Estimation of linkage disequilibrium levels and allele frequency distribution in crossbred Vrindavani cattle using 50K SNP data

The objective of this study was to calculate the extent and decay of linkage disequilibrium (LD) in 96 crossbred Vrindavani cattle genotyped with Bovine SNP50K Bead Chip. After filtering, 43,821 SNPs were retained for final analysis, across 2500.3 Mb of autosome. A significant percentage of SNPs was having minor allele frequency of less than 0.20. The extent of LD between autosomal SNPs up to 10 Mb apart across the genome was measured using r2 statistic. The mean r2 value was 0.43, if pairwise distance of marker was less than10 kb and it decreased further to 0.21 for 25–50 kb markers distance. Further, the effect of minor allele frequency and sample size on LD estimate was investigated. The LD value decreased with the increase in inter-marker distance, and increased with the increase of minor allelic frequency. The estimated inbreeding coefficient and effective population size were 0.04, and 46 for present generation, which indicated small and unstable population of Vrindavani cattle. These findings suggested that a denser or breed specific SNP panel would be required to cover all genome of Vrindavani cattle for genome wide association studies (GWAS).

SNP Development in Penaeus vannamei via Next-Generation Sequencing and DNA Pool Sequencing

Next-generation sequencing and pool sequencing have been widely used in SNP (single-nucleotide polymorphism) detection and population genetics research; however, there are few reports on SNPs related to the growth of Penaeus vannamei. The purpose of this study was to call SNPs from rapid-growing (RG) and slow-growing (SG) individuals’ transcriptomes and use DNA pool sequencing to assess the reliability of SNPs. Two parameters were applied to detect SNPs. One parameter was the p-values generated using Fisher’s exact test, which were used to calculate the significance of allele frequency differences between RG and SG. The other one was the AFI (minor allele frequency imbalance), which was defined to highlight the fold changes in MAF (minor allele frequency) values between RG and SG. There were 216,015 hypothetical SNPs, which were obtained based on the transcriptome data. Finally, 104 high-quality SNPs and 96,819 low-quality SNPs were predicted. Then, 18 high-quality SNPs and 17 low-quality SNPs were selected to assess the reliability of the detection process. Here, 72.22% (13/18) accuracy was achieved for high-quality SNPs, while only 52.94% (9/17) accuracy was achieved for low-quality SNPs. These SNPs enrich the data for population genetics studies of P. vannamei and may play a role in the development of SNP markers for future breeding studies.

Distribution of rare LOXL1 missense alleles, haplotypes and diplotypes suggests association with reduced risk of glaucoma-related exfoliation syndrome.

Purpose: Common LOXL1 protein-altering variants are significant genetic risk factors for exfoliation syndrome (XFS) and the related secondary glaucoma (XFG). A rare LOXL1 missense allele has been associated with protective effects in a Japanese cohort, suggesting that other rare alleles may also exhibit protective effects. The goal of this study was to assess the contributions of rare LOXL1 variants to XFS/XFG risk in cases and controls from the United States. Methods: LOXL1 rare (minor allele frequency less than 1%) variants were identified from Humanexome BeadArray (Illumina) data for 1118 XFS/XFG cases and 3661 controls. Distribution of rare variants, haplotypes (defined using IMPUTE2) and diplotypes were examined using the Fisher exact test. Rare variant allele distribution was confirmed in an independent set of primary open angle glaucoma (POAG) controls and multi-ethnic datasets. Correlation of LOXL1 common allele homozygosity with disease risk used data from gnomAD (gnomad.broadinstitute.org) and an existing multi-ethnic meta-analysis. Results: Four rare LOXL1 missense alleles were identified, and all were more common in controls (combined P= 7.6E-4), with two of these located in a LOXL1 intrinsic disordered region (IDR) known to be involved in LOXL1 aggregation. Haplotypes that included the rare or minor variants were more common in controls compared to cases (OR= 0.33, P=1.7E-8). Heterozygous diplotypes were significantly associated with reduced risk overall (OR= 0.45 P= 1.7 E-89) with the largest effects observed for diplotypes with more than one heterozygous genotype (OR= 0.05, P= 1.0E-39). A homozygous diplotype was associated with increased disease risk (OR= 6.8, P= 4.7E-157) and homozygosity was correlated with disease risk for common LOXL1 variants across multi-ethnic populations (Pearson= 0.92, P<0.001). Conclusions: Using exome array data from XFS/XFG cases and controls from the United States, we identify 4 rare protective LOXL1 missense variants and show that the distribution of the corresponding haplotypes and diplotypes are associated with reduced risk of XFS/XFG. The diplotype results also demonstrate that LOXL1 allelic heterozygosity is protective while homozygosity is associated with increased disease risk. These results suggest that LOXL1 minor allele frequency variation among populations, with corresponding variation in genotype heterozgyosity and homozygosity, determines the XFS/XFG association effects and that genotypic effects may also impact protein aggregation involving intrinsic disordered regions.

