Exon Capture Research Articles

3D models related to the publication: Exon capture museomics deciphers the nine-banded armadillo species complex and identifies a new species endemic to the Guiana Shield.

3D models related to the publication: Exon capture museomics deciphers the nine-banded armadillo species complex and identifies a new species endemic to the Guiana Shield.

Simultaneous high throughput genotyping of 36 blood group systems using NGS based on probe capture technology

Human blood group antigen has important biological functions, and transfusion of incompatible blood can cause alloimmunization and may lead to serious hemolytic reactions. Currently, serological methods are most commonly used in blood group typing. However, this technique has certain limitations and cannot fully meet the increasing demand for the identification of blood group antigens. This study describes a next-generation sequencing (NGS) technology platform based on exon and flanking region capture probes to detect full coding exon and flanking intron regions of the 36 blood group systems, providing a new high-throughput method for the identification of blood group antigens. The 871 capture probes were designed for the exon and flanking intron sequences of 36 blood group system genes, and synchronization analysis for 36 blood groups was developed. The library for NGS was tested using the MiSeq Sequencing Reagent Kit (v2, 300 cycles) by Illumina NovaSeq, and the data were analyzed by the CLC Genomics Workbench 21.0 software. A total of 199 blood specimens have been sequenced for the 41 genes from 36 blood groups. Among them, heterozygote genotypes were found in the ABO, Rh, MNS, Lewis, Duffy, Kidd, Diego, Gerbich, Dombrock, Globoside, JR, LAN, and Landsteiner-Wiene blood group systems. Only the homozygous genotype was found in the remaining 22 blood group systems. The obtained data in the NGS method shows a good correlation (99.98 %) with those of the polymerase chain reaction-sequence-based typing. An NGS technology platform for 36 blood group systems genotyping was successfully established, which has the characteristics of high accuracy, high throughput, and wide coverage.

Exon capture museomics deciphers the nine-banded armadillo species complex and identifies a new species endemic to the Guiana Shield.

The nine-banded armadillo (Dasypus novemcinctus) is the most widespread xenarthran species across the Americas. Recent studies have suggested it is composed of four morphologically and genetically distinct lineages of uncertain taxonomic status. To address this issue, we used a museomic approach to sequence 80 complete mitogenomes and capture 997 nuclear loci for 71 Dasypus individuals sampled across the entire distribution. We carefully cleaned up potential genotyping errors and cross contaminations that could blur species boundaries by mimicking gene flow. Our results unambiguously support four distinct lineages within the D. novemcinctus complex. We found cases of mito-nuclear phylogenetic discordance but only limited contemporary gene flow confined to the margins of the lineage distributions. All available evidence including the restricted gene flow, phylogenetic reconstructions based on both mitogenomes and nuclear loci, and phylogenetic delimitation methods consistently supported the four lineages within D. novemcinctus as four distinct species. Comparable genetic differentiation values to other recognized Dasypus species further reinforced their status as valid species. Considering congruent morphological results from previous studies, we provide an integrative taxonomic view to recognise four species within the D. novemcinctus complex: D. novemcinctus, D. fenestratus, D. mexicanus, and D. guianensis sp. nov., a new species endemic of the Guiana Shield that we describe here. The two available individuals of D. mazzai and D. sabanicola were consistently nested within D. novemcinctus lineage and their status remains to be assessed. The present work offers a case study illustrating the power of museomics to reveal cryptic species diversity within a widely distributed and emblematic species of mammals.

Development of a Target Enrichment Probe Set for Conifer (REMcon).

Conifers are an ecologically and economically important seed plant group that can provide significant insights into the evolution of land plants. Molecular phylogenetics has developed as an important approach in evolutionary studies, although there have been relatively few studies of conifers that employ large-scale data sourced from multiple nuclear genes. Target enrichment sequencing (target capture, exon capture, or Hyb-Seq) has developed as a key approach in modern phylogenomic studies. However, until now, there has been no bait set that specifically targets the entire conifer clade. REMcon is a target sequence capture probe set intended for family- and species-level phylogenetic studies of conifers that target c. 100 single-copy nuclear loci. We tested the REMcon probe set using 69 species, including 44 conifer genera across six families and four other gymnosperm taxa, to evaluate the efficiency of target capture to efficiently generate comparable DNA sequence data across conifers. The recovery of target loci was high, with, on average, 94% of the targeted regions recovered across samples with high read coverage. A phylogenetic analysis of these data produced a well-supported topology that is consistent with the current understanding of relationships among conifers. The REMcon bait set will be useful in generating relatively large-scale nuclear data sets consistently for any conifer lineage.

