Specific Copy Number Variations Research Articles

Clinical and neurobehavioral phenotype in children with autism and intragenic copy number duplications in CNTN4: Case series report

Copy Number Variation (CNV) of contactin genes (CNTNs) - CNTN3, CNTN4, CNTN5, and CNTN6 - have been associated with various neurodevelopmental disorders, including Autism Spectrum Disorder (ASD). However, the literature on the associated phenotypes to specific copy number variants of these genes is still scarce. Therefore, limiting clinicians' and researchers' understanding of the impact of these CNVs makes genetic counselling regarding recurrence risks more challenging. In this study, we report on five patients with rare CNVs involving the CNTN4 gene and the associated clinical and neurobehavioral phenotype. Overall, the patients exhibited stereotypic motor symptoms, including finger and hand mannerisms (4/5), and repetitive use of objects (4/5), as well as sensory symptoms, including unusual sensory interests or hypersensitivity (4/5). One child of the cohort had epilepsy, and (4/5) had Intellectual Disability. All cases fulfilled DSM-5 criteria for diagnosis of Autism Spectrum Disorder (ASD). However, larger cohorts are needed for unbiased characterization of the phenotypic features associated with the genetic variations in CNTN4.

Copy number variant risk loci for schizophrenia converge on the BDNF pathway

Objectives Schizophrenia genetics is intricate, with common and rare variants’ contributions not fully understood. Certain copy number variations (CNVs) elevate risk, pivotal for understanding mental disorder models. Despite CNVs’ genome-wide distribution and variable gene and protein effects, we must explore beyond affected genes to interaction partners and molecular pathways. Methods In this study, we developed machine-readable interactive pathways to enable analysis of functional effects of genes within CNV loci and identify ten common pathways across CNVs with high schizophrenia risk using the WikiPathways database, schizophrenia risk gene collections from GWAS studies, and a gene-disease association database. Results For CNVs that are pathogenic for schizophrenia, we found overlapping pathways, including BDNF signalling, cytoskeleton, and inflammation. Common schizophrenia risk genes identified by different studies are found in all CNV pathways, but not enriched. Conclusions Our findings suggest that specific pathways - BDNF signalling - are critical contributors to schizophrenia risk conferred by rare CNVs. Our approach highlights the importance of not only investigating deleted or duplicated genes within pathogenic CNV loci, but also study their direct interaction partners, which may explain pleiotropic effects of CNVs on schizophrenia risk and offer a broader field for interventions.

A genomic biomarker for the rapid identification of the rob(1;29) translocation in beef cattle breeds.

Robertsonian translocations, specifically rob(1;29) translocation, have reportedly been the most prevalent chromosomal abnormalities in cattle, affecting various breeds and leading to a decrease in fertility and reproductive value. Currently, the identification of rob(1;29) carriers relies on cytogenetic analysis that has limitations in terms of accessibility, cost, and sample requirements. To address these limitations, a novel genomic biomarker was developed in this study for the rapid and precise identification of rob(1;29) carriers. Using q-PCR, a specific copy number variation associated with translocation was targeted, which effectively distinguished between wild-type, homozygous and heterozygous carriers. Crucially, the biomarker can be applied to DNA extracted from various biological matrices, such as semen, embryos, oocytes, milk, saliva, coat, and muscle, and it is compatible with fresh, refrigerated, or frozen samples. Furthermore, this approach offers significant reductions in cost compared to those associated with traditional cytogenetic analysis and provides results within a short turnaround time. The successful development of this genomic biomarker has considerable potential for widespread adoption in screening programs. It facilitates timely identification and management of rob(1;29) carriers while mitigating economic losses and preserving genetic integrity in bovine populations.

Genetic outcomes in children with developmental language disorder: a systematic review.

Developmental language disorder (DLD) is a common childhood condition negatively influencing communication and psychosocial development. An increasing number of pathogenic variants or chromosomal anomalies possibly related to DLD have been identified. To provide a base for accurate clinical genetic diagnostic work-up for DLD patients, understanding the specific genetic background is crucial. This study aims to give a systematic literature overview of pathogenic variants or chromosomal anomalies causative for DLD in children. We conducted a systematic search in PubMed and Embase on available literature related to the genetic background of diagnosed DLD in children. Included papers were critically appraised before data extraction. An additional search in OMIM was performed to see if the described DLD genes are associated with a broader clinical spectrum. The search resulted in 15,842 papers. After assessing eligibility, 47 studies remained, of which 25 studies related to sex chromosome aneuploidies and 15 papers concerned other chromosomal anomalies (SCAs) and/or Copy Number Variants (CNVs), including del15q13.1-13.3 and del16p11.2. The remaining 7 studies displayed a variety of gene variants. 45 (candidate) genes related to language development, including FOXP2, GRIN2A, ERC1, and ATP2C2. After an additional search in the OMIM database, 22 of these genes were associated with a genetic disorder with a broader clinical spectrum, including intellectual disability, epilepsy, and/or autism. Our study illustrates that DLD can be related to SCAs and specific CNV's. The reported (candidate) genes (n = 45) in the latter category reflect the genetic heterogeneity and support DLD without any comorbidities and syndromic language disorder have an overlapping genetic etiology.

