Strategies for phasing and imputation in a population isolate

In the search for genetic associations with complex traits, population isolates offer the advantage of reduced genetic and environmental heterogeneity. In addition, cost‐efficient next‐generation association approaches have been proposed in these populations where only a subsample of representative individuals is sequenced and then genotypes are imputed into the rest of the population. Gene mapping in such populations thus requires high‐quality genetic imputation and preliminary phasing. To identify an effective study design, we compare by simulation a range of phasing and imputation software and strategies. We simulated 1,115,604 variants on chromosome 10 for 477 members of the large complex pedigree of Campora, a village within the established isolate of Cilento in southern Italy. We assessed the phasing performance of identical by descent based software ALPHAPHASE and SLRP, LD‐based software SHAPEIT2, SHAPEIT3, and BEAGLE, and new software EAGLE that combines both methodologies. For imputation we compared IMPUTE2, IMPUTE4, MINIMAC3, BEAGLE, and new software PBWT. Genotyping errors and missing genotypes were simulated to observe their effects on the performance of each software. Highly accurate phased data were achieved by all software with SHAPEIT2, SHAPEIT3, and EAGLE2 providing the most accurate results. MINIMAC3, IMPUTE4, and IMPUTE2 all performed strongly as imputation software and our study highlights the considerable gain in imputation accuracy provided by a genome sequenced reference panel specific to the population isolate.     

Reproducibility of sonographic measurement of the substantia nigra

The aim of this study was to evaluate inter-reader, intra-investigator and inter-investigator reproducibility and correlations in the assessment of substantia nigra (SN) echogenicity and area measurement by a physician-sonographer (PS), a sonographic laboratory assistant (SLA) and a physician without sonographic experience (PN). A total of 22 patients with extrapyramidal symptoms were examined using transcranial sonography (TCS). SN images were encoded and evaluated by the three readers. A second TCS examination was performed after 7+/-2 d. A second investigator performed TCS examination 1 mo later. Spearman rank correlation and Pearson's correlation coefficient were used when assessing the agreement between readers. All three readers identified the same 15 patients with SN echogenicity III or more. Inter-reader SN echogenicity and area measurement correlations were r=0.55 to 0.82 and r=0.31 to 0.74 between PS and SLA and r=0.55 to 0.77 and 0.49 to 0.62 between PS and PN, respectively (p<0.05 in all cases). Intra-reader echogenicity and area measurement correlations (r=0.85 to 0.96 and r=0.51 to 0.69) were statistically significant only for PS (p<0.001). All intra- and inter-investigator correlations of SN area measurement (r=0.69 to 0.88 and r=0.5 to 0.61) and SN echogenicity (r=0.64 to 0.92 and r=0.51 to 0.69) were statistically significant (p<0.05). Semiquantitative evaluation of SN echogenicity and area using TCS is highly dependent on the experience of the sonographer. Only an experienced sonographer was able to produce very reproducible results with statistically significant correlations; SLA and PN intra-reader correlations were poor.     

Anti‐ACSA‐2 defines a novel monoclonal antibody for prospective isolation of living neonatal and adult astrocytes

Astrocytes are the most abundant cell type of the central nervous system and cover a broad range of functionalities. We report here the generation of a novel monoclonal antibody, anti‐astrocyte cell surface antigen‐2 (Anti‐ACSA‐2). Flow cytometry, immunohistochemistry and immunocytochemistry revealed that Anti‐ACSA‐2 reacted specifically with a not yet identified glycosylated surface molecule of murine astrocytes at all developmental stages. It did not show any labeling of non‐astroglial cells such as neurons, oligodendrocytes, NG2⁺ cells, microglia, endothelial cells, leukocytes, or erythrocytes. Co‐labeling studies of GLAST and ACSA‐2 showed largely overlapping expression. However, there were also notable differences in protein expression levels and frequencies of single‐positive subpopulations of cells in some regions of the CNS such as cerebellum, most prominently at early postnatal stages. In the neurogenic niches, the dentate gyrus of the hippocampus and the subventricular zone (SVZ), again a general overlap with slight differences in expression levels were observed. ACSA‐2 was unlike GLAST not sensitive to papain‐based tissue dissociation and allowed for a highly effective, acute, specific, and prospective purification of viable astrocytes based on a new rapid sorting procedure using Anti‐ACSA‐2 directly coupled to superparamagnetic MicroBeads. In conclusion, ACSA‐2 appears to be a new surface marker for astrocytes, radial glia, neural stem cells and bipotent glial progenitor cells which opens up the possibility of further dissecting the characteristics of astroglial subpopulations and lineages.     

Use of an automated multiple-locus, variable-number tandem repeat-based method for rapid and high-throughput genotyping of Staphylococcus aureus isolates

Fast and reliable genotyping methods that allow real-time epidemiological surveillance would be instrumental to monitoring of the spread of methicillin-resistant Staphylococcus aureus. We describe an automated variable-number tandem repeat-based method for the rapid genotyping of Staphylococcus aureus. Multiplex PCR amplifications with eight primer pairs that target gene regions with variable numbers of tandem repeats were resolved by microcapillary electrophoresis and automatically assessed by cluster analysis. This genotyping technique was evaluated for its discriminatory power and reproducibility with clinical isolates of various origins, including a panel of control strains previously characterized by several typing methods and collections from either long-term carriers or defined nosocomial outbreaks. All steps of this new procedure were developed to ensure a rapid turnaround time and moderate cost. The results obtained suggest that this rapid approach is a valuable tool for the genotyping of S. aureus isolates in real time.