Paralogue Ratio Test Research Articles

Genotyping complex structural variation at the malaria-associated human glycophorin locus using a PCR-based strategy.

Structural variation in the human genome can affect risk of disease. An example is a complex structural variant of the human glycophorin gene cluster, called DUP4, which is associated with a clinically significant level of protection against severe malaria. The human glycophorin gene cluster harbours at least 23 distinct structural variants, and accurate genotyping of this complex structural variation remains a challenge. Here, we use a polymerase chain reaction-based strategy to genotype structural variation at the human glycophorin gene cluster, including the alleles responsible for the U- blood group. We validate our approach, based on a triplex paralogue ratio test, on publically available samples from the 1000 Genomes project. We then genotype 574 individuals from a longitudinal birth cohort (Tori-Bossito cohort) using small amounts of DNA at low cost. Our approach readily identifies known deletions and duplications, and can potentially identify novel variants for further analysis. It will allow exploration of genetic variation at the glycophorin locus, and investigation of its relationship with malaria, in large sample sets at minimal cost, using standard molecular biology equipment.

Association between complement 4 copy number variation and systemic lupus erythematosus: a meta-analysis.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by multiple genetic mutations. Complement 4 (C4) copy number variation (CNV) is a target-of-interest located on chromosome 6. C4 encodes for either of the two C4 paralogs, C4A or C4B, and low C4 levels have been associated with SLE activity. In this study, we conducted a meta-analysis to comprehensively understand the role of C4 CNV in SLE. Three databases (PubMed, Embase, and Web of Science) were searched for relevant studies. Two investigators independently extracted and evaluated data from eligible studies. Associations between C4 CNV and SLE were estimated by odds ratios (OR) and 95% confidence intervals (95% CI). Further analysis was conducted using the STATA 12.0 software. A total of eight case-control studies were included in the analysis with 4107 SLE patients and 5889 healthy controls. Six studies used TaqMan real-time PCR to genotype C4 CNV, with 1 study used paralog ratio test and other one used multiplex ligation-dependent probe amplification (MLPA). Lower total C4 CNV and C4A CNV were associated with SLE in the overall analysis (pooled OR: 1.55, 95% CI: 1.23-1.95; pooled OR: 1.86, 95% CI: 1.51-2.29). The subgroup analysis found that total C4 CNV and lower C4A CNV were significantly associated with SLE in Caucasians (pooled OR: 1.84, 95% CI: 1.60-2.12; pooled OR: 2.23, 95% CI:1.92-2.59). However, the association was not detected in East Asians. Lastly, SLE was not associated with C4B CNV, long C4 CNV, or short C4 CNV. The meta-analysis confirmed that lower total C4 CNV and lower C4A CNV are associated with SLE in certain populations. Future studies should consider other ethnic groups to further investigate the relationship between the C4 gene and SLE.

Population-specific profiling of CCL3L1 copy number of the three major ethnic groups in Malaysia and the implication on HIV susceptibility.

CC chemokine ligand 3 like-1 (CCL3L1) encodes for CCL3L1 protein, which is a human immunodeficiency virus (HIV) suppressive chemokine and a potent ligand of HIV CCR5 co-receptor. CCL3L1 exhibits variation in the gene copy number (CN) and could influence HIV susceptibility through gene dosage effect. The study aims to determine the distribution of CCL3L1 CN among HIV subjects of Malay, Chinese, and Indian ethnics in Malaysia and to evaluate the impact of CCL3L1 CN on susceptibility to HIV. This study involved 182 HIV patients who attended outpatient clinics of three hospitals in Malaysia and 150 non-HIV (control) subjects. Typing of CCL3L1 CN was conducted via multiplex paralogue ratio tests (PRTs), followed by validation of the CCL3L1 CN by microsatellite analyses. Both Malay and Indian HIV subjects had the CN mode of two, while the CN mode for the Chinese was four. The CCL3L1 gene CN was found to be strongly associated with ethnicity (p<0.001) with the diverse distribution of CCL3L1 CN between the Malay (range=0-6), Chinese (range=0-9), and Indian (range=1-4) ethnic groups. CCL3L1 CN higher than and equal to the average was associated with reduced HIV susceptibility among the Malays (p<0.05). However, the negative results found for the Indian and Chinese need to be further analysed in a larger sample size.