GATK-gCNV enables the discovery of rare copy number variants from exome sequencing data.

Copy number variants (CNVs) are major contributors to genetic diversity and disease. While standardized methods, such as the genome analysis toolkit (GATK), exist for detecting short variants, technical challenges have confounded uniform large-scale CNV analyses from whole-exome sequencing (WES) data. Given the profound impact of rare and de novo coding CNVs on genome organization and human disease, we developed GATK-gCNV, a flexible algorithm to discover rare CNVs from sequencing read-depth information, complete with open-source distribution via GATK. We benchmarked GATK-gCNV in 7,962 exomes from individuals in quartet families with matched genome sequencing and microarray data, finding up to 95% recall of rare coding CNVs at a resolution of more than two exons. We used GATK-gCNV to generate a reference catalog of rare coding CNVs in WES data from 197,306 individuals in the UK Biobank, and observed strong correlations between per-gene CNV rates and measures of mutational constraint, as well as rare CNV associations with multiple traits. In summary, GATK-gCNV is a tunable approach for sensitive and specific CNV discovery in WES data, with broad applications.

Noninvasive early differential diagnosis and progression monitoring of ovarian cancer using the copy number alterations of plasma cell-free DNA

Ovarian cancer (OV) is the most lethal gynecological malignancy and requires improved early detection methods and more effective intervention to achieve a better prognosis. The lack of sensitive and noninvasive biomarkers with clinical utility remains a challenge. Here, we conducted a genome-wide copy number variation (CNV) profiling analysis using low-coverage whole genome sequencing (LC-WGS) of plasma cfDNA in patients with nonmalignant and malignant ovarian tumors and identified 10 malignancy-specific and 12 late-stage-specific CNV markers from plasma cfDNA LC-WGS data. Concordance analysis indicated a significant correlation of identified CNV markers between CNV profiles of plasma cfDNA and tissue DNA (Pearson's r = 0.64, P = 0.006 for the TCGA cohort and r = 0.51, P = 0.04 for the Dariush cohort). By leveraging these specific CNV markers and machine learning algorithms, we developed robust predictive models showing excellent performance in distinguishing between malignant and nonmalignant ovarian tumors with F1-scores of 0.90 and ranging from 0.75 to 0.99, and prediction accuracy of 0.89 and ranging from 0.66 to 0.98, respectively, as well as between early- and late-stage ovarian tumors with F1-scores of 0.84 and ranging from 0.61 to 1.00, and prediction accuracy of 0.82 and ranging from 0.63 to 0.96 in our institute cohort and other external validation cohorts. Furthermore, we also discovered and validated certain CNV features associated with survival outcomes and platinum-based chemotherapy response in multicenter cohorts. In conclusion, our study demonstrated the clinical utility of CNV profiling in plasma cfDNA using LC-WGS as a cost-effective and accessible liquid biopsy for OV.

Microdeletions on chromosomes 2 and 20 are associated with equine early pregnancy loss

