Detection of Genomic Structural Variants from Next-Generation Sequencing Data

Structural variants are genomic rearrangements larger than 50?bp accounting for around 1% of the variation among human genomes. They impact on phenotypic diversity and play a role in various diseases including neurological/neurocognitive disorders and cancer development and progression. Dissecting structural variants from next-generation sequencing data presents several challenges and a number of approaches have been proposed in the literature. In this mini review, we describe and summarize the latest tools ? and their underlying algorithms ? designed for the analysis of whole-genome sequencing, whole-exome sequencing, custom captures, and amplicon sequencing data, pointing out the major advantages/drawbacks. We also report a summary of the most recent applications of third-generation sequencing platforms. This assessment provides a guided indication ? with particular emphasis on human genetics and copy number variants ? for researchers involved in the investigation of these genomic events

Epilepsy with auditory features: a heterogeneous clinico-molecular disease

Objective: To identify novel genes implicated in epilepsy with auditory features (EAF) in phenotypically heterogeneous families with unknown molecular basis.Methods: We identified 15 probands with EAF in whom an LGI1 mutation had been excluded. We performed electroclinical phenotyping on all probands and available affected relatives. We used whole-exome sequencing (WES) in 20 individuals with EAF (including all the probands and 5 relatives) to identify single nucleotide variants, small insertions/deletions, and copy number variants.Results: WES revealed likely pathogenic variants in genes that had not been previously associated with EAF: a CNTNAP2 intragenic deletion, 2 truncating mutations of DEPDC5, and a missense SCN1A change.Conclusions: EAF is a clinically and molecularly heterogeneous disease. The association of EAF with CNTNAP2, DEPDC5, and SCN1A mutations widens the phenotypic spectrum related to these genes. CNTNAP2 encodes CASPR2, a member of the voltage-gated potassium channel complex in which LGI1 plays a role. The finding of a CNTNAP2 deletion emphasizes the importance of this complex in EAF and shows biological convergence

CNVScan: detecting border- line copy number variations in NGS data via scan statistics

Background. Next Generation Sequencing (NGS) data has been extensively exploited in the last decade to analyse genome variations and to understand the role of genome variations in complex diseases. Copy number variations (CNVs) are genomic structural variants estimated to account for about 1.2% of the total variation in humans. CNVs in coding or regulatory regions may have an impact on the gene expression, often also at a functional level, and contribute to cause different diseases like cancer, autism and cardiovascular diseases. Computational methods developed for detection of CNVs from NGS data and based on the depth of coverage are limited to the identification of medium/large events and heavily influenced by the level of coverage. Result. In this paper we propose, CNVScan a CNV detection method based on scan statistics that overcomes limitations of previous read count (RC) based approaches mainly by being a window-less approach. The scans statistics have been used before mainly in epidemiology and ecology studies, but never before was applied to the CNV detection problem to the best of our knowledge. Since we avoid window- ing we do not have to choose an optimal window-size which is a key step in many previous approaches. Extensive simulated experiments with single read data in extreme situations (low coverage, short reads, homo/heterozygoticity) show that this approach is very effective for a range of small CNV (200-500 bp) for which previous state-of-the-art methods are not suitable. Conclusion. The scan statistics technique is applied and adapted in this paper for the first time to the CNV detection problem. Comparison with state-of-the art methods shows the approach is quite effective in discovering shortCNVin rather extreme situations in which previous methods fail or have degraded performance. CNVScan thus extends the range of CNV sizes and types that can be detected via read count with single read data

miR-26a targets identification in prostate cancer cell lines using miRNA pull-out assay

