Assessment of alternative genotyping strategies to maximize imputation accuracy at minimal cost

Background

Commercial breeding programs seek to maximise the rate of genetic gain while minimizing the costs of attaining that gain. Genomic information offers great potential to increase rates of genetic gain but it is expensive to generate. Low-cost genotyping strategies combined with genotype imputation offer dramatically reduced costs. However, both the costs and accuracy of imputation of these strategies are highly sensitive to several factors. The objective of this paper was to explore the cost and imputation accuracy of several alternative genotyping strategies in pedigreed populations.

Methods

Pedigree and genotype data from a commercial pig population were used. Several alternative genotyping strategies were explored. The strategies differed in the density of genotypes used for the ancestors and the individuals to be imputed. Parents, grandparents, and other relatives that were not descendants, were genotyped at high-density, low-density, or extremely low-density, and associated costs and imputation accuracies were evaluated.

Results

Imputation accuracy and cost were influenced by the alternative genotyping strategies. Given the mating ratios and the numbers of offspring produced by males and females, an optimized low-cost genotyping strategy for a commercial pig population could involve genotyping male parents at high-density, female parents at low-density (e.g. 3000 SNP), and selection candidates at very low-density (384 SNP).

Conclusions

Among the selection candidates, 95.5 % and 93.5 % of the genotype variation contained in the high-density SNP panels were recovered using a genotyping strategy that costs respectively, $24.74 and $20.58 per candidate.     

The orphan nuclear receptor small heterodimer partner is required for thiazolidinedione effects in leptin-deficient mice

Background

Small heterodimer partner (SHP, NR0B2) is involved in diverse metabolic pathways, including hepatic bile acid, lipid and glucose homeostasis, and has been implicated in effects on the peroxisome proliferator-activated receptor γ (PPARγ), a master regulator of adipogenesis and the receptor for antidiabetic drugs thiazolidinediones (TZDs). In this study, we aim to investigate the role of SHP in TZD response by comparing TZD-treated leptin-deficient (ob/ob) and leptin-, SHP-deficient (ob/ob;Shp^−/−) double mutant mice.

Results

Both ob/ob and double mutant ob/ob;Shp^−/− mice developed hyperglycemia, insulin resistance, and hyperlipidemia, but hepatic fat accumulation was decreased in the double mutant ob/ob;Shp^−/− mice. PPARγ2 mRNA levels were markedly lower in ob/ob;Shp^−/− liver and decreased to a lesser extent in adipose tissue. The TZD troglitazone did not reduce glucose or circulating triglyceride levels in ob/ob;Shp^−/− mice. Expression of the adipocytokines, such as adiponectin and resistin, was not stimulated by troglitazone treatment. Expression of hepatic lipogenic genes was also reduced in ob/ob;Shp^−/− mice. Moreover, overexpression of SHP by adenovirus infection increased PPARγ2 mRNA levels in mouse primary hepatocytes.

Conclusions

Our results suggest that SHP is required for both antidiabetic and hypolipidemic effects of TZDs in ob/ob mice through regulation of PPARγ expression.     

High-resolution melting molecular signatures for rapid identification of human papillomavirus genotypes

Background

Genotyping of human papillomarvirus (HPV) is crucial for patient management in a clinical setting. This study accesses the combined use of broad-range real-time PCR and high-resolution melting (HRM) analysis for rapid identification of HPV genotypes.

Methods

Genomic DNA sequences of 8 high-risk genotypes (HPV16/18/39/45/52/56/58/68) were subject to bioinformatic analysis to select for appropriate PCR amplicon. Asymmetric broad-range real-time PCR in the presence of HRM dye and two unlabeled probes specific to HPV16 and 18 was employed to generate HRM molecular signatures for HPV genotyping. The method was validated via assessment of 119 clinical HPV isolates.

Results

A DNA fragment within the L1 region was selected as the PCR amplicon ranging from 215–221 bp for different HPV genotypes. Each genotype displayed a distinct HRM molecular signature with minimal inter-assay variability. According to the HRM molecular signatures, HPV genotypes can be determined with one PCR within 3 h from the time of viral DNA isolation. In the validation assay, a 91% accuracy rate was achieved when the genotypes were in the database. Concomitantly, the HRM molecular signatures for additional 6 low-risk genotypes were established.

