DNA Sequence Variation Research Articles

Evaluating the Potential Fitness Effects of Chinook Salmon (Oncorhynchus tshawytscha) Aquaculture Using Non-Invasive Population Genomic Analyses of MHC Nucleotide Substitution Spectra

Genetic diversity plays a vital role in the adaptability of salmon to changing environmental conditions that can introduce new selective pressures on populations. Variability among local subpopulations may increase the chance that certain advantageous genes are passed down to future generations to mitigate susceptibility to novel diseases, warming oceans, loss of genetic stocks, and ocean acidification. Class I and II genes of the major histocompatibility complex (MHC) are crucial for the fitness of Chinook salmon due to the role they play in disease and pathogen resistance. The objective of this study was to assess the DNA sequence variability among wild and hatchery populations of Alaskan Chinook salmon at the class I α1 and class II β1 exons of the MHC. We hypothesized that the 96 wild samples taken from the Deshka River would display greater levels of observed heterozygosity (Ho) relative to expected heterozygosity (He) in suggesting that individuals with similar phenotypes mate with one another more frequently than would be expected under random mating patterns. Conversely, since no mate selection occurs in the William Jack Hernandez Sport Fish hatchery, we would not expect to see this discrepancy (He = Ho) in the 96 hatchery fish tested in this study. Alternatively, we hypothesized that post-mating selection is driving higher levels of observed heterozygosity as opposed to mate selection. If this is the case, we will observe higher than expected levels of heterozygosity among hatchery salmon. Both populations displayed higher levels of observed heterozygosity than expected heterozygosity at the Class I and II loci but genetic differentiation between the spatially distinct communities was minimal. Class I sequences showed evidence of balancing selection, despite high rates of non-synonymous substitutions observed, specifically at the peptide binding regions of both MHC genes.

Molecular phylogenetics shed light on polyploid speciation in gorses (Ulex, Fabaceae: Genisteae) and on the origin of the invasive Ulex europaeus

Abstract Hybridization and polyploidy are key evolutionary forces in plant diversification, and their co-occurrence in the context of allopolyploid speciation is often associated with increased ability to colonize new environments and invasiveness. In the genus Ulex (Fabaceae), the European gorse (Ulex europaeus subsp. europaeus) is the only invasive and the only polyploid that has recently spread in different eco-geographical regions across the world. Understanding what confers such ecological advantages to this species, compared to its diploid and polyploid congeners, first requires clarification of the ecogeographical and evolutionary context of its formation. To achieve this, the geographical distributions of all Ulex spp. were estimated from species occurrence records, and phylogenetic analyses including all Ulex spp. were performed based on four nuclear (ITS and ETS nrDNA) and plastid (rps12 intron and trnK-matK) regions. The resulting trees were dated using a secondary calibration. Patterns of DNA sequence variation and dated phylogenetic trees were then interpreted in light of previous knowledge of chromosome numbers in Ulex to infer past events of polyploid speciation in the genus. We show that: (1) most current Ulex spp. radiated in the Iberian Peninsula during the past 1–2 Myr; (2) the history of Ulex was punctuated by multiple whole-genome duplication events; and (3) U. europaeus subsp. europaeus is the only gorse taxon that was formed by hybridization of two well-differentiated lineages (which separated c. 5 Mya) with wide climatic ranges (currently represented by Ulex minor and Ulex europaeus subsp. latebracteatus), possibly contributing to the invasive nature and wider climatic range of U. europaeus subsp. europaeus. These findings provide a much-needed evolutionary framework in which to explore the adaptive consequences of genome mergers and duplication in Ulex.