Copy number variants (CNVs) are chromosomal microdeletions and microduplications greater than 1000 bp and can confer disease risk, if they impact gene dosage. Specific pathogenic CNVs have been described as associated with miscarriage and stillbirth in women but are unexplored in equine pregnancy. This study aimed to identify and characterize pathogenic CNVs associated with equine early embryonic loss (EEL) and early fetal losses collectively referred to as early pregnancy loss (EPL). Conceptuses were submitted to the laboratory by the attending veterinarians following clinical confirmation of pregnancy failure between gestation days 15 and 65. DNA was isolated from the allantochorion (ALC) (n=52) and fetus (n=13) of EPL pregnancies. As a control, DNA was isolated from allantochorion and fetuses of manually terminated clinically normal pregnancies (CNP n=20), and peripheral blood mononuclear cells (PBMCs) were isolated from adult horses (n=371). DNA was hybridized to 96 sample Axiom Equine 670K Arrays. DNAcopy was used to identify autosomal CNVs >1000 bp in length after excluding samples with <95% call rate, MAPD <0.35, log2 intensity between -0.2 and +0.2, and a significance of p>0.05. Two microdeletions were found in at least two EPL conceptuses that were absent in all control samples. A review of the literature confirmed both microdeletions were unreported to date (n=3040 individuals). In this study, PCR confirmed the deletions(del2:91608493-91610830 and del20:30652320-30656312). Bioinformatic analysis of del2:91608493-91610830 using ENSEMBL revealed that the 2.3 kb deletion region contained part of exon1 and intron1 of the mastermind like transcriptional coactivator 3 (MAML3) gene, a co-activator of Notch signaling known to play a key role in early development. This deletion in MAML3 was identified in the ALC of two EPLs lost at 42 and 64 days of gestation. Del20:30652320-30656312 was identified in seven EPLs, including both the fetal and ALC genome of two EPLs and only the fetal genome of an additional five EPLs. Bioinformatic analysis revealed the deletion in ECA20 encompassed a 4 kb region that included exon1 and partial intron1 of both transcripts for tubulin beta class 1 (TUBB), which is essential to neurogenesis. Five of the seven EPLs failed at day 35-40, and three of the fetuses had abnormalities of the central nervous system, including one failure of neural tube closure. Whilst causality can't be confirmed from these experiments, the rarity of microdeletions in viable horses, and the biological plausibility of the deleted gene impacting early development is supportive of a role for both microdeletions in lethality of the conceptus in the first two months of pregnancy. Further experiments are required to determine if the deletions are acquired through the germline.

Maximizing the Detection of Copy Number Variants in the Highly Homologous Deafness-Infertility Syndrome Locus in Standard-of-care Testing

Genome-wide chromosomal microarrays (CMA) can identify pathogenic copy number variants (CNVs) associated with patients’ clinical indications but also have the potential to detect incidental findings and carrier status for recessive disorders. Patients can benefit from maximizing the detection and reporting of potentially clinically significant CNVs from this standard-of-care genome-wide test in accordance with professional guidelines (1); however, accurate detection of specific CNVs can be limited by the technology used and the complexity of the genomic regions under consideration. One example of this complexity is in the detection of disease-causing variants in the STRC gene associated with DFNB16, the second most frequent cause of autosomal recessive nonsyndromic hearing loss (NSHL), which is a common sensory disorder with high genetic heterogeneity. DFNB16 is estimated to account for up to 16% of individuals with NSHL (2). Most pathogenic STRC alterations are deletions encompassing all or part of the gene. These deletions often extend into the neighboring gene CATSPER2, which is associated with male fertility. Homozygous, contiguous deletions involving both STRC and CATSPER2 cause deafness-infertility syndrome (DIS), a contiguous gene deletion syndrome characterized by nonsyndromic, congenital to post-lingual onset of mild to moderate bilateral sensorineural hearing loss in both males and females and exclusive male infertility. Several studies have reported that 1%–2% of individuals are asymptomatic heterozygous carriers for a deletion of STRC with or without CATSPER2 (2, 3). The high frequency of STRC deletions has also led to reports of “pseudodominant inheritance,” in which a parent and child both have hearing loss as a result of homozygous STRC deletions (3, 4).

Abstract 260: Long RNA and DNA sequencing reveals HEK293 diversity between commercial sources

Abstract HEK293 cells are used extensively in cancer research, with more than 4800 articles between 2017 and 2022 (PubMed search “cancer[MeSH Major Topic] and hek293”). Studies involving transfection to overexpress, knock down, or modify proteins in these cells are common. While HEK293 cells are relatively easy to grow and maintain, early use of high throughput sequencing Yao-Cheng et. al. identified genomic variability between different sources of cell lines and revealed unexpected details about their developmental lineage (likely adrenal, not kidney)[1]. Genomic or transcriptomic reorganization may complicate interpretation of results from targeted intervention experiments. Here, we evaluate HEK293 cells from various commercial sources using long read sequencing technology. Oxford Nanopore direct cDNA profiling was used to compare isoform usage and examine gene fusions. Long DNA libraries were prepared using a high molecular weight isolation method, sequenced on Oxford Nanopore, and assembled to produce long contiguous fragments. Genome assemblies generated from long read sequencing support the genomic diversity reported by Yao-Cheng et.al, though the specific copy number variants are distinct from those previously reported. Assembled genomes and transcriptomes for these specific lots of HEK293 cells will be available at NCBI. Given the plastic nature of HEK293 cells, we recommend confirmation of genomic and transcriptomic structure of the specific cells in use before and after intervention. [1] Lin, Yao-Cheng, Morgane Boone, Leander Meuris, Irma Lemmens, Nadine Van Roy, Arne Soete, Joke Reumers, et al. “Genome Dynamics of the Human Embryonic Kidney 293 Lineage in Response to Cell Biology Manipulations.” Nature Communications 5, no. 1 (September 3, 2014): 4767. https://doi.org/10.1038/ncomms5767. Citation Format: Laura Blum, Brittany Sexton, Jonathan C. Sanford, Evan Janzen, Luo Sun, Paul A. Koetsier, Keerthana Krishnan, Lynne Apone, Bradley W. Langhorst. Long RNA and DNA sequencing reveals HEK293 diversity between commercial sources [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 260.

