Low-pass whole genome sequencing as a cost-effective alternative to chromosomal microarray analysis for low- and middle-income countries.

Low-pass whole genome sequencing (LP-WGS) has been applied as alternative method to detect copy number variants (CNVs) in the clinical setting. Compared with chromosomal microarray analysis (CMA), the sequencing-based approach provides a similar resolution of CNV detection at a lower cost. In this study, we assessed the efficiency and reliability of LP-WGS as a more affordable alternative to CMA. A total of 1363 patients with unexplained neurodevelopmental delay/intellectual disability, autism spectrum disorders, and/or multiple congenital anomalies were enrolled. Those patients were referred from 15 nonprofit organizations and university centers located in different states in Brazil. The analysis of LP-WGS at 1x coverage (>50kb) revealed a positive testing result in 22% of the cases (304/1363), in which 219 and 85 correspond to pathogenic/likely pathogenic (P/LP) CNVs and variants of uncertain significance (VUS), respectively. The 16% (219/1363) diagnostic yield observed in our cohort is comparable to the 15%-20% reported for CMA in the literature. The use of commercial software, as demonstrated in this study, simplifies the implementation of the test in clinical settings. Particularly for countries like Brazil, where the cost of CMA presents a substantial barrier to most of the population, LP-WGS emerges as a cost-effective alternative for investigating copy number changes in cytogenetics.

A Benchmark of In-House Homologous Recombination Repair Deficiency Testing Solutions for High-Grade Serous Ovarian Cancer Diagnosis.

Homologous recombination deficiency (HRD) has become an important prognostic and predictive biomarker for patients with high-grade serous ovarian cancer who may benefit from poly-ADP ribose polymerase inhibitors (PARPi) and platinum-based therapies. HRD testing provides relevant information to personalize patients' treatment options and has been progressively incorporated into diagnostic laboratories. Here, we assessed the performance of an in-house HRD testing system deployable in a diagnostic clinical setting, comparing results from two commercially available next-generation sequencing (NGS)-based tumor tests (SOPHiA DDMTM HRD Solution and AmoyDx® (HRD Focus Panel)) with the reference assay from Myriad MyChoice® (CDx). A total of 85 ovarian cancer samples were subject to HRD testing. An overall strong correlation was observed across the three assays evaluated, regardless of the different underlying methods employed to assess genomic instability, with the highest pairwise correlation between Myriad and SOPHiA (R = 0.87, p-value = 3.39 × 10-19). The comparison of the assigned HRD status to the reference Myriad's test revealed a positive predictive value (PPV) and negative predictive value (NPV) of 90.9% and 96.3% for SOPHiA's test, while AmoyDx's test achieved 75% PPV and 100% NPV. This is the largest HRD testing evaluation using different methodologies and provides a clear picture of the robustness of NGS-based tests currently offered in the market. Our data shows that the implementation of in-house HRD testing in diagnostic laboratories is technically feasible and can be reliably performed with commercial assays. Also, the turnaround time is compatible with clinical needs, making it an ideal alternative to offer to a broader number of patients while maintaining high-quality standards at more accessible price tiers.

Differential microRNA expression profile in blood of children with Down syndrome suggests a role in immunological dysfunction.

Down syndrome (DS), caused by trisomy of chromosome 21 (HSA21), results in a broad range of phenotypes. However, the determinants contributing to the complex and variable phenotypic expression of DS are still not fully known. Changes in microRNAs (miRNAs), short non-coding RNA molecules that regulate gene expression post-transcriptionally, have been associated with some DS phenotypes. Here, we investigated the genome-wide mature miRNA expression profile in peripheral blood mononuclear cells (PBMCs) of children with DS and controls and identified biological processes and pathways relevant to the DS pathogenesis. The expression of 754 mature miRNAs was profiled in PBMCs from six children with DS and six controls by RT-qPCR using TaqMan® Array Human MicroRNA Cards. Functions and signaling pathways analyses were performed using DIANA-miRPath v.3 and DIANA-microT-CDS software. Children with DS presented six differentially expressed miRNAs (DEmiRs): four overexpressed (miR-378a-3p, miR-130b-5p, miR-942-5p, and miR-424-3p) and two downregulated (miR-452-5p and miR-668-3p). HSA21-derived miRNAs investigated were not found to be differentially expressed between the groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed potential target genes involved in biological processes and pathways pertinent to immune response, e.g., toll-like receptors (TLRs) signaling, Hippo, and transforming growth factor ß (TGF-ß) signaling pathways. These results suggest that altered miRNA expression could be contributing to the well-known immunological dysfunction observed in individuals with DS.

