Benign Copy Number Variants Research Articles

Detection of Copy Number Variants by Next-Generation Sequencing in Fetuses with Congenital Heart Disease

Objective: To report the molecular findings of 89 fetuses with prenatal ultrasound diagnosis of congenital heart disease (CHD) and a normal karyotype through next-generation sequencing (NGS) in an attempt to improve our understanding of submicroscopic abnormalities present in malformed fetuses. Method: High-throughput NGS was carried out in fetal cord blood samples. All potential cytogenetic alterations detected on NGS platforms were matched against the known copy number variant (CNV) database. Results: A total of 204 CNVs were identified in the entire population of 89 fetuses with CHD. Eleven cases had no deletions or duplications, five cases (5.6%) had pathogenic CNVs, 13 cases had CNVs that were likely to be pathogenic, 42 cases had CNVs of uncertain significance, and 18 cases had benign and/or likely benign CNVs. All pathogenic CNVs were found only in fetuses with conotruncal heart defects. Conclusion: NGS can facilitate etiological diagnosis in a high proportion of fetuses with CHD and a normal karyotype and can be implemented as a diagnostic tool in the prenatal setting to complement karyotyping for evaluation of genomic defects in fetuses with CHD.

Smith‑Magenis syndrome in monozygotic twin fetuses presenting with discordant phenotypes and uteroplacental insufficiency.

Smith‑Magenis syndrome (SMS) is a rare condition with multiple congenital malformations caused by the haploinsufficiency of RAI1 (deletion or mutation of RAI1). However, the correlation between genotype and phenotype is not well understood. The present study describes the prenatal diagnosis of monozygotic twins with a 17p11.2 deletion, which is indicative of SMS, who presented with discordant phenotypes and uteroplacental insufficiency. A high‑resolution genome‑wide single nucleotide polymorphism array revealed a 3.7‑Mb deletion in the 17p11.2 chromosome region. Accurate breakpoints of the deletion in these patients were used to identify correlations between SMS and the concomitant phenotypes, particularly uteroplacental insufficiency, which has rarely been investigated in SMS. In addition, no exonic mutations were identified in or affected known disease‑associated loci that could explain the congenital anomalies, according to a model that accounts for the possibility of incomplete penetrance. Furthermore, a novel benign copy number variation (a duplication of 195 kb at 13q12.13) was identified but was unlikely to be clinically significant in the discordant phenotypes of the twins. The present study showed that multiple interacting genetic and environmental factors are involved in determining the variance of the SMS phenotype.

Subtelomeric chromosomal anomalies in infantile epileptic encephalopathies

The infantile epileptic encephalopathies are a heterogeneous group of disorders where in about 30% of cases the cause remains unknown. Chromosomal aberrations, identified by karyotype analysis, constitute one of the major causes for these disorders. The new molecular cytogenetic techniques such as fluorescence in situ hybridization, multiplex ligation-dependent probe amplification (MLPA) and single nucleotide polymorphism array analyses may allow us to discover additional submicroscopic chromosomal rearrangements in this group of patients. Herein, we describe a girl with infantile epileptic encephalopathy. Fluorescence in situ hybridization, MLPA and single nucleotide polymorphism array analyses revealed a microdeletion and microduplication at the 1p36 region. Because of that, we further screened a cohort of 38 patients with infantile epileptic encephalopathy by using MLPA analysis to exclude the presence of subtelomeric microdeletions or microduplications. One patient was shown to harbor a microduplication at 11q25, which is however most likely a benign copy number variant derived from the healthy mother. No disease-causing dose changes were identified, indicating that unbalanced rearrangements at the subtelomeric regions may not be a common cause for infantile epileptic encephalopathy. However, when the patient has infantile epileptic encephalopathy and other features such as dysmorphic facies or major congenital anomalies, molecular cytogenetic analysis is warranted.

The clustering of functionally related genes contributes to CNV-mediated disease.