Conclusions

This assay provides a novel approach for HPV genotyping in a rapid and cost-effective manner.     

Interaction between Age and Obesity on Cardiomyocyte Contractile Function: Role of Leptin and Stress Signaling

Objectives

This study was designed to evaluate the interaction between aging and obesity on cardiac contractile and intracellular Ca²⁺ properties.

Methods

Cardiomyocytes from young (4-mo) and aging (12- and 18-mo) male lean and the leptin deficient ob/ob obese mice were treated with leptin (0.5, 1.0 and 50 nM) for 4 hrs in vitro. High fat diet (45% calorie from fat) and the leptin receptor mutant db/db obesity models at young and older age were used for comparison. Cardiomyocyte contractile and intracellular Ca²⁺ properties were evaluated including peak shortening (PS), maximal velocity of shortening/relengthening (± dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR₉₀), intracellular Ca²⁺ levels and decay. O₂ ⁻ levels were measured by dihydroethidium fluorescence.

Results

Our results revealed reduced survival in ob/ob mice. Aging and obesity reduced PS, ± dL/dt, intracellular Ca²⁺ rise, prolonged TR₉₀ and intracellular Ca²⁺ decay, enhanced O₂ ⁻ production and p ^47phox expression without an additive effect of the two, with the exception of intracellular Ca²⁺ rise. Western blot analysis exhibited reduced Ob-R expression and STAT-3 phosphorylation in both young and aging ob/ob mice, which was restored by leptin. Aging and obesity reduced phosphorylation of Akt, eNOS and p38 while promoting pJNK and pIκB. Low levels of leptin reconciled contractile, intracellular Ca²⁺ and cell signaling defects as well as O₂ ⁻ production and p ^47phox upregulation in young but not aging ob/ob mice. High level of leptin (50 nM) compromised contractile and intracellular Ca²⁺ response as well as O₂ ⁻ production and stress signaling in all groups. High fat diet-induced and db/db obesity displayed somewhat comparable aging-induced mechanical but not leptin response.

Conclusions

Taken together, our data suggest that aging and obesity compromise cardiac contractile function possibly via phosphorylation of Akt, eNOS and stress signaling-associated O₂ ⁻ release.     

Non-Invasive Bioluminescence Imaging of β-Cell Function in Obese-Hyperglycemic [ob/ob] Mice

Background

Type 2 diabetes results from failure of the β-cells to compensate for increased insulin demand due to abnormal levels of metabolic factors. The ob/ob(lep-/-) mouse has been extensively studied as an animal model of type 2 diabetes. Previous studies have shown a correlation between β-cell function and bioluminescent imaging in lean genetically engineered mice. The ability to noninvasively monitor β-cell function in ob/ob mice could provide new information on β-cell regulation in type 2 diabetes.

Methods

To create the B6 Albino ob/ob MIP-luc mice (ob/ob-luc), the ob/ob mouse was crossed with the CD1 MIP-luc mouse. All mice were backcrossed over multiple generations to ensure the genetic background of the transgenic mice was over 96% similar to the background of the original ob/ob mouse. Animal weight, blood glucose levels, insulin in plasma, and in vivo bioluminescence (BLI) were monitored weekly or biweekly for up to 70 weeks of age. BL imaging was performed using IVIS Spectrum (Perkin Elmer) and calculated by integrating the bioluminescence signal between 5 and 10 min after i.v. injection of D-luciferin. Insulin immunohistochemistry determined islet beta cell count and insulin secretion assay determined islet insulin function.

Results

There were significant increases in BLI and insulin levels as the ob/ob-luc mice aged while glucose levels gradually decreased. Ob/ob-luc were sacrificed at different time points to determine ex vivo BLI, islet function and total β-cell numbers using a cell counting training algorithm developed for the Vectra image analysis system (Perkin Elmer). The number of β-cells increased as the mice aged and all three ex vivo measurements correlated with BLI.

Conclusions

The ob/ob-luc mice can serve as a model of metabolic stress, similar to human type 2 diabetes using BLI as a surrogate marker for β-cell function.     

