Complex Variants Research Articles

SVision: a deep learning approach to resolve complex structural variants.

Complex structural variants (CSVs) encompass multiple breakpoints and are often missed or misinterpreted. We developed SVision, a deep-learning-based multi-object-recognition framework, to automatically detect and haracterize CSVs from long-read sequencing data. SVision outperforms current callers at identifying the internal structure of complex events and has revealed 80 high-quality CSVs with 25 distinct structures from an individual genome. SVision directly detects CSVs without matching known structures, allowing sensitive detection of both common and previously uncharacterized complex rearrangements.

Exonization of a deep intronic long interspersed nuclear element in Becker muscular dystrophy.

The precise identification of pathogenic DMD variants is sometimes rather difficult, mainly due to complex structural variants (SVs) and deep intronic splice-altering variants. We performed genomic long-read whole DMD gene sequencing in a boy with asymptomatic hyper-creatine kinase-emia who remained genetically undiagnosed after standard genetic testing, dystrophin protein and DMD mRNA studies, and genomic short-read whole DMD gene sequencing. We successfully identified a novel pathogenic SV in DMD intron 1 via long-read sequencing. The deep intronic SV consists of a long interspersed nuclear element-1 (LINE-1) insertion/non-tandem duplication rearrangement causing partial exonization of the LINE-1, establishing a genetic diagnosis of Becker muscular dystrophy. Our study expands the genetic spectrum of dystrophinopathies and highlights the significant role of disease-causing LINE-1 insertions in monogenic diseases.

Which MAPF Model Works Best for Automated Warehousing?

Multi-Agent Path Finding (MAPF) algorithms and their variants can find high-quality collision-free plans for automated warehousing under simplified assumptions about the robot dynamics. However, these simplifying assumptions pose challenging implementational issues as the robots cannot follow the plans precisely. Various robust execution frameworks, such as the Action Dependency Graph (ADG) framework, have been proposed to enable the real-world execution of MAPF plans. Under such a framework, it is unclear how the simplifying assumptions affect the performance of the robots. In this work, we first argue that the ADG framework provides the same robustness guarantees as the single-agent framework (where plans are generated independently for each robot and collisions are avoided through a reservation table), which is widely used in industry. We then improve the efficiency of the ADG framework by integrating it with the Rolling-Horizon Collision-Resolution framework to solve MAPF problems with a persistent stream of online tasks. Using the integrated framework, we compare the standard MAPF model with many of its more complex variants, such as MAPF with rotation, k-robust MAPF, and continuous-time MAPF (taking robot dynamics into account). We examine their effectiveness in improving throughput through realistic simulations of warehouse settings with the Gazebo simulator.

Understanding the assumptions of an SEIR compartmental model using agentization and a complexity hierarchy

Equation-based and agent-based models are popular methods in understanding disease dynamics. Although there are many types of equation-based models, the most common is the SIR compartmental model that assumes homogeneous mixing and populations. One way to understand the effects of these assumptions is by agentization. Equation-based models can be agentized by creating a simple agent-based model that replicates the results of the equation-based model, then by adding complexity to these agentized models it is possible to break the assumptions of homogeneous mixing and populations and test how breaking these assumptions results in different outputs. We report a set of experiments comparing the outputs of an SEIR model and a set of agent-based models of varying levels of complexity, using as a case study a measles outbreak in a town in Ireland. We define and use a six level complexity hierarchy for agent-based models to create a set of progressively more complex variants of an agentized SEIR model for the spread of infectious disease. We then compare the results of the agent-based model at each level of complexity with results of the SEIR model to determine when the agentization breaks. Our analysis shows this occurs on the fourth step of complexity, when scheduled movements are added into the model. When agents networks and behaviours are complex the peak of the outbreak is shifted to the right and is lower than in the SEIR model suggesting that heterogeneous populations and mixing patterns lead to slower outbreaks compared homogeneous populations and mixing patterns.