Copy number variation of CCL3L1 among three major ethnic groups in Malaysia

BackgroundC-C motif Chemokine Ligand 3 Like 1 (CCL3L1) is a multiallelic copy number variable, which plays a crucial role in immunoregulatory and hosts defense through the production of macrophage inflammatory protein (MIP)-1α. Variable range of the CCL3L1 copies from 0 to 14 copies have been documented in several different populations. However, there is still lack of report on the range of CCL3L1 copy number exclusively among Malaysians who are a multi-ethnic population. Thus, this study aims to extensively examine the distribution of CCL3L1 copy number in the three major populations from Malaysia namely Malay, Chinese and Indian. A diploid copy number of CCL3L1 for 393 Malaysians (Malay = 178, Indian = 90, and Chinese = 125) was quantified using Paralogue Ratio Tests (PRTs) and then validated with microsatellites analysis.ResultsTo our knowledge, this is the first report on the CCL3L1 copy number that has been attempted among Malaysians and the Chinese ethnic group exhibits a diverse pattern of CCL3L1 distribution copy number from the Malay and Indian (p < 0.0001). The CCL3L1 ranged from 0 to 8 copies for both the Malay and Indian ethnic groups while 0 to 10 copies for the Chinese ethnic. Consequently, the CCL3L1 copy number among major ethnic groups in the Malaysian population is found to be significantly varied when compared to the European population (p < 0.0001). The mean/median reported for the Malay, Chinese, Indian, and European are 2.759/2.869, 3.453/3.290, 2.437/1.970 and 2.001/1.940 respectively.ConclusionThis study reveals the existence of genetic variation of CCL3L1 in the Malaysian population, and suggests by examining genetic diversity on the ethnicity, and specific geographical region could help in reconstructing human evolutionary history and for the prediction of disease risk related to the CCL3L1 copy number.

No Evidence for Association of BMI with Salivary Amylase Gene Copy Number in the UK 1958 Birth Cohort.

In a 2014 publication, evidence was presented supporting the association of BMI with the copy number of the salivary amylase 1 (AMY1) gene, with an unprecedented effect size of -0.15kg/m2 (SE 0.02) per copy of AMY1. Most well-powered attempts to reproduce these findings have not been successful. However, because of different study designs, a significant association may still apply under restricted conditions such as in particular age groups. This study specifically tested the BMI-AMY1 association at different age points in the same individuals using longitudinal BMI information from participants in the UK 1958 Birth Cohort study. This study measured the AMY1 copy number by paralogue ratio tests in genomic DNA and by using array comparative genomic hybridization data. BMI data from 1958 Birth Cohort participants were available from eight different age points between 7 and 50years. No evidence, even at nominal significance, was found for association of the AMY1 copy number with BMI at any age point in approximately 1,400 members of the 1958 Birth Cohort or in 2,835 people from two disease cohorts from the Wellcome Trust Case Control Consortium. The results do not support an association between BMI and AMY1 copy number at any age point.

Understanding Determinants of Carbohydrate Metabolism and Their Contribution to Metabolic Health; The Impact of AMY1 CNV (P21-015-19)

ObjectivesSalivary α- amylase (AMY1) is responsible for the breakdown of starch into oligosaccharides, tri and di-saccharides giving a start to the starch digestion in the oral cavity on food consumption. Several studies recently reported contradicting results regarding the link between AMY1 copy numbers (CNs) and obesity and type 2 diabetes.Objective: We investigated whether CN in the AMY1 gene was associated with differential anthropometrics and glycaemic outcomes in obese individuals who underwent a dietary plan varying in macronutrient intake, as a part of weight loss and weight maintenance program. We also investigated whether there existed an interaction between nutrient intakes and AMY1 CNs and if AMY1 CNs have influence on body weight, body composition and glycemic trajectories during dietary interventions. MethodsUsing the Paralogue Ratio Test, we accurately measured the AMY1 CNs in 761 obese individuals from the Diogenes study. Subjects underwent first an 8-week low-caloric diet (LCD, at 800 kcal/d) and those achieving >8% weight loss were then randomized to a 6-month weight maintenance dietary (WMD) intervention. The association between AMY1 CNs and weight- and glycemic- parameters was tested at baseline and following each intervention phase (LCD, WMD) with the use of linear mixed effect models adjusting for gender, age, center and total energy intake. ResultsAt baseline, a modest association between AMY1 CN and BMI (P = 0.04) was observed. AMY1 CN was not associated with baseline glycemic variables. Additionally, AMY1 CN was not associated with anthropometric or glycemic-outcomes following either LCD or WMD. Interaction analyses between AMY1 CN and nutrient intake did not reveal significant association with any clinical parameters (at baseline and following LCD or WMD) or when testing gene x WMD interactions during the WMD phase ConclusionsIn the absence of association with weight trajectories or glycemic improvements, the AMY1 CN cannot be considered as an important biomarker for response to a clinical weight loss and weight maintenance programs in overweight/obese subjects. Funding SourcesEuropean Commission, Food Quality and Safety Priority of the Sixth Framework Program (FP6-2005-513946), Nestlé Institute of Health Sciences. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Salivary α-amylase copy number is not associated with weight trajectories and glycemic improvements following clinical weight loss: results from a 2-phase dietary intervention study

