Relevant Copy Number Variants Research Articles

Array Comparative Genomic Hybridization (aCGH) Results among Patients Referred to Invasive Prenatal Testing after First-Trimester Screening: A Comprehensive Cohort Study

Introduction: Invasive prenatal testing with chromosomal microarray analysis after first-trimester screening is a relevant option but there is still debate regarding the indications. Therefore, we evaluated the prevalence of numerical chromosomal aberrations detected by classic karyotype and clinically relevant copy number variants (CNVs) in prenatal samples using array comparative genomic hybridization (aCGH) stratified to NT thickness: <the 95th percentile, the 95th percentile–2.9 mm, 3.0–3.4 mm, 3.5–3.9 mm, 4.0–4.5 mm, and >4.5 mm, and by the presence/absence of associated structural anomalies detected by ultrasonography. Materials and Methods: Retrospective cohort study carried out at two tertiary Polish centers for prenatal diagnosis (national healthcare system) in central and south regions from January 2018 to December 2021. A total of 1746 prenatal samples were received. Indications for invasive prenatal testing included high risk of Down syndrome in the first-trimester combined test (n = 1484) and advanced maternal age (n = 69), and, in 193 cases, other reasons, such as parental request, family history of congenital defects, and genetic mutation carrier, were given. DNA was extracted directly from amniotic fluid (n = 1582) cells and chorionic villus samples (n = 164), and examined with classic karyotype and aCGH. Results: Of the entire cohort of 1746 fetuses, classical karyotype revealed numerical chromosomal aberrations in 334 fetuses (19.1%), and aCGH detected CNV in 5% (n = 87). The frequency of numerical chromosomal aberrations increased with NT thickness from 5.9% for fetuses with NT < p95th to 43.3% for those with NT > 4.5 mm. The highest rate of numerical aberrations was observed in fetuses with NT > 4.5 mm having at least one structural anomaly (50.2%). CNVs stratified by NT thickness were detected in 2.9%, 2.9%, 3.5%, 4.3%, 12.2%, and 9.0% of fetuses with NT < 95th percentile, 95th percentile–2.9 mm, 3.0–3.4 mm, 3.5–3.9 mm, 4.0–4.5 mm, and >4.5 mm, respectively. After exclusion of fetuses with structural anomalies and numerical aberrations, aCGH revealed CNVs in 2.0% of fetuses with NT < 95th percentile, 1.5% with NTp95–2.9 mm, 1.3% with NT 3.0–3.4 mm, 5.4% with NT 3.5–3.9 mm, 19.0% with NT 4.0–4.5 mm, and 14.8% with NT > 4.5 mm. Conclusions: In conclusion, our study indicates that performing aCGH in samples referred to invasive prenatal testing after first-trimester screening provides additional clinically valuable information over conventional karyotyping, even in cases with normal NT and anatomy.

Contribution of clinically relevant copy number variations in long QT syndrome: a large cohort study

Abstract Background Copy number variations (CNVs) have increasingly been recognized in the cardiovascular setting. Their role in hypertrophic cardiomyopathy or sudden cardiac death has been described, but their involvement in Long QT syndrome (LQTS) has been scarcely studied. Most of these descriptions are confined to case reports or small series. Purpose The aim of this study is to investigate the proportion of clinically relevant CNVs among a large cohort of index cases referred for genetic testing due to LQTS. Methods Retrospective study in which the presence of likely pathogenic (LP) or pathogenic (P) CNVs was evaluated in a cohort of index cases referred for genetic study with LQTS phenotype. Index cases were genotyped with customized libraries through next generation sequencing (NGS). CNVs analysis was conducted by a proprietary method by means of comparative analysis of normalized read depth data. Clinically relevant (LP or P) CNVs were then confirmed by other techniques. Results 1183 index cases were referred for genetic testing to our laboratory with long QT syndrome phenotype. Some of these index cases were referred for suspicion of Jervell-Lange-Nielsen syndrome (JNLS) or for LQTS associated with congenital deafness (n=10) or Anderson-Tawil syndrome (n=1). Around 27% (n=322) of the index cases had a positive result. CNVs analysis was run on 1071 (91%) of the index cases. Most of the CNVs were considered negative (n=994, 93%), 59 could not be analysed (6%) and in 11 patients (1%), a LP/P CNV was identified. All CNVs but three (72.72%), were confirmed by orthogonal molecular techniques (Sanger sequencing or multiplex ligation-dependent probe amplification (MLPA)). In all, but two, the genetic study was considered positive due to the CNV findings. One of these patients carried a pathogenic lost-of-function (LOF) CNV in the LDLR gene (incidental finding) but the report was negative because he was referred for suspicion of JLNS. In another case a CNV in the DSP gene was identified in mosaicism (30-40%). However, in this case, the positive report was attributed to the presence of an additional pathogenic variant in CACNA1C. Regarding the other index cases with LP/P CNVs: 4 carried a deletion in the KCNH2 gene, 3 carried a deletion in KCNQ1 gene (one of them in homozygosis and was studied for suspicion of JLNS) and 1 carried a duplication in the KCQN1 (LOF variant). In addition, one patient carried a deletion of exon 3 in the RYR2 gene. Even if the patient was referred for suspicion of LQTS, the report was considered positive. These clinically relevant CNVs represented around 3% of the positive genetic results. Conclusions In our large cohort of patients referred for genetic testing for LQTS, around 1% of clinically relevant CNVs were identified. As such large genomic rearrangements underlie a non-neglectable portion of cases, we consider that their analysis should be performed as part of the routine genetic testing.Deletion exons 6-14 in KCNH2 gene (CNV)MLPA with deletion exons 6-14 in KCNH2