Molecular Characterization and Genotype-Phenotype Correlation of G6PD Mutations in Five Ethnicities of Northern Vietnam.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme disorder and is caused by G6PD gene mutations. To date, more than 400 variants in the G6PD gene have been discovered, and about 160 identified variants are associated with a significant decrease in the G6PD enzyme activity. However, the molecular characterization and epidemiological study of G6PD deficiency are still limited in Vietnam. Therefore, we conducted this study to determine the G6PD variants among the Vietnamese populations and evaluate their correlation to G6PD enzyme activity. A total of 339 patients (302 males and 37 females) were enrolled in this study. The G6PD variants were identified by Sanger sequencing. Our results indicate that males are more severely deficient in G6PD than females. This enzyme activity in males (1.27 ± 1.06 IU/g·Hb) is significantly lower than in females (2.98 ± 1.57 IU/g·Hb) (p < 0.0001). The enzyme activity of the heterozygous-homozygous females and heterozygous females-hemizygous males was found to be significantly different (p < 0.05), which is interpreted due to random X-inactivation. For G6PD molecular characteristics, Viangchan (c.871G>A), Canton (c.1376G>T) and Kaiping (c.1388G>A) variants were the most dominant, accounting for 24.48%, 17.70%, and 22.42%, respectively, whereas the highest frequency of complex variants was observed in Viangchan/Silent with 20.35%. In terms of G6PD activity, the Union variant presented the lowest mean value (1.03 IU/g·Hb) compared to the other variants (p < 0.05). Computational analysis using Polyphen-2 tool investigated that all variants were relative to G6PD deficiency and separated the levels as benign and damaged. The result will establish effective methods to screen G6PD variants in Vietnam.

Intra-host genetic population diversity: Role in emergence and persistence of drug resistance among Mycobacterium tuberculosis complex minor variants.

Drug resistant tuberculosis (DR-TB) is an important public health issue in different parts of the world. Mycobacterium tuberculosis complex variants (MTBC vars) preferentially infect certain hosts, limiting their distribution to different ecosystems. However, MTBC vars can infect other hosts beyond their preferred target potentially contributing to persistence of drug resistance (DR) in other niches. Here, we performed a comprehensive intra-host genetic analysis for the identification of DR-related mutations among all MTBC minor vars whole genome sequences (8,095 strains) publicly available worldwide. High confidence drug-resistance mutations in katG (isoniazid), rpsL (streptomycin), pncA (pyrazinamide), rpoB (rifampicin) and gyrA (fluoroquinolones) genes were identified among intrahost minor sub-populations in 197 different strains (2.43%) belonging to vars africanum, bovis, caprae, microti, orygis and pinnipedii. In addition, a three-dimensional structure modeling analysis to assess the role of novel mutations was also performed. Our findings highlight the importance of detecting discrete intra-host populations carrying DR mutations.

First Identification of Rare Exonic and Deep Intronic Splice-Altering Variants in Patients With Beta-Sarcoglycanopathy.

BackgroundThe precise genetic diagnosis of a sarcoglycanopathy or dystrophinopathy is sometimes extremely challenging, as pathogenic non-coding variants and/or complex structural variants do exist in DMD or sarcoglycan genes. This study aimed to determine the genetic diagnosis of three patients from two unrelated families with a suspected sarcoglycanopathy or dystrophinopathy based on their clinical, radiological, and pathological features, for whom routine genomic detection approaches failed to yield a definite genetic diagnosis.MethodsMuscle-derived reverse transcription-polymerase chain reaction analysis and/or TA cloning of DMD, SGCA, SGCB, SGCD, and SGCG mRNA were performed to identify aberrant transcripts. Genomic Sanger sequencing around the aberrant transcripts was performed to detect possible splice-altering variants. Bioinformatic and segregation studies of the detected genomic variants were performed in both families.ResultsIn patients F1-II1 and F1-II2, we identified two novel pathogenic compound heterozygous variants in SGCB. One is a deep intronic splice-altering variant (DISV), c.243 + 1558C > T in intron 2 causing the activation of an 87-base pair (bp) pseudoexon, and the other one is a non-canonical splicing site variant, c.243 + 6T > A leading to the partial intron inclusion of 10-bp sequence. A novel DISV, c.243 + 1576C > G causing a 106-bp pseudoexon activation, and a nonsense variant in SGCB were identified in compound heterozygous state in patient F2-II1. Unexpectedly, the predicted nonsense variant, c.334C > T in exon 3, created a new donor splice site in exon 3 that was stronger than the natural one, resulting in a 97-bp deletion of exon 3 (r.333_429del).ConclusionThis is the first identification of rare exonic and DISVs in the SGCB gene.

