Exploring the gonad transcriptome of two extreme male pigs with RNA-seq

Background: Although RNA-seq greatly advances our understanding of complex transcriptome landscapes, such as those found in mammals, complete RNA-seq studies in livestock and in particular in the pig are still lacking. Here, we used high-throughput RNA sequencing to gain insight into the characterization of the poly-A RNA fraction expressed in pig male gonads. An expression analysis comparing different mapping approaches and detection of allele specific expression is also discussed in this study. Results: By sequencing testicle mRNA of two phenotypically extreme pigs, one Iberian and one Large White, we identified hundreds of unannotated protein-coding genes (PcGs) in intergenic regions, some of them presenting orthology with closely related species. Interestingly, we also detected 2047 putative long non-coding RNA (lncRNA), including 469 with human homologues. Two methods, DEGseq and Cufflinks, were used for analyzing expression. DEGseq identified 15% less expressed genes than Cufflinks, because DEGseq utilizes only unambiguously mapped reads. Moreover, a large fraction of the transcriptome is made up of transposable elements (14500 elements encountered), as has been reported in previous studies. Gene expression results between microarray and RNA-seq technologies were relatively well correlated (r = 0.71 across individuals). Differentially expressed genes between Large White and Iberian showed a significant overrepresentation of gamete production and lipid metabolism gene ontology categories. Finally, allelic imbalance was detected in ~ 4% of heterozygous sites. Conclusions: RNA-seq is a powerful tool to gain insight into complex transcriptomes. In addition to uncovering many unnanotated genes, our study allowed us to determine that a considerable fraction is made up of long non-coding transcripts and transposable elements. Their biological roles remain to be determined in future studies. In terms of differences in expression between Large White and Iberian pigs, these were largest for genes involved in spermatogenesis and lipid metabolism, which is consistent with phenotypic extreme differences in prolificacy and fat deposition between these two breeds

Methylenetetrahydrofolate reductase C677T polymorphism in patients with gastric and colorectal cancer in a Korean population

<p>Abstract</p> <p>Background</p> <p>This study was designed to investigate an association between the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism and the risk of gastric and colorectal cancer in the Korean population.</p> <p>Methods</p> <p>We conducted a population-based large-scale case-control study involving 2,213 patients with newly diagnosed gastric cancer, 1,829 patients with newly diagnosed colorectal cancer, and 1,700 healthy controls. Genotyping was performed with peripheral blood DNA for MTHFR C677T polymorphisms. The statistical significance was estimated by logistic regression analysis.</p> <p>Results</p> <p>The MTHFR C677T frequencies of CC, CT, and TT genotypes were 35.2%, 47.5%, and 17.3% among stomach cancer, 34%, 50.5%, and 15.5% in colorectal cancer, and 31.8%, 50.7%, and 17.5% in the controls, respectively. The MTHFR 677TT genotype showed a weak opposite association with colorectal cancer compared to the homozygous CC genotype [adjusted age and sex odds ratio (OR) = 0.792, 95% confidence interval (CI) = 0.638-0.984, <it>P </it>= 0.035]. Subjects with the MTHFR 677CT showed a significantly reduced risk of gastric cancer compared whose with the 677CC genotype (age- and sex-adjusted OR = 0.810; 95% CI = 0.696-0.942, <it>P </it>= 0.006). We also observed no significant interactions between the MTHFR C677T polymorphism and smoking or drinking in the risk of gastric and colorectal cancer.</p> <p>Conclusions</p> <p>The T allele was found to provide a weak protective association with gastric cancer and colorectal cancer.</p

Global assessment of genomic variation in cattle by genome resequencing and high-throughput genotyping

<p>Abstract</p> <p>Background</p> <p>Integration of genomic variation with phenotypic information is an effective approach for uncovering genotype-phenotype associations. This requires an accurate identification of the different types of variation in individual genomes.</p> <p>Results</p> <p>We report the integration of the whole genome sequence of a single Holstein Friesian bull with data from single nucleotide polymorphism (SNP) and comparative genomic hybridization (CGH) array technologies to determine a comprehensive spectrum of genomic variation. The performance of resequencing SNP detection was assessed by combining SNPs that were identified to be either in identity by descent (IBD) or in copy number variation (CNV) with results from SNP array genotyping. Coding insertions and deletions (indels) were found to be enriched for size in multiples of 3 and were located near the N- and C-termini of proteins. For larger indels, a combination of split-read and read-pair approaches proved to be complementary in finding different signatures. CNVs were identified on the basis of the depth of sequenced reads, and by using SNP and CGH arrays.</p> <p>Conclusions</p> <p>Our results provide high resolution mapping of diverse classes of genomic variation in an individual bovine genome and demonstrate that structural variation surpasses sequence variation as the main component of genomic variability. Better accuracy of SNP detection was achieved with little loss of sensitivity when algorithms that implemented mapping quality were used. IBD regions were found to be instrumental for calculating resequencing SNP accuracy, while SNP detection within CNVs tended to be less reliable. CNV discovery was affected dramatically by platform resolution and coverage biases. The combined data for this study showed that at a moderate level of sequencing coverage, an ensemble of platforms and tools can be applied together to maximize the accurate detection of sequence and structural variants.</p

