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Background Targeted resequencing has become the most used and cost-effective approach for identifying causative mutations of Mendelian diseases both for diagnostics and research purposes. Due to very rapid technological progress, NGS laboratories are expanding their capabilities to address the increasing number of analyses. Several open source tools are available to build a generic variant calling pipeline, but a tool able to simultaneously execute multiple analyses, organize, and categorize the samples is still missing. Results Here we describe VarGenius, a Linux based command line software able to execute customizable pipelines for the analysis of multiple targeted resequencing data using parallel computing. VarGenius provides a database to store the output of the analysis (calling quality statistics, variant annotations, internal allelic variant frequencies) and sample information (personal data, genotypes, phenotypes). VarGenius can also perform the “joint analysis” of hundreds of samples with a single command, drastically reducing the time for the configuration and execution of the analysis. VarGenius executes the standard pipeline of the Genome Analysis Tool-Kit (GATK) best practices (GBP) for germinal variant calling, annotates the variants using Annovar, and generates a user-friendly output displaying the results through a web page. VarGenius has been tested on a parallel computing cluster with 52 machines with 120GB of RAM each. Under this configuration, a 50 M whole exome sequencing (WES) analysis for a family was executed in about 7 h (trio or quartet); a joint analysis of 30 WES in about 24 h and the parallel analysis of 34 single samples from a 1 M panel in about 2 h. Conclusions We developed VarGenius, a “master” tool that faces the increasing demand of heterogeneous NGS analyses and allows maximum flexibility for downstream analyses. It paves the way to a different kind of analysis, centered on cohorts rather than on singleton. Patient and variant information are stored into the database and any output file can be accessed programmatically. VarGenius can be used for routine analyses by biomedical researchers with basic Linux skills providing additional flexibility for computational biologists to develop their own algorithms for the comparison and analysis of data. The software is freely available at: https://github.com/frankMusacchia/VarGenius

The influence of the N-terminal residues 203–214 and the linker domain on motions in the human topoisomerase I-DNA complex has been investigated by comparing the molecular dynamics simulations of the system with (topo70) or without (topo58/6.3) these regions. Topo58/6.3 is found to fluctuate more than topo70, indicating that the presence of the N-terminal residues and the linker domain dampen the core and C-terminal fluctuations. The simulations also show that residues 203–207 and the linker domain participate in a network of correlated movements with key regions of the enzyme, involved in the human topoisomerase I catalytic cycle, providing a structural-dynamical explanation for the better DNA relaxation activity of topo70 when compared to topo58/6.3. The data have been examined in relation to a wealth of biochemical, site-directed mutagenesis and crystallographic data on human topoisomerase I. The simulations finally show the occurrence of a network of direct and water mediated hydrogen bonds in the proximity of the active site, and the presence of a water molecule in the appropriate position to accept a proton from the catalytic Tyr-723 residue, suggesting that water molecules have an important role in the stabilization and function of this enzyme.

Targeted resequencing has become the most used and cost-effective approach for identifying causative mutations of Mendelian diseases both for diagnostics and research purposes. Due to very rapid technological progress, NGS laboratories are expanding their capabilities to address the increasing number of analyses. Several open source tools are available to build a generic variant calling pipeline, but a tool able to simultaneously execute multiple analyses, organize, and categorize the samples is still missing. Here we describe VarGenius, a Linux based command line software able to execute customizable pipelines for the analysis of multiple targeted resequencing data using parallel computing. VarGenius provides a database to store the output of the analysis (calling quality statistics, variant annotations, internal allelic variant frequencies) and sample information (personal data, genotypes, phenotypes). VarGenius can also perform the \"joint analysis\" of hundreds of samples with a single command, drastically reducing the time for the configuration and execution of the analysis. We developed VarGenius, a \"master\" tool that faces the increasing demand of heterogeneous NGS analyses and allows maximum flexibility for downstream analyses. It paves the way to a different kind of analysis, centered on cohorts rather than on singleton. Patient and variant information are stored into the database and any output file can be accessed programmatically. VarGenius can be used for routine analyses by biomedical researchers with basic Linux skills providing additional flexibility for computational biologists to develop their own algorithms for the comparison and analysis of data.

Background Coffin–Siris syndrome (CSS) is characterized by intellectual disability, dysmorphic facial features, growth deficiency, microcephaly, and abnormalities of the fifth fingers/toes. CSS is caused by mutations in several genes of the BRG1‐associated factor pathway including SMARCA4. Methods Whole‐exome sequencing was performed on a 14‐year‐old female individual who presented with mild intellectual disability and dysmorphic features, tooth abnormalities, and short stature. She had brachydactyly but no aplasia or hypoplasia of the distal phalanx or nail of the fifth digit. She was also found to have retinal dystrophy that has not been previously reported in CSS. Results The individual presented herein was found to harbor a previously unreported de novo variant in SMARCA4. Conclusion This case expands the phenotypic spectrum of CSS manifestations. We report an individual harboring a previously unreported de novo variant in SMARCA4, who presented with mild dysmorphic features and retinopathy, which expand the phenotypic spectrum of Coffin‐Siris syndrome manifestations.

