Next-generation sequencing for endocrine cancers : Recent advances and challenges

Contemporary molecular biology research tools have enriched numerous areas of biomedical research that address challenging diseases, including endocrine cancers (pituitary, thyroid, parathyroid, adrenal, testicular, ovarian, and neuroendocrine cancers). These tools have placed several intriguing clues before the scientific community. Endocrine cancers pose a major challenge in health care and research despite considerable attempts by researchers to understand their etiology. Microarray analyses have provided gene signatures from many cells, tissues, and organs that can differentiate healthy states from diseased ones, and even show patterns that correlate with stages of a disease. Microarray data can also elucidate the responses of endocrine tumors to therapeutic treatments. The rapid progress in next-generation sequencing methods has overcome many of the initial challenges of these technologies, and their advantages over microarray techniques have enabled them to emerge as valuable aids for clinical research applications (prognosis, identification of drug targets, etc.). A comprehensive review describing the recent advances in next-generation sequencing methods and their application in the evaluation of endocrine and endocrine-related cancers is lacking. The main purpose of this review is to illustrate the concepts that collectively constitute our current view of the possibilities offered by next-generation sequencing technological platforms, challenges to relevant applications, and perspectives on the future of clinical genetic testing of patients with endocrine tumors. We focus on recent discoveries in the use of next-generation sequencing methods for clinical diagnosis of endocrine tumors in patients and conclude with a discussion on persisting challenges and future objectives

Next-Generation Sequencing — An Overview of the History, Tools, and “Omic” Applications

Next-generation sequencing (NGS) technologies using DNA, RNA, or methylation sequencing have impacted enormously on the life sciences. NGS is the choice for large-scale genomic and transcriptomic sequencing because of the high-throughput production and outputs of sequencing data in the gigabase range per instrument run and the lower cost compared to the traditional Sanger first-generation sequencing method. The vast amounts of data generated by NGS have broadened our understanding of structural and functional genomics through the concepts of “omics” ranging from basic genomics to integrated systeomics, providing new insight into the workings and meaning of genetic conservation and diversity of living things. NGS today is more than ever about how different organisms use genetic information and molecular biology to survive and reproduce with and without mutations, disease, and diversity within their population networks and changing environments. In this chapter, the advances, applications, and challenges of NGS are reviewed starting with a history of first-generation sequencing followed by the major NGS platforms, the bioinformatics issues confronting NGS data storage and analysis, and the impacts made in the fields of genetics, biology, agriculture, and medicine in the brave, new world of ”omics.

The potential for liquid biopsies in the precision medical treatment of breast cancer.

Currently the clinical management of breast cancer relies on relatively few prognostic/predictive clinical markers (estrogen receptor, progesterone receptor, HER2), based on primary tumor biology. Circulating biomarkers, such as circulating tumor DNA (ctDNA) or circulating tumor cells (CTCs) may enhance our treatment options by focusing on the very cells that are the direct precursors of distant metastatic disease, and probably inherently different than the primary tumor's biology. To shift the current clinical paradigm, assessing tumor biology in real time by molecularly profiling CTCs or ctDNA may serve to discover therapeutic targets, detect minimal residual disease and predict response to treatment. This review serves to elucidate the detection, characterization, and clinical application of CTCs and ctDNA with the goal of precision treatment of breast cancer

Cancer Genome Sequencing and Its Implications for Personalized Cancer Vaccines

New DNA sequencing platforms have revolutionized human genome sequencing. The dramatic advances in genome sequencing technologies predict that the $1,000 genome will become a reality within the next few years. Applied to cancer, the availability of cancer genome sequences permits real-time decision-making with the potential to affect diagnosis, prognosis, and treatment, and has opened the door towards personalized medicine. A promising strategy is the identification of mutated tumor antigens, and the design of personalized cancer vaccines. Supporting this notion are preliminary analyses of the epitope landscape in breast cancer suggesting that individual tumors express significant numbers of novel antigens to the immune system that can be specifically targeted through cancer vaccines

