Recurrent Copy Number Variations Research Articles

Abstract PO4-01-02: High throughput analysis in HER2 positive locally advanced breast cancer: pCR and mutational status

Abstract Background Pathologic complete response (pCR) after neoadjuvant treatment (NAD) is a strong prognostic biomarker associated with improved survival in breast cancer patients, especially with HER2-positive and Triple negative subtypes. PIK3CA is mutated in up to 20% of HER2 positive breast cancers, contributes to anti-HER-2 resistance and may be predictive of the lack of response to anti HER2 NAD. PIK3CA mutations in breast cancer occur primarily at hotspots E545K at exon 9 and H1047R at exon 20. Next generation sequencing has improved our knowledge regarding the biology of the mechanisms behind treatment resistance. The aim of this study is to evaluate the genomic landscape of tissue samples obtained from locally advanced breast cancer patients treated in an neoadjuvant setting, in order to correlate the genomic analysis to response and clinical outcome. Methods DNA extracted from the archival 84 formalin-fixed paraffin-embedded (FFPE) samples from core needle biopsies was subjected to deep sequencing using the TruSight Oncology (TSO) 500 panel (Illumina, San Diego, USA; 523 genes, size: 1.94 Mb), following the manufacturer’s protocol, which assesses microsatellite instability (MSI) status, tumor mutation burden (TMB), recurrent somatic copy number variations (sCNV), somatic single nucleotide variants (SNVs). Libraries were sequenced on a NovaSeq 6000 instrument (Illumina) to reach a minimum of 500 × read depth. All samples were evaluated for RNA single cell transcriptional profile. 73 of 84 patients were treated with traditional neoadjuvant chemotherapy plus trastuzumab meanwhile 13 of 84 received neoadjuvant chemotherapy plus trastuzumab and pertuzumab. Statistical analysis was performed to detect the genes whose mutation involved in the progression and/or non-response of the disease using the Statistical Package for Social Science (SPSS), release 23.0. Continuous variables were expressed as mean ± SD or median [range], categorical variables were displayed as frequencies and the appropriate parametric (Student‘s t-test) non-parametric test (X2-test or Mann-Whitney test) was used to assess significance of the differences between subgroups (patients with or without pCR). Results In total we analyzed samples of 84 patients. The median age was 48 years. 64 of 84 (76.2%) were triple positive, 66 (78.6%) with G3 and 29 (34.5%) with stage III. The most common frequently mutated genes were PIK3CA (16/84, 19%) and BRCA1 (7/84, 8.3%). The pathogenic variant of BRCA1 (sBRCA1) had a significant association with resistance to treatment: we found a pCR in only 14.3% of sBRCA1 versus 62.3% in sBRCA1 wild type (wt) patients (p=0.014). We also identified 11 somatic mutations in hereditary genes (ATM, BRCA1, BRCA2, PALB2, CHEK2, RAD51C, RAD51D). From a germline standpoint we detected 7 pathogenic variants (two affecting BRCA1, two affecting CHEK2, one affecting PALB2, 7/84, 8.3%), which will be clinically confirmed. Among patients with a mutated PI3KCA, 43.8% obtained a pCR versus 61.8% of patients with PI3KCA wt (p=0.19). This data probably presents an imbalance due to the sample size A PTEN mutation was identified in 2% of pts (2/84) which obtained no benefit in terms of pCR after NAD versus 59.8% in wt patients (p=0.09). Conclusions In this preliminary report, genetic drivers such as BRCA1 status may have a clinical implication for prognosis and treatment response after NAD in locally advanced HER2 positive breast cancer. More samples are needed to evaluate the significance of PI3K and PTEN mutations in this patient setting. We are waiting for the results of the RNA single cell transcriptional profile of these samples. Event free survival data is not yet mature at this moment. We would like to acknowledge the contribution of Multi-specialistic Biobank Research Core Facility G-STeP, Fondazione Policlinico Universitario “A. Gemelli” IRCCS (Biobank-FPG) who provided the bio-resources. Citation Format: Ida Paris, Angelo Minucci, Daniele Generali, simona Nanni, Tatiana D'angelo, Francesco Pavese, Alessandra Fabi, Maria De Bonis, Katia Cannita, Alessia Piermattei, Giordana Tiberi, Paola Fuso, Luisa Carbognin, Silvia Rotondaro, Camilla Nero, Flavia Giacomini, Tina Pasciuto, Maria Marrazzo, Luciano Giaco', Antonino Mulè, Riccardo Masetti, Gianluca Franceschini, Giorgia Garganese, Diana Giannarelli, Giovanni Scambia. High throughput analysis in HER2 positive locally advanced breast cancer: pCR and mutational status [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-01-02.

Association of behavioural and social-communicative profiles in children with 16p11.2 copy number variants: a multi-site study.

Despite the established knowledge that recurrent copy number variants (CNVs) at the 16p11.2 locus BP4-BP5 confer risk for behavioural and language difficulties, limited research has been conducted on the association between behavioural and social-communicative profiles. The current study aims to further delineate the prevalence, nature and severity of, and the association between, behavioural and social-communicative features of school-aged children with 16p11.2 deletion syndrome (16p11.2DS) and 16p11.2 duplication (16p11.2Dup). A total of 68 individuals (n=47 16p11.2DS and n=21 16p11.2Dup) aged 6-17years participated. Standardised intelligence tests were administered, and behavioural and social-communicative skills were assessed by standardised questionnaires. Scores of both groups were compared with population norms and across CNVs. The influence of confounding factors was investigated, and correlation analyses were performed. Compared with the normative sample, children with 16p11.2DS showed high rates of social responsiveness (67%) and communicative problems (69%), while approximately half (52%) of the patients displayed behavioural problems. Children with 16p11.2Dup demonstrated even higher rates of social-communicative problems (80-90%) with statistically significantly more externalising and overall behavioural challenges (89%). In both CNV groups, there was a strong positive correlation between behavioural and social-communicative skills. School-aged children with 16p11.2 CNVs show high rates of behavioural, social responsiveness and communicative problems compared with the normative sample. These findings point to the high prevalence of autistic traits and diagnoses in these CNV populations. Moreover, there is a high comorbidity between behavioural and social-communicative problems. Patients with difficulties in both domains are vulnerable and need closer clinical follow-up and care.