Abstract 2935: Whole genome sequence of triple negative breast cancer (TNBC) tumors of African descent

Abstract In the US, BC incidence has recently converged among self-reported European Americans (EA) and African Americans (AA), however BC mortality remains approximately 40% higher among AA compared to EA and other SRR groups. The initial divergence in mortality curves between these groups occurred at the advent of hormone receptor (HR) targeted treatment options, unmasking the heterogeneity of BC diagnoses. Here, we present Whole Genomes Sequencing of 30 Triple Negative Breast Cancers of African women presenting to the clinics in New York. These were collected as part of the ICSBCS cohort to identify ancestry related mutational signatures including complex structural variants which could help better explain etiology of cancer in these population and that we may leveraged for treatment or prognostic purposes. Somatic mutational landscape included higher frequency of TP53 (73%), BRCA1 (40%), ATK1 (37%), CDH1(37%), and CTCF (33%). We confirmed African ancestry of patients through ADMIXTURE and observed (&amp;gt;70%) African ancestry. Comparison between our cohort and TCGA BRCA showed higher tumor mutational burden in our cohort which could be accounted for by greater sensitivity in our updated sequencing technology and analysis pipelines. Mutational signature analysis showed cosmic Signature 3 suggesting evidence of defective homologous recombination and signature 16 with unknown etiology. In addition, we also observed evidence deletions and structural variants such as rigma, pyrgo and typhonas. Ongoing efforts are investigating complex structural variants in our cohort compared to similar populations from other studies while waiting on additional samples. Citation Format: Samuel Terkper Ahuno, Yajas Shah, Rachel Martini, Andrea Sboner, Nicolas Robine, Olivier Elemento, Marcin Imielinski, Lisa Newman, Melissa Davis. Whole genome sequence of triple negative breast cancer (TNBC) tumors of African descent [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2935.

Abstract 3346: BBT-207, a novel, 4th generation, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) with broad-spectrum activity to both treatment-emergent and drug-naïve mutants for the treatment of NSCLC

Abstract Background: Activating mutations in EGFR, e.g., L858R (L) and Del19 (D), are observed in 14.1-32.3% of NSCLC worldwide and defines a druggable entity. Single agent EGFR TKI treatment was established in clinical trials. However, a treatment-emergent mutation of T790M (T) was identified as a major source of drug resistance. Irreversible 2nd and 3rd generation EGFR TKIs were shown effective. Mutant-specific inhibition by 3rd generation EGFR TKIs reduced adverse events such as skin toxicities. The emergence of a missense mutation at C797 (C), to which irreversible TKIs covalently bind, limited their efficacy. Patients harboring treatment-emergent C mutations are left with few therapeutic options. Despite anecdotal reports of efficacy from 1st generation TKIs, these complex molecular variants involving C797 are an increasing clinical concern. To prevent the emergence of drug-resistant subclones, a broad-spectrum EGFR TKI against both treatment-emergent and drug-naïve mutants is required. Such a 4th generation EGFR TKI must also enable monotherapy, allowing precise dose adjustment in case of toxicity. Method: Derived from BBT-176 which is under clinical evaluation (NCT04820023), we synthesized BBT-207 and other related molecules. Using Promega ADP-Glo™ kinase assay, we tested compounds against wild-type (WT) and mutated EGFR proteins. Ba/F3 cells harboring WT and mutated EGFR genes were created and cell viability was measured by CellTiter-Glo® and SpectraMax iD3®. In vivo anti-tumor activity was evaluated in Ba/F3 cell-derived xenograft models and an Osimertinib-resistant patient-derived xenograft model. Single dose pharmacokinetic (PK) study was performed in animals. Results: BBT-207 showed IC50 values &amp;lt; 10 nM against EGFR triple mutants (DTC and LTC) and comparable IC50 values against double mutants (DC and LC), while Osimertinib was ineffective in both groups. BBT-207 was also as active as Gefitinib to single mutants (D and L). Double mutants containing T790M (DT and LT) were inhibited effectively by BBT-207 and Osimertinib at similar concentrations, while not by Gefitinib. In in vivo models to which Osimertinib was inactive (LC and DTC), significant tumor growth inhibition was observed from BBT-207 alone, which was enhanced by the addition of Osimertinib. PK studies in mouse, rat, dog and monkey were consistent with high exposure and sustained drug concentrations above the IC50 values, with evidence of penetration into the central nervous system. Conclusion: BBT-207 is a reversible, mutant-specific, broad-spectrum TKI, active to clinically observed mutations of EGFR and is expected to be compatible with monotherapy of QD schedule in humans. BBT-207 is well-positioned to augment the treatment of EGFR mutated NSCLC, either for acquired drug resistance or in earlier line. Citation Format: Taiguang Jimmy Jin, Sang-Uk Kang, Chulwon Kim, Jehrod Brenneman, Mihee Song, Pavel Printsev, Bo-Bae Seo, Yong-Hee Lee, Sang-Yoon Lee. BBT-207, a novel, 4th generation, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) with broad-spectrum activity to both treatment-emergent and drug-naïve mutants for the treatment of NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3346.