A cohort study of neurodevelopmental disorders and/or congenital anomalies using high resolution chromosomal microarrays in southern Brazil highlighting the significance of ASD

Chromosomal microarray (CMA) is the reference in evaluation of copy number variations (CNVs) in individuals with neurodevelopmental disorders (NDDs), such as intellectual disability (ID) and/or autism spectrum disorder (ASD), which affect around 3–4% of the world’s population. Modern platforms for CMA, also include probes for single nucleotide polymorphisms (SNPs) that detect homozygous regions in the genome, such as long contiguous stretches of homozygosity (LCSH). These regions result from complete or segmental chromosomal homozygosis and may be indicative of uniparental disomy (UPD), inbreeding, population characteristics, as well as replicative DNA repair events. In this retrospective study, we analyzed CMA reading files requested by geneticists and neurologists for diagnostic purposes along with available clinical data. Our objectives were interpreting CNVs and assess the frequencies and implications of LCSH detected by Affymetrix CytoScan HD (41%) or 750K (59%) platforms in 1012 patients from the south of Brazil. The patients were mainly children with NDDs and/or congenital anomalies (CAs). A total of 206 CNVs, comprising 132 deletions and 74 duplications, interpreted as pathogenic, were found in 17% of the patients in the cohort and across all chromosomes. Additionally, 12% presented rare variants of uncertain clinical significance, including LPCNVs, as the only clinically relevant CNV. Within the realm of NDDs, ASD carries a particular importance, owing to its escalating prevalence and its growing repercussions for individuals, families, and communities. ASD was one clinical phenotype, if not the main reason for referral to testing, for about one-third of the cohort, and these patients were further analyzed as a sub-cohort. Considering only the patients with ASD, the diagnostic rate was 10%, within the range reported in the literature (8–21%). It was higher (16%) when associated with dysmorphic features and lower (7%) for "isolated" ASD (without ID and without dysmorphic features). In 953 CMAs of the whole cohort, LCSH (≥ 3 Mbp) were analyzed not only for their potential pathogenic significance but were also explored to identify common LCSH in the South Brazilians population. CMA revealed at least one LCSH in 91% of the patients. For about 11.5% of patients, the LCSH suggested consanguinity from the first to the fifth degree, with a greater probability of clinical impact, and in 2.8%, they revealed a putative UPD. LCSH found at a frequency of 5% or more were considered common LCSH in the general population, allowing us to delineate 10 regions as potentially representing ancestral haplotypes of neglectable clinical significance. The main referrals for CMA were developmental delay (56%), ID (33%), ASD (33%) and syndromic features (56%). Some phenotypes in this population may be predictive of a higher probability of indicating a carrier of a pathogenic CNV. Here, we present the largest report of CMA data in a cohort with NDDs and/or CAs from the South of Brazil. We characterize the rare CNVs found along with the main phenotypes presented by each patient and show the importance and usefulness of LCSH interpretation in CMA results that incorporate SNPs, as well as we illustrate the value of CMA to investigate CNV in ASD.

Evaluation of Circulating Tumor DNA as a Liquid Biomarker in Uveal Melanoma.

