Constitutional Copy Number Variants Research Articles

A large-scale retrospective analysis of copy number variations in single center using ACMG-ClinGen latest guidelines

Through a retrospective large sample analysis of copy number variants in single center, we explored the technical standards for the interpretation and reporting of constitutional copy-number variants (CNVs) jointly proposed by the American College of Medical Genetics and Genomics (ACMG) and the Clinical Genome Resource (ClinGen) in 2019, analyzing its impact on CNVs ratings and the improvement in the consistency of the classification of CNVs in clinical laboratories. 236 CNVs that assessed as pathogenic, uncertain significant (including likely pathogenic, uncertain and likely benign) by the 2011 ACMG guidelines between August 2018 and December 2019 in our center were re-analyzed. Four working group members of the center reclassified and evaluated 235 CNVs according to 2019 ACMG guidelines. The consistency of clinical significance classification of CNVs was 91% and the α test coefficient was 0.98 among four working group members. Compared with the 2011 and 2019 ACMG technical standards for the CNVs classification, evaluation of pathogenicity and uncertain significant is basically consistent. 90% (45/50) of likely pathogenic and likely benign CNVs were Re-evaluated as variants of uncertain significance, and the difference is significant. The new version ACMG/ClinGen guidelines for the evaluation of CNVs developed semi-quantitative point-based scoring system and help to improve the consistency in clinical classifications. It can also make the interpretation of CNVs more standardized and transparent.

OP041: Implementation of 2019 ACMG technical standards for the interpretation and reporting of constitutional CNVs: Experiences from an academic reference laboratory

Classification and reporting of constitutional copy-number variants (CNVs) in the clinical setting can be challenging and may result in discrepancies in interpretation for various reasons. In 2019, the ACMG and ClinGen published technical standards (2019 CNV guidelines) to guide the evaluation of constitutional CNVs. To improve consistency and accuracy in classification and reporting practices at ARUP Laboratories, we began using the 2019 CNV guidelines for genomic microarray testing beginning in 2020, including the adoption of the 5-tier variant classification system, incorporation of the scoring metrics into our CNV investigation workflow, and changing our reporting practices for CNVs involving reduced penetrance or autosomal recessive (AR) disease risk.

Standards for the interpretation of constitutional copy number gain: Recommendation from the American College of Medical Genetics and Genomics (ACMG) and Clinical Genome Resource (ClinGen)

Copy number variants (CNVs) are common causes of human genetic diseases. CNVs detection has become a routine component of genetic testing, especially for pediatric neurodevelopmental disorders, multiple congenital abnormalities, prenatal evaluation of fetuses with structural anomalies detected by ultrasound. Although the technologies for CNVs detection are continuously improving, the interpretation is still challenging, with significant discordance across different laboratories. In 2020, the American College of Medical Genetics and Genomics (ACMG) and the Clinical Genome Resource (ClinGen) developed a guideline for the interpreting and reporting of constitutional copy number variants, which introduced a quantitative, evidence-based scoring framework. Here, we detailed the key points of interpreting the copy number gain based on the guideline, used six examples of different categories to illuminate the scoring process and principles. We encourage a professional understanding and application of this guideline for the detected copy number gains in China in order to further improve the clinical evaluation accuracy and consistency across different laboratories.

AutoCNV: a semiautomatic CNV interpretation system based on the 2019 ACMG/ClinGen Technical Standards for CNVs

BackgroundThe American College of Medical Genetics and Genomics (ACMG) and the Clinical Genome Resource (ClinGen) presented technical standards for interpretation and reporting of constitutional copy-number variants in 2019 (the standards). Although ClinGen developed a web-based CNV classification calculator based on scoring metrics, it can only track and tally points that have been assigned based on observed evidence. Here, we developed AutoCNV (a semiautomatic automated CNV interpretation system) based on the standards, which can automatically generate predictions on 18 and 16 criteria for copy number loss and gain, respectively.ResultsWe assessed the performance of AutoCNV using 72 CNVs evaluated by external independent reviewers and 20 illustrative case examples. Using AutoCNV, it showed that 100 % (72/72) and 95 % (19/20) of CNVs were consistent with the reviewers’ and ClinGen-verified classifications, respectively. AutoCNV only required an average of less than 5 milliseconds to obtain the result for one CNV with automated scoring. We also applied AutoCNV for the interpretation of CNVs from the ClinVar database and the dbVar database. We also developed a web-based version of AutoCNV (wAutoCNV).ConclusionsAutoCNV may serve to assist users in conducting in-depth CNV interpretation, to accelerate and facilitate the interpretation process of CNVs and to improve the consistency and reliability of CNV interpretation.

