The X-chromosome activity states of 11 manifesting carriers of dystrophinopathies, all with normal karyotypes, were estimated by restriction fragment length polymorphism (RFLP)-methylation analysis with the probes M27 beta (DXS255), p2-19(DXS605) and pSPT/PGK (PGK1) to test the role of skewed X-inactivation ratios as the cause of their affected phenotypes. In eight cases preferential inactivation of the putative X chromosome carrying the normal dystrophin allele in > or = 90% of their peripheral lymphocytes was observed, two cases showed non-apparent deviant ratios (60:40 and 70:30) from the theoretically expected values around the mean of 50% and in one case the three markers employed yielded no information. The analysis of the X-inactivation ratio in six mother-daughter pairs, all non-manifesting Duchenne muscular dystrophy (DMD) carriers, and in the close female relatives of the patients showed: (a) neither of the two X chromosomes was preferentially inactivated with respect to their parental origin; (b) a high concordance among the activation ratios of mothers and daughters, a result difficult to explain just in terms of random X-chromosome inactivation.

The rapid spread of the use of new 24-color karyotyping techniques has preceded their standardization. This is best documented by the fact that the exact resolution limits have not yet been defined. Indeed, it is shown here that a substantial proportion of interchromosomal aberrations will be missed by all multicolor karyotyping systems currently in use. We demonstrate that both the sensitivity and the specificity of 24-color karyotyping critically depend on the fluorochrome composition of chromosomes involved in an interchromosomal rearrangement. As a solution, we introduce a conceptual change in probe labeling. Seven-fluorochrome sets that overcome many of the current limitations are described, and examples of their applications are shown. The criteria presented here for an optimized probe-set design and for the estimation of resolution limits should have important consequences for pre- and postnatal diagnostics and for research applications.

Background Routine application of multicolor fluorescence in situ hybridization (M‐FISH) technology for molecular cytogenetic diagnostics has been hampered by several technical limitations. First, when using chromosome‐specific painting probes, there is a limit in cytogenetic resolution of approximately 2–3 Mb, which can mask hidden structural abnormalities that have a significant clinical effect. Second, using whole chromosome painting probes, intrachromosomal rearrangements cannot be detected and the exact localization of breakpoints is often not possible. Methods We suggest the use of multiplex‐labeled region or locus‐ specific probes in combination with an optimal probe design to improve the sensitivity and resolution of the M‐FISH technology. To allow the application of this assay in routine diagnostics, we developed a multipurpose image analysis system. Results goldFISH was applied to the study of cryptic translocations in mental retardation patients and to the study of high‐resolution breakpoint mapping in non‐small cell lung cancer patients. For an individual with mental retardation, who had an apparently normal karyotype by G‐banding, we detected an unbalanced translocation involving chromosomes 2 and 7. Conclusions In combination with optimally designed probe kits, goldFISH overcomes most of the present limitations of the M‐FISH technology and results in virtually 100% reliability for detecting interchromosomal and intrachromosomal rearrangements. Cytometry 44:7–15, 2001. © 2001 Wiley‐Liss, Inc.

Rotaviruses were prospectively studied in 51 rural Costa Rican children from birth to two years. Samples of feces were collected weekly over a 33-month period. Rotavirus was detected in 45 (1.04%) of 4,317 fecal specimens; 39 infections were documented (an incidence of 0.5 infection per child-year), only five of which were associated with diarrhea (a pathogenicity of 12.8%). Secretory antibody in fecal extracts, detected in six of 39 infections, was short lived and did not protect against reinfection. Serum antibody was present in 69.6% of two-year-old children, but was not detected in 18.8% with documented infections. On the other hand, serum antibody was present in six of 14 children in whom rotavirus was not detected, thus increasing the overall incidence to 0.6 infection per child-year. The combination of prolonged breast-feeding, exposure to a lower infecting dose (compared with urban children), and a higher standard of hygiene than expected may explain the low incidence and low pathogenicity of rotavirus among these rural children.The epidemiology of rotavirus infection in populations observed in their own environment needs further elucidation, particularly in less-developed countries where diarrheal disease takes its highest toll. In Costa Rica, rotavirus was demonstrated as the most important etiologic agent of acute diarrhea among outpatients at the National Children's Hospital in San Jose [1]. Hospital-based studies are of limited epidemiological value, however, because they are cross-sectional in nature; cover, by definition, the most serious cases that require hospital treatment; and thus, do not reflect the situation in the general population. Consequently, a cohort study was designed in order to study patterns of infection and immunity to rotavirus under natural conditions. This longitudinal study permitted investigation of the