Genome-wide Copy Number Variation Research Articles

MLIP genotype as a predictor of pharmacological response in primary open-angle glaucoma and ocular hypertension

Predicting the therapeutic response to ocular hypotensive drugs is crucial for the clinical treatment and management of glaucoma. Our aim was to identify a possible genetic contribution to the response to current pharmacological treatments of choice in a white Mediterranean population with primary open-angle glaucoma (POAG) or ocular hypertension (OH). We conducted a prospective, controlled, randomized, partial crossover study that included 151 patients of both genders, aged 18 years and older, diagnosed with and requiring pharmacological treatment for POAG or OH in one or both eyes. We sought to identify copy number variants (CNVs) associated with differences in pharmacological response, using a DNA pooling strategy of carefully phenotyped treatment responders and non-responders, treated for a minimum of 6 weeks with a beta-blocker (timolol maleate) and/or prostaglandin analog (latanoprost). Diurnal intraocular pressure reduction and comparative genome wide CNVs were analyzed. Our finding that copy number alleles of an intronic portion of the MLIP gene is a predictor of pharmacological response to beta blockers and prostaglandin analogs could be used as a biomarker to guide first-tier POAG and OH treatment. Our finding improves understanding of the genetic factors modulating pharmacological response in POAG and OH, and represents an important contribution to the establishment of a personalized approach to the treatment of glaucoma.

Combination of Genome-Wide Polymorphisms and Copy Number Variations of Pharmacogenes in Koreans.

For predicting phenotypes and executing precision medicine, combination analysis of single nucleotide variants (SNVs) genotyping with copy number variations (CNVs) is required. The aim of this study was to discover SNVs or common copy CNVs and examine the combined frequencies of SNVs and CNVs in pharmacogenes using the Korean genome and epidemiology study (KoGES), a consortium project. The genotypes (N = 72,299) and CNV data (N = 1000) were provided by the Korean National Institute of Health, Korea Centers for Disease Control and Prevention. The allele frequencies of SNVs, CNVs, and combined SNVs with CNVs were calculated and haplotype analysis was performed. CYP2D6 rs1065852 (c.100C>T, p.P34S) was the most common variant allele (48.23%). A total of 8454 haplotype blocks in 18 pharmacogenes were estimated. DMD ranked the highest in frequency for gene gain (64.52%), while TPMT ranked the highest in frequency for gene loss (51.80%). Copy number gain of CYP4F2 was observed in 22 subjects; 13 of those subjects were carriers with CYP4F2*3 gain. In the case of TPMT, approximately one-half of the participants (N = 308) had loss of the TPMT*1*1 diplotype. The frequencies of SNVs and CNVs in pharmacogenes were determined using the Korean cohort-based genome-wide association study.

Analysis of Reelin signaling and neurodevelopmental trajectory in primary cultured cortical neurons with RELN deletion identified in schizophrenia

Reelin, an extracellular matrix protein, is secreted by Cajal-Retzius cells and plays crucial roles in the development of brain structures and neuronal functions. Reductions in Reelin cause the brain dysfunctions associated with mental disorders, such as schizophrenia. A recent genome-wide copy number variation analysis of Japanese schizophrenia patients identified a novel deletion in RELN encoding Reelin. To clarify the pathophysiological role of the RELN deletion, we developed transgenic mice carrying the RELN deletion (Reln-del) and found abnormalities in their brain structures and social behavior. In the present study, we performed an in vitro analysis of Reelin expression, intracellular Reelin signaling, and the morphology of primary cultured cortical neurons from wild-type (WT) and Reln-del mice. Reelin protein levels were lower in Reln-del neurons than in WT neurons. Dab1 expression levels were significantly higher in Reln-del neurons than in WT neurons, suggesting that Reelin signaling was decreased in Reln-del neurons. Reelin was mainly expressed in γ-aminobutyric acid (GABA)-ergic inhibitory neurons, but not in parvalbumin (PV)-positive neurons. A small proportion of Ca2+/calmodulin-dependent protein kinase II α subunit (CaMKIIα)-positive excitatory neurons also expressed Reelin. In comparisons with WT neurons, significant decreases were observed in neurite lengths and branch points as well as in the number of postsynaptic density protein 95 (PSD95) immunoreactive puncta in Reln-del neurons. A disintegrin and metalloproteinase with thrombospondin motifs-3 (ADAMTS-3) is a protease that inactivates Reelin by cleavage at the N-t site. The knockdown of ADAMTS-3 by short hairpin RNAs suppressed Reelin cleavage in conditioned medium and reduced Dab1 expression, indicating that Reelin signaling was enhanced in the primary cultured cortical neurons of WT and heterozygous Reln-del. Accordingly, the inhibition of ADAMTS-3 may be a potential candidate in the clinical treatment of schizophrenia by enhancing Reelin signaling in the brain.