Museum Skins Enable Identification of Introgression Associated with Cytonuclear Discordance.

Increased sampling of genomes and populations across closely related species has revealed that levels of genetic exchange during and after speciation are higher than previously thought. One obvious manifestation of such exchange is strong cytonuclear discordance, where the divergence in mitochondrial DNA (mtDNA) differs from that for nuclear genes more (or less) than expected from differences between mtDNA and nuclear DNA (nDNA) in population size and mutation rate. Given genome-scale data sets and coalescent modeling, we can now confidently identify cases of strong discordance and test specifically for historical or recent introgression as the cause. Using population sampling, combining exon capture data from historical museum specimens and recently collected tissues we showcase how genomic tools can resolve complex evolutionary histories in the brachyotis group of rock-wallabies (Petrogale). In particular, applying population and phylogenomic approaches we can assess the role of demographic processes in driving complex evolutionary patterns and assess a role of ancient introgression and hybridization. We find that described species are well supported as monophyletic taxa for nDNA genes, but not for mtDNA, with cytonuclear discordance involving at least 4 operational taxonomic units across 4 species which diverged 183-278 kya. ABC modeling of nDNA gene trees supports introgression during or after speciation for some taxon pairs with cytonuclear discordance. Given substantial differences in body size between the species involved, this evidence for gene flow is surprising. Heterogenous patterns of introgression were identified but do not appear to be associated with chromosome differences between species. These and previous results suggest that dynamic past climates across the monsoonal tropics could have promoted reticulation among related species.

Abstract 2796: Colibactin mutation signatures are associated with younger age of onset in colorectal cancer

Abstract Escherichia coli (E. coli) strains carrying the polyketide synthase (pks) island produce the genotoxin colibactin associated with pathogenesis of colorectal cancer (CRC). Colibactin binds directly to DNA, causing alkylating damage leading to double stranded DNA breaks. Colibactin mutagenesis results in mutations in hexameric A-T rich DNA motifs that can be identified by whole genome sequencing (WGS) as mutation signatures SBS28 and SBS88 (SBS-pks). Since WGS is not routinely used clinically, we developed an approach to specifically identify SBS-pks using a clinical targeted exon capture assay, MSK-IMPACT. In an institutional pan-cancer clinical cohort of 78,905 tumors we identified 15,967 samples with > 20 mutations detected by MSK-IMPACT. We identified 150/1,884 samples with microsatellite stable CRC with >10% mutations attributable to SBS-pks. Examining clinicopathologic and genomic features of SBS-pks+ compared to SBS-pks- CRC tumors, we found that SBS-pks+ signatures are significantly enriched in younger age of onset compared to SBS-pks- CRC patients (median age 55 vs median age 59, p= 0.0028). SBS-pks+ CRC exhibit similar driver mutations and tumor mutational burden (TMB) compared to SBS-pks- CRC but are associated with increased fraction of genome altered (FGA). To mechanistically investigate colibactin-associated damage we developed a novel long-term human colon organoid-microbe co-culture model system that takes advantage of reversal of polarity in suspension culture. WGS of human CRC organoids co-cultured for three months with pks+ and delta-pks E. coli NC101 confirmed induction of pks+ specific SBS-pks signatures, validating the model. Leveraging this model and a biobank of normal colon organoids from donors aged </> 50 years, we are investigating the molecular mechanisms underlying the association of SBS-pks with younger age of onset CRC. Citation Format: Stefanie Gerstberger, Melissa Lumish, Saskia Hartner, Farheen Shah, Seongmin Choi, Anisha Luthra, Qingwen Jiang, Hyung Jun Woo, Ahmed Mahmoud, Henry Walch, Simran Asawa, Mark Donoghue, Andrea Cercek, Rona Yaeger, Andrew McPherson, Francisco Sanchez-Vega, Karuna Ganesh. Colibactin mutation signatures are associated with younger age of onset in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2796.