Genomic markers of recurrence risk in atypical meningioma following gross total resection.

Meningiomas are the most common primary central nervous system (CNS) tumor in adults and CNS World Health Organization grade 2 (atypical) meningiomas show an intermediate risk of recurrence/progression. Molecular parameters are needed to better inform management following gross total resection (GTR). We performed comprehensive genomic analysis of tumor tissue from 63 patients who underwent radiologically confirmed GTR of a primary grade 2 meningioma, including a CLIA-certified target next-generation sequencing panel (n = 61), chromosomal microarray (n = 63), genome-wide methylation profiling (n = 62), H3K27me3 immunohistochemistry (n = 62), and RNA-sequencing (n = 19). Genomic features were correlated with long-term clinical outcomes (median follow-up: 10 years) using Cox proportional hazards regression modeling and published molecular prognostic signatures were evaluated. The presence of specific copy number variants (CNVs), including -1p, -10q, -7p, and -4p, was the strongest predictor of decreased recurrence-free survival (RFS) within our cohort (P < .05). NF2 mutations were frequent (51%) but did not show a significant association with RFS. DNA methylation-based classification assigned tumors to DKFZ Heidelberg benign (52%) or intermediate (47%) meningioma subclasses and was not associated with RFS. H3K27 trimethylation (H3K27me3) was unequivocally lost in 4 tumors, insufficient for RFS analysis. Application of published integrated histologic/molecular grading systems did not improve prediction of recurrence risk over the presence of -1p or -10q alone. CNVs are strong predictors of RFS in grade 2 meningiomas following GTR. Our study supports incorporation of CNV profiling into clinical evaluation to better guide postoperative patient management, which can be readily implemented using existing, clinically validated technologies.

A Single Next Generation Sequencing Assay for Detection of Driver Mutations, Translocations and Copy Number Alterations in Patients with Multiple Myeloma

