Multi-site Technical Performance and Concordance of Optical Genome Mapping: Constitutional Postnatal Study for SV, CNV, and Repeat Array Analysis

Background The standard of care (SOC) cytogenetic testing methods, such as chromosomal microarray (CMA) and Fragile-X syndrome (FXS) testing, have been employed for the detection of copy number variations (CNVs), and tandem repeat expansions/contractions that contribute towards a sizable portion of genetic abnormalities in constitutional disorders. However, CMA is unable to detect balanced structural variations (SVs) or determine the precise location or orientation of copy number gains. Karyotyping, albeit with lower resolution, has been used for the detection of balanced SVs. Other molecular methods such as PCR and Southern blotting, either simultaneously or in a tiered fashion have been used for FXS testing, adding time, cost, and complexity to reach an accurate diagnosis in affected individuals. Optical genome mapping (OGM), innovative technology in the cytogenomics arena enables a direct, high-resolution view of ultra-long DNA molecules (>150 kbp), which are then assembled de novo to detect germline SVs ranging from 500 bp insertions and deletions to complex chromosomal rearrangements. The purpose of this study was to evaluate the performance of OGM in comparison to the current SOC methods and assess the intra- and inter-site reproducibility of the OGM technique. We report the largest retrospective dataset to date on OGM performed at five laboratories (multi-site) to assess the robustness, QC performance, and analytical validation (multi-operator, and multi-instrument) in detecting SVs and CNVs associated with constitutional disorders compared to SOC technologies. Methods This multi-center IRB-approved, double-blinded, study includes a total of 331 independent flow cells run (including replicates), representing 202 unique retrospective samples, including but not limited to pediatric-onset neurodevelopmental disorders. This study included affected individuals with either a known genetic abnormality or no known genetic diagnosis. Control samples (n=42) were also included. Briefly, OGM was performed on either peripheral blood samples or cell lines using the Saphyr system. The OGM assay results were compared to the human reference genome (GRCh38) to detect different types of SVs (CNV, insertions, inversions, translocations). A unique coverage-based CNV calling algorithm was also used to complement the SV calls. Analysis of heterozygous SVs was performed to assess the absence of heterozygosity (AOH) regions in the genome. For specific clinical indications of FSHD1 and FXS, the EnFocus FXS and FSHD1 tools were used to generate the region-specific reports. OGM data was analyzed and visualized using Access software (version 1.7), where the SVs were filtered using an OGM specific internal control database. The samples were analyzed by laboratory analysts at each site in a blinded fashion using ACMG guidelines for SV interpretation and further reviewed by expert geneticists to assess concordance with SOC testing results. Results Of the first 331 samples run between five sites, 99.1% of sample runs were completed successfully. Of the 331 datasets, 219 were assessed for concordance by the time of this publication; these were samples that harbored known variants, of which 214/219 were detected by OGM resulting in a concordance of 97.7% compared to SOC testing. 47 samples were also run in intra- and inter-site replicate and showed 100% concordance for pathogenic CNVs and SVs and 100% concordance for pathogenic FMR1 repeat expansions. Conclusion The results from this study demonstrate the potential of OGM as an alternative to existing SOC methods in detecting SVs of clinical significance in constitutional postnatal genetic disorders. The outstanding technical performance of OGM across multiple sites demonstrates the robustness and reproducibility of the OGM technique as a rapid cytogenomics testing tool. Notably, OGM detected all classes of SVs in a single assay, which allows for a faster result in cases with diverse and heterogeneous clinical presentations. OGM demonstrated 100% concordance for pathogenic variants previously identified including FMR1 repeat expansions (full mutation range), pathogenic D4Z4 repeat contractions (FSHD1 cases), aneuploidies, interstitial deletions, interstitial duplications, intragenic deletions, balanced translocations, and inversions. Based on our large dataset and high technical performance we recommend OGM as an alternative to the existing SOC tests for the rapid detection and diagnosis of postnatal constitutional disorders.

