Enhanced diagnostic yield in Meckel-Gruber and Joubert syndrome through exome sequencing supplemented with split-read mapping

Background The widespread adoption of high-throughput sequencing technologies by genetic diagnostic laboratories has enabled significant expansion of their testing portfolios. Rare autosomal recessive conditions have been a particular focus of many new services. Here we report a cohort of 26 patients referred for genetic analysis of Joubert (JBTS) and Meckel-Gruber (MKS) syndromes, two clinically and genetically heterogeneous neurodevelopmental conditions that define a phenotypic spectrum, with MKS at the severe end. Methods Exome sequencing was performed for all cases, using Agilent SureSelect v5 reagents and Illumina paired-end sequencing. For two cases medium-coverage (9×) whole genome sequencing was subsequently undertaken. Results Using a standard analysis pipeline for the detection of single nucleotide and small insertion or deletion variants, molecular diagnoses were confirmed in 12 cases (4 %). Seeking to determine whether our cohort harboured pathogenic copy number variants (CNV), in JBTS- or MKS-associated genes, targeted comparative read-depth analysis was performed using FishingCNV. These analyses identified a putative intragenic AHI1 deletion that included three exons spanning at least 3.4 kb and an intergenic MPP4 to TMEM237 deletion that included exons spanning at least 21.5 kb. Whole genome sequencing enabled confirmation of the deletion-containing alleles and precise characterisation of the mutation breakpoints at nucleotide resolution. These data were validated following development of PCR-based assays that could be subsequently used for “cascade” screening and/or prenatal diagnosis. Conclusions Our investigations expand the AHI1 and TMEM237 mutation spectrum and highlight the importance of performing CNV screening of disease-associated genes. We demonstrate a robust increasingly cost-effective CNV detection workflow that is applicable to all MKS/JBTS referrals

Interrogating Microfinance Performance beyond Products, Clients and the Environment. Insights from the work of BRAC in Tanzania

The performance of microfinance organisations can depend upon many factors. Current research emphasizes factors pertaining to clients, products, or broader environments. But researchers have paid less attention to the workings and internal systems of microfinance organisations. We explore how variation in performance within an organisation can alter the consequences of loans and their popularity among clients and potential clients. We illustrate with data from BRAC in Tanzania, where the arrival and rapid expansion of BRAC’s microfinance programme provides an apposite case study

A LC-MS metabolomics approach to investigate the effect of raw apple intake in the rat plasma metabolome

Fruit and vegetable consumption has been associated with several health benefits; however the mechanisms are largely unknown at the biochemical level. Our research aims to investigate whether plasma metabolome profiling can reflect biological effects after feeding rats with raw apple by using an untargeted UPLC-ESI-TOF-MS based metabolomics approach in both positive and negative mode. Eighty young male rats were randomised into groups receiving daily 0, 5 or 10 g fresh apple slices, respectively, for 13 weeks. During weeks 3-6 some of the animals were receiving 4 mg/ml 1,2-dimethylhydrazine dihydrochloride (DMH) once a week. Plasma samples were taken at the end of the intervention and among all groups, about half the animals were 12 h fasted. An initial ANOVA-simultaneous component analysis with a three-factor or two-factor design was employed in order to isolate potential metabolic variations related to the consumption of fresh apples. Partial least squares-discriminant analysis was then applied in order to select discriminative features between plasma metabolites in control versus apple fed rats and partial least squares modelling to reveal possible dose response. The findings indicate that in laboratory rats apple feeding may alter the microbial amino acid fermentation, lowering toxic metabolites from amino acids metabolism and increasing metabolism into more protective products. It may also delay lipid and amino acid catabolism, gluconeogenesis, affect other features of the transition from the postprandial to the fasting state and affect steroid metabolism by suppressing the plasma level of stress corticosteroids, certain mineralocorticoids and oxidised bile acid metabolites. Several new hypotheses regarding the cause of health effects from apple intake can be generated from this study for further testing in humans. © 2013 Springer Science+Business Media New York

Immunization of young heifers with staphylococcal immune evasion proteins before natural exposure to Staphylococcus aureus induces a humoral immune response in serum and milk

Background: Staphylococcus aureus, a leading cause of mastitis in dairy cattle, causes severe mastitis and/or chronic persistent infections with detrimental effects on the cows' wellbeing, lifespan and milk production. Despite years of research there is no effective vaccine against S. aureus mastitis. Boosting of non-protective pre-existing immunity to S. aureus, induced by natural exposure to S. aureus, by vaccination may interfere with vaccine efficacy. The aim was to assess whether experimental immunization of S. aureus naïve animals results in an immune response that differs from immunity following natural exposure to S. aureus. Results: First, to define the period during which calves are immunologically naïve for S. aureus, Efb, LukM, and whole-cell S. aureus specific serum antibodies were measured in a cohort of newborn calves by ELISA. Rising S. aureus specific antibodies indicated that from week 12 onward calves mounted an immune response to S. aureus due to natural exposure. Next, an experimental immunization trial was set up using 8-week-old heifer calves (n = 16), half of which were immunized with the immune evasion molecules Efb and LukM. Immunization was repeated after one year and before parturition and humoral and cellular immunity specific for Efb and LukM was determined throughout the study. Post-partum, antibody levels against LukM and EfB were significantly higher in serum, colostrum and milk in the experimentally immunized animals compared to animals naturally exposed to S. aureus. LukM specific IL17a responses were also significantly higher in the immunized cows post-partum. Conclusions: Experimental immunization with staphylococcal immune evasion molecules starting before natural exposure resulted in significantly higher antibody levels against Efb and LukM around parturition in serum as well as the site of infection, i.e. in colostrum and milk, compared to natural exposure to S. aureus. This study showed that it is practically feasible to vaccinate S. aureus naïve cattle and that experimental immunization induced a humoral immune response that differed from that after natural exposure only.</p