Genome-Wide Copy Number Variation of Circulating Cell-Free DNA As a Biomarker in Head and Neck Cancer Patients Treated With Immunotherapy

Genome-Wide Copy Number Variation of Circulating Cell-Free DNA As a Biomarker in Head and Neck Cancer Patients Treated With Immunotherapy

Short somatic alterations at the site of copy number variation in breast cancer.

Copy number variation (CNV) is a polymorphism in the human genome involving DNA fragments larger than 1 kb. Copy number variation sites provide hotspots of somatic alterations in cancers. Herein, we examined somatic alterations at sites of CNV in DNA from 20 invasive breast cancers using a Comparative Genomic Hybridization array specifically designed to detect the genome‐wide CNV status of approximately 412 000 sites. Somatic copy number alterations (CNAs) were detected in 39.9% of the CNV probes examined. The most frequently altered regions were gains of 1q21‐22 (90%), 8q21‐24 (85%), 1q44 (85%), and 3q11 (85%) or losses of 16q22‐24 (80%). Gene ontology analyses of genes within the CNA fragments revealed that cascades related to transcription and RNA metabolism correlated significantly with human epidermal growth factor receptor 2 positivity and menopausal status. Thirteen of 20 tumors showed CNAs in more than 35% of sites examined and a high prevalence of CNAs correlated significantly with estrogen receptor (ER) negativity, higher nuclear grade (NG), and higher Ki‐67 labeling index. Finally, when CNA fragments were categorized according to their size, CNAs smaller than 10 kb correlated significantly with ER positivity and lower NG, whereas CNAs exceeding 10 Mb correlated with higher NG, ER negativity, and a higher Ki‐67 labeling index. Most of these findings were confirmed or supported by quantitative PCR of representative DNA fragments in 72 additional breast cancers. These results suggest that most CNAs are caused by gain or loss of large chromosomal fragments and correlate with NG and several malignant features, whereas solitary CNAs of less than 10 kb could be involved in ER‐positive breast carcinogenesis.

Genome-Wide Analysis of Copy Number Variation in Latin American Parkinson's Disease Patients.

Parkinson's disease is the second most common neurodegenerative disorder and affects people from all ethnic backgrounds, yet little is known about the genetics of Parkinson's disease in non-European populations. In addition, the overall identification of copy number variants at a genome-wide level has been understudied in Parkinson's patients. The objective of this study was to understand the genome-wide burden of copy number variants in Latinos and its association with Parkinson's disease. We used genome-wide genotyping data from 747 Parkinson's disease patients and 632 controls from the Latin American Research Consortium on the Genetics of Parkinson's disease. Genome-wide copy number burden analysis showed that patients were significantly enriched for copy number variants overlapping known Parkinson's disease genes compared with controls (odds ratio, 3.97; 95%CI, 1.69-10.5; P = 0.018). PRKN showed the strongest copy number burden, with 20 copy number variant carriers. These patients presented an earlier age of disease onset compared with patients with other copy number variants (median age at onset, 31 vs 57 years, respectively; P = 7.46 × 10-7 ). We found that although overall genome-wide copy number variant burden was not significantly different, Parkinson's disease patients were significantly enriched with copy number variants affecting known Parkinson's disease genes. We also identified that of 250 patients with early-onset disease, 5.6% carried a copy number variant on PRKN in our cohort. Our study is the first to analyze genome-wide copy number variant association in Latino Parkinson's disease patients and provides insights about this complex disease in this understudied population. © 2020 International Parkinson and Movement Disorder Society.

