Genomic Hotspots Research Articles

Standardized Targeted Sequencing of 29 Genes with Recurrent Mutations in Chronic Lymphocytic Leukemia (CLL) Helps to Better Define Clinical Correlates and Genomic Hotspots

Standardized Targeted Sequencing of 29 Genes with Recurrent Mutations in Chronic Lymphocytic Leukemia (CLL) Helps to Better Define Clinical Correlates and Genomic Hotspots

Chromosomal characteristics of salt stress heritable gene expression in the rice genome

BackgroundGene expression is potentially an important heritable quantitative trait that mediates between genetic variation and higher-level complex phenotypes through time and condition-dependent regulatory interactions. Therefore, we sought to explore both the genomic and condition-specific characteristics of gene expression heritability within the context of chromosomal structure.ResultsHeritability was estimated for biological gene expression using a diverse, 84-line, Oryza sativa (rice) population under optimal and salt-stressed conditions. Overall, 5936 genes were found to have heritable expression regardless of condition and 1377 genes were found to have heritable expression only during salt stress. These genes with salt-specific heritable expression are enriched for functional terms associated with response to stimulus and transcription factor activity. Additionally, we discovered that highly and lowly expressed genes, and genes with heritable expression are distributed differently along the chromosomes in patterns that follow previously identified high-throughput chromosomal conformation capture (Hi-C) A/B chromatin compartments. Furthermore, multiple genomic hot-spots enriched for genes with salt-specific heritability were identified on chromosomes 1, 4, 6, and 8. These hotspots were found to contain genes functionally enriched for transcriptional regulation and overlaps with a previously identified major QTL for salt-tolerance in rice.ConclusionsInvestigating the heritability of traits, and in-particular gene expression traits, is important towards developing a basic understanding of how regulatory networks behave across a population. This work provides insights into spatial patterns of heritable gene expression at the chromosomal level.

Single Cell Transcriptomic and Genomic Characterization of Brugada Syndrome Associated Genes

Brugada syndrome (BrS) is a rare, inherited arrhythmia with high risk of sudden cardiac death. Complex genetic background of BrS primarily involves mutations in genes encoding ion channels and regulatory/structural proteins. However, the molecular pathways and single cell expression patterns linked to clinically associated genes in BrS are not well defined. To evaluate the convergence of key genes, domains, motifs, molecular pathways, and single cell expression associated with BrS, we collected 733 mutations represented by 16 sodium, calcium, potassium channels, regulatory and structural genes related to BrS reported between 1999-2019.266 of the clinically relevant mutations (splice site, stop, frame shift and deleterious missense) in genes occurred once and 88 mutations were recurrent in nature, which appeared at two to forty-three instances. We observed an over-representation of clinically relevant mutations (~80%) in SCN5A gene, and also identified several candidate genes, including GPD1L, TRPM4, and SCN10A. Single cell transcriptomic analysis revealed restrictive expression of highly mutated BrS genes. Furthermore, protein domain enrichment analysis revealed that a large proportion of the mutations impacted ion-transport domains in multiple genes, SCN5A, TRPM4 and SCN10A. Gene enrichment analysis identified the action potential pathway as the most significant pathway involving the mutated genes in BrS. A comparative protein domain analysis of the SCN5A further established a significant (p=0.04) relation between mutation distribution and ion-transport domain. A strong association (p=0.00003) was also observed between mutation distribution across the ion transport domain and early/late onset of BrS. The significant role of the mutations in the ion transport domain leading to BrS manifestation was further established by window-based and MEME motif analysis which identified residue 1321-1380 (part of ion transport domain) as one of the significant (p=0.02) hotspot regions. Our study identified genomic and proteomic hotspots and a convergent pathway (ion transport) that provides in-depth insight into the complex pathophysiology of BrS. Single cell transcriptome profile of heart tissue shows that highly mutated genes (SCN5A and GPD1L) in BrS are restrictively expressed in ventricular cardiomyocyte cell type.

Single-cell transcriptomics trajectory and molecular convergence of clinically relevant mutations in Brugada syndrome.