Uveal melanoma (UM) has a high propensity to metastasize. Prognosis is associated with specific driver mutations and copy number variations (CNVs), but limited primary tumor tissue is available for molecular characterization due to eye-sparing irradiation treatment. This study aimed to assess the rise in circulating tumor DNA (ctDNA) levels in UM and evaluate its efficacy for CNV-profiling of patients with UM. In a pilot study, we assessed ctDNA levels in the blood of patients with UM (n = 18) at various time points, including the time of diagnosis (n = 13), during fractionated stereotactic radiotherapy (fSRT) treatment (n = 6), and upon detection of metastatic disease (n = 13). Shallow whole-genome sequencing (sWGS) combined with in silico size-selection was used to identify prognostically relevant CNVs in patients with UM (n = 26) from peripheral blood retrieved at the time of diagnosis (n = 9), during fSRT (n = 5), during post-treatment follow-up (n = 4), metastasis detection (n = 6), and metastasis follow-up (n = 4). A total of 34 patients had blood analyzed for ctDNA detection (n = 18) and/or CNV analysis (n = 26) at various time points. At the time of diagnosis, 5 of 13 patients (38%) had detectable ctDNA (median = 0 copies/mL). Upon detection of metastatic disease, ctDNA was detected in 10 of 13 patients (77%) and showed increased ctDNA levels (median = 24 copies/mL, P < 0.01). Among the six patients analyzed during fSRT, three (50%) patients had detectable ctDNA at baseline and three of six (50%) patients had undetectable levels of ctDNA. During the fSRT regimen, ctDNA levels remained unchanged (P > 0.05). The ctDNA fractions were undetectable to low in localized disease, and sWGS did not elucidate chromosome 3 status from blood samples. However, in 7 of 10 (70%) patients with metastases, the detection of chromosome 3 loss corresponded to the high metastatic-risk class. The rise in ctDNA levels observed in patients with UM harboring metastases suggests its potential utility for CNV profiling. These findings highlight the potential of using ctDNA for metastasis detection and patient inclusion in therapeutic studies targeting metastatic UM.

Streamlined identification of clinically and functionally relevant genetic regulators of lower-tract urogenital development

Congenital anomalies of the lower genitourinary (LGU) tract are frequently comorbid due to genetically linked developmental pathways, and are among the most common yet most socially stigmatized congenital phenotypes. Genes involved in sexual differentiation are prime candidates for developmental anomalies of multiple LGU organs, but insufficient prospective screening tools have prevented the rapid identification of causative genes. Androgen signaling is among the most influential modulators of LGU development. The present study uses SpDamID technology in vivo to generate a comprehensive map of the pathways actively regulated by the androgen receptor (AR) in the genitalia in the presence of the p300 coactivator, identifying wingless/integrated (WNT) signaling as a highly enriched AR-regulated pathway in the genitalia. Transcription factor (TF) hits were then assayed for sexually dimorphic expression at two critical time points and also cross-referenced to a database of clinically relevant copy number variations to identify 252 TFs exhibiting copy variation in patients with LGU phenotypes. A subset of 54 TFs was identified for which LGU phenotypes are statistically overrepresented as a proportion of total observed phenotypes. The 252 TF hitlist was then subjected to a functional screen to identify hits whose silencing affects genital mesenchymal growth rates. Overlap of these datasets results in a refined list of 133 TFs of both functional and clinical relevance to LGU development, 31 of which are top priority candidates, including the well-documented renal progenitor regulator, Sall1. Loss of Sall1 was examined in vivo and confirmed to be a powerful regulator of LGU development.

Impact of copy number variants in epilepsy plus neurodevelopment disorders

IntroductionEpilepsy, a neurological disorder characterized by recurring unprovoked seizures due to excessive neuronal excitability, is primarily attributed to genetic factors, accounting for an estimated 70 % of cases. Array-comparative genomic hybridization (aCGH) is a crucial genetic test for detecting copy number variants (CNVs) associated with epilepsy. This study aimed to analyze a cohort of epilepsy patients with CNVs detected through aCGH to enhance our understanding of the genetic underpinnings of epilepsy. MethodsA retrospective cross-sectional study was conducted using the aCGH database from the Genetics Department of the Faculty of Medicine of the University of Porto, encompassing 146 patients diagnosed with epilepsy, epileptic encephalopathy, or seizures. Clinical data were collected, and aCGH was performed following established guidelines. CNVs were classified based on ACMG standards, and patients were categorized into four groups according to their clinical phenotype. ResultsAmong the 146 included patients, 94 (64 %) had at least one CNV, with 22 (15.1 %) classified as pathogenic or likely pathogenic. Chromosomes 1, 2, 16, and X were frequently implicated, with Xp22.33 being the most reported region (8 CNVs). The phenotype "Epilepsy and global developmental delay/intellectual disability" showed the highest prevalence of clinically relevant CNVs. Various CNVs were identified across different groups, suggesting potential roles in epilepsy. ConclusionsThis study highlights the significance of aCGH in unraveling the genetic basis of epilepsy and tailoring treatment strategies. It contributes valuable insights to the expanding knowledge in the field, emphasizing the need for research to elucidate the diverse genetic causes of epilepsy.

Validating Clinical Exome Sequencing (CES) Test for Copy Number Variation (CNV) Analysis in Patients with Autism Spectrum Disorder

A routine methodologies to detect germline copy number variants (CNVs) are ArrayCGH, and SNParray, while clinical exome sequencing (CES) it is not a standard methods used in clinical settings. This study aims to investigate the efficiency of CNVs identification by CES in the diagnosis of Autism spectrum disorder (ASD) based on a Italian cohort. In this cohort study, 50 patients with ASD diagnosis were recruited for ASD screening. Simultaneous CNVs and single nucleotide variation (SNV) analysis was conducted to identify the anomalies that can cause ASD phenotype. A total of 13 patients were identified with a causative CNVs among 15 (86.67%) SNP array positive patients. Showing that CES data can be used for detecting clinically relevant CNVs with high sensitivity for use in clinical diagnostic settings.

