Analysis of copy number variation at DMBT1 and age-related macular degeneration

BACKGROUND: DMBT1 is a gene that shows extensive copy number variation (CNV) that alters the number of bacteria-binding domains in the protein and has been shown to activate the complement pathway. It lies next to the ARMS2/HTRA1 genes in a region of chromosome 10q26, where single nucleotide variants have been strongly associated with age-related macular degeneration (AMD), the commonest cause of blindness in Western populations. Complement activation is thought to be a key factor in the pathogenesis of this condition. We sought to investigate whether DMBT1 CNV plays any role in the susceptibility to AMD. METHODS: We analysed long-range linkage disequilibrium of DMBT1 CNV1 and CNV2 with flanking single nucleotide polymorphisms (SNPs) using our previously published CNV and HapMap Phase 3 SNP data in the CEPH Europeans from Utah (CEU). We then typed a large cohort of 860 AMD patients and 419 examined age-matched controls for copy number at DMBT1 CNV1 and CNV2 and combined these data with copy numbers from a further 480 unexamined controls. RESULTS: We found weak linkage disequilibrium between DMBT1 CNV1 and CNV2 with the SNPs rs1474526 and rs714816 in the HTRA1/ARMS2 region. By directly analysing copy number variation, we found no evidence of association of CNV1 or CNV2 with AMD. CONCLUSIONS: We have shown that copy number variation at DMBT1 does not affect risk of developing age-related macular degeneration and can therefore be ruled out from future studies investigating the association of structural variation at 10q26 with AMD

A Comparison of Assays for Accurate Copy Number Measurement of the Low-Affinity Fc Gamma Receptor Genes FCGR3A and FCGR3B

The FCGR3 locus encoding the low affinity activating receptor FcγRIII, plays a vital role in immunity triggered by cellular effector and regulatory functions. Copy number of the genes FCGR3A and FCGR3B has previously been reported to affect susceptibility to several autoimmune diseases and chronic inflammatory conditions. However, such genetic association studies often yield inconsistent results; hence require assays that are robust with low error rate. We investigated the accuracy and efficiency in estimating FCGR3 CNV by comparing Sequenom MassARRAY and paralogue ratio test-restriction enzyme digest variant ratio (RT-REDVR). In addition, since many genetic association studies of FCGR3B CNV were carried out using real-time quantitative PCR, we have also included the evaluation of that method’s performance in estimating the multi-allelic CNV of FCGR3B. The qPCR assay exhibited a considerably broader distribution of signal intensity, potentially introducing error in estimation of copy number and higher false positive rates. Both Sequenom and PRT-REDVR showed lesser systematic bias, but Sequenom skewed towards copy number normal (CN = 2). The discrepancy between Sequenom and PRT-REDVR might be attributed either to batch effects noise in individual measurements. Our study suggests that PRT-REDVR is more robust and accurate in genotyping the CNV of FCGR3, but highlights the needs of multiple independent assays for extensive validation when performing a genetic association study with multi-allelic CNVs

Identification of a Rare Coding Variant in Complement 3 Associated with Age-related Macular Degeneration

Macular degeneration is a common cause of blindness in the elderly. To identify rare coding variants associated with a large increase in risk of age-related macular degeneration (AMD), we sequenced 2,335 cases and 789 controls in 10 candidate loci (57 genes). To increase power, we augmented our control set with ancestry-matched exome sequenced controls. An analysis of coding variation in 2,268 AMD cases and 2,268 ancestry matched controls revealed two large-effect rare variants; previously described R1210C in the CFH gene (fcase = 0.51%, fcontrol = 0.02%, OR = 23.11), and newly identified K155Q in the C3 gene (fcase = 1.06%, fcontrol = 0.39%, OR = 2.68). The variants suggest decreased inhibition of C3 by Factor H, resulting in increased activation of the alternative complement pathway, as a key component of disease biology

Complement receptor 1 gene (CR1) intragenic duplication and risk of Alzheimer’s disease

