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.

Increased neurotransmitter release at the neuromuscular junction in a mouse model of polyglutamine disease.

In Huntington's disease (HD), the expansion of polyglutamine (polyQ) repeats at the N terminus of the ubiquitous protein huntingtin (htt) leads to neurodegeneration in specific brain areas. Neurons degenerating in HD develop synaptic dysfunctions. However, it is unknown whether mutant htt impacts synaptic function in general. To investigate that, we have focused on the nerve terminals of motor neurons that typically do not degenerate in HD. Here, we have studied synaptic transmission at the neuromuscular junction of transgenic mice expressing a mutant form of htt (R6/1 mice). We have found that the size and frequency of miniature endplate potentials are similar in R6/1 and control mice. In contrast, the amplitude of evoked endplate potentials in R6/1 mice is increased compared to controls. Consistent with a presynaptic increase of release probability, synaptic depression under high-frequency stimulation is higher in R6/1 mice. In addition, no changes were detected in the size and dynamics of the recycling synaptic vesicle pool. Moreover, we have found increased amounts of the synaptic vesicle proteins synaptobrevin 1,2/VAMP 1,2 and cysteine string protein-α, and the SNARE protein SNAP-25, concomitant with normal levels of other synaptic vesicle markers. Our results reveal that the transgenic expression of a mutant form of htt leads to an unexpected gain of synaptic function. That phenotype is likely not secondary to neurodegeneration and might be due to a primary deregulation in synaptic protein levels. Our findings could be relevant to understand synaptic toxic effects of proteins with abnormal polyQ repeats.

A large study of androgen receptor germline variants and their relation to sex hormone levels and prostate cancer risk. Results from the National Cancer Institute Breast and Prostate Cancer Cohort Consortium

Background: Androgens are key regulators of prostate gland maintenance and prostate cancer growth, and androgen deprivation therapy has been the mainstay of treatment for advanced prostate cancer for many years. A long-standing hypothesis has been that inherited variation in the androgen receptor (AR) gene plays a role in prostate cancer initiation. However, studies to date have been inconclusive and often suffered from small sample sizes. Objective and Methods: We investigated the association of AR sequence variants with circulating sex hormone levels and prostate cancer risk in 6058 prostate cancer cases and 6725 controls of Caucasian origin within the Breast and Prostate Cancer Cohort Consortium. We genotyped a highly polymorphic CAG microsatellite in exon 1 and six haplotype tagging single nucleotide polymorphisms and tested each genetic variant for association with prostate cancer risk and with sex steroid levels. Results: We observed no association between AR genetic variants and prostate cancer risk. However, there was a strong association between longer CAG repeats and higher levels of testosterone (P = 4.73 × 10−5) and estradiol (P = 0.0002), although the amount of variance explained was small (0.4 and 0.7%, respectively). Conclusions: This study is the largest to date investigating AR sequence variants, sex steroid levels, and prostate cancer risk. Although we observed no association between AR sequence variants and prostate cancer risk, our results support earlier findings of a relation between the number of CAG repeats and circulating levels of testosterone and estradiol.

Colonization with multiple Staphylococcus aureus strains among patients in European intensive care units.

Patients and healthy individuals intermittently and inconsistently carry different methicillin-resistant Staphylococcus aureus (MRSA) subtypes. In the present study, we assessed the clonality of methicillin-susceptible S. aureus (MSSA) and MRSA strains in patients admitted to 1 of 6 intensive care units (ICUs), using spa typing and multilocus variable number of tandem repeats analysis (MLVA).

Functional Variants in NOS1 and NOS2A Are Not Associated with Progressive Hearing Loss in Ménière's Disease in a European Caucasian Population

Hearing loss in Meniere's disease (MD) is associated with loss of spiral ganglion neurons and hair cells. In a guinea pig model of endolymphatic hydrops, nitric oxide synthases (NOS) and oxidative stress mediate loss of spiral ganglion neurons. To test the hypothesis that functional variants of NOS1 and NOS2A are associated with MD, wed genotyped three functional variants of NOS1 (rs41279104,rs2682826, and a cytosine-adenosine microsatellite repeat in exon 1f) and the CCTTT repeat in the promoter of NOS2A gene (rs3833912) in two independent MD sets(273 patients in total) and 550 controls. A third cohort of American patients was genotyped as replication cohort for the CCTTT repeat. Neither allele nor genotype frequencies of rs41279104 and rs2682826 were associated with MD, although longer alleles of the cytosine-adenosine microsatellite repeat were marginally significant (corrected p = 0.05) in the Mediterranean cohort but not in a second Galicia cohort. Shorter numbers of the CCTTT repeat in NOS2A were significantly more frequent in Galicia controls (OR = 0.37 [CI, 0.18-0.76], corrected p =0.04), but this finding could not be replicated in Mediterranean or American case-control populations. Meta-analysis did not support an association between CCTTT repeats and risk for MD. Severe hearing loss (>75 dB) was also not associated with any functional variants studied. Functional variants of NOS1 and and NOS2A do not confer susceptibility for MD.

Prospective universal application of mycobacterial interspersed repetitive-unit-variable-number tandem-repeat genotyping to characterize Mycobacterium tuberculosis isolates for fast identification of clustered and orphan cases.