Brugada syndrome (BrS) is a rare, inherited arrhythmia with high risk of sudden cardiac death. To evaluate the molecular convergence of clinically relevant mutations and to identify developmental cardiac cell types that are associated with BrS etiology, we collected 733 mutations represented by 16 sodium, calcium, potassium channels, and regulatory and structural genes related to BrS. Among the clinically relevant mutations, 266 are unique singletons and 88 mutations are recurrent. We observed an over-representation of clinically relevant mutations (∼80%) in SCN5A gene and also identified several candidate genes, including GPD1L, TRPM4, and SCN10A. Furthermore, protein domain enrichment analysis revealed that a large proportion of the mutations impacted ion transport domains in multiple genes, including SCN5A, TRPM4, and SCN10A. A comparative protein domain analysis of SCN5A further established a significant (P = 0.04) enrichment of clinically relevant mutations within ion transport domain, including a significant (P = 0.02) mutation hotspot within 1321-1380 residue. The enrichment of clinically relevant mutations within SCN5A ion transport domain is stronger (P = 0.00003) among early onset of BrS. Our spatiotemporal cellular heart developmental (prenatal to adult) trajectory analysis applying single-cell transcriptome identified the most frequently BrS-mutated genes (SCN5A and GPD1L) are significantly upregulated in the prenatal cardiomyocytes. A more restrictive cellular expression trajectory is prominent in the adult heart ventricular cardiomyocytes compared to prenatal. Our study suggests that genomic and proteomic hotspots in BrS converge into ion transport pathway and cardiomyocyte as a major BrS-associated cell type that provides insight into the complex genetic etiology of BrS.NEW & NOTEWORTHY Brugada syndrome is a rare inherited arrhythmia with high risk of sudden cardiac death. We present the findings for a molecular convergence of clinically relevant mutations and identification of a single-cell transcriptome-derived cardiac cell types that are associated with the etiology of BrS. Our study suggests that genomic and proteomic hotspots in BrS converge into ion transport pathway and cardiomyocyte as a major BrS-associated cell type that provides insight into the complex genetic etiology of BrS.

Coronavirus Antigens: Efficient Human Cell Coexpression System and Its Application to the Production of Multiple Coronavirus Antigens (Adv. Biology 4/2021)

The production of biomolecules that require human-specific lipid environments is extremely useful for basic research and medical applications. In article number 2000154, Seong-Jun Kim, Jae-Sung Woo, Sangsu Bae, and co-workers integrate multiple proteins or virus antigens into defined transcriptional hotspots in the human genome via a homology-independent targeted insertion method using CRISPR nucleases. This system is similar to a production pipeline of biomolecules in a factory controlled by CRISPR.

DEEPGENTM—A Novel Variant Calling Assay for Low Frequency Variants

Detection of genetic variants in clinically relevant genomic hot-spot regions has become a promising application of next-generation sequencing technology in precision oncology. Effective personalized diagnostics requires the detection of variants with often very low frequencies. This can be achieved by targeted, short-read sequencing that provides high sequencing depths. However, rare genetic variants can contain crucial information for early cancer detection and subsequent treatment success, an inevitable level of background noise usually limits the accuracy of low frequency variant calling assays. To address this challenge, we developed DEEPGENTM, a variant calling assay intended for the detection of low frequency variants within liquid biopsy samples. We processed reference samples with validated mutations of known frequencies (0%–0.5%) to determine DEEPGENTM’s performance and minimal input requirements. Our findings confirm DEEPGENTM’s effectiveness in discriminating between signal and noise down to 0.09% variant allele frequency and an LOD(90) at 0.18%. A superior sensitivity was also confirmed by orthogonal comparison to a commercially available liquid biopsy-based assay for cancer detection.

An extended APOBEC3A mutation signature in cancer

APOBEC mutagenesis, a major driver of cancer evolution, is known for targeting TpC sites in DNA. Recently, we showed that APOBEC3A (A3A) targets DNA hairpin loops. Here, we show that DNA secondary structure is in fact an orthogonal influence on A3A substrate optimality and, surprisingly, can override the TpC sequence preference. VpC (non-TpC) sites in optimal hairpins can outperform TpC sites as mutational hotspots. This expanded understanding of APOBEC mutagenesis illuminates the genomic Twin Paradox, a puzzling pattern of closely spaced mutation hotspots in cancer genomes, in which one is a canonical TpC site but the other is a VpC site, and double mutants are seen only in trans, suggesting a two-hit driver event. Our results clarify this paradox, revealing that both hotspots in these twins are optimal A3A substrates. Our findings reshape the notion of a mutation signature, highlighting the additive roles played by DNA sequence and DNA structure.