Several studies recently reported contradicting results regarding the link between amylase 1 (AMY1) copy numbers (CNs), obesity, and type 2 diabetes. The aim of this study was to assess the impact of AMY1 CN on anthropometrics and glycemic outcomes in obese individuals following a 2-phase dietary weight loss intervention. Using the paralog ratio test, AMY1 CNs were accurately measured in 761 obese individuals from the DiOGenes study. Subjects first underwent an 8-wk low-calorie diet (LCD, at 800 kcal/d) and then were randomly assigned to a 6-mo weight maintenance dietary (WMD) intervention with arms having different glycemic loads. At baseline, a modest association between AMY1 CN and BMI (P=0.04) was observed. AMY1 CN was not associated with baseline glycemic variables. In addition, AMY1 CN was not associated with anthropometric or glycemic outcomes following either LCD or WMD. Interaction analyses between AMY1 CN and nutrient intake did not reveal any significant association with clinical parameters (at baseline and following LCD or WMD) or when testing gene × WMD interactions during the WMD phase. In the absence of association with weight trajectories or glycemic improvements, the AMY1 CN cannot be considered as an important biomarker for response to a clinical weight loss and weight maintenance programs in overweight/obese subjects. This trial was registered at www.clinicaltrials.gov as NCT00390637.

Human CCL3L1 copy number variation, gene expression, and the role of the CCL3L1-CCR5 axis in lung function.

Background: The CCL3L1-CCR5 signaling axis is important in a number of inflammatory responses, including macrophage function, and T-cell-dependent immune responses. Small molecule CCR5 antagonists exist, including the approved antiretroviral drug maraviroc,and therapeutic monoclonal antibodies are in development. Repositioning of drugs and targets into new disease areas can accelerate the availability of new therapies and substantially reduce costs. As it has been shown that drug targets with genetic evidence supporting their involvement in the disease are more likely to be successful in clinical development, using genetic association studies to identify new target repurposing opportunities could be fruitful. Here we investigate the potential of perturbation of the CCL3L1-CCR5 axis as treatment for respiratory disease. Europeans typically carry between 0 and 5 copies of CCL3L1 and this multi-allelic variation is not detected by widely used genome-wide single nucleotide polymorphism studies. Methods: We directly measured the complex structural variation of CCL3L1 using the Paralogue Ratio Test and imputed (with validation) CCR5del32 genotypes in 5,000 individuals from UK Biobank, selected from the extremes of the lung function distribution, and analysed DNA and RNAseq data for CCL3L1 from the 1000 Genomes Project. Results: We confirmed the gene dosage effect of CCL3L1 copy number on CCL3L1 mRNA expression levels. We found no evidence for association of CCL3L1 copy number or CCR5del32 genotype with lung function. Conclusions: These results suggest that repositioning CCR5 antagonists is unlikely to be successful for the treatment of airflow obstruction.

Human CCL3L1 copy number variation, gene expression, and the role of the CCL3L1-CCR5 axis in lung function