P.739 High-throughput proteomics for suicide biomarker discovery in patients with bipolar disorder

The genetic basis of suicide attempts (SA) remains unclear. Especially the role of copy number variations (CNVs) remains to be elucidated. The present study aimed to identify susceptibility variants associated with SA among Chinese with major depressive disorder (MDD), covering both CNVs and single-nucleotide polymorphisms (SNPs).We conducted a genome-wide association study (GWAS) on MDD patients with and without SA and top results were tested in a replication study. A genome-wide CNV study was also performed. Subsequently, a validation assay using qRT-PCR technology was performed to confirm any associated CNVs and then applied to the entire cohort to examine the association.Although GWAS did not identify any SNPs reaching genome-wide significance, we identified TPH2 as the top susceptibility gene (p-value = 2.75e−05) in gene-based analysis, which is a strong biological candidate for its role in the serotonergic system. As for CNV analysis, we found that the global rate of CNV was higher in SA than that in non-SA subjects (p-value = 0.023). Genome-wide CNV study revealed an SA-associated CNV region that achieved genome-wide significance (corrected p-value = 0.014). The associated CNV was successfully validated with a more rigorous qRT-PCR assay and identified to be a common variant in this cohort. Its deletion rate was higher in SA subjects [OR = 2.05 (1.02–4.12), adjusted p-value = 0.045]. Based on the GTEx database, genetic variants that probed this CNV were significantly associated with the expression level of ZNF33B in two brain regions (p-value < 4.2e−05). In stratified analysis, the CNV showed a significant effect [OR = 2.58 (1.06–6.27), p-value = 0.039] in those with high neuroticism but not in those with average or low neuroticism.We identified a new common CNV likely involved in the etiology of SA. This finding sheds light on an important role of common CNVs in the pathophysiology of SA, suggesting a new promising avenue for investigating its genetic architecture.

Comparative analyses of copy number variations between Bos taurus and Bos indicus

BackgroundBos taurus and Bos indicus are two main sub-species of cattle. However, the differential copy number variations (CNVs) between them are not yet well studied.ResultsBased on the new high-quality cattle reference genome ARS-UCD1.2, we identified 13,234 non-redundant CNV regions (CNVRs) from 73 animals of 10 cattle breeds (4 Bos taurus and 6 Bos indicus), by integrating three detection strategies. While 6990 CNVRs (52.82%) were shared by Bos taurus and Bos indicus, large CNV differences were discovered between them and these differences could be used to successfully separate animals into two subspecies. We found that 2212 and 538 genes uniquely overlapped with either indicine-specific CNVRs and or taurine-specific CNVRs, respectively. Based on FST, we detected 16 candidate lineage-differential CNV segments (top 0.1%) under selection, which overlapped with eight genes (CTNNA1, ENSBTAG00000004415, PKN2, BMPER, PDE1C, DNAJC18, MUSK, and PLCXD3). Moreover, we obtained 1.74 Mbp indicine-specific sequences, which could only be mapped on the Bos indicus reference genome UOA_Brahman_1. We found these sequences and their associated genes were related to heat resistance, lipid and ATP metabolic process, and muscle development under selection. We further analyzed and validated the top significant lineage-differential CNV. This CNV overlapped genes related to muscle cell differentiation, which might be generated from a retropseudogene of CTH but was deleted along Bos indicus lineage.ConclusionsThis study presents a genome wide CNV comparison between Bos taurus and Bos indicus. It supplied essential genome diversity information for understanding of adaptation and phenotype differences between the Bos taurus and Bos indicus populations.

Genome variation and population structure among 1142 mosquitoes of the African malaria vector species Anopheles gambiae and Anopheles coluzzii.

Mosquito control remains a central pillar of efforts to reduce malaria burden in sub-Saharan Africa. However, insecticide resistance is entrenched in malaria vector populations, and countries with a high malaria burden face a daunting challenge to sustain malaria control with a limited set of surveillance and intervention tools. Here we report on the second phase of a project to build an open resource of high-quality data on genome variation among natural populations of the major African malaria vector species Anopheles gambiae and Anopheles coluzzii. We analyzed whole genomes of 1142 individual mosquitoes sampled from the wild in 13 African countries, as well as a further 234 individuals comprising parents and progeny of 11 laboratory crosses. The data resource includes high-confidence single-nucleotide polymorphism (SNP) calls at 57 million variable sites, genome-wide copy number variation (CNV) calls, and haplotypes phased at biallelic SNPs. We use these data to analyze genetic population structure and characterize genetic diversity within and between populations. We illustrate the utility of these data by investigating species differences in isolation by distance, genetic variation within proposed gene drive target sequences, and patterns of resistance to pyrethroid insecticides. This data resource provides a foundation for developing new operational systems for molecular surveillance and for accelerating research and development of new vector control tools. It also provides a unique resource for the study of population genomics and evolutionary biology in eukaryotic species with high levels of genetic diversity under strong anthropogenic evolutionary pressures.