The pressure towards innovation and creation of new model systems in regenerative medicine and cancer research has fostered the development of novel potential therapeutic applications. Kidney injuries provoke a high request of organ transplants making it the most demanding system in the field of regenerative medicine. Furthermore, renal cancer frequently threaten patients’ life and aggressive forms still remain difficult to treat. Ethical issues related to the use of embryonic stem cells, has fueled research on adult, patient-specific pluripotent stem cells as a model for discovery and therapeutic development, but to date, normal and cancerous renal experimental models are lacking. Several research groups are focusing on the development of organoid cultures. Since organoids mimic the original tissue architecture in vitro, they represent an excellent model for tissue engineering studies and cancer therapy testing. We established normal and tumor renal cell carcinoma organoids previously maintained in a heterogeneous multi-clone stem cell-like enriching medium. Starting from adult normal kidney specimens, we were able to isolate and propagate organoid 3D-structures composed of both differentiated and undifferentiated cells while expressing nephron specific markers. Furthermore, we were capable to establish organoids derived from cancer tissues although with a success rate inferior to that of their normal counterpart. Cancer cultures displayed epithelial and mesenchymal phenotype while retaining tumor specific markers. Of note, tumor organoids recapitulated neoplastic masses when orthotopically injected into immunocompromised mice. Our data suggest an innovative approach of long-term establishment of normal- and cancer-derived renal organoids obtained from cultures of fleshly dissociated adult tissues. Our results pave the way to organ replacement pioneering strategies as well as to new models for studying drug-induced nephrotoxicity and renal diseases. Along similar lines, deriving organoids from renal cancer patients opens unprecedented opportunities for generation of preclinical models aimed at improving therapeutic treatments.

Quiescent cancer cells (QCCs) are a common feature of solid tumors, representing a major obstacle to the long-term success of cancer therapies. We isolated QCCs ex vivo from non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) xenografts with a label-retaining strategy and compared QCCs gene expression profiles to identify a shared “quiescence signature”. Principal Component Analysis (PCA) revealed a specific component neatly discriminating quiescent and replicative phenotypes in NSCLC and CRC. The discriminating component showed significant overlapping, with 688 genes in common including ZEB2, a master regulator of stem cell plasticity and epithelial-to-mesenchymal transition (EMT). Gene set enrichment analysis showed that QCCs of both NSCLC and CRC had an increased expression of factors related to stemness/self renewal, EMT, TGF-β, morphogenesis, cell adhesion and chemotaxis, whereas proliferating cells overexpressed Myc targets and factors involved in RNA metabolism. Eventually, we analyzed in depth by means of a complex network approach, both the ‘morphogenesis module’ and the subset of differentially expressed genes shared by NCSLC and CRC. This allowed us to recognize different gene regulation network wiring for quiescent and proliferating cells and to underpin few genes central for network integration that may represent new therapeutic vulnerabilities. Altogether, our results highlight common regulatory pathways in QCCs of lung and colorectal tumors that may be the target of future therapeutic interventions.

A class of retinal dystrophies known as retinitis pigmentosa (RP) is caused by the loss of photoreceptor cells. RP can be genetically transmitted as an autosomal dominant, autosomal recessive, or X-linked trait. About one-third of genes implicated in retinal degeneration encode for proteins whose functional dysregulation affects the “connecting cilium” in photoreceptors, altering its structure and function. Here we report on a 33-year-old woman who was referred for clinical genetic testing following a previous diagnosis of degenerative retinopathy, which was not informative. She was enrolled in a research program dedicated to undiagnosed retinal disorders, where a whole genome sequencing approach was employed to understand the underlying genetic basis. The genomic analysis documented the occurrence of compound heterozygosity for two functionally relevant missense variants in BAIAP3, which encodes a protein with a well-documented role in SNARE-mediated trafficking and ciliogenesis. Confocal microscopy analysis showed elongated cilia in patient-derived and BAIAP3-depleted fibroblasts compared to control cells. Real-time PCR analyses showed a consistent significant reduction of GLI1 mRNA levels in patient-derived and BAIAP3-depleted cells, both in basal conditions and after treatment with Smoothened agonist, SAG, indicating Sonic hedgehog signaling dysregulation. Collectively, these data suggest that biallelic loss-of-function variants of BAIAP3 may cause photoreceptor degeneration and underlie isolated RP.

