variants' frequency. CONCLUSION: By describing two large families with paradigmatic complexity of FMF genetics, we conclude that established concepts in assessing the causative role of variants identified in mutation screening cannot be easily translated into appropriate genetic counselling in FMF. Furthermore, we demonstrate that variants frequently associated with severe disease are not predicted to significantly impact protein function using in silico algorithms.

The mechanism of alternative splicing of the X-linked NDUFB11 gene of the respiratory chain complex I, impact of rotenone treatment in neuroblastoma cells.

A study is presented on the regulation of alternative splicing (AS) of the Ndufb11 gene of complex I of the mitochondrial respiratory chain and the impact on this process of rotenone treatment in neuroblastoma cells. In physiological conditions the Ndufb11 gene produces at high level a short transcript isoform encoding for a 153 aa protein. This subunit is essential for the assembly of a functional and stable mammalian complex I. The gene produces also, at low level, a longer transcript isoform encoding for a 163 aa protein whose role is unknown. Evidence is presented here showing that the level of the two isoforms is regulated by three DGGGD ESS elements located in exon 2 which can bind the hnRNPH1 protein. In neuronal cells rotenone treatment affects the Ndufb11 alternative splicing pathway, with the increase of the 163/153 mRNAs ratio. This effect appears to be due to the down-regulation of the hnRNPH1 protein. Since rotenone induces apoptosis in neuronal cells, the post-transcriptional regulation of the Ndufb11 gene can be involved in the programmed cell death process.

Synthesis of a 2,4,6-trisubstituted 5-cyano-pyrimidine library and evaluation of its immunosuppressive activity in a Mixed Lymphocyte Reaction assay.

A series of novel pyrimidine analogues were synthesized and evaluated for immunosuppressive activity in the Mixed Lymphocyte Reaction assay, which is well-known as the in vitro model for in vivo rejection after organ transplantation. Systematic variation of the substituents at positions 2, 4 and 6 of the pyrimidine scaffold led to the discovery of 2-benzylthio-5-cyano-6-(4-methoxyphenyl)-4-morpholinopyrimidine with an IC(50) value of 1.6 µM in the MLR assay.

Metabolic dysfunction-associated gallstone disease: expecting more from critical care manifestations.

About 20% of adults worldwide have gallstones which are solid conglomerates in the biliary tree made of cholesterol monohydrate crystals, mucin, calcium bilirubinate, and protein aggregates. About 20% of gallstone patients will definitively develop gallstone disease, a condition which consists of gallstone-related symptoms and/or complications requiring medical therapy, endoscopic procedures, and/or cholecystectomy. Gallstones represent one of the most prevalent digestive disorders in Western countries and patients with gallstone disease are one of the largest categories admitted to European hospitals. About 80% of gallstones in Western countries are made of cholesterol due to disturbed cholesterol homeostasis which involves the liver, the gallbladder and the intestine on a genetic background. The incidence of cholesterol gallstones is dramatically increasing in parallel with the global epidemic of insulin resistance, type 2 diabetes, expansion of visceral adiposity, obesity, and metabolic syndrome. In this context, gallstones can be largely considered a metabolic dysfunction-associated gallstone disease, a condition prone to specific and systemic preventive measures. In this review we discuss the key pathogenic and clinical aspects of gallstones, as the main clinical consequences of metabolic dysfunction-associated disease.

Intraabdominal sporadic desmoid tumors and inflammation: an updated literature review and presentation and insights on pathogenesis of synchronous sporadic mesenteric desmoid tumors occurring after surgery for necrotizing pancreatitis.