Optical Genome Mapping in Routine Human Genetic Diagnostics—Its Advantages and Limitations

In recent years, optical genome mapping (OGM) has developed into a highly promising method of detecting large-scale structural variants in human genomes. It is capable of detecting structural variants considered difficult to detect by other current methods. Hence, it promises to be feasible as a first-line diagnostic tool, permitting insight into a new realm of previously unknown variants. However, due to its novelty, little experience with OGM is available to infer best practices for its application or to clarify which features cannot be detected. In this study, we used the Saphyr system (Bionano Genomics, San Diego, CA, USA), to explore its capabilities in human genetic diagnostics. To this end, we tested 14 DNA samples to confirm a total of 14 different structural or numerical chromosomal variants originally detected by other means, namely, deletions, duplications, inversions, trisomies, and a translocation. Overall, 12 variants could be confirmed; one deletion and one inversion could not. The prerequisites for detection of similar variants were explored by reviewing the OGM data of 54 samples analyzed in our laboratory. Limitations, some owing to the novelty of the method and some inherent to it, were described. Finally, we tested the successful application of OGM in routine diagnostics and described some of the challenges that merit consideration when utilizing OGM as a diagnostic tool.

Trypsin Genes Are Regulated through the miRNA Bantam and Associated with Drug Sensitivity in the Sea Louse Caligus rogercresseyi

The role of trypsin genes in pharmacological sensitivity has been described in numerous arthropod species, including the sea louse Caligus rogercresseyi. This ectoparasite species is mainly controlled by xenobiotic drugs in Atlantic salmon farming. However, the post-transcriptional regulation of trypsin genes and the molecular components involved in drug response remain unclear. In particular, the miRNA bantam family has previously been associated with drug response in arthropods and is also found in C. rogercresseyi, showing a high diversity of isomiRs. This study aimed to uncover molecular interactions among trypsin genes and bantam miRNAs in the sea louse C. rogercresseyi in response to delousing drugs. Herein, putative mRNA/miRNA sequences were identified and localized in the C. rogercresseyi genome through genome mapping and blast analyses. Expression analyses were obtained from the mRNA transcriptome and small-RNA libraries from groups with differential sensitivity to three drugs used as anti-sea lice agents: azamethiphos, deltamethrin, and cypermethrin. The validation was conducted by qPCR analyses and luciferase assay of selected bantam and trypsin genes identified from in silico transcript prediction. A total of 60 trypsin genes were identified in the C. rogercresseyi genome, and 39 bantam miRNAs were differentially expressed in response to drug exposure. Notably, expression analyses and correlation among values obtained from trypsin and bantam revealed an opposite trend and potential binding sites with significant ΔG values. The luciferase assay showed a reduction of around 50% in the expression levels of the trypsin 2-like gene, which could imply that this gene is a potential target for bantam. The role of trypsin genes and bantam miRNAs in the pharmacological sensitivity of sea lice and the use of miRNAs as potential markers in these parasites are discussed in this study.

The whole-genome sequencing in predicting Mycobacterium tuberculosis drug susceptibility and resistance in Papua, Indonesia

Background Tuberculosis is one of the deadliest disease caused by Mycobacterium tuberculosis. Its treatment still becomes a burden for many countries including Indonesia. Drug resistance is one of the problems in TB treatment. However, a development in the molecular field through Whole-genome sequencing (WGS) can be used as a solution in detecting mutations associated with TB- drugs. This investigation intended to implement this data for supporting the scientific community in deeply understanding any TB epidemiology and evolution in Papua along with detecting any mutations in genes associated with TB-Drugs. Result A whole-genome sequencing was performed on the random samples from TB Referral Laboratory in Papua utilizing MiSeq 600 cycle Reagent Kit (V3). Furthermore, TBProfiler was used for genome analysis, RAST Server was employed for annotation, while Gview server was applied for BLAST genome mapping and a Microscope server was implemented for Regions of Genomic Plasticity (RGP). The largest genome of M. tuberculosis obtained was at the size of 4,396,040 bp with subsystems number at 309 and the number of coding sequences at 4326. One sample (TB751) contained one RGP. The drug resistance analysis revealed that several mutations associated with TB-drug resistance existed. In details, mutations of rpoB gene which were identified as S450L, D435Y, H445Y, L430P, and Q432K had caused the reduced effectiveness of rifampicin; while the mutases in katG (S315T), kasA (312S), inhA (I21V), and Rv1482c-fabG1 (C-15 T) genes had contributed to the resistance in isoniazid. In streptomycin, the resistance was triggered by the mutations in rpsL (K43R) and rrs (A514C, A514T) genes, and, in Amikacin, its resistance was led by mutations in rrs (A514C) gene. Additionally, in Ethambutol and Pyrazinamide, their reduced effectiveness was provoked by embB gene mutases (M306L, M306V, D1024N) and pncA (W119R). Conclusions The results from whole-genome sequencing of TB clinical sample in Papua, Indonesia could contribute to the surveillance of TB-drug resistance. In the drug resistance profile, there were 15 Multi Drugs Resistance (MDR) samples. However, Extensively Drug-resistant (XDR) samples have not been found, but samples were resistant to only Amikacin, a second-line drug.