Background: The CCL3L1-CCR5 signaling axis is important in a number of inflammatory responses, including macrophage function, and T-cell-dependent immune responses. Small molecule CCR5 antagonists exist, including the approved antiretroviral drug maraviroc, and therapeutic monoclonal antibodies are in development. Repositioning of drugs and targets into new disease areas can accelerate the availability of new therapies and substantially reduce costs. As it has been shown that drug targets with genetic evidence supporting their involvement in the disease are more likely to be successful in clinical development, using genetic association studies to identify new target repurposing opportunities could be fruitful. Here we investigate the potential of perturbation of the CCL3L1-CCR5 axis as treatment for respiratory disease. Europeans typically carry between 0 and 5 copies of CCL3L1 and this multi-allelic variation is not detected by widely used genome-wide single nucleotide polymorphism studies. Methods: We directly measured the complex structural variation of CCL3L1 using the Paralogue Ratio Test and imputed (with validation) CCR5d32 genotypes in 5,000 individuals from UK Biobank, selected from the extremes of the lung function distribution, and analysed DNA and RNAseq data for CCL3L1 from the 1000 Genomes Project. Results: We confirmed the gene dosage effect of CCL3L1 copy number on CCL3L1 mRNA expression levels. We found no evidence for association of CCL3L1 copy number or CCR5d32 genotype with lung function. Conclusions: These results suggest that repositioning CCR5 antagonists is unlikely to be successful for the treatment of airflow obstruction.

Complement receptor 1 gene (CR1) intragenic duplication and risk of Alzheimer\u2019s 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.

Human CCL3L1 copy number variation, gene expression, and the role of the CCL3L1-CCR5 axis in lung function

Background: The CCL3L1-CCR5 signaling axis is important in a number of inflammatory responses, including macrophage function, and T-cell-dependent immune responses. Small molecule CCR5 antagonists exist, including the approved antiretroviral drug maraviroc, and therapeutic monoclonal antibodies are in development. Repositioning of drugs and targets into new disease areas can accelerate the availability of new therapies and substantially reduce costs. As it has been shown that drug targets with genetic evidence supporting their involvement in the disease are more likely to be successful in clinical development, using genetic association studies to identify new target repurposing opportunities could be fruitful. Here we investigate the potential of perturbation of the CCL3L1-CCR5 axis as treatment for respiratory disease. Europeans typically carry between 0 and 5 copies of CCL3L1 and this multi-allelic variation is not detected by widely used genome-wide single nucleotide polymorphism studies. Methods: We directly measured the complex structural variation of CCL3L1 using the Paralogue Ratio Test and imputed (with validation) CCR5del32 genotypes in 5,000 individuals from UK Biobank, selected from the extremes of the lung function distribution, and analysed DNA and RNAseq data for CCL3L1 from the 1000 Genomes Project. Results: We confirmed the gene dosage effect of CCL3L1 copy number on CCL3L1 mRNA expression levels. We found no evidence for association of CCL3L1 copy number or CCR5del32 genotype with lung function. Conclusions: These results suggest that repositioning CCR5 antagonists is unlikely to be successful for the treatment of airflow obstruction.

Copy number variation of functional RBMY1 is associated with sperm motility: an azoospermia factor-linked candidate for asthenozoospermia.

What is the influence of copy number variation (CNV) in functional RNA binding motif protein Y-linked family 1 (RBMY1) on spermatogenic phenotypes? The RBMY1 functional copy dosage is positively correlated with sperm motility, and dosage insufficiency is an independent risk factor for asthenozoospermia. RBMY1, a multi-copy gene expressed exclusively in the adult testis, is one of the most important candidates for male infertility in the azoospermia factor (AZF) region of the Y-chromosome. RBMY1 encodes an RNA-binding protein that serves as a pre-mRNA splicing regulator during spermatogenesis, and male mice deficient in Rbmy are sterile. A total of 3127 adult males were recruited from 2009 to 2016; of this group, the dosage of RBMY1 functional copy were investigated in 486 fertile males. In the remaining 2641 males with known spermatogenesis status, 1070 Y-chromosome haplogroup (Y-hg) O3* or O3e carriers without chromosomal aberration or known AZF structure mutations responsible for spermatogenic impairment, including 506 men with normozoospermia and 564 men with oligozoospermia or/and asthenozoospermia, were screened, and the RBMY1 functional copy dosage and copy conversion were determined to explore their associations with sperm phenotypes. The correlation between RBMY1 dosage and its mRNA level or RBMY1 protein level and the correlation between sperm RBMY1 level and motility were analysed in 15 testis tissue samples and eight semen samples. Ten additional semen samples were used to confirm the subcellular localization of RBMY1 in individual sperm. All the Han volunteers donating whole blood, semen and testis tissue were from southwest China. RBMY1 copy number, copy conversion, mRNA/protein amount and protein location in sperm were detected using the AccuCopy® assay method, paralog ratio test, quantitative PCR, western blotting and immunofluorescence staining methods, respectively. This study identified Y-hg-independent CNV of functional RBMY1 in the enrolled population. A difference in the distribution of RBMY1 copy number was observed between the group with normal sperm motility and the group with asthenozoospermia. A positive correlation between the RBMY1 copy dosage and sperm motility was identified, and the males with fewer than six copies of RBMY1 showed an elevated risk for asthenozoospermia relative to those with six RBMY1 copies, the most common dosage in the population. The RBMY1 copy dosage was positively correlated with its mRNA and protein level in the testis. Sperm with high motility were found to carry more RBMY1 protein than those with relatively low motility. The RBMY1 protein was confirmed to predominantly localize in the neck and mid-piece region of sperm as well as the principal piece of the sperm tail. Our population study completes a chain of evidence suggesting that RBMY1 influences the susceptibility of males to asthenozoospermia by modulating sperm motility. High sequence similarity between the RBMY1 functional copies and a large number of pseudogenes potentially reduces the accuracy of the copy number detection. The mechanism underlying the CNV in RBMY1 is still unclear, and the effect of the structural variations in the RBMY1 copy cluster on the copy dosage of other protein-coding genes located in the region cannot be excluded, which may potentially bias our observations. Asthenozoospermia is a multi-factor complex disease with a limited number of proven susceptibility genes. This study identified a novel genomic candidate independently contributing to the condition, enriching our understanding of the role of AZF-linked genes in male reproduction. Our finding provides insight into the physiological and pathological characteristics of RBMY1 in terms of sperm motility, supplies persuasive evidence of the significance of RBMY1 copy number analysis in the clinical counselling of male infertility resulting from asthenozoospermia. This work was funded by the National Natural Science Foundation of China (Nos. 81370748 and 30971598). The authors have no conflicts of interest.