53. Re-analysis of reported constitutional copy number variants using the newly published 2019 ACMG/ClinGen Technical Standards reveals few significant interpretation changes

Cytogenomic microarray analysis (CMA) is a powerful tool to identify submicroscopic copy number variants (CNVs) in patients with developmental delay and/or congenital anomalies, detecting CNVs in roughly 20% of this population. It is challenging to establish many CNVs as definitively pathogenic or benign, leading to a significant proportion of CNVs reported as variants of uncertain significance (VOUS). In a 6 year period, our laboratory reported a CNV in 20% of approximately 5,000 patients. Of these CNVs, 40% were interpreted as VOUS, with limited benefit to the patient.

28. Standards for the classification and reporting of constitutional copy number variants: A ClinGen/ACMG joint consensus recommendation

To assist clinical laboratories in performing consistent, transparent classification of constitutional CNVs, irrespective of the technology used to identify them, the American College of Medical Genetics and Genomics (ACMG), in collaboration with the NIH-funded Clinical Genome Resource (ClinGen) project, has updated existing professional standards. While the existing ACMG guidelines provide a high-level conceptual framework for evaluating constitutional CNVs in diagnostic testing, this proposed update will provide point-based, hierarchical scoring systems for both copy number losses and copy number gains to systematically evaluate relevant evidence. Multiple categories are used to help interpret CNVs including: overlap with CNVs reported in clinically affected individuals, overlap with CNVs reported in unaffected individuals, case-control studies, the presence of known dosage-sensitive genes, case reports with segregation data, de novo occurrence of CNVs, and the number of protein-coding genes included in the CNV. These metrics were tested on a set of 111 CNVs previously evaluated by clinical genetic laboratories. Each CNV was evaluated by 2 independent reviewers. Using the metrics, reviewers came to concordant classifications 73.0% of the time. Other significant updates include implementing a 5-tier classification system parallel to that used for interpreting sequence variants, and the recommendation to uncouple the evidence-based classification of a variant from its potential implications for a particular individual (i.e., not using clinical context to justify classifying the same variant differently in different individuals). These updated standards will have broad impact in the clinical community by providing a robust system to support the consistent classification of CNVs.

The Role of Constitutional Copy Number Variants in Breast Cancer.

Constitutional copy number variants (CNVs) include inherited and de novo deviations from a diploid state at a defined genomic region. These variants contribute significantly to genetic variation and disease in humans, including breast cancer susceptibility. Identification of genetic risk factors for breast cancer in recent years has been dominated by the use of genome-wide technologies, such as single nucleotide polymorphism (SNP)-arrays, with a significant focus on single nucleotide variants. To date, these large datasets have been underutilised for generating genome-wide CNV profiles despite offering a massive resource for assessing the contribution of these structural variants to breast cancer risk. Technical challenges remain in determining the location and distribution of CNVs across the human genome due to the accuracy of computational prediction algorithms and resolution of the array data. Moreover, better methods are required for interpreting the functional effect of newly discovered CNVs. In this review, we explore current and future application of SNP array technology to assess rare and common CNVs in association with breast cancer risk in humans.

Impact of constitutional copy number variants on biological pathway evolution

BackgroundInherited Copy Number Variants (CNVs) can modulate the expression levels of individual genes. However, little is known about how CNVs alter biological pathways and how this varies across different populations. To trace potential evolutionary changes of well-described biological pathways, we jointly queried the genomes and the transcriptomes of a collection of individuals with Caucasian, Asian or Yoruban descent combining high-resolution array and sequencing data.ResultsWe implemented an enrichment analysis of pathways accounting for CNVs and genes sizes and detected significant enrichment not only in signal transduction and extracellular biological processes, but also in metabolism pathways. Upon the estimation of CNV population differentiation (CNVs with different polymorphism frequencies across populations), we evaluated that 22% of the pathways contain at least one gene that is proximal to a CNV (CNV-gene pair) that shows significant population differentiation. The majority of these CNV-gene pairs belong to signal transduction pathways and 6% of the CNV-gene pairs show statistical association between the copy number states and the transcript levels.ConclusionsThe analysis suggested possible examples of positive selection within individual populations including NF-kB, MAPK signaling pathways, and Alu/L1 retrotransposition factors. Altogether, our results suggest that constitutional CNVs may modulate subtle pathway changes through specific pathway enzymes, which may become fixed in some populations.