Clusters of functionally related genes can be disrupted by a single copy number variant (CNV). We demonstrate that the simultaneous disruption of multiple functionally related genes is a frequent and significant characteristic of de novo CNVs in patients with developmental disorders (P = 1 × 10−3). Using three different functional networks, we identified unexpectedly large numbers of functionally related genes within de novo CNVs from two large independent cohorts of individuals with developmental disorders. The presence of multiple functionally related genes was a significant predictor of a CNV's pathogenicity when compared to CNVs from apparently healthy individuals and a better predictor than the presence of known disease or haploinsufficient genes for larger CNVs. The functionally related genes found in the de novo CNVs belonged to 70% of all clusters of functionally related genes found across the genome. De novo CNVs were more likely to affect functional clusters and affect them to a greater extent than benign CNVs (P = 6 × 10−4). Furthermore, such clusters of functionally related genes are phenotypically informative: Different patients possessing CNVs that affect the same cluster of functionally related genes exhibit more similar phenotypes than expected (P < 0.05). The spanning of multiple functionally similar genes by single CNVs contributes substantially to how these variants exert their pathogenic effects.

Copy number variations in cryptogenic cerebral palsy.

To determine the prevalence and characteristics of copy number variations (CNVs) in children with cerebral palsy (CP) of unknown etiology, comprising approximately 20% of the CP population. Fifty-two participants (age 10.5 ± 7.8 years; Gross Motor Function Classification System scale 2.8 ± 1.3) with nonprogressive pyramidal and/or extrapyramidal signs since infancy and no identified etiology were enrolled. Individuals with evidence of acquired causes were excluded. Participants underwent neurologic and clinical genetic examinations before the genomic testing. Chromosomal microarray analysis to detect CNVs was performed using the Affymetrix platform. CNVs identified were classified as pathogenic, likely pathogenic, likely benign, or benign. Only pathogenic and likely pathogenic CNVs were defined as clinically significant. Thirty-nine CNVs were found in 25 of 52 participants (48%). Sixteen participants (31%) had clinically significant CNVs: 10 pathogenic and 6 likely pathogenic, of which 7 were not previously associated with motor disability. Nine participants had likely benign CNVs. Clinically significant CNVs were more frequently de novo (12/16; p < 0.001) including in 5 of 8 individuals who had a first- or second-degree relative with a major neurologic disorder. Dysmorphic features and nonmotor comorbidities were more prevalent in individuals with clinically significant CNVs (p < 0.05 for both). CNVs, most frequently de novo, are common in individuals with cryptogenic CP. We recommend CNV testing in individuals with CP of unknown etiology.

Recurrent benign copy number variants & issues in interpretation of variants of unknown significance identified by cytogenetic microarray in Indian patients with intellectual disability.

Background & objectives:Cytogenetic microarray (CMA) is now recommended as a first-tier clinical diagnostic test in cases with idiopathic intellectual disability and/or developmental delay (ID/DD). Along with clinically relevant variants, CMA platforms also identify variants of unknown significance (VUS). This study was done to look for utility and various issues in interpretation of copy number variants (CNVs) in Indian patients with ID/DD.Methods:The CMA was performed in 86 Indian patients with idiopathic ID/DD with or without dysmorphic features. CNV was reported if copy number gain was >400 kb in size and copy number loss was > 200 kb in size.Results:Pathogenic CNVs were found in 18 of 86 (20.9%) patients. One large (14 Mb size) de novo heterozygous copy number gain was found in one patient. VUS (total 31) were present in 17 of 86 (19.7%) patients. Five novel recurrent benign CNVs were also present in our patients.Interpretation & conclusions:Our findings highlight the difficulties in interpretation of CNVs identified by CMA. More Indian data on VUS and recurrent benign CNVs will be helpful in the interpretation of CMA in patients with ID/DD.

Genomic Copy Number Variation Affecting Genes Involved in the Cell Cycle Pathway: Implications for Somatic Mosaicism.

