Validation Of Single Nucleotide Polymorphisms Research Articles

Validation of tag SNPs for multiple sclerosis HLA risk alleles across the 1000 genomes panel

Currently, the genetic variants strongly associated with risk for Multiple Sclerosis (MS) are located in the Major Histocompatibility Complex. This includes DRB1*15:01 and DRB1*15:03 alleles at the HLA-DRB1 locus, the latter restricted to African populations; the DQB1*06:02 allele at the HLA-DQB1 locus which is in high linkage disequilibrium (LD) with DRB1*15:01; and protective allele A*02:01 at the HLA-A locus. HLA allele identification is facilitated by co-inherited (‘tag’) single nucleotide polymorphisms (SNPs); however, SNP validation is not typically done outside of the discovery population. We examined 19 SNPs reported to be in high LD with these alleles in 2,502 healthy subjects included in the 1000 Genomes panel having typed HLA data. Examination of 3 indices (LD R2 values, sensitivity and specificity, minor allele frequency) revealed few SNPs with high tagging performance. All SNPs examined that tag DRB1*15:01 were in perfect LD in the British population; three showed high tagging performance in 4 of the 5 European, and 2 of the 4 American populations. For DQB1*06:02, with no previously validated tag SNPs, we show that rs3135388 has high tagging performance in one South Asian, one American, and one European population. We identify for the first time that rs2844821 has high tagging performance for A*02:01 in 5 of 7 African populations including African Americans, and 4 of the 5 European populations. These results provide a basis for selecting SNPs with high tagging performance to assess HLA alleles across diverse populations, for MS risk as well as for other diseases and conditions.

Unraveling the Molecular Basis of Stabilizing Selection by Experimental Evolution

Abstract Stabilizing selection provides a challenge to molecular population genetics. Although stabilizing selection is ubiquitous, its genomic signature is difficult to distinguish from demographic signals. Experimental evolution provides a promising approach to characterize genomic regions exposed to stabilizing selection. A recent experimental evolution study of Aedes aegypti populations evolving either with or without sexual selection found a pattern of genetic differentiation suggestive of relaxed stabilizing selection. I argue that this study could not have detected the signal of relaxed stabilizing selection. I highlight why incorrect statistical methods resulted in a high number of false positive candidate single nucleotide polymorphism (SNPs) and discuss the fallacy of functional validation of candidate SNPs for polygenic traits by RNA-mediated knockdown.

Prospective Validation of Single Nucleotide Polymorphisms as Predictors of Gastrointestinal, Genitourinary, and Sexual Patient-Reported Outcomes Following Radiotherapy for Prostate Cancer.

Radiotherapy (RT) for localized prostate cancer (PC) can adversely impact gastrointestinal (GI), genitourinary (GU), and sexual quality of life (QoL). Biomarkers are needed to accurately predict individualized risk of toxicity and enable tailoring of therapy. Single nucleotide polymorphisms (SNPs) have been reported as potential predictors of RT-related toxicities, but have not been associated with patient-report outcomes (PRO) in prospective cohorts. In this study, we sought to validate these SNPs in a prospective registry study of RT for PC, with high-quality prospectively collected PRO data. Men with low/intermediate risk PC were consented to a multicenter companion registry study associated with the PARTIQoL Phase III Randomized Trial of proton vs. photon RT. Patients received RT to prostate/seminal vesicles per protocol. Androgen deprivation therapy and pelvic lymph node RT were not allowed. 95 patients enrolled between 2014-2020 at a single institution had blood specimens available for germline DNA analysis. 172 SNPs previously reported to correlate with GI, GU, and/or sexual RT toxicities were genotyped. PRO data through the Expanded Prostate Cancer Index Composite (EPIC) were collected prior to RT and at prespecified follow-up (FU) time points. Change of the EPIC score from baseline was compared between genotypes of previously identified SNPs using a two-sample t-test. Significant clinically meaningful QoL differences were identified by an effect size of at least 0.4σ with two-sided p<0.05, where σ represents the standard deviation of the score change. Median FU was 39 months (r, 6-89). Median age was 68 years (r, 52-83). Features at diagnosis include: 77% T1c, median PSA 5.8 (r, 1.43-15.1), 53% Gleason 7, median prostate volume 45.5cc (r, 16-142). 43% received proton RT; 53% had a rectal spacer. 40% received 79.2 Gy/44 fractions; 60% received 70 Gy/28 fractions. Between 6 to 24 months post-RT, there were 19 SNPs that were significantly associated with clinically meaningful decreases in GI QoL scores, 19 that were significantly associated with clinically meaningful decreases in GU QoL scores, and 10 SNPs that were significantly associated with clinically meaningful decreases in sexual QoL scores. Three BRCA2 SNPs (rs1801439, rs1801499, rs1799944) were significantly associated with clinically meaningful decreases in both GI and GU QoL scores. Of the 172 SNPs previously reported to be associated with GI, GU, and/or sexual toxicity after prostate RT, 23% were validated for domain-specific QoL detriment. Ongoing analyses include integrated modeling of dosimetry and SNP data for prediction of toxicities and PRO, and evaluation of potential interactions between RT modality (proton/photon) and QoL-associated SNPs.