The use of molecular tools for genotyping Mycobacterium tuberculosis isolates in epidemiological surveys in order to identify clustered and orphan strains requires faster response times than those offered by the reference method, IS6110 restriction fragment length polymorphism (RFLP) genotyping. A method based on PCR, the mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) genotyping technique, is an option for fast fingerprinting of M. tuberculosis, although precise evaluations of correlation between MIRU-VNTR and RFLP findings in population-based studies in different contexts are required before the methods are switched. In this study, we evaluated MIRU-VNTR genotyping (with a set of 15 loci [MIRU-15]) in parallel to RFLP genotyping in a 39-month universal population-based study in a challenging setting with a high proportion of immigrants. For 81.9% (281/343) of the M. tuberculosis isolates, both RFLP and MIRU-VNTR types were obtained. The percentages of clustered cases were 39.9% (112/281) and 43.1% (121/281) for RFLP and MIRU-15 analyses, and the numbers of clusters identified were 42 and 45, respectively. For 85.4% of the cases, the RFLP and MIRU-15 results were concordant, identifying the same cases as clustered and orphan (kappa, 0.7). However, for the remaining 14.6% of the cases, discrepancies were observed: 16 of the cases clustered by RFLP analysis were identified as orphan by MIRU-15 analysis, and 25 cases identified as orphan by RFLP analysis were clustered by MIRU-15 analysis. When discrepant cases showing subtle genotypic differences were tolerated, the discrepancies fell from 14.6% to 8.6%. Epidemiological links were found for 83.8% of the cases clustered by both RFLP and MIRU-15 analyses, whereas for the cases clustered by RFLP or MIRU-VNTR analysis alone, links were identified for only 30.8% or 38.9% of the cases, respectively. The latter group of cases mainly comprised isolates that could also have been clustered, if subtle genotypic differences had been tolerated. MIRU-15 genotyping seems to be a good alternative to RFLP genotyping for real-time interventional schemes. The correlation between MIRU-15 and IS6110 RFLP findings was reasonable, although some uncertainties as to the assignation of clusters by MIRU-15 analysis were identified.

Optimized molecular resolution of cross-contamination alerts in clinical mycobacteriology laboratories

BACKGROUND. The phenomenon of misdiagnosing tuberculosis (TB) by laboratory cross-contamination when culturing Mycobacterium tuberculosis (MTB) has been widely reported and it has an obvious clinical, therapeutic and social impact. The final confirmation of a cross-contamination event requires the molecular identification of the same MTB strain cultured from both the potential source of the contamination and from the false-positive candidate. The molecular tool usually applied in this context is IS6110-RFLP which takes a long time to provide an answer, usually longer than is acceptable for microbiologists and clinicians to make decisions. Our purpose in this study is to evaluate a novel PCR-based method, MIRU-VNTR as an alternative to assure a rapid and optimized analysis of cross-contamination alerts. RESULTS. MIRU-VNTR was prospectively compared with IS6110-RFLP for clarifying 19 alerts of false positivity from other laboratories. MIRU-VNTR highly correlated with IS6110-RFLP, reduced the response time by 27 days and clarified six alerts unresolved by RFLP. Additionally, MIRU-VNTR revealed complex situations such as contamination events involving polyclonal isolates and a false-positive case due to the simultaneous cross-contamination from two independent sources. CONCLUSION. Unlike standard RFLP-based genotyping, MIRU-VNTR i) could help reduce the impact of a false positive diagnosis of TB, ii) increased the number of events that could be solved and iii) revealed the complexity of some cross-contamination events that could not be dissected by IS6110-RFLP.

Real-time molecular epidemiology of tuberculosis by direct genotyping of smear-positive clinical specimens.

We applied MIRU-VNTR (mycobacterial interspersed repetitive-unit-variable-number tandem-repeat typing) to directly analyze the bacilli present in 61 stain-positive specimens from tuberculosis patients. A complete MIRU type (24 loci) was obtained for all but one (no amplification in one locus) of the specimens (98.4%), and the allelic values fully correlated with those obtained from the corresponding cultures. Our study is the first to demonstrate that real-time genotyping of Mycobacterium tuberculosis can be achieved, fully transforming the way in which molecular epidemiology techniques can be integrated into control programs.

Molecular testing for fragile X: analysis of 5062 tests from 1105 fragile X families--performed in 12 clinical laboratories in Spain.

Fragile X syndrome is the most common inherited form of intellectual disability. Here we report on a study based on a collaborative registry, involving 12 Spanish centres, of molecular diagnostic tests in 1105 fragile X families comprising 5062 individuals, of whom, 1655 carried a full mutation or were mosaic, three cases had deletions, 1840 had a premutation, and 102 had intermediate alleles. Two patients with the full mutation also had Klinefelter syndrome. We have used this registry to assess the risk of expansion from parents to children. From mothers with premutation, the overall rate of allele expansion to full mutation is 52.5%, and we found that this rate is higher for male than female offspring (63.6% versus 45.6%; P < 0.001). Furthermore, in mothers with intermediate alleles (45-54 repeats), there were 10 cases of expansion to a premutation allele, and for the smallest premutation alleles (55-59 repeats), there was a 6.4% risk of expansion to a full mutation, with 56 repeats being the smallest allele that expanded to a full mutation allele in a single meiosis. Hence, in our series the risk for alleles of <59 repeats is somewhat higher than in other published series. These findings are important for genetic counselling.