PDE4B Proposed as a High Myopia Susceptibility Gene in Chinese Population

Myopia is the most common cause of refractive error worldwide. High myopia is a severe type of myopia, which usually accompanies pathological changes in the fundus. To identify high myopia susceptibility genes, DNA-pooling based genome-wide association analysis was used to search for a correlation between single nucleotide polymorphisms and high myopia in a Han Chinese cohort (cases vs. controls in discovery stage: 507 vs. 294; replication stage 1: 991 vs. 1,025; replication stage 2: 1,021 vs. 52,708). Three variants (rs10889602T/G, rs2193015T/C, rs9676191A/C) were identified as being significantly associated with high myopia in the discovery, and replication stage. rs10889602T/G is located at the third intron of phosphodiesterase 4B (PDE4B), whose functional assays were performed by comparing the effects of rs10889602T/T deletion of this risk allele on PDE4B and COL1A1 gene and protein expression levels in the rs10889602T/Tdel/del, rs10889602T/Tdel/wt, and normal control A549 cell lines. The declines in the PDE4B and COL1A1 gene expression levels were larger in the rs10889602T/T deleted A549 cells than in the normal control A549 cells (one-way ANOVA, p < 0.001). The knockdown of PDE4B by siRNA in human scleral fibroblasts led to downregulation of COL1A1. This correspondence between the declines in rs10889602 of the PDE4B gene, PDE4B knockdown, and COL1A1 protein expression levels suggest that PDE4B may be a novel high myopia susceptibility gene, which regulates myopia progression through controlling scleral collagen I expression levels. More studies are needed to determine if there is a correlation between PDE4B and high myopia in other larger sample sized cohorts.

168 Use of Genomic Tools to Improve Production Efficiency, Health Resilience and Carbon Footprint of Beef Production

The aim is to present validation studies that demonstrate the benefits of genomic retained heterozygosity, genomic enhanced expected progeny differences (gEPDs) for feed efficiency and carcass traits, as well as DNA pooling technologies, to the beef industry. Team members of Livestock Gentec are global leaders in beef genomics research as evidenced by their leadership roles on the Canadian Cattle Genome Project, 1,000 Bull Genomes Project, gEPDs for Commercial Cattle Project and the Functional Annotation of ANimal Genomes initiative. These large-scale projects have created databases of 380 whole bovine sequence genomes, >24,000 cattle genotypes imputed to sequence variants using Run 6 genotypes from 1000 Bull Genomes project, and >20,000 cattle with associated phenotypes for feed efficiency, carcass quality, cow fertility and methane emissions. The use of admixture analysis, genome wide association studies, and genomic prediction have resulted in new genomic tools that aid in mate selection, improve herd heterosis, female fertility, lifetime productivity and health resilience, and improve accuracy (acc. >0.36) of gEPDs for 18 traits in crossbred cattle. Genomic retained heterozygosity has a benefit of $161/female over five parities while decreasing morbidity of calves and improving the carbon intensity of beef production. Multi-trait selection studies using gEPDs for residual feed intake (acc. > 0.35) have demonstrated annual rates of genetic progress of 0.7%. Validation studies have reported that sires with superior gEPDs for increased marbling, decreased grade fat, increased ribeye and increased carcass weight (acc. > 0.45) produced progeny with improved AAA retail cut yield (59.9 vs 56.7%). DNA pooling shows potential for cheaper genotyping while providing information on pooled records related to sire contribution, heterosis and performance as influenced by genetics. The application of these genomic tools has potential to improve calf crop percentage, health resilience, and retail cut yield while decreasing the carbon footprint of beef production.