Evaluation of Supervised Machine Learning Algorithms and Computational Structural Validation of Single Nucleotide Polymorphisms Related to Acute Liver Injury with Paracetamol.

To identify single nucleotide polymorphisms (SNPs) of paracetamol-metabolizing enzymes that can predict acute liver injury. Paracetamol is a commonly administered analgesic/antipyretic in critically ill and chronic renal failure patients and several SNPs influence the therapeutic and toxic effects. To evaluate the role of machine learning algorithms (MLAs) and bioinformatics tools to delineate the predictor SNPs as well as to understand their molecular dynamics. A cross-sectional study was undertaken by recruiting critically ill patients with chronic renal failure and administering intravenous paracetamol as a standard of care. Serum concentrations of paracetamol and the principal metabolites were estimated. Following SNPs were evaluated: CYP2E1*2, CYP2E1_-1295G>C, CYP2D6*10, CYP3A4*1B, CYP3A4*2, CYP1A2*1K, CYP1A2*6, CYP3A4*3, and CYP3A5*7. MLAs were used to identify the predictor genetic variable for acute liver failure. Bioinformatics tools such as Predict SNP2 and molecular docking (MD) were undertaken to evaluate the impact of the above SNPs with binding affinity to paracetamol. CYP2E1*2 and CYP1A2*1C genotypes were identified by MLAs to significantly predict hepatotoxicity. The predictSNP2 revealed that CYP1A2*3 was highly deleterious in all the tools. MD revealed binding energy of -5.5 Kcal/mol, -6.9 Kcal/mol, and -6.8 Kcal/mol for CYP1A2, CYP1A2*3, and CYP1A2*6 against paracetamol. MD simulations revealed that CYP1A2*3 and CYP1A2*6 missense variants in CYP1A2 affect the binding ability with paracetamol. In-silico techniques found that CYP1A2*2 and CYP1A2*6 are highly harmful. MD simulations revealed CYP3A4*2 (A>G) had decreased binding energy with paracetamol than CYP3A4, and CYP3A4*2(A>T) and CYP3A4*3 both have greater binding energy with paracetamol. Polymorphisms in CYP2E1, CYP1A2, CYP3A4, and CYP3A5 significantly influence paracetamol's clinical outcomes or binding affinity. Robust clinical studies are needed to identify these polymorphisms' clinical impact on the pharmacokinetics or pharmacodynamics of paracetamol.

Identification of KASP markers and candidate genes for drought tolerance in wheat using 90K SNP array genotyping of near-isogenic lines targeting a 4BS quantitative trait locus.

