InnoGenomics (United States)

Pseudogenes are non-functional copies of protein-coding genes that arise through genomic duplication or retrotransposition. Processed pseudogenes (PPs) is the most abundant class of pseudogenes, which is generated via mRNA reverse transcription and subsequent cDNA integration. Presence of PPs complicates the analysis of short read sequencing data due to high similarity with parental gene and frequent absence from reference genome. Here we demonstrate that the presence of non-reference (absent from reference genome) PPs leads to the very distinctive artefact of germline variant calling - long insertions on exon-intron boundaries, which sequences could be mapped to other exons of the same gene. We showed that by detecting these artifacts it is possible to identify non-reference PPs existence based on the cohort summary statistics without analysing samplelevel data. We used identified signature of PPs presence to systematically mine the gnomAD database which currently contains over 70,000 whole-genome and 1 over 700,000 exome samples to describe novel non-reference PPs. Our approach uncovered 1498 non-reference PPs of which 1268 were novel and absent in the latest GENCODE release. This resource enhances the accuracy of variant interpretation and contributes to a deeper understanding of pseudogenes diversity across human populations.

MutationAssessor (MA) helps researchers evaluate the likely functional impact of somatic and germline mutations in cancer. It provides an evolution-based functional impact score (FIS) to classify mutations based on their likely effect on protein function. FIS scores are based on analysis of patterns of conservation in protein families (conserved residues) and subfamilies (specificity residues). In this new version (r4) we have (1) refined the combinatorial entropy analysis of conservation patterns, (2) recalculated full-length protein multiple sequence alignments covering a larger fraction of human proteins and making use of the explosive growth of protein sequence data, (3) compared predicted functional impact with the pathogenic-benign classification of sequence variants in curated knowledge bases, such as ClinVar, (4) observed the inverse relationship between predicted high functional impact and variant frequency in germline genome sequences and (5) explore the evaluation of switch-of-function mutational effects. Functional impact of ~4 million somatic amino-acid changing mutations across more than 320K human tumor samples are now available in the widely used cBioPortal for Cancer Genomics.

Introduction: Vulto-van Silfhout-de Vries Syndrome (VSVS; OMIM#615828) is a rare hereditary disease associated with impaired intellectual development and speech, delayed psychomotor development, and behavioral anomalies, including autistic behavioral traits and poor eye contact. To date, 27 patients with VSVS have been reported in the literature. Materials and Methods: We describe a 23-year-old male patient with autism spectrum disorder (ASD) who was admitted to the gastroenterological hospital with signs of pseudomembranous colitis. ASD was first noted in the patient at the age of 2.5 years. Later, he developed epileptic seizures and important growth retardation. Prior to the hospitalization, chromosomal aberrations, Fragile X syndrome, and aminoacidopathies/aminoacidurias associated with ASD were excluded. Whole-genome sequencing (WGS) was prescribed to the patient at 23 years old. Results: The patient had a heterozygous carrier of “de novo” variant c.662C > T (p.S221L) in exon 4 of the DEAF1 gene. c.662C > T had not been previously described in genomic databases. According to the ACMG criteria, this missense variant was considered to be pathogenic. VSVS was diagnosed in the patient. Conclusions: The phenotype of the patient is very similar to the data presented in the world literature. However, growth retardation and cachexia, which have not been described previously in the articles, are of interest.