Chromosomal microarray testing yield in 829 cases of microcephaly: a clinical characteristics-based analysis for prenatal and postnatal cases.

Microcephaly, characterized by abnormal head growth, can often serve as an initial indicator of congenital, genetic, or acquired disorders. In this study, we sought to evaluate the effectiveness of chromosomal microarray (CMA) testing in detecting abnormalities in both prenatal and postnatal cases of microcephaly. CMA Testing: We conducted CMA testing on 87 prenatally-detected microcephaly cases and 742 postnatal cases at a single laboratory. We evaluated the CMA yield in relation to specific clinical characteristics. In prenatal cases, pathogenic and likely pathogenic (LP) results were identified in 4.6% of cases, a significantly higher rate compared to low-risk pregnancies. The male-to-female ratio in this cohort was 3, and the CMA yield was not influenced by gender or other clinical parameters. For postnatal cases, the CMA yield was 15.0%, with a significantly higher detection rate associated with dysmorphism, hypotonia, epilepsy, congenital heart malformations (CHM), learning disabilities (LD), and a history of Fetal growth restriction (FGR). No specific recurrent copy number variations (CNVs) were observed, and the rate of variants of unknown significance was 3.9%. The yield of CMA testing in prenatal microcephaly is lower than in postnatal cases (4.6% vs. 15%). The presence of microcephaly, combined with dysmorphism, hypotonia, epilepsy, CHD, LD, and FGR, significantly increases the likelihood of an abnormal CMA result.

The role of copy number variants in the genetic architecture of common familial epilepsies.

Copy number variants (CNVs) contribute to genetic risk and genetic etiology of both rare and common epilepsies. Whereas many studies have explored the role of CNVs in sporadic or severe cases, fewer have been done in familial generalized and focal epilepsies. We analyzed exome sequence data from 267 multiplex families and 859 first-degree relative pairs with a diagnosis of genetic generalized epilepsies or nonacquired focal epilepsies to predict CNVs. Validation and segregation studies were performed using an orthogonal method when possible. We identified CNVs likely to contribute to epilepsy risk or etiology in the probands of 43 of 1116 (3.9%) families, including known recurrent CNVs (16p13.11 deletion, 15q13.3 deletion, 15q11.2 deletion, 16p11.2 duplication, 1q21.1 duplication, and 5-Mb duplication of 15q11q13). We also identified CNVs affecting monogenic epilepsy genes, including four families with CNVs disrupting the DEPDC5 gene, and a de novo deletion of HNRNPU in one affected individual from a multiplex family. Several large CNVs (>500 kb) of uncertain clinical significance were identified, including a deletion in 18q, a large duplication encompassing the SCN1A gene, and a 15q13.3 duplication (BP4-BP5). The overall CNV landscape in common familial epilepsies is similar to that of sporadic epilepsies, with large recurrent deletions at 15q11, 15q13, and 16p13 contributing in 2.5%-3% of families. CNVs that interrupt known epilepsy genes and rare, large CNVs were also identified. Multiple etiologies were found in a subset of families, emphasizing the importance of genetic testing for multiple affected family members. Rare CNVs found in a single proband remain difficult to interpret and require larger cohorts to confirm their potential role in disease. Overall, our work indicates that CNVs contribute to the complex genetic architecture of familial generalized and focal epilepsies, supporting the role for clinical testing in affected individuals.

Cell-Free DNA from Blood or Bone Marrow as Alternatives to Bone Marrow Cells for Molecular Diagnostics and Monitoring of Multiple Myeloma