Copy number variant hotspots in Han Taiwanese population induced pluripotent stem cell lines - lessons from establishing the Taiwan human disease iPSC Consortium Bank

BackgroundThe Taiwan Human Disease iPSC Service Consortium was established to accelerate Taiwan’s growing stem cell research initiatives and provide a platform for researchers interested in utilizing induced pluripotent stem cell (iPSC) technology. The consortium has generated and characterized 83 iPSC lines: 11 normal and 72 disease iPSC lines covering 21 different diseases, several of which are of high incidence in Taiwan. Whether there are any reprogramming-induced recurrent copy number variant (CNV) hotspots in iPSCs is still largely unknown.MethodsWe performed genome-wide copy number variant screening of 83 Han Taiwanese iPSC lines and compared them with 1093 control subjects using an Affymetrix genome-wide human SNP array.ResultsIn the iPSCs, we identified ten specific CNV loci and seven “polymorphic” CNV regions that are associated with the reprogramming process. Additionally, we established several differentiation protocols for our iPSC lines. We demonstrated that our iPSC-derived cardiomyocytes respond to pharmacological agents and were successfully engrafted into the mouse myocardium demonstrating their potential application in cell therapy.ConclusionsThe CNV hotspots induced by cell reprogramming have successfully been identified in the current study. This finding may be used as a reference index for evaluating iPSC quality for future clinical applications. Our aim was to establish a national iPSC resource center generating iPSCs, made available to researchers, to benefit the stem cell community in Taiwan and throughout the world.

Chromosomal microarray analysis of benign mesenchymal tumors with RB1 deletion

Spindle cell lipomas/pleomorphic lipomas, mammary-type myofibroblastomas, and cellular angiofibromas are benign mesenchymal tumors that demonstrate histologically overlapping features but with varying anatomic locations and an uncertain etiologic relationship. These tumors have also been found to have an overlapping molecular profile with shared 13q14 deletions, which is the location of the tumor suppressor gene RB1 that encodes the retinoblastoma protein. Molecular studies thus far have largely focused on the RB1 locus, using primarily immunohistochemistry and fluorescence in situ hybridization to characterize RB1 status. However, further characterization of the molecular profile of these lesions, including genome-wide copy number variation, remains to be well defined. The goal of this study is to further characterize the specific RB1 deletions seen in spindle cell lipomas/pleomorphic lipomas, cellular angiofibromas, and mammary-type myofibroblastomasas well as to evaluate these neoplasms for additional molecular abnormalities using the OncoScan™ CNV Plus Assay, which is used for clinical use as a whole-genome copy number microarray-based assay. Ten of eleven cases demonstrated deletion of the RB1 gene with varying deletion size and breakpoints. The majority of additional genetic alterations were chromosomal losses and loss of heterozygosity with rare chromosomal gains. Although only a small subset of mesenchymal neoplasms was evaluated, the principle of creating a novel pairing of the molecular method with the tumor type represents a promising avenue for further study in a variety of tumors.

ARHGAP10, which encodes Rho GTPase-activating protein 10, is a novel gene for schizophrenia risk