This study identified a novel SNP and developed a highly efficient KASP marker for drought tolerance in wheat by genotyping NILs targeting a major QTL for drought tolerance using an SNP array and validation with commercial varieties. Common wheat (Triticum aestivum L.) is an important winter crop worldwide and a typical allopolyploid with a large and complex genome. With global warming, the environmental volatility and incidence of drought in wheat-producing areas will increase. Molecular markers for drought tolerance are urgently needed to enhance drought tolerance breeding. Here, we genotyped four near-isogenic line (NIL) pairs targeting a major QTL qDSI.4B.1 on wheat chromosome arm 4BS for drought tolerance using the 90K SNP Illumina iSelect array and discovered a single nucleotide polymorphism (SNP) (Excalibur_c100336_106) with consistent genotype-phenotype associations among all four NIL pairs and their parents. Then, we converted the SNP into a Kompetitive Allele-Specific PCR (KASP) marker, with an accuracy of 100% for the four NIL pairs and their parents and as high as 81.8% for the 44 tested wheat lines with known phenotypes collected from Australia and China. Two genes near this SNP were suggested as candidate genes for drought tolerance in wheat after checking the Chinese Spring reference genome annotation version 1.1. One gene, TraesCS4B02G085300, encodes an F-box protein reportedly related to the ABA network, a main pathway for drought tolerance, and another gene, TraesCS4B02G085400, encodes a calcineurin-like metallophos-phoesterase transmembrane protein, which participates in Ca2+-dependent phosphorylation regulatory system. Based on this work and previous research on pre-harvest sprouting, we established a quick and efficient general SQV-based approach for KASP marker development, integrating genotyping by SNP arrays (S) using NILs targeting major QTL for a specific trait (Q) and validating them with commercial varieties (V). The identified SNP and developed KASP marker could be applied to marker-assisted selection in drought breeding, and further study of the candidate genes may improve our understanding of drought tolerance in wheat.

Significant association between a non-synonymous snp in IGFBP5 gene and the growth of striped catfish (Pangasianodon hypophthalmus, Sauvage, 1878)

Insulin-like growth factor binding protein 5 (IGFBP5) is the highest conserved member of IGFBP family, and has the broad range of biological activities effecting on the cell growth. This study aims to investigate the association between genetic variation in IGFBP5 gene and the growth of striped catfish (Pangasianodon hypophthalmus). Single nucleotide polymorphisms (SNPs) were discovered and validated in IGFBP5 gene from two growth-selected populations (fast- and slow- growing fish). For SNP discovery, the fragments of IGFBP5 from sample sets of 10 fast- growing fish and 10 slow- growing fish were directly sequenced by Sanger sequencing. In this stage, 4 exonic SNPs were discovered, including a non-synonymous SNP 525 T&gt;A (p. Val16Glu) in exon 1, and three synonymous SNPs (8859 G&gt;A, 11713 C&gt;A, 11992 T&gt;C) in exon 4. The non-synonymous SNP 525 T&gt;A (p.Val16Glu) was filtered to the next step of SNP validation. For validation, the SNP was individually genotyped in the test populations of 70 fast- growing fish and 70 slow- growing fish by single base extension method. Data analysis from the total SNPs which were collected from 80 fast- growing fish and 80 slow- growing fish indicated that non-synonymous SNP 525 T&gt;A (p.Val16Glu) was significantly associated to the growth of striped catfish (p-value &lt;0.001). Analysis of genetic diversity parameters (PIC, MAF) suggested that this SNP is a common variant, contributes significantly to the genetic variance. The non-synonymous SNP 525 T&gt;A (p.Val16Glu) in IGFBP5 gene would become a SNP marker candidate for marker assisted selection (MAS) that can be used in pangasius breeding.

Discovery of SNP Molecular Markers and Candidate Genes Associated with Sacbrood Virus Resistance in Apis cerana cerana Larvae by Whole-Genome Resequencing.

Sacbrood virus (SBV) is a significant problem that impedes brood development in both eastern and western honeybees. Whole-genome sequencing has become an important tool in researching population genetic variations. Numerous studies have been conducted using multiple techniques to suppress SBV infection in honeybees, but the genetic markers and molecular mechanisms underlying SBV resistance have not been identified. To explore single nucleotide polymorphisms (SNPs), insertions, deletions (Indels), and genes at the DNA level related to SBV resistance, we conducted whole-genome resequencing on 90 Apis cerana cerana larvae raised in vitro and challenged with SBV. After filtering, a total of 337.47 gigabytes of clean data and 31,000,613 high-quality SNP loci were detected in three populations. We used ten databases to annotate 9359 predicted genes. By combining population differentiation index (FST) and nucleotide polymorphisms (π), we examined genome variants between resistant (R) and susceptible (S) larvae, focusing on site integrity (INT < 0.5) and minor allele frequency (MAF < 0.05). A selective sweep analysis with the top 1% and top 5% was used to identify significant regions. Two SNPs on the 15th chromosome with GenBank KZ288474.1_322717 (Guanine > Cytosine) and KZ288479.1_95621 (Cytosine > Thiamine) were found to be significantly associated with SBV resistance based on their associated allele frequencies after SNP validation. Each SNP was authenticated in 926 and 1022 samples, respectively. The enrichment and functional annotation pathways from significantly predicted genes to SBV resistance revealed immune response processes, signal transduction mechanisms, endocytosis, peroxisomes, phagosomes, and regulation of autophagy, which may be significant in SBV resistance. This study presents novel and useful SNP molecular markers that can be utilized as assisted molecular markers to select honeybees resistant to SBV for breeding and that can be used as a biocontrol technique to protect honeybees from SBV.