Phylogenomics of Neogastropoda: The Backbone Hidden in the Bush.

The molluskan order Neogastropoda encompasses over 15,000 almost exclusively marine species playing important roles in benthic communities and in the economies of coastal countries. Neogastropoda underwent intensive cladogenesis in the early stages of diversification, generating a "bush" at the base of their evolutionary tree, which has been hard to resolve even with high throughput molecular data. In the present study to resolve the bush, we use a variety of phylogenetic inference methods and a comprehensive exon capture dataset of 1817 loci (79.6% data occupancy) comprising 112 taxa of 48 out of 60 Neogastropoda families. Our results show consistent topologies and high support in all analyses at (super)family level, supporting monophyly of Muricoidea, Mitroidea, Conoidea, and, with some reservations, Olivoidea and Buccinoidea. Volutoidea and Turbinelloidea as currently circumscribed are clearly paraphyletic. Despite our analyses consistently resolving most backbone nodes, 3 prove problematic: First, the uncertain placement of Cancellariidae, as the sister group to either a Ficoidea-Tonnoidea clade or to the rest of Neogastropoda, leaves monophyly of Neogastropoda unresolved. Second, relationships are contradictory at the base of the major "core Neogastropoda" grouping. Third, coalescence-based analyses reject monophyly of the Buccinoidea in relation to Vasidae. We analyzed phylogenetic signal of targeted loci in relation to potential biases, and we propose the most probable resolutions in the latter 2 recalcitrant nodes. The uncertain placement of Cancellariidae may be explained by orthology violations due to differential paralog loss shortly after the whole genome duplication, which should be resolved with a curated set of longer loci.

The effect of missing data on evolutionary analysis of sequence capture bycatch, with application to an agricultural pest

Sequence capture is a genomic technique that selectively enriches target sequences before high throughput next-generation sequencing, to generate specific sequences of interest. Off-target or ‘bycatch’ data are often discarded from capture experiments, but can be leveraged to address evolutionary questions under some circumstances. Here, we investigated the effects of missing data on a variety of evolutionary analyses using bycatch from an exon capture experiment on the global pest moth, Helicoverpa armigera. We added > 200 new samples from across Australia in the form of mitogenomes obtained as bycatch from targeted sequence capture, and combined these into an additional larger dataset to total > 1000 mitochondrial cytochrome c oxidase subunit I (COI) sequences across the species’ global distribution. Using discriminant analysis of principal components and Bayesian coalescent analyses, we showed that mitogenomes assembled from bycatch with up to 75% missing data were able to return evolutionary inferences consistent with higher coverage datasets and the broader literature surrounding H. armigera. For example, low-coverage sequences broadly supported the delineation of two H. armigera subspecies and also provided new insights into the potential for geographic turnover among these subspecies. However, we also identified key effects of dataset coverage and composition on our results. Thus, low-coverage bycatch data can offer valuable information for population genetic and phylodynamic analyses, but caution is required to ensure the reduced information does not introduce confounding factors, such as sampling biases, that drive inference. We encourage more researchers to consider maximizing the potential of the targeted sequence approach by examining evolutionary questions with their off-target bycatch where possible—especially in cases where no previous mitochondrial data exists—but recommend stratifying data at different genome coverage thresholds to separate sampling effects from genuine genomic signals, and to understand their implications for evolutionary research.

Genetic mixing in conservation translocations increases diversity of a keystone threatened species, Bettongia lesueur.