Introduction: Molecular profiling of bone marrow (BM) tumor cells in multiple myeloma (MM) can be limited by low tumor cell content for analysis by multiple assays. We therefore evaluated the concordance of molecular profiles of cancer cells enriched from BM versus cell-free DNA (cfDNA) derived from peripheral blood (PB) and BM plasma collected at diagnosis. Use of cfDNA has the potential to enable the prioritization of scarce cancer cells for single-cell RNA-sequencing or other applications that require intact cells. Methods: The MM Molecular Monitoring (M4) Study is a prospective cohort study of newly diagnosed MM patients (pts) receiving standard-of-care treatment at 12 sites across Canada. From 27 pts in this study, we analyzed diagnostic BM-derived CD138+ enriched plasma cell (PC) DNA (n=27, 15-20 mL BM per pt), BM plasma cfDNA (n=26 same tube as BM sampling), PB cfDNA (n=27, 15-20 mL), and PB buffy coat DNA (n=27, 4-6mL) using a combination of a targeted next generation sequencing (NGS) panel (10,000X coverage) and shallow (0.1-1X) whole-genome sequencing (sWGS). PB and BM samples collected in STRECK and EDTA tubes, respectively incurred overnight shipping to a central lab for initial processing. The targeted panel includes exons from 37 genes of potential clinical relevance, all immunoglobulin (Ig) loci, and 5 MM-specific translocation hotspots. CD138+ PCs were profiled by clinical FISH for recurrent MM translocations and copy number variations (CNVs). Results: All PB and BM cfDNA samples yielded sufficient DNA quantity for analyses (>20ng), yet only 19/26 BM cfDNA samples passed quality control and were sequenced. Various strategies such as shearing, dilution, and the use of heparinase were not effective in salvaging the failed BM cfDNA. However, the high success rate in a separate local study (24/25 BM cfDNA samples) suggests shipping in non-cell stabilizing tubes may have contributed to failures. The average DNA yield for PB and BM cfDNA samples was 1408ng (sd = 2752ng) and 2462ng (sd = 4301ng), respectively. We estimated tumor fraction (TF) by examining the proportion of genome altered by CNVs using ichorCNA in samples with sWGS. 14/24 (58%) of PB cfDNA samples and 10/18 (56%) of BM cfDNA samples had a high TF (>5%). TF in PB and BM cfDNA were significantly correlated (rho = 0.85, p < 0.001, n = 18 pairs), but were consistently higher in PB cfDNA (p = 0.03, n = 18 pairs). We first compared mutations between BM-derived PCs and cfDNA. We found 12 distinct somatic mutations (variant allele fraction >1%) that were previously reported to have a pathogenic effect in 11/27 (41%) PC samples. This consisted of 10 single nucleotide variants (SNVs) and 2 deletions across NRAS, BRAF, KRAS, CYLD and PRDM1. We found 9/12 mutations in matched PB cfDNA samples [sensitivity (SN)=75%] and 4/8 mutations in matched BM cfDNA (SN = 50%). We next searched for FISH-validated chromosome rearrangements among enriched BM PCs. From these, we correctly detected 5/8 (63%) fusions using the targeted panel that were called out by clinical FISH. These were further detected in matched PB cfDNA (4/5) and BM cfDNA (2/4) samples. Recurrent MM CNVs (amplification at chromosome 1q or deletions at chromosomes 1p, 13p or 17p) were identified in 18/27 (67%) PC samples by sWGS. These CNVs were successfully detected in 8/9 matched PB cfDNA samples with high TF (by sWGS) and in 2/7 samples with low TF. In BM cfDNA, CNVs were successfully detected in 7/8 high TF samples and 1/6 low TF samples. Lastly, we evaluated BCR Ig rearrangements. We detected a clonal Ig rearrangement in 26/27 (96%) BM-derived PC samples. These clones were detected in 25/26 (96%) matched PC to PB cfDNA pairs and in 16/18 (89%) PC to BM cfDNA pairs. The proportion of fragments at Ig loci which corresponded to productive Ig rearrangements was significantly correlated with TF in PB cfDNA (rho = 0.62, p = 0.002, n = 23) but not in BM cfDNA (rho = 0.37, p = 0.14, n = 17). Conclusions: PB cfDNA yielded 78% of the aberrations detected in BM-derived PCs, whereas BM cfDNA yielded 62%. The lower rate of detection in BM cfDNA may reflect DNA contamination from dying white blood cells as a result of shipping BM samples in non-cell stabilizing preservative tubes. BM and PB cfDNA contain highly concordant CNV calls to PCs when cfDNA TF estimates were >5%. Tumor Ig rearrangements were more readily detected in cfDNA compared to other sources of variation and may provide a less-invasive alternative for disease monitoring.

CNVs Numbers Scoring System Was a Good Prognostic Risk Stratification for NDMM with Whole Genomic Mapping Array

Multiple myeloma (MM) is a genetically heterogeneous disease characterized by plasma cell clonal proliferation. Due to the potential complex cytogenetic abnormalities, the clinical progresses are heterogeneous. Thus, genome-wide identification of cytogenetic abnormalities is necessary given the limitations of traditional cytogenetic detecting methods. In this study, we utilized whole genomic mapping methods Cytoscan 750K array to analyze the genomic copy number variations (CNVs) of 611 Newly Diagnosed MM (NDMM) from The First Affiliated Hospital of Soochow University. The results showed that 95.3% of NDMM patients harbored the chromosome alterations including structural or numerical abnormalities, and most of them (67.3%) were accompanied both, including 23.7% with only structural abnormalities and 4.3% with only numerical abnormalities. The average number of CNV per patient was 9. In addition, we have identified 34 common CNV events with a frequency of ≥5%, 5 of which were high frequency (≥20%), including dup(1)(q21q44) (50.2%), del(14)(q22q32) (24.9%), del(1)(p31p12) (21.9%), del(13)(q12q22) (21.9%) and dup(19)(p13p12) (20.6%). Additionally, 6 CNVs with median frequency (≥15% and <20%) included dup(6)(p25p12) (16.9%), dup(9)(q21q34) (18.2%), del(16)(q12q24) (18.7%), del(8)(p23p12) (17.5%), dup(11)(q13q25) (15.7%) and del(22)(q11q12) (15.4%). And the other 23 CNVs were low frequency (<15%), including del(6)(q24q27) (14.6%), del(X)(p22q25) (14.1%), dup(X)(q28) (13.7%), dup(8) (q11q24) (12.6%), del(17)(p13p12) (11%) and so on. Most CNVs were closely related with clinical risk factors of MM such as LDH. Prognostic analysis based on the VRD treatment (n=353, the median follow-up was 22 months) showed that patients with only numerical abnormalities had a better progress free survival (PFS) outcome than that with structural abnormalities only or with both, although they seemed to have the similar overall survival (OS) rate. Furthermore, univariate analysis showed that 6 CNVs were correlated with poor PFS including 1q+, 4q-, 6q-, 12p-, 16q-, 17p-, and 5 CNVs were correlated with poor OS including 1p-, 4q-, 7p-, 12p-, 17p-. For the effect of counts of CNV events on MM prognosis, the receiver operating characteristic (ROC) curve analysis was performed to calculate the best cutoff, with a result of 23 CNV events. Therefore, MM patients were divided into 4 subgroups according to the counts of CNV they harbored, including high complexity group (CNVs≥23, n=32) which excluded patients with chromothripsis (more than 10 CNVs on the same chromosome) , median complexity group (10≤CNVs<23, n=168), low complexity group (1≤CNVs<10, n=324), and no CNVs group (n=73). The patients in high complexity group had the worst prognosis of PFS and OS (media PFS 18 months, media OS 29 months, P<0.001). As expected, some common high risk clinical factors such as 1q+, 17p-, DS III, ISS III, serum Ca++,β2-MG and plasma cells in BM were gradually increased among the group from low to high complexity, but decreased in the rate of complete remission (CR), which suggested that the risk classification system was reliable. Importantly, univariate and multivariate COX regression analysis showed that CNV≥23 was an independent prognostic risk factor affecting both PFS and OS in NDMM patients. Over all, we utilized a large MM cohort to identify recurrent and potential driver CNV events, and their correlation with prognosis. In addition, the count of CNVs in NDMM patient was creatively considered to be the basis for prognostic risk stratification.