Prospective validation of single nucleotide polymorphisms as predictors of gastrointestinal, genitourinary, and sexual toxicities following radiation therapy for prostate cancer.

385 Background: Radiation therapy (RT) for localized prostate cancer (PC) may result in long-term gastrointestinal (GI), genitourinary (GU), and sexual toxicities that adversely impact quality of life. Biomarkers are needed to accurately predict individualized RT toxicity risk and enable tailoring of treatment. Single nucleotide polymorphisms (SNPs) have been reported as predictors of RT-related toxicities, but have not been independently validated in prospective, prostate-specific cohorts. In this study, we sought to validate these SNPs in a prospective registry study of RT for PC, with high-quality prospectively collected toxicity data. Methods: Men with low/intermediate risk PC were consented to a multicenter companion registry study associated with the PARTIQoL Phase III Randomized Trial of proton vs. photon RT. Patients received RT to prostate/seminal vesicles per protocol. Androgen deprivation therapy/pelvic lymph node RT were not allowed. 95 patients enrolled between 2014-2020 at a single institution had whole blood specimens available for analysis. 172 SNPs previously reported to correlate with GI, GU, and/or sexual RT toxicities were genotyped. CTCAEv4 physician-reported toxicity was collected at prespecified follow-up (FU) time points. Association of previously identified SNP genotypes with the worst toxicity grade was analyzed using two-sided Fisher’s exact test. Results: Median FU was 39 months (r, 6-89). Median age was 68 years (r, 52-83). Features at diagnosis include: 77% T1c, median PSA 5.8 (r, 1.43-15.1), 53% Gleason 7, median prostate volume 45.5cc (r, 16-142). 43% received proton RT; 53% had a rectal spacer. 40% received 79.2 Gy/44 fractions; 60% received 70 Gy/28 fractions. By 24 months post-RT, 21 (22%) patients experienced grade 1-2 Gl toxicity, 33 (35%) experienced grade 1-2 GU toxicity, and 68 (72%) experienced grade 1-3 sexual toxicity. Of the 172 SNPs examined, significant associations were detected for rs1805794 with a higher incidence of grade 1-2 late GI toxicity (P=0.006), rs1800872 with a higher incidence of grade 1-2 late GU effects (P=0.005) and rs25489 with a higher incidence of grade 1-3 sexual dysfunction (P=0.003). rs3749191, rs4073, rs2243250, and rs7356945 were significantly associated with grade 2 late GU toxicity (all P≤0.008). Significant associations were also observed between other SNPs and late toxicity: 4 SNPs GI, 9 SNPs GU, 7 SNPs sexual (all 0.01≤P≤0.05). Conclusions: Of the 172 SNPs previously reported to be associated with GI, GU, and/or sexual toxicity after prostate RT, 16% were validated by conventional statistical criteria. Ongoing analyses include integrated modeling of dosimetry and SNP data for prediction of toxicities and PROs, and evaluation of potential interactions between RT modality (proton/photon) and toxicity-associated SNPs.

High-Throughput Association Mapping in Brassica napus L.: Methods and Applications.

Oil seed rape (Braasica napus L.) is ranked second among oil seed crops cultivated globally for edible oil for human, and seed cake for animal consumption. Recent genetic and genomics advancements highlighted the diversity that exists within B. napus, which is largely discovered using the most promising genetic markers called single nucleotide polymorphism (SNP). Their calling rate is also enhanced to ~100 folds after the continuous advancements in the next generation sequencing (NGS) technologies. As the high throughput of NGS resulted in multi-Giga bases data, the detailed quality control (QC) prior to downstream analyses is a pre-requisite. It mainly involved the removal of false positives, missing proportions, filtering of low-quality SNPs, and adjustments of minor-allele frequency and heterozygosity. After marker-trait association, for conformation of target SNPs, validations of SNPs can be performed using various methods, especially allele-specific PCR assay-based methods have been utilized for SNP genotyping of genes targeting agronomic traits and somaclonal variations occurred during transgenic studies. In the present study, the authors mainly argue on the genotypic progress, and pipelines/methods that are being used for detection, calling, filtering, and validation of SNPs. Also, insight is provided into the application of SNPs in linkage and association mapping, including QTL mapping and genome-wide association studies targeting mainly developmental traits related to the root system and plant architecture, flowering time, silique, and oil quality. Briefly, the present study provides the recent information and recommendations on the SNP genotyping methods and its applications, which can be useful for marker-assisted breeding in B. napus and other crops.

