Menu
April 21, 2020

Schizophrenia risk variants influence multiple classes of transcripts of sorting nexin 19 (SNX19).

Genome-wide association studies (GWAS) have identified many genomic loci associated with risk for schizophrenia, but unambiguous identification of the relationship between disease-associated variants and specific genes, and in particular their effect on risk conferring transcripts, has proven difficult. To better understand the specific molecular mechanism(s) at the schizophrenia locus in 11q25, we undertook cis expression quantitative trait loci (cis-eQTL) mapping for this 2 megabase genomic region using postmortem human brain samples. To comprehensively assess the effects of genetic risk upon local expression, we evaluated multiple transcript features: genes, exons, and exon-exon junctions in multiple brain regions-dorsolateral prefrontal cortex (DLPFC), hippocampus, and caudate. Genetic risk variants strongly associated with expression of SNX19 transcript features that tag multiple rare classes of SNX19 transcripts, whereas they only weakly affected expression of an exon-exon junction that tags the majority of abundant transcripts. The most prominent class of SNX19 risk-associated transcripts is predicted to be overexpressed, defined by an exon-exon splice junction between exons 8 and 10 (junc8.10) and that is predicted to encode proteins that lack the characteristic nexin C terminal domain. Risk alleles were also associated with either increased or decreased expression of multiple additional classes of transcripts. With RACE, molecular cloning, and long read sequencing, we found a number of novel SNX19 transcripts that further define the set of potential etiological transcripts. We explored epigenetic regulation of SNX19 expression and found that DNA methylation at CpG sites near the primary transcription start site and within exon 2 partially mediate the effects of risk variants on risk-associated expression. ATAC sequencing revealed that some of the most strongly risk-associated SNPs are located within a region of open chromatin, suggesting a nearby regulatory element is involved. These findings indicate a potentially complex molecular etiology, in which risk alleles for schizophrenia generate epigenetic alterations and dysregulation of multiple classes of SNX19 transcripts.


April 21, 2020

TIN2 Functions with TPP1/POT1 To Stimulate Telomerase Processivity.

TIN2 is an important regulator of telomere length, and mutations in TINF2, the gene encoding TIN2, cause short-telomere syndromes. While the genetics underscore the importance of TIN2, the mechanism through which TIN2 regulates telomere length remains unclear. Here, we tested the effects of human TIN2 on telomerase activity. We identified a new isoform in human cells, TIN2M, that is expressed at levels similar to those of previously studied TIN2 isoforms. All three TIN2 isoforms localized to and maintained telomere integrity in vivo, and localization was not disrupted by telomere syndrome mutations. Using direct telomerase activity assays, we discovered that TIN2 stimulated telomerase processivity in vitro All of the TIN2 isoforms stimulated telomerase to similar extents. Mutations in the TPP1 TEL patch abrogated this stimulation, suggesting that TIN2 functions with TPP1/POT1 to stimulate telomerase processivity. We conclude from our data and previously published work that TIN2/TPP1/POT1 is a functional shelterin subcomplex. Copyright © 2019 Pike et al.


April 21, 2020

Mitochondrial DNA Variants in Patients with Liver Injury Due to Anti-Tuberculosis Drugs.

Hepatotoxicity is the most severe adverse effect of anti-tuberculosis therapy. Isoniazid’s metabolite hydrazine is a mitochondrial complex II inhibitor. We hypothesized that mitochondrial DNA variants are risk factors for drug-induced liver injury (DILI) due to isoniazid, rifampicin or pyrazinamide.We obtained peripheral blood from tuberculosis (TB) patients before anti-TB therapy. A total of 38 patients developed DILI due to anti-TB drugs. We selected 38 patients with TB but without DILI as controls. Next-generation sequencing detected point mutations in the mitochondrial DNA genome. DILI was defined as ALT =5 times the upper limit of normal (ULN), or ALT =3 times the ULN with total bilirubin =2 times the ULN.In 38 patients with DILI, the causative drug was isoniazid in eight, rifampicin in 14 and pyrazinamide in 16. Patients with isoniazid-induced liver injury had more variants in complex I’s NADH subunit 5 and 1 genes, more nonsynonymous mutations in NADH subunit 5, and a higher ratio of nonsynonymous to total substitutions. Patients with rifampicin- or pyrazinamide-induced liver injury had no association with mitochondrial DNA variants.Variants in complex I’s subunit 1 and 5 genes might affect respiratory chain function and predispose isoniazid-induced liver injury when exposed to hydrazine, a metabolite of isoniazid and a complex II inhibitor.