COMP-angiopoietin-1 recovers molecular biomarkers of neuropathy and improves vascularisation in sciatic nerve of ob/ob mice

Background

Leptin-deficient ob/ob mice are a model of type 2 diabetes induced peripheral neuropathy. Ob/ob mice exhibit obesity, insulin resistance, hyperglycaemia, and alterations of peripheral nerve fibres and endoneural microvessels. Here we test the hypothesis that cartilage oligomeric matrix protein (COMP)-Ang-1, a soluble and stabile form of Ang-1 which promotes angiogenesis and nerve growth, improves regeneration of nerve fibres and endoneural microvessels in ob/ob mice.

Methods and Findings

COMP-Ang-1 (100 ng/ml) or NaCl were intraperitoneally (i.p.) injected into male (N = 184), 3-month old, ob/ob or ob/+ mice for 7 and 21 days. We measured expression of Nf68, GAP43, Cx32, Cx26, Cx43, and TNFα in sciatic nerves using Western blot analysis. To investigate the inflammation in sciatic nerves, numbers of macrophages and T-cells were counted after immunofluorescence staining. In ultrathin section, number of myelinated/non-mylinated nerve fibers, g-ratio, the thickness of Schwann cell basal lamina and microvessel endothelium were investigated.Endoneural microvessels were reconstructed with intracardial FITC injection. Treatment with COMP-Ang-1 over 21 days significantly reduced fasting blood glucose and plasma cholesterol concentrations compared to saline treated ob/ob mice. In addition, COMP-Ang-1 treatment: 1) up-regulated expression of Nf68 and GAP43; 2) improved expression of gap junction proteins including connexin 32 and 26; 3) suppressed the expression of TNFα and Cx43 and 4) led to decreased macrophage and T-cell infiltration in sciatic nerve of ob/ob mice. The significant changes of sciatic nerve ultrastructure were not observed after 21-day long COMP-Ang-1 treatment. COMP-Ang-1 treated ob/ob mice displayed regeneration of small-diameter endoneural microvessels. Effects of COMP-Ang-1 corresponded to increased phosphorylation of Akt and p38 MAPK upon Tie-2 receptor.

Conclusions

COMP-Ang-1 recovers molecular biomarkers of neuropathy, promotes angiogenesis and suppresses inflammation in sciatic nerves of ob/ob mice suggesting COMP-Ang-1 as novel treatment option to improve morphologic and protein expression changes associated with diabetic neuropathy.     

Cannabinoid CB2 Receptor Potentiates Obesity-Associated Inflammation,Insulin Resistance and Hepatic Steatosis

Background

Obesity-associated inflammation is of critical importance in the development of insulin resistance and non-alcoholic fatty liver disease. Since the cannabinoid receptor CB2 regulates innate immunity, the aim of the present study was to investigate its role in obesity-induced inflammation, insulin resistance and fatty liver.

Methodology

Murine obesity models included genetically leptin-deficient ob/ob mice and wild type (WT) mice fed a high fat diet (HFD), that were compared to their lean counterparts. Animals were treated with pharmacological modulators of CB2 receptors. Experiments were also performed in mice knock-out for CB2 receptors (Cnr2 −/−).

Principal Findings

In both HFD-fed WT mice and ob/ob mice, Cnr2 expression underwent a marked induction in the stromal vascular fraction of epididymal adipose tissue that correlated with increased fat inflammation. Treatment with the CB2 agonist JWH-133 potentiated adipose tissue inflammation in HFD-fed WT mice. Moreover, cultured fat pads isolated from ob/ob mice displayed increased Tnf and Ccl2 expression upon exposure to JWH-133. In keeping, genetic or pharmacological inactivation of CB2 receptors decreased adipose tissue macrophage infiltration associated with obesity, and reduced inductions of Tnf and Ccl2 expressions. In the liver of obese mice, Cnr2 mRNA was only weakly induced, and CB2 receptors moderately contributed to liver inflammation. HFD-induced insulin resistance increased in response to JWH-133 and reduced in Cnr2 −/− mice. Finally, HFD-induced hepatic steatosis was enhanced in WT mice treated with JWH-133 and blunted in Cnr2 −/− mice.