PATH-13. APPLICATIONS OF SPATIAL TRANSCRIPTOMICS FOR THE DIAGNOSTICS OF GLIOMAS

Abstract Due to their infiltrative growth, diffuse gliomas can pose a major challenge for neuropathologists. Lack of specific immunohistochemistry markers for some gliomas like IDH-wildtype glioblastoma can make it hard to identify the tumour cell content or distinguish between tumour and reactive gliosis. Other hurdles are high fractions of DNA from healthy cells in molecular diagnostic analysis, or scarce tissue, which can result in a failed DNA extraction. To overcome these limitations, we evaluated the application of spatial transcriptomics on 14 glioblastoma bulk resections and 18 stereotactic biopsies of 7 glioblastoma, IDH wildtype, 4 oligodendroglioma IDH-mutant and 7 astrocytoma IDH-mutant samples with the aim to generate a diagnostic report containing all information needed by the neuropathologist and clinician. Using the SPATA2 R package, we were able to infer the copy number variation (CNV) profiles for each spot. When comparing the inferred CNVs to the CNVs calculated by the 850K methylome analysis, a correlation between CNVs identified from the 850K data and the inferred CNVs could be seen. For diagnostic relevant CNVs of chr. 7, 10, 1p and 19q, the inferred CNVs were concordant with the 850K data in 100% of the cases with confirmed tumour tissue. CNVs from spatial transcriptomics were able to reveal sub-clones, which could not be detected by the 850K array data. The CNVs could then be used in combination with a deconvolution approach to determine the tumour burden of the tissue. Using RNA expression, the immunohistochemistry staining of important diagnostic markers like NeuN, GFAP and Ki67 was predicted. In addition, the spatial gene expression could be used to identify the glioblastoma subtype based on the gene expression of different methylation subtypes. All the above-mentioned analysis also generated meaningful results for tissue fragments smaller than 1mm in diameter, which are too small for molecular diagnostic analysis.

Low-pass whole genome sequencing is a reliable and cost-effective approach for copy number variant analysis in the clinical setting.

Next generation sequencing technology has greatly reduced the cost and time required for sequencing a genome. An approach that is rapidly being adopted as an alternative method for CNV analysis is the low-pass whole genome sequencing (LP-WGS). Here, we evaluated the performance of LP-WGSto detect copy number variants (CNVs) in clinical cytogenetics. DNA samples with known CNVs detected by chromosomal microarray analyses (CMA) were selected for comparison and used as positive controls; our panel included 44 DNA samples (12 prenatal and 32 postnatal), comprising a total of 55 chromosome imbalances. The selected cases were chosen to provide a wide range of clinically relevant CNVs, the vast majority being associated with intellectual disability or recognizable syndromes. The chromosome imbalances ranged in size from 75kb to 90.3Mb, including aneuploidies and two cases of mosaicism. All CNVs were successfully detected by LP-WGS, showing a high level of consistency and robust performance of the sequencing method. Notably, the size of chromosome imbalances detected by CMA and LP-WGS were compatible between the two different platforms, which indicates that the resolution and sensitivity of the LP-WGS approach are at least similar to those provided by CMA. Our data show the potential use of LP-WGS to detect CNVs in clinical diagnosis and confirm the method as an alternative for chromosome imbalances detection. The diagnostic effectiveness and feasibility of LP-WGS, in this technical validation study, were evidenced by a clinically representative dataset of CNVs that allowed a systematic assessment of the detection power and the accuracy of the sequencing approach. Further, since the software used in this study is commercially available, the method can easily be tested and implemented in a routine diagnostic setting.

Diagnostic yield and novel candidate genes for neurodevelopmental disorders by exome sequencing in an unselected cohort with microcephaly

ObjectivesMicrocephaly is caused by reduced brain volume and most usually associated with a variety of neurodevelopmental disorders (NDDs). To provide an overview of the diagnostic yield of whole exome sequencing (WES) and promote novel candidates in genetically unsolved families, we studied the clinical and genetic landscape of an unselected Chinese cohort of patients with microcephaly.MethodsWe performed WES in an unselected cohort of 103 NDDs patients with microcephaly as one of the features. Full evaluation of potential novel candidate genes was applied in genetically undiagnosed families. Functional validations of selected variants were conducted in cultured cells. To augment the discovery of novel candidates, we queried our genomic sequencing data repository for additional likely disease-causing variants in the identified candidate genes.ResultsIn 65 families (63.1%), causative sequence variants (SVs) and clinically relevant copy number variants (CNVs) with a pathogenic or likely pathogenic (P/LP) level were identified. By incorporating coverage analysis to WES, a pathogenic or likely pathogenic CNV was detected in 15 families (16/103, 15.5%). In another eight families (8/103, 7.8%), we identified variants in newly reported gene (CCND2) and potential novel neurodevelopmental disorders /microcephaly candidate genes, which involved in cell cycle and division (PWP2, CCND2), CDC42/RAC signaling related actin cytoskeletal organization (DOCK9, RHOF), neurogenesis (ELAVL3, PPP1R9B, KCNH3) and transcription regulation (IRF2BP1). By looking into our data repository of 5066 families with NDDs, we identified additional two cases with variants in DOCK9 and PPP1R9B, respectively.ConclusionOur results expand the morbid genome of monogenic neurodevelopmental disorders and support the adoption of WES as a first-tier test for individuals with microcephaly.