Spermatozoal sensitive biomarkers to defective protaminosis and fragmented DNA

Human sperm DNA damage may have adverse effects on reproductive outcome. Infertile men possess substantially more spermatozoa with damaged DNA compared to fertile donors. Although the extent of this abnormality is closely related to sperm function, the underlying etiology of ensuing male infertility is still largely controversial. Both intra-testicular and post-testicular events have been postulated and different mechanisms have been proposed to explain the presence of damaged DNA in human spermatozoa. Three among them, i.e. abnormal chromatin packaging, oxidative stress and apoptosis, are the most studied and discussed in the present review. Furthermore, results from numerous investigations are presented, including our own findings on these pathological conditions, as well as the techniques applied for their evaluation. The crucial points of each methodology on the successful detection of DNA damage and their validity on the appraisal of infertile patients are also discussed. Along with the conventional parameters examined in the standard semen analysis, evaluation of damaged sperm DNA seems to complement the investigation of factors affecting male fertility and may prove an efficient diagnostic tool in the prediction of pregnancy outcome

Genome-wide association studies of cancer: current insights and future perspectives.

Genome-wide association studies (GWAS) provide an agnostic approach for investigating the genetic basis of complex diseases. In oncology, GWAS of nearly all common malignancies have been performed, and over 450 genetic variants associated with increased risks have been identified. As well as revealing novel pathways important in carcinogenesis, these studies have shown that common genetic variation contributes substantially to the heritable risk of many common cancers. The clinical application of GWAS is starting to provide opportunities for drug discovery and repositioning as well as for cancer prevention. However, deciphering the functional and biological basis of associations is challenging and is in part a barrier to fully unlocking the potential of GWAS

Concomitant Aficamten and Disopyramide in Symptomatic Obstructive Hypertrophic Cardiomyopathy

Background Disopyramide, used in obstructive hypertrophic cardiomyopathy (oHCM) for its negative inotropic properties mediated by its reduction in cytosolic calcium, has been recommended for decades as an option to relieve resistant obstruction. Aficamten is a selective cardiac myosin inhibitor that reduces hypercontractility directly by reducing myosin-actin interaction. Objectives This study aims to investigate the safety and efficacy of concomitant use and withdrawal of disopyramide in patients with symptomatic oHCM receiving aficamten. Methods Patients with oHCM enrolled in REDWOOD-HCM Cohort 3 (open-label), SEQUOIA-HCM (placebo-controlled), and FOREST-HCM (open-label) were analyzed. The authors identified 4 groups, each with patients symptomatic despite background therapy with disopyramide who received: 1) disopyramide plus aficamten and subsequent aficamten withdrawal per protocol (Diso-Afi Withdrawal); 2) disopyramide plus placebo (Diso-Pbo); 3) aficamten plus disopyramide with subsequent disopyramide withdrawal (Afi-Diso Withdrawal); and 4) continued both disopyramide and aficamten (Diso+Afi Continuous). Assessments were performed at baseline, after aficamten or placebo add-on therapy, and after washout (except at week 24 for Diso+Afi Continuous group). Results Overall, 50 unique patients from 3 trials enrolled, resulting in 93 subjects (segments) across 4 groups: Diso-Afi Withdrawal (n = 29), Diso-Pbo (n = 20), Afi-Diso Withdrawal (n = 17), and Diso+Afi Continuous (n = 27); mean disopyramide dose was 331 ± 146 mg/d. The addition of aficamten to disopyramide alleviated left ventricular outflow tract (LVOT) obstruction (resting: change [Δ] in least squares mean −27.0 ± 3.6, Valsalva: Δ least squares mean −39.2 ± 5.0, both P < 0.0001), symptoms (≥1 NYHA functional class improvement: 77.8% [95% CI: 61.0-94.5]; P < 0.0001; Kansas City Cardiomyopathy Questionnaire–Clinical Summary Score: 12.3 ± 3.3 [P < 0.001]), and reduced N-terminal pro–B-type natriuretic peptide ratio: 0.35 [95% CI: 0.26-0.48]; P < 0.0001, and there was no significant change with placebo. Withdrawal of aficamten while on disopyramide resulted in return of LVOT obstruction, worsening of symptoms, and increase in NT-proBNP to baseline values. Conversely, withdrawal of disopyramide while on aficamten did not impact efficacy. There were no safety events associated with aficamten or disopyramide withdrawal, and no episodes of atrial fibrillation after disopyramide withdrawal. Conclusions In this cohort of patients with symptomatic oHCM with persistent LVOT obstruction, combination therapy with aficamten and disopyramide was safe and well tolerated but did not enhance clinical efficacy vs aficamten alone. For such oHCM patients, aficamten treatment may be considered with an option to discontinue disopyramide. (Dose-finding Study to Evaluate the Safety, Tolerability, PK, and PD of CK-3773274 in Adults With HCM [REDWOOD-HCM]; NCT04219826) (Aficamten vs Placebo in Adults With Symptomatic Obstructive Hypertrophic Cardiomyopathy [SEQUOIA-HCM]; NCT05186818) (Open-label Extension Study to Evaluate the Long-term Safety and Tolerability of Aficamten in Adults With HCM [FOREST-HCM]; NCT04848506