Single nucleotide variants (SNVs) within and surrounding the complement receptor 1 (CR1) gene show some of the strongest genome-wide association signals with late-onset Alzheimer’s disease. Some studies have suggested that this association signal is due to a duplication allele (CR1-B) of a low copy repeat (LCR) within the CR1 gene, which increases the number of complement C3b/C4b-binding sites in the mature receptor. In this study, we develop a triplex paralogue ratio test assay for CR1 LCR copy number allowing large numbers of samples to be typed with a limited amount of DNA. We also develop a CR1-B allele-specific PCR based on the junction generated by an historical non-allelic homologous recombination event between CR1 LCRs. We use these methods to genotype CR1 and measure CR1-B allele frequency in both late-onset and early-onset cases and unaffected controls from the United Kingdom. Our data support an association of late-onset Alzheimer’s disease with the CR1-B allele, and confirm that this allele occurs most frequently on the risk haplotype defined by SNV alleles. Furthermore, regression models incorporating CR1-B genotype provide a better fit to our data compared to incorporating the SNV-defined risk haplotype, supporting the CR1-B allele as the variant underlying the increased risk of late-onset Alzheimer’s disease

Technical considerations for genotyping multi-allelic copy number variation (CNV), in regions of segmental duplication

BACKGROUND: Intrachromosomal segmental duplications provide the substrate for non-allelic homologous recombination, facilitating extensive copy number variation in the human genome. Many multi-copy gene families are embedded within genomic regions with high levels of sequence identity (>95%) and therefore pose considerable analytical challenges. In some cases, the complexity involved in analyzing such regions is largely underestimated. Rapid, cost effective analysis of multi-copy gene regions have typically implemented quantitative approaches, however quantitative data are not an absolute means of certainty. Therefore any technique prone to degrees of measurement error can produce ambiguous results that may lead to spurious associations with complex disease. RESULTS: In this study we have focused on testing the accuracy and reproducibility of quantitative analysis techniques. With reference to the C-C Chemokine Ligand-3-like-1 (CCL3L1) gene, we performed analysis using real-time Quantitative PCR (QPCR), Multiplex Ligation-dependent Probe Amplification (MLPA) and Paralogue Ratio Test (PRT). After controlling for potential outside variables on assay performance, including DNA concentration, quality, preparation and storage conditions, we find that real-time QPCR produces data that does not cluster tightly around copy number integer values, with variation substantially greater than that of the MLPA or PRT systems. We find that the method of rounding real-time QPCR measurements can potentially lead to mis-scoring of copy number genotypes and suggest caution should be exercised in interpreting QPCR data. CONCLUSIONS: We conclude that real-time QPCR is inherently prone to measurement error, even under conditions that would seem favorable for association studies. Our results indicate that potential variability in the physicochemical properties of the DNA samples cannot solely explain the poor performance exhibited by the real-time QPCR systems. We recommend that more robust approaches such as PRT or MLPA should be used to genotype multi-allelic copy number variation in disease association studies and suggest several approaches which can be implemented to ensure the quality of the copy number typing using quantitative methods

Almost total protection from age-related macular degeneration by haplotypes of the Regulators of Complement Activation

Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries. It has been proposed that the polymorphism encoding Y402H (T1277C) in the complement factor H gene (CFH) is one of the main determinants of disease. We genotyped the polymorphism at a number of loci in the region encompassing the Regulators of Complement Activation (RCA) on chromosome 1, including T1277C SNP, in 187 patients and 146 controls. Haplotypes have been classified as protective (P) or susceptible (S) with respect to AMD. This included the identification of an S haplotype with a T at 1277. The results show that no single locus should be assumed to be directly responsible for AMD, but rather argue for the existence of RCA haplotypes, which can be assigned meaningful predictive values for AMD. We conclude that the critical sequences are within a region 450. kb centromeric to 128. kb telomeric of CFH

Multiallelic copy number variation in the complement component 4A (C4A) gene is associated with late-stage age-related macular degeneration (AMD)

Molecular Technology and Informatics for Personalised Medicine and Healt

Recurrent Inversion Toggling and Great Ape Genome Evolution

Inversions play an important role in disease and evolution but are difficult to characterize because their breakpoints map to large repeats. We increased by sixfold the number (n = 1,069) of previously reported great ape inversions by using single-cell DNA template strand and long-read sequencing. We find that the X chromosome is most enriched (2.5-fold) for inversions, on the basis of its size and duplication content. There is an excess of differentially expressed primate genes near the breakpoints of large (>100 kilobases (kb)) inversions but not smaller events. We show that when great ape lineage-specific duplications emerge, they preferentially (approximately 75%) occur in an inverted orientation compared to that at their ancestral locus. We construct megabase-pair scale haplotypes for individual chromosomes and identify 23 genomic regions that have recurrently toggled between a direct and an inverted state over 15 million years. The direct orientation is most frequently the derived state for human polymorphisms that predispose to recurrent copy number variants associated with neurodevelopmental disease