ETV4 plays a role on the primary events during the adenoma-adenocarcinoma progression in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the most common cancers worldwide; it is the fourth leading cause of death in the world and the third in Brazil. Mutations in the APC, DCC, KRAS and TP53 genes have been associated with the progression of sporadic CRC, occurring at defined pathological stages of the tumor progression and consequently modulating several genes in the corresponding signaling pathways. Therefore, the identification of gene signatures that occur at each stage during the CRC progression is critical and can present an impact on the diagnosis and prognosis of the patient. In this study, our main goal was to determine these signatures, by evaluating the gene expression of paired colorectal adenoma and adenocarcinoma samples to identify novel genetic markers in association to the adenoma-adenocarcinoma stage transition. METHODS: Ten paired adenoma and adenocarcinoma colorectal samples were subjected to microarray gene expression analysis. In addition, mutations in APC, KRAS and TP53 genes were investigated by DNA sequencing in paired samples of adenoma, adenocarcinoma, normal tissue, and peripheral blood from ten patients. RESULTS: Gene expression analysis revealed a signature of 689 differentially expressed genes (DEG) (fold-change> 2, p< 0.05), between the adenoma and adenocarcinoma paired samples analyzed. Gene pathway analysis using the 689 DEG identified important cancer pathways such as remodeling of the extracellular matrix and epithelial-mesenchymal transition. Among these DEG, the ETV4 stood out as one of the most expressed in the adenocarcinoma samples, further confirmed in the adenocarcinoma set of samples from the TCGA database. Subsequent in vitro siRNA assays against ETV4 resulted in the decrease of cell proliferation, colony formation and cell migration in the HT29 and SW480 colorectal cell lines. DNA sequencing analysis revealed KRAS and TP53 gene pathogenic mutations, exclusively in the adenocarcinomas samples. CONCLUSION: Our study identified a set of genes with high potential to be used as biomarkers in CRC, with a special emphasis on the ETV4 gene, which demonstrated involvement in proliferation and migration.

Differential Expression of Inflammation-Related Genes in Children with Down Syndrome.

OBJECTIVE: The aim of the study was to investigate the expression patterns of a specific set of genes involved in the inflammation process in children with Down Syndrome (DS) and children without the syndrome (control group) to identify differences that may be related to the immune abnormalities observed in DS individuals. METHOD: RNA samples were obtained from peripheral blood, and gene expression was quantified using the TaqMan® Array Plate Human Inflammation Kit, which facilitated the investigation into 92 inflammation-related genes and four reference genes using real-time polymerase chain reaction (qPCR). RESULTS: Twenty genes showed differential expression in children with DS; 12 were overexpressed (PLA2G2D, CACNA1D, ALOX12, VCAM1, ICAM1, PLCD1, ADRB1, HTR3A, PDE4C, CASP1, PLA2G5, and PLCB4), and eight were underexpressed (LTA4H, BDKRB1, ADRB2, CD40LG, ITGAM, TNFRSF1B, ITGB1, and TBXAS1). After statistically correcting for the false discovery rate, only the genes BDKRB1 and LTA4H showed differential expression, and both were underexpressed within the DS group. CONCLUSION: DS children showed differential expression of inflammation-related genes that were not located on chromosome 21 compared with children without DS. The BDKRB1 and LTA4H genes may differentiate the case and control groups based on the inflammatory response, which plays an important role in DS pathogenesis.

Altered expression of immune-related genes in children with Down syndrome.

Individuals with Down syndrome (DS) have a high incidence of immunological alterations with increased susceptibility to bacterial and viral infections and high frequency of different types of hematologic malignancies and autoimmune disorders. In the current study, we profiled the expression pattern of 92 immune-related genes in peripheral blood mononuclear cells (PBMCs) of two different groups, children with DS and control children, to identify differentially expressed genes that might be of pathogenetic importance for the development and phenotype of the immunological alterations observed in individuals with DS. PBMCs samples were obtained from six DS individuals with karyotypically confirmed full trisomy 21 and six healthy control individuals (ages 2-6 years). Gene expression was profiled in duplicate according to the manufacturer's instructions provided by commercially available TaqMan Human Immune Array representing 92 immune function genes and four reference genes on a 96-plex gene card. A set of 17 differentially expressed genes, not located on chromosome 21 (HSA21), involved in immune and inflammatory pathways was identified including 13 genes (BCL2, CCL3, CCR7, CD19, CD28, CD40, CD40LG, CD80, EDN1, IKBKB, IL6, NOS2 and SKI) significantly down-regulated and four genes (BCL2L1, CCR2, CCR5 and IL10) significantly up-regulated in children with DS. These findings highlight a list of candidate genes for further investigation into the molecular mechanism underlying DS pathology and reinforce the secondary effects of the presence of a third copy of HSA21.

Brief report: The lincRNA Hotair is required for epithelial-to-mesenchymal transition and stemness maintenance of cancer cell lines.

Hotair is a member of the recently described class of noncoding RNAs called lincRNA (large intergenic noncoding RNA). Various studies suggest that Hotair acts regulating epigenetic states by recruiting chromatin-modifying complexes to specific target sequences that ultimately leads to suppression of several genes. Although Hotair has been associated with metastasis and poor prognosis in different tumor types, a deep characterization of its functions in cancer is still needed. Here, we investigated the role of Hotair in the scenario of epithelial-to-mesenchymal transition (EMT) and in the arising and maintenance of cancer stem cells (CSCs). We found that treatment with TGF-ß1 resulted in increased Hotair expression and triggered the EMT program. Interestingly, ablation of Hotair expression by siRNA prevented the EMT program stimulated by TGF-ß1, and also the colony-forming capacity of colon and breast cancer cells. Furthermore, we observed that the colon CSC subpopulation (CD133(+)/CD44(+)) presents much higher levels of Hotair when compared with the non-stem cell subpopulation. These results indicate that Hotair acts as a key regulator that controls the multiple signaling mechanisms involved in EMT. Altogether, our data suggest that the role of Hotair in tumorigenesis occurs through EMT triggering and stemness acquisition.