Sporadic intra-abdominal desmoid tumors are rare and known to potentially occur after trauma including previous surgery, although knowledge of the underlying pathogenetic mechanism is still limited. We reviewed the recent literature on sporadic intraabdominal desmoids and inflammation as we investigated the mutational and epigenetic makeup of a case of multiple synchronous mesenterial desmoids occurring after necrotizing pancreatitis. A 62-year-old man had four mesenteric masses up to 4.8 cm diameter detected on CT eighteen months after laparotomy for peripancreatic collections from necrotizing pancreatitis. All tumors were excised and diagnosed as mesenteric desmoids. DNA from peripheral blood was tested for a multigene panel. The tumour DNA was screened for three most frequent ß-catenin gene mutations T41A, S45F and S45P. Expression levels of miR-21-3p and miR-197-3-p were compared between the desmoid tumors and other wild-type sporadic desmoids. The T41A CTNNB1 mutation was present in all four desmoid tumors. miR-21-3p and miR-197-3p were respectively upregulated and down-regulated in the mutated sporadic mesenteric desmoids, with respect to wild-type lesions. The patient is free from recurrence 34 months post-surgery. The literature review did not show similar studies. To our knowledge, this is the first study to interrogate genetic and epigenetic signature of multiple intraabdominal desmoids to investigate potential association with abdominal inflammation following surgery for necrotizing pancreatitis. We found mutational and epigenetic features that hint at potential activation of inflammation pathways within the desmoid tumor.

Genetic and clinical features of familial mediterranean fever (FMF) in a homogeneous cohort of patients from South-Eastern Italy.

Familial Mediterranean Fever (FMF) is linked with the MEFV gene and is the commonest among monogenic autoinflammatory diseases, with high prevalence in the Mediterranean basin. Although the clinical presentation of FMF has a major role in diagnosis, genotype/phenotype correlations and the role of "benign" gene variants (as R202Q) appear highly variable and incompletely clear, making difficult to select the most effective strategy in the management of patients. Aim of the present study was to investigate the clinical presentation and the genetic background in a homogenous cohort of patients from Apulia (south eastern Italy). We investigated 217 patients with a clinical suspect of autoinflammatory diseases, who were characterized for the occurrence of specific symptoms and with next generation sequencing by a 4-gene panel including MEFV, MVK, NLRP3 and TNFRSF1A. A genetic change was identified in 122 (53.7%) patients, with 161 different MEFV variants recorded in 100 individuals, 10 variants in NLRP3, and 6 each in TNFRSF1A and MVK. The benign variant R202Q was largely prevalent (41.6% of all MEFV variants). When patients were selected according the number of pathogenic MEFV variants (0, 1, or 2 pathogenic variants), results failed to show significant links between the frequency of symptoms and the number of pathogenic variants. Only family history and Pras score (indicative for severity of disease) predicted the presence of pathogenic variants, as compared with carriers of variants considered of uncertain significance or benign. Fever >38 °C and arthralgias appeared more frequently in R202Q-positive patients than in non-R202Q carriers. These two subgroups showed comparable duration of fever, occurrence of myalgia, abdominal and chest pain, Pras, and IFFS scores. In conclusion, results confirm that FMF manifests in mild form in non-middle eastern patients. This possibility partly affects the reliability of clinical criteria/scores. Furthermore, the presence of the R202Q variant might not be completely neutral in selected groups of patients.

Impact of High-to-Moderate Penetrance Genes on Genetic Testing: Looking over Breast Cancer.

Breast cancer (BC) is the most common cancer and the leading cause of cancer death in women worldwide. Since the discovery of the highly penetrant susceptibility genes BRCA1 and BRCA2, many other predisposition genes that confer a moderate risk of BC have been identified. Advances in multigene panel testing have allowed the simultaneous sequencing of BRCA1/2 with these genes in a cost-effective way. Germline DNA from 521 cases with BC fulfilling diagnostic criteria for hereditary BC were screened with multigene NGS testing. Pathogenic (PVs) and likely pathogenic (LPVs) variants in moderate penetrance genes were identified in 15 out of 521 patients (2.9%), including 2 missense, 7 non-sense, 1 indel, and 3 splice variants, as well as two different exon deletions, as follows: ATM (n = 4), CHEK2 (n = 5), PALB2 (n = 2), RAD51C (n = 1), and RAD51D (n = 3). Moreover, the segregation analysis of PVs and LPVs into first-degree relatives allowed the detection of CHEK2 variant carriers diagnosed with in situ melanoma and clear cell renal cell carcinoma (ccRCC), respectively. Extended testing beyond BRCA1/2 identified PVs and LPVs in a further 2.9% of BC patients. In conclusion, panel testing yields more accurate genetic information for appropriate counselling, risk management, and preventive options than assessing BRCA1/2 alone.