Abstract 2128: Single-cell replication dynamics in genomically unstable cancers

Abstract Background: Single-cell whole genome sequencing (scWGS) methods such as direct library preparation (DLP) provide amplification-free capture of cells in all cycle phases and have enabled rich interrogation into the cell to cell genomic diversity of cancer genomes. Previous DLP-driven clonal evolution studies removed S-phase cells as replicated loci confounded phylogenetic inference from somatic copy number aberrations (CNAs), leaving single-cell replication timing (scRT) as an unstudied property. We thus developed a method that assigned S-phase cells to phylogenetic clones and used such assignments to infer scRT profiles in aneuploid cell lines and tumors. We applied this method to determine the relative proliferation rate between clones and obtain a single-cell picture of DNA replication fork progression for genomically unstable cancers. Methods: S-phase cells were assigned to phylogenetic clones based on copy number profile similarity and subsequently scRT profiles were inferred by normalizing out the effects of clonal, subclonal, and cell-specific CNAs before binarizing each genomic bin as replicated or unreplicated. scRT heterogeneity was quantified via a summary statistic representing the aggregate difference between expected and observed replication times (T-width). The scRT profiles were grouped into clone- and sample-level pseudobulks (aggregations of single cells) to determine genomic regions with differential replication timing. We then assessed the ability of S-phase enrichment to predict clonal expansions on time-series patient-derived xenografts of HER2+ and triple negative breast cancers that were either treatment-naive or exposed to cisplatin. In addition, we studied genetically engineered cell lines with inactivated DNA damage response genes (TP53, BRCA1, BRCA2) and primary high-grade serous ovarian cancer samples to quantify scRT heterogeneity at different states of genomic instability. Results: We analyzed 15 datasets containing over 25,000 cells with ~1/3 of cells in S-phase. Time-series datasets revealed that S-phase enriched clones were highly proliferative in the treatment-naive setting and were most sensitive to cisplatin. T-width values calculated from inferred scRT profiles increased as a function of genomic instability, with the degree of heterogeneity varied between genomic regions. Clone-level pseudobulk replication timing profiles revealed that whole-chromosome aneuploidies were more likely to have unperturbed replication timing compared to complex structural variants such as translocations, inversions, and micronuclei. Conclusions: Our time-series results show that S-phase cells enable the prediction of clone-specific proliferation and chemosensitivity using scWGS data from a single time point. The quantification of scRT heterogeneity across our collection of data implicates dysfunctional DNA replication as both driver and consequence of genomic instability. Citation Format: Adam C. Weiner, Andrew McPherson, Sohrab P. Shah. Single-cell replication dynamics in genomically unstable cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2128.