Multiple myeloma (MM) is a plasma cell malignancy characterized by several genetic abnormalities that impact risk stratification, outcome, and response to therapy; with fluorescence in situ hybridization (FISH) considered the gold-standard assay to detect the presence of chromosomal translocations and copy-number variations (CNV). Here, we evaluate the performance of a single clinically actionable targeted sequencing (NGS) assay for detecting cytogenetic abnormalities in addition to recurrent driver mutations in MM. Patient-derived bone marrow aspirates were obtained and enriched for CD138+ plasma cells. Targeted sequencing was performed using a 2.3 Mb custom capture panel covering 139 genes and regions surrounding MYC, immunoglobulin heavy chain (IgH), and immunoglobulin light chain kappa (IgK) and lambda (IgL) loci. Chromosomal translocations were identified using an in-house algorithm designed specifically for targeted capture DNA sequencing data analyses. Structural variants were called based on discordant read pairs as well as junction split reads, solving the challenges from both the overlapping paired-end reads due to the longer read lengths (150 bp) and the mapping ambiguities in the Ig regions. CNV were assessed using both on and off target reads, using a 200 kb bin size for segmentation covering the entire genome. Clinical annotations and FISH panel reports were abstracted. We sequenced 264 MM patient samples, 94% of which had available FISH information. A median of two somatic mutated genes per sample (range 0-13) was found. The most commonly mutated genes per sample were: KRAS (35%), NRAS (25%), FAM46C (15%), DIS3 (13%), BRAF (13%), ATM (11%), and TP53 (11%). Comparing NGS with FISH, we found a perfect concordance for the identification of t(4;14), t(6;14), t(14;16), and t(14;20); a 100% specificity and 97% sensitivity for t(11;14); and sensitivity >90% and an average specificity of 85% for trisomies of chromosomes 3, 7, 9, 11, and 15. The NGS approach was also able to detect gain(1q) and mono(13) with a sensitivity and specificity >90%, and del(17p) with a 60% sensitivity and 100% specificity. The reduced sensitivity for del(17p) detection was mostly due to focal deletions and low tumor purity. Because the TP53 gene is included in the custom capture panel, we increased the sensitivity by determining six separate regions of coverage using on-target probes, which enabled the detection of focal del(17p), increasing the sensitivity to 78%. The NGS approach also allowed the characterization of 16 (6%) samples with unavailable FISH. Of the 27 samples with del(17p) detected by FISH, NGS was able to identify TP53 mutations (i.e., biallelic inactivation) in 14 samples (52%). Due to the existence of multiple and promiscuous chromosomal partners involved in IgH translocation, the identification of the chromosomal partners is limited to the clinical FISH probe sets; in fact, 19 study samples presented abnormal IgH without a known partner by FISH. Our NGS assay was able to identify these partners, confirming MYC-IGH translocations in seven (37%) of these cases. Additionally, we detected MYC translocations with immunoglobulin light chains: 14 (5%) cases with IGK, and eight (3%) with IGL. Although IgL translocations, particularly IgL-MYC translocations, have been recently associated to poor prognosis, they are not routinely evaluated in FISH assays. The risk stratification and clinical utility of the NGS assay was demonstrated by the shorter overall survival observed in samples classified as high risk (mSMART 3.0; hazard ratio [HR]= 2.81; CI:1.89-4.17; p<0.0001), which was also observed with risk assessment by FISH (HR = 2.48; CI:1.72-3.58; p<0.0001). Therefore, in addition to the detection of clinically relevant somatic mutations, the targeted NGS approach showed robust detection of recurrent MM specific IgH translocations and CNV. Furthermore, the detection of relevant abnormalities not routinely assessed in most clinical labs, such as TP53 bi-allelic inactivation, IgL and MYC rearrangements, supports the use of this small, targeted panel as a clinically relevant, single molecular assay, for the comprehensive profiling of MM.

Clinical implementation of integrated molecular-morphologic risk prediction for meningioma.

Risk prediction for meningioma tumors was until recently almost exclusively based on morphological features of the tumor. To improve risk prediction, multiple models have been established that incorporate morphological and molecular features for an integrated risk prediction score. One such model is the integrated molecular-morphologic meningioma integrated score (IntS), which allocates points to the histological grade, epigenetic methylation family and specific copy-number variations. After publication of the IntS, questions arose in the neuropathological community about the practical and clinical implementation of the IntS, specifically regarding the calling of CNVs, the applicability of the newly available version (v12.5) of the brain tumor classifier and the need for incorporation of TERT-promoter and CDKN2A/B status analysis in the IntS calculation. To investigate and validate these questions additional analyses of the discovery (n=514), retrospective validation (n=184) and prospective validation (n=287) cohorts used for IntS discovery and validation were performed. Our findings suggest that any loss over 5% of the chromosomal arm suffices for the calling of a CNV, that input from the v12.5 classifier is as good or better than the dedicated meningioma classifier (v2.4) and that there is most likely no need for additional testing for TERT-promoter mutations and/or homozygous losses of CDKN2A/B when defining the IntS for an individual patient. The findings from this study help facilitate the clinical implementation of IntS-based risk prediction for meningioma patients.