Development and Validation of SNP and InDel Markers for Pod-Shattering Tolerance in Soybean.

Pod-shattering causes a significant yield loss in many soybean cultivars. Shattering-tolerant cultivars provide the most effective approach to minimizing this loss. We developed molecular markers for pod-shattering and validated them in soybeans with diverse genetic backgrounds. The genes Glyma.16g141200, Glyma.16g141500, and Glyma.16g076600, identified in our previous study by quantitative trait locus (QTL) mapping and whole-genome resequencing, were selected for marker development. The whole-genome resequencing of three parental lines (one shattering-tolerant and two shattering-susceptible) identified single nucleotide polymorphism (SNP) and/or insertion/deletion (InDel) regions within or near the selected genes. Two SNPs and one InDel were converted to Kompetitive Allele-Specific PCR (KASP) and InDel markers, respectively. The accuracy of the markers was examined in the two recombinant inbred line populations used for the QTL mapping, as well as the 120 varieties and elite lines, through allelic discrimination and phenotyping by the oven-drying method. Both types of markers successfully discriminated the pod shattering-tolerant and shattering-susceptible genotypes. The prediction accuracy, which was as high as 90.9% for the RILs and was 100% for the varieties and elite lines, also supported the accuracy and usefulness of these markers. Thus, the markers can be used effectively for genetic and genomic studies and the marker-assisted selection for pod-shattering tolerance in soybean.

Validation of somatic cell score-associated SNPs from Holstein cattle in Sudanese Butana and Butana\u2009\xd7\u2009Holstein crossbred cattle

The Bos indicus zebu cattle Butana is the most commonly used indigenous dairy cattle breed in Sudan. In the last years, high-yielding Holstein dairy cattle were introgressed into Butana cattle to improve their milk yield and simultaneously keep their good adaption to extreme environmental conditions. With the focus on the improvement of milk production, other problems arose such as an increased susceptibility to mastitis. Thus, genetic selection for mastitis resistance should be considered to maintain healthy and productive cows. In this study, we tested 10 single nucleotide polymorphisms (SNPs) which had been associated with somatic cell score (SCS) in Holstein cattle for association with SCS in 37 purebred Butana and 203 Butana × Holstein crossbred cattle from Sudan. Animals were genotyped by competitive allele-specific PCR assays and association analysis was performed using a linear mixed model. All 10 SNPs were segregating in the crossbred Butana × Holstein populations, but only 8 SNPs in Sudanese purebred Butana cattle. The SNP on chromosome 13 was suggestively associated with SCS in the Butana × Holstein crossbred population (rs109441194, 13:79,365,467, PBF = 0.054) and the SNP on chromosome 19 was significantly associated with SCS in both populations (rs41257403, 19:50,027,458, Butana: PBF = 0.003, Butana × Holstein: PBF = 6.2 × 10−16). The minor allele of both SNPs showed an increase in SCS. Therefore, selection against the disadvantageous minor allele could be used for genetic improvement of mastitis resistance in the studied populations. However, investigations in a bigger population and across the whole genome are needed to identify additional genomic loci.

SNP characteristics and validation success in genome wide association studies.