April 21, 2020

Centromeric Satellite DNAs: Hidden Sequence Variation in the Human Population.

The central goal of medical genomics is to understand the inherited basis of sequence variation that underlies human physiology, evolution, and disease. Functional association studies currently ignore millions of bases that span each centromeric region and acrocentric short arm. These regions are enriched in long arrays of tandem repeats, or satellite DNAs, that are known to vary extensively in copy number and repeat structure in the human population. Satellite sequence variation in the human genome is often so large that it is detected cytogenetically, yet due to the lack of a reference assembly and informatics tools to measure this variability, contemporary high-resolution disease association studies are unable to detect causal variants in these regions. Nevertheless, recently uncovered associations between satellite DNA variation and human disease support that these regions present a substantial and biologically important fraction of human sequence variation. Therefore, there is a pressing and unmet need to detect and incorporate this uncharacterized sequence variation into broad studies of human evolution and medical genomics. Here I discuss the current knowledge of satellite DNA variation in the human genome, focusing on centromeric satellites and their potential implications for disease.


April 21, 2020

Uncovering Missing Heritability in Rare Diseases.

The problem of ‘missing heritability’ affects both common and rare diseases hindering: discovery, diagnosis, and patient care. The ‘missing heritability’ concept has been mainly associated with common and complex diseases where promising modern technological advances, like genome-wide association studies (GWAS), were unable to uncover the complete genetic mechanism of the disease/trait. Although rare diseases (RDs) have low prevalence individually, collectively they are common. Furthermore, multi-level genetic and phenotypic complexity when combined with the individual rarity of these conditions poses an important challenge in the quest to identify causative genetic changes in RD patients. In recent years, high throughput sequencing has accelerated discovery and diagnosis in RDs. However, despite the several-fold increase (from ~10% using traditional to ~40% using genome-wide genetic testing) in finding genetic causes of these diseases in RD patients, as is the case in common diseases-the majority of RDs are also facing the ‘missing heritability’ problem. This review outlines the key role of high throughput sequencing in uncovering genetics behind RDs, with a particular focus on genome sequencing. We review current advances and challenges of sequencing technologies, bioinformatics approaches, and resources.


April 21, 2020

Single-Molecule Real-Time (SMRT) Full-Length RNA-Sequencing Reveals Novel and Distinct mRNA Isoforms in Human Bone Marrow Cell Subpopulations.

Hematopoietic cells are continuously replenished from progenitor cells that reside in the bone marrow. To evaluate molecular changes during this process, we analyzed the transcriptomes of freshly harvested human bone marrow progenitor (lineage-negative) and differentiated (lineage-positive) cells by single-molecule real-time (SMRT) full-length RNA-sequencing. This analysis revealed a ~5-fold higher number of transcript isoforms than previously detected and showed a distinct composition of individual transcript isoforms characteristic for bone marrow subpopulations. A detailed analysis of messenger RNA (mRNA) isoforms transcribed from the ANXA1 and EEF1A1 loci confirmed their distinct composition. The expression of proteins predicted from the transcriptome analysis was evaluated by mass spectrometry and validated previously unknown protein isoforms predicted e.g., for EEF1A1. These protein isoforms distinguished the lineage negative cell population from the lineage positive cell population. Finally, transcript isoforms expressed from paralogous gene loci (e.g., CFD, GATA2, HLA-A, B, and C) also distinguished cell subpopulations but were only detectable by full-length RNA sequencing. Thus, qualitatively distinct transcript isoforms from individual genomic loci separate bone marrow cell subpopulations indicating complex transcriptional regulation and protein isoform generation during hematopoiesis.


April 21, 2020

A Genome-Wide Epstein-Barr Virus Polyadenylation Map and Its Antisense RNA to EBNA.