Schizophrenia (SCZ) is known to be a heritable disorder; however, its multifactorial nature has significantly hampered attempts to establish its pathogenesis. Therefore, in this study, we performed genome-wide copy-number variation (CNV) analysis of 2940 patients with SCZ and 2402 control subjects and identified a statistically significant association between SCZ and exonic CNVs in the ARHGAP10 gene. ARHGAP10 encodes a member of the RhoGAP superfamily of proteins that is involved in small GTPase signaling. This signaling pathway is one of the SCZ-associated pathways and may contribute to neural development and function. However, the ARHGAP10 gene is often confused with ARHGAP21, thus, the significance of ARHGAP10 in the molecular pathology of SCZ, including the expression profile of the ARHGAP10 protein, remains poorly understood. To address this issue, we focused on one patient identified to have both an exonic deletion and a missense variant (p.S490P) in ARHGAP10. The missense variant was found to be located in the RhoGAP domain and was determined to be relevant to the association between ARHGAP10 and the active form of RhoA. We evaluated ARHGAP10 protein expression in the brains of reporter mice and generated a mouse model to mimic the patient case. The model exhibited abnormal emotional behaviors, along with reduced spine density in the medial prefrontal cortex (mPFC). In addition, primary cultured neurons prepared from the mouse model brain exhibited immature neurites in vitro. Furthermore, we established induced pluripotent stem cells (iPSCs) from this patient, and differentiated them into tyrosine hydroxylase (TH)-positive neurons in order to analyze their morphological phenotypes. TH-positive neurons differentiated from the patient-derived iPSCs exhibited severe defects in both neurite length and branch number; these defects were restored by the addition of the Rho-kinase inhibitor, Y-27632. Collectively, our findings suggest that rare ARHGAP10 variants may be genetically and biologically associated with SCZ and indicate that Rho signaling represents a promising drug discovery target for SCZ treatment.

Genome-wide characterization of copy number variations in the host genome in genetic resistance to Marek\u2019s disease using next generation sequencing

BackgroundMarek’s disease (MD) is a highly neoplastic disease primarily affecting chickens, and remains as a chronic infectious disease that threatens the poultry industry. Copy number variation (CNV) has been examined in many species and is recognized as a major source of genetic variation that directly contributes to phenotypic variation such as resistance to infectious diseases. Two highly inbred chicken lines, 63 (MD-resistant) and 72 (MD-susceptible), as well as their F1 generation and six recombinant congenic strains (RCSs) with varied susceptibility to MD, are considered as ideal models to identify the complex mechanisms of genetic and molecular resistance to MD.ResultsIn the present study, to unravel the potential genetic mechanisms underlying resistance to MD, we performed a genome-wide CNV detection using next generation sequencing on the inbred chicken lines with the assistance of CNVnator. As a result, a total of 1649 CNV regions (CNVRs) were successfully identified after merging all the nine datasets, of which 90 CNVRs were overlapped across all the chicken lines. Within these shared regions, 1360 harbored genes were identified. In addition, 55 and 44 CNVRs with 62 and 57 harbored genes were specifically identified in line 63 and 72, respectively. Bioinformatics analysis showed that the nearby genes were significantly enriched in 36 GO terms and 6 KEGG pathways including JAK/STAT signaling pathway. Ten CNVRs (nine deletions and one duplication) involved in 10 disease-related genes were selected for validation by using quantitative real-time PCR (qPCR), all of which were successfully confirmed. Finally, qPCR was also used to validate two deletion events in line 72 that were definitely normal in line 63. One high-confidence gene, IRF2 was identified as the most promising candidate gene underlying resistance and susceptibility to MD in view of its function and overlaps with data from previous study.ConclusionsOur findings provide valuable insights for understanding the genetic mechanism of resistance to MD and the identified gene and pathway could be considered as the subject of further functional characterization.

Abstract B04: Three distinct subgroups of Wilms’ tumors with novel molecular features and important clinical implications are defined by genome-wide DNA methylation profiles

