Two new RHD alleles with deletions spanning multiple exons.

BACKGROUND: The most common large-deletion RHD allele (RHD*01N.01) includes the entire coding sequence, intervening regions and untranslated regions. The rest of large-deletion RHD alleles reported to-date consist of single-exon deletions, such as RHD*01N.67 which includes exon 1. MATERIALS AND METHODS: Samples from two donors with RhD-negative serology yielded unclear or inconclusive results when subject to confirmatory testing on RHD genotyping arrays. To determine their RHD genotypes, genomic DNA was analyzed with a combination of allele-specific PCR, long-range PCR, Sanger sequencing, and next-generation sequencing assays. RESULTS: Allele-specific PCR failed to detect products for RHD exons 1 to 3 in one sample and RHD exons 1 to 5 in the other. A quantitative next-generation sequencing assay confirmed deletion of exons 1 to 3 and 1 to 5 respectively, and detected the absence of an RHD gene in trans in both samples. Long-range PCR and Sanger sequencing enabled identification of the breakpoints for both alleles. Both deletions start within the 5' Rhesus box (upstream of the identity region for the 1-to-3 deletion, downstream of it for the 1-to-5 deletion), and end within introns. CONCLUSIONS: Resolution of unclear or inconclusive results from targeted genotyping arrays often leads to the discovery of new alleles. The 5' Rhesus box may be a hot spot for genetic recombination events, such as the large deletions described in this report.

Three missense mutations found in the KEL gene lead to K(mod) or K0 red blood cell phenotypes.

BACKGROUND: The KEL gene is highly polymorphic. It presents two major alleles, KEL1(K) and KEL2(k), but a variety of mutations give rise to weakened (K(mod) phenotype) or lack (K0 phenotype) of Kell antigen expression. Recently, the use of advanced DNA-based techniques has greatly increased our understanding of the Kell blood group system. STUDY DESIGN AND METHODS: Three blood samples that had shown discordant results between the serologic and molecular typing for k were investigated by DNA sequencing. Two of these samples were also subjected to studies of adsorption and elution. RESULTS: After sequencing the whole KEL gene, we found three new missense mutations: c.455A>G (p.Tyr152Cys) at Exon 5, c.2111A>C (p.Pro704His) at Exon 19, and c.1726G>C (p.Gly576Arg) at Exon 16. So far, no known clinical implications are associated with these mutations. Further investigation by adsorption and elution methods has defined that c.455A>G and c.1726G>C resulted in K0 phenotype, while c.2111A>C encoded a K(mod) phenotype. CONCLUSION: Molecular investigation is an important complement to routine serologic analyses of Kell antigens. Discrepancies between genotype and phenotype may reveal the presence of K(mod) or K0 phenotypes. Our description of three new KEL alleles suggests a role for a wider diagnostic approach to typing of the Kell system.

Application of high-resolution melting to large-scale, high-throughput SNP genotyping: a comparison with the TaqMan method.

Because of the wide use of single-nucleotide polymorphisms (SNPs) as markers of genetic variation, several high-throughput genotyping methods have been developed and applied during the past decades. High-resolution melting (HRM) is a very attractive, advanced, fast, and cost-effective SNP genotyping technology based on the analysis of the melting profile of PCR products, using intercalating fluorescent dyes to monitor the transition from unmelted to melted DNA. The authors used HRM for genotyping 215 human DNA samples for SNPs in the ABCB1, NQO1, and SLC19A1 genes and 96 samples for SNPs in the IL1A and IL12B genes with the aim of assessing HRM sensitivity and accuracy in comparisons with the TaqMan((R)) assay in view of large-scale, high-throughput SNP-typing applications. The potential effect of PCR product size, T(M), GC content, and SNP position on HRM performances was explored with amplicons that were heterogeneous for these factors. Discrimination power ranged from 91.4% to 98.4%, being significantly lower only when the number of rare homozygotes dropped to 1 or few units. The availability of specific and validated assays, in addition to a better standardization of HRM experimental conditions, can considerably reduce time and costs of large-scale genotyping studies with a negligible risk of failure or misclassification.

A novel Real Time PCR strategy to detect SOD3 SNP using LNA probes.

Extracellular superoxide dismutase (SOD3) is the primary enzymatic antioxidant defence of the vascular wall. The physiopathological role of SOD3 has been examined in vascular-related diseases, atherosclerosis, hypertension, diabetes, ischaemia-reperfusion injury, lung disease, various inflammatory conditions, and neurological diseases. An important single nucleotide polymorphism (SNP), nt.760 G>C of the SOD3 gene (rs#1799895) leads to the amino acid substitution Arg(213)Gly (R213G) in the center of the heparin-binding domain and consequently to a lowered affinity for the endothelium. This mutation, which occurs with a relatively high frequency in the population (4% of Swedish, 3% of Australian and 6% of Japanese people), is associated with decreased tissue antioxidant defences and increased risk of ischaemic heart disease. The identification of patients carrying this mutation is therefore of great interest in order to highlight lowered antioxidant defences at a vascular level which could lead to increased susceptibility toward coronary artery disease and atherogenesis. Here we describe a method to detect the 760 G>C single nucleotide polymorphism based on Real Time PCR strategy using locked nucleic acid (LNA) probes. This technique, a modification of classic TaqMan probes SNP genotyping, amplifies and detects the mutation in a single reaction tube. Moreover, the implementation of LNA probes remarkably increases the specificity of the reaction. The proposed method enables unambigous and rapid discrimination of wild type and mutant genotype both in plasmid and genomic DNA samples. In light of the role of SOD3 polymorphism, the genotyping of 760 G>C mutant has important clinical implications. The proposed assay combines rapidity, high specificity, can be easily automated and overall reduces labor and cost of analyses. Moreover, identification of patients with lowered vascular antioxidant defences could address pharmacogenomical approaches to the therapy of cardiovascular diseases.