Background: Estrogen receptors alpha (ERα) and beta (ERβ) are transcription factors (TFs) that mediate estrogen signaling and define the hormone-responsive phenotype of breast cancer (BC). The two receptors can be found co-expressed and play specific, often opposite, roles, with ERβ being able to modulate the effects of ERα on gene transcription and cell proliferation. ERβ is frequently lost in BC, where its presence generally correlates with a better prognosis of the disease. The identification of the genomic targets of ERβ in hormone-responsive BC cells is thus a critical step to elucidate the roles of this receptor in estrogen signaling and tumor cell biology. Results: Expression of full-length ERβ in hormone-responsive, ERα-positive MCF-7 cells resulted in a marked reduction in cell proliferation in response to estrogen and marked effects on the cell transcriptome. By ChIP-Seq we identified 9702 ERβ and 6024 ERα binding sites in estrogen-stimulated cells, comprising sites occupied by either ERβ, ERα or both ER subtypes. A search for TF binding matrices revealed that the majority of the binding sites identified comprise one or more Estrogen Response Element and the remaining show binding matrixes for other TFs known to mediate ER interaction with chromatin by tethering, including AP2, E2F and SP1. Of 921 genes differentially regulated by estrogen in ERβ+ vs ERβ- cells, 424 showed one or more ERβ site within 10 kb. These putative primary ERβ target genes control cell proliferation, death, differentiation, motility and adhesion, signal transduction and transcription, key cellular processes that might explain the biological and clinical phenotype of tumors expressing this ER subtype. ERβ binding in close proximity of several miRNA genes and in the mitochondrial genome, suggests the possible involvement of this receptor in small non-coding RNA biogenesis and mitochondrial genome functions. Conclusions: Results indicate that the vast majority of the genomic targets of ERβ can bind also ERα, suggesting that the overall action of ERβ on the genome of hormone-responsive BC cells depends mainly on the relative concentration of both ERs in the cell.

Background: Inherited retinal dystrophies, including Retinitis Pigmentosa and Leber Congenital Amaurosis among others, are a group of genetically heterogeneous disorders that lead to variable degrees of visual deficits. They can be caused by mutations in over 100 genes and there is evidence for the presence of as yet unidentified genes in a significant proportion of patients. We aimed at identifying a novel gene for an autosomal recessive form of early onset severe retinal dystrophy in a patient carrying no previously described mutations in known genes. Methods: An integrated strategy including homozygosity mapping and whole exome sequencing was used to identify the responsible mutation. Functional tests were performed in the medaka fish (Oryzias latipes) model organism to gain further insight into the pathogenic role of the ADAMTS18 gene in eye and central nervous system (CNS) dysfunction. Results: This study identified, in the analyzed patient, a homozygous missense mutation in the ADAMTS18 gene, which was recently linked to Knobloch syndrome, a rare developmental disorder that affects the eye and the occipital skull. In vivo gene knockdown performed in medaka fish confirmed both that the mutation has a pathogenic role and that the inactivation of this gene has a deleterious effect on photoreceptor cell function. Conclusion: This study reveals that mutations in the ADAMTS18 gene can cause a broad phenotypic spectrum of eye disorders and contribute to shed further light on the complexity of retinal diseases.

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. 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. 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

We performed a clinical and genetic characterization of a pediatric cohort of patients with inherited retinal dystrophy (IRD) to identify the most suitable cases for gene therapy. The cohort comprised 43 patients, aged between 2 and 18 years, with severe isolated IRD at the time of presentation. The ophthalmological characterization also included assessment of the photoreceptor layer integrity in the macular region (ellipsoid zone (EZ) band). In parallel, we carried out a targeted, next-generation sequencing (NGS)-based analysis using a panel that covers over 150 genes with either an established or a candidate role in IRD pathogenesis. Based on the ophthalmological assessment, the cohort was composed of 24 Leber congenital amaurosis, 14 early onset retinitis pigmentosa, and 5 achromatopsia patients. We identified causative mutations in 58.1% of the cases. We also found novel genotype-phenotype correlations in patients harboring mutations in the CEP290 and CNGB3 genes. The EZ band was detectable in 40% of the analyzed cases, also in patients with genotypes usually associated with severe clinical manifestations. This study provides the first detailed clinical-genetic assessment of severe IRDs with infantile onset and lays the foundation of a standardized protocol for the selection of patients that are more likely to benefit from gene replacement therapeutic approaches.

Inherited retinal diseases (IRDs) are often associated with variable clinical expressivity (VE) and incomplete penetrance (IP). Underlying mechanisms may include environmental, epigenetic, and genetic factors. Cis-acting expression quantitative trait loci (cis-eQTLs) can be implicated in the regulation of genes by favoring or hampering the expression of one allele over the other. Thus, the presence of such loci elicits allelic expression imbalance (AEI) that can be traced by massive parallel sequencing techniques. In this study, we performed an AEI analysis on RNA-sequencing (RNA-seq) data, from 52 healthy retina donors, that identified 194 imbalanced single nucleotide polymorphisms(SNPs) in 67 IRD genes. Focusing on SNPs displaying AEI at a frequency higher than 10%, we found evidence of AEI in several IRD genes regularly associated with IP and VE (BEST1, RP1, PROM1, and PRPH2). Based on these SNPs commonly undergoing AEI, we performed pyrosequencing in an independent sample set of 17 healthy retina donors in order to confirm our findings. Indeed, we were able to validate CDHR1, BEST1, and PROM1 to be subjected to cis-acting regulation. With this work, we aim to shed light on differentially expressed alleles in the human retina transcriptome that, in the context of autosomal dominant IRD cases, could help to explain IP or VE.

Mendelian disorders are mostly caused by single mutations in the DNA sequence of a gene, leading to a phenotype with pathologic consequences. Whole Exome Sequencing of patients can be a cost-effective alternative to standard genetic screenings to find causative mutations of genetic diseases, especially when the number of cases is limited. Analyzing exome sequencing data requires specific expertise, high computational resources and a reference variant database to identify pathogenic variants.