STEM-12. DISCOVERY THE ORIGIN-CELLS FOR HUMAN GLIOBLASTOMA GENESIS IN SUBVENTRICULAR ZONE

Abstract Human glioblastoma (GBM), originating from the subventricular zone (SVZ), occurs due to molecular disruptions in chromosomes. Most GBM tissues exhibit definitive chromosomal patterns: copy-number-variations (CNV) in chromosome 7 (gain) and 10 (loss), known as the earliest molecular events. Herein, we hypothesised that the origin-cells in SVZ of GBM patients can provide clues regarding these chromosomal alterations. We compared bulk RNA sequencing (RNAseq) data of GBM tumor tissue (n=126), tumour free GBM SVZ (n=40), and tumor-free control SVZ of non-glial tumor (n=9). Paired single-cell-level RNAseq samples of tumor free GBM SVZ (n=7) and GBM tumor tissue (n=10), were done to see cell specific CNVs. Using human SVZ and GBM samples as a background, we generated origin-cell and origin-cell-derived tumour cell using CRISPR/Cas9. In this work, we identified two GBM-origin-cell types with stem-cell signatures during single-cell level analyses of 60 SVZ tissue samples obtained from tumor-free regions of GBM patients. Furthermore, single-cell level analysis revealed that two origin-cell types in SVZ harbor ongoing patterns of CNV alterations. Among the origin-cells found in the SVZ of GBM patients, NO-like cells showed neural progenitor plus oligodendrocyte progenitor (NO) signature, while AN-like cells showed astrocyte plus neural stem cell (AN) signature. For the interconnectedness, we subjected single-cell-level RNA-seq data to ligand-receptor connection analysis. In the stem cell mode, NO-like cells was connected to AN-like cells in SVZ samples and while in the tumor samples, cycling cell was connected to AN-like cells. NO-like cells was common in TERT promoter wildtype GBM and AN-like cells was more common in TERT promoter mutant GBM. CRISPR/Cas9 models revealed accumulation copy-number alterations from non-tumorigenic origin-cells to tumor cells. These two origin-cells (NO-like cells and AN-like cells) derived from the SVZ of the adult human brain will facilitate the understanding of GBM genesis and development of potential novel therapeutic targets.

ParseCNV2: efficient sequencing tool for copy number variation genome-wide association studies.

Improved copy number variation (CNV) detection remains an area of heavy emphasis for algorithm development; however, both CNV curation and disease association approaches remain in its infancy. The current practice of focusing on candidate CNVs, where researchers study specific CNVs they believe to be pathological while discarding others, refrains from considering the full spectrum of CNVs in a hypothesis-free GWAS. To address this, we present a next-generation approach to CNV association by natively supporting the popular VCF specification for sequencing-derived variants as well as SNP array calls using a PennCNV format. The code is fast and efficient, allowing for the analysis of large (>100,000 sample) cohorts without dividing up the data on a compute cluster. The scripts are condensed into a single tool to promote simplicity and best practices. CNV curation pre and post-association is rigorously supported and emphasized to yield reliable results of highest quality. We benchmarked two large datasets, including the UK Biobank (n > 450,000) and CAG Biobank (n > 350,000) both of which are genotyped at >0.5 M probes, for our input files. ParseCNV has been actively supported and developed since 2008. ParseCNV2 presents a critical addition to formalizing CNV association for inclusion with SNP associations in GWAS Catalog. Clinical CNV prioritization, interactive quality control (QC), and adjustment for covariates are revolutionary new features of ParseCNV2 vs. ParseCNV. The software is freely available at: https://github.com/CAG-CNV/ParseCNV2 .

The Dynamic Mutational Landscape of Cerebrospinal Fluid Circulating Tumor DNA can Predict Survival after Proton Craniospinal Irradiation for Leptomeningeal Metastasis

<h3>Purpose/Objective(s)</h3> Solid tumor leptomeningeal metastasis (LM) is a devastating disease with poor survival. Proton craniospinal irradiation (pCSI) is a promising treatment for LM, and there is a need for biomarkers for patient selection. Understanding the genetic variation of LM pre- and post-pCSI can elucidate differences in response to radiotherapy. We analyzed the observed mutations in cerebrospinal fluid (CSF) circulating tumor DNA (ctDNA) of LM patients treated with pCSI and explored whether the variant allele fraction (VAF) can predict survival. <h3>Materials/Methods</h3> We examined 14 patients from a phase I trial pCSI for solid tumor LM (NCT03281889) with pre- and post-pCSI ctDNA assessment. Mutations were identified using an institutional targeted-sequencing panel, MSK-IMPACT, and allele specific copy number variation was used to determine mutation clonality. Primary tumors or solid tumor metastases, and ctDNA in the plasma was also analyzed. VAF and cancer cell fraction (CCF) were calculated. An independent clinical sequencing cohort of 50 LM patients who did not receive pCSI was used for comparison. Kaplan Meier analysis was used to associate mutation measures and survival outcomes. <h3>Results</h3> The median overall survival for the cohort was 9 months (IQR: 5-21 months). The median number of mutations observed in the CSF ctDNA pre- pCSI was 10 (range:0-25) and post-pCSI was 9 (range:0-42). We observed the clonal evolution between solid tissue and ctDNA of the CSF and plasma, and we identified mutations that are unique by compartment, with more mutations seen in the CSF compared to plasma. A higher VAF pre-pCSI (VAF_pre) was associated with worse OS (8 months for VAF_pre≥0.32 [median] vs 21 months for VAF_pre<0.32, p=0.05). Patients with increased VAF after pCSI had significantly worse survival (6 months vs 18 months, p=0.008). The CCF of novel subclonal mutations after pCSI showed that higher values were associated with worse OS (8 months vs 17 months, p=0.05). In the comparison cohort, VAF≥0.37 [median] was associated with worse OS after CNS-directed RT (8 months vs 9 months, p=0.05). <h3>Conclusion</h3> In LM patients undergoing pCSI, there are unique baseline mutation profiles and distinct responses to pCSI that may be able to predict survival after radiotherapy. Analysis of the ctDNA of LM patients before and after pCSI shows that improved outcomes are associated with lower frequency of mutations. The quantification of these mutations has important implications for patient stratification in CNS-directed radiotherapy and escalation of therapy after pCSI.