Abstract 6400: JBrowse 2: An extensible open-source platform for modern genome analysis

Abstract Introduction: Genome browsers are useful tools for research in human genetics, able to display and integrate biological information such as long and short read sequencing data, variant calls, and annotations. While traditional linear genome browsers have demonstrated broad utility for many applications, there is an increasing need for visualizing complex structural variants (SVs) beyond the linear view. To meet these challenges, we created JBrowse 2: an extensible open-source platform for visualizing and integrating genomic data. Results: JBrowse 2 is a flexible platform that provides the foundation for the development of applications and web dashboards that combine novel views and representations extending beyond linear displays in genomic reference coordinates. For example, a flagship JBrowse 2 application is the SV Inspector, which lets users open a list of structural variants in a data table and see the results in a whole-genome circular view. Clicking on a given variant in the table or circular view opens up a linear view that displays the read evidence supporting the SV, even across complex breakpoints like translocations. Dotplot and synteny views are built-in, enabling “long read vs reference” dotplot visualizations, or alignments of de-novo assembled contigs to the genome. These views are integrated with each other, so that (for example) a long read is just a few clicks away from the corresponding dotplot. In addition to including these new views, the JBrowse 2 platform is designed from the ground up to enable third-party plugins to add new views, data adapters, and track types, which has facilitated the creation of new plugins that address some very specific use cases. These include our MSA view plugin for viewing multiple sequence alignments, data adapters that download data from UCSC and CIVIC APIs, and the Quantseq plugin for viewing quantitative motif scores as a genome browser track. JBrowse 2 is available as both a web app or a local desktop app. We also offer re-usable components on NPM, and users of R can programmatically create an instance of JBrowse 2 with the JBrowseR package on CRAN. JBrowse 2 can also generate high quality SVG snapshots from inside the app, and we also created a CLI tool called jb2export to perform automated or bulk exports of JBrowse 2 visualizations. We anticipate that JBrowse 2 will better serve genome scientists with its structural variant visualization capabilities, and will provide the flexibility to adapt to new visualization and analysis challenges as they emerge in the coming years. Citation Format: Scott Cain, Robin Haw, Caroline Bridge, Junjun Zhang, Robert Buels, Colin Diesh, Garrett Stevens, Teresa Martinez, Peter K. Xie, Elliot Hershberg, Shihab Dider, Lincoln Stein, Ian Holmes. JBrowse 2: An extensible open-source platform for modern genome analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6400.

Surrogate-assisted evolutionary multi-objective optimisation applied to a pressure swing adsorption system

The complexity of chemical plant systems (CPS) makes optimising their design and operation challenging tasks. This complexity also results in analytical and numerical simulation models of these systems having high computational costs. Research demonstrates the benefits of using machine learning models as surrogates or substitutes for these computationally expensive simulation models during CPS optimisation. This paper presents the results of our study, extending recent research into optimising chemical plant design and operation. The study explored the original surrogate-assisted genetic algorithms (SA-GA) in more complex variants of the plant design and operation optimisation problem. The more complex plant design variants include additional parallel and feedback components. The study also proposes a novel multivariate extension, surrogate-assisted NSGA (SA-NSGA), to the original univariate SA-GA algorithm. The study evaluated the SA-NSGA extension on the popular pressure swing adsorption (PSA) system. This paper outlines our extensive experimentation, comparing various meta-heuristic optimisation techniques and numerous machine learning models as surrogates. The results in both more complex plant design variants and the PSA case show the suitability of genetic algorithms combined with surrogate models as an optimisation framework for CPS design and operation in single and multi-objective scenarios. The analysis further confirms that combining a genetic algorithm framework with machine learning surrogate models as a substitute for long-running simulation models yields significant computational efficiency improvements, 1.7–1.84 times speedup for the increased complexity examples and a 2.7 times speedup for the pressure swing adsorption system. The discussion successfully concludes that surrogate-assisted evolutionary algorithms can be scaled to increasingly complex CPS with parallel and feedback components.