The re-expression of tumor suppressor (TS) miRNAs in cancer cells in which they are downregulated (miRNA replacement therapy) has been evaluated as a promising approach to inhibit tumor proliferation both in in vitro and in vivo models. As a single miRNA simultaneously targets hundreds of genes, it derives that the TS-miRNA re-expression gives rise to a broad inhibition of pro-tumorigenic genes and pathways. In this work we investigated the molecular mechanism at the basis of the antiproliferative potential of the TS-miR-26a by identifying all its targets in prostate cancer (PCa) cell lines using the miRNA pull-out assay. miR-26a was transfected using the appropriate transfectant for each tumor cell lines. Cell proliferation was detected with crystal violet staining. miRNA pull-out assay was performed using biotinylated synthetic version of miR- 26a and the miRNA/target complexes isolated with streptvidine sepharose high performance (GE Health care). RNA-seq was performed with TruSeq stranded total RNA sample preparation kit and sequenced with HiSeq 2000 (Illumina). We rst demonstrated that miR-26a was downregulated in several tumor cell lines (including PCa cell lines). By overexpressing miR-26a in some of these tumor cell lines we established its tumor suppressor activity in that it was able to inhibit cell proliferation. In particular, it was able to affect cell proliferation in both PC-3 and DU-145 PCa cells. To identify the miR-26a targets in PCa, the miRNA pull-out assay was performed in DU-145 cells. Using this approach we were able to isolate the miRNA/target complexes that we sequenced using high-throughput technology. We obtained 1423 transcripts and we found that 85% of them presented canonical miRNA binding sites predicted by more than one predictive algorithms, suggesting that the miRNA/targets isolation was successful. These results were reinforced by the fact that some of the identi ed transcripts were miR-26a targets already validated in other biological contests. Finally, the isolated targets were signi cantly enriched of transcript belonging to biological processes relevant for cancer proliferation. The results indicate that the TS-miRNA pull-out assay protocol may be useful for the identi cation of TS-miRNA targets involved in key anti-tumorigenic processes and for that possible targets for anticancer therapy

MicroRNA 19a replacement partially rescues fin and cardiac defects in zebrafish model of Holt Oram syndrome

Holt-Oram Syndrome (HOS) is an autosomal dominant heart-hand syndrome caused by mutations in the TBX5 gene, a transcription factor capable of regulating hundreds of cardiac-specific genes through complex transcriptional networks. Here we show that, in zebrafish, modulation of a single miRNA is sufficient to rescue the morphogenetic defects generated by HOS. The analysis of miRNA-seq profiling revealed a decreased expression of miR-19a in Tbx5-depleted zebrafish embryos compared to the wild type. We revealed that the transcription of the miR-17-92 cluster, which harbors miR-19a, is induced by Tbx5 and that a defined dosage of miR-19a is essential for the correct development of the heart. Importantly, we highlighted that miR-19a replacement is able to rescue cardiac and pectoral fin defects and to increase the viability of HOS zebrafish embryos. We further observed that miR-19a replacement shifts the global gene expression profile of HOS-like zebrafish embryos towards the wild type condition, confirming the ability of miR-19a to rescue the Tbx5 phenotype. In conclusion our data demonstrate the importance of Tbx5/miR-19a regulatory circuit in heart development and provide a proof of principle that morphogenetic defects associated with HOS can be rescued by transient miRNA modulation

Circulating microRNAs in metastatic colorectal cancer (mCRC) patients (pts) treated with regorafenib

Introduction: Regorafenib is indicated for the treatment of mCRC patients who have failed all other therapies. Nevertheless a substantial percentage of patients experiences rapid disease progression (PD) and serious adverse events may occur. For these reasons, clinical and/or molecular markers able to improve the cost/benefit ratio are urgently needed. Circulating microRNAs (c-miRNAs) have been recognized as possible prognostic and diagnostic markers in mCRC. The aim of this study was to describe the early changes in plasma levels of 10 selected c-miRNAs during the treatment with regorafenib and to investigate their correlation with clinical outcome.Methods: Plasma samples of patients treated with regorafenib at our Institution were collected at baseline (D1) and after 15 days of treatment (D15). Plasma levels of c-miR-17, c-miR-21, c-miR-29, c-miR-34, c-miR-92, c-miR-126, c-miR-141, c-miR-221, c-miR-601, c-miR-760 were analysed by means of real-time PCR. Paired levels at D1 and D15 were compared by means of Wilcoxon test for each c-miRNA. C-miRNAs showing significant changes were further analysed in order to identify possible correlations with outcome.Results: Thirty-four patients were included in the present study. Main characteristics were the following: M/F = 50%/50%; median age = 65 (range 48-78 years); ECOG-PS 0/1-2 = 71%/29%; time from diagnosis of metastases </ ? 18 months 15%/85%. Median PFS and OS were 2.4 and 6.5 months, respectively. One (3%) patient achieved a response and 16 (47%) had disease stabilization (disease control rate: 50%). As compared to D1, the following c-miRNAs increased at D15: c-miR-601 (p = 0.01), c-miR-141 (p = 0.04) and c-miR-21 (p = 0.06). Despite a median increase in the overall population, 12 (35%) out of 34 patients showed reduced level of c-miR-21 at D15. Nine out of 12 (75%) patients with reduced levels of c-miR-21 achieved disease control, as compared to 8 out of 23 (35%) patients with increased levels (Fisher \u27s Exact Test, p = 0.035). Median PFS of patients with increased and decreased level of levels of c-miR-21 were 2.1 and 3.9 months, respectively (HR = 1.89 95%CI 0.92-4.14 p = 0.08). Data on OS are not yet mature. Early modifications of c-miR-21 levels showed a sensitivity of 82% in predicting benefit from regorafenib.Conclusion: The early modulation of c-miR-21 levels may predict benefit from regorafenib in terms of disease control. These results need validation in independent series