Structural Variation Evolution at the 15q11-q13 Disease-Associated Locus.

The impact of segmental duplications on human evolution and disease is only just starting to unfold, thanks to advancements in sequencing technologies that allow for their discovery and precise genotyping. The 15q11-q13 locus is a hotspot of recurrent copy number variation associated with Prader-Willi/Angelman syndromes, developmental delay, autism, and epilepsy and is mediated by complex segmental duplications, many of which arose recently during evolution. To gain insight into the instability of this region, we characterized its architecture in human and nonhuman primates, reconstructing the evolutionary history of five different inversions that rearranged the region in different species primarily by accumulation of segmental duplications. Comparative analysis of human and nonhuman primate duplication structures suggests a human-specific gain of directly oriented duplications in the regions flanking the GOLGA cores and HERC segmental duplications, representing potential genomic drivers for the human-specific expansions. The increasing complexity of segmental duplication organization over the course of evolution underlies its association with human susceptibility to recurrent disease-associated rearrangements.

Recurrent copy number variations in the human fungal pathogen Candida parapsilosis.

Candida parapsilosis is an opportunistic fungal pathogen with increasing incidence in hospital settings worldwide; however, we lack a comprehensive understanding of the mechanisms promoting its virulence and drug resistance. Bergin et al. systematically quantify the frequency and effect of copy number variation (CNV) across 170 diverse clinical and environmental isolates of C. parapsilosis (Bergin SA, Zhao F, Ryan AP, Müller CA, Nieduszynski CA, Zhai B, Rolling T, Hohl TM, Morio F, Scully J, Wolfe KH, Butler G, 2022, mBio, https://doi.org/10.1128/mbio.01777-22). Using a combination of both short- and long-read whole genome sequencing techniques, they determine the structure and copy number of two CNVs that arose recurrently throughout the evolution of these isolates. Each CNV predominantly amplifies one coding sequence (ARR3 or RTA3); however, the amplitude and recombination breakpoints are variable across the isolates. Amplification of RTA3 correlates with drug resistance and deletion causes drug susceptibility. This study highlights the need for further research into the mechanisms and dynamics of CNV formation and the impact of these CNVs on virulence and drug resistance across diverse fungal pathogens.

Integrative analysis of multi-omics data reveals a pseudouridine-related lncRNA signature for prediction of glioma prognosis and chemoradiotherapy sensitivity

BackgroundGlioblastoma is the most common type of glioma with a high incidence and poor prognosis, and effective medical treatment remains challenging. Pseudouridine (Ψ) is the first post-transcriptional modification discovered and one of the most abundant modifications to RNA. However, the prognostic value of Ψ-related lncRNAs (ΨrLs) for glioma patients has never been systematically evaluated. This study aims to construct a risk model based on ΨrLs signature and to validate the predictive efficiency of the model. MethodTranscriptomic data, genomic data, and relevant clinical data of glioma patients were extracted from the Cancer Genome Atlas (TCGA) and the Chinese Glioma Genome Atlas (CGGA). ΨrLs with significant correlation with Ψ-related genes were identified, and univariate Cox regression, least absolute shrinkage and selection operator (LASSO) regression, and multivariate Cox regression were used to further select biomarkers and construct a ΨrLs signature risk model. Then, the expression of lncRNAs of ΨrLs signature in multiple glioma cell lines was detected by qPCR. Further, ROC analysis, stratification analysis, correlation analysis, survival analysis, nomogram, enrichment analysis, immune infiltration analysis, chemoradiotherapy sensitivity analysis, somatic mutation, and recurrent copy number variation (CNV) analysis were used to validate the predictive efficiency of ΨrLs signature in TCGA and CGGA datasets. ResultsA four-lncRNA ΨrLs signature (DNAJC27-AS1, GDNF-AS1, ZBTB20-AS4, and DNMBP-AS1) risk model was constructed. By ROC analysis, stratified analysis, correlation analysis, survival analysis, and nomogram, the signature showed satisfactory predictive efficiency. Functional enrichment analysis revealed the differences in immune-related biological processes between high- and low-risk groups. Immune infiltration analysis showed that the high-risk group had lower tumor purity and higher stromal, immune and ESTIMATE scores. Mitoxantrone was identified as effective drug for low-risk group of glioma patients. Key genes in glioma development, including IDH1, EGFR, PTEN, etc., were differentially mutated between risk groups. The main recurrent CNVs in low-risk groups were 19q13.42 deletion and 7q34 amplification; 10q23.31 deletion and 12q14.1 in the high-risk group. ConclusionsOur study identified a four-lncRNA ΨrLs signature that effectively predicts the prognosis of glioma patients and may serve as a diagnostic tool. Risk scores of glioma patients generated by the signature is associated with immune-related biological processes and chemoradiotherapy sensitivity. These findings may inform the development of more targeted and effective therapies for glioma patients.