Phloem wedges in Malpighiaceae: origin, structure, diversification, and systematic relevance

BackgroundPhloem wedges furrowing the wood are one of the most notorious, widespread types of cambial variants in Angiosperms. Many lianas in Malpighiaceae show these variations in the arrangement of the secondary tissues. Here we explore their ontogeny, structure, and evolution in Malpighiaceae, where phloem wedges appeared multiple times, showing how they have contributed to the anatomical diversification of the family. Using a broad sampling with 143 species from 50 genera, covering all major lineages in Malpighiaceae, we crossed data from ontogeny, stem anatomy, and phylogenetic comparative methods to determine ontogenetic trajectories, final anatomical architectures, and evolution within the most recent phylogeny for the family.ResultsPhloem wedges appeared exclusively in lianas and disappeared in shrub lineages nested within liana lineages. At the onset of development, the vascular cambium is regular, producing secondary tissues homogeneously across its girth, but soon, portions of the cambium in between the leaf insertions switch their activity producing less wood and more phloem, initially generating phloem arcs, which progress into phloem wedges. In the formation of these wedges, two ontogenetic trajectories were found, one that maintains the continuity of the cambium, and another where the cambium gets dissected. Phloem wedges frequently remain as the main cambial variant in several lineages, while in others there are additional steps toward more complex cambial variants, such as fissured stems, or included phloem wedges, the latter a novel type of interxylary phloem first described for the family.ConclusionsPhloem wedges evolved exclusively in lianas, with two different ontogenies explaining the 10 independent origins of phloem wedges in Malpighiaceae. The presence of phloem wedges has favored the evolution of even more complex cambial variants such as fissured stems and interxylary phloem.

Somatic whole genome dynamics of precancer in Barrett\u2019s esophagus reveals features associated with disease progression

While the genomes of normal tissues undergo dynamic changes over time, little is understood about the temporal-spatial dynamics of genomes in premalignant tissues that progress to cancer compared to those that remain cancer-free. Here we use whole genome sequencing to contrast genomic alterations in 427 longitudinal samples from 40 patients with stable Barrett’s esophagus compared to 40 Barrett’s patients who progressed to esophageal adenocarcinoma (ESAD). We show the same somatic mutational processes are active in Barrett’s tissue regardless of outcome, with high levels of mutation, ESAD gene and focal chromosomal alterations, and similar mutational signatures. The critical distinction between stable Barrett’s versus those who progress to cancer is acquisition and expansion of TP53−/− cell populations having complex structural variants and high-level amplifications, which are detectable up to six years prior to a cancer diagnosis. These findings reveal the timing of common somatic genome dynamics in stable Barrett’s esophagus and define key genomic features specific to progression to esophageal adenocarcinoma, both of which are critical for cancer prevention and early detection strategies.

Epidemiology, Impact, and Management Strategies of Anti-Glomerular Basement Membrane Disease.

Anti-glomerular basement membrane (anti-GBM) disease is a rare but serious autoimmune disease, which is characterized by the development of pathogenic antibodies to type IV collagen antigens in the glomerular and alveolar basement membranes. This results in rapidly progressive glomerulonephritis (GN), alveolar hemorrhage, or both. A variety of environmental factors can trigger the disease in genetically predisposed patients. Temporal associations with influenza, SARS-CoV-2 infection, and COVID-19 vaccination have been described although there is insufficient evidence to suggest causality. Anti-GBM disease accounts for approximately 20% of the cases of rapidly progressive GN cases secondary to crescentic GN, but is an uncommon cause of end-stage kidney disease. Early diagnosis by detection of circulating antibodies, increased awareness of atypical as well as complex clinical variants of the disease, and combined therapy with immunosuppression and plasma exchange has improved the prognosis of patients with this potentially fatal disease. Progress has been hampered by the rarity of anti-GBM disease, but new agents and therapeutic regimens are emerging.

Rapid Identification of Mycobacterium tuberculosis Complex Using Mass Spectrometry: A Proof of Concept.