Chromosomal Aberrations Accumulate during Metastasis of Virus-Negative Merkel Cell Carcinoma

Merkel cell carcinoma is a rare, aggressive skin tumor initiated by polyomavirus integration or UV light DNA damage. In New Zealand, there is a propensity toward the UV-driven form (31 of 107, 29% virus positive). Using archival formalin-fixed, paraffin-embedded tissues, we report targeted DNA sequencing covering 246 cancer genes on 71 tumor tissues and 38 nonmalignant tissues from 37 individuals, with 33 of 37 being negative for the virus. Somatic variants of New Zealand virus-negative Merkel cell carcinomas partially overlapped with those reported overseas, including TP53 variants in all tumors and RB1, LRP1B, NOTCH1, and EPHA3/7 variants each found in over half of the cohort. Variants in genes not analyzed or reported in previous studies were also found. Cataloging variants in TP53 and RB1 from published datasets revealed a broad distribution across these genes. Chr 1p gain and Chr 3p loss were identified in around 50% of New Zealand virus-negative Merkel cell carcinomas, and RB1 loss of heterozygosity was found in 90% of cases. Copy number variants accumulate in most metastases. Virus-negative Merkel cell carcinomas have complex combinations of somatic DNA-sequence variants and copy number variants. They likely carry the small genomic changes permissive for metastasis from early tumor development; however, chromosomal alterations may contribute to driving metastatic progression.

Molecular systematic analysis demonstrates that the threatened southern bell frog, Litoria raniformis (Anura: Pelodryadidae) of eastern Australia, comprises two sub-species

In south-eastern Australia, the pelodryadid Litoria aurea Group (sensu Tyler & Davies 1978) comprises three species: Litoria aurea (Lesson, 1829), Litoria raniformis (Keferstein, 1867), and Litoria castanea (Steindachner, 1867). All three species have been subject to declines over recent decades and taxonomic uncertainty persists among populations on the tablelands in New South Wales. We address the systematics of the Group by analysing mitochondrial and nuclear DNA sequences to assess divergence in the Litoria raniformis from across its current range in New South Wales (NSW), Victoria, South Australia (SA) and Tasmania. We also included samples of Litoria castanea from a recently rediscovered population in the southern tablelands of NSW. Our phylogenetic and population genetic analyses show that Litoria raniformis comprises northern and southern lineages, showing deep mitochondrial DNA sequence divergence (7% net average sequence divergence) and can be diagnosed by fixed allelic differences at more than 4,000 SNP loci. Samples of the northern lineage were collected from the Murray-Darling Basin while those of the southern lineage were collected from south-eastern South Australia, southern and south-eastern Victoria and Tasmania. Analysis of the morphology and bioacoustics did not unequivocally delineate the two lineages. The presence of a hybrid backcross individual in western Victoria at the northern margin of the southern lineage, leads us to assign sub-species status to the two lineages, L. r. raniformis for the northern lineage and L. r. major for the southern lineage. Our data do not unequivocally resolve the taxonomic status of L. castanea which will require molecular genetic analyses of museum vouchers from those parts of the range where L. castanea and L. raniformis are no longer extant. Our data also suggest that human mediated movement of frogs may have occurred over the past 50 years. Our genotyping of vouchers collected in the 1970s from the Mount Lofty Ranges in South Australia detected mitochondrial haplotypes of both sub-species and SNP analysis showed that a single Tasmanian specimen was a backcross with L. r. raniformis ancestry. Movement of L. r. raniformis into Tasmania and both sub-species into the Mount Lofty Ranges are not likely due to passive movements of animals through agricultural commerce, but due to the attractiveness of the species as pets and subsequent escapes or releases, potentially of the larval life stage.

Defining the mutational profile of lower-risk myelodysplastic neoplasm patients with respect to disease progression using next-generation sequencing and pyrosequencing.