Low copy numbers of FCGR3A and FCGR3B associated with Chinese patients with SLE and AASV.

Low-affinity Fcγ receptors (FcγR) act as key mediators of the pathogenic effects of autoantibodies. In this study, we aimed to determine whether copy number variations (CNVs) in FCGR3A and FCGR3B were associated with systemic lupus nephritis (SLE) and ANCA-associated systemic vasculitis (AASV) in Chinese individuals. A total of 1118 individuals were enrolled, including 415 SLE patients, 139 AASV patients, and 564 healthy controls. FCGR3A and FCGR3B copy numbers (CNs) were determined by both a paralogue ratio test and TaqMan quantitative PCR assay. In the susceptibility associations, a low FCGR3B CN was significantly associated with SLE ( p = 5.01 × 10-3; odds ratio (OR) 1.71; 95% confidence interval (CI) 1.17-2.48) and AASV ( p = 0.04; OR = 1.72; 95% CI 1.02-2.88). A low FCGR3A CN was also significantly associated with SLE ( p = 6.02 × 10-3; OR 2.72; 95% CI 1.30-5.71) and AASV ( p = 0.042; OR 2.64; 95% CI 1.00-6.93). Further subphenotype analysis revealed that low CNs of FCGR3A and FCGR3B were significantly associated with clinical manifestations in SLE and AASV patients. Therefore, in this case-control study, we identified low CNs of FCGR2A and FCGR3B to be common risk factors for SLE and AASV.

Human β-Defensin 2 in Primary Sclerosing Cholangitis.

Objectives:Primary sclerosing cholangitis (PSC) is a chronic inflammatory disease of the bile ducts frequently associated with inflammatory bowel disease (IBD), suggesting an important role for the gut–liver axis. Defensins are small (3.5–4.5 kDa) anti-microbial peptides that contribute to innate immunity at mucosal surfaces and have been implicated in IBD. The aim of this study was to investigate copy number variation of the gene (DEFB4) encoding human β-defensin 2 (HBD2) and protein expression of HBD2 in PSC.Methods:US and Italian PSC cases and unaffected controls (US PSC patients n=89, US controls n=87; Italian PSC patients n=46, Italian controls n=84) were used to estimate HBD2 gene copy number by both quantitative real-time PCR and paralog ratio test. Serum levels of HBD2 were measured by enzyme-linked immunosorbent assay and liver expression was analyzed by immunohistochemistry.Results:Mean serum levels of HBD2 were significantly greater in PSC (1,086±1,721 ng/μl) compared with primary biliary cholangitis (544±754 ng/μl), ulcerative colitis (417±506 ng/μl), and healthy controls (514±731 ng/μl) (P=0.02). However, no significant differences between the frequencies of high DEFB4 gene copy number, defined by >4 copies, and PSC were found in the US, Italian, or combined cohorts. Importantly, a high number of biliary ducts were found immunopositive in PSC samples compared with controls.Conclusions:Our data show that HBD2 serum levels and tissue expression are increased in PSC subjects, suggesting that this arm of innate immunity may be important in the etiopathogenesis of PSC.