Genome wide association studies (GWASs) have identified tens of thousands of single nucleotide polymorphisms (SNPs) associated with human diseases and characteristics. A significant fraction of GWAS findings can be false positives. The gold standard for true positives is an independent validation. The goal of this study was to identify SNP features associated with validation success. Summary statistics from the Catalog of Published GWASs were used in the analysis. Since our goal was an analysis of reproducibility, we focused on the diseases/phenotypes targeted by at least 10 GWASs. GWASs were arranged in discovery-validation pairs based on the time of publication, with the discovery GWAS published before validation. We used four definitions of the validation success that differ by stringency. Associations of SNP features with validation success were consistent across the definitions. The strongest predictor of SNP validation was the level of statistical significance in the discovery GWAS. The magnitude of the effect size was associated with validation success in a non-linear manner. SNPs with risk allele frequencies in the range 30-70% showed a higher validation success rate compared to rarer or more common SNPs. Missense, 5'UTR, stop gained, and SNPs located in transcription factor binding sites had a higher validation success rate compared to intergenic, intronic and synonymous SNPs. There was a positive association between validation success and the level of evolutionary conservation of the sites. In addition, validation success was higher when discovery and validation GWASs targeted the same ethnicity. All predictors of validation success remained significant in a multivariate logistic regression model indicating their independent contribution. To conclude, we identified SNP features predicting validation success of GWAS hits. These features can be used to select SNPs for validation and downstream functional studies.

Development of an SNP Assay for Marker-Assisted Selection of Soil-Borne Rhizoctonia solani AG-2-2-IIIB Resistance in Sugar Beet.

Rhizoctonia solani, causing Rhizoctonia crown and root rot, is a major risk to sugar beet (Beta vulgaris L.) cultivation. The development of resistant varieties accelerated by marker-assisted selection is a priority of breeding programs. We report the identification of a single-nucleotide polymorphism (SNP) marker linked to Rhizoctonia resistance using restriction site-associated DNA (RAD) sequencing of two geographically discrete sets of plant materials with different degrees of resistance/susceptibility to enable a wider selection of superior genotypes. The variant calling pipeline utilized SAMtools for variant calling and the resulting raw SNPs from RAD sequencing (15,988 and 22,439 SNPs) were able to explain 13.40% and 25.45% of the phenotypic variation in the two sets of material from different sources of origin, respectively. An association analysis was carried out independently on both the datasets and mutually occurring significant SNPs were filtered depending on their contribution to the phenotype using principal component analysis (PCA) biplots. To provide a ready-to-use marker for the breeding community, a systematic molecular validation of significant SNPs distributed across the genome was undertaken to combine high-resolution melting, Sanger sequencing, and rhAmp SNP genotyping. We report that RsBv1 located on Chromosome 6 (9,000,093 bp) is significantly associated with Rhizoctonia resistance (p < 0.01) and able to explain 10% of the phenotypic disease variance. The related SNP assay is thus ready for marker-assisted selection in sugar beet breeding for Rhizoctonia resistance.

Polygenic Risk Scores for Prediction of Gastric Cancer Based on Bioinformatics Screening and Validation of Functional lncRNA SNPs.

INTRODUCTION:Single-nucleotide polymorphisms (SNPs) are used to stratify the risk of gastric cancer. However, no study included gastric cancer–related long noncoding RNA (lncRNA) SNPs into the risk model for evaluation. This study aimed to replicate the associations of 21 lncRNA SNPs and to construct an individual risk prediction model for gastric cancer.METHODS:The bioinformatics method was used to screen gastric cancer–related lncRNA functional SNPs and verified in population. Gastric cancer risk prediction models were constructed using verified SNPs based on polygenic risk scores (PRSs).RESULTS:Twenty-one SNPs were screened, and the multivariate unconditional logistic regression analysis showed that 14 lncRNA SNPs were significantly associated with gastric cancer. In the distribution of genetic risk score in cases and controls, the mean value of PRS in cases was higher than that in controls. Approximately 20.1% of the cases was caused by genetic variation (P = 1.9 × 10−34) in optimal PRS model. The individual risk of gastric cancer in the lowest 10% of PRS was 82.1% (95% confidence interval [CI]: 0.102, 0.314) lower than that of the general population. The risk of gastric cancer in the highest 10% of PRS was 5.75-fold that of the general population (95% CI: 3.09, 10.70). The introduction of family history of tumor (area under the curve, 95% CI: 0.752, 0.69–0.814) and Helicobacter pylori infection (area under the curve, 95% CI: 0.773, 0.702–0.843) on the basis of PRS could significantly improve the recognition ability of the model.DISCUSSION:PRSs based on lncRNA SNPs could identify individuals with high risk of gastric cancer and combined with risk factors could improve the stratification.