Preliminary study of noninvasive prenatal screening for 22q11.2 deletion/duplication syndrome using multiplex dPCR assay

ObjectiveThis study aimed to establish a cell-free fetal DNA (cffDNA) assay using multiplex digital PCR (dPCR) for identifying fetuses at increased risk of 22q11.2 deletion/duplication syndrome.MethodsSix detection sites and their corresponding probes were designed for the 22q11.2 recurrent region. A dPCR assay for the noninvasive screening of 22q11.2 deletion/duplication syndrome was established. A total of 130 plasma samples from pregnant women (including 15 samples with fetal 22q11.2 deletion/duplication syndrome) were blindly tested for evaluating the sensitivity and specificity of the established assay.ResultsDNA with different sizes of 22q11.2 deletion/duplication was detected via dPCR, indicating that the designed probes and detection sites were reasonable and effective. In the retrospective clinical samples, 11 out of 15 samples of pregnant women with 22q11.2 deletion/duplication were detected during the cffDNA assay, and accurate regional localization was achieved. Among the 115 normal samples, 111 were confirmed to be normal. Receiver operating characteristic curves were used for assessing the cut-off values and AUC for these samples. The sensitivity, specificity, and positive as well as negative predictive values were 73.3%, 96.5%, 73.3%, and 96.5%, respectively.ConclusionThe cffDNA assay based on dPCR technology for the noninvasive detection of 22q11.2 recurrent copy number variants in fetuses detected most affected cases, including smaller but relatively common nested deletions, with a low false-positive rate. It is a potential, efficient and simple method for the noninvasive screening of 22q11.2 deletion/duplication syndrome.

Clinical exome sequencing efficacy and phenotypic expansions involving anomalous pulmonary venous return.

Anomalous pulmonary venous return (APVR) frequently occurs with other congenital heart defects (CHDs) or extra-cardiac anomalies. While some genetic causes have been identified, the optimal approach to genetic testing in individuals with APVR remains uncertain, and the etiology of most cases of APVR is unclear. Here, we analyzed molecular data from 49 individuals to determine the diagnostic yield of clinical exome sequencing (ES) for non-isolated APVR. A definitive or probable diagnosis was made for 8 of those individuals yielding a diagnostic efficacy rate of 16.3%. We then analyzed molecular data from 62 individuals with APVR accrued from three databases to identify novel APVR genes. Based on data from this analysis, published case reports, mouse models, and/or similarity to known APVR genes as revealed by a machine learning algorithm, we identified 3 genes-EFTUD2, NAA15, and NKX2-1-for which there is sufficient evidence to support phenotypic expansion to include APVR. We also provide evidence that 3 recurrent copy number variants contribute to the development of APVR: proximal 1q21.1 microdeletions involving RBM8A and PDZK1, recurrent BP1-BP2 15q11.2 deletions, and central 22q11.2 deletions involving CRKL. Our results suggest that ES and chromosomal microarray analysis (or genome sequencing) should be considered for individuals with non-isolated APVR for whom a genetic etiology has not been identified, and that genetic testing to identify an independent genetic etiology of APVR is not warranted in individuals with EFTUD2-, NAA15-, and NKX2-1-related disorders.

Exome sequencing identified rare recurrent copy number variants and hereditary breast cancer susceptibility.

Copy number variants (CNVs) are a major source of genetic variation and can disrupt genes or affect gene dosage. They are known to be causal or underlie predisposition to various diseases. However, the role of CNVs in inherited breast cancer susceptibility has not been thoroughly investigated. To address this, we performed whole-exome sequencing based analysis of rare CNVs in 98 high-risk Northern Finnish breast cancer cases. After filtering, selected candidate alleles were validated and characterized with a combination of orthogonal methods, including PCR-based approaches, optical genome mapping and long-read sequencing. This revealed three recurrent alterations: a 31 kb deletion co-occurring with a retrotransposon insertion (delins) in RAD52, a 13.4 kb deletion in HSD17B14 and a 64 kb partial duplication of RAD51C. Notably, all these genes encode proteins involved in pathways previously identified as essential for breast cancer development. Variants were genotyped in geographically matched cases and controls (altogether 278 hereditary and 1983 unselected breast cancer cases, and 1229 controls). The RAD52 delins and HSD17B14 deletion both showed significant enrichment among cases with indications of hereditary disease susceptibility. RAD52 delins was identified in 7/278 cases (2.5%, P = 0.034, OR = 2.86, 95% CI = 1.10-7.45) and HSD17B14 deletion in 8/278 cases (2.9%, P = 0.014, OR = 3.28, 95% CI = 1.31-8.23), the frequency of both variants in the controls being 11/1229 (0.9%). This suggests a role for RAD52 and HSD17B14 in hereditary breast cancer susceptibility. The RAD51C duplication was very rare, identified only in 2/278 of hereditary cases and 2/1229 controls (P = 0.157, OR = 4.45, 95% CI = 0.62-31.70). The identification of recurrent CNVs in these genes, and especially the relatively high frequency of RAD52 and HSD17B14 alterations in the Finnish population, highlights the importance of studying CNVs alongside single nucleotide variants when searching for genetic factors underlying hereditary disease predisposition.

Association of prenatal thoracic ultrasound abnormalities with copy number variants at a single Chinese tertiary center.