Mycobacteria that form the Mycobacterium tuberculosis complex are responsible for deadly tuberculosis in animals and patients. Identification of these pathogens at the species level is of primary importance for treatment and source tracing and currently relies on DNA analysis, including whole genome sequencing (WGS), which requires a whole day. In this study, we report the unprecedented discrimination of M. tuberculosis complex species using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS), with WGS as the comparative reference standard. In the first step, optimized peptide extraction applied to 36 isolates otherwise identified in five of the 11 M. tuberculosis complex variants by WGS yielded 139 MALDI-TOF spectra, which were used to identify biomarkers of interest that facilitate differentiation between variants. In a second step, 70/80 (88%) other isolates were correctly classified by an algorithm based on specific peaks. This study is the first to report a MALDI-TOF-MS method for discriminating M. tuberculosis complex mycobacteria that is easily implemented in clinical microbiology laboratories.

Multi-omic cross-sectional cohort study of pre-malignant Barrett\u2019s esophagus reveals early structural variation and retrotransposon activity

Barrett’s esophagus is a pre-malignant lesion that can progress to esophageal adenocarcinoma. We perform a multi-omic analysis of pre-cancer samples from 146 patients with a range of outcomes, comprising 642 person years of follow-up. Whole genome sequencing reveals complex structural variants and LINE-1 retrotransposons, as well as known copy number changes, occurring even prior to dysplasia. The structural variant burden captures the most variance across the cohort and genomic profiles do not always match consensus clinical pathology dysplasia grades. Increasing structural variant burden is associated with: high levels of chromothripsis and breakage-fusion-bridge events; increased expression of genes related to cell cycle checkpoint, DNA repair and chromosomal instability; and epigenetic silencing of Wnt signalling and cell cycle genes. Timing analysis reveals molecular events triggering genomic instability with more clonal expansion in dysplastic samples. Overall genomic complexity occurs early in the Barrett’s natural history and may inform the potential for cancer beyond the clinically discernible phenotype.

EP261: The spectrum of CFTR and PRSS1 pathogenic variants in chronic pancreatitis

Chronic pancreatitis is defined as persistent inflammation of the pancreas that is ongoing for 6 months or longer. Chronic pancreatitis often develops by early adulthood in affected individuals and can lead to permanent tissue damage and loss of pancreatic function, including both exocrine and endocrine pancreatic insufficiency. Chronic pancreatitis increases the risk for diabetes and pancreatic cancer, more so with smoking and use of alcohol. Variants in several genes have been identified to be causative for, or induce susceptibility to, chronic pancreatitis. Genetic testing for hereditary chronic pancreatitis can aid in differential diagnosis of chronic pancreatitis from other disorders which also present with pancreatitis and may inform treatment strategy. This study reports the spectrum of pathogenic variants in individuals referred for chronic pancreatitis panel testing. This study included 747 individuals who underwent chronic pancreatitis panel testing at a single clinical laboratory (PreventionGenetics) between June 15, 2018 and September 24, 2021. Clinical information was obtained from provider-completed test requisition forms. Next generation sequencing (NGS) with copy number variant (CNV) detection was performed on patient-derived DNA using the Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA) and Sanger sequencing as necessary. Testing provided 100% coverage for the coding regions of the CASR, CFTR, CTRC, PRSS1, and SPINK1 genes plus ∼10 bases of non-coding DNA flanking each exon, as well as non-coding regions in which pathogenic variants had been identified by the clinical testing laboratory or reported elsewhere. The test also included sequencing of CFTR regions within intron 11 and intron 21, along with a complete analysis of the compound (TG)n(T)n sequence (5T/TG tract) in intron 8. Variations in the 5T/TG tract have been reported to cause congenital absence of the vas deferens, non-classic cystic fibrosis, and classic cystic fibrosis. At time of testing, the patients ranged in age from 1 week to 87 years, with a median age of 32 years. Of the patients tested, 68% were negative for variants in genes included on the panel that are likely to cause disease while 27% were found to be indeterminate, where variants were identified but the results were not diagnostic. Overall, 5% of patients were positive, with a clear molecular diagnosis identified. Pathogenic and likely pathogenic variants in CFTR and PRSS1 accounted for 90% of positive cases overall, with 42% of positive cases being attributed to CFTR-related pancreatitis (1 homozygous or 2 heterozygous causative variants identified) and 33% of positive cases attributed to PRSS1-related pancreatitis (1 heterozygous causative variant). An additional 15% of positive cases were found to have pathogenic or likely pathogenic variants in both CFTR and PRSS1 (1 heterozygous causative variant in both CFTR and PRSS1). The CFTR c.1210-34TG[12]T[5] variant (more commonly referred to as 5T/12TG) contributed to ∼21% of CFTR-related pancreatitis. Importantly, ∼72% of pathogenic and likely pathogenic variants identified in this patient cohort are approved as therapy responsive variants for FDA approved CFTR modulators. In approximately 40% of patients with PRSS1-related pancreatitis, we detected a ∼600kb copy number gain encompassing the whole PRSS1 gene, compared to an estimated 6% of patients in the literature. These data indicate that variants in CFTR are a common cause of chronic pancreatitis and highlights the clinic utility of including complete analysis of the compound (TG)n(T)n sequence (5T/TG tract) in intron 8 of CFTR in this patient cohort. Additionally, the high frequency of treatment responsive variants suggests that a molecular diagnosis in patients with chronic pancreatitis may alter treatment strategy, particularly in the context of CFTR-related pancreatitis. Furthermore, these data suggest that whole gene deletions/duplications may be more frequent that previously thought in this patient cohort and that complex copy number variants in the PRSS1 gene may escape detection by other NGS testing strategies.