Translocation programmes are increasingly being informed by genetic data to monitor and enhance conservation outcomes for both natural and established populations. These data provide a window into contemporary patterns of genetic diversity, structure and relatedness that can guide managers in how to best source animals for their translocation programmes. The inclusion of historical samples, where possible, strengthens monitoring by allowing assessment of changes in genetic diversity over time and by providing a benchmark for future improvements in diversity via management practices. Here, we used reduced representation sequencing (ddRADseq) data to report on the current genetic health of three remnant and seven translocated boodie (Bettongia lesueur) populations, now extinct on the Australian mainland. In addition, we used exon capture data from seven historical mainland specimens and a subset of contemporary samples to compare pre-decline and current diversity. Both data sets showed the significant impact of population founder source (whether multiple or single) on the genetic diversity of translocated populations. Populations founded by animals from multiple sources showed significantly higher genetic diversity than the natural remnant and single-source translocation populations, and we show that by mixing the most divergent populations, exon capture heterozygosity was restored to levels close to that observed in pre-decline mainland samples. Relatedness estimates were surprisingly low across all contemporary populations and there was limited evidence of inbreeding. Our results show that a strategy of genetic mixing has led to successful conservation outcomes for the species in terms of increasing genetic diversity and provides strong rationale for mixing as a management strategy.

Initiation of speciation across multiple dimensions in a rock-restricted, tropical lizard.

Population isolation and concomitant genetic divergence, resulting in strong phylogeographical structure, is a core aspect of speciation initiation. If and how speciation then proceeds and ultimately completes depends on multiple factors that mediate reproductive isolation, including divergence in genomes, ecology and mating traits. Here we explored these multiple dimensions in two young (Plio-Pleistocene) species complexes of gekkonid lizards (Heteronotia) from the Kimberley-Victoria River regions of tropical Australia. Using mitochondrial DNA screening and exon capture phylogenomics, we show that the rock-restricted Heteronotia planiceps exhibits exceptional fine-scale phylogeographical structure compared to the codistributed habitat generalist Heteronotia binoei. This indicates pervasive population isolation and persistence in the rock-specialist, and thus a high rate of speciation initiation across this geographically complex region, with levels of genomic divergence spanning the "grey zone" of speciation. Proximal lineages of H.planiceps were often separated by different rock substrates, suggesting a potential role for ecological isolation; however, phylogenetic incongruence and historical introgression were inferred between one such pair. Ecomorphological divergence among lineages within both H.planiceps and H.binoei was limited, except that limestone-restricted lineages of H.planiceps tended to be larger than rock-generalists. By contrast, among-lineage divergence in the chemical composition of epidermal pore secretions (putative mating trait) exceeded ecomorphology in both complexes, but with less trait overlap among lineages in H.planiceps. This system-particularly the rock-specialist H.planiceps-highlights the role of multidimensional divergence during incipient speciation, with divergence in genomes, ecomorphology and chemical signals all at play at very fine spatial scales.

RF17 | PMON23 Argentinian multicentric genetic study of pituitary hormonal deficiencies using a custom panel based on single molecule molecular inversion probes

Abstract Hypopituitarism with deficiency of one or more pituitary hormones (combined pituitary hormone deficiency or CPHD) can vary in severity and age at presentation. Additionally, the hormone abnormalities may evolve with time necessitating frequent evaluation. These hormonal deficits can also be present as part of a syndrome, with patients showing extrapituitary abnormalities such as eye or forebrain malformations. Over the past decade, there has been an explosion in the knowledge of the genetic cascade that orchestrates hypothalamic and pituitary development. Several transcription factors and signaling molecules are critical for cell differentiation and proliferation at a very early stage of gestation. However, more genes remain to be identified in order to provide patients with a definitive aetiology. We studied 95 children with congenital hypopituitarism from seven pediatric hospitals from Argentina. Children with non-endocrine, non-familial idiopathic short stature served as a control group (n=100). A custom exon capture panel based on single-molecule molecular inversion probes sequencing (MIPS) was performed in probands and parents. This panel tests 104 genes: including a selection of reported and candidate genes chosen from our knowledge of pituitary development in mice and PROP1 interacting proteins. We found at least 1 variant in 50 probands. The prevalence of known variants in the pituitary transcription factor genes PROP1 and PIT1 (frequently mutated in CPHD) was low in our cohort. A significant number of disease-causing variants were found in LHX3, LHX4, GLI2 and OTX2. An important advance of our study is the identification of pathogenic variants in recently discovered and novel genes. We found an heterozygous variant in the Roundabout (ROBO) receptor homolog 1 gene: ROBO1;p.Arg1504* in a patient with CPHD and septo-optic dysplasia (SOD); two variants in Steroid Receptor SRA1: p. Ile179Thr and p.Val110delinsArgLeu in a patient with CPHD and hypogonadism hypogonadotropic and a missense variant in Semaphorin 3A SEMA3A: p.Val588Leu in a case of CPHD and aortic coarctation. Our findings support evidence that the etiology of congenital hypopituitarism is highly heterogeneous and may be infrequently monogenic with full penetrance, underlying a more complex pathogenesis. Presentation: Sunday, June 12, 2022 12:36 p.m. - 12:41 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Phylogenomics and classification of Notropis and related shiners (Cypriniformes: Leuciscidae) and the utility of exon capture on lower taxonomic groups.