Epstein-Barr virus (EBV) is a ubiquitous human pathogen associated with Burkitt’s lymphoma and nasopharyngeal carcinoma. Although the EBV genome harbors more than a hundred genes, a full transcription map with EBV polyadenylation profiles remains unknown. To elucidate the 3′ ends of all EBV transcripts genome-wide, we performed the first comprehensive analysis of viral polyadenylation sites (pA sites) using our previously reported polyadenylation sequencing (PA-seq) technology. We identified that EBV utilizes a total of 62?pA sites in JSC-1, 60 in Raji, and 53 in Akata cells for the expression of EBV genes from both plus and minus DNA strands; 42 of these pA sites are commonly used in all three cell lines. The majority of identified pA sites were mapped to the intergenic regions downstream of previously annotated EBV open reading frames (ORFs) and viral promoters. pA sites lacking an association with any known EBV genes were also identified, mostly for the minus DNA strand within the EBNA locus, a major locus responsible for maintenance of viral latency and cell transformation. The expression of these novel antisense transcripts to EBNA were verified by 3′ rapid amplification of cDNA ends (RACE) and Northern blot analyses in several EBV-positive (EBV+) cell lines. In contrast to EBNA RNA expressed during latency, expression of EBNA-antisense transcripts, which is restricted in latent cells, can be significantly induced by viral lytic infection, suggesting potential regulation of viral gene expression by EBNA-antisense transcription during lytic EBV infection. Our data provide the first evidence that EBV has an unrecognized mechanism that regulates EBV reactivation from latency.IMPORTANCE Epstein-Barr virus represents an important human pathogen with an etiological role in the development of several cancers. By elucidation of a genome-wide polyadenylation landscape of EBV in JSC-1, Raji, and Akata cells, we have redefined the EBV transcriptome and mapped individual polymerase II (Pol II) transcripts of viral genes to each one of the mapped pA sites at single-nucleotide resolution as well as the depth of expression. By unveiling a new class of viral lytic RNA transcripts antisense to latent EBNAs, we provide a novel mechanism of how EBV might control the expression of viral latent genes and lytic infection. Thus, this report takes another step closer to understanding EBV gene structure and expression and paves a new path for antiviral approaches.This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.


April 21, 2020

The Versatility of SMRT Sequencing.

The adoption of single molecule real-time (SMRT) sequencing [1] is becoming widespread, not only in basic science, but also in more applied areas such as agricultural, environmental, and medical research. SMRT sequencing offers important advantages over current short-read DNA sequencing technologies, including exceptionally long read lengths (20 kb or more), unparalleled consensus accuracy, and the ability to sequence native, non-amplified, DNA molecules. These sequencing characteristics enable creation of highly accurate de novo genome assemblies, characterization of complex structural variation, direct characterization of nucleotide base modifications, full-length RNA isoform sequencing, phasing of genetic variants, low frequency mutation detection, and clonal evolution determination [2,3]. This Special Issue of Genes is a collection of articles showcasing the latest developments and the breadth of applications enabled by SMRT sequencing technology.


April 21, 2020

Critical length in long-read resequencing

Long-read sequencing has substantial advantages for structural variant discovery and phasing of vari- ants compared to short-read technologies, but the required and optimal read length has not been as- sessed. In this work, we used long reads simulated from human genomes and evaluated structural vari- ant discovery and variant phasing using current best practicebioinformaticsmethods.Wedeterminedthatoptimal discovery of structural variants from human genomes can be obtained with reads of minimally 20 kb. Haplotyping variants across genes only reaches its optimum from reads of 100 kb. These findings are important for the design of future long-read sequenc- ing projects.


April 21, 2020

Adaptive archaic introgression of copy number variants and the discovery of previously unknown human genes