MicroRNA 19a replacement partially rescues fin and cardiac defects in zebrafish model of Holt Oram syndrome

Holt-Oram Syndrome (HOS) is an autosomal dominant heart-hand syndrome caused by mutations in the TBX5 gene, a transcription factor capable of regulating hundreds of cardiac-specific genes through complex transcriptional networks. Here we show that, in zebrafish, modulation of a single miRNA is sufficient to rescue the morphogenetic defects generated by HOS. The analysis of miRNA-seq profiling revealed a decreased expression of miR-19a in Tbx5-depleted zebrafish embryos compared to the wild type. We revealed that the transcription of the miR-17-92 cluster, which harbors miR-19a, is induced by Tbx5 and that a defined dosage of miR-19a is essential for the correct development of the heart. Importantly, we highlighted that miR-19a replacement is able to rescue cardiac and pectoral fin defects and to increase the viability of HOS zebrafish embryos. We further observed that miR-19a replacement shifts the global gene expression profile of HOS-like zebrafish embryos towards the wild type condition, confirming the ability of miR-19a to rescue the Tbx5 phenotype. In conclusion our data demonstrate the importance of Tbx5/miR-19a regulatory circuit in heart development and provide a proof of principle that morphogenetic defects associated with HOS can be rescued by transient miRNA modulation

Characterization and identification of hidden rare variants in the human genome

BackgroundBy examining the genotype calls generated by the 1000 Genomes Project we discovered that the human reference genome GRCh37 contains almost 20,000 loci in which the reference allele has never been observed in healthy individuals and around 70,000 loci in which it has been observed only in the heterozygous state.ResultsWe show that a large fraction of this rare reference allele (RRA) loci belongs to coding, functional and regulatory elements of the genome and could be linked to rare Mendelian disorders as well as cancer. We also demonstrate that classical germline and somatic variant calling tools are not capable to recognize the rare allele when present in these loci. To overcome such limitations, we developed a novel tool, named RAREVATOR, that is able to identify and call the rare allele in these genomic positions. By using a small cancer dataset we compared our tool with two state-of-the-art callers and we found that RAREVATOR identified more than 1,500 germline and 22 somatic RRA variants missed by the two methods and which belong to significantly mutated pathways.ConclusionsThese results show that, to date, the investigation of around 100,000 loci of the human genome has been missed by re-sequencing experiments based on the GRCh37 assembly and that our tool can fill the gap left by other methods. Moreover, the investigation of the latest version of the human reference genome, GRCh38, showed that although the GRC corrected almost all insertions and a small part of SNVs and deletions, a large number of functionally relevant RRAs still remain unchanged. For this reason, also future resequencing experiments, based on GRCh38, will benefit from RAREVATOR analysis results. RAREVATOR is freely available at http://sourceforge.net/projects/rarevator

miR-182-5p is an Evolutionarily Conserved Tbx5 Effector that Impacts Cardiac Development and Electrical Activity in Zebrafish

To dissect the TBX5 regulatory circuit, we focused on microRNAs (miRNAs) that collectively contribute to make TBX5 a pivotal cardiac regulator. We profiled miRNAs in hearts isolated from wild-type, CRE, Tbx5lox/+and Tbx5del/+ mice using a Next Generation Sequencing (NGS) approach. TBX5 deficiency in cardiomyocytes increased the expression of the miR-183 cluster family that is controlled by Kruppel-like factor 4, a transcription factor repressed by TBX5. MiR-182-5p, the most highly expressed miRNA of this family, was functionally analyzed in zebrafish. Transient overexpression of miR-182-5p affected heart morphology, calcium handling and the onset of arrhythmias as detected by ECG tracings. Accordingly, several calcium channel proteins identified as putative miR-182-5p targets were downregulated in miR-182-5p overexpressing hearts. In stable zebrafish transgenic lines, we demonstrated that selective miRNA-182-5p upregulation contributes to arrhythmias. Moreover, cardiac-specific down-regulation of miR-182-5p rescued cardiac defects in a zebrafish model of Holt-Oram syndrome. In conclusion, miR-182-5p exerts an evolutionarily conserved role as a TBX5 effector in the onset of cardiac propensity for arrhythmia, and constitutes a relevant target for mediating the relationship between TBX5, arrhythmia and heart development