Aficamten and Cardiopulmonary Exercise Test Performance

Importance Impaired exercise capacity is a cardinal manifestation of obstructive hypertrophic cardiomyopathy (HCM). The Phase 3 Trial to Evaluate the Efficacy and Safety of Aficamten Compared to Placebo in Adults With Symptomatic Obstructive HCM (SEQUOIA-HCM) is a pivotal study characterizing the treatment effect of aficamten, a next-in-class cardiac myosin inhibitor, on a comprehensive set of exercise performance and clinical measures. Objective To evaluate the effect of aficamten on exercise performance using cardiopulmonary exercise testing with a novel integrated measure of maximal and submaximal exercise performance and evaluate other exercise measures and clinical correlates. Design, Setting, and Participants This was a prespecified analysis from SEQUOIA-HCM, a double-blind, placebo-controlled, randomized clinical trial. Patients were recruited from 101 sites in 14 countries (North America, Europe, Israel, and China). Individuals with symptomatic obstructive HCM with objective exertional intolerance (peak oxygen uptake [pVO2] ≤90% predicted) were included in the analysis. Data were analyzed from January to March 2024. Interventions Randomized 1:1 to aficamten (5-20 mg daily) or matching placebo for 24 weeks. Main Outcomes and Measures The primary outcome was change from baseline to week 24 in integrated exercise performance, defined as the 2-component z score of pVO2 and ventilatory efficiency throughout exercise (minute ventilation [VE]/carbon dioxide output [VCO2] slope). Response rates for achieving clinically meaningful thresholds for change in pVO2 and correlations with clinical measures of treatment effect (health status, echocardiographic/cardiac biomarkers) were also assessed. Results Among 282 randomized patients (mean [SD] age, 59.1 [12.9] years; 115 female [40.8%], 167 male [59.2%]), 263 (93.3%) had core laboratory–validated exercise testing at baseline and week 24. Integrated composite exercise performance improved in the aficamten group (mean [SD] z score, 0.17 [0.51]) from baseline to week 24, whereas the placebo group deteriorated (mean [SD] z score, −0.19 [0.45]), yielding a placebo-corrected improvement of 0.35 (95% CI, 0.25-0.46; P &amp;amp;lt;.001). Further, aficamten treatment demonstrated significant improvements in total workload, circulatory power, exercise duration, heart rate reserve, peak heart rate, ventilatory efficiency, ventilatory power, and anaerobic threshold (all P &amp;amp;lt;.001). In the aficamten group, large improvements (≥3.0 mL/kg per minute) in pVO2 were more common than large reductions (32% and 2%, respectively) compared with placebo (16% and 11%, respectively). Improvements in both components of the primary outcome, pVO2 and VE/VCO2 slope throughout exercise, were significantly correlated with improvements in symptom burden and hemodynamics (all P &amp;amp;lt;.05). Conclusions and Relevance This prespecified analysis of the SEQUOIA-HCM randomized clinical trial found that aficamten treatment improved a broad range of exercise performance measures. These findings offer valuable insight into the therapeutic effects of aficamten. Trial Registration ClinicalTrials.gov Identifier: NCT0518681