As they migrated out of Africa and into Europe and Asia, anatomically modern humans interbred with archaic hominins, such as Neanderthals and Denisovans. The result of this genetic introgression on the recipient populations has been of considerable interest, especially in cases of selection for specific archaic genetic variants. Hsieh et al. characterized adaptive structural variants and copy number variants that are likely targets of positive selection in Melanesians. Focusing on population-specific regions of the genome that carry duplicated genes and show an excess of amino acid replacements provides evidence for one of the mechanisms by which genetic novelty can arise and result in differentiation between human genomes.Science, this issue p. eaax2083INTRODUCTIONCharacterizing genetic variants underlying local adaptations in human populations is one of the central goals of evolutionary research. Most studies have focused on adaptive single-nucleotide variants that either arose as new beneficial mutations or were introduced after interbreeding with our now-extinct relatives, including Neanderthals and Denisovans. The adaptive role of copy number variants (CNVs), another well-known form of genomic variation generated through deletions or duplications that affect more base pairs in the genome, is less well understood, despite evidence that such mutations are subject to stronger selective pressures.RATIONALEThis study focuses on the discovery of introgressed and adaptive CNVs that have become enriched in specific human populations. We combine whole-genome CNV calling and population genetic inference methods to discover CNVs and then assess signals of selection after controlling for demographic history. We examine 266 publicly available modern human genomes from the Simons Genome Diversity Project and genomes of three ancient homininstextemdasha Denisovan, a Neanderthal from the Altai Mountains in Siberia, and a Neanderthal from Croatia. We apply long-read sequencing methods to sequence-resolve complex CNVs of interest specifically in the Melanesianstextemdashan Oceanian population distributed from Papua New Guinea to as far east as the islands of Fiji and known to harbor some of the greatest amounts of Neanderthal and Denisovan ancestry.RESULTSConsistent with the hypothesis of archaic introgression outside Africa, we find a significant excess of CNV sharing between modern non-African populations and archaic hominins (P = 0.039). Among Melanesians, we observe an enrichment of CNVs with potential signals of positive selection (n = 37 CNVs), of which 19 CNVs likely introgressed from archaic hominins. We show that Melanesian-stratified CNVs are significantly associated with signals of positive selection (P = 0.0323). Many map near or within genes associated with metabolism (e.g., ACOT1 and ACOT2), development and cell cycle or signaling (e.g., TNFRSF10D and CDK11A and CDK11B), or immune response (e.g., IFNLR1). We characterize two of the largest and most complex CNVs on chromosomes 16p11.2 and 8p21.3 that introgressed from Denisovans and Neanderthals, respectively, and are absent from most other human populations. At chromosome 16p11.2, we sequence-resolve a large duplication of >383 thousand base pairs (kbp) that originated from Denisovans and introgressed into the ancestral Melanesian population 60,000 to 170,000 years ago. This large duplication occurs at high frequency (>79%) in diverse Melanesian groups, shows signatures of positive selection, and maps adjacent to Homo sapienstextendashspecific duplications that predispose to rearrangements associated with autism. On chromosome 8p21.3, we identify a Melanesian haplotype that carries two CNVs, a ~6-kbp deletion, and a ~38-kbp duplication, with a Neanderthal origin and that introgressed into non-Africans 40,000 to 120,000 years ago. This CNV haplotype occurs at high frequency (44%) and shows signals consistent with a partial selective sweep in Melanesians. Using long-read sequencing genomic and transcriptomic data, we reconstruct the structure and complex evolutionary history for these two CNVs and discover previously undescribed duplicated genes (TNFRSF10D1, TNFRSF10D2, and NPIPB16) that show an excess of amino acid replacements consistent with the action of positive selection.CONCLUSIONOur results suggest that large CNVs originating in archaic hominins and introgressed into modern humans have played an important role in local population adaptation and represent an insufficiently studied source of large-scale genetic variation that is absent from current reference genomes.Large adaptive-introgressed CNVs at chromosomes 8p21.3 and 16p11.2 in Melanesians.The magnifying glasses highlight structural differences between the archaic (top) and reference (bottom) genomes. Neanderthal (red) and Denisovan (blue) haplotypes encompassing large CNVs occur at high frequencies in Melanesians (44 and 79%, respectively) but are absent (black) in all non-Melanesians. These CNVs create positively selected genes (TNFRSF10D1, TNFRSF10D2, and NPIPB16) that are absent from the reference genome.Copy number variants (CNVs) are subject to stronger selective pressure than single-nucleotide variants, but their roles in archaic introgression and adaptation have not been systematically investigated. We show that stratified CNVs are significantly associated with signatures of positive selection in Melanesians and provide evidence for adaptive introgression of large CNVs at chromosomes 16p11.2 and 8p21.3 from Denisovans and Neanderthals, respectively. Using long-read sequence data, we reconstruct the structure and complex evolutionary history of these polymorphisms and show that both encode positively selected genes absent from most human populations. Our results collectively suggest that large CNVs originating in archaic hominins and introgressed into modern humans have played an important role in local population adaptation and represent an insufficiently studied source of large-scale genetic variation.


April 21, 2020

A personalized platform identifies trametinib plus zoledronate for a patient with KRAS-mutant metastatic colorectal cancer.

Colorectal cancer remains a leading source of cancer mortality worldwide. Initial response is often followed by emergent resistance that is poorly responsive to targeted therapies, reflecting currently undruggable cancer drivers such as KRAS and overall genomic complexity. Here, we report a novel approach to developing a personalized therapy for a patient with treatment-resistant metastatic KRAS-mutant colorectal cancer. An extensive genomic analysis of the tumor’s genomic landscape identified nine key drivers. A transgenic model that altered orthologs of these nine genes in the Drosophila hindgut was developed; a robotics-based screen using this platform identified trametinib plus zoledronate as a candidate treatment combination. Treating the patient led to a significant response: Target and nontarget lesions displayed a strong partial response and remained stable for 11 months. By addressing a disease’s genomic complexity, this personalized approach may provide an alternative treatment option for recalcitrant disease such as KRAS-mutant colorectal cancer.