Application of next generation sequencing in genetic and genomic studies

Genetic variants that spread along the human genome play vital roles in determining our traits, affecting development and potentially causing disorders. Most common disorders have complex underlying mechanisms involving genetic or environmental factors and the interaction between them. Over the past decade, genome-wide association studies (GWAS) have identified thousands of common variants that contribute to complex disorders and partially explain the heritability. However, there is still a large portion that is unexplained and the missing heritability may be caused by several factors, such as rare or low-frequency variants with high effect that are not covered by GWAS and linkage analysis. With the development of next generation sequencing (NGS), it is possible to rapidly detect large amount of novel rare and low-frequency variants simultaneously at a low cost. This new technology provides vast information on studying the association of genetic variations and complex disorders. Once the susceptibility gene is mapped, model organisms such as zebrafish (Danio rerio) are popular for further investigating the possible function of diseaseassociated gene in determining the phenotype. However, the genome annotation of zebrafish is not complete, which affects the characterization of gene functions. Accordingly, highthroughput RNA sequencing can be employed for identifying new transcripts. In our studies, pooled DNA samples were used for whole genome sequencing (WGS) and exome sequencing. In Paper I, we evaluated minor allele frequency (MAF) estimates using three variant detection tools with two sets of pooled exome sequencing and one set of pooled WGS data. The MAFs from the pooled sequencing data demonstrated high concordance (r = 0.88-0.94) with those from the individual genotyping data. In Paper II, exome sequencing implementing pooling strategy was performed on 100 idiopathic scoliosis (IS) patients for mapping susceptibility genes. After validating 20 candidate single nucleotide variants (SNVs), we did not find associations between them and IS. However, the previously reported common variant rs11190870 near LBX1 was validated in a large Scandinavian cohort. In Paper III, we analyzed WGS of pooled DNA samples performed on 19 affected individuals who shared a phenotype-linked haplotype in a dyslexic Finish family. Two of the individuals were sequenced for the whole genome individually as well. The screen for causative variants was narrowed down to a rare SNV, which might affect the binding affinity of LHX2 that regulated dyslexia associated gene ROBO1. In Paper IV, RNA sequencing (RNA-seq) data were analyzed for identifying novel transcripts in zebrafish early development using an inhouse pipeline. We discovered 152 novel transcribed regions (NTRs), validated more than 10 NTRs and quantified their expression in early developmental stages. In our studies, we evaluated and applied a pooling approach for identifying variants susceptible to disease using high-throughput DNA sequencing. Based on RNA sequencing data, we provided new information for genome annotation on model organism zebrafish, which is valuable for studying the function of disease causative genes. In summary, the whole series of studies demonstrate how NGS can be applied in studying the genetic basis of complex disorders and assisting in follow-up functional studies in model organisms

AMY-tree: an algorithm to use whole genome SNP calling for Y chromosomal phylogenetic applications

BACKGROUND: Due to the rapid progress of next-generation sequencing (NGS) facilities, an explosion of human whole genome data will become available in the coming years. These data can be used to optimize and to increase the resolution of the phylogenetic Y chromosomal tree. Moreover, the exponential growth of known Y chromosomal lineages will require an automatic determination of the phylogenetic position of an individual based on whole genome SNP calling data and an up to date Y chromosomal tree. RESULTS: We present an automated approach, ‘AMY-tree’, which is able to determine the phylogenetic position of a Y chromosome using a whole genome SNP profile, independently from the NGS platform and SNP calling program, whereby mistakes in the SNP calling or phylogenetic Y chromosomal tree are taken into account. Moreover, AMY-tree indicates ambiguities within the present phylogenetic tree and points out new Y-SNPs which may be phylogenetically relevant. The AMY-tree software package was validated successfully on 118 whole genome SNP profiles of 109 males with different origins. Moreover, support was found for an unknown recurrent mutation, wrong reported mutation conversions and a large amount of new interesting Y-SNPs. CONCLUSIONS: Therefore, AMY-tree is a useful tool to determine the Y lineage of a sample based on SNP calling, to identify Y-SNPs with yet unknown phylogenetic position and to optimize the Y chromosomal phylogenetic tree in the future. AMY-tree will not add lineages to the existing phylogenetic tree of the Y-chromosome but it is the first step to analyse whole genome SNP profiles in a phylogenetic framework

Next generation sequencing approaches in rare diseases: the study of four different families