Genome sequencing demonstrates high diagnostic yield in children with undiagnosed global developmental delay/intellectual disability: A prospective study.

Genome sequencing (GS) has been used in the diagnosis of global developmental delay (GDD)/intellectual disability (ID). However, the performance of GS in patients with inconclusive results from chromosomal microarray analysis (CMA) and exome sequencing (ES) is unknown. We recruited 100 pediatric GDD/ID patients from multiple sites in China from February 2018 to August 2020 for GS. Patients have received at least one genomic diagnostic test before enrollment. Reanalysis of their CMA/ES data was performed. The yield of GS was calculated and explanations for missed diagnoses by CMA/ES were investigated. Clinical utility was assessed by interviewing the parents by phone. The overall diagnostic yield of GS was 21%. Seven cases could have been solved with reanalysis of ES data. Thirteen families were missed by previous CMA/ES due to improper methodology. Two remained unsolved after ES reanalysis due to complex variants missed by ES, and a CNV in untranslated regions. Follow-up of the diagnosed families revealed that nine families experienced changes in clinical management, including identification of targeted treatments, cessation of unnecessary treatment, and considerations for family planning. GS demonstrated high diagnostic yield and clinical utility in this undiagnosed GDD/ID cohort, detecting a wide range of variant types of different sizes in a single workflow.

Third-generation sequencing: A novel tool detects complex variants in the α-thalassemia gene

ObjectiveThalassemia is a monogenic disorder with a high carrier rate in the southern region of China. Most laboratories currently follow the protocol of testing hematologic indicators in individuals with positive hematologic indicators and then using the hot-spot mutation test kit. A novel thalassemia gene test is performed if there is a mismatch between the hematology and hot-spot mutation test results. However, due to the large population in southern China, some individuals carry complex α-globin gene cluster (CAGC) variants in NG_000006.1, which are difficult to detect using conventional thalassemia genetic analysis protocols, leading to missed or false genetic test results for individuals carrying these complex α-globin gene cluster variants. When an individual carries a complex α-thalassemia gene variant, and an individual carries a β- thalassemia gene variant, there may be clinical symptoms that might complicate clinical consultation and prenatal diagnosis if not accurately detected. Third-generation sequencing (TGS) enables long-read single-molecule sequencing with high detection accuracy, and long-length DNA chain reads in high-fidelity reads mode. TGS can be used to analyze high homology and rich GC DNA sequences. ResultsFour samples that showed abnormalities in the thalassemia genetic test were studied using TGS, revealing that they carried genotypes with complex α-globin gene cluster variants, one of which was a complex variant αα anti3.7 α anti3.7 α 17.2. ConclusionsTGS detects complex α-globin gene cluster variants. This study may provide a reference protocol for the use of TGS for the detection of complex α-globin gene cluster variants. TGS can reveal individuals with complex α-thalassemia genotypes in the population and improve the accuracy of genetic counseling and prenatal diagnosis.