Conclusion/Significance

These data unravel a previously unrecognized contribution of CB2 receptors to obesity-associated inflammation, insulin resistance and non-alcoholic fatty liver disease, and suggest that CB2 receptor antagonists may open a new therapeutic approach for the management of obesity-associated metabolic disorders.     

The utility of low-density genotyping for imputation in the Thoroughbred horse

Background

Despite the dramatic reduction in the cost of high-density genotyping that has occurred over the last decade, it remains one of the limiting factors for obtaining the large datasets required for genomic studies of disease in the horse. In this study, we investigated the potential for low-density genotyping and subsequent imputation to address this problem.

Results

Using the haplotype phasing and imputation program, BEAGLE, it is possible to impute genotypes from low- to high-density (50K) in the Thoroughbred horse with reasonable to high accuracy. Analysis of the sources of variation in imputation accuracy revealed dependence both on the minor allele frequency of the single nucleotide polymorphisms (SNPs) being imputed and on the underlying linkage disequilibrium structure. Whereas equidistant spacing of the SNPs on the low-density panel worked well, optimising SNP selection to increase their minor allele frequency was advantageous, even when the panel was subsequently used in a population of different geographical origin. Replacing base pair position with linkage disequilibrium map distance reduced the variation in imputation accuracy across SNPs. Whereas a 1K SNP panel was generally sufficient to ensure that more than 80% of genotypes were correctly imputed, other studies suggest that a 2K to 3K panel is more efficient to minimize the subsequent loss of accuracy in genomic prediction analyses. The relationship between accuracy and genotyping costs for the different low-density panels, suggests that a 2K SNP panel would represent good value for money.

Conclusions

Low-density genotyping with a 2K SNP panel followed by imputation provides a compromise between cost and accuracy that could promote more widespread genotyping, and hence the use of genomic information in horses. In addition to offering a low cost alternative to high-density genotyping, imputation provides a means to combine datasets from different genotyping platforms, which is becoming necessary since researchers are starting to use the recently developed equine 70K SNP chip. However, more work is needed to evaluate the impact of between-breed differences on imputation accuracy.     

A phasing and imputation method for pedigreed populations that results in a single-stage genomic evaluation

Background

Efficient, robust, and accurate genotype imputation algorithms make large-scale application of genomic selection cost effective. An algorithm that imputes alleles or allele probabilities for all animals in the pedigree and for all genotyped single nucleotide polymorphisms (SNP) provides a framework to combine all pedigree, genomic, and phenotypic information into a single-stage genomic evaluation.

Methods

An algorithm was developed for imputation of genotypes in pedigreed populations that allows imputation for completely ungenotyped animals and for low-density genotyped animals, accommodates a wide variety of pedigree structures for genotyped animals, imputes unmapped SNP, and works for large datasets. The method involves simple phasing rules, long-range phasing and haplotype library imputation and segregation analysis.

Results

Imputation accuracy was high and computational cost was feasible for datasets with pedigrees of up to 25 000 animals. The resulting single-stage genomic evaluation increased the accuracy of estimated genomic breeding values compared to a scenario in which phenotypes on relatives that were not genotyped were ignored.

Conclusions

The developed imputation algorithm and software and the resulting single-stage genomic evaluation method provide powerful new ways to exploit imputation and to obtain more accurate genetic evaluations.     

FTO Is a Relevant Factor for the Development of the Metabolic Syndrome in Mice

The metabolic syndrome is a worldwide problem mainly caused by obesity. FTO was found to be a obesity-risk gene in humans and FTO deficiency in mice led to reduction in adipose tissue. Thus, FTO is an important factor for the development of obesity. Leptin-deficient mice are a well characterized model for analysing the metabolic syndrome. To determine the relevance of FTO for the development of the metabolic syndrome we analysed different parameters in combined homozygous deficient mice (Lep^ob/ob;Fto^−/−). Lep^ob/ob;Fto^−/− mice showed an improvement in analysed hallmarks of the metabolic syndrome in comparison to leptin-deficient mice wild type or heterozygous for Fto. Lep^ob/ob;Fto^−/− mice did not develop hyperglycaemia and showed an improved glucose tolerance. Furthermore, extension of beta-cell mass was prevented in Lep^ob/ob;Fto^−/−mice and accumulation of ectopic fat in the liver was reduced. In conclusion this study demonstrates that FTO deficiency has a protective effect not only on the development of obesity but also on the metabolic syndrome. Thus, FTO plays an important role in the development of metabolic disorders and is an interesting target for therapeutic agents.     