Implementation of Nanopore sequencing as a pragmatic workflow for copy number variant confirmation in the clinic

BackgroundDiagnosis of rare genetic diseases can be a long, expensive and complex process, involving an array of tests in the hope of obtaining an actionable result. Long-read sequencing platforms offer the opportunity to make definitive molecular diagnoses using a single assay capable of detecting variants, characterizing methylation patterns, resolving complex rearrangements, and assigning findings to long-range haplotypes. Here, we demonstrate the clinical utility of Nanopore long-read sequencing by validating a confirmatory test for copy number variants (CNVs) in neurodevelopmental disorders and illustrate the broader applications of this platform to assess genomic features with significant clinical implications.MethodsWe used adaptive sampling on the Oxford Nanopore platform to sequence 25 genomic DNA samples and 5 blood samples collected from patients with known or false-positive copy number changes originally detected using short-read sequencing. Across the 30 samples (a total of 50 with replicates), we assayed 35 known unique CNVs (a total of 55 with replicates) and one false-positive CNV, ranging in size from 40 kb to 155 Mb, and assessed the presence or absence of suspected CNVs using normalized read depth.ResultsAcross 50 samples (including replicates) sequenced on individual MinION flow cells, we achieved an average on-target mean depth of 9.5X and an average on-target read length of 4805 bp. Using a custom read depth-based analysis, we successfully confirmed the presence of all 55 known CNVs (including replicates) and the absence of one false-positive CNV. Using the same CNV-targeted data, we compared genotypes of single nucleotide variant loci to verify that no sample mix-ups occurred between assays. For one case, we also used methylation detection and phasing to investigate the parental origin of a 15q11.2-q13 duplication with implications for clinical prognosis.ConclusionsWe present an assay that efficiently targets genomic regions to confirm clinically relevant CNVs with a concordance rate of 100%. Furthermore, we demonstrate how integration of genotype, methylation, and phasing data from the Nanopore sequencing platform can potentially simplify and shorten the diagnostic odyssey.

Should Prenatal Chromosomal Microarray Analysis Be Offered for Pulmonary Atresia? A Single-Center Retrospective Study in China.

(1) Objective: To evaluate the application of chromosomal microarray analysis (CMA) in fetuses with pulmonary atresia (PA) and to explore the risk factors for predicting chromosomal imbalances and adverse perinatal outcomes. (2) Methods: This study investigated 428 cases of PA singleton pregnancies that were tested using CMA and quantitative fluorescent polymerase chain reaction (QF-PCR) as first-line genetic testing. The PA cases were divided into two groups: an isolated group and a non-isolated group. (3) Results: CMA revealed clinically relevant copy number variations (CNVs) in 9/139 (6.47%) PA fetuses, i.e., pathogenic copy number variations (pCNVs) in 8/139 (5.76%) fetuses and likely pathogenic CNVs in 1/139 (0.72%) fetuses. Stratified analysis showed that the incidence of clinically significant variants was higher in non-isolated PA fetuses than in isolated PA fetuses (12.50%, 6/48 vs. 3.30%, 3/91, p = 0.036). Regression analysis showed that a combination of other structural abnormalities at diagnosis of PA represented the principal risk factor for chromosomal imbalances (OR = 2.672). A combination of other structural abnormalities and a high maternal age increased the risk of adverse pregnancy outcomes in PA cases, including intrauterine fetal death (IUFD), termination of pregnancy (TOP), and preterm delivery. (4) Conclusions: The value of CMA for locating imbalanced genetic variations in fetuses with PA was highlighted by this study, particularly when combined with additional structural abnormalities.

Construction of copy number variation landscape and characterization of associated genes in a Bangladeshi cohort of neurodevelopmental disorders.