Somatic genome variations (mosaicism) seem to represent a common mechanism for human intercellular/interindividual diversity in health and disease. However, origins and mechanisms of somatic mosaicism remain a matter of conjecture. Recently, it has been hypothesized that zygotic genomic variation naturally occurring in humans is likely to predispose to nonheritable genetic changes (aneuploidy) acquired during the lifetime through affecting cell cycle regulation, genome stability maintenance, and related pathways. Here, we have evaluated genomic copy number variation (CNV) in genes implicated in the cell cycle pathway (according to Kyoto Encyclopedia of Genes and Genomes/KEGG) within a cohort of patients with intellectual disability, autism, and/or epilepsy, in which the phenotype was not associated with genomic rearrangements altering this pathway. Benign CNVs affecting 20 genes of the cell cycle pathway were detected in 161 out of 255 patients (71.6%). Among them, 62 individuals exhibited >2 CNVs affecting the cell cycle pathway. Taking into account the number of individuals demonstrating CNV of these genes, a support for this hypothesis appears to be presented. Accordingly, we speculate that further studies of CNV burden across the genes implicated in related pathways might clarify whether zygotic genomic variation generates somatic mosaicism in health and disease.

In silico molecular cytogenetics: a bioinformatic approach to prioritization of candidate genes and copy number variations for basic and clinical genome research.

BackgroundThe availability of multiple in silico tools for prioritizing genetic variants widens the possibilities for converting genomic data into biological knowledge. However, in molecular cytogenetics, bioinformatic analyses are generally limited to result visualization or database mining for finding similar cytogenetic data. Obviously, the potential of bioinformatics might go beyond these applications. On the other hand, the requirements for performing successful in silico analyses (i.e. deep knowledge of computer science, statistics etc.) can hinder the implementation of bioinformatics in clinical and basic molecular cytogenetic research. Here, we propose a bioinformatic approach to prioritization of genomic variations that is able to solve these problems.ResultsSelecting gene expression as an initial criterion, we have proposed a bioinformatic approach combining filtering and ranking prioritization strategies, which includes analyzing metabolome and interactome data on proteins encoded by candidate genes. To finalize the prioritization of genetic variants, genomic, epigenomic, interactomic and metabolomic data fusion has been made. Structural abnormalities and aneuploidy revealed by array CGH and FISH have been evaluated to test the approach through determining genotype-phenotype correlations, which have been found similar to those of previous studies. Additionally, we have been able to prioritize copy number variations (CNV) (i.e. differentiate between benign CNV and CNV with phenotypic outcome). Finally, the approach has been applied to prioritize genetic variants in cases of somatic mosaicism (including tissue-specific mosaicism).ConclusionsIn order to provide for an in silico evaluation of molecular cytogenetic data, we have proposed a bioinformatic approach to prioritization of candidate genes and CNV. While having the disadvantage of possible unavailability of gene expression data or lack of expression variability between genes of interest, the approach provides several advantages. These are (i) the versatility due to independence from specific databases/tools or software, (ii) relative algorithm simplicity (possibility to avoid sophisticated computational/statistical methodology) and (iii) applicability to molecular cytogenetic data because of the chromosome-centric nature. In conclusion, the approach is able to become useful for increasing the yield of molecular cytogenetic techniques.

Copy number variation in Thai population.

Copy number variation (CNV) is a major genetic polymorphism contributing to genetic diversity and human evolution. Clinical application of CNVs for diagnostic purposes largely depends on sufficient population CNV data for accurate interpretation. CNVs from general population in currently available databases help classify CNVs of uncertain clinical significance, and benign CNVs. Earlier studies of CNV distribution in several populations worldwide showed that a significant fraction of CNVs are population specific. In this study, we characterized and analyzed CNVs in 3,017 unrelated Thai individuals genotyped with the Illumina Human610, Illumina HumanOmniexpress, or Illumina HapMap550v3 platform. We employed hidden Markov model and circular binary segmentation methods to identify CNVs, extracted 23,458 CNVs consistently identified by both algorithms, and cataloged these high confident CNVs into our publicly available Thai CNV database. Analysis of CNVs in the Thai population identified a median of eight autosomal CNVs per individual. Most CNVs (96.73%) did not overlap with any known chromosomal imbalance syndromes documented in the DECIPHER database. When compared with CNVs in the 11 HapMap3 populations, CNVs found in the Thai population shared several characteristics with CNVs characterized in HapMap3. Common CNVs in Thais had similar frequencies to those in the HapMap3 populations, and all high frequency CNVs (>20%) found in Thai individuals could also be identified in HapMap3. The majorities of CNVs discovered in the Thai population, however, were of low frequency, or uniquely identified in Thais. When performing hierarchical clustering using CNV frequencies, the CNV data were clustered into Africans, Europeans, and Asians, in line with the clustering performed with single nucleotide polymorphism (SNP) data. As CNV data are specific to origin of population, our population-specific reference database will serve as a valuable addition to the existing resources for the investigation of clinical significance of CNVs in Thais and related ethnicities.