North American minnows of the Shiner Clade, within the family Leuciscidae, represent one of the most taxonomically complex clades of the order Cypriniformes due to the large number of taxa coupled with conserved morphologies. Species within this clade were moved between genera and subgenera until the community decided to lump many of the unclassified taxa with similar morphologies into one genus, Notropis, which has held up to 325 species. Despite phylogentic studies that began to re-elevate some genera merged into Notropis, such as Cyprinella, Luxilus, Lythrurus, and Pteronotropis, the large genus Notropis remained as a taxonomic repository for many shiners of uncertain placement. Recent molecular advances in sequencing technologies have provided the opportunity to re-examine the Shiner Clade using phylogenomic markers. Using a fish probe kit, we sequenced 90 specimens in 87 species representing 16 genera included in the Shiner Clade, with a resulting dataset of 1,004 loci and 286,455 base pairs. Despite the large dataset, only 32,349 bp (11.29%) were phylogenetically informative. In our maximum likelihood tree, 78% of nodes are 100% bootstrap supported demonstrating the utility of the phylogenomic markers at lower taxonomic levels. Unsurprisingly, species within Notropis as well as Hudsonius, Luxilus, and Alburnops are not resolved as monophyletic groups. Cyprinella is monophyletic if Cyprinella callistia is excluded, and Pteronotropis is monophyletic if it includes Hudsonius cummingsae. Taxonomic changes we propose are: restriction of species included in Alburnops and Notropis, elevation of the subgenus Hydrophlox, expansion of species included in Miniellus, movement of Hudsonius cummingsae to Pteronotropis, and resurrection of the genera Coccotis and Paranotropis. We additionally had two specimens of three species, Notropis atherinoides, Ericymba amplamala, and Pimephales vigilax and found signficant differences between the localities (1,086, 1,424, and 845 nucleotides respectively).

Phylogenetic resolution of the fly superfamily Ephydroidea-Molecular systematics of the enigmatic and diverse relatives of Drosophilidae.

The schizophoran superfamily Ephydroidea (Diptera: Cyclorrhapha) includes eight families, ranging from the well-known vinegar flies (Drosophilidae) and shore flies (Ephydridae), to several small, relatively unusual groups, the phylogenetic placement of which has been particularly challenging for systematists. An extraordinary diversity in life histories, feeding habits and morphology are a hallmark of fly biology, and the Ephydroidea are no exception. Extreme specialization can lead to “orphaned” taxa with no clear evidence for their phylogenetic position. To resolve relationships among a diverse sample of Ephydroidea, including the highly modified flies in the families Braulidae and Mormotomyiidae, we conducted phylogenomic sampling. Using exon capture from Anchored Hybrid Enrichment and transcriptomics to obtain 320 orthologous nuclear genes sampled for 32 species of Ephydroidea and 11 outgroups, we evaluate a new phylogenetic hypothesis for representatives of the superfamily. These data strongly support monophyly of Ephydroidea with Ephydridae as an early branching radiation and the placement of Mormotomyiidae as a family-level lineage sister to all remaining families. We confirm placement of Cryptochetidae as sister taxon to a large clade containing both Drosophilidae and Braulidae–the latter a family of honeybee ectoparasites. Our results reaffirm that sampling of both taxa and characters is critical in hyperdiverse clades and that these factors have a major influence on phylogenomic reconstruction of the history of the schizophoran fly radiation.