Abstract A qPCR multiplex that utilizes two types of multi-copy retrotransposable elements (RE) in the human genome, as target loci, has been developed and validated. The single-well, qPCR reaction amplifies 80bp, 265bp and an internal-positive-control simultaneously, within 2 hours and with high PCR efficiencies. The selected RE targets comprise over 1500 copies in the genome and allow robust detection of low level cell-free (cf) DNA pg/µL) from <0.5mL plasma. The 80bp target measures total cfDNA. The 265bp target is used to assess cfDNA fragmentation and is presented as the DNA Integrity Index (DII=265/80) which is a ratio of the 265bp quantity divided by 80bp quantity. cfDNA concentration and integrity assessment has shown great promise as a highly sensitive, minimally invasive blood biomarker for multiple cancer types. InnoGenomics has developed an accurate, extremely sensitive liquid biopsy blood test for improved management of cancer patient care. Utilizing standard qPCR platforms for rapid and cost-effective processing, this test can reliably obtain results from small blood volumes (<0.5 mL of plasma), and possesses exceptionally high analytical sensitivity with a detection capability of < 0.1 pg of DNA (LOD). The developed assay demonstrated high predictive capacity in discriminating a group of 39 CRC stage IV patients from 40 healthy controls using cfDNA concentration and DNA Integrity Index (DII-265/80) values. All markers displayed high Area Under the receiver operating characteristic Curve (AUC) values - 80bp target: 0.9891, 265bp target: 0.9859, and DII-265/80: 0.8603. The DNA integrity ratio (DII) showed great potential as a prognostic marker for progression free survival (P=0.019). A retrospective cohort study of 3 patients demonstrated the potential use of this RE-qPCR cfDNA assay for cancer treatment monitoring. NCI SBIR Phase I studies showed high predictive capacity in discriminating metastatic colorectal cancer (CRC) patients from non-cancer healthy controls, with a significant increase in sensitivity and specificity compared to the CEA test, a serum biomarker currently used for CRC patient monitoring. Retrospective patient case studies demonstrated the test's ability to detect resistance to therapy and disease progression more accurately than CEA results and earlier than CT imaging. These results suggest strong potential clinical utility in several applications: (1) treatment monitoring for CRC stage IV patients, (2) recurrence surveillance for CRC stage I-III patients, (3) assessment of residual disease and (4) as a prognostic tool to help predict CRC patient outcomes which can serve as a tool to guide personalized/effective treatment regimens. A detailed prospective study will be undertaken during Phase II research, in collaboration with MD Anderson Cancer Center, to clinically validate the test. ACKNOWLEDGEMENT: This project was funded by an SBIR Phase I contract from NCI. Citation Format: Sudhir K. Sinha, Hiromi Brown, Zhide Fang, Mathilde Couetoux, Karen Gambaro, Gerald Batist. A multiplexed RE-qPCR cell-free DNA assay to assess response and resistance to cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5575.

Objectives: Retrotransposable elements (REs), consisting of long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs), are a group of markers that can be useful for human identity testing. Until now, however, due to the inherent size difference (up to 6 kb in some instances) associated with insertion and null alleles (or INNULs), the use of REs for facilitated population studies has not been sought or practical. The size of the insertion elements (from a few hundred to several thousand bp) has proven to limit their utility as a marker because of the inefficient amplicon yield with PCR. A novel primer design now facilitates INNUL marker testing. A preliminary panel of single-locus markers was developed to evaluate the potential of typing these insertion elements. Nine INNULs (5 Alu and 4 LINEs) were typed in three major North American populations and analyzed for population genetic features. In addition, the variation of each marker among the sample populations provides insight of its potential use as individual identification or ancestral marker. Methods: INNUL markers were developed into fluorescently labeled single-loci PCR. Nine markers were developed with amplicons that were less than 180 bp in length, and, depending on the locus amplicons of the INNULs, alleles varied in size from 50 to 1 bp. This allele size is noteworthy because the insertion alleles of the 9 loci range in size from 297 to 6,195 bp. The allele distribution of the INNULs was assessed and analyzed in three major North American populations. Results: Upon observation of the distribution of the alleles in three major North American populations, the markers generally met Hardy-Weinberg expectations, and there was little evidence of detectable levels of linkage disequilibrium. Due to varying distributions of the alleles in the major population groups tested, some of the markers might be better suited for use as an individual identification marker, while others are better suited for bio-ancestral studies. Conclusions: Using the primer design strategy described in our work, SINEs and (for the first time, to our knowledge) LINEs can be utilized as markers for studying population genetic variation that is more amenable to the limitations of the PCR technique. This study lays the foundation for future work of developing a multiplex panel of INNUL markers that can be used as a single-tube assay for human identity testing utilizing small amplicons (<180 bp), which could be useful for ancient or degraded forensic DNA samples.