Resource-efficient pooled sequencing expands translational impact in solid tumors

Intratumoral genetic heterogeneity (ITH) poses a significant challenge to utilizing sequencing for decision making in the management of cancer. Although sequencing of multiple tumor regions can address the pitfalls of ITH, it does so at a significant increase in cost and resource utilization. We propose a pooled multiregional sequencing strategy, whereby DNA aliquots from multiple tumor regions are mixed prior to sequencing, as a cost-effective strategy to boost translational value by addressing ITH while preserving valuable residual tissue for secondary analysis. Focusing on kidney cancer, we demonstrate that DNA pooling from as few as two regions significantly increases mutation detection while reducing clonality misattribution. This leads to an increased fraction of patients identified with therapeutically actionable mutations, improved patient risk stratification, and improved inference of evolutionary trajectories with an accuracy comparable to bona fide multiregional sequencing. The same approach applied to non-small-cell lung cancer data substantially improves tumor mutational burden (TMB) detection. Our findings demonstrate that pooled DNA sequencing strategies are a cost-effective alternative to address intrinsic genetic heterogeneity in clinical settings.

Resource-efficient pooled sequencing expands translational impact in solid tumors

Intratumoral genetic heterogeneity (ITH) poses a significant challenge to utilizing sequencing for decision making in the management of cancer. Although sequencing of multiple tumor regions can address the pitfalls of ITH, it does so at a significant increase in cost and resource utilization. We propose a pooled multiregional sequencing strategy, whereby DNA aliquots from multiple tumor regions are mixed prior to sequencing, as a cost-effective strategy to boost translational value by addressing ITH while preserving valuable residual tissue for secondary analysis. Focusing on kidney cancer, we demonstrate that DNA pooling from as few as two regions significantly increases mutation detection while reducing clonality misattribution. This leads to an increased fraction of patients identified with therapeutically actionable mutations, improved patient risk stratification, and improved inference of evolutionary trajectories with an accuracy comparable to bona fide multiregional sequencing. The same approach applied to non-small-cell lung cancer data substantially improves tumor mutational burden (TMB) detection. Our findings demonstrate that pooled DNA sequencing strategies are a cost-effective alternative to address intrinsic genetic heterogeneity in clinical settings.

MLIP genotype as a predictor of pharmacological response in primary open-angle glaucoma and ocular hypertension

Predicting the therapeutic response to ocular hypotensive drugs is crucial for the clinical treatment and management of glaucoma. Our aim was to identify a possible genetic contribution to the response to current pharmacological treatments of choice in a white Mediterranean population with primary open-angle glaucoma (POAG) or ocular hypertension (OH). We conducted a prospective, controlled, randomized, partial crossover study that included 151 patients of both genders, aged 18 years and older, diagnosed with and requiring pharmacological treatment for POAG or OH in one or both eyes. We sought to identify copy number variants (CNVs) associated with differences in pharmacological response, using a DNA pooling strategy of carefully phenotyped treatment responders and non-responders, treated for a minimum of 6 weeks with a beta-blocker (timolol maleate) and/or prostaglandin analog (latanoprost). Diurnal intraocular pressure reduction and comparative genome wide CNVs were analyzed. Our finding that copy number alleles of an intronic portion of the MLIP gene is a predictor of pharmacological response to beta blockers and prostaglandin analogs could be used as a biomarker to guide first-tier POAG and OH treatment. Our finding improves understanding of the genetic factors modulating pharmacological response in POAG and OH, and represents an important contribution to the establishment of a personalized approach to the treatment of glaucoma.

Genetic analysis of dormancy and shattering traits in the backcross inbred lines derived from Oryza sativa cv. Swarna / O. nivara Ac. CR100008