To systematically evaluate the association of prenatal thoracic ultrasound abnormalities with copy number variants (CNVs). Chromosomal microarray (CMA) data and clinical characteristics from fetuses with thoracic ultrasound abnormalities were retrieved and analyzed. Thoracic ultrasound findings were mainly isolated except for fetal pleural effusion (FPE) and pulmonary hypoplasia. The diagnostic yield of CMA for thoracic anomaly was 9.66%, and FPE (17/68, 25%), pulmonary hypoplasia (1/8, 12.5%), and congenital diaphragmatic hernia (CDH) (6/79, 7.59%) indicated relatively high pathogenic/likely pathogenic (P/LP) CNV findings. The detection rate for P/LP CNVs was obviously increased in non-isolated thoracic anomalies (27.91% vs. 1.96%, P < 0.0001), non-isolated FPE (37.78% vs. 0%, P = 0.0007) and non-isolated congenital pulmonary airway malformation (CPAM) (27.27% vs. 0%, P < 0.0001), and significantly different among thoracic anomalies. Additionally, the rate of termination of pregnancy in cases with non-isolated thoracic anomalies (58.49% vs. 12.34%, P < 0.0001) and P/LP CNVs (85.71% vs. 24.15%, P < 0.0001) was obviously increased. The present study expanded phenotype spectrums for particular recurrent CNVs. FPE, CDH, and pulmonary hypoplasia indicated relatively high P/LP CNV findings among common thoracic ultrasound abnormalities, CPAM associated with other ultrasound abnormalities increased the incidence of diagnostic CNVs, while bronchopulmonary sequestration might not be associated with positive CNVs. The present data recommended CMA application for cases with prenatal thoracic ultrasound abnormalities, especially non-isolated FPE, non-isolated CPAM, CDH, and pulmonary hypoplasia.

Integrated multiomics analysis of chromosome 19 miRNA cluster in bladder cancer

With 46 microRNAs (miRNAs) embedded tandemly over a distance of ~100 kb, chromosome 19 microRNA cluster (C19MC) is the largest miRNA cluster in the human genome. The C19MC is transcribed from a long noncoding genomic region and is usually expressed simultaneously at a higher level. Hence, we performed an integrative multiomics data analysis to examine C19MC regulation, expression patterns, and their impact on bladder cancer (BCa). We found that 43 members of C19MC were highly expressed in BCa. However, its co-localization with recurrent copy number variation (CNV) gain was not statistically significant to implicate its upregulation. It has been reported that C19MC expression is regulated by a well-established CpG island situated 17.6 kb upstream of the transcription start site, but we found that CpG probes at this island were hypomethylated, which was not statistically significant in the BCa cohort. In addition, the promoter region of C19MC is strongly regulated by a group of seven transcription factors (NR2F6, SREBF1, TBP, GATA3, GABPB1, ETV4, and ZNF444) and five chromatin modifiers (SMC3, KDMA1, EZH2, RAD21, and CHD7). Interestingly, these 12 genes were found to be overexpressed in BCa patients. Further, C19MC targeted 42 tumor suppressor (TS) genes that were downregulated, of which 15 were significantly correlated with patient survival. Our findings suggest that transcription factors and chromatin modifiers at the promoter region may regulate C19MC overexpression. The upregulated C19MC members, transcription regulators, and TS genes can be further exploited as potential diagnostic and prognostic indicators as well as for therapeutic management of BCa.

Proximal 1q21 duplication: A syndrome or a susceptibility locus?

Proximal 1q21 microduplication is an incomplete penetrance and variable expressivity syndrome. This study reports 28 new cases and summarizes data on phenotype, gender, and parental origin. Data on isolated proximal 1q21.1 microduplications (g. chr1:145,394,956-145,762,959 GRCh37/hg19) was retrieved in postnatal and prenatal "clinical cases" group, and prenatal "control group." The "clinical cases" cases included cases where chromosomal microarray (CMA) was performed due to congenital anomalies, autism spectrum disorder, seizures, and developmental delay/intellectual disability. The "control group" cases consisted of fetal CMA performed upon parental request despite normal nuchal translucency and anatomical second trimester fetal scans. We analyzed a local database of 27,990 cases and another cohort of 80,000 cases (including both indicated and non-indicated cases) for population frequency analysis. A total of 62 heterozygous cases were found, including 28 index cases and 34 family members. Among the index cases, 13 (9 males, 4 females) were identified in the "clinical cases" group, of which 10 had developmental abnormalities. Parental origin was tested in 9/13 cases, and all were found to be maternally inherited. In the "control group," which comprised non-affected cases, of 15 cases (10 males, 5 females), only 5/11 were maternally inherited. Four cases with clinical follow-up showed no reported neurodevelopmental abnormalities. No de-novo cases were detected, and the population frequency in both cohorts was 1:1000. Proximal 1q21.1 microduplication is a recurrent copy number variant, associated with neurodevelopmental abnormalities. It has a greater impact on males inheriting it from their mothers than females from their fathers.