OS10.6.A What is the initial cell in the subventricular zone for human glioblastoma genesis?

Abstract Background We all have a fundamental question about why glioblastoma (GBM) develops. In order to find the answer to this fundamental question, if you find out what the first cell is, you will get closer to the answer. GBM arises from the subventricular zone (SVZ). GBM is one of the most devastating tumour of human brain as the most optimal treatment barely prolongs the survival, and it does not cure the disease. As the majority of GBM tissues show copy number variations (CNV) of co-altered chromosomal 7 gain and 10 loss, we hypothesized the origin cell (Oc) of SVZ may be traced back with these markers. The cellular identity of the Oc is still unknown and it is different from the tumour-derived progenitor-like cells. We aimed to define these cells from the SVZ that have a potential to get activated into GBM. Material and Methods We compared bulk RNA sequencing (RNAseq) data of IDHwt GBM tumor tissue (n=122), tumor free SVZ from GBM patients (n=40), tumor-free control SVZ of non-glial tumor (n=9). Pared single nucleus RNAseq (snRNAseq) or single cell RNAseq (scRNAseq) samples of tumor free SVZ (n=11) and GBM tumor (n=8), were done to see cell specific CNVs. We developed genetically engineered mouse models for GBM genesis introducing three driver mutations (TP53, PTEN, and EGFRviii) into SVZ to isolate mouse Oc (mOc) and mouse cancer cells (mCc). The biological characteristics of separated mOc and mCc were compared. Bulk RNAseq and scRNAseq were performed on these cells (mOc, mCc), and their cellular state was compared with the human gene set. Results In this work, we found two types of the Oc in the RNA sequencing of 60 human tumour free-SVZ samples. Furthermore, single-cell level analysis revealed that two Oc types in SVZ harbor ongoing patterns of CNV co-alterations from Oc1 to Oc2, and finally to GBM. The Oc1 type cells contained the CNV signature of Oc2 ancestor with neural progenitor cell (NPC) signature. Oc2 type cells expressed a high level of EGFR than other cells with astrocyte-like cell signature. Both of these cells expressed oligodendrocyte progenitor cell (OPC)-like signatures in the SVZ. We validated the human-based findings by using the P53/PTEN/EGFR-mutant mouse model with EGFR/tdTomato overexpression and P53/PTEN knockout in the SVZ cells. As a result, non-tumourigenic and highly motile Oc-like cell-states are found in the mouse models, supporting the firework-like migration pattern from the SVZ. Conclusion Our results demonstrate how members of Oc preoccupy the SVZ, known as the stem cell niche and give rise to the tumour. We anticipate that a new therapy may emerge by targeting the Oc in the SVZ.

Whole exome sequencing of high-risk neuroblastoma identifies novel non-synonymous variants.

Neuroblastoma (NBL), one of the main death-causing cancers in children, is known for its remarkable genetic heterogeneity and varied patient outcome spanning from spontaneous regression to widespread disease. Specific copy number variations and single gene rearrangements have been proven to be associated with biological behavior and prognosis; however, there is still an unmet need to enlarge the existing armamentarium of prognostic and therapeutic targets. We performed whole exome sequencing (WES) of samples from 18 primary tumors and six relapse samples originating from 18 NBL patients. Our cohort consists of 16 high-risk, one intermediate, and one very low risk patient. The obtained results confirmed known mutational hotspots in ALK and revealed other non-synonymous variants of NBL-related genes (TP53, DMD, ROS, LMO3, PRUNE2, ERBB3, and PHOX2B) and of genes cardinal for other cancers (KRAS, PIK3CA, and FLT3). Beyond, GOSeq analysis determined genes involved in biological adhesion, neurological cell-cell adhesion, JNK cascade, and immune response of cell surface signaling pathways. We were able to identify novel coding variants present in more than one patient in nine biologically relevant genes for NBL, including TMEM14B, TTN, FLG, RHBG, SHROOM3, UTRN, HLA-DRB1, OR6C68, and XIRP2. Our results may provide novel information about genes and signaling pathways relevant for the pathogenesis and clinical course in high-risk NBL.