Impacto da toxémia de gestação em cabras na sobrevivência dos cabritos

Dissertação de Mestrado Integrado em Medicina VeterináriaDe entre as principais doenças que afetam os pequenos ruminantes, a toxémia de gestação (TG) apresenta-se como a afeção metabólica mais frequente dos mesmos. Caracteriza-se por ter uma elevada mortalidade e, como tal, representa um grave problema económico nas explorações. O objetivo deste estudo foi determinar qual a taxa de sobrevivência dos cabritos, provenientes de cabras com TG, nascidos de cesariana, e compará-la com a taxa de sobrevivência dos cabritos nascidos por parto natural, sem auxílio humano, provenientes de cabras saudáveis. Outro objetivo deste trabalho foi determinar quais os parâmetros sanguíneos que estão associados a uma menor taxa de sobrevivência dos cabritos. Este estudo envolveu 8 cabras com TG (cujos cabritos nasceram de cesariana) e 9 cabras saudáveis (cujos cabritos nasceram de parto natural). Uma amostra de sangue foi recolhida da veia jugular até 15min, no máximo, após o nascimento, dos cabritos e foram determinados os seguintes parâmetros: Na+, K+, Cl-, HCO3 -, glucose, pH, excesso de base (BE), pCO2, anion gap (AG), ureia sanguínea (BUN, do inglês blood urea nitrogen), L-lactato, proteína total e hematócrito (Htc). Foi ainda registado o peso de cada cabrito. Nasceram 13 cabritos de parto natural, todos vivos ao sétimo dia, o que contrastou com os 21 cabritos nascidos de cesariana, dos quais 6 morreram, equivalendo a uma taxa de mortalidade de 28,5%. Foram encontradas diferenças estatisticamente significativas entre os valores dos 21 cabritos nascidos de cesariana e os dos 13 nascidos de parto natural nos seguintes parâmetros: peso, Na+, pH, HCO3 -, BE, BUN e L-lactato. Ocorreram também diferenças estatisticamente significativas nos 15 cabritos, nascidos de cesariana, que sobreviveram e os 6 cabritos que não sobreviveram nos valores de pH, BE, pCO2, BUN e L-lactato. Concluiu-se que a cetoacidose materna devida à TG teve um impacto negativo na taxa de sobrevivência dos cabritos, que apresentavam uma acidose metabólica e, nos casos fatais, com uma componente respiratória.ABSTRACT - The impact of pregnancy toxemia in goats on the survival rate of newborn kids - Amongst the main diseases that affect small ruminants, pregnancy toxemia (PT) presents itself as the most frequent metabolic disease. It is characterized by a high mortality rate and therefore it represents a serious economic problem for the farms. The main goal of this study was to determine the survival rate of the offspring of goats with PT, born from a caesarean section and compare it to the survival rate of the offspring that was naturally delivered, from healthy goats. Another goal was to determine which blood parameters were associated to a lower survival rate of the kids. This study involved 8 goats with PT (whose kids were born from caesarean section) and 9 healthy goats (whose kids were delivered naturally). A blood sample was obtained from the kids’ jugular vein 15 minutes (maximal) after they were born and the following parameters were determined: Na+, K+, Cl-, HCO3 -, glucose, pH, base excess (BE), pCO2, anion gap (AG), blood urea nitrogen (BUN), L-lactate, total protein and packed cell volume (PCV). The weight of each kid was also determined and registered. There were 13 kids delivered naturally, all alive at the 7th day of life, differing from the 21 kids that were born from a caesarean section, of which 6 died, giving a final mortality rate of 28.5% in this last group. There were statistically significant differences between the weight and blood values of the 21 kids born from caesarean section and the 13 kids delivered naturally in the following parameters: Na+, pH, HCO3 -, BE, BUN and L-lactate. There were also statistically significant differences between the 15 kids born from caesarean section that survived and the ones also born from caesarean section who did not survive, in the following parameters: pH, BE, pCO2, BUN and L-lactate. In conclusion, maternal ketoacidosis due to PT had a negative impact on the survival rate of the offspring. In the most severe cases the new-born kids had both a metabolic and respiratory acidosis.N/