P–541 Identification of novel variants and candidate genes in women with familial idiopathic premature ovarian failure using whole-exome sequencing

Study question Is it possible to identify a genetic cause of familial premature ovarian failure (POF) with whole-exome sequencing (WES)? Summary answer Whole-exome sequencing is the most efficient strategy to identify probably pathogenic mutations in different genes in pathologies of polygenic etiology such as premature ovarian failure. What is known already Premature ovarian failure is the loss of ovarian function before the age of 40, and it is a common cause of infertility in women. This pathology has a heterogeneous etiology. Some chromosomal and genetic alterations have been described, and could explain approximately 20% of cases. However, in most patients the origin remains unknown. Recent studies with next-generation sequencing (NGS) have identified new variants in candidate genes related with premature ovarian insufficiency (POI) or premature ovarian failure (POF). These genes are not only involved in processes such as folliculogenesis, but also with DNA damage repair, homologous recombination, and meiosis. Study design, size, duration Fourteen women, from 7 families, affected by idiopathic POF were included in the study from October 2019 to September 2020. Seven POF patients were recruited when they came to our clinic to undergo assisted reproductive treatment. In the anamnesis, it was found that they had relatives with a diagnosis of POF, who were also recruited for the study. The inclusion criteria were amenorrhea before 38 years old and analytical and ultrasound signs of ovarian failure. Participants/materials, setting, methods WES was performed using TrusightOne (Illumina®). Sequenced data were aligned through BWA tool and GATK algorithm was used for SNVs/InDel identification. VCF files were annotated using Variant Interpreter software. Only the variants shared by each family were extracted for analysis and these criteria were followed: (1) Exonic/splicing variants in genes related with POF or involved in biological ovarian functions (2) Variants with minor allele frequency (MAF) ≤0.05 and (3) having potentially moderate/strong functional effects. Main results and the role of chance Seventy-nine variants possibly related with the POF phenotype were identified in the seven families. All these variants had a minor allele frequency (MAF) ≤0.05 in the gnomAD database and 1000 genomes project. Among these candidate variants, two were nonsense, six splice region, one frameshift, two inframe deletion and 68 missense. Thirty-two of the missense variants were predicted to have deleterious effects by minimum two of the four in silico algorithms used (SIFT, PolyPhen–2, MutationTaster and PROVEAN). All variants were heterozygous, and all the families carried three or more candidate variants. Altogether, 43 probably damaging genetic variants were identified in 39 genes expressed in the ovary and related with POF/POI or linked to ovarian physiology. We have described genes that have never been associated to POF pathology, however they may be involved in key biological processes for ovarian function. Moreover, some of these genes were found in two families, for example DDX11, VWF, PIWIL3 and HSD3B1. DDX11 may function at the interface of replication-coupled DNA repair and sister chromatid cohesion. VWF gene is suggested to be associated with follicular atresia in previous studies. PIWIL3 functions in development and maintenance of germline stem cells, and HSD3B1 is implicated in ovarian steroidogenesis. Limitations, reasons for caution Whole-exome sequencing has some limitations: does not cover noncoding regions of the genome, it also cannot detect large rearrangements, copy-number variants (large deletions/duplications), mosaic mutations, mutations in repetitive or high GC rich regions and mutations in genes with corresponding pseudogenes or other highly homologous sequences. Wider implications of the findings: WES has previously shown to be an efficient tool to identify genes as cause of POF, and has demonstrated the polygenic etiology. Although some studies have focused on it, and many genes are identified, this study proposes new candidate genes and variants, having potentially moderate/strong functional effects, associated with POF. Trial registration number Not applicable

MO275RARE VARIANTS OF COMPLEMENT FACTOR H AND THROMBOMODULIN ARE OVER-REPRESENTED IN A COHORT OF SEVERE IGA NEPHROPATHY PATIENTS

