Genome-wide mapping and functional analysis of copy number variation in the human genome

Copy Number Variation (CNV) has emerged as a major source of human genomic variation comprising benign and pathological variants. These deletions and duplications of genomic regions form a size continuum from small indels to whole chromosomal aneuploidies, and have been mapped by dozens of studies employing diverse methods. Accurate and efficient genome-wide detection of copy number variants (CNVs) is essential for understanding human genomic variation, genome-wide CNV association type studies, cytogenetics research and diagnostics, and independent validation of CNVs identified from sequencing based technologies. There are several microarray-based technologies for mapping CNVs utilizing array Comparative Genome Hybridization (aCGH), Single Nucleotide Polymorphism (SNP) genotyping and combination platforms. We developed methods to refine the mapping of CNVs in the human genome using high-resolution and high-throughput microarray technologies. We developed several aCGH platforms targeted to specific genomic regions in order to map CNVs in these regions of interest with high breakpoint accuracy. We also attempted to develop a multiplexed aCGH protocol to allow four different samples to be hybridized to the same array, thereby increasing the array CNV mapping efficiency. Alongside our efforts, several commercial array- based CNV detection platforms became available. We quantitatively assessed the abilities of twelve leading genome-wide CNV detection platforms to accurately detect Gold Standard sets of CNVs in the genome of HapMap CEU sample NA12878 (Haraksingh et al. 2011). We found significant differences in performance and that sensitivity, total number, size range, and breakpoint resolution of CNV calls were highest for CNV focused arrays. Despite the rapidly growing appreciation for the extent of CNV in the human genome, evidence for their functional consequences remains limited. It is clear, that CNVs are theoretically capable of reorganizing functional elements of the genome by altering gene dosage, coding segments, and regulatory regions. Recently, several association studies have suggested that CNVs significantly impact certain disease phenotypes. Performing CNV-phenotype association studies requires cost-effective, unbiased, genome-wide, high-resolution mapping of common and rare CNVs. We used some of the best performing array-based technologies from our comparison to investigate the association of CNVs with various phenotypes; the NimbleGen 2.1 M CNV array for hereditary hearing loss, our custom NimbleGen Functional Elements and Variable Regions (FEVR) array for melanoma, our custom NimbleGen lexinome array for dyslexia, and the NimbleGen 2.1 M WG array for basal cell carcinoma. We found a relatively strong association between a deletion on chromosome 16 and hearing loss (Odds Ratio = 3.41). In addition, we investigated whether certain pathways were enriched for CNVs in cases versus controls, and whether the cases had a higher CNV load than the controls. Both of these analyses showed no difference in CNV load between the cases and controls. We found several other CNVs in genes already known to be associated with hearing loss, indicating the existence of multiple causative alleles in this sample set. We also found weaker CNV associations to melanoma and basal cell carcinoma. Finally, we attempted to measure the direct effects of CNVs on transcription using RNA-seq on 42 lymphoblastoid cell lines each containing one of three large CNVs known to be associated with Schizophrenia. We found that copy number within these large CNVs is generally not predictive of transcriptional activity indicating that complex dosage compensation mechanisms may exist. This work highlights the importance of high-resolution mapping of CNVs to understand their role in human genomic variation and their biological relevance

Prevalence of CYP2C19*2 and CYP2C19*3 Allelic Variants and Clopidogrel Use in Patients with Cardiovascular Disease in Trinidad & Tobago

Abstract Introduction Trinidad & Tobago has the highest prevalence of cardiovascular disease (CVD) in the Caribbean and clopidogrel is a ubiquitously used treatment. Yet, the extent of genetically mediated clopidogrel resistance is unknown. To determine this, we investigated whether the association between CYP2C19*2 and CYP2C19*3 genetic variants and clopidogrel resistance holds, and calculated the frequencies of these in the Trinidadian CVD population. Methods Demographic data, clinical data, and a saliva sample were collected under informed consent from 22 patients with CVD on dual anti-platelet therapy whose biochemical resistance to clopidogrel is known, and a further 162 patients accessing the main public CVD clinic in Trinidad and who are either currently being treated or are likely to be treated with clopidogrel. A polymerase chain reaction (PCR) and restriction enzyme digestion procedure was used to genotype each patient for the CYP2C19*2 and CYP2C19*3 allelic variants. Genotype was compared to known clopidogrel resistance in the 22 patients, and to disease status and clopidogrel usage in the larger cohort. Results CYP2C19*2 genotype was concordant with clopidogrel resistance. CYP2C19*2 was detected in 61.1% (99/162) of patients and CYP2C19*3 was undetected. Clopidogrel was the most prescribed antiplatelet therapy (42%). A total of 120 people presented with coronary artery disease (CAD) and 52.5% of these (n = 63/120) are currently prescribed clopidogrel. 63.5% (40/63) of patients with CAD who are prescribed clopidogrel carry the CYP2C19*2 allele; ten homozygous and 30 heterozygous. Indian patients comprised 65% of the cohort and were four times more likely to carry the CYP2C19*2 allele than African patients. Conclusions A large proportion of Trinidadian patients with CVD who are prescribed or may be prescribed clopidogrel carry genetic variants associated with clopidogrel resistance. These results emphasize the clinical need for further investigation into whether CYP2C19*2 genotype should guide clopidogrel use for the cardiovascular disease population in Trinidad & Tobago. A slide deck is available for this article

Prevalence of CYP2C19*2 and CYP2C19*3 Allelic Variants and Clopidogrel Use in Patients with Cardiovascular Disease in Trinidad & Tobago

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