Abstract A017: Molecular analysis of matched PIN, invasive prostate cancer, and adjacent normal prostate tissue samples reveal distinct transcriptional signatures and clonal relationships

Abstract Background: Prostate cancer (PCa) is highly heterogeneous, and while some tumors remain indolent throughout a patient's lifetime, others are highly aggressive, leading to significant morbidity and death. The current methods for PCa diagnosis and prognosis are highly suboptimal, resulting in over-diagnosis and overtreatment of many clinically insignificant tumors. To understand the nature of PCa heterogeneity, we conducted comprehensive genomic analyses on matched samples of adjacent normal prostate tissue, prostate intraepithelial neoplasia (PIN), and invasive PCa. Methods: To investigate the changes cells undergo during PCa development and progression, we analyzed genomic and transcriptomic alterations in 92 adjacent normal, 80 PIN, and 99 PCa samples from 34 patients. For each sample type, both epithelial and stromal components were analyzed separately using laser capture microdissection of formalin fixed paraffin embedded tissue. Transcriptomic analysis was performed using smart3-SEQ and copy number analysis was performed by low-pass whole genome sequencing (WGS). The samples were analyzed as three separate data sets. Results: We studied the spectrum of molecular changes present in both epithelial and stromal samples. A negative-binomial regression model was used to identify differentially expressed genes between adjacent normal prostate tissue and invasive PCa in epithelium and stroma separately using the discovery data set. The resulting gene lists were used to perform non-negative matrix factorization clustering of all samples in the three datasets separately. This analysis identified two epithelial and two stromal clusters with distinct RNA expression profiles in all three data sets. For invasive PCa samples, all except three epithelial samples and five stromal samples clustered in the ‘invasive-like’ clusters. Similarly for adjacent normal samples, all except three of the epithelial and six of the stromal samples clustered as ‘normal-like’ across the three datasets. For PIN samples, 54% of epithelial and 62% of stromal samples classified as ‘normal-like’. We performed phylogenetic analysis of WGS data and identified PIN lesions with and without clear clonal relation to invasive PCa lesions. Recurrent copy number variations were identified in both PIN and PCa lesions. Conclusion. We identified two distinct epithelial and two distinct stromal expression clusters with ‘invasive-like’ and ‘normal-like’ signatures. WGS analysis revealed recurrent copy number alterations in PIN and PCa lesions, and evidence of clonal relationships between PIN and invasive PCa. These studies provide new insight into PIN biology and the relationship between PIN and invasive PCa. Citation Format: Siri H. Strand, Okyaz Eminaga, Sujay Vennam, Chunfang Zhu, Jason Wang, Sushama Varma, Rosalie Nolley, Christian Kunder, Jonathan Pollack, Andreas Roeder, Karina D. Sorensen, James D. Brooks, Robert B. West. Molecular analysis of matched PIN, invasive prostate cancer, and adjacent normal prostate tissue samples reveal distinct transcriptional signatures and clonal relationships [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A017.

Clinical features and magnesium levels: Novel insights in 15q11.2 BP1-BP2 copy number variants.

Investigating copy number variations (CNVs) such as microdeletions or microduplications can significantly contribute to discover the aetiology of neurodevelopmental disorders. 15q11.2 genomic region, including NIPA1 and NIPA2 genes, contains a recurrent but rare CNV, flanked by the break points BP1 and BP2. Both BP1-BP2 microdeletion and microduplication have been associated with intellectual disability (ID), neuropsychiatric/behavioural disturbances and mild clinical features, even if with incomplete penetrance and variable expressivity. The pathogenic role of this CNV is quite unclear though. Unknown variants in other DNA regions and parent-of-origin effect (POE) are some of the mechanisms that have been proposed as an explanation of the wide phenotypic variability. As NIPA1 and NIPA2 encode for proteins that mediate magnesium (Mg2+ ) metabolism, it has been suggested that urinary Mg2+ levels could potentially represent informative and affordable biomarkers for a rapid screening of 15q11.2 duplications or deletions. Furthermore, magnesium supplementation has been proposed as possible therapeutic strategy. Thirty one children with ID and/or other neurodevelopmental disorders carrying either a duplication or a deletion in 15q11.2 BP1-BP2 region have been recruited. When available, blood samples from parents have been analysed to identify the CNV origin. All participants underwent family and medical data collection, physical examination and neuropsychiatric assessment. Electroencephalogram (EEG) and brain magnetic resonance imaging (MRI) scan were performed in 15 children. In addition, 11 families agreed to participate to the assessment of blood and urinary Mg2+ levels. We observed a highly variable phenotypic spectrum of developmental issues encompassing ID in most subjects as well as a variety of behavioural disorders such as autism and attention-deficit disorder/attention-deficit hyperactivity disorder. Dysmorphic traits and malformations were detected only in a minority of the participants, and no clear association with growth anomalies was found. Abnormal brain MRI and/or EEG were reported respectively in 64% and 92% of the subjects. Inheritance assessment highlighted an excess of duplication of maternal origin, while cardiac alterations were detected only in children with 15q11.2 CNV inherited from the father. We found great variability in Mg2+ urinary values, without correlation with 15q11.2 copy numbers. However, the variance of urinary Mg2+ levels largely increases in individuals with 15q11.2 deletion/duplication. This study provides further evidence that 15q11.2 BP1-BP2 CNV is associated with a broad spectrum of neurodevelopmental disorders and POE might be an explanation for clinical variability. However, some issues may question the real impact of 15q11.2 CNV on the phenotype in the carriers: DNA sequencing could be useful to exclude other pathogenic gene mutations. Our results do not support the possibility that urinary Mg2+ levels can be used as biomarkers to screen children with neurodevelopmental disorders for 15q11.2 duplication/deletion. However, there are evidences of correlations between 15q11.2 BP1-BP2 CNV and Mg2+ metabolism and future studies may pave the way to new therapeutic options.