Introduction: Copy number variations (CNVs) play a critical role in the pathogenesis of neurodevelopmental disorders (NDD) among children. In this study, we aim to identify clinically relevant CNVs, genes and their phenotypic characteristics in an ethnically underrepresented homogenous population of Bangladesh. Methods: We have conducted chromosomal microarray analysis (CMA) for 212 NDD patients with male to female ratio of 2.2:1.0 to identify rare CNVs. To identify candidate genes within the rare CNVs, gene constraint metrics [i.e., "Critical-Exon Genes (CEGs)"] were applied to the population data. Autism Diagnostic Observation Schedule-Second Edition (ADOS-2) was followed in a subset of 95 NDD patients to assess the severity of autism and all statistical tests were performed using the R package. Results: Of all the samples assayed, 12.26% (26/212) and 57.08% (121/212) patients carried pathogenic and variant of uncertain significance (VOUS) CNVs, respectively. While 2.83% (6/212) patients' pathogenic CNVs were found to be located in the subtelomeric regions. Further burden test identified females are significant carriers of pathogenic CNVs compared to males (OR = 4.2; p = 0.0007). We have observed an increased number of Loss of heterozygosity (LOH) within cases with 23.85% (26/109) consanguineous parents. Our analyses on imprinting genes show, 36 LOH variants disrupting 69 unique imprinted genes and classified these variants as VOUS. ADOS-2 subset shows severe social communication deficit (p = 0.014) and overall ASD symptoms severity (p = 0.026) among the patients carrying duplication CNV compared to the CNV negative group. Candidate gene analysis identified 153 unique CEGs in pathogenic CNVs and 31 in VOUS. Of the unique genes, 18 genes were found to be in smaller (<1 MB) focal CNVs in our NDD cohort and we identified PSMC3 gene as a strong candidate gene for Autism Spectrum Disorder (ASD). Moreover, we hypothesized that KMT2B gene duplication might be associated with intellectual disability. Conclusion: Our results show the utility of CMA for precise genetic diagnosis and its integration into the diagnosis, therapy and management of NDD patients.

Copy Number Variant Analysis of Spinocerebellar Ataxia Genes in a Cohort of Dutch Patients With Cerebellar Ataxia.

The spinocerebellar ataxias (SCAs) are a genetically heterogeneous group of neurodegenerative disorders generally caused by single nucleotide variants (SNVs) or indels in coding regions or by repeat expansions in coding and noncoding regions of SCA genes. Copy number variants (CNVs) have now also been reported for 3 genes-ITPR1, FGF14, and SPTBN2-but not all SCA genes have been screened for CNVs as the underlying cause of the disease in patients. In this study, we aim to assess the prevalence of CNVs encompassing 36 known SCA genes. A cohort of patients with cerebellar ataxia who were referred to the University Medical Center Groningen for SCA genetic diagnostics was selected for this study. Genome-wide single nucleotide polymorphism (SNP) genotyping was performed using the Infinium Global Screening Array. Following data processing, genotyping data were uploaded into NxClinical software to perform CNV analysis per patient and to visualize identified CNVs in 36 genes with allocated SCA symbols. The clinical relevance of detected CNVs was determined using evidence from studies based on PubMed literature searches for similar CNVs and phenotypic features. Of the 338 patients with cerebellar ataxia, we identified putative clinically relevant CNV deletions in 3 patients: an identical deletion encompassing ITPR1 in 2 patients, who turned out to be related, and a deletion involving PPP2R2B in another patient. Although the CNV deletion in ITPR1 was clearly the underlying cause of SCA15 in the 2 related patients, the clinical significance of the deletion in PPP2R2B remained unknown. We showed that CNVs detectable with the limited resolution of SNP array are a very rare cause of SCA. Nevertheless, we suggest adding CNV analysis alongside SNV analysis to SCA gene diagnostics using next-generation sequencing approaches, at least for ITPR1, to improve the genetic diagnostics for patients.

Genetic and epigenetic analysis of hepatocellular adenomas with atypical morphological features.

Hepatocellular adenoma (HCA) is a rare liver tumour, which can have atypical morphological features such as cytological atypia, pseudoglandular architecture, and altered reticulin framework. Little is known about the genetic and epigenetic alterations of such HCAs and whether they show the alterations classically found in hepatocellular carcinoma (HCC) or in HCA without atypical morphology. We analysed five HCAs with atypical morphological features and one HCA with transition to HCC. Every tumour was subtyped by immunohistochemistry, sequenced by a targeted NGS panel, and analysed on a DNA methylation microarray. Subtyping of the five HCAs with atypical features revealed two β-catenin mutated HCA (b-HCA), two β-catenin mutated inflammatory HCA (b-IHCA), and one sonic hedgehog activated HCA (shHCA). None of them showed mutations typically found in HCC, such as, e.g. TERT or TP53 mutations. The epigenomic pattern of HCAs with atypical morphological features clustered with reference data for HCAs without atypical morphological features but not with HCC. Similarly, phyloepigenetic trees using the DNA methylation data reproducibly showed that HCAs with morphological atypia are much more similar to nonmalignant samples than to malignant samples. Finally, atypical HCAs showed no relevant copy number variations (CNV). In our series, mutational, DNA methylation, as well as CNV analyses, supported a relationship of atypical HCAs with nonatypical HCAs rather than with HCC. Therefore, in cases with difficult differential diagnosis between HCC and HCA, it might be advisable to perform targeted sequencing and/or combined methylation/copy number profiling.