266 (PB046) - Evaluating the performance of two NGS-based copy number aberration detection tools in patient-derived organoids

Introduction: NGS sequencing technology, including whole genome sequencing (WGS) and whole exome sequencing (WES), has become a primary tool to study genetic variants of tumors. Many tools have been developed for NGS-based copy number aberration (CNA) detection including software GATK (Broad Institute) and CopyWriteR, which both demonstrate reliable performance. GATK software can be applied to WES and WGS data, using target region depth and germline mutation allele frequency for copy number detection. CopyWriteR software uses only WES data, leveraging on the off-target region depth. In this study the advantages and disadvantages of these two tools are compared. Methods: CNA of 23 pairs of patient-derived organoid (PDO) models were analyzed using WGS data with the GATK software, and 18 pairs of PDO models using WES data with both the GATK and CopyWriteR software. CNA was inferred in the presence and absence of paired WGS and WES-based CNA results were compared to evaluate the influence of NGS data type. The WES-based CNA data by GATK and CopyWriteR were compared to evaluate the two algorithms. Results: The number of amplified/deleted regions and genes are comparable for the three combinations (GATK + WGS, GATK + WES, and CopyWriteR + WES) when matched controls were used. WES-based combination detected significantly more CNA events in the absence of matched controls when compared to WGS-based combination. GATK software was prone to unexpected results when the WES capture efficiency was not equal in library preparation. CopyWriteR software missed the presence of some highly amplified genes. Conclusions: WGS data was found to be a superior source for CNA detection when compared to WES data. CopyWriteR software has shortage on detection of high gene amplification events and for WES data, with uneven exon capture rate, it demonstrated superior performance over GATK. These results suggest it is integrating the WES-based results from both software. No conflict of interest.

Composition of a chemical signalling trait varies with phylogeny and precipitation across an Australian lizard radiation.

The environment presents challenges to the transmission and detection of animal signalling systems, resulting in selective pressures that can drive signal divergence amongst populations in disparate environments. For chemical signals, climate is a potentially important selective force because factors such as temperature and moisture influence the persistence and detection of chemicals. We investigated an Australian lizard radiation (Heteronotia) to explore relationships between a sexually dimorphic chemical signalling trait (epidermal pore secretions) and two key climate variables: temperature and precipitation. We reconstructed the phylogeny of Heteronotia with exon capture phylogenomics, estimated phylogenetic signal in amongst-lineage chemical variation and assessed how chemical composition relates to temperature and precipitation using multivariate phylogenetic regressions. High estimates of phylogenetic signal indicate that the composition of epidermal pore secretions varies amongst lineages in a manner consistent with Brownian motion, although there are deviations to this, with stark divergences coinciding with two phylogenetic splits. Accounting for phylogenetic non-independence, we found that amongst-lineage chemical variation is associated with geographic variation in precipitation but not temperature. This contrasts somewhat with previous lizard studies, which have generally found an association between temperature and chemical composition. Our results suggest that geographic variation in precipitation can affect the evolution of chemical signalling traits, possibly influencing patterns of divergence amongst lineages and species.

Association of DNA damage repair (DDR) mutations (mts) and clinical outcomes in CALGB 90601 (Alliance).