Cytogenomic mapping and bioinformatic mining reveal interacting brain expressed genes for intellectual disability

BackgroundMicroarray analysis has been used as the first-tier genetic testing to detect chromosomal imbalances and copy number variants (CNVs) for pediatric patients with intellectual and developmental disabilities (ID/DD). To further investigate the candidate genes and underlying dosage-sensitive mechanisms related to ID, cytogenomic mapping of critical regions and bioinformatic mining of candidate brain-expressed genes (BEGs) and their functional interactions were performed. Critical regions of chromosomal imbalances and pathogenic CNVs were mapped by subtracting known benign CNVs from the Databases of Genomic Variants (DGV) and extracting smallest overlap regions with cases from DatabasE of Chromosomal Imbalance and Phenotype in Humans using Ensembl Resources (DECIPHER). BEGs from these critical regions were revealed by functional annotation using Database for Annotation, Visualization, and Integrated Discovery (DAVID) and by tissue expression pattern from Uniprot. Cross-region interrelations and functional networks of the BEGs were analyzed using Gene Relationships Across Implicated Loci (GRAIL) and Ingenuity Pathway Analysis (IPA).ResultsOf the 1,354 patients analyzed by oligonucleotide array comparative genomic hybridization (aCGH), pathogenic abnormalities were detected in 176 patients including genomic disorders in 66 patients (37.5%), subtelomeric rearrangements in 45 patients (25.6%), interstitial imbalances in 33 patients (18.8%), chromosomal structural rearrangements in 17 patients (9.7%) and aneuploidies in 15 patients (8.5%). Subtractive and extractive mapping defined 82 disjointed critical regions from the detected abnormalities. A total of 461 BEGs was generated from 73 disjointed critical regions. Enrichment of central nervous system specific genes in these regions was noted. The number of BEGs increased with the size of the regions. A list of 108 candidate BEGs with significant cross region interrelation was identified by GRAIL and five significant gene networks involving cell cycle, cell-to-cell signaling, cellular assembly, cell morphology, and gene expression regulations were denoted by IPA.ConclusionsThese results characterized ID related cross-region interrelations and multiple networks of candidate BEGs from the detected genomic imbalances. Further experimental study of these BEGs and their interactions will lead to a better understanding of dosage-sensitive mechanisms and modifying effects of human mental development.

219: Novel benign copy number variations (CNVs) detected by SNP array in prenatal diagnosis in Southern China

219: Novel benign copy number variations (CNVs) detected by SNP array in prenatal diagnosis in Southern China

CNVinspector: a web-based tool for the interactive evaluation of copy number variations in single patients and in cohorts

ObjectivesMany genetic disorders are caused by copy number variations (CNVs) in the human genome. However, the large number of benign CNV polymorphisms makes it difficult to delineate causative variants for...

Complex reorganization and predominant non-homologous repair following chromosomal breakage in karyotypically balanced germline rearrangements and transgenic integration

We defined the genetic landscape of balanced chromosomal rearrangements at nucleotide resolution by sequencing 141 breakpoints from cytogenetically-interpreted translocations and inversions. We confirm that the recently described phenomenon of “chromothripsis” (massive chromosomal shattering and reorganization) is not unique to cancer cells but also occurs in the germline where it can resolve to a karyotypically balanced state with frequent inversions. We detected a high incidence of complex rearrangements (19.2%) and substantially less reliance on microhomology (31%) than previously observed in benign CNVs. We compared these results to experimentally-generated DNA breakage-repair by sequencing seven transgenic animals, and revealed extensive rearrangement of the transgene and host genome with similar complexity to human germline alterations. Inversion is the most common rearrangement, suggesting that a combined mechanism involving template switching and non-homologous repair mediates the formation of balanced complex rearrangements that are viable, stably replicated and transmitted unaltered to subsequent generations.