Lower-risk myelodysplastic neoplasms (LR-MDS) comprise the majority of MDS. Despite favourable prognoses, some patients remain at risk of rapid progression. We aimed to define the mutational profile of LR-MDS using next-generation sequencing (NGS), Sanger Sequencing (SSeq), and pyrosequencing. Samples from 5 primary LR-MDS (67 exons of SF3B1, U2AF1, SRSF2, ZRSR2, TET2, ASXL1, DNMT3A, TP53, and RUNX1 genes) were subjected to NGS. Next, a genomic study was performed to test for the presence of identified DNA sequence variants on a larger group of LR-MDS patients (25 bone marrow [BM], 3 saliva [SAL], and one peripheral blood [PB] sample/s). Both SSeq (all selected DNA sequence variants) and pyrosequencing (9 selected DNA sequence variants) were performed. Next-generation sequencing results identified 13 DNA sequence variants in 7 genes, comprising 8 mutations in 6 genes (ASXL1, DNMT3A, RUNX1, SF3B1, TET2, ZRSR2) in LR-MDS. The presence of 8 DNA variants was detected in the expanded LR-MDS group using SSeq and pyrosequencing. Mutation acquisition was observed during LR-MDS progression. Four LR-MDS and one acute myeloid leukaemia myelodysplasia-related patient exhibited the presence of at least one mutation. ASXL1 and SF3B1 alterations were most commonly observed (2 patients). Five DNA sequence variants detected in BM (patients: 9, 13) were also present in SAL. We suggest using NGS to determine the LR-MDS mutational profile at diagnosis and suspicion of disease progression. Moreover, PB and SAL molecular testing represent useful tools for monitoring LR-MDS at higher risk of progression. However, the results need to be confirmed in a larger group.

Genetic Approaches for Sex Determination of Chinese Female with Male Pseudohermaphroditism

Abstract Male pseudohermaphroditism is a rare disorders of sex development (DSD) that is manifested by a female-like appearance or incompletely differentiated external genitalia in an individual with a Y chromosome. In this paper, we report our investigation of the case of a 33-year-old Chinese female who was diagnosed with a malignant mixed germ-cell tumor of the ovary. To confirm the sex of the female, we utilized genetic approaches to detect amelogenin and Y-STR loci. Y chromosome microdeletion was performed to identify existing deletions in the AZF regions and SRY. Chromosome karyotyping and whole-exome sequencing (WES) were then applied to reveal the deletion of sex chromosome segments and pathogenic variations in DNA sequences. Using DNA-STR genotyping, we detected both AMEL-X and AMEL-Y fragments. We also found haplotype Y-STR loci and detected all alleles. Furthermore, no microdeletion was detected in the AZF regions and SRY. The chromosome karyotyping was 46, XY. WES revealed a transversion mutation of 58T → C in the androgen receptor exon 1, which could be the pathogenic variant in this case of abnormal sexual development. Sex determination in forensic DNA typing is based on the amelogenin system. It is important that forensic biologists should master various genetic approaches to overcome the issue of gender ambiguity caused by DSDs.

Heritable Cas9-induced nonhomologous recombination in C. elegans.

Identification of the genetic basis of phenotypic variation within species remains challenging. In species with low recombination rates, such as Caenorhabditis elegans , genomic regions linked to a phenotype of interest by genetic mapping studies are often large, making it difficult to identify the specific genes and DNA sequence variants that underlie phenotypic differences. Here, we introduce a method that enables researchers to induce heritable targeted recombination in C. elegans with Cas9. We demonstrate that high rates of targeted nonhomologous recombination can be induced by Cas9 in a genomic region in which naturally occurring meiotic recombination events are exceedingly rare. We anticipate that Cas9-induced nonhomologous recombination (CINR) will greatly facilitate high-resolution genetic mapping in this species.

From genetic variation to precision medicine.

Genetics has been an important tool for discovering new aspects of biology across life. In humans, there is growing momentum behind the application of this knowledge to drive innovation in clinical care, most notably through developments in precision medicine. Nowhere has the impact of genetics on clinical practice been more striking than in the field of rare disorders. For most of these conditions, individual disease susceptibility is influenced by DNA sequence variation in a single or a small number of genes. In contrast, most common disorders are multifactorial and are caused by a complex interplay of multiple genetic, environmental and stochastic factors. The longstanding division of human disease genetics into rare and common components has obscured the continuum of human traits and echoes aspects of the century-old debate between the Mendelian and biometric views of human genetics. In this article, we discuss the differences in data and concepts between rare and common disease genetics. Opportunities to unify these two areas are noted and the importance of adopting a holistic perspective that integrates diverse genetic and environmental factors is discussed.