Characterizing the Impact of Cancer Mutations in DNA Base Editing Enzymes

We have developed a comprehensive approach to find and characterize the impact of cancer mutants on any protein, and have applied it to DNA modification enzymes. This approach is an extension of conventional genome wide-association studies (GWAS). Our approach employs a new method for discovery and statistical validation of single nucleotide polymorphisms (SNPs) on specific genes called HyDn-SNP-S, followed by atomistic simulations via molecular dynamics (MD) and/or quantum mechanics/molecular mechanics (QM/MM) techniques. We will present the details of our mutant discovery and characterization approach, as well as examples of cancer mutations on three different DNA modification enzymes. These examples include a breast cancer mutation on DNA polymerase l, a prostate cancer mutation on the DNA dealkylase ALKBH7, and a lung cancer mutation on the cytidine deaminaseAPOBEC3H. Our simulations provide atomistic details about how these mutations affect the specific protein structure and/or function, which have been further validated by experimental methods.

Identification and validation of SNPs in the phytoene synthase 2 (psy2) gene associated with yellow color of the root in cassava (Manihot esculenta Crantz) accessions of the Brazilian Amazon

The biofortification of cassava (Manihot esculenta Crantz) roots is one of the actual goals of genetic breeding, with the development of roots richer in carotenoids. In order to evaluate the natural genetic variation of cassava, this study analyzed 78 accessions from the Brazilian Amazon to verify the presence of single nucleotide polymorphism (SNP) molecular markers in the phytoene synthase 2 (psy2) gene associated with root color. From the sequencing of 294 bp containing part of the intron 2 and the exon 3 of the psy2 gene, three SNPs located in the coding region and the existence of eight haplotypes were identified. The c.1434G > C (SNP1) and c.1485C > A (SNP3) variations were found in yellow and cream root accessions and nucleotide polymorphism resulted in amino acid substitution. SNP2 (c.1462T > C) was identified in accessions with white roots and its allelic variation did not result in amino acid substitution. Four haplotypes that presented SNP3 were widely distributed in the states of Para, Amapa, Rondonia and Amazonas. All accessions of yellow or cream root that presented the SNP3 were in heterozygosity, confirming the dominance of the SNP, except for one accession, that could be used in genetic breeding. The effect of mutations on the structure and function of PSY2 was in silico evaluated by molecular dynamics and it was indicated that the aminoacid substitutions affected the active site of the protein depending on the distance. The results were compatible with the phenotype detected experimentally.

A combination of genome-wide association study and transcriptome analysis in leaf epidermis identifies candidate genes involved in cuticular wax biosynthesis in Brassica napus

BackgroundBrassica napus L. is one of the most important oil crops in the world. However, climate-change-induced environmental stresses negatively impact on its yield and quality. Cuticular waxes are known to protect plants from various abiotic/biotic stresses. Dissecting the genetic and biochemical basis underlying cuticular waxes is important to breed cultivars with improved stress tolerance.ResultsHere a genome-wide association study (GWAS) of 192 B. napus cultivars and inbred lines was used to identify single-nucleotide polymorphisms (SNPs) associated with leaf waxes. A total of 202 SNPs was found to be significantly associated with 31 wax traits including total wax coverage and the amounts of wax classes and wax compounds. Next, epidermal peels from leaves of both high-wax load (HW) and low-wax load (LW) lines were isolated and used to analyze transcript profiles of all GWAS-identified genes. Consequently, 147 SNPs were revealed to have differential expressions between HW and LW lines, among which 344 SNP corresponding genes exhibited up-regulated while 448 exhibited down-regulated expressions in LW when compared to those in HW. According to the gene annotation information, some differentially expressed genes were classified into plant acyl lipid metabolism, including fatty acid-related pathways, wax and cutin biosynthesis pathway and wax secretion. Some genes involved in cell wall formation and stress responses have also been identified.ConclusionsCombination of GWAS with transcriptomic analysis revealed a number of directly or indirectly wax-related genes and their associated SNPs. These results could provide clues for further validation of SNPs for marker-assisted breeding and provide new insights into the genetic control of wax biosynthesis and improving stress tolerance of B. napus.