Addressing Darwin's dilemma: Can pseudo-overdominance explain persistent inbreeding depression and load?

Darwin spent years investigating the effects of self-fertilization, concluding that "nature abhors perpetual self-fertilization." Given that selection purges inbred populations of strongly deleterious mutations and drift fixes mild mutations, why does inbreeding depression (ID) persist in highly inbred taxa and why do no purely selfing taxa exist? Background selection, associations and interference among loci, and drift within small inbred populations all limit selection while often increasing fixation. These mechanisms help to explain why more inbred populations in most species consistently show more fixed load. This drift load is manifest in the considerable heterosis regularly observed in between-population crosses. Such heterosis results in subsequent high ID, suggesting a mechanism by which small populations could retain variation and inbreeding load. Multiple deleterious recessive mutations linked in repulsion generate pseudo-overdominance. Many tightly linked load loci could generate a balanced segregating load high enough to sustain ID over many generations. Such pseudo-overdominance blocks (or "PODs") are more likely to occur in regions of low recombination. They should also result in clear genetic signatures including genomic hotspots of heterozygosity; distinct haplotypes supporting alleles at intermediate frequency; and high linkage disequilibrium in and around POD regions. Simulation and empirical studies tend to support these predictions. Additional simulations and comparative genomic analyses should explore POD dynamics in greater detail to resolve whether PODs exist in sufficient strength and number to account for why ID and load persist within inbred lineages.

Efficient Human Cell Coexpression System and Its Application to the Production of Multiple Coronavirus Antigens.

Coproduction of multiple proteins at high levels in a single human cell line would be extremely useful for basic research and medical applications. Here, a novel strategy for the stable expression of multiple proteins by integrating the genes into defined transcriptional hotspots in the human genome is presented. As a proof-of-concept, it is shown that EYFP is expressed at similar levels from hotspots and that the EYFP expression increases proportionally with the copy number. It is confirmed that three different fluorescent proteins, encoded by genes integrated at different loci, can be coexpressed at high levels. Further, a stable cell line is generated, producing antigens from different human coronaviruses: MERS-CoV and HCoV-OC43. Antibodies raised against these antigens, which contain human N-glycosylation, show neutralizing activities against both viruses, suggesting that the coexpression system provides a quick and predictable way to produce multiple coronavirus antigens, such as the recent 2019 novel human coronavirus.

Structure and contingency determine mutational hotspots for flower color evolution.

Evolutionary genetic studies have uncovered abundant evidence for genomic hotspots of phenotypic evolution, as well as biased patterns of mutations at those loci. However, the theoretical basis for this concentration of particular types of mutations at particular loci remains largely unexplored. In addition, historical contingency is known to play a major role in evolutionary trajectories, but has not been reconciled with the existence of such hotspots. For example, do the appearance of hotspots and the fixation of different types of mutations at those loci depend on the starting state and/or on the nature and direction of selection? Here, we use a computational approach to examine these questions, focusing the anthocyanin pigmentation pathway, which has been extensively studied in the context of flower color transitions. We investigate two transitions that are common in nature, the transition from blue to purple pigmentation and from purple to red pigmentation. Both sets of simulated transitions occur with a small number of mutations at just four loci and show strikingly similar peaked shapes of evolutionary trajectories, with the mutations of the largest effect occurring early but not first. Nevertheless, the types of mutations (biochemical vs. regulatory) as well as their direction and magnitude are contingent on the particular transition. These simulated color transitions largely mirror findings from natural flower color transitions, which are known to occur via repeated changes at a few hotspot loci. Still, some types of mutations observed in our simulated color evolution are rarely observed in nature, suggesting that pleiotropic effects further limit the trajectories between color phenotypes. Overall, our results indicate that the branching structure of the pathway leads to a predictable concentration of evolutionary change at the hotspot loci, but the types of mutations at these loci and their order is contingent on the evolutionary context.