Background and Aims IgA nephropathy is the most frequent primary glomerulonephritis leading to end stage renal disease (ESRD) in about 30% of cases within 20 years after diagnosis. Complement activation through alternative and lectin pathways has been described to impact the pathogeny of the disease. We hypothesized in this study that rare variants of alternative pathways regulatory genes could be overrepresented and could play a role at initiating the disease and could harm the prognosis of IgA Nephropathy. Method Patients with biopsy proven IgA nephropathy with markers of severity comprising an evolution through ESRD and/or a proteinuria &gt;0.5g/day with available DNA sample were included. All coding sequences of CFH, CFI, MCP, C3, Factor B THBD and CFHR5 genes were analyzed by next generation sequencing. We defined a variant as rare when its minor allele frequency was below 0.1% in the general population. Frequencies were compared to a French volonteers cohort (n=80) and a European large cohort (n=503) Results We screened 128 patients with IgA N, with following characteristics at diagnosis: median age 42.4 yo, proteinuria (median) 1.4g/day, hypertension 66%, median eGFR 48.7 mL/min/1.73m². The median follow-up was 99 months and 58% of patients progressed to ESRD. We identified rare variants with MAF&lt;0.1% in 10.2 % (n=13) including 1 patient with two rare variants. The functional consequences of the 12 out the 14 variants are unknown. Two variants in CFH are located in function domains and are pathogenic. Patients with IgA N have high rates of rare variants in CFH (n=9/128 ; 7 %) versus normal controls (n=9/503 ; 1.8%) (p=0.004); Pathogenic Variants with minor allele frequency &lt;0.1% in CFH were found in 2 IgA N (2 out of 128, 1.5%) versus 1 European controls (1 out of 503) In total, 11 % (14/128), 3.8 % (5/128) and 0.8 % (1/128) of the 128 patients were homozygous for the at-risk haplotype MCP ggaac, CFH tgtgt or both, respectively (versus 6.2 % (5/80), 3.8 % (3/80) and 0% in the controls) 6 patients carried the pathogenic variant in THDM gene p.Ala43Thr (6/128) versus 5 in 508 controls population (p=0.01). No difference in term of hypertension, proteinuria, eGFR, Oxford classification, vascular score at diagnosis was noticed between patients without any rare variant compared to patients with at least one rare variant. The progression through ESRD was not different between groups. Conclusion In this cohort of Caucasian IgA nephropathy patients, rare variants of CFH and THBD were found significantly overrepresented compared to a French and European control cohort. Rare variants of alternative pathway regulatory genes were not associated with particular severity or prognosis.

Association of NOS3 Genetic Polymorphism With the Predisposition to Diabetes and Pre-Diabetes, Retrospective Study

Background: Endothelial nitric oxide synthetase (eNOS) encoded by NOS3 gene has an important role in modulating vascular endothelial function. Many studies reported a possible role of NOS3 in the pathogenesis of diabetes mellitus (DM). This study investigated the association of NOS3 (G&gt;T) rs1799983 genetic polymorphism with DM, pre-diabetes (pre-DM), and insulin resistance (IR). Methods: A random sample of 220 subjects (DM & pre-DM) compared with 220 healthy subjects. Sample obtained from Palestinian adults who consented to genetic and biochemical testing. All subjects genotyped for NOS3 (G &gt; T) rs1799983 SNP using ARMS PCR. Fasting blood sugar (FBS) and triglyceride (TGA) levels were obtained for all subjects. Triglyceride glucose index (TyG) was used as a surrogate marker for IR. Regression analysis adjusted for age and body mass index (BMI) was performed to investigate the association between DM & Pre-DM status, FBS, and TyG with NOS3 genetic polymorphism. Results: NOS3 minor allele frequency positively correlated with FBS levels after controlling for age and BMI (P-value 0.006). DM & pre-DM were more frequent in homozygous NOS3 subjects with an odds ratio of 2.04 (P = 0.05). NOS3 minor allele frequency positively correlated with TyG but not statistically significant association (P = 0.061). Discussion: Many studies reported a potential role of NOS3 genetic polymorphism in DM and IR pathogenesis. In this study, NOS3 minor allele frequency positivity correlated with FBS levels. Homozygous NOS3 was associated with a 2-fold increase in the prevalence of DM & pre-DM. NOS3 genetic polymorphism didn’t show a statistically significant correlation with TyG (P = 0.061). With the increasing availability of genetic testing, NOS3 may serve as an early screening tool to identify subjects with a high risk for elevated FBS. Further studies are required to understand the exact role of NOS3 genetic polymorphism in the pathogenesis of DM, and to evaluate the clinical efficacy and cost-effectiveness of genetic testing. Conclusion: NOS3 genetic polymorphism has a statistically significant relationship with the FBS level. Further studies are required to confirm the association between NOS3 and DM.