Abstract Introduction: Wilms’ tumor (WT) is the most common childhood renal cancer. Although many children are cured with standard therapy, survivors frequently suffer chronic diseases as a result of their treatment. Historical evidence suggests that a number of children could be cured with surgery alone; however, many of these children are currently difficult to identify. Although the majority of mutations described in this disease are not prognostic, the commonest single molecular alteration in WT is epigenetic—loss of imprinting at chromosome 11p15. We therefore examined genome-wide patterns of DNA methylation in WTs to identify groups of patients with low risk of relapse. Methods: We enrolled two cohorts of patients: a discovery cohort consisting of 32 tumors and 21 matched kidneys was recruited at the time of surgery at our hospital. A validation cohort included 40 tumor-normal pairs from the Children’s Oncology Group (COG) biobank and an additional 11 pairs from our hospital. All samples had genome-wide DNA methylation and copy number variation data generated. The samples from COG additionally had whole-exome sequencing while 22 local tumors had RNA sequencing. Results: Unsupervised clustering of DNA methylation data demonstrated three DNA methylation subgroups that could be reproduced in the validation cohort using a “signature” of selected differentially methylated sites. One group (“PRO”) had genome-wide loss of methylation with specific gain of methylation at renal development genes and tumor-suppressor genes. This group also had increased expression of genes related to cell proliferation. Mutations in microRNA processing genes were found exclusively in this group and were associated with dysregulation of microRNA expression. All known cases of relapse were found in this group. A second group (“DIFF”) had a DNA methylation profile similar to normal kidney, no copy number alterations, reduced expression of genes associated with early renal development, and mutations only in WT1 or CTNNB1. No relapses were known in this group, but there was an increased incidence of bilateral disease. A third group (“INT”) had intermediate methylation values at all signature sites, a gene expression profile similar to the DIFF group, and increased microRNA dysregulation. All tumors in the INT group had either mutations in TRIM28, epithelial histology, or both. Discussion: Genome-wide DNA methylation profiling demonstrates three subgroups of WT with distinct molecular and clinical characteristics. The DIFF group has molecular features consistent with a differentiation process occurring. These patients may be candidates for therapy reduction. The INT group suggests that some patients with wild-type TRIM28 share features with tumors with mutations in the gene—known to be associated with low risk of relapse. This group may also be a candidate for therapy reduction. We are now investigating the features that drive increased proliferation in the PRO group as these may be targets of future therapy for these higher-risk patients. Citation Format: Jack Brzezinski, Sanaa Choufani, Rodrigo Romao, Cheryl Shuman, Haiying Chen, Ronald Grant, Armando Lorenzo, Rosanna Weksberg. Three distinct subgroups of Wilms’ tumors with novel molecular features and important clinical implications are defined by genome-wide DNA methylation profiles [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B04.

Molecular karyotyping and gene expression analysis in childhood cancer patients

The genetic etiology of sporadic childhood cancer cases remains unclear. We recruited a cohort of 20 patients who survived a childhood malignancy and then developed a second primary cancer (2N), and 20 carefully matched patients who survived a childhood cancer without developing a second malignancy (1N). Twenty matched cancer-free (0N) and additional 1000 (0N) GHS participants served as controls. Aiming to identify new candidate loci for cancer predisposition, we compared the genome-wide DNA copy number variations (CNV) with the RNA-expression data obtained after in vitro irradiation of primary fibroblasts. In 2N patients, we detected a total of 142 genes affected by CNV. A total of 53 genes of these were not altered in controls. Six genes (POLR3F, SEC23B, ZNF133, C16orf45, RRN3, and NTAN1) that we found to be overexpressed after irradiation were also duplicated in the genome of the 2N patients. For the 1N collective, 185 genes were affected by CNV and 38 of these genes were not altered in controls. Five genes (ZCWPW2, SYNCRIP, DHX30, DHRS4L2, and THSD1) were located in duplicated genomic regions and exhibited altered RNA expression after irradiation. One gene (ABCC6) was partially duplicated in one 1N and one 2N patient. Analysis of methylation levels of THSD1 and GSTT2 genes which were detected in duplicated regions and are frequently aberrantly methylated in cancer showed no changes in patient’s fibroblasts. In summary, we describe rare and radiation-sensitive genes affected by CNV in childhood sporadic cancer cases, which may have an impact on cancer development.Key messages• Rare CNV’s may have an impact on cancer development in sporadic, non-familial, non-syndromic childhood cancer cases.• In our cohort, each patient displayed a unique pattern of cancer-related gene CNVs, and only few cases shared similar CNV.• Genes that are transcriptionally regulated after radiation can be located in CNVs in cancer patients and controls.• THSD1 and GSTT2 methylation is not altered by CNV.

Epilepsy subtype-specific copy number burden observed in a genome-wide study of 17 458 subjects.