Rapid parallel adaptation despite gene flow in silent crickets

Gene flow is predicted to impede parallel adaptation via de novo mutation, because it can introduce pre-existing adaptive alleles from population to population. We test this using Hawaiian crickets (Teleogryllus oceanicus) in which ‘flatwing’ males that lack sound-producing wing structures recently arose and spread under selection from an acoustically-orienting parasitoid. Morphometric and genetic comparisons identify distinct flatwing phenotypes in populations on three islands, localized to different loci. Nevertheless, we detect strong, recent and ongoing gene flow among the populations. Using genome scans and gene expression analysis we find that parallel evolution of flatwing on different islands is associated with shared genomic hotspots of adaptation that contain the gene doublesex, but the form of selection differs among islands and corresponds to known flatwing demographics in the wild. We thus show how parallel adaptation can occur on contemporary timescales despite gene flow, indicating that it could be less constrained than previously appreciated.

Development of recombinase-based targeted integration systems for production of exogenous proteins using transposon-mediated landing pads

Abstract Current methods for stably expressing recombinant protein therapeutics in CHO cells often rely on random or semi-random genomic integration events which result in a widely heterogeneous cell population. Consequently, a significant portion of cell line development efforts involves extensive pool and clone screening to identify clones with high expression, growth, and product quality. In this study, we developed two targeted integration systems that express high levels of recombinant protein in CHO cells. We first generated two clonal cell lines stably expressing enhanced green fluorescent protein (eGFP) reporter landing pads in genomic hot spots. We then demonstrated functional integration of several donor vectors encoding both monoclonal antibodies and a Fc-fusion molecule using Cre or PhiC31 recombinase mediated integration. TLA and PCR characterization of integrated cell lines showed correct targeting of landing pads. Post-integration enrichment for fully saturated landing pads using GCV increased recombinant protein titer by 2–2.5-fold and specific productivity by ∼3.4-fold with observed potential off-target random integration detected only following GCV enrichment. By targeting predefined genomic locations that are known to support high expression of an exogenous protein, several cell lines expressing different recombinant protein therapeutics can easily be established with a high degree of specificity and reproducibility.

Promiscuous Structural Variants Drive Myeloma Initiation and Progression.

A comprehensive genomic analysis of structural variants in multiple myeloma in this issue highlights the key role of these events, involving primarily the immunoglobulin heavy chain locus in disease initiation and the MYC locus in disease progression. However, the current study reveals the large number of genomic hotspots, oncogenes, tumor suppressor genes, and recombination mechanisms that contribute to multiple myeloma heterogeneity. See related article by Rustad et al., p. 258.

Diverse Horizontally-Acquired Gene Clusters Confer Sucrose Utilization to Different Lineages of the Marine Pathogen Photobacterium damselae subsp. damselae.

The ability to metabolize sucrose is a variable trait within the family Vibrionaceae. The marine bacterium Photobacterium damselae subsp. damselae (Pdd), pathogenic for marine animals and humans, is generally described as negative for sucrose utilization (Scr−). Previous studies have reported sucrose-utilizing isolates (Scr+), but the genetic basis of this variable phenotype remains uncharacterized. Here, we carried out the genome sequencing of five Scr+ and two Scr− Pdd isolates and conducted a comparative genomics analysis with sixteen additional Pdd genomes sequenced in previous studies. We identified two different versions of a four-gene cluster (scr cluster) exclusive of Scr+ isolates encoding a PTS system sucrose-specific IIBC component (scrA), a fructokinase (scrK), a sucrose-6-phosphate hydrolase (scrB), and a sucrose operon repressor (scrR). A scrA deletion mutant did not ferment sucrose and was impaired for growth with sucrose as carbon source. Comparative genomics analyses suggested that scr clusters were acquired by horizontal transfer by different lineages of Pdd and were inserted into a recombination hot-spot in the Pdd genome. The incongruence of phylogenies based on housekeeping genes and on scr genes revealed that phylogenetically diverse gene clusters for sucrose utilization have undergone extensive horizontal transfer among species of Vibrio and Photobacterium.

Genetic Architecture Underpinning Yield Components and Seed Mineral-Nutrients in Sesame.