Diversity and phylogeny of the tick-borne bacterial genus Candidatus Allocryptoplasma (Anaplasmataceae)

The family Anaplasmataceae includes tick-borne bacteria of major public and veterinary health interest, as best illustrated by members of the genera Anaplasma and Ehrlichia. Recent epidemiological surveys have also reported on the presence of a novel putative genus in the Anaplasmataceae, Candidatus Allocryptoplasma, previously described as Candidatus Cryptoplasma in the western black-legged tick, Ixodes pacificus. However, the genetic diversity of Ca. Allocryptoplasma and its phylogenetic relationship with other Anaplasmataceae remain unclear. In this study, we developed a multi-locus sequence typing approach, examining the DNA sequence variation at five genes of Ca. Allocryptoplasma found in ticks. Combining this multi-locus sequence typing and genetic data available on public databases, we found that substantial genetic diversity of Ca. Allocryptoplasma is present in Ixodes, Amblyomma and Haemaphysalis spp. ticks on most continents. Further analyses confirmed that the Ca. Allocryptoplasma of ticks, the Ca. Allocryptoplasma of lizards and some Anaplasma-like bacteria of wild mice cluster into a monophyletic genus, divergent from all other genera of the family Anaplasmataceae. Candidatus Allocryptoplasma appears as a sister genus of Anaplasma and, with the genera Ehrlichia and Neoehrlichia, they form a monophyletic subgroup of Anaplasmataceae associated with tick-borne diseases. The detection of genetically distinct Ca. Allocryptoplasma in ticks of significant medical or veterinary interest supports the hypothesis that it is an emergent genus of tick-borne pathogens of general concern.

Phylogenetic assessment of endangered and look-alike Pigtoe species in a freshwater mussel diversity hotspot.

The Green River in Kentucky in the eastern United States is a freshwater mussel biodiversity hotspot, with 71 known species. Among them, the endangered Pleurobema plenum coexists with other morphologically similar species in the genera Fusconaia and Pleurobema, known colloquially as "pigtoes." Identification of species in these genera is challenging even for mussel experts familiar with them. In our study, the correct identification of these species by experts ranged from 57% to 83%. We delineated taxonomic boundaries among seven species and tested for cryptic biodiversity among these look-alike mussels utilizing mitochondrial and nuclear DNA sequence variation. Phylogenetic analysis of combined (1215 bp) mitochondrial DNA cytochrome oxidase I (COI) and NADH dehydrogenase 1 (ND1) genes showed five well-diverged groups that included F.flava, F.subrotunda, P.cordatum, and P.plenum as distinct clades, with P.sintoxia and P.rubrum grouped into a single clade. While our mitochondrial DNA analyses did not distinguish P.sintoxia and P.rubrum as phylogenetically distinct species, the typical shell forms of these two nominal taxa are very distinct. Further phylogenetic analysis using nuclear ribosomal transcribed spacer region subunit I (ITS1) DNA sequences also showed that P.sintoxia and P.rubrum were not distinct lineages. No cryptic species were detected in the Fusconaia and Pleurobema samples analyzed from the Green River. The highest haplotype diversity (h), average number of nucleotide differences (k), and nucleotide diversity (π) were observed for F.subrotunda at both the COI (h=0.896, k=3.805, π=0.00808) and ND1 (h=0.984, k=6.595, π =0.00886) markers, with similarly high genetic diversity in the other taxa. Our results give managers confidence that cryptic taxa do not occur within or among these morphologically similar species in the Green River, and populations appear genetically diverse, indicative of large and healthy populations.