Beyond BRCA1/2: Homologous Recombination Repair Genetic Profile in a Large Cohort of Apulian Ovarian Cancers.

BACKGROUND: Pathogenic variants in homologous recombination repair (HRR) genes other than BRCA1/2 have been associated with a high risk of ovarian cancer (OC). In current clinical practice, genetic testing is generally limited to BRCA1/2. Herein, we investigated the mutational status of both BRCA1/2 and 5 HRR genes in 69 unselected OC, evaluating the advantage of multigene panel testing in everyday clinical practice. METHODS: We analyzed 69 epithelial OC samples using an NGS custom multigene panel of the 5 HRR pathways genes, beyond the genetic screening routine of BRCA1/2 testing. RESULTS: Overall, 19 pathogenic variants (27.5%) were detected. The majority (21.7%) of patients displayed a deleterious mutation in BRCA1/2, whereas 5.8% harbored a pathogenic variant in one of the HRR genes. Additionally, there were 14 (20.3%) uncertain significant variants (VUS). The assessment of germline mutational status showed that a small number of variants (five) were not detected in the corresponding blood sample. Notably, we detected one BRIP1 and four BRCA1/2 deleterious variants in the low-grade serous and endometrioid histology OC, respectively. CONCLUSION: We demonstrate that using a multigene panel beyond BRCA1/2 improves the diagnostic yield in OC testing, and it could produce clinically relevant results.

Analysis of telomere dynamics in peripheral blood cells from patients with Lynch syndrome.

BACKGROUND: In patients with Lynch syndrome, germline mutations in DNA mismatch repair (MMR) genes cause a high risk of developing a broad spectrum of cancers. To date, the management of patients with Lynch syndrome has represented a major challenge because of large variations in age at cancer onset. Several factors, including genetic anticipation, have been proposed to explain this phenotypic heterogeneity, but the molecular mechanisms remain unknown. Telomere shortening is a common event in tumorigenesis and also has been observed in different familial cancers. In this study, the authors investigated the possibility of a relation between telomere length and cancer onset in patients with Lynch syndrome. METHODS: The mean telomere length was measured using quantitative polymerase chain reaction in peripheral blood samples from a control group of 50 individuals, from 31 unaffected mutation carriers, and from 43 affected patients, and the results were correlated with both gene mutation and cancer occurrence. In affected patients, telomere attrition was correlated with age at cancer onset. In all patients, a t test was used to assess the linearity of the regression. RESULTS: A significant correlation between telomere length and age was observed in both affected and unaffected mutation carriers (P = .0016 and P = .004, respectively) and in mutS homolog 2 (MSH2) mutation carriers (P = .0002) but not in mutL homolog 1 (MLH1) mutation carriers. Telomere attrition was correlated significantly with age at onset in MSH2 carriers (P = .004), whereas an opposite trend toward longer telomeres in patients with delayed onset was observed in MLH1 carriers. CONCLUSIONS: The current data suggested that telomere dynamics differ between MLH1 and MSH2 mutation carriers. It is possible that subtle, gene-specific mechanisms can be linked to cancer onset and anticipation in patients with Lynch syndrome.

Synthesis and antibacterial evaluation of a novel series of 2-(1,2-dihydro-3-oxo-3H-pyrazol-2-yl)benzothiazoles.

The 2-(1,2-dihydro-3-oxo-3H-pyrazol-2-yl)benzothiazole scaffold was selected as a central core structure for the discovery of novel antibacterial compounds. A systematic variation of the substituents on the oxo-pyrazole moiety, as well as on the benzo moiety, led to the creation of a small and focused library of benzothiazoles that was subjected to antibacterial screening. In a first round of screening, activity of the compounds against six representative microorganisms was established. For the most potent congeners, MIC values against S. aureus and P. aeruginosa were determined. The structure-activity relationship study clearly revealed that subtle structural variations influence the antibacterial activity to a large extent. The most potent congeners displayed MIC values of 3.30 µM.