Absence of adipose differentiation related protein upregulates hepatic VLDL secretion,relieves hepatosteatosis,and improves whole body insulin resistance in leptin-deficient mice

We previously showed that adipose differentiation related protein (Adfp)-deficient mice display a 60% reduction in hepatic triglyceride (TG) content. In this study, we investigated the role of ADFP in lipid and glucose homeostasis in a genetic obesity model, Lep^ob/ob mice. We bred Adfp^−/− mice with Lep^ob/ob mice to create Lep^ob/ob/Adfp^−/− and Lep^ob/ob/Adfp^+/+ mice and analyzed the hepatic lipids, lipid droplet (LD) morphology, LD protein composition and distribution, lipogenic gene expression, and VLDL secretion, as well as insulin sensitivity of the two groups of mice. Compared with Lep^ob/ob/Adfp^+/+ mice, Lep^ob/ob/Adfp^−/− mice displayed an increased VLDL secretion rate, a 25% reduction in hepatic TG associated with improvement in fatty liver grossly and microscopically with a change of the size of LDs in a proportion of the hepatocytes and a redistribution of major LD-associated proteins from the cytoplasmic compartment to the LD surface. There was no detectable change in lipogenic gene expression. Lep^ob/ob/Adfp^−/− mice also had improved glucose tolerance and insulin sensitivity in both liver and muscle. The alteration of LD size in the liver of Lep^ob/ob/Adfp^−/− mice despite the relocation of other LDPs to the LD indicates a nonredundant role for ADFP in determining the size and distribution of hepatic LDs.     

Hepatic Macrosteatosis Is Partially Converted to Microsteatosis by Melatonin Supplementation in ob/ob Mice Non-Alcoholic Fatty Liver Disease

Background

Obesity is a common risk factor for non-alcoholic fatty liver disease (NAFLD). Currently, there are no specific treatments against NAFLD. Thus, examining any molecule with potential benefits against this condition emerged melatonin as a molecule that influences metabolic dysfunctions. The aim of this study was to determine whether melatonin would function against NAFDL, studying morphological, ultrastuctural and metabolic markers that characterize the liver of ob/ob mice.

Methods

Lean and ob/ob mice were supplemented with melatonin in the drinking water for 8 weeks. Histology and stereology were performed to assess hepatic steatosis and glycogen deposition. Ultrastructural features of mitochondria, endoplasmic reticulum (ER) and their juxtapositions were evaluated in livers of all experimental groups. Furthermore, hepatic distribution and expression of markers of ER and mitochondria (calnexin, ATP sintase β, GRP78 and CHOP) and metabolic dysfunction (RPB4, β-catenin) and cellular longevity (SIRT1) were analyzed.

Results

Melatonin significantly reduced glycemia, identified also by a decrease of hepatic RBP4 expression, reversed macrosteatosis in microsteatosis at the hepatic pericentral zone, enlarged ER-mitochondrial distance and ameliorated the morphology and organization of these organelles in ob/ob mouse liver. Furthermore, in ob/ob mice, calnexin and ATP synthase β were partially restored, GRP78 and CHOP decreased in periportal and midzonal hepatocytes and β-catenin expression was, in part, restored in peripheral membranes of hepatocytes. Melatonin supplementation to ob/ob mice improves hepatic morphological, ultrastructural and metabolic damage that occurs as a result of NAFLD.

Conclusions

Melatonin may be a potential adjuvant treatment to limit NAFLD and its progression into irreversible complications.     

A low-marker density implementation of genomic selection in aquaculture using within-family genomic breeding values

Background

Genomic selection can increase genetic gain within aquaculture breeding programs, but the high costs related to high-density genotyping of a large number of individuals would make the breeding program expensive. In this study, a low-cost method using low-density genotyping of pre-selected candidates and their sibs was evaluated by stochastic simulation.