ERG11 Gene Variability and Azole Susceptibility in Malassezia pachydermatis

Malassezia pachydermatis is part of the normal skin microbiota of various animal species but under certain circumstances becomes an opportunistic pathogen producing otitis and dermatitis. Commonly these Malassezia diseases are effectively treated using azoles. However, some cases of treatment failure have been reported. Alterations in the ERG11 gene have been associated with in vitro azole resistance in M. pachydermatis. In the present study, in vitro antifungal susceptibility of 89 different strains of M. pachydermatis isolated from different animal species and health status was studied. The susceptibility to fluconazole (FLZ), itraconazole (ITZ), ketoconazole and amphotericin B was tested by a disk diffusion method and 17 strains were also subjected to an ITZ E-test. Mueller–Hinton supplemented with 2% glucose and methylene blue was used as culture medium in both susceptibility assays. Multilocus sequence typing was performed in 30 selected strains using D1D2, ITS, CHS2 and β-tubulin genes. Also, ERG11 gene was sequenced. The four antifungals tested were highly effective against most of the strains. Only two strains showed no inhibition zone to antifungals and a strain showed an increased MIC to ITZ. The study of the ERG11 sequences revealed a high diversity of DNA sequences and a total of 23 amino acid substitutions, from which only two have been previously described. Also, three deleterious substitutions (A302T, G459D and G461D) previously associated with azole resistance in this yeast were recovered. A correlation between certain genotypes and ERG11 mutations was observed. Some of the ERG11 mutations recovered were correlated with a reduced susceptibility to azoles.

Review: Genetic and protein variants of milk caseins in goats.

The milk casein genes in goats, are highly polymorphic genes with numerous synonymous and non-synonymous mutations. So far, 20 protein variants have been reported in goats for alpha-S1-casein, eight for beta-casein, 14 for alpha-S2-casein, and 24 for kappa-casein. This review provides a comprehensive overview on identified milk casein protein variants in goat and non-coding DNA sequence variants with some affecting the expression of the casein genes. The high frequency of some casein protein variants in different goat breeds and geographical regions might reflect specific breeding goals with respect to milk processing characteristics, properties for human nutrition and health, or adaptation to the environment. Because protein names, alongside the discovery of protein variants, go through a historical process, we linked old protein names with new ones that reveal more genetic variability. The haplotypes across the cluster of the four genetically linked casein genes are recommended as a valuable genetic tool for discrimination between breeds, managing genetic diversity within and between goat populations, and breeding strategies. The enormous variation in the casein proteins and genes is crucial for producing milk and dairy products with different properties for human health and nutrition, and for genetic improvement depending on local breeding goals.

CONGENITAL POSTERIOR POLAR CHORIORETINAL HYPOPLASIA: Expansion of the Clinical Spectrum, Mutation, and Its Association With PRDM13.

To describe a new ocular phenotype in a single Egyptian family associated with a heterozygous noncoding mutation in the North Carolina macular dystrophy (NCMD/MCDR1) locus, likely affecting the PRDM13 gene. A retrospective, clinical chart review of 11 members of a four-generation family. Comprehensive ophthalmic examinations included visual acuity, refraction, fundus imaging, spectral-domain optical coherence tomography, and full-field electroretinography. Molecular genetic analysis of the MCDR1 region was performed using whole genome and targeted sequencing. The main outcome measures were DNA sequence variants, clinical, retinal imaging, and electroretinography findings. The five affected adult family members tested carried a single heterozygous mutation in a noncoding region (Chr6:100,046,783A>C) located 7.8 kb upstream of PRDM13. Visual acuity ranged from 20/200 to 20/400. All members had extensive chorioretinal absence/thinning extending outside of the maculae with extensive posterior bowing of the choroid and sclera centered in the macula giving a large macular coloboma-like appearance. Two additional members had cystoid fluid, and one had macular detachment. Full-field electroretinography revealed reduced cone and rod responses in all affected members. The phenotype of this disease falls between the spectrum of progressive bifocal chorioretinal atrophy and NCMD. The findings are most consistent with progressive bifocal chorioretinal atrophy with the exception that there is no bifocal nature to the appearance nor is it progressive. Another view is that the phenotype seems to be an extremely severe form of NCMD. Given that this disease falls in between progressive bifocal chorioretinal atrophy and NCMD, we propose calling it congenital posterior polar chorioretinal hypoplasia.

A common transcriptional mechanism involving R-loop and RNA abasic site regulates an enhancer RNA of APOE.