Hereditary spastic paraplegias (HSP) are clinical and genetic heterogeneous diseases with more than 80 disease genes identified thus far. Studies on large cohorts of HSP patients showed that, by means of current technologies, the percentage of genetically solved cases is close to 50%. Notably, the percentage of molecularly confirmed diagnoses decreases significantly in sporadic patients. To describe our diagnostic molecular genetic approach on patients with pediatric-onset pure and complex HSP, 47 subjects with HSP underwent molecular screening of 113 known and candidate disease genes by targeted capture and massively parallel sequencing. Negative cases were successively analyzed by multiplex ligation-dependent probe amplification (MLPA) analysis for the SPAST gene and high-resolution SNP array analysis for genome-wide CNV detection. Diagnosis was molecularly confirmed in 29 out of 47 (62%) patients, most of whom had clinical diagnosis of cHSP. Although SPG11 and SPG4 remain the most frequent cause of, respectively, complex and pure HSP, a large number of pathogenic variants were disclosed in POLR3A, FA2H, DDHD2, ATP2B4, ENTPD1, ERLIN2, CAPN1, ALS2, ADAR1, RNASEH2B, TUBB4A, ATL1, and KIF1A. In a subset of these disease genes, phenotypic expansion and novel genotype-phenotype correlations were recognized. Notably, SNP array analysis did not provide any significant contribution in increasing the diagnostic yield. Our findings document the high diagnostic yield of targeted sequencing for patients with pediatric-onset, complex, and pure HSP. MLPA for SPAST and SNP array should be limited to properly selected cases based on clinical suspicion.

Colorectal cancer stem cell (CSC)‐enriched cultures were obtained efficiently from primary tumor specimens by optimizing a CSC‐isolation protocol. Based on in vitro and in vivo validation, genetic characterization, and drug sensitivity analysis, panels of CSC lines were generated with defined patterns of genetic mutations and therapy sensitivity. An analysis of CSC response to EGFR‐targeted therapy in vivo and an overview of factors implicated in therapy response/resistance are presented. Colorectal cancer (CRC) therapy mainly relies on the use of conventional chemotherapeutic drugs combined, in a subset of patients, with epidermal growth factor receptor [EGFR]‐targeting agents. Although CRC is considered a prototype of a cancer stem cell (CSC)‐driven tumor, the effects of both conventional and targeted therapies on the CSC compartment are largely unknown. We have optimized a protocol for colorectal CSC isolation that allowed us to obtain CSC‐enriched cultures from primary tumor specimens, with high efficiency. CSC isolation was followed by in vitro and in vivo validation, genetic characterization, and drug sensitivity analysis, thus generating panels of CSC lines with defined patterns of genetic mutations and therapy sensitivity. Colorectal CSC lines were polyclonal and maintained intratumor heterogeneity in terms of somatically acquired mutations and differentiation state. Such CSC‐enriched cultures were used to investigate the effects of both conventional and targeted therapies on the CSC compartment in vivo and to generate a proteomic picture of signaling pathways implicated in sensitivity/resistance to anti‐EGFR agents. We propose CSC lines as a sound preclinical framework to test the effects of therapies in vitro and in vivo and to identify novel determinants of therapy resistance. Significance Colorectal cancer stem cells (CSCs) have been shown to be responsible for tumor propagation, metastatic dissemination, and relapse. However, molecular pathways present in CSCs, as well as mechanisms of therapy resistance, are mostly unknown. Taking advantage of genetically characterized CSC lines derived from colorectal tumors, this study provides an extensive analysis of CSC response to EGFR‐targeted therapy in vivo and an overview of factors implicated in therapy response or resistance. Furthermore, the implementation of a biobank of molecularly annotated CSC lines provides an innovative resource for future investigations in colorectal cancer.

Background: Dominantly acting variants in TUBB2B have primarily been associated with cortical dysplasia complex with other brain malformations 7 (CDCBM7), a disorder in which cortical brain abnormalities are typically linked to developmental delay/intellectual disability (DD/ID) and seizures. While the majority of TUBB2B pathogenic variants have been linked to isolated CDCBM7, only one family with CDCBM7 and congenital fibrosis of the extraocular muscles (CFEOM) has been reported so far. We describe a second individual with a severe phenotype of CFEOM combined with CDCBM7 carrying a pathogenic TUBB2B missense variant previously reported in two individuals with isolated CDCBM7. Methods: A trio-based WGS analysis was performed. The structural impact of the identified substitution was assessed by using the UCSF Chimera (v.1.17.3) software and PyMOL docking plugin DockingPie tool. Results: WGS analysis identified a de novo missense TUBB2B variant (p.Ile202Thr, NM_178012.5), previously associated with isolated CDCBM7. Structural analysis and docking simulations revealed that Ile202 contributes to establishing a proper hydrophobic environment required to stabilize GTP/GDP in the β-tubulin pocket. p.Ile202Thr was predicted to disrupt these interactions. Conclusions: Our findings broaden the mutational spectrum of TUBB2B-related CFEOM, targeting a different functional domain of the protein, and further document the occurrence of phenotypic heterogeneity. We also highlight the limitations of exome sequencing in accurately mapping TUBB2B coding exons due to its high sequence homology with TUBB2A and suggest targeted or genome analyses when clinical suspicion is strong.