Copy number variation analysis in 189 Romanian patients with global developmental delay/intellectual disability

BackgroundDevelopmental delay and intellectual disability represent a common pathology in general population, involving about 3% of the pediatric age population, the genetic etiology being often involved. The aim of this study was to determine the clinically relevant copy number variants in patients diagnosed with global developmental delay/intellectual disability in our population, using the chromosomal microarray analysis.MethodsWe analyzed 189 patients diagnosed with global developmental delay/intellectual disability, presented in Clinical Emergency Hospital for Children, Cluj-Napoca. The patients were completely clinically investigated, including dysmorphic and internal malformations evaluation, psychiatric, neuropsychological and metabolic evaluation, standard karyotyping. Genomic analysis was done using chromosomal microarray analysis.ResultsPathogenic findings (including uniparental disomy) and variants of unknown significance were detected in 53 of 189 patients (28.04%). Pathogenic copy number variants and uniparental disomy were observed in 35 of 189 patients (18.51%). Two patients presented uniparental disomy for chromosome 15, one with clinical phenotype of Prader-Willi syndrome and the other with clinical phenotype with Angelman syndrome. Within the category of pathogenic findings, the recurrent copy number variants were seen in 21 of 35 patients (60%).ConclusionsThe increased percentage of pathogenic structural variants observed in patients with global developmental delay/intellectual disability analyzed by chromosomal microarray technique supports its use in patients with a non-specific phenotype such as these neurodevelopmental disorders. The high percentage of recurrent pathogenic variants between these findings is a finding that support their initial evaluation when a genetic testing algorithm could be a useful option.

Accurate Detection of Clinically Actionable Copy Number Variants in Diverse Hematological Neoplasms By Routine Targeted Sequencing: A Comparative Performance Study

Background: Accurate genotyping of Copy Number Variations (CNVs) is an essential part of the clinical evaluation of diverse hematologic malignancies. Conventional karyotyping and FISH are current gold standards to assess CNVs, but both have some limitations. Next-generation targeted sequencing panels are commonly incorporated into current clinical practice in most cancer centers. We examined the utility of CANARy (Copy number ANomaly Assessment and Recovery), a CNV profiling method for three hematological malignancies using targeted panel sequencing data. Patients and Methods: We studied patients with myelodysplastic syndrome (MDS), chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), and plasma cell disorders (PCD) identified from a cohort of 4752 hematological specimens profiled at Stanford University by conventional cytogenetics and next-generation sequencing (NGS) between 2018 and 2021. We curated a cohort of 200 patients diagnosed with MDS (n=72), CLL (n=46), and PCDs including MM, AL amyloidosis, and MGUS (PCD, n=82), along with 50 cytogenetically normal controls. Specimens were bone marrow aspirates, peripheral blood, or FFPE tissue. All samples were characterized by conventional metaphase karyotyping and/or FISH and we focused on the analysis of clinically relevant CNV (-5q, -7/-7q, -20q, +8 for MDS; -11q22, -13q14, -17p13, +12 for CLL; and +1q21, -1p32, -13q14, -17p.13 for PCD). The specimens separately had clinical NGS (164 genes, Heme-STAMP) performed no more than four weeks from standard cytogenetic analysis. We applied multiple CNV enumeration tools to sequencing data for CNV genotyping at these loci, including our recently developed method CANARy (Chabon et al, Nature 2020). CANARy is a read depth-based, genome-wide CNV caller from targeted sequencing data that combines information from on-target and off-target fragments to assign a final Z-score and copy number index to a set of pre-defined genomic regions. We benchmarked CANARy alongside three others publicly available CNV callers: CNVkit (Talevich et al, PLoS Comput Biol. 2016), ichorCNA (Adalsteinsson et al, Nat Commun. 2017), and WISECONDOR (Straver et al, Nucleic Acids Res. 2014). All tools were run using the default parameters and following author recommendations. We calculated the accuracy and other statistics parameters for each CNV caller, considering karyotype/FISH as the true reference. We estimated tumor fraction using two strategies: the variant allele fraction (VAF) of disease-related driver mutations and using the ichorCNA estimate. Results: A total of 211 CNVs were identified by karyotype and/or FISH, with disease-specific distribution summarized in Fig. A. CANARy detected 148/211 (70.1%), CNVkit 88/211 (41.7%), ichorCNA 116/211 (55%) and WISECONDOR 158/211 (74.9%). The overall accuracy for CANARy, CNVkit, ichorCNA and WISECONDOR was 86.3%, 81.8%, 84.3% and 83.1% respectively. CANARy had both the highest overall accuracy (86.3%) and overall F1-score (0.73) (Fig.B). WISECONDOR had the highest number of false positives (83 vs. CANARy = 48). Interestingly, all CNV tools performed better in MDS specimens, with 92% accuracy for CANARy in MDS samples, likely related to the higher neoplastic purity of these samples. The median ichorCNA tumor fraction estimates for MDS, CLL and PCD were 22% (0-99%), 14% (0-70%), and 7% (0-94%), respectively. In addition, CNV calling was inferior in PCD tumors than other tumor types. Within CLL, -11q22 and -13q14 lesions were more challenging to detect with any of the four methods, with CANARy sensitivity of 50% and 31.3% respectively, likely related to the smaller minimal deletion regions of these lesions. The 17p deletion in CLL cases, known for its poor prognostic impact, was best identified by CANARy; with a 100% sensitivity and a 97.6% specificity. The only non-concordant result for -17p was a false positive that was detected by all 4 NGS CNV genotypers but not FISH, possibly suggesting a false negative FISH result. Conclusions: CANARy accurately identifies clinically significant CNVs in common hematologic neoplasms and could thus serve as a complementary approach to the current cytogenetic evaluation. CANARy uses data already generated from targeted sequencing panels and therefore does not impose additional work or cost. Low tumor fraction could be a limitation for CNV calling. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Detection of copy number variants and genes by chromosomal microarray in an Emirati neurodevelopmental disorders cohort.