Seed dormancy and shattering are important traits influencing the economics of rice farming. The genetic basis of dormancy and shattering traits were investigated in 174 Backcross Inbred Lines (BILs) derived from Oryza sativa cv. Swarna and O. nivara ac. CR100008. Significant variation was observed among the BILs for dormancy and shattering traits. Dormancy of 4-40 days was observed among BILs harvested at 35 days after heading and all the BILs attained > 80% germination by 6th week. Among all the BILs, least dormancy period (4 days) was found in SN-1, 13, 23, 25 and SN-28. Highest dormancy period (40 days) was found in 4 BILs i.e., SN-108, SN-116, SN-117 and SN-122 (40 days). None of the BILs were found to have non-shattering trait, while 2 BILs (SN-38 and SN-163) showed low shattering and 18 BILs were found with very high grain shattering percent. Of the 312 SSRs screened, 94 were polymorphic between the parents. A strategy of combining the DNA pooling from phenotypic extremes and genotyping was employed to detect the putative markers associated with dormancy and shattering traits. Single marker analysis revealed co-segregation of two putative markers RM488 on chromosome 1 and RM247 on chromosome 12 were with dormancy and shattering traits respectively. The putative marker RM488 identified is suitable for the marker-assisted transfer of the dormancy shown by O. nivara accession CR100008 for addressing pre harvest sprouting in modern cultivars. Interestingly, O. nivara type allele at RM247 was observed in BILs with low shattering phenotype.

Selection and implementation of SNP markers for parentage analysis in a Chinese crossbred cattle population

Combining direct sequencing method in polymerase chain reaction (PCR) products of deoxyribonucleic acid (DNA) pooling and matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS) genotyping method in individuals, a panel consisting of 50 highly informative single nucleotide polymorphisms (SNPs) for parentage analysis was developed in a crossbred Chinese cattle population. The average minor allele frequency (MAF) was 0.43 and the cumulative exclusion probability for single-parent and both-parent inference met 0.99797 and 0.999999, respectively. The maker-set was then used for parentage verification in a group of 81 trios with the likelihood-based parentage-assignment program of Cervus software. Compared with on-farm records, the results showed that this 50-SNP system could provide sufficient and reliable information for parentage testing with the parental mistakes for mother-offspring and sire-offspring being 8.6% and 18.5%, respectively. Knowledge of these results, we provided one low-cost and efficient method of SNP assays for running paternity testing in crossbred cattle population of Simmental and Holstein in China.

Association of Polymorphisms in Candidate Genes with the Litter Size in Two Sheep Breeds

Hu sheep and Small-tailed Han sheep are the most widely raised and most famous maternal sheep breeds in China, which are known for precocious puberty, perennial oestrus and high fecundity (1–6 lambs each parity). Therefore, it is crucial to increase litter size of these two breeds for intensive sheep industry. The objective of this study was to identify potential genetic markers linked with sheep litter size located at ten genes. This study collected blood sample of 537 Hu sheep and 420 Small-tailed Han sheep with litter size of first parity. The average litter sizes in Hu sheep and Small-tailed Han sheep were 2.21 and 1.93. DNA-pooling sequencing method was used for detecting the potential single nucleotide polymorphisms (SNPs) in ten genes related to follicle development and female reproduction. SNPscan® was used for individually genotyping. As a result, a total of 78 putative SNPs in nine out of ten candidate genes (except NOG) were identified. In total, 50 SNPs were successfully genotyped in Hu sheep and Small-tailed Han sheep. After quality control, a total of 42 SNPs in Hu sheep and 44 SNPs in Small-tailed Han sheep were finally used for further analysis. Association analysis revealed that nine SNPs within six genes (KIT: g.70199073A>G, KITLG: g.124520653G>C, ADAMTS1: g.127753565T>C, ADAMTS1: g.127754640G>T, NCOA1: g.31928165C>T, NCOA1: g.32140565G>A, LIFR: g.35862868C>T, LIFR: g.35862947G>T and NGF: g.91795933T>C) were significantly associated with litter size in Hu sheep or Small-tailed Han sheep. A combined haplotypes analysis of the two loci (LIFR: g.35862868C>T and LIFR: g.35862947G>T) revealed that H2H3 (CTTT) combined haplotypes had the largest litter size than the rest combined haplotypes and more than those with either mutation alone in Small-tailed Han sheep. Taken together, our study suggests that nine significant SNPs in six genes can be served as useful genetic markers for MAS in sheep.