The genetic architecture of medication-use

Genomics has been forecasted to revolutionise human health by improving medical treatment through a better understanding of the molecular mechanisms of human diseases. Despite great successes of the last decade’s genome-wide association studies (GWAS), the results have to a limited extent been translated to genomic medicine. We propose, that one route to get closer to improved medical treatment is by understanding the genetics of medication-use. Here we obtained entire medication profiles from 335,744 individuals from the UK Biobank and performed a GWAS to identify which common genetic variants are major drivers of medication-use. We analysed 9 million imputed genetic variants, estimated SNP heritability, partitioned the genomic variance across functional categories, and constructed genetic scores for medication-use. In total, 59 independent loci were identified for medication-use and approximately 18% of the total variation was attributable to common genetic (minor allele frequency >0.01) variants. The largest fraction of variance was captured by variants with low to medium minor allele frequency. In particular coding and conserved regions, as well as transcription start sites, displayed significantly enrichment of heritability. The average correlation between medication-use and predicted genetic scores was 0.14. These results demonstrate that medication-use per se is a highly polygenic complex trait and that individuals with higher genetic liability are on average more diseased and have a higher risk for adverse drug reactions. These results provide an insight into the genetic architecture of medication use and pave the way for developments of multicomponent genetic risk models that includes the genetically informed medication-use.

Molecular Screening of PROKR2 Gene in Girls with Idiopathic Central Precocious Puberty.

Background: Prokineticin receptor 2 (PROKR2) loss of function mutations have been described as cause of hypogonadotropic hypogonadism. In 2017 a first case of central precocious puberty (CPP) caused by PROKR2 heterozygous gain of function mutation was described in a 3.5-year-old girl. No other cases have been reported yet. This study performs a molecular screening in girls with early onset CPP (breast budding before 6 years of age) in order to identify possible alterations in PROKR2. Methods: We analyzed DNA of 31 girls with idiopathic CPP diagnosed via basal LH levels >0.3 IU/L or peak-LH>5IU/L after stimulation, negative for MKRN3 mutations. The Fisher exact test was used to compare allele frequency of polymorphism found to genome aggregation database (gnomAD).Results: No rare variants were identified. Five polymorphisms were found (rs6076809, rs8116897, rS3746684, rs3746682, rs3746683). All except one (i.e. rs3746682) had a minor allele frequency similar to that reported in literature. rs3746682 presented a minor allele frequency higher than described in the gnomAD (0.84 in our population vs 0.25 from gnomAD). Conclusions: As for other G-protein-coupled receptors (i.e. GPR54), mutations in PROKR2 do not seem to be a frequent cause of CPP in girls.

Regarding the F-word: the effects of data Filtering on inferred genotype-environment associations

Genotype-environment association (GEA) methods have become part of the standard landscape genomics toolkit, yet, we know little about how to filter genotype-by-sequencing data to provide robust inferences for environmental adaptation. In many cases, default filtering thresholds for minor allele frequency and missing data are applied regardless of sample size, having unknown impacts on the results. These effects could be amplified in downstream predictions, including management strategies. Here, we investigate the effects of filtering on GEA results and the potential implications for adaptation to environment. Using empirical and simulated datasets derived from two widespread tree species to assess the effects of filtering on GEA outputs. Critically, we find that the level of filtering of missing data and minor allele frequency affect the identification of true positives. Even slight adjustments to these thresholds can change the rate of true positive detection. Using conservative thresholds for missing data and minor allele frequency substantially reduces the size of the dataset, lessening the power to detect adaptive variants (i.e. simulated true positives) with strong and weak strength of selections. Regardless, strength of selection was a good predictor for GEA detection, but even SNPs under strong selection went undetected. We further show that filtering can significantly impact the predictions of adaptive capacity of species in downstream analyses. We make several recommendations regarding filtering for GEA methods. Ultimately, there is no filtering panacea, but some choices are better than others, depending largely on the study system, availability of genomic resources, and desired objectives of the study.

Mutation profile of over 4500 SARS-CoV-2 isolations reveals prevalent cytosine-to-uridine deamination on viral RNAs

Aim: The inference of coronavirus evolution is largely based on mutations in SARS-CoV-2 genome. Misinterpretation of these mutations would mislead people about the evolution of SARS-CoV-2. Materials & methods: With 4521 lines of SARS-CoV-2, we obtained 3169 unique point mutation sites. We counted the numbers and calculated the minor allele frequency (MAF) of each mutation type. Results: Nearly half of the point mutations are C–T mismatches and 20% are A–G mismatches. The MAF of C–T and A–G mismatches is significantly higher than MAF of other mutation types. Conclusion: The excessive C–T mismatches do not resemble the random mutation profile. They are likely to be caused by the cytosine-to-uridine deamination system in hosts.