Identification of novel rare copy number variants associated with sporadic tetralogy of Fallot and clinical implications

Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart disease. Highly penetrant copy number variants (CNVs) and genes related to the etiology of TOF likely exist with differences among populations. We aimed to identify CNV contributions to sporadic TOF cases in Han Chinese. Genomic DNA was extracted from peripheral blood in 605 subjects (303 sporadic TOF and 302 unaffected Han Chinese [Control] from cardiac centers in China) and analyzed by genome-wide association study (GWAS). The GWAS results were compared with existing Database of Genetic Variants. These CNVs were further validated by qPCR. Bioinformatics analyses were performed with protein-protein interaction (PPI) network and KEGG pathway enrichment. Across all chromosomes 119 novel "TOF-specific CNVs" were identified with prevalence of CNVs of 21.5% in chromosomes 1-20 and 37.0% including Chr21/22. In chromosomes 1-20, CNVs on 11q25 (encompasses genes ACAD8, B3GAT1, GLB1L2, GLB1L3, IGSF9B, JAM3, LOC100128239, LOC283177, MIR4697, MIR4697HG, NCAPD3, OPCML, SPATA19, THYN1, and VPS26B) and 14q32.33 (encompasses genes THYN1, OPCML, and NCAPD3) encompass genes most likely to be associated with TOF. Specific CNVs found on the chromosome 21 (6.3%) and 22(11.9%) were also identified in details. PPI network analysis identified the genes covering the specific CNVs related to TOF and the signaling pathways. This study for first time identified novel TOF-specific CNVs in the Han Chinese with higher frequency than in Caucasians and with 11q25 and 14q32.33 not reported in TOF of Caucasians. These novel CNVs identify new candidate genes for TOF and provide new insights into genetic basis of TOF.

CNest: A novel copy number association discovery method uncovers 862 new associations from 200,629 whole-exome sequence datasets in the UK Biobank

Copy number variation (CNV) is known to influence human traits, having a rich history of research into common and rare genetic disease, and although CNV is accepted as an important class of genomic variation, progress on copy-number-based genome-wide association studies (GWASs) from next-generation sequencing (NGS) data has been limited. Here we present a novel method for large-scale copy number analysis from NGS data generating robust copy number estimates and allowing copy number GWASs (CN-GWASs) to be performed genome-wide in discovery mode. We provide a detailed analysis in the UK Biobank resource and a specifically designed software package. We use these methods to perform CN-GWAS analysis across 78 human traits, discovering over 800 genetic associations that are likely to contribute strongly to trait distributions. Finally, we compare CNV and SNP association signals across the same traits and samples, defining specific CNV association classes.

Clinical impact of variants in non-coding regions of SHOX - Current knowledge.

The short stature homeobox-containing (SHOX) is the most frequently analysed gene in patients classified as short stature patients (ISS) or diagnosed with Leri-Weill dyschondrosteosis (LWD), Langer mesomelic dysplasia (LMD), or Madelung deformity (MD). However, clinical testing of this gene focuses primarily on single nucleotide variants (SNV) in its coding sequences and copy number variants (CNV) overlapping SHOX gene. This review summarizes the clinical impact of variants in noncoding regions of SHOX. RECENT FINDINGS: CNV extending exclusively into the regulatory elements (i.e., not interrupting the coding sequence) are found more frequently in downstream regulatory elements of SHOX. Further, duplications are more frequent than deletions. Interestingly, downstream duplications are more common than deletions in patients with ISS or LWD but no such differences exist for upstream CNV. Moreover, the presence of specific CNVs in the patient population suggests the involvement of additional unknown factors. Some of its intronic variants, notably NM_000451.3(SHOX):c.-9delG and c.-65C>A in the 5'UTR, have unclear clinical roles. However, these intronic SNV may increase the probability that other CNV will arise de novo in the SHOX gene based on homologous recombination or incorrect splicing of mRNA. SUMMARY: This review highlights the clinical impact of noncoding changes in the SHOX gene and the need to apply new technologies and genotype-phenotype correlation in their analysis.