Methods

A breeding scheme with selection for two traits, one measured on candidates and one on sibs was simulated. Genomic breeding values were estimated within families and combined with conventional family breeding values for candidates that were pre-selected based on conventional BLUP breeding values. This strategy was compared with a conventional breeding scheme and a full genomic selection program for which genomic breeding values were estimated across the whole population. The effects of marker density, level of pre-selection and number of sibs tested and genotyped for the sib-trait were studied.

Results

Within-family genomic breeding values increased genetic gain by 15% and reduced rate of inbreeding by 15%. Genetic gain was robust to a reduction in marker density, with only moderate reductions, even for very low densities. Pre-selection of candidates down to approximately 10% of the candidates before genotyping also had minor effects on genetic gain, but depended somewhat on marker density. The number of test-individuals, i.e. individuals tested for the sib-trait, affected genetic gain, but the fraction of the test-individuals genotyped only affected the relative contribution of each trait to genetic gain.

Conclusions

A combination of genomic within-family breeding values, based on low-density genotyping, and conventional BLUP family breeding values was shown to be a possible low marker density implementation of genomic selection for species with large full-sib families for which the costs of genotyping must be kept low without compromising the effect of genomic selection on genetic gain.     

Genotyping on ALDH2: Comparison of Four Different Technologies

Objectives

This study aimed to compare the accuracy and performance of four genotyping methods for detecting single nucleotide polymorphisms (SNPs) in aldehyde dehydrogenase-2 (ALDH2), which is the principal enzyme involved in alcohol metabolism.

Design and Methods

We genotyped rs671 of ALDH2 in 96 coronary heart disease (CHD) patients with four methods including high resolution melting analysis (HRM), TaqMan allelic discrimination assay (TaqMan), allele-specific PCR (AS-PCR) and pyrosequencing. Meanwhile, we compared the accuracy and performance of these methods.

Results

All selected patients were successfully genotyped with referred methods. The results of these four assays showed 100% concordant results and had 100% accuracy as verified by Sanger sequencing.

Conclusions

All of the referred methods can be used for genotyping ALDH2 rs671 with the same accuracy compared to Sanger sequencing. In small size of clinical samples, HRM and AS-PCR outperform over others due to their lower cost and less hands-on operation, which are suitable for clinical application.     

In Vitro vs In Silico Detected SNPs for the Development of a Genotyping Array: What Can We Learn from a Non-Model Species?

Background

There is considerable interest in the high-throughput discovery and genotyping of single nucleotide polymorphisms (SNPs) to accelerate genetic mapping and enable association studies. This study provides an assessment of EST-derived and resequencing-derived SNP quality in maritime pine (Pinus pinaster Ait.), a conifer characterized by a huge genome size (∼23.8 Gb/C).

Methodology/Principal Findings

A 384-SNPs GoldenGate genotyping array was built from i/ 184 SNPs originally detected in a set of 40 re-sequenced candidate genes (in vitro SNPs), chosen on the basis of functionality scores, presence of neighboring polymorphisms, minor allele frequencies and linkage disequilibrium and ii/ 200 SNPs screened from ESTs (in silico SNPs) selected based on the number of ESTs used for SNP detection, the SNP minor allele frequency and the quality of SNP flanking sequences. The global success rate of the assay was 66.9%, and a conversion rate (considering only polymorphic SNPs) of 51% was achieved. In vitro SNPs showed significantly higher genotyping-success and conversion rates than in silico SNPs (+11.5% and +18.5%, respectively). The reproducibility was 100%, and the genotyping error rate very low (0.54%, dropping down to 0.06% when removing four SNPs showing elevated error rates).

Conclusions/Significance

This study demonstrates that ESTs provide a resource for SNP identification in non-model species, which do not require any additional bench work and little bio-informatics analysis. However, the time and cost benefits of in silico SNPs are counterbalanced by a lower conversion rate than in vitro SNPs. This drawback is acceptable for population-based experiments, but could be dramatic in experiments involving samples from narrow genetic backgrounds. In addition, we showed that both the visual inspection of genotyping clusters and the estimation of a per SNP error rate should help identify markers that are not suitable to the GoldenGate technology in species characterized by a large and complex genome.     