Comparison of Multiple Methods for Determination of FCGR3A/B Genomic Copy Numbers in HapMap Asian Populations with Two Public Databases.

Low FCGR3 copy numbers (CNs) has been associated with susceptibility to several systemic autoimmune diseases. However, inconsistent associations were reported and errors caused by shaky methods were suggested to be the major causes. In large scale case control association studies, robust copy number determination method is thus warranted, which was the main focus of the current study. In the present study, FCGR3 CNs of 90 HapMap Asians were firstly checked using four assays including paralog ratio test combined with restriction enzyme digest variant ratio (PRT-REDVR), real-time quantitative (qPCR) using TaqMan assay, real-time qPCR using SYBR Green dye and short tenden repeat (STR). To improve the comparison precision reproductively, the results were compared with those from recently released sequencing data from 1000 genomes project as well as whole-genome tiling BAC array data. The tendencies of inconsistent samples by these methods were also characterized. Refined in-home TaqMan qPCR assay showed the highest correlation with array-CGH results (r = 0.726, p < 0.001) and the highest concordant rate with 1000 genome sequencing data (FCGR3A 91.76%, FCGR3B 85.88%, and FCGR3 81.18%). For samples with copy number variations, comprehensive analysis of multiple methods was required in order to improve detection accuracy. All these method were prone to detect copy number to be higher than that from direct sequencing. All the four PCR based CN determination methods (qPCR using TaqMan probes or SYBR Green, PRT, STR) were prone to higher estimation errors and thus may lead to artificial associations in large-scale case-control association studies. But different to previous reports, we observed that properly refined TaqMan qPCR assay was not inferior to or even more accurate than PRT when using sequencing data as the reference.

Analysis of Copy Number Variation Using the Paralogue Ratio Test (PRT).

Copy number variation (CNV), where a segment of DNA differs in copy number between different individuals, is an extensive and often underappreciated source of genetic variation within species. However, reliably determining copy number of a particular DNA sequence for a large number of samples can be challenging. Here, I describe and review the paralogue ratio test (PRT) in detail. PRT was developed to robustly type the CNV of the beta-defensin locus using small amounts of genomic DNA in a high-throughput manner, and has been applied successfully at many other loci. I discuss the strategies for designing successful PRT assays using both manual and bioinformatics methods, how to optimize experimental conditions, and approaches for analyzing the data. I discuss strengths and weaknesses of the approach, and how to troubleshoot results, as well as the range of problems to which PRT can be a potential solution.

Determining multiallelic complex copy number and sequence variation from high coverage exome sequencing data.

BackgroundCopy number variation (CNV) is a major component of genomic variation, yet methods to accurately type genomic CNV lag behind methods that type single nucleotide variation. High-throughput sequencing can contribute to these methods by using sequence read depth, which takes the number of reads that map to a given part of the reference genome as a proxy for copy number of that region, and compares across samples. Furthermore, high-throughput sequencing also provides information on the sequence differences between copies within and between individuals.MethodsIn this study we use high-coverage phase 3 exome sequences of the 1000 Genomes project to infer diploid copy number of the beta-defensin genomic region, a well-studied CNV that carries several beta-defensin genes involved in the antimicrobial response, signalling, and fertility. We also use these data to call sequence variants, a particular challenge given the multicopy nature of the region.ResultsWe confidently call copy number and sequence variation of the beta-defensin genes on 1285 samples from 26 global populations, validate copy number using Nanostring nCounter and triplex paralogue ratio test data. We use the copy number calls to verify the genomic extent of the CNV and validate sequence calls using analysis of cloned PCR products. We identify novel variation, mostly individually rare, predicted to alter amino-acid sequence in the beta-defensin genes. Such novel variants may alter antimicrobial properties or have off-target receptor interactions, and may contribute to individuality in immunological response and fertility.ConclusionsGiven that 81 % of identified sequence variants were not previously in dbSNP, we show that sequence variation in multiallelic CNVs represent an unappreciated source of genomic diversity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2123-y) contains supplementary material, which is available to authorized users.