4521 Background: Platinum-based chemotherapy is the standard 1st-line therapy for metastatic urothelial cancer (mUC). C90601 was a randomized phase III trial testing gemcitabine and cisplatin (GC) with bevacizumab (B) or placebo (P) in patients (pts) with untreated mUC. Median overall survival (OS) for GCB vs GCP was 14.5 months (mo) vs 14.3 mo (p=0.14) and median progression-free survival (PFS) was 8 vs 6.7 mo, respectively. DDR mts have been implicated in response and survival in mUC and were investigated in this negative trial. Methods: C90601 enrolled 506 pts randomized 1:1 to GCB or GCP from 7/15/09-12/2/14, with stratification for prior chemotherapy and visceral metastases. Consenting pts submitted archival FFPE tumor specimens and blood for matched germline (g)DNA. Tumor and gDNA were sequenced by MSK-IMPACT, a 468-gene exon capture assay, to detect mts in select DDR genes. The proportional hazards model was used to correlate mts in the DNA helicase ERCC2 (pre-specified hypothesis) and additional DDR gene panels being explored in prospective trials in muscle-invasive disease with OS and PFS, adjusting for tumor mt burden and stratification factors. Mts were categorized as deleterious (del) or non-del using pre-defined published criteria. Results: 208 pts underwent DNA sequencing. Clinical features and PFS/OS were comparable to the 506-pt cohort. Median sequencing coverage was 497X. Median mutation count was 13.2 and 8.8 for DDR mt and wild-type tumors, respectively. A non-significant improvement in OS and PFS was seen in pts with ERCC2 mts (HR 0.70), but the 5.3% frequency of ERCC2 mts was lower than in historical series. Neither del mts (table) nor any mts in DDR genes were associated with PFS/OS. Conclusions: DDR mts were not associated with improved outcomes in C90601. The reliance on archival specimens, lower-than-expected ERCC2 mt frequency, small sample sizes, and tumor genomic heterogeneity may have influenced the predictive capacity of DDR mts in this cohort. Similar analyses are underway in pts who received neoadjuvant chemotherapy prior to cystectomy from completed prospective trials. Support: U10CA180821, U10CA180882, Genentech.[Table: see text]

Association of DNA damage response (DDR) gene mutations (mts) and response to neoadjuvant cisplatin-based chemotherapy (chemo) in muscle-invasive bladder cancer (MIBC) patients (pts) enrolled onto SWOG S1314.

4522 Background: Neoadjuvant cisplatin-based chemo followed by radical cystectomy (RC) is a standard of care treatment for pts with MIBC. DDR gene mts, including within ERCC2, a DNA helicase implicated in cisplatin sensitivity in MIBC, have been associated with higher pathologic (path) downstaging ( < pT2) and complete response (pT0) at RC and improved overall survival (OS) in retrospective series. S1314 randomized pts to one of 2 chemo regimens (dose dense MVAC or Gem/Cis) followed by RC. We sought to correlate ERCC2 and other DDR gene mts with response and survival in MIBC pts enrolled onto this prospective trial. Methods: Tumor and matched germline DNA from evaluable pts enrolled onto S1314 underwent exon capture sequencing of 505 cancer-associated genes (MSK-IMPACT). Both deleterious (del) mts and any mts in 9 DDR genes (ERCC2, ERCC5, BRCA1, BRCA2, RECQL4, ATM, ATR, RAD51C, FANCC) were correlated with clinical outcomes. The prespecified analyses included the association of mts with < pT2 and pT0 by logistic regression analysis and with progression-free survival (PFS) and OS by Cox proportional hazards regression. Results: 179 patients (median 61 years, 85% male, 87% white, and 87% clinical stage T2) who received >2 cycles of chemo and were evaluable for path response were included in the analysis. The pT0 rate was 28% and < pT2 was 41%. Del mts in ERCC2 were detected in 26 (14%) pts followed by ATM (n = 12, 7%), ATR (n = 3) and BRCA2 (n = 2). ERCC2 mts were associated with statistically significantly higher path responses with a 54% pT0 rate and 62% downstaging rate. Patients with any del mts had higher path response rates (51% pT0, 56% < pT2) and better PFS (Table) with a median follow-up of 53 months. There was a non-significant trend towards improved OS. Conclusions: In pts managed with neoadjuvant chemo and RC on S1314, both ERCC2 mts and del DDR gene mts correlated with pathologic response. Any del DDR gene mt was associated with improved PFS. These results are in line with retrospective analyses displaying a correlation between DDR gene mts and neoadjuvant chemosensitivity in MIBC and support ongoing genomically-informed organ sparing trials.[Table: see text]

Parallel decay of vision genes in subterranean water beetles

In the framework of neutral theory of molecular evolution, genes specific to the development and function of eyes in subterranean animals living in permanent darkness are expected to evolve by relaxed selection, ultimately becoming pseudogenes. However, definitive empirical evidence for the role of neutral processes in the loss of vision over evolutionary time remains controversial. In previous studies, we characterized an assemblage of independently-evolved water beetle (Dytiscidae) species from a subterranean archipelago in Western Australia, where parallel vision and eye loss have occurred. Using a combination of transcriptomics and exon capture, we present evidence of parallel coding sequence decay, resulting from the accumulation of frameshift mutations and premature stop codons, in eight phototransduction genes (arrestins, opsins, ninaC and transient receptor potential channel genes) in 32 subterranean species in contrast to surface species, where these genes have open reading frames. Our results provide strong evidence to support neutral evolutionary processes as a major contributing factor to the loss of phototransduction genes in subterranean animals, with the ultimate fate being the irreversible loss of a light detection system.