Practical guidelines for interpreting copy number gains detected by high-resolution array in routine diagnostics

The correct interpretation of copy number gains in patients with developmental delay and multiple congenital anomalies is hampered by the large number of copy number variations (CNVs) encountered in healthy individuals. The variable phenotype associated with copy number gains makes interpretation even more difficult. Literature shows that inheritence, size and presence in healthy individuals are commonly used to decide whether a certain copy number gain is pathogenic, but no general consensus has been established. We aimed to develop guidelines for interpreting gains detected by array analysis using array CGH data of 300 patients analysed with the 105K Agilent oligo array in a diagnostic setting. We evaluated the guidelines in a second, independent, cohort of 300 patients. In the first 300 patients 797 gains of four or more adjacent oligonucleotides were observed. Of these, 45.4% were de novo and 54.6% were familial. In total, 94.8% of all de novo gains and 87.1% of all familial gains were concluded to be benign CNVs. Clinically relevant gains ranged from 288 to 7912 kb in size, and were significantly larger than benign gains and gains of unknown clinical relevance (P < 0.001). Our study showed that a threshold of 200 kb is acceptable in a clinical setting, whereas heritability does not exclude a pathogenic nature of a gain. Evaluation of the guidelines in the second cohort of 300 patients revealed that the interpretation guidelines were clear, easy to follow and efficient.

23-chromosome single nucleotide polymorphism (SNP) microarray detects genomic aberrations that may be missed by comparative genomic hybridization (CGH) arrays in preimplantation genetic screening (PGS)

23-chromosome single nucleotide polymorphism (SNP) microarray detects genomic aberrations that may be missed by comparative genomic hybridization (CGH) arrays in preimplantation genetic screening (PGS)

23-chromosome single nucleotide polymorphism (SNP) microarray preimplantation genetic screening (PGS) on blastocysts, versus day-3 embryos, results in significantly higher clinical pregnancy rates

To determine the clinical pregnancy rate using SNP PGS in women primarily with recurrent pregnancy loss (RPL) undergoing in vitro fertilization (IVF) Retrospective. A retrospective review was conducted of all embryos that underwent PGS by 23 chromosome SNP microarrays from 1/1/2010 to 1/25/2011. Patients underwent standard in vitro fertilization (IVF) and PGS due to primarily a history of > 2 spontaneous miscarriages. Embryo biopsy was performed at either the cleavage or blastocyst stage. Sample DNA was amplified and analyzed using HumanCytoSNP-12 DNA beadchips and GenomeStudio and KaryoStudio software. Parental DNA was analyzed and compared to embryonic cells to determine the presence of benign copy number variations. Embryos derived from parents with known translocations or inversions were excluded from the study. Binomial confidence intervals for proportions were calculated by the modified Wald method. 2,976 embryos from 317 clinical IVF cycles were tested. 2,704 (90.8%) of these embryos, derived from 249 (78.5%) IVF cycles, were at Day-3 of development (D3 group) when biopsy was performed. Conversely, 272 (9.2%) of embryos, derived from 68 (21.5%) IVF cycles, were at the blastocyst stage (D5 group) when biopsy was performed. Aneuploidy was observed in 59% of the Day 3 embryos compared with only 41% of Day 5 embryos.The clinical pregnancy rates were significantly higher (86%, P<.05) in the D5 group compared to the D3 group (65%). The miscarriage rate in the D3 and D5 groups were 6% and 9% respectively. These miscarriage rates were not statistically different. PGS using 23-chromosome SNP microarrays is practical in improving clinical pregnancy rates in couples with RPL. In addition, PGS performed on blastocysts, versus Day-3 embryos, results in significantly higher clinical pregnancy rates in similar patient populations.