Genetic dissection of yield components and seed mineral-nutrient is crucial for understanding plant physiological and biochemical processes and alleviate nutrient malnutrition. Sesame (Sesamum indicum L.) is an orphan crop that harbors rich allelic repertoire for seed mineral–nutrients. Here, we harness this wide diversity to study the genetic architecture of yield components and seed mineral–nutrients using a core-collection of worldwide genotypes and segregating mapping population. We also tested the association between these traits and the effect of seed nutrients concentration on their bio-accessibility. Wide genetic diversity for yield components and seed mineral–nutrients was found among the core-collection. A high-density linkage map consisting of 19,309 markers was constructed and used for genetic mapping of 84 QTL associated with yield components and 50 QTL for seed minerals. To the best of our knowledge, this is the first report on mineral–nutrients QTL in sesame. Genomic regions with a cluster of overlapping QTL for several morphological and nutritional traits were identified and considered as genomic hotspots. Candidate gene analysis revealed potential functional associations between QTL and corresponding genes, which offers unique opportunities for synchronous improvement of mineral–nutrients. Our findings shed-light on the genetic architecture of yield components, seed mineral–nutrients and their inter- and intra- relationships, which may facilitate future breeding efforts to develop bio-fortified sesame cultivars.

Genomic Diversity and Hotspot Mutations in 30,983 SARS-CoV-2 Genomes: Moving Toward a Universal Vaccine for the "Confined Virus"?

The COVID-19 pandemic has been ongoing since its onset in late November 2019 in Wuhan, China. Understanding and monitoring the genetic evolution of the virus, its geographical characteristics, and its stability are particularly important for controlling the spread of the disease and especially for the development of a universal vaccine covering all circulating strains. From this perspective, we analyzed 30,983 complete SARS-CoV-2 genomes from 79 countries located in the six continents and collected from 24 December 2019, to 13 May 2020, according to the GISAID database. Our analysis revealed the presence of 3206 variant sites, with a uniform distribution of mutation types in different geographic areas. Remarkably, a low frequency of recurrent mutations has been observed; only 169 mutations (5.27%) had a prevalence greater than 1% of genomes. Nevertheless, fourteen non-synonymous hotspot mutations (>10%) have been identified at different locations along the viral genome; eight in ORF1ab polyprotein (in nsp2, nsp3, transmembrane domain, RdRp, helicase, exonuclease, and endoribonuclease), three in nucleocapsid protein, and one in each of three proteins: Spike, ORF3a, and ORF8. Moreover, 36 non-synonymous mutations were identified in the receptor-binding domain (RBD) of the spike protein with a low prevalence (<1%) across all genomes, of which only four could potentially enhance the binding of the SARS-CoV-2 spike protein to the human ACE2 receptor. These results along with intra-genomic divergence of SARS-CoV-2 could indicate that unlike the influenza virus or HIV viruses, SARS-CoV-2 has a low mutation rate which makes the development of an effective global vaccine very likely.

Genome-wide pQTL analysis of protein expression regulatory networks in the human liver

BackgroundPrevious expression quantitative trait loci (eQTL) studies have identified thousands of genetic variants to be associated with gene expression at the mRNA level in the human liver. However, protein expression often correlates poorly with mRNA levels. Thus, protein quantitative trait loci (pQTL) study is required to identify genetic variants that regulate protein expression in human livers.ResultsWe conducted a genome-wide pQTL study in 287 normal human liver samples and identified 900 local pQTL variants and 4026 distant pQTL variants. We further discovered 53 genome hotspots of pQTL variants. Transcriptional region mapping analysis showed that 1133 pQTL variants are in transcriptional regulatory regions. Genomic region enrichment analysis of the identified pQTL variants revealed 804 potential regulatory interactions among 595 predicted regulators (e.g., non-coding RNAs) and 394 proteins. Moreover, pQTL variants and trait-variant integration analysis implied several novel mechanisms underlying the relationships between protein expression and liver diseases, such as alcohol dependence. Notably, over 2000 of the identified pQTL variants have not been reported in previous eQTL studies, suggesting extensive involvement of genetic polymorphisms in post-transcriptional regulation of protein expression in human livers.ConclusionsWe have partially established protein expression regulation networks in human livers and generated a wealth of pQTL data that could serve as a valuable resource for the scientific community.