A lack of association between genetic polymorphisms in beta-defensins and susceptibility of psoriasis in Taiwanese: A case–control study

Abstract Background Genetic predisposition of the inflammatory-host response may affect the development of psoriasis. Previous studies have shown that copy number variations (CNVs) of β-defensin genes ( DEFB ) are associated with the susceptibility of psoriasis in Caucasian populations. Objectives This study aimed to assess the role of the CNVs of the DEFB4 gene and functional variants in the DEFB1 gene in Taiwanese patients with psoriasis. Methods In total, 196 patients with psoriasis and 196 control individuals were analyzed for the presence of the DEFB4 CNVs using the paralogue ratio test, and also for the DEFB1 polymorphisms rs11362, rs1800972, and rs1799946, using a polymerase chain reaction. Results None of the polymorphisms were found to be associated with psoriasis. The distribution of DEFB4 genomic CNVs did not significantly differ between the control group and psoriasis group. The frequencies of patients who carried a greater than the median (≥ 5) number of copies did not significantly differ in patients with psoriasis and controls. The multivariate analysis similarly revealed that the DEFB4 CNVs were not associated with psoriasis (odds ratio = 1.03, 95% confidence interval = 0.89–1.19, p = 0.720). No significant difference was detected in the genotype and allele distribution for any of the individual DEFB1 polymorphisms between the cases and the controls. Finally, the overall haplotype frequency profiles derived from the three polymorphisms did not significantly differ between the cases and the controls. Conclusion Our results do not suggest that these genetic variants of the β-defensin genes contribute to the genetic background of psoriasis in Taiwanese patients.

Genotyping of common SIRPB1 copy number variant using Paralogue Ratio Test coupled to MALDI-MS quantification

Copy number variant (CNV) regions have been proven to have a significant impact on gene expression. Some of them have been also found to be associated to different human diseases. CNV genotyping is often prone to error and cross-validation with independent methods is frequently required. The platform of choice depends on whether it is a genome-wide discovery screening or a candidate CNV study, the cohort size and the number of CNVs included in the assay and, finally, the budget available. Here we illustrate a affordable approach to determine the CNV genotype using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and based on the quantitative determination of single nucleotide duplicated mismatches (SNDM) mapping the CNV region and a paralogue genomic region that is used as a two-copy reference. We have genotyped nsv436327, a common CNV mapping SIRPB1 intron 1 that has been associated to human personality behavior. SIRP cluster region was subjected to several ancestral duplication events what makes SIRPB1 CNV genotyping technically challenging. We designed three sets of primer pairs that amplified paralogue regions inside and outside the CNV, containing three SNDMs. Post-PCR extension analyses of sequencing oligonucleotides mapping immediately upstream each SNDM allowed us to quantify using MALDI-MS the proportion of PCR products derived from the CNV region versus the external reference. In contrast to other approaches, setting up this genotyping method requires an affordable investment.

Obesity, starch digestion and amylase: association between copy number variants at human salivary (AMY1) and pancreatic (AMY2) amylase genes.

The human salivary amylase genes display extensive copy number variation (CNV), and recent work has implicated this variation in adaptation to starch-rich diets, and in association with body mass index. In this work, we use paralogue ratio tests, microsatellite analysis, read depth and fibre-FISH to demonstrate that human amylase CNV is not a smooth continuum, but is instead partitioned into distinct haplotype classes. There is a fundamental structural distinction between haplotypes containing odd or even numbers of AMY1 gene units, in turn coupled to CNV in pancreatic amylase genes AMY2A and AMY2B. Most haplotypes have one copy each of AMY2A and AMY2B and contain an odd number of copies of AMY1; consequently, most individuals have an even total number of AMY1. In contrast, haplotypes carrying an even number of AMY1 genes have rearrangements leading to CNVs of AMY2A/AMY2B. Read-depth and experimental data show that different populations harbour different proportions of these basic haplotype classes. In Europeans, the copy numbers of AMY1 and AMY2A are correlated, so that phenotypic associations caused by variation in pancreatic amylase copy number could be detected indirectly as weak association with AMY1 copy number. We show that the quantitative polymerase chain reaction (qPCR) assay previously applied to the high-throughput measurement of AMY1 copy number is less accurate than the measures we use and that qPCR data in other studies have been further compromised by systematic miscalibration. Our results uncover new patterns in human amylase variation and imply a potential role for AMY2 CNV in functional associations.