April 21, 2020

Genomic inversions and GOLGA core duplicons underlie disease instability at the 15q25 locus.

Human chromosome 15q25 is involved in several disease-associated structural rearrangements, including microdeletions and chromosomal markers with inverted duplications. Using comparative fluorescence in situ hybridization, strand-sequencing, single-molecule, real-time sequencing and Bionano optical mapping analyses, we investigated the organization of the 15q25 region in human and nonhuman primates. We found that two independent inversions occurred in this region after the fission event that gave rise to phylogenetic chromosomes XIV and XV in humans and great apes. One of these inversions is still polymorphic in the human population today and may confer differential susceptibility to 15q25 microdeletions and inverted duplications. The inversion breakpoints map within segmental duplications containing core duplicons of the GOLGA gene family and correspond to the site of an ancestral centromere, which became inactivated about 25 million years ago. The inactivation of this centromere likely released segmental duplications from recombination repression typical of centromeric regions. We hypothesize that this increased the frequency of ectopic recombination creating a hotspot of hominid inversions where dispersed GOLGA core elements now predispose this region to recurrent genomic rearrangements associated with disease.


April 21, 2020

Collateral damage and CRISPR genome editing.

The simplicity and the versatility of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR-Cas) systems have enabled the genetic modification of virtually every organism and offer immense therapeutic potential for the treatment of human disease. Although these systems may function efficiently within eukaryotic cells, there remain concerns about the accuracy of Cas endonuclease effectors and their use for precise gene editing. Recently, two independent reports investigating the editing accuracy of the CRISPR-Cas9 system were published by separate groups at the Wellcome Sanger Institute; our study-Iyer and colleagues [1]-defined the landscape of off-target mutations, whereas the other by Kosicki and colleagues [2] detailed the existence of on-target, potentially deleterious deletions. Although both studies found evidence of large on-target CRISPR-induced deletions, they reached seemingly very different conclusions.


April 21, 2020

Amplification-free long-read sequencing of TCF4 expanded trinucleotide repeats in Fuchs Endothelial Corneal Dystrophy.

Amplification of a CAG trinucleotide motif (CTG18.1) within the TCF4 gene has been strongly associated with Fuchs Endothelial Corneal Dystrophy (FECD). Nevertheless, a small minority of clinically unaffected elderly patients who have expanded CTG18.1 sequences have been identified. To test the hypothesis that the CAG expansions in these patients are protected from FECD because they have interruptions within the CAG repeats, we utilized a combination of an amplification-free, long-read sequencing method and a new target-enrichment sequence analysis tool developed by Pacific Biosciences to interrogate the sequence structure of expanded repeats. The sequencing was successful in identifying a previously described interruption within an unexpanded allele and provided sequence data on expanded alleles greater than 2000 bases in length. The data revealed considerable heterogeneity in the size distribution of expanded repeats within each patient. Detailed analysis of the long sequence reads did not reveal any instances of interruptions to the expanded CAG repeats, but did reveal novel variants within the AGG repeats that flank the CAG repeats in two of the five samples from clinically unaffected patients with expansions. This first examination of the sequence structure of CAG repeats in CTG18.1 suggests that factors other than interruptions to the repeat structure account for the absence of disease in some elderly patients with repeat expansions in the TCF4 gene.


April 21, 2020

Reconstruction of the genomes of drug-resistant pathogens for outbreak investigation through metagenomic sequencing

Culture-independent methods that target genome fragments have shown promise in identifying certain pathogens, but the holy grail of comprehensive pathogen genome detection from microbiologically complex samples for subsequent forensic analyses remains a challenge. In the context of an investigation of a nosocomial outbreak, we used shotgun metagenomic sequencing of a human fecal sample and a neural network algorithm based on tetranucleotide frequency profiling to reconstruct microbial genomes and tested the same approach using rectal swabs from a second patient. The approach rapidly and readily detected the genome of Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae in the patient fecal specimen and in the rectal swab sample, achieving a level of strain resolution that was sufficient for confident transmission inference during a highly clonal outbreak. The analysis also detected previously unrecognized colonization of the patient by vancomycin-resistant Enterococcus faecium, another multidrug-resistant bacterium.IMPORTANCE The study results reported here perfectly demonstrate the power and promise of clinical metagenomics to recover genome sequences of important drug-resistant bacteria and to rapidly provide rich data that inform outbreak investigations and treatment decisions, independently of the need to culture the organisms.


Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.