The A to I editing landscape in melanoma and its relation to clinical outcome.

RNA editing refers to non-transient RNA modifications that occur after transcription and prior to translation by the ribosomes. RNA editing is more widespread in cancer cells than in non-transformed cells and is associated with tumorigenesis of various cancer tissues. However, RNA editing can also generate neo-antigens that expose tumour cells to host immunosurveillance. Global RNA editing in melanoma and its relevance to clinical outcome currently remain poorly characterized. The present study compared RNA editing as well as gene expression in tumour cell lines from melanoma patients of short or long metastasis-free survival, patients relapsing or not after immuno- and targeted therapy and tumours harbouring BRAF or NRAS mutations. Overall, our results showed that NTRK gene expression can be a marker of resistance to BRAF and MEK inhibition and gives some insights of candidate genes as potential biomarkers. In addition, this study revealed an increase in Adenosine-to-Inosine editing in Alu regions and in non-repetitive regions, including the hyperediting of the MOK and DZIP3 genes in relapsed tumour samples during targeted therapy and of the ZBTB11 gene in NRAS mutated melanoma cells. Therefore, RNA editing could be a promising tool for identifying predictive markers, tumour neoantigens and targetable pathways that could help in preventing relapses during immuno- or targeted therapies.

A Novel 31.1 kb α-Thalassemia Deletion (- -MEX3) Found in a Mexican Family.

α-Thalassemia (α-thal), a genetic disease characterized by microcytosis, hypochromia and anemia, is predominantly caused by deletions of the α-globin genes, HBA2 and HBA1. In this study, we describe a novel 31.1 kb α-thal deletion, - -MEX3 (NC_000016.10: g.151479_182582del), observed in a Mexican family, probably originated from non homologous recombination between two Alu sequences; the 5' Alu element has been involved in at least two other α-thal deletions [- -FIL (NG_000006.1: g.11684_43534del) and - -KOL] and possesses a core homologous sequence next to the - -MEX3 breakpoint. In addition, a 286 bp insertion in an Alu sequence downstream to the - -MEX3 3' breakpoint was found in the studied family, - -FIL carriers, and healthy subjects, suggesting a common genetic variation in the Mexican population. We highlight the involvement of Alu elements and their core sequence in the origin of deletions in the α-globin gene cluster, and the importance of characterizing rare mutations, to better understand DNA rearrangement origins.

Targeted long-read sequencing identifies and characterizes structural variants in cases of inherited platelet disorders.

BACKGROUND: Genetic diagnosis of inherited platelet disorders (IPDs) is mainly performed by high-throughput sequencing (HTS). These short-read-based sequencing methods sometimes fail to characterize the genetics of the disease. OBJECTIVES: To evaluate nanopore long-read DNA sequencing for characterization of structural variants (SVs) in patients with IPDs. METHODS: Four patients with a clinical and laboratory diagnosis of Glanzmann thrombasthenia (GT) (P1 and P2) and Hermansky-Pudlak syndrome (HPS) (P3 and P4) in whom HTS missed the underlying molecular cause were included. DNA was analyzed by both standard HTS and nanopore sequencing on a MinION device (Oxford Nanopore Technologies) after enrichment of DNA spanning regions covering GT and HPS genes. RESULTS: In patients with GT, HTS identified only 1 heterozygous ITGB3 splice variant c.2301+1G>C in P2. In patients with HPS, a homozygous deletion in HPS5 was suspected in P3, and 2 heterozygous HPS3 variants, c.2464C>T (p.Arg822∗) and a deletion affecting 2 exons, were reported in P4. Nanopore sequencing revealed a complex SV affecting exons 2 to 6 in ITGB3 (deletion-inversion-duplication) in homozygosity in P1 and compound heterozygosity with the splice variant in P2. In the 2 patients with HPS, nanopore defined the length of the SVs, which were characterized at nucleotide resolution. This allowed the identification of repetitive Alu elements at the breakpoints and the design of specific polymerase chain reactions for family screening. CONCLUSION: The nanopore technology overcomes the limitations of standard short-read sequencing techniques in SV characterization. Using nanopore, we characterized novel defects in ITGB3, HPS5, and HPS3, highlighting the utility of long-read sequencing as an additional diagnostic tool in IPDs.