Deletions and duplications identified by 23 chromosome single nucleotide polymorphism (SNP) microarray are associated with aneuploidy

Deletions and duplications identified by 23 chromosome single nucleotide polymorphism (SNP) microarray are associated with aneuploidy

Tandem configurations of variably duplicated segments of 22q11.2 confirmed by fiber-FISH analysis

22q11.2 duplication syndrome has recently been established as a new syndrome manifesting broad clinical phenotypes including mental retardation. It is reciprocal to DiGeorge (DGS)/velo-cardio-facial syndrome (VCFS), in which the same portion of the chromosome is hemizygously deleted. Deletions and duplications of the 22q11.2 region are facilitated by the low-copy repeats (LCRs) flanking this region. In this study, we aimed to identify the directions of the duplicated segments of 22q11.2 to better understand the mechanism of chromosomal duplication. To achieve this aim, we accumulated samples from four patients with 22q11.2 duplications. One of the patients had an atypically small (741 kb) duplication of 22q11.2. The centromeric end of the breakpoint was on LCR22A, but the telomeric end was between LCR22A and B. Therefore, the duplicated segment did not include T-box 1 gene (TBX1), the gene primarily responsible for the DGS/VCFS. As this duplication was shared by the patient's healthy mother, this appears to be a benign copy-number variation rather than a disease-causing alteration. The other three patients showed 3.0 or 4.0 Mb duplications flanked by LCRs. The directions of the duplicated segments were investigated by fiber-fluorescence in situ hybridization analysis. All samples showed tandem configurations. These results support the hypothesized mechanism of non-allelic homologous recombination with flanking LCRs and add additional evidence that many interstitial duplications are aligned as tandem configurations.

Identification of Copy Number Variants on Human Chromosome 22 in Patients with a Variety of Clinical Findings

The aims of this study were to create a copy number variant (CNV) profile of human chromosome 22 and to establish a genotype-phenotype correlation for patients with genomic abnormalities on chromosome 22. Thus, 1,654 consecutive pediatric patients with a diversity of clinical findings were evaluated by high-resolution chromosomal microarray analysis (CMA). We identified 25 individuals with abnormal CNVs on chromosome 22, representing 1.5% of the cases analyzed in this cohort. Meanwhile, we detected 1,298 benign CNVs on this chromosome in these individuals. Twenty-one of the 25 abnormal CNVs and the majority of the benign CNVs occurred through involvement of the 8 unstable genomic regions enriched with low copy repeats (LCR22A–H). The highly dynamic status of LCR22s within the 22q11 region facilitates the formation of diverse genomic abnormalities. This CNV profile provides a general perspective of the spectrum of chromosome 22 genomic imbalances and subsequently improves the CNV-phenotype correlations.

Clinical application of whole-genome array CGH during prenatal diagnosis: Study of 25 selected pregnancies with abnormal ultrasound findings or apparently balanced structural aberrations

BackgroundThe purpose of the study was the application and evaluation of array Comparative Genomic Hybridization (array CGH) in selected cases during prenatal diagnosis. Array CGH was applied in 25 fetal samples out of which 15 had normal karyotypes and abnormal ultrasound findings and 10 had apparently balanced structural aberrations with or without abnormal ultrasound findings. DNA was extracted from peripheral blood, chorionic villi samples (CV) and amniotic fluid. Bacterial Artificial Chromosome (BAC) array CGH (Cytochip, BlueGnome Ltd.) of 1 Mb was applied and results were confirmed with either Fluorescence In Situ Hybridization (FISH), Multiplex Ligation-dependant Probe Amplification (MLPA) or Real-Time PCR.ResultsThree out of 25 samples (12%), referred for prenatal array CGH, were found to carry copy number alterations. The number of cases with clinically significant alterations was 2/25 (8%), while one (4%) was of uncertain clinical significance. Two benign Copy Number Variations (CNVs) were also found in 1/25 cases (4%).ConclusionsThe outcome of this study indicates the ability of array CGH to identify chromosomal abnormalities which cannot be detected during routine prenatal cytogenetic analysis, therefore increasing the overall detection rate.