RNA is modified by hundreds of chemical reactions and folds into innumerable shapes. However, the regulatory role of RNA sequence and structure and how dysregulation leads to diseases remain largely unknown. Here, we uncovered a mechanism where RNA abasic sites in R-loops regulate transcription by pausing RNA polymerase II. We found an enhancer RNA, AANCR, that regulates the transcription and expression of apolipoprotein E (APOE). In some human cells such as fibroblasts, AANCR is folded into an R-loop and modified by N-glycosidic cleavage; in this form, AANCR is a partially transcribed nonfunctional enhancer and APOE is not expressed. In contrast, in other cell types including hepatocytes and under stress, AANCR does not form a stable R-loop as its sequence is not modified, so it is transcribed into a full-length enhancer that promotes APOE expression. DNA sequence variants in AANCR are associated significantly with APOE expression and Alzheimer's Disease, thus AANCR is a modifier of Alzheimer's Disease. Besides AANCR, thousands of noncoding RNAs are regulated by abasic sites in R-loops. Together our data reveal the essentiality of the folding and modification of RNA in cellular regulation and demonstrate that dysregulation underlies common complex diseases such as Alzheimer's disease.

Epigenetic Fine Mapping and Functional Dissection of Inherited Non-Coding Variation Impacting the Pharmacogenomics of Acute Lymphoblastic Leukemia Treatment

Acute lymphoblastic leukemia (ALL) is the most prevalent childhood cancer. Despite cure rates exceeding 90%, relapsed ALL is the 5th most common pediatric cancer. Although chemotherapeutic drug resistance remains the primary cause of treatment failure and relapse, it remains unclear to what extent non-coding inherited DNA sequence variants impact gene regulatory programs that contribute to differences in chemotherapeutic drug resistance and clinical outcome in childhood ALL. To address this knowledge gap we performed epigenetic fine mapping on inherited non-coding DNA sequence variants associated with ex vivo resistance to six antileukemic agents (dexamethasone, prednisolone, l-asparaginase, vincristine, 6-mercaptopurine and 6-thioguanine) in primary ALL cells from pediatric patients and inherited non-coding DNA sequence variants associated with clinical outcome (persistence of minimal residual disease and relapse) in pediatric patient cohorts using chromatin accessibility data from over 130 primary ALL cells, ALL cell lines and ALL xenografts. This analysis fine mapped over 1700 variants (sentinel variants and variants in high linkage disequilibrium; r2>0.8) to cis-regulatory elements in the ALL genome. To comprehensively dissect the functional effects of these fine mapped variants on gene regulatory activity we performed massively parallel reporter assays (MPRAs) in 10 human ALL cell lines of both B- and T-cell lineages. MPRAs utilized 23 DNA barcodes per variant allele and tested variants at bidirectional promoters using both sequence orientations. In total, 98,168 oligonucleotides each containing 175-bp of genomic sequence centered on each variant allele and a unique 10-bp barcode sequence were evaluated for gene regulatory activity. MPRA identified 558 variants with significant and concordant allele-specific activities in three or more ALL cell lines. Expression quantitative trait loci (eQTL) mapping and HiChIP three-dimensional promoter loop profiling identified candidate target genes for a subset of functional variants. We additionally evaluated the functional effects of top ranked variants using luciferase reporter assays and CRISPR/Cas9 genome editing. These variants were associated with the expression of B3GNT5, CACUL1, HERC1, KDM4C and VRK3. Notably, two of these functional variants (rs10411204 and rs1247117) impacted transcription factor (TF) consensus DNA motifs for AP-1 family TFs and PU.1. Collectively, these data suggest that many inherited non-coding variants associated with pharmacogenomic traits in childhood ALL have significant gene regulatory effects. Importantly, the identification of chemotherapeutic drug resistance-associated molecular pathways perturbed by inherited non-coding variants can improve our ability to circumvent drug resistance with novel therapeutic approaches.