Abstract 1447: Investigating susceptibility to sporadic osteosarcoma by genome-wide copy number analysis

Abstract Constitutional copy-number variants (CNVs) frequently overlap genomic regions that contain genes associated with cancer. In addition, copy number alterations (CNAs) in tumors often coincide with sites of CNVs and may arise through progression of CNVs. Osteosarcoma (OS), the most common human bone malignancy, can arise in familial or sporadic forms. While germline mutations of tumor suppressor genes have been implicated in a minority of OS cases, little is known about genetic changes that contribute to the etiology of sporadic OS. Using high-resolution SNP/CNV arrays, we observed a higher global germline CNV frequency in patients with sporadic OS compared to healthy controls. Given the early age of onset and the extensive constitutional CNVs, we hypothesize that these genomic alterations are linked to susceptibility to OS. Here we investigate whether specific constitutional CNVs are related to susceptibility to sporadic OS and whether OS tumor CNAs arise via progression of constitutional CNVs in these patients. Blood derived DNA samples from patients with sporadic OS with wild-type germline p53 sequence and gene dosage were selected for analysis on Affymetrix GW6.0 arrays with paired pre-therapy tumor biopsies. Initial results from a discovery set of samples (n=43 pairs) reveal a copy number loss at 1q43, in a region that overlaps several genes that have a role in bone formation, microtubules and cytoskeletal organization. This loss was present in 43% of the blood-derived DNA samples and 44% of the tumor-derived samples, whereas this variant was noted in only 1.2% of controls. Furthermore, the germline 1q43 deletion correlates with poor survival of the OS patients (p=0.04, Long Rank). Analysis of an independent validation set (n=32 pairs) is currently underway to confirm the initial finding and further characterize specific constitutional CNVs among sporadic OS patients. The findings of this study will open new avenues for the discovery of novel loci implicated in susceptibility to OS with potential for improved diagnostic and prognostic options as well as further insight into disease pathogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1447. doi:1538-7445.AM2012-1447

American College of Medical Genetics standards and guidelines for interpretation and reporting of postnatal constitutional copy number variants

Genomic microarrays used to assess DNA copy number are now recommended as first-tier tests for the postnatal evaluation of individuals with intellectual disability, autism spectrum disorders, and/or multiple congenital anomalies. Application of this technology has resulted in the discovery of widespread copy number variation in the human genome, both polymorphic variation in healthy individuals and novel pathogenic copy number imbalances. To assist clinical laboratories in the evaluation of copy number variants and to promote consistency in interpretation and reporting of genomic microarray results, the American College of Medical Genetics has developed the following professional guidelines for the interpretation and reporting of copy number variation. These guidelines apply primarily to evaluation of constitutional copy number variants detected in the postnatal setting.

Abstract 882: Copy number variation analysis identified multiple genetic variants related to nasopharyngeal carcinoma predisposition

Abstract Nasopharyngeal carcinoma (NPC) is a multi-factorial malignancy closely associated with genetic factors and Epstein-Barr virus infection. Recently, we have conducted a genome-wide association study (GWAS) in 277 NPC patients and 285 healthy controls within the Taiwanese population, analyzing 480,365 single-nucleotide polymorphisms (SNPs). Twelve statistically significant SNPs were identified and mapped to chromosome 6p21.3 in the major histocompatibility complex region. In addition to HLA-A gene, we detected novel significant associations between NPC and two genes: specifically, gamma aminobutyric acid b receptor 1 and HLA-F. To our knowledge, it is the first GWAS report of NPC showing that multiple loci (HLA-A, HLA-F, and GABBR1) within chromosome 6p21.3 are associated with NPC. Following the GWAS study, we made an effort to search systematically for copy number variations (CNVs) that confer increased risk to NPC using the same dataset. We generated CNV calls using four different softwares and used 15 internal duplicated samples to look for best filtering criteria. CNVs were selected if same CNVs were identified from two independent softwares to increase credibility of prediction. We identified eight CNVs that overrepresented in NPC patients compared to healthy controls. Six deletions on chromosomes 3, 6, 7, 8 and 19, as well as 2 duplications on chromosomes 7 and 12 were found. One of the most significant CNV on chromosome 6p21.3 was further replicated in an independent case-control dataset, using a deletion-specific polymerase-chain reaction. To the best of our knowledge, it is the first study of genome-wide constitutional CNV in NPC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 882. doi:10.1158/1538-7445.AM2011-882

Abstract 1146: Three-color array CGH differentiates constitutional from acquired CNVs in cancer genomes

