Copy number variation (CNV) is a common feature of eukaryotic genomes, and a growing body of evidence suggests that genes affected by CNV are enriched in processes associated with environmental responses. Traditionally, the analysis of next-generation sequencing (NGS) and SNP microarray datasets has focused on the analysis of single-nucleotide polymorphism (SNPs), and small insertions and deletions (INDELS). However, by employing different analytical techniques CNV can be identified in these same datasets. Using NGS data from domesticated apple (Malus x Domestica Borkh) accessions, and SNP microarray data from participants with gout and publicly available controls, I investigated, for the first time, the impact that CNV has within the genome of the domesticated apple and on gout. Although seemingly unrelated, gout disease and resistance to pathogens and infection in apples both involve the immune sys- tem. This is important because genomic regions harbouring CNVs are enriched in immune system functions within all eukaryotes investigated to date, making the apple genome and gout disease novel contexts where an investigation of CNV would be worthwhile.Genome-wide association studies in gout have identified a number of common associated SNPs. Many of these variants have been implicated in uric acid metabolism while the genetic factors involved in the inflammatory response have remained elusive. Since CNVs are enriched in immune-related functions, they represent a potential source for the genetic components of the immune response in gout. Using Immunochip SNP array data from a sample set con- sisting of 468 participants with gout and 1,000 publicly-available controls, I performed an analysis of CNV in gout. While no overall burden of rare (fre- quency < 1%) CNV was observed, a deletion on chromosome 5 between po- sitions 686,123-731,394 was discovered to be protective for gout (Pdeletions=0.02, Pcorrected=0.09). Using qPCR, this association was replicated across the full range of integer copy-numbers at this region (ORinteger−copy−number=1.28, P=0.04). Interestingly, there is functional connection between TPPP, which is a gene that overlapped the CNV, and Colchicine, a compound that is used to treat gout. Specifically, TPPP promotes acetylation of α-tubulin by inhibiting HDAC6 and SIRT2, and Colchicine prevents the polymerisation of tubulin, indirectly reducing the levels of acetylated α-tubulin. A non-additive interaction between the CNV region at chr5:686123-731394 and rs2231142 was detected (Interaction term OR=0.43, P=0.018). This provided evidence that this CNV region influences microtubule dynamics. TPPP has been implicated in diseases of the brain, such as Alzheimer’s and Parkinson’s. Since gout and/or high uric acid levels are protective risk factors for these disorders, CNV of chr5:686123-731394 may provide insight into the molecular mechanisms underpinning this epidemiological observation.The copy-number variable regions (CNVRs) of the apple genome were identified using NGS data derived from 30 accessions of apple. This was achieved by analysing this data using the read-depth method, as implemented in the CN- Vrd2 software. To improve the reliability of the results, a quality control and analysis procedure was developed. This involved checking for organelle DNA, not repeat masking, and the determination of CNVR identity using a permutation testing procedure. Overall, I identified 876 CNVRs, which spanned 3.5% of the apple genome. To verify that detected CNVRs were not artifacts, I analysed the B-allele-frequencies (BAF) within a single nucleotide polymorphism (SNP) array dataset derived from a screening of 185 individual apple accessions and found the CNVRs were enriched for SNPs having aberrant BAFs (P < 1 × 10−13, Fisher’s Exact test). Putative CNVRs overlapped 845 gene models and were enriched for resistance (R) gene models (P < 1 × 10−22, Fisher’s exact test). Of note was a cluster of resistance gene models on chromosome 2 near a region containing multiple major gene loci conferring resistance to apple scab. I present the first analysis and catalog of CNVRs in the M. x domestica genome. The enrichment of the CNVRs with R gene models and their overlap with gene loci of agricultural significance draw attention to a form of unexplored genetic variation in apple.While these investigations of CNV in apple and gout represent significant advances in their respective research areas, they also improve our understanding of CNV more broadly within the eukaryotes. In apple, resistance-genes were enriched in CNVRs, and the only CNV that was associated with gout is hypothesised to be involved in immunity. This reiterates just how crucial CNV is to the survival of many eukaryotic species.
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The impact of copy number variation on gout, and the apple genome
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