The main purpose of this PhD project was to study the molecular bases of rare Mendelian diseases through Next Generation Sequencing (NGS), finding the most appropriate NGS technology and data analysis approach. To this aim, we enrolled at Umberto I General Hospital and Sapienza University of Rome four different families with a phenotype due to a supposed genetic cause, in order to find the causative gene/genes. The selection of the experimental strategy, the number of subjects to sequence (the most distant family members, trio or singleton) and the data analysis approach were dictated by considerations on the diagnostic potential of each sequencing strategy and its feasibility and cost: the diagnostic rate, the possibility to re-evaluate the NGS data periodically, the management of NGS data, the functional interpretation of coding and non coding variants and the number of secondary findings were some of the criteria driving the choice of the NGS test. The choice was also influenced by specific features of each case, e.g. the supposed mode of inheritance, the available samples and the information about the phenotype. Whole exome sequencing (WES) and clinical exome sequencing (CES) experiments were performed in our laboratory or by outer companies. We analysed sequencing data through a dedicated bioinformatic pipeline and we filtered and prioritized the variants according to several parameters, specific for each case. Then, we validated the selected variant/variants through Sanger sequencing on the proband and the other family members, to study their segregation in the family, and we investigated the functional link between the candidate variant/variants and the phenotype. To study the molecular bases of the complex phenotypes regarding canine agenesis and eruption anomalies in the family A, we performed a WES approach on three first degree cousins. Different data analyses, based on different shared genetic causes, allowed us to identify several candidate variants: two missense variants in EDARADD and COL5A1, previously associated with tooth agenesis and a syndromic phenotype including dental anomalies, respectively; three missense variants in RSPO4, T and NELL1, genes functionally related to tooth morphogenesis. The segregation analyses pointed to two different signaling pathways as responsible for the phenotypes, one of them (i.e. EDA) for the canine agenesis, and the other (i.e. WNT) for the less severe canine eruption anomalies. To find the cause of the isolated brachydactyly observed in family B, we used a WES approach on the proband and his grandfather. We identified a shared frameshift variant in the GDF5 gene, encoding for a secreted ligand of TGF-β and predominantly expressed in long bones during embryonic development. This was important for genetic counselling as it is causative of a mild phenotype in heterozygous state, but also of a very severe phenotype in homozygous state. To find the cause of the corpus callosum anomaly observed in the proband of family C, we chose a trio approach. We performed a CES, using an enrichment kit that included 171 of 180 genes reported in literature as causative of corpus callosum malformations. We identified in the proband a supposedly de novo nonsense variant in the ARX gene, critical for early development and formation of a normal brain. Segregation analysis disclosed the presence of the same variant also in the fetus of a previous pregnancy, suggesting a gonadal or gonosomal mosaicism in one of the parents. The identification of this variant was important for genetic counselling as there is an increased recurrence risk for the couple to have a child with the same disorder. It was also important for the proband’s clinical prognosis and to properly calculate the risk to transmit the mutation, which is associated with different clinical outcomes depending on the sex. To investigate the molecular bases of the recurrent Dandy- Walker malformation observed in the family D, we performed WES only of the proband. We identified a homozygous missense variant in FKTN gene, encoding a glycosyltransferase with a role in brain development. In order to test the pathogenicity of the variant, we also performed a structural modeling of FKTN. This result allowed to properly redefine the clinical diagnosis as a Muscular Dystrophy-Dystroglycanopathy Type A, with implications on recurrence risk for the couple and on reproductive choices. The adopted experimental and data analysis strategies allowed us to identify the molecular causes of phenotypes involving different systems and belonging to different clinical pictures, with significant impact on diagnosis, prognosis and genetic counselling. These results show how NGS is revolutionizing medical genetics, accelerating the research about rare-genetic diseases, facilitating clinical diagnosis and leading us to the personalized medicine

Exploring molecular biology in sequence space: the road to next-generation single-molecule biophysics

Next-generation sequencing techniques have led to a new quantitative dimension in the biological sciences. In particular, integrating sequencing techniques with biophysical tools allows sequence-dependent mechanistic studies. Using the millions of DNA clusters that are generated during sequencing to perform high-throughput binding affinity and kinetics measurements enabled the construction of energy landscapes in sequence space, uncovering relationships between sequence, structure, and function. Here, we review the approaches to perform ensemble fluorescence experiments on next-generation sequencing chips for variations of DNA, RNA, and protein sequences. As the next step, we anticipate that these fluorescence experiments will be pushed to the single-molecule level, which can directly uncover kinetics and molecular heterogeneity in an unprecedented high-throughput fashion. Molecular biophysics in sequence space, both at the ensemble and single-molecule level, leads to new mechanistic insights. The wide spectrum of applications in biology and medicine ranges from the fundamental understanding of evolutionary pathways to the development of new therapeutics.Biological and Soft Matter Physic

Human muscle-derived CLEC14A-positive cells regenerate muscle independent of PAX7

Skeletal muscle stem cells, called satellite cells and defined by the transcription factor PAX7, are responsible for postnatal muscle growth, homeostasis and regeneration. Attempts to utilize the regenerative potential of muscle stem cells for therapeutic purposes so far failed. We previously established the existence of human PAX7-positive cell colonies with high regenerative potential. We now identified PAX7-negative human muscle-derived cell colonies also positive for the myogenic markers desmin and MYF5. These include cells from a patient with a homozygous PAX7 c.86-1G > A mutation (PAX7null). Single cell and bulk transcriptome analysis show high intra- and inter-donor heterogeneity and reveal the endothelial cell marker CLEC14A to be highly expressed in PAX7null cells. All PAX7-negative cell populations, including PAX7null, form myofibers after transplantation into mice, and regenerate muscle after reinjury. Transplanted PAX7neg cells repopulate the satellite cell niche where they re-express PAX7, or, strikingly, CLEC14A. In conclusion, transplanted human cells do not depend on PAX7 for muscle regeneration