Genetic and Epigenetic Cis-Regulatory Disruptions Impacting the Canonical Wnt Signaling Repressor TLE1 Gene Promote Glucocorticoid Resistance in Childhood Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most prevalent childhood cancer and despite improved survival rates, more children die of ALL than any other cancer. Glucocorticoids (GCs) are a mainstay of contemporary, multi-drug chemotherapy in the treatment of childhood ALL. Resistance to GCs is predictive of ALL relapse and poor clinical outcomes, and therefore represents a major hurdle limiting further improvements in survival rates. While advances have been made in identifying genes implicated in GC resistance, there remains an insufficient understanding of the impact of cis-regulatory disruptions in GC resistance. This is particularly relevant for GCs, which function through the activation of the glucocorticoid receptor (GR) nuclear receptor transcription factor. To better understand the role of cis-regulatory disruptions in GC resistance we comprehensively mapped the dynamic gene regulatory response to GCs in two human ALL cell lines using functional genomic (RNA-seq, ChIP-seq, ATAC-seq and HiChIP) and massively parallel reporter (ATAC-STARR-seq) assays at multiple time points within a 24-hour window. In total, over 100 independent datasets were generated and integrated. We identified thousands of GR binding sites and GC-responsive changes to chromatin state, including the formation of over 250 GC-responsive super-enhancers and depletion of AP-1 bound cis-regulatory elements implicated in cell proliferation and anti-apoptotic processes. Our results further show that GR mediates gene regulatory effects in ALL cells at GC-responsive chromatin sites through long-range looping interactions with GC-responsive gene promoters. Notably, GC-responsive chromatin sites were also enriched for functionally active cis-regulatory elements. By integrating our GC response maps with inherited DNA sequence variants and differentially accessible chromatin sites associated with GC resistance in primary ALL cells from patients enrolled on St. Jude clinical trials, we identified two GR-bound cis-regulatory elements at the canonical Wnt signaling repressor TLE1 gene locus that were genetically or epigenetically disrupted in GC-resistant patient samples. Functional evaluation of these two GR binding sites using CRISPR genome or CRISPR interference epigenome editing validated their impact on GC resistance and TLE1 gene expression. To better understand the relationship between GC signaling and canonical Wnt signaling, we further performed TOP-flash luciferase reporter assays, cellular drug viability assays and transcriptomic drug response profiling which identified mutual antagonism between these two signaling pathways. Collectively, these data uncovered genetic and epigenetic cis-regulatory disruptions of canonical Wnt signaling as a novel mechanism impacting GC resistance in childhood ALL and further suggest that therapeutic inhibition of canonical Wnt signaling could improve GC sensitivity in patients with resistant disease.

Chromatin Accessibility Landscapes of B-Cell Acute Lymphoblastic Leukemia Identify Extensive Epigenomic Reprogramming and Heterogeneity Among Subtypes

Acute lymphoblastic leukemia (ALL) is the most common malignancy in pediatric patients and represents about a quarter of all pediatric malignancies. Past work has characterized B-cell lineage ALL (B-ALL) into molecular subtypes spanning a range of aberrant chromosomal rearrangements and oncogene chimeric fusions driving malignancy. While transcriptional and DNA methylome profiling of these subtypes has been extensively examined, the accompanying chromatin landscape and corresponding gene regulatory repertoire are not well characterized for many B-ALL subtypes. To better profile the B-ALL epigenome and gene regulatory networks we examined chromatin accessibility of 11 distinct molecular subtypes (BCR-ABL1, DUX4/ERG, ETV6-RUNX1, Hyperdiploid, Low hypodiploid, KMT2A-rearranged, Ph-like, PAX5-altered, TCF3-PBX1, ZNF384-rearranged and B-other) using 154 primary patient samples from across the United States with assay for transposase-accessible chromatin and high-throughput sequencing (ATAC-seq). We mapped extensive chromatin reprogramming when comparing B-ALL and normal B-cells that drive oncogenic signaling pathways and inhibit normal B-cell functions. Strikingly, we also uncovered chromatin accessibility sites in B-ALL (42,457 sites) with distinct subtype-specific enrichment. A classification model utilizing subtype distinct loci was able to accurately predict B-ALL subtypes with 87% cross-validation accuracy. Transcription factor (TF) footprint profiling and transcriptomic information further identified candidate subtype-enriched TFs, such as DUX4, ZNF384, HOXA9, MEIS1, GATA3 and RARA, along with subtype distinct gene regulatory networks. In connecting these findings with genetic variation, we identified 9080 inherited DNA sequence variants that act as ATAC-seq chromatin accessibility quantitative trait loci (ATAC-QTLs) and contribute to variability in chromatin accessibility among patient samples. Nearly one-third of ATAC-QTLs map to TF footprints and commonly altered DNA consensus motifs impacting chromatin accessibility include ETS family TFs (PU.1, SPI-B, ELF3, EHF), ZNF family (ZNF263, ZNF460) and CTCF. Collectively, using the largest B-ALL chromatin accessibility dataset to date, our investigation offers a unique window into the gene regulatory landscape that highlights the complexity and heterogeneity of chromatin accessibility among B-ALL molecular subtypes.