Rare recurrent copy number variations in metabotropic glutamate receptor interacting genes in children with neurodevelopmental disorders

BackgroundNeurodevelopmental disorders (NDDs), such as attention deficit hyperactivity disorder (ADHD) and autism spectrum disorder (ASD), are examples of complex and partially overlapping phenotypes that often lack definitive corroborating genetic information. ADHD and ASD have complex genetic associations implicated by rare recurrent copy number variations (CNVs). Both of these NDDs have been shown to share similar biological etiologies as well as genetic pleiotropy.MethodsPlatforms aimed at investigating genetic-based associations, such as high-density microarray technologies, have been groundbreaking techniques in the field of complex diseases, aimed at elucidating the underlying disease biology. Previous studies have uncovered CNVs associated with genes within shared candidate genomic networks, including glutamate receptor genes, across multiple different NDDs. To examine shared biological pathways across two of the most common NDDs, we investigated CNVs across 15,689 individuals with ADHD (n = 7920), ASD (n = 4318), or both (n = 3,416), as well as 19,993 controls. Cases and controls were matched by genotype array (i.e., Illumina array versions). Three case–control association studies each calculated and compared the observed vs. expected frequency of CNVs across individual genes, loci, pathways, and gene networks. Quality control measures of confidence in CNV-calling, prior to association analyses, included visual inspection of genotype and hybridization intensity.ResultsHere, we report results from CNV analysis in search for individual genes, loci, pathways, and gene networks. To extend our previous observations implicating a key role of the metabotropic glutamate receptor (mGluR) network in both ADHD and autism, we exhaustively queried patients with ASD and/or ADHD for CNVs associated with the 273 genomic regions of interest within the mGluR gene network (genes with one or two degrees protein–protein interaction with mGluR 1–8 genes). Among CNVs in mGluR network genes, we uncovered CNTN4 deletions enriched in NDD cases (P = 3.22E − 26, OR = 2.49). Additionally, we uncovered PRLHR deletions in 40 ADHD cases and 12 controls (P = 5.26E − 13, OR = 8.45) as well as clinically diagnostic relevant 22q11.2 duplications and 16p11.2 duplications in 23 ADHD + ASD cases and 9 controls (P = 4.08E − 13, OR = 15.05) and 22q11.2 duplications in 34 ADHD + ASD cases and 51 controls (P = 9.21E − 9, OR = 3.93); those control samples were not with previous 22qDS diagnosis in their EHR records.ConclusionTogether, these results suggest that disruption in neuronal cell-adhesion pathways confers significant risk to NDDs and showcase that rare recurrent CNVs in CNTN4, 22q11.2, and 16p11.2 are overrepresented in NDDs that constitute patients predominantly suffering from ADHD and ASD.Trial registrationClinicalTrials.gov Identifier: NCT02286817 First Posted: 10 November 14, ClinicalTrials.gov Identifier: NCT02777931 first posted: 19 May 2016, ClinicalTrials.gov Identifier: NCT03006367 first posted: 30 December 2016, ClinicalTrials.gov Identifier: NCT02895906 first posted: 12 September 2016.

Antenatal ultrasound features of isolated recurrent copy number variation in 7q11.23 (Williams syndrome and 7q11.23 duplication syndrome).

We aimed to gather fetal cases carrying a 7q11.23 copy number variation (CNV) and collect precise clinical data to broaden knowledge of antenatal features in these syndromes. We retrospectively recruited unrelated cases with 7q11.23 deletion, known as Williams-Beuren syndrome (WBS), or 7q11.23 duplication who had prenatal ultrasound findings. We collected laboratory and clinical data, fetal ultrasound, cardiac ultrasound and fetal autopsy reports from 18 prenatal diagnostic centers throughout France. 40 fetuses with WBS were collected and the most common features were intra-uterine growth retardation (IUGR) (70.0%, 28/40), cardiovascular defects (30.0%, 12/40), polyhydramnios (17.5%, 7/40) and protruding tongue (15.0%, 6/40). Fetal autopsy reports were available for 11 cases and were compared with ultrasound prenatal features. Four cases of fetuses with 7q11.23 microduplication were collected and prenatal ultrasound signs were variable and often isolated. This work strengthens the fact that 7q11.23 CNVs are associated with a broad spectrum of antenatal presentations. IUGR and cardiovascular defects were the most frequent ultrasound signs. By reporting the biggest series of antenatal WBS, we aim to better delineate distinctive signs in fetuses with 7q11.23 CNVs.

Impact of Copy Number Variants and Polygenic Risk Scores on Psychopathology in the UK Biobank

BackgroundOur understanding of the impact of copy number variants (CNVs) on psychopathology and their joint influence with polygenic risk scores (PRSs) remains limited. MethodsThe UK Biobank recruited 502,534 individuals ages 37 to 73 years living in the United Kingdom between 2006 and 2010. After quality control, genotype data from 459,855 individuals were available for CNV calling. A total of 61 commonly studied recurrent neuropsychiatric CNVs were selected for analyses and examined individually and in aggregate (any CNV, deletion, or duplication). CNV risk scores were used to quantify intolerance of CNVs to haploinsufficiency. Major depressive disorder and generalized anxiety disorder PRSs were generated for White British individuals (N = 408,870). Mood/anxiety factor scores were generated using item-level questionnaire data (N = 501,289). ResultsCNV carriers showed higher mood/anxiety scores than noncarriers, with the largest effects seen for intolerant deletions. A total of 11 individual deletions and 8 duplications were associated with higher mood/anxiety. Carriers of the 9p24.3 (DMRT1) duplication showed lower mood/anxiety. Associations remained significant for most CNVs when excluding individuals with psychiatric diagnoses. Nominally significant CNV × PRS interactions provided preliminary evidence that associations between select individual CNVs, but not CNVs in aggregate, and mood/anxiety may be modulated by PRSs. ConclusionsCNVs associated with risk for psychiatric disorders showed small to large effects on dimensional mood/anxiety scores in a general population cohort, even when excluding individuals with psychiatric diagnoses. CNV × PRS interactions showed that associations between select CNVs and mood/anxiety may be modulated by PRSs.