Abstract In recent years microarray-based comparative genomic hybridisation (CGH) has become the assay of choice for high-resolution screening of genetic aberrations in research and clinical diagnostics, and has revealed a plethora of small, constitutional copy number variations (CNVs) in healthy individuals. The appearance of these benign CNVs on the array profile affects the ability to identify CNVs that are acquired and pathogenic, in particular if these are also small in size. It is possible to eliminate constitutional CNVs by using a matched self-reference for each sample, but this compromises ability to compare array profiles between different individuals. In addition, the ability to link specific constitutional CNVs or combinations of these to disease susceptibility is lost. We have developed a new 3-color microarray CGH assay which allows identification of constitutional CNVs while retaining the ability to compare between different samples by simultaneous application of two distinctly labelled reference samples. The new 3-color microarray CGH assay uses three spectrally distinct fluorescent dyes to label the test sample, matched self-reference, and generic reference, for simultaneous hybridization to the same array platform. We have applied this new protocol to the analysis of samples from healthy individuals, leukemia patients, and the leukemia-derived cell line K562, and performed pair-wise comparison of 26 array-CGH profiles from the new 3-color array with corresponding profiles from conventional 2-color arrays. Our results demonstrate that introducing the third dye has no adverse effect on array assay noise, as measured by paired ttest of the standard deviation of the log2 ratio of all clones on the 2-color and 3-color array profile. Copy number calls from the new 3-color array assays show a consistency of up to 97% with the corresponding conventional 2-color array assays, equivalent to the level of consistency that was achieved when repeating array-CGH on the same sample using the same protocol. The prevalence-adjusted Cohen Kappa test indicates almost perfect agreement between the new 3-color assay and conventional 2-color assay. Application of the new 3-color microarray assay has enabled us to identify 44 constitutional CNVs in 57% of our leukemia samples. At least one third of these CNVs have been reported as sites of normal copy number variation and are listed in The Human Genome Variation Database. Introducing a third sample into the microarray assay is a new and more economical approach for high resolution genome-wide comparison of DNA, and therefore potentially amenable to uptake in both research and diagnostics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1146.

The Impact of Constitutional Copy Number Variants in Myeloma

Single nucleotide polymorphisms (SNPs) have been long regarded as being important in determining variation and disease predisposition. Recently, chromosomal structural variation in the form of deletions, insertions and duplifications have been identified frequently in the genome of the general population. Such copy number variations (CNVs) have been shown to contribute to a range of human diseases. In recent studies we have utilized Affymetrix 50K and 500K arrays to identify acquired copy number change in myeloma tumor samples. In those studies we had access to paired constitutional DNA and in the present study have been able to report for the first time a CNV map of the constitutional genome of myeloma patients. Affymetrix 500K mapping arrays were used to identify copy number changes in 63 paired samples using DNA from peripheral blood and CD138 selected plasma cells. Tumor samples were analyzed in CNAG using both a paired and unpaired analysis to distinguish between inherited and acquired copy number change. Constitutional DNA was analyzed by both CNAG and GEMCA using 90 Caucasian samples from the Hapmap database as a reference set. For maximum calling accuracy, only those regions identified by both algorithms were called as CNVs. As with similar studies, overlapping CNVs identified using this approach were merged to generate a list of CNV regions (CNVRs) characteristic of the constitutional DNA of these myeloma cases. Using this approach, we identified 292 CNVs across 63 cases, with a median of 4 regions per sample. There were 155 discrete CNVRs, of which 46 were recurrent. The recurrent CNVRs were found most frequently in the pericentric regions of chromosome 14 and 15 in keeping with other studies. We then compared these recurrent CNVRs with a comparable dataset of normal individuals generated using Affymetrix 500K arrays. In this analysis, 25/46 recurrent CNVRs in the myeloma cases were novel. The two most frequent novel CNVRs in the myeloma cases were gains on chromosome 21 and 15. We also compared the characteristics of the constitutional CNVs with the acquired copy number changes in the corresponding tumor samples and identified that the constitutional CNVs were generally considerably smaller. However, using unpaired analysis it was possible to determine the presence of the constitutional CNV in the tumor sample, providing validation of the CNVs. We were also able to demonstrate that acquired copy number change in the tumor cells can either exaggerate or ameliorate the effect of the inherited CNV in the tumor genome, such as cases with acquired trisomy 15 and deletion or gain of regions of 15q in the constitutional DNA. These findings also reinforce the need for paired non-tumor DNA when undertaking copy number analysis of tumor DNA using SNP arrays. In this study we have been able to identify for the first time the presence of CNVs in the constitutional genome of individuals with myeloma. We have been able to systematically catalogue these CNVRs. These results provide the basis for future studies aimed at identifying how this type of genomic variation may influence the development of and outcome of myeloma and a broad range of other hematological conditions.