210P Ultra-sensitive monitoring of leukemia patients using superRCA mutation detection assays

Rare tumor-specific mutations in patient samples serve as excellent markers to monitor the course of malignant disease and responses to therapy in clinical routine, and improved assay techniques are needed for broad adoption. We describe herein - superRCA assays - which provides for rapid and highly specific detection of DNA sequence variants present at very low frequencies in DNA samples. Using a standard flow cytometer we demonstrate precise, ultra-sensitive detection of single-nucleotide mutant sequences from malignant cells against a 100,000-fold excess of DNA, to follow the course of patients treated for acute myeloid leukemia (AML).

Using molecular phylogenetic and stable isotopic analysis to identify species, geographical origin and production method of mullet roes

The flathead grey mullet, Mugil cephalus (Mugilidae), is one of the most commercially important fish species in Taiwan since mullet roe is considered as a delicacy with a high economic value. Farmed, wild-caught, and imported mullet roes are often not properly labelled in retail stores and merchants can make unethical profits from selling mislabeled products. Therefore, there is an urgent need to identify the provenance and production method of the mullet roes. In this study, we conducted a molecular phylogenetic analysis so as to identify the species and geographical origin of the mullet roes and we used stable isotopic values as a tool to differentiate between the wild-caught and farmed mullet roes. DNA sequence variations of a mitochondrial COI gene from the sampled mullets and mullet roes of wild-caught and farmed individuals, as well as the roe products sold in retail stores in Taiwan, were analyzed within the phylogenetic framework. The results showed that the samples were from four M. cephalus lineages (or putative Mugil species B, C, I, and H) occurring in the Caribbean Sea, Northwestern Pacific, and Southeastern Australia, and the M. liza in the Western Atlantic, respectively. The M. liza samples were from the salted roe products purchased from two retail stores, in which one claimed that the roes were imported from Brazil, whereas the other claimed that the roes were from locally reared flathead grey mullets. The farmed mullet roes had significantly lower δ13C values and less variation in δ15N values when compared with the wild-caught and imported mullet roes. Quadratic discriminant analysis based on the δ13C and δ15N values successfully discriminated the farmed mullet roes from the wild-caught and imported roes with an approximate error rate of 2%. Our study demonstrated that the combined approach of genetic and stable isotopic analyses can effectively authenticate the mullet roe origin and production methods.

Borgs are giant genetic elements with potential to expand metabolic capacity

Anaerobic methane oxidation exerts a key control on greenhouse gas emissions1, yet factors that modulate the activity of microorganisms performing this function remain poorly understood. Here we discovered extraordinarily large, diverse DNA sequences that primarily encode hypothetical proteins through studying groundwater, sediments and wetland soil where methane production and oxidation occur. Four curated, complete genomes are linear, up to approximately 1 Mb in length and share genome organization, including replichore structure, long inverted terminal repeats and genome-wide unique perfect tandem direct repeats that are intergenic or generate amino acid repeats. We infer that these are highly divergent archaeal extrachromosomal elements with a distinct evolutionary origin. Gene sequence similarity, phylogeny and local divergence of sequence composition indicate that many of their genes were assimilated from methane-oxidizing Methanoperedens archaea. We refer to these elements as ‘Borgs’. We identified at least 19 different Borg types coexisting with Methanoperedens spp. in four distinct ecosystems. Borgs provide methane-oxidizing Methanoperedens archaea access to genes encoding proteins involved in redox reactions and energy conservation (for example, clusters of multihaem cytochromes and methyl coenzyme M reductase). These data suggest that Borgs might have previously unrecognized roles in the metabolism of this group of archaea, which are known to modulate greenhouse gas emissions, but further studies are now needed to establish their functional relevance.