N-nitroso compounds (NOCs) are suspected human carcinogens and relevant in human exposure. NOCs also induce micronuclei (MN) formation in vivo. Since lymphocytic MN represent a validated biomarker of human cancer risk, establishing a link between NOC exposure and MN frequency in humans and concurrently investigating associated transcriptomic responses may provide crucial information on underlying molecular mechanisms that predispose to carcinogenicity. We used lymphocytes, from adult females participating in the pan-European biomarker research project NewGeneris, as a surrogate tissue for analysing such potentially carcinogenic gene expression and MN formation events in target organs. To assess NOC exposure, urine samples were analysed for marker nitrosamines. NOC excretion levels and MN frequency were subsequently linked to peripheral blood transcriptomics. We demonstrated a significant association between MN frequency and urinary NOCs (r = 0.41, P = 0.025) and identified modifications in among others cytoskeleton remodeling, cell cycle, apoptosis and survival, signal transduction, immune response, G-protein signaling and development pathways, which indicate a response to NOC-induced genotoxicity. Moreover, we established a network of genes, the most important ones of which include FBXW7, BUB3, Caspase 2, Caspase 8, SMAD3, Huntingtin and MGMT, which are involved in processes relevant in carcinogenesis. The modified genetic processes and genes found in this study may be of interest for future investigations into the potential carcinogenic risk associated with NOC exposure in humans.

(2010). Professor Akira Yazawa. Mineral Processing and Extractive Metallurgy: Vol. 119, No. 4, pp. 249-250.

Microarrays have been used to evaluate the expression of thousands of genes in various tissues. However, few studies have investigated the change in gene expression profiles in one of the most easily accessible tissues, whole blood. We utilized an acute inflammation model to investigate the possibility of using a cDNA microarray to measure the gene expression profile in the cells of whole blood. Blood was collected from male Sprague-Dawley rats at 2 and 6 h after treatment with 5 mg/kg (ip) LPS. Hematology showed marked neutrophilia accompanied by lymphopenia at both time points. TNF-alpha and IL-6 levels were markedly elevated at 2 h, indicating acute inflammation, but by 6 h the levels had declined. Total RNA was isolated from whole blood and hybridized to the National Institute of Environmental Health Sciences Rat Chip v.3.0. LPS treatment caused 226 and 180 genes to be differentially expressed at 2 and 6 h, respectively. Many of the differentially expressed genes are involved in inflammation and the acute phase response, but differential expression was also noted in genes involved in the cytoskeleton, cell adhesion, oxidative respiration, and transcription. Real-time RT-PCR confirmed the differential regulation of a representative subset of genes. Principal component analysis of gene expression discriminated between the acute inflammatory response apparent at 2 h and the observed recovery underway at 6 h. These studies indicate that, in whole blood, changes in gene expression profiles can be detected that are reflective of inflammation, despite the adaptive shifts in leukocyte populations that accompany such inflammatory processes.

In response to acute and chronic stresses, the heart frequently undergoes a remodeling process that is accompanied by myocyte hypertrophy, impaired contractility, and pump failure, often culminating in sudden death. The existence of redundant signaling pathways that trigger heart failure poses challenges for therapeutic intervention. Cardiac remodeling is associated with the activation of a pathological gene program that weakens cardiac performance. Thus, targeting the disease process at the level of gene expression represents a potentially powerful therapeutic approach. In this review, we describe strategies for normalizing gene expression in the failing heart with small molecules that control signal transduction pathways directed at transcription factors and associated chromatin-modifying enzymes.