Evaluation of the new advanced 15-loci MIRU-VNTR genotyping tool in Mycobacterium tuberculosis molecular epidemiology studies

Background. During the last few years, PCR-based methods have been developed to simplify and reduce the time required for genotyping Mycobacterium tuberculosis (MTB) by standard approaches based on IS6110-Restriction Fragment Length Polymorphism (RFLP). Of these, MIRU-12-VNTR (Mycobacterial interspersed repetitive units- variable number of tandem repeats) (MIRU-12) has been considered a good alternative. Nevertheless, some limitations and discrepancies with RFLP, which are minimized if the technique is complemented with spoligotyping, have been found. Recently, a new version of MIRU-VNTR targeting 15 loci (MIRU-15) has been proposed to improve the MIRU-12 format. Results. We evaluated the new MIRU-15 tool in two different samples. First, we analyzed the same convenience sample that had been used to evaluate MIRU-12 in a previous study, and the new 15-loci version offered higher discriminatory power (Hunter-Gaston discriminatory index [HGDI]: 0.995 vs 0.978; 34.4% of clustered cases vs 57.5%) and better correlation (full or high correlation with RFLP for 82% of the clusters vs 47%). Second, we evaluated MIRU-15 on a population-based sample and, once again, good correlation with the RFLP clustering data was observed (for 83% of the RFLP clusters). To understand the meaning of the discrepancies still found between MIRU-15 and RFLP, we analyzed the epidemiological data for the clustered patients. In most cases, splitting of RFLP-clustered patients by MIRU-15 occurred for those without epidemiological links, and RFLP-clustered patients with epidemiological links were also clustered by MIRU-15, suggesting a good epidemiological background for clustering defined by MIRU-15. Conclusion. The data obtained by MIRU-15 suggest that the new design is very efficient at assigning clusters confirmed by epidemiological data. If we add this to the speed with which it provides results, MIRU-15 could be considered a suitable tool for real-time genotyping.

Novel (60%) and recurrent (40%) androgen receptor gene mutations in a series of 59 patients with a 46,XY disorder of sex development.

BACKGROUND Androgen receptor (AR) gene mutations are the most frequent cause of 46,XY disorders of sex development (DSD) and are associated with a variety of phenotypes, ranging from phenotypic women [complete androgen insensitivity syndrome (CAIS)] to milder degrees of undervirilization (partial form or PAIS) or men with only infertility (mild form or MAIS). OBJECTIVE The aim of the study was to characterize the contribution of the AR gene to the molecular cause of 46,XY DSD in a series of Spanish patients. SETTING We studied a series of 133 index patients with 46,XY DSD in whom gonads were differentiated as testes, with phenotypes including varying degrees of undervirilization, and in whom the AR gene was the first candidate for a molecular analysis. METHODS The AR gene was sequenced (exons 1 to 8 with intronic flanking regions) in all patients and in family members of 61% of AR-mutated gene patients. RESULTS AR gene mutations were found in 59 individuals (44.4% of index patients), of whom 46 (78%) were CAIS and 13 (22%) PAIS. Fifty-seven different mutations were found: 21.0% located in exon 1, 15.8% in exons 2 and 3, 57.9% in exons 4-8, and 5.3% intronic. Twenty-three mutations (40.4%) had been previously described and 34 (59.6%) were novel. CONCLUSIONS AR gene mutation is the most frequent cause of 46,XY DSD, with a clearly higher frequency in the complete phenotype. Mutations spread along the whole coding sequence, including exon 1. This series shows that 60% of mutations detected during the period 2002-2009 were novel.