Copy number variations (CNVs) are highly implicated in the etiology of neurodevelopmental disorders (NDDs), and chromosomal microarray analysis (CMA) has been recommended as a first-tier test for many NDDs. We undertook a study to identify clinically relevant CNVs and genes in an ethnically homogenous population of the United Arab Emirates. We genotyped 98 patients with NDDs using genome-wide chromosomal microarray analysis, and observed 47.1% deletion and 52.9% duplication CNVs, of which 11.8% are pathogenic, 23.5% are likely pathogenic, and 64.7% VOUS. The average size of copy number losses (3.9Mb) was generally higher than of gains (738.4kb). Analysis of VOUS CNVs for constrained genes (enrichment for brain critical exons and high pLI genes) yielded 7 unique genes. Among these 7 constrained genes, we propose FNTA and PXK as potential candidate genes for neurodevelopmental disorders, which warrants further investigation. Thirty-two overlapping CNVs (Decipher and ClinVar) containing the FNTA gene were previously identified in NDD patients and 6 overlapping CNVs (Decipher and ClinVar) containing the PXK gene were previously identified in NDD patients. Our study supports the utility of CMA for CNV profiling which aids in precise genetic diagnosis and its integration into therapeutics and management of NDD patients.

EP443: Fetal fraction amplification within NIPS enables detection of clinically-relevant genome-wide copy-number variants to 1Mb resolution

While clinically relevant copy number variants (CNVs) occur in 6-8% of pregnancies with fetal structural anomalies, they are typically difficult to detect via noninvasive prenatal screening (NIPS) because of the low fetal fraction in cell free DNA (cfDNA) isolated from maternal plasma. By leveraging fetal fraction amplification, a method to preferentially sequence shorter fragments that are enriched for fetal-derived cfDNA, we can detect small CNVs with high sensitivity. Here we describe an analytical validation of an NIPS that can detect clinically relevant CNVs across the genome.

Chromosomal microarray analysis of 410 Han Chinese patients with autism spectrum disorder or unexplained intellectual disability and developmental delay

Copy number variants (CNVs) are recognized as a crucial genetic cause of neurodevelopmental disorders (NDDs). Chromosomal microarray analysis (CMA), the first-tier diagnostic test for individuals with NDDs, has been utilized to detect CNVs in clinical practice, but most reports are still from populations of European ancestry. To contribute more worldwide clinical genomics data, we investigated the genetic etiology of 410 Han Chinese patients with NDDs (151 with autism and 259 with unexplained intellectual disability (ID) and developmental delay (DD)) using CMA (Affymetrix) after G-banding karyotyping. Among all the NDD patients, 109 (26.6%) carried clinically relevant CNVs or uniparental disomies (UPDs), and 8 (2.0%) had aneuploidies (6 with trisomy 21 syndrome, 1 with 47,XXY, 1 with 47,XYY). In total, we found 129 clinically relevant CNVs and UPDs, including 32 CNVs in 30 ASD patients, and 92 CNVs and 5 UPDs in 79 ID/DD cases. When excluding the eight patients with aneuploidies, the diagnostic yield of pathogenic and likely pathogenic CNVs and UPDs was 20.9% for all NDDs (84/402), 3.3% in ASD (5/151), and 31.5% in ID/DD (79/251). When aneuploidies were included, the diagnostic yield increased to 22.4% for all NDDs (92/410), and 33.6% for ID/DD (87/259). We identified a de novo CNV in 14.9% (60/402) of subjects with NDDs. Interestingly, a higher diagnostic yield was observed in females (31.3%, 40/128) compared to males (16.1%, 44/274) for all NDDs (P = 4.8 × 10−4), suggesting that a female protective mechanism exists for deleterious CNVs and UPDs.