Cytogenic testing is routinely applied in most neurological centres for severe paediatric epilepsies. However, which characteristics of copy number variants (CNVs) confer most epilepsy risk and which epilepsy subtypes carry the most CNV burden, have not been explored on a genome-wide scale. Here, we present the largest CNV investigation in epilepsy to date with 10 712 European epilepsy cases and 6746 ancestry-matched controls. Patients with genetic generalized epilepsy, lesional focal epilepsy, non-acquired focal epilepsy, and developmental and epileptic encephalopathy were included. All samples were processed with the same technology and analysis pipeline. All investigated epilepsy types, including lesional focal epilepsy patients, showed an increase in CNV burden in at least one tested category compared to controls. However, we observed striking differences in CNV burden across epilepsy types and investigated CNV categories. Genetic generalized epilepsy patients have the highest CNV burden in all categories tested, followed by developmental and epileptic encephalopathy patients. Both epilepsy types also show association for deletions covering genes intolerant for truncating variants. Genome-wide CNV breakpoint association showed not only significant loci for genetic generalized and developmental and epileptic encephalopathy patients but also for lesional focal epilepsy patients. With a 34-fold risk for developing genetic generalized epilepsy, we show for the first time that the established epilepsy-associated 15q13.3 deletion represents the strongest risk CNV for genetic generalized epilepsy across the whole genome. Using the human interactome, we examined the largest connected component of the genes overlapped by CNVs in the four epilepsy types. We observed that genetic generalized epilepsy and non-acquired focal epilepsy formed disease modules. In summary, we show that in all common epilepsy types, 1.5-3% of patients carry epilepsy-associated CNVs. The characteristics of risk CNVs vary tremendously across and within epilepsy types. Thus, we advocate genome-wide genomic testing to identify all disease-associated types of CNVs.

Genome-wide copy number variation-, validation- and screening study implicates a new copy number polymorphism associated with suicide attempts in major depressive disorder.

The genetic basis of suicide attempts (SA) remains unclear. Especially the role of copy number variations (CNVs) remains to be elucidated. The present study aimed to identify susceptibility variants associated with SA among Chinese with major depressive disorder (MDD), covering both CNVs and single-nucleotide polymorphisms (SNPs). We conducted a genome-wide association study (GWAS) on MDD patients with and without SA and top results were tested in a replication study. A genome-wide CNV study was also performed. Subsequently, a validation assay using qRT-PCR technology was performed to confirm any associated CNVs and then applied to the entire cohort to examine the association. Although GWAS did not identify any SNPs reaching genome-wide significance, we identified TPH2 as the top susceptibility gene (p-value=2.75e-05) in gene-based analysis, which is a strong biological candidate for its role in the serotonergic system. As for CNV analysis, we found that the global rate of CNV was higher in SA than that in non-SA subjects (p-value=0.023). Genome-wide CNV study revealed an SA-associated CNV region that achieved genome-wide significance (corrected p-value=0.014). The associated CNV was successfully validated with a more rigorous qRT-PCR assay and identified to be a common variant in this cohort. Its deletion rate was higher in SA subjects [OR=2.05 (1.02-4.12), adjusted p-value=0.045]. Based on the GTEx database, genetic variants that probed this CNV were significantly associated with the expression level of ZNF33B in two brain regions (p-value<4.2e-05). In stratified analysis, the CNV showed a significant effect [OR=2.58 (1.06-6.27), p-value=0.039] in those with high neuroticism but not in those with average or low neuroticism. We identified a new common CNV likely involved in the etiology of SA. This finding sheds light on an important role of common CNVs in the pathophysiology of SA, suggesting a new promising avenue for investigating its genetic architecture.

Genomic Analysis of Circulating Tumor Cells at the Single-Cell Level

Circulating tumor cells (CTCs) have a great potential for noninvasive diagnosis and real-time monitoring of cancer. A comprehensive evaluation of four whole genome amplification (WGA)/next-generation sequencing workflows for genomic analysis of single CTCs, including PCR-based (GenomePlex and Ampli1), multiple displacement amplification (Repli-g), and hybrid PCR- and multiple displacement amplification–based [multiple annealing and loop-based amplification cycling (MALBAC)] is reported herein. To demonstrate clinical utilities, copy number variations (CNVs) in single CTCs isolated from four patients with squamous non–small-cell lung cancer were profiled. Results indicate that MALBAC and Repli-g WGA have significantly broader genomic coverage compared with GenomePlex and Ampli1. Furthermore, MALBAC coupled with low-pass whole genome sequencing has better coverage breadth, uniformity, and reproducibility and is superior to Repli-g for genome-wide CNV profiling and detecting focal oncogenic amplifications. For mutation analysis, none of the WGA methods were found to achieve sufficient sensitivity and specificity by whole exome sequencing. Finally, profiling of single CTCs from patients with non–small-cell lung cancer revealed potentially clinically relevant CNVs. In conclusion, MALBAC WGA coupled with low-pass whole genome sequencing is a robust workflow for genome-wide CNV profiling at single-cell level and has great potential to be applied in clinical investigations. Nevertheless, data suggest that none of the evaluated single-cell sequencing workflows can reach sufficient sensitivity or specificity for mutation detection required for clinical applications.