Identification and Molecular Characterization of a Novel Large-Scale Variant (Exons 4_18 Loss) in the LDLR Gene as a Cause of Familial Hypercholesterolaemia in an Italian Family.

Next-generation sequencing (NGS) is nowadays commonly used for clinical purposes, and represents an efficient approach for the molecular diagnosis of familial hypercholesterolemia (FH). Although the dominant form of the disease is mostly due to the low-density lipoprotein receptor (LDLR) small-scale pathogenic variants, the copy number variations (CNVs) represent the underlying molecular defects in approximately 10% of FH cases. Here, we reported a novel large deletion in the LDLR gene involving exons 4-18, identified by the bioinformatic analysis of NGS data in an Italian family. A long PCR strategy was employed for the breakpoint region analysis where an insertion of six nucleotides (TTCACT) was found. Two Alu sequences, identified within intron 3 and exon 18, could underlie the identified rearrangement by a nonallelic homologous recombination (NAHR) mechanism. NGS proved to be an effective tool suitable for the identification of CNVs, together with small-scale alterations in the FH-related genes. For this purpose, the use and implementation of this cost-effective, efficient molecular approach meets the clinical need for personalized diagnosis in FH cases.

Replicative Senescence-Associated LINE1 Methylation and LINE1-Alu Expression Levels in Human Endothelial Cells.

One of the main challenges of current research on aging is to identify the complex epigenetic mechanisms involved in the acquisition of the cellular senescent phenotype. Despite some evidence suggested that epigenetic changes of DNA repetitive elements, including transposable elements (TE) sequences, are associated with replicative senescence of fibroblasts, data on different types of cells are scarce. We previously analysed genome-wide DNA methylation of young and replicative senescent human endothelial cells (HUVECs), highlighting increased levels of demethylated sequences in senescent cells. Here, we aligned the most significantly demethylated single CpG sites to the reference genome and annotated their localization inside TE sequences and found a significant hypomethylation of sequences belonging to the Long-Interspersed Element-1 (LINE-1 or L1) subfamilies L1M, L1P, and L1HS. To verify the hypothesis that L1 demethylation could be associated with increased transcription/activation of L1s and/or Alu elements (non-autonomous retroelements that usually depend on L1 sequences for reverse transcription and retrotransposition), we quantified the RNA expression levels of both L1 (generic L1 elements or site-specific L1PA2 on chromosome 14) and Alu elements in young and senescent HUVECs and human dermal fibroblasts (NHDFs). The RNA expression of Alu and L1 sequences was significantly increased in both senescent HUVECs and NHDFs, whereas the RNA transcript of L1PA2 on chromosome 14 was not significantly modulated in senescent cells. Moreover, we found an increased amount of TE DNA copies in the cytoplasm of senescent HUVECs and NHDFs. Our results support the hypothesis that TE, which are significantly increased in senescent cells, could be retrotranscribed to DNA sequences.

Embryonated Chicken Tumor Xenografts Derived from Circulating Tumor Cells as a Relevant Model to Study Metastatic Dissemination: A Proof of Concept.

Patient-Derived Xenografts (PDXs) in the Chorioallantoic Membrane (CAM) are a representative model for studying human tumors. Circulating Tumor Cells (CTCs) are involved in cancer dissemination and treatment resistance mechanisms. To facilitate research and deep analysis of these few cells, significant efforts were made to expand them. We evaluated here whether the isolation of fresh CTCs from patients with metastatic cancers could provide a reliable tumor model after a CAM xenograft. We enrolled 35 patients, with breast, prostate, or lung metastatic cancers. We performed microfluidic-based CTC enrichment. After 48-72 h of culture, the CTCs were engrafted onto the CAM of embryonated chicken eggs at day 9 of embryonic development (EDD9). The tumors were resected 9 days after engraftment and histopathological, immunochemical, and genomic analyses were performed. We obtained in ovo tumors for 61% of the patients. Dedifferentiated small tumors with spindle-shaped cells were observed. The epithelial-to-mesenchymal transition of CTCs could explain this phenotype. Beyond the feasibility of NGS in this model, we have highlighted a genomic concordance between the in ovo tumor and the original patient's tumor for constitutional polymorphism and somatic alteration in one patient. Alu DNA sequences were detected in the chicken embryo's distant organs, supporting the idea of dedifferentiated cells with aggressive behavior. To our knowledge, we performed the first chicken CAM CTC-derived xenografts with NGS analysis and evidence of CTC dissemination in the chicken embryo.