Pooled genome-wide analysis to identify novel risk loci for pediatric allergic asthma

Background

Genome-wide association studies of pooled DNA samples were shown to be a valuable tool to identify candidate SNPs associated to a phenotype. No such study was up to now applied to childhood allergic asthma, even if the very high complexity of asthma genetics is an appropriate field to explore the potential of pooled GWAS approach.

Methodology/Principal Findings

We performed a pooled GWAS and individual genotyping in 269 children with allergic respiratory diseases comparing allergic children with and without asthma. We used a modular approach to identify the most significant loci associated with asthma by combining silhouette statistics and physical distance method with cluster-adapted thresholding. We found 97% concordance between pooled GWAS and individual genotyping, with 36 out of 37 top-scoring SNPs significant at individual genotyping level. The most significant SNP is located inside the coding sequence of C5, an already identified asthma susceptibility gene, while the other loci regulate functions that are relevant to bronchial physiopathology, as immune- or inflammation-mediated mechanisms and airway smooth muscle contraction. Integration with gene expression data showed that almost half of the putative susceptibility genes are differentially expressed in experimental asthma mouse models.

Conclusion/Significance

Combined silhouette statistics and cluster-adapted physical distance threshold analysis of pooled GWAS data is an efficient method to identify candidate SNP associated to asthma development in an allergic pediatric population.     

Assignment of SNP allelic configuration in polyploids using competitive allele-specific PCR: application to citrus triploid progeny

Background

Polyploidy is a major component of eukaryote evolution. Estimation of allele copy numbers for molecular markers has long been considered a challenge for polyploid species, while this process is essential for most genetic research. With the increasing availability and whole-genome coverage of single nucleotide polymorphism (SNP) markers, it is essential to implement a versatile SNP genotyping method to assign allelic configuration efficiently in polyploids.

Scope

This work evaluates the usefulness of the KASPar method, based on competitive allele-specific PCR, for the assignment of SNP allelic configuration. Citrus was chosen as a model because of its economic importance, the ongoing worldwide polyploidy manipulation projects for cultivar and rootstock breeding, and the increasing availability of SNP markers.

Conclusions

Fifteen SNP markers were successfully designed that produced clear allele signals that were in agreement with previous genotyping results at the diploid level. The analysis of DNA mixes between two haploid lines (Clementine and pummelo) at 13 different ratios revealed a very high correlation (average = 0·9796; s.d. = 0·0094) between the allele ratio and two parameters [θ angle = tan⁻¹ (y/x) and y′ = y/(x + y)] derived from the two normalized allele signals (x and y) provided by KASPar. Separated cluster analysis and analysis of variance (ANOVA) from mixed DNA simulating triploid and tetraploid hybrids provided 99·71 % correct allelic configuration. Moreover, triploid populations arising from 2n gametes and interploid crosses were easily genotyped and provided useful genetic information. This work demonstrates that the KASPar SNP genotyping technique is an efficient way to assign heterozygous allelic configurations within polyploid populations. This method is accurate, simple and cost-effective. Moreover, it may be useful for quantitative studies, such as relative allele-specific expression analysis and bulk segregant analysis.     

Quantitative Analysis of Single Nucleotide Polymorphisms within Copy Number Variation

Background

Single nucleotide polymorphisms (SNPs) have been used extensively in genetics and epidemiology studies. Traditionally, SNPs that did not pass the Hardy-Weinberg equilibrium (HWE) test were excluded from these analyses. Many investigators have addressed possible causes for departure from HWE, including genotyping errors, population admixture and segmental duplication. Recent large-scale surveys have revealed abundant structural variations in the human genome, including copy number variations (CNVs). This suggests that a significant number of SNPs must be within these regions, which may cause deviation from HWE.

Results

We performed a Bayesian analysis on the potential effect of copy number variation, segmental duplication and genotyping errors on the behavior of SNPs. Our results suggest that copy number variation is a major factor of HWE violation for SNPs with a small minor allele frequency, when the sample size is large and the genotyping error rate is 0∼1%.

Conclusions

Our study provides the posterior probability that a SNP falls in a CNV or a segmental duplication, given the observed allele frequency of the SNP, sample size and the significance level of HWE testing.