Marfan Syndrome Caused by Disruption of the FBN1 Gene due to A Reciprocal Chromosome Translocation

Marfan syndrome (MFS) is a hereditary connective tissue disease caused by heterozygous mutations in the fibrillin-1 gene (FBN1) located on chromosome 15q21.1. A complex chromosomal rearrangement leading to MFS has only been reported in one case so far. We report on a mother and daughter with marfanoid habitus and no pathogenic variant in the FBN1 gene after next generation sequencing (NGS) analysis, both showing a cytogenetically reciprocal balanced translocation between chromosomes 2 and 15. By means of fluorescence in situ hybridization of Bacterial artificial chromosome (BAC) clones from the breakpoint area on chromosome 15 the breakpoint was narrowed down to a region of approximately 110 kb in FBN1. With the help of optical genome mapping (OGM), the translocation breakpoints were further refined on chromosomes 2 and 15. Sequencing of the regions affected by the translocation identified the breakpoint of chromosome 2 as well as the breakpoint of chromosome 15 in the FBN1 gene leading to its disruption. To our knowledge, this is the first report of patients with typical clinical features of MFS showing a cytogenetically reciprocal translocation involving the FBN1 gene. Our case highlights the importance of structural genome variants as an underlying cause of monogenic diseases and the useful clinical application of OGM in the elucidation of structural variants.

High-Throughput Characterization of Cytogenomic Heterogeneity of MDS Using High-Resolution Optical Genome Mapping