Evaluation of the Interplay between the ADAR Editome and Immunotherapy in Melanoma.

BACKGROUND: RNA editing is a highly conserved posttranscriptional mechanism that contributes to transcriptome diversity. In mammals, it includes nucleobase deaminations that convert cytidine (C) into uridine (U) and adenosine (A) into inosine (I). Evidence from cancer studies indicates that RNA-editing enzymes promote certain mechanisms of tumorigenesis. On the other hand, recoding editing in mRNA can generate mutations in proteins that can participate in the Major Histocompatibility Complex (MHC) ligandome and can therefore be recognized by the adaptive immune system. Anti-cancer treatment based on the administration of immune checkpoint inhibitors enhance these natural anti-cancer immune responses. RESULTS: Based on RNA-Seq datasets, we evaluated the editome of melanoma cell lines generated from patients pre- and post-immunotherapy with immune checkpoint inhibitors. Our results reveal a differential editing in Arthrobacter luteus (Alu) sequences between samples pre-therapy and relapses during therapy with immune checkpoint inhibitors. CONCLUSION: These data pave the way towards the development of new diagnostics and therapies targeted to editing that could help in preventing relapses during immunotherapies.

Genesis of Non-Coding RNA Genes in Human Chromosome 22-A Sequence Connection with Protein Genes Separated by Evolutionary Time.

A small phylogenetically conserved sequence of 11,231 bp, termed FAM247, is repeated in human chromosome 22 by segmental duplications. This sequence forms part of diverse genes that span evolutionary time, the protein genes being the earliest as they are present in zebrafish and/or mice genomes, and the long noncoding RNA genes and pseudogenes the most recent as they appear to be present only in the human genome. We propose that the conserved sequence provides a nucleation site for new gene development at evolutionarily conserved chromosomal loci where the FAM247 sequences reside. The FAM247 sequence also carries information in its open reading frames that provides protein exon amino acid sequences; one exon plays an integral role in immune system regulation, specifically, the function of ubiquitin-specific protease (USP18) in the regulation of interferon. An analysis of this multifaceted sequence and the genesis of genes that contain it is presented.

Regulatory-Compliant Validation of a Highly Sensitive qPCR for Biodistribution Assessment of Hemophilia A Patient Cells.

The investigation of the biodistribution profile of a cell-based medicinal product is a pivotal prerequisite to allow a factual benefit-risk assessment within the non-clinical to clinical translation in product development. Here, a qPCR-based method to determine the amount of human DNA in mouse DNA was validated according to the guidelines of the European Medicines Agency and the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. Furthermore, a preclinical worst-case scenario study was performed in which this method was applied to investigate the biodistribution of 2 × 106 intravenously administered, genetically modified, blood outgrowth endothelial cells from hemophilia A patients after 24 h and 7 days. The validation of the qPCR method demonstrated high accuracy, precision, and linearity for the concentration interval of 1:1 × 103 to 1:1 × 106 human to mouse DNA. The application of this method in the biodistribution study resulted in the detection of human genomes in four out of the eight investigated organs after 24 h. After 7 days, no human DNA was detected in the eight organs analyzed. This biodistribution study provides mandatory data on the toxicokinetic safety profile of an actual candidate cell-based medicinal product. The extensive evaluation of the required validation parameters confirms the applicability of the qPCR method for non-clinical biodistribution studies.

Cell-free DNA integrity for the monitoring of breast cancer: Future perspectives?

Breast cancer (BC) is the most common cancer and the second cause of death in women worldwide. Therapeutic options are increasing, but the response to treatments is not always efficient and the risk of recurrence covers decades. In this perspective, the need to have a proper follow-up for the therapeutic responses and for anticipating recurrence it is urgent in the clinical setting. Liquid biopsy provides the basic principle for a non-invasive method for the routinely monitoring of BC. However, due to the heterogeneity of tumors during onset and progression, the search for tumor DNA mutations of targeted genes in plasma/serum is a limiting factor. A possible approach overtaking this problem comes from the measurement of cell-free DNA integrity, which is an independent factor from the mutational status and theoretically is representative of all tumors. This review summarizes the state-of-the-art of cell-free DNA integrity researches in BC, the controversies and the future perspective.