Validation of variants using cost effective highresolution melting (HRM) analysis predicted from target re-sequencing in Eucalyptus

The advent of next-generation sequencing has facilitated large-scale discovery and mapping of genomic variants for high-throughput genotyping. Several research groups working in tree species are presently employing next generation sequencing (NGS) platforms for marker discovery, since it is a cost effective and time saving strategy. However, most trees lack a chromosome level genome map and validation of variants for downstream application becomes obligatory. The cost associated with identifying potential variants from the enormous amount of sequence data is a major limitation. In the present study, high resolution melting (HRM) analysis was optimized for rapid validation of single nucleotide polymorphisms (SNPs), insertions or deletions (InDels) and simple sequence repeats (SSRs) predicted from exome sequencing of parents and hybrids of Eucalyptus tereticornis Sm. ? Eucalyptus grandis Hill ex Maiden generated from controlled hybridization. The cost per data point was less than 0.5 USD, providing great flexibility in terms of cost and sensitivity, when compared to other validation methods. The sensitivity of this technology in variant detection can be extended to other applications including Bar-HRM for species authentication and TILLING for detection of mutants.

Targeted genotype analyses of GWAS-derived lean body mass and handgrip strength-associated single-nucleotide polymorphisms in elite master athletes.

Recent large genome-wide association studies (GWAS) have independently identified a set of genetic loci associated with lean body mass (LBM) and handgrip strength (HGS). Evaluation of these candidate single-nucleotide polymorphisms (SNPs) may be useful to investigate genetic traits of populations at higher or lower risk of muscle dysfunction. As such, we investigated associations between six SNPs linked to LBM or HGS in a population of elite master athletes (MA) and age-matched controls as a representative population of older individuals with variable maintenance of muscle mass and function. Genomic DNA was isolated from buffy coat samples of 96 individuals [consisting of 48 MA (71 ± 6 yr, age-graded performance 83 ± 9%) and 48 older controls (75 ± 6 yr)]. SNP validation and sample genotyping were conducted using the tetra-primer amplification refractory mutation system (ARMS). For the three SNPs analyzed that were previously associated with LBM (FTO, IRS1, and ADAMTSL3), multinomial logistic regression revealed a significant association of the ADAMTSL3 genotype with %LBM (P < 0.01). For the three HGS-linked SNPs, neither GBF1 nor GLIS1 showed any association with HGS, but for TGFA, multinomial logistic regression revealed a significant association of genotype with HGS (P < 0.05). For ADAMTSL3, there was an enrichment of the effect allele in the MA (P < 0.05, Fisher's exact test). Collectively, of the six SNPs analyzed, ADAMTSL3 and TGFA showed significant associations with LBM and HGS, respectively. The functional relevance of the ADAMTSL3 SNP in body composition and of TGFA in strength may highlight a genetic component of the elite MA phenotype.

Piloting a surveillance system to monitor the global patterns of drug efficacy and the emergence of anthelmintic resistance in soil-transmitted helminth control programs: a Starworms study protocol

To eliminate soil-transmitted helminth (STH) infections as a public health problem, the administration of benzimidazole (BZ) drugs to children has recently intensified. But, as drug pressure increases, the development of anthelmintic drug resistance (AR) becomes a major concern. Currently, there is no global surveillance system to monitor drug efficacy and the emergence of AR. Consequently, it is unclear what the current efficacy of the used drugs is and whether AR is already present. The aim of this study is to pilot a global surveillance system to assess anthelmintic drug efficacy and the emergence of AR in STH control programs. For this, we will incorporate drug efficacy trials into national STH control programs of eight countries (Bangladesh, Cambodia, Lao PDR, Vietnam, Ghana, Rwanda, Senegal and a yet to be defined country in the Americas). In each country, one trial will be performed in one program implementation unit to assess the efficacy of BZ drugs against STHs in school-aged children by faecal egg count reduction test. Stool samples will be collected before and after treatment with BZs for Kato-Katz analysis and preserved to purify parasite DNA. The presence and frequency of known single nucleotide polymorphisms (SNPs) in the β-tubulin genes of the different STHs will subsequently be assessed. This study will provide a global pattern of drug efficacy and emergence of AR in STH control programs. The results will provide complementary insights on the validity of known SNPs in the ß-tubulin gene as a marker for AR in human STHs as well as information on the technical and financial resources required to set up a surveillance system. Finally, the collected stool samples will be an important resource to validate different molecular technologies for the detection of AR markers or to identify novel potential molecular markers associated with AR in STH.