DNA methylation and copy number variation profiling of T-cell lymphoblastic leukemia and lymphoma

Despite having common overlapping immunophenotypic and morphological features, T-cell lymphoblastic leukemia (T-ALL) and lymphoma (T-LBL) have distinct clinical manifestations, which may represent separate diseases. We investigated and compared the epigenetic and genetic landscape of adult and pediatric T-ALL (n = 77) and T-LBL (n = 15) patient samples by high-resolution genome-wide DNA methylation and Copy Number Variation (CNV) BeadChip arrays. DNA methylation profiling identified the presence of CpG island methylator phenotype (CIMP) subgroups within both pediatric and adult T-LBL and T-ALL. An epigenetic signature of 128 differentially methylated CpG sites was identified, that clustered T-LBL and T-ALL separately. The most significant differentially methylated gene loci included the SGCE/PEG10 shared promoter region, previously implicated in lymphoid malignancies. CNV analysis confirmed overlapping recurrent aberrations between T-ALL and T-LBL, including 9p21.3 (CDKN2A/CDKN2B) deletions. A significantly higher frequency of chromosome 13q14.2 deletions was identified in T-LBL samples (36% in T-LBL vs. 0% in T-ALL). This deletion, encompassing the RB1, MIR15A and MIR16-1 gene loci, has been reported as a recurrent deletion in B-cell malignancies. Our study reveals epigenetic and genetic markers that can distinguish between T-LBL and T-ALL, and deepen the understanding of the biology underlying the diverse disease localization.

Population Structure, and Selection Signatures Underlying High-Altitude Adaptation Inferred From Genome-Wide Copy Number Variations in Chinese Indigenous Cattle.

Copy number variations (CNVs) have been demonstrated as crucial substrates for evolution, adaptation and breed formation. Chinese indigenous cattle breeds exhibit a broad geographical distribution and diverse environmental adaptability. Here, we analyzed the population structure and adaptation to high altitude of Chinese indigenous cattle based on genome-wide CNVs derived from the high-density BovineHD SNP array. We successfully detected the genome-wide CNVs of 318 individuals from 24 Chinese indigenous cattle breeds and 37 yaks as outgroups. A total of 5,818 autosomal CNV regions (683 bp–4,477,860 bp in size), covering ~14.34% of the bovine genome (UMD3.1), were identified, showing abundant CNV resources. Neighbor-joining clustering, principal component analysis (PCA), and population admixture analysis based on these CNVs support that most Chinese cattle breeds are hybrids of Bos taurus taurus (hereinafter to be referred as Bos taurus) and Bos taurus indicus (Bos indicus). The distribution patterns of the CNVs could to some extent be related to the geographical backgrounds of the habitat of the breeds, and admixture among cattle breeds from different districts. We analyzed the selective signatures of CNVs positively involved in high-altitude adaptation using pairwise Fst analysis within breeds with a strong Bos taurus background (taurine-type breeds) and within Bos taurus×Bos indicus hybrids, respectively. CNV-overlapping genes with strong selection signatures (at top 0.5% of Fst value), including LETM1 (Fst = 0.490), TXNRD2 (Fst = 0.440), and STUB1 (Fst = 0.420) within taurine-type breeds, and NOXA1 (Fst = 0.233), RUVBL1 (Fst = 0.222), and SLC4A3 (Fst=0.154) within hybrids, were potentially involved in the adaptation to hypoxia. Thus, we provide a new profile of population structure from the CNV aspects of Chinese indigenous cattle and new insights into high-altitude adaptation in cattle.