Introduction Introduction of next-generation sequencing has defined the somatic mutational landscape in MDS. Comprehensive high-throughput structural variant profiling (SVP) is as important as mutation profiling in characterizing MDS clonal architecture since these large genomic aberrations have already shown to be critical for diagnosis and risk-stratification of MDS. A subset (MECOM, KMT2A rearrangements) are therapeutic targets in clinical trials. At this time, technical advances in SVP for copy number alterations (CNAs) and fusions have not been congruent with mutation profiling due to the inability of short-read (150bp) NGS to detect SVs. Currently available long-read (10-20Kbp) and whole genome sequencing cannot detect all SVs due to the presence of repeat sequences. Hence, conventional karyotyping (CK) remains the gold standard. Optical genome mapping (OGM) is a novel single-platform technique that measures ultra-long-range sequence patterns (>300Kbp), thereby unaffected by repeat sequences, enabling unbiased evaluation of all types of SVs at a high resolution. Here, we performed comprehensive SVP and mutation profiling in a large well-characterized cohort of MDS. Methods We selected samples with available fresh/frozen BM cells from consecutive treatment-naïve MDS pts who also underwent standard-of-care tests (CK, FISH, targeted 81-gene NGS for mutations). For OGM, ultra-high-molecular-weight-DNA was extracted, followed by labeling, linearization and imaging of DNA (Saphyr, Bionano) [median coverage:>300X]. The results were analyzed using de novo (>500 bp), rare variant (>5000 bp) and copy number (>500,000 bp) pipelines. The data was compared against 200 healthy controls to exclude common germline SVs. Clinical significance of the SVs was determined based on the location/overlap with the coding region of myeloid malignancy associated genes. The detection sensitivity was 10%. Results There were 76 treatment naïve MDS patients. Baseline characteristics, comprehensive cytogenetic scoring system (CCSS) and R-IPPS risk categories and somatic mutations are in Fig 1. OGM identified all clonal abnormalities detected by CK [CNAs, inversions, inter/intra-chromosomal translocations, dicentric, complex derivative chromosomes]. Precise mapping of SVs by OGM at gene-level allowed determining the status of clinically informative biomarkers such as TET2, MECOM, TP53 and KMT2A, without the need for confirmatory assays. Detailed gene-level characterization of different SVs included KMT2A-ELL [t(11;19)] in MDS with WT1 mut, t(9;11) with SYTL2 fusion (and not KMT2A), der(1;7) leading to del(7q) in MDS with GATA2 mut/IDH2 mutand t(1;3)(p36;q21) rearrangements with potential PRDM16 disruption in SF3B1 mut/RUNX1 mutMDS, among others. Using OGM, we mapped the sequence patterns in both samples with IM with high level of confidence. Additionally, OGM identified 23 cryptic, clinically significant SVs in 14 (18%) of 76 pts. These included deletions of TET2, KMT2A, and del(5q), KMT2A amplification in MDS with FLT3-ITD/DNMT3A mut/RAS mut, NUP98-PRRX2, MECOM rearrangement in TET mut mutated NK-MDS. In addition, there were SVs of uncertain significance: duplications of chr1 (PDE41P), deletions of chr21 (involving RUNX1), chr2 (DNMT3A, ASXL2), chr12 (ETV6) and chr22 (EP300) and der(16)t(12;16)(q21.1;q12.1). These cryptic SVs were noted across all R-IPSS risk categories (highest yield in very-low and low R-IPSS) and across all cytogenetic risk-groups (very-good to very-poor). In complex karyotype setting, OGM could resolve the markers and additional genetic material, and in most cases, showed a much higher the degree of complexity within the genome than was apparent by CK. Four pts showed SV patterns typical of chromothripsis/chromoplexy. The median number of mutations per pt was 1 (0-6). When compared to mutation subsets, cryptic SVs were only identified in pts with ≤3 mutations. Majority represented either MDS with TP53 mut (6, 29%) or SF3B1 mut/TET mut (deletions of TET2, KMT2A, NOTCH1 and EP300 genes). Conclusions Unbiased, high-throughput whole genome SVP revealed cryptic, clinically significant SVs in ~18% of MDS pts. OGM is a single-platform cytogenomic tool that can facilitate SVP at a gene-level resolution. This study provides strong support for further validation in expanded cohorts to guide clinical implementation and integration of SVP for routine work-up. Figure 1 Figure 1. Disclosures Wei: Daiichi Sanko: Research Funding. Kantarjian: Ipsen Pharmaceuticals: Honoraria; Amgen: Honoraria, Research Funding; Astellas Health: Honoraria; Astra Zeneca: Honoraria; AbbVie: Honoraria, Research Funding; KAHR Medical Ltd: Honoraria; NOVA Research: Honoraria; Ascentage: Research Funding; Aptitude Health: Honoraria; Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Jazz: Research Funding; Immunogen: Research Funding; Daiichi-Sankyo: Research Funding; BMS: Research Funding; Precision Biosciences: Honoraria; Taiho Pharmaceutical Canada: Honoraria.

Enhancer Promoter Interactome and Mendelian Randomization Identify Network of Druggable Vascular Genes in Coronary Artery Disease

Coronary artery disease (CAD) is a multifactorial disorder, which is partly heritable. Herein, we implemented a mapping of CAD-associated candidate genes by using genome-wide enhancer-promoter conformation (H3K27ac-HiChIP) and expression quantitative trait loci (eQTL). Enhancer-promoter anchor loops from human coronary artery smooth muscle cells (HCASMC) explained 22% of the heritability for CAD. 3D enhancer-promoter genome mapping of CAD-genes in HCASMC was enriched in vascular eQTL genes. By using colocalization and Mendelian randomization analyses, we identified 58 causal candidate vascular genes including some druggable targets (MAP3K11, CAMK1D, PDGFD, IPO9 and CETP). A network analysis of causal candidate genes was enriched in TGF beta and MAPK pathways. The pharmacologic inhibition of causal candidate gene MAP3K11 in vascular SMC reduced the expression of athero-relevant genes and lowered cell migration, a cardinal process in CAD. Genes connected to enhancers are enriched in vascular eQTL and druggable genes causally associated with CAD.