Genetic Analysis and Functional Study of a Pedigree With Bruck Syndrome Caused by PLOD2 Variant.

BackgroundBruck syndrome (BS) is a rare autosomal recessive inherited osteogenesis imperfecta disease characterized by increased bone fragility and joint contracture. The pathogenic gene of type I BS is FKBPl0, whereas that of type II BS is PLOD2. No significant difference has been found in the clinical phenotype between the two types of BS. In this study, we performed genetic analysis of a BS pedigree caused by PLOD2 variant and studied the corresponding cellular function.MethodsSerum biochemistry, parathyroid hormone (PTH), 25-hydroxyvitamin D [25-(OH) D], osteocalcin, and 24-h urinary calcium levels of a family member with BS was assessed. The genes of the proband were analyzed by second-generation sequencing and exon capture techniques. Sanger sequencing was also performed for the suspected responsible variant of the family member. Wild- and variant-type lentivirus plasmids were constructed by gene cloning and transfected into HEK293T cells. Cell function was verified by real-time quantitative polymerase chain reaction, western blotting, and immunofluorescence detection.ResultsIn this pedigree, the proband was found to have a homozygous variant c.1856G > A (p.Arg619His) in exon 17 of PLOD2 (NM_182943.3). His consanguineous parents and sisters were p.Arg619His heterozygous carriers. The mRNA expression of PLOD2 in the constructed p.Arg619His variant cells was significantly upregulated, while the expression of PLOD2 and collagen I protein in the cell lysate was significantly downregulated. Immunofluorescence revealed that the wild-type PLOD2 was mainly located in the cytoplasm, and the expression of the PLOD2 protein after c.1856G > A variant was significantly downregulated, with almost no expression, aligning with the western blot results. The serum sodium, potassium, calcium, phosphorus, magnesium, alkaline phosphatase, PTH, 25-(OH) D, osteocalcin, and 24 h urinary calcium levels of the proband, his parents, and sisters were normal.ConclusionThrough gene and cell function analyses, PLOD2 Arg619His missense variant was preliminarily confirmed to cause BS by reducing protein expression.

Analysis of a Family with Brugada Syndrome and Sudden Cardiac Death Caused by a Novel Mutation of SCN5A.

Background Brugada syndrome is a hereditary cardiac disease associated with mutations in ion channel genes. The clinical features include ventricular fibrillation, syncope, and sudden cardiac death. A family with Brugada syndrome with sudden cardiac death was analyzed to locate the associated mutation in the SCN5A gene. Methods and Results Three generations of a Han Chinese family with Brugada syndrome were recruited in the study; their clinical phenotype data were collected and DNA samples extracted from the peripheral blood. Next-generation sequencing was carried out in the proband, and candidate genes and mutations were screened using the full exon capture technique. The family members who participated in the survey were tested for possible mutations using Sanger sequencing. Six family members were diagnosed with Brugada syndrome, including four asymptomatic patients. A newly discovered heterozygous mutation in the proband was located in exon 25 of SCN5A (NM_000335.5) at c.4313dup(p.Trp1439ValfsTer32). Among the surviving family members, only those with a Brugada wave on their electrocardiogram carried the c.4313dup(p.Trp1439ValfsTer32) variant. Bioinformatics prediction revealed that the frameshift of the c.4313dup (p.Trp1439ValfsTer32) mutant led to a coding change of 32 amino acids, followed by a stop codon, resulting in a truncated protein product. Conclusion The newly discovered mutation site c.4313dup(p.Trp1439ValfsTer32) in exon 25 of SCN5A may be the molecular genetic basis of the family with Brugada syndrome.