Association Analysis of ANRIL Polymorphisms and Haplotypes with Autism Spectrum Disorders.

Autism spectrum disorder (ASD) has been shown to have a complex inheritance. Several single-nucleotide polymorphisms (SNPs) have been shown to be associated with risk of this neurodevelopmental disorder. In the current study, we genotyped four SNPs in a genomic hotspot for human disorders. The selected SNPs were located in adjacency of the antisense noncoding RNA in the INK4 locus (ANRIL) and have been shown to be associated with a number of human disorders. Genotyping was performed in 420 ASD cases and 420 normally developed children. After correction of P values for multiple comparisons, there was no significant difference in frequencies of rs1333045, rs1333048, rs4977574, and rs10757278 alleles, genotypes, or haplotypes between ASD children and children with normal development. However, one estimated haplotype (T A A A haplotype corresponding to rs1333045, rs1333048, rs4977574, and rs10757278 SNPs, respectively) tended to be more prevalent among cases compared with controls (OR (95% CI) = 1.77 (1.19-2.64), adjusted P value = 0.07). Besides, the T A G G tended to be less common among ASD cases compared with controls (OR (95% CI) = 0.64 (0.47-0.87), adjusted P value = 0.07). Although we could not detect significant difference in alleles, genotypes, or haplotypes frequencies between cases and controls, the trend toward association between two haplotypes and ASD risk implies that there might be a putative causative variant in the mentioned haplotypes whose association with ASD could be determined in larger cohorts of patients.

Disorders Associated With Diverse, Recurrent Deletions and Duplications at 1q21.1.

The subchromosomal region 1q21.1 is one of the hotspots in the human genome for deletions and reciprocal duplications, owing to the existence of hundreds of segmental duplications. Recurrent deletions and duplications in this region are thought to be causative in patients with variable clinical manifestations. Based on the genomic locations, deletions and duplications at the 1q21.1 locus have been associated with distinguishable syndromes: chromosome 1q21.1 deletion syndrome, chromosome 1q21.1 duplication syndrome, and thrombocytopenia-absent radius (TAR) syndrome, which is partially due to deletions at the proximal 1q21.1 region. We report here diverse, recurrent deletions and duplications at the 1q21.1 locus in 36 patients from a cohort of 5,200 individuals. Among the 36 patients, 18 patients carry 1q21.1 deletions, nine individuals have reciprocal duplications at 1q21.1, two patients share an identical short deletion, and the remaining seven possess variable sizes of duplications at the proximal 1q21.1 region. Furthermore, we provide cytogenetic characterization and detailed clinical features for each patient. Notably, duplications at the proximal 1q21.1 region have not been associated with a defined disorder in publications. However, recurrent duplications at the proximal 1q21.1 region among the seven patients strongly suggested that the variants are likely pathogenic. The common phenotypical features of those disorders are also summarized to facilitate clinical diagnoses and genetic counseling.

Abstract 44: Monitoring of ctDNA burden from noncoding DNA

Abstract Our understanding of mutations in noncoding DNA is still nascent across most cancer types. Similarly, the potential clinical utility of noncoding DNA in cancer liquid biopsy assays remains mostly unexplored. Here, we identify high-frequency noncoding mutation hotspots in whole genomes from hundreds of gastrointestinal tumors and show that these mutations can be detected in liquid biopsy samples from cancer patients. We demonstrate how profiling of noncoding mutation hotspots can significantly increase the accuracy of ctDNA burden estimation, with minimal impact on sequencing cost, when paired with existing targeted cfDNA assays. Our results suggest that targeted NGS liquid biopsy assays should target both protein-coding and noncoding mutations when estimation and monitoring of ctDNA burden is a key clinical endpoint. Citation Format: Guo Yu, Zhong Wee Poh, Guanhua Zhu, Iain Tan, Sarah Ng, Patrick Tan, Anders Skanderup. Monitoring of ctDNA burden from noncoding DNA [abstract]. In: Proceedings of the AACR Special Conference on Advancing Precision Medicine Drug Development: Incorporation of Real-World Data and Other Novel Strategies; Jan 9-12, 2020; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_1):Abstract nr 44.