Development and Validation of Kompetitive Allele-Specific PCR Assays for Erucic Acid Content in Indian Mustard [Brassica juncea (L.) Czern and Coss.]

Brassica juncea L. is the most widely cultivated oilseed crop in Indian subcontinent. Its seeds contain oil with very high concentration of erucic acid (≈50%). Of late, there is increasing emphasis on the development of low erucic acid varieties because of reported association of the consumption of high erucic acid oil with cardiac lipidosis. Erucic acid is synthesized from oleic acid by an elongation process involving two cycles of four sequential steps. Of which, the first step is catalyzed by β-ketoacyl-CoA synthase (KCS) encoded by the fatty acid elongase 1 (FAE1) gene in Brassica. Mutations in the coding region of the FAE1 lead to the loss of KCS activity and consequently a drastic reduction of erucic acid in the seeds. Molecular markers have been developed on the basis of variation available in the coding or promoter region(s) of the FAE1. However, majority of these markers are not breeder friendly and are rarely used in the breeding programs. Present studies were planned to develop robust kompetitive allele-specific PCR (KASPar) assays with high throughput and economics of scale. We first cloned and sequenced FAE1.1 and FAE1.2 from high and low erucic acid (<2%) genotypes of B. juncea (AABB) and its progenitor species, B. rapa (AA) and B. nigra (BB). Sequence comparisons of FAE1.1 and FAE1.2 genes for low and high erucic acid genotypes revealed single nucleotide polymorphisms (SNPs) at 8 and 3 positions. Of these, three SNPs for FAE1.1 and one SNPs for FAE1.2 produced missense mutations, leading to amino acid modifications and inactivation of KCS enzyme. We used SNPs at positions 735 and 1,476 for genes FAE1.1 and FAE1.2, respectively, to develop KASPar assays. These markers were validated on a collection of diverse genotypes and a segregating backcross progeny. KASPar assays developed in this study will be useful for marker-assisted breeding, as these can track recessive alleles in their heterozygous state with high reproducibility.

Polymorphisms of Pro-Inflammatory IL-6 and IL-1β Cytokines in Ascending Aortic Aneurysms as Genetic Modifiers and Predictive and Prognostic Biomarkers

Background: Previous studies have demonstrated that polymorphisms involved in immune genes can affect the risk, pathogenesis, and outcome of thoracic ascending aortic aneurysms (TAAA). Here, we explored the potential associations of five functional promoter polymorphisms in interleukin-6 (IL-6), IL-1B, IL-1A, IL-18, and Tumor necrosis factor (TNF)A genes with TAAA. Methods: 144 TAAA patients and 150 age/gender matched controls were typed using KASPar assays. Effects on telomere length and levels of TAAA related histopathological and serological markers were analyzed. Results: Significant associations with TAAA risk were obtained for IL-6 rs1800795G>C and IL-1B rs16944C>T SNPs. In addition, the combined rs1800795C/rs16944T genotype showed a synergic effect on TAAA pathogenesis and outcome. The combined rs1800795C/rs16944T genotype was significantly associated with: (a) higher serum levels of both cytokines and MMP-9 and -2; (b) a significant CD3+CD4+CD8+ CD68+CD20+ cell infiltration in aorta aneurysm tissues; (c) a significant shorter telomere length and alterations in telomerase activity. Finally, it significantly correlated with TAAA aorta tissue alterations, including elastic fragmentation, medial cell apoptosis, cystic medial changes, and MMP-9 levels. Conclusions: the combined rs1800795C/rs16944T genotype appears to modulate TAAA risk, pathogenesis, and outcome, and consequently can represent a potential predictive and prognostic TAAA biomarker for individual management, implementation of innovative treatments, and selection of the more proper surgical timing and approaches.

Association of the ACE rs4646994 and rs4341 polymorphisms with the progression of carotid atherosclerosis in slovenian patients with type 2 diabetes mellitus

The current study was designed to reveal possible associations between the angiotensin-converting-enzyme (ACE) gene polymorphisms (rs4646994 and rs4341) with markers of carotid atherosclerosis in patients with type 2 diabetes mellitus (T2DM) in a 4-year-long follow-up study. Five hundred and ninety-five T2DM subjects and 200 control subjects were enrolled. Genotyping of ACE polymorphisms was performed using KASPar assays, and ultrasound examinations were performed twice (at the enrollment and at follow-up). With regard to the progression of atherosclerosis in subjects with T2DM, statistically significant differences were demonstrated in the change of the sum of carotid plaques thickness for the rs4646994 polymorphism. We did not demonstrate an association between the tested polymorphisms (rs4646994 and rs4341) and either carotid intima media thickness (CIMT) or CIMT progression in a 3.8-year period. In our study, we demonstrated that subjects with T2DM with the DD genotype of the rs4646994 [ACE insertion/deletion (I/D)] polymorphism had faster progression of atherosclerosis in comparison to subjects with other genotypes.