Mutation analysis of feline Niemann-Pick C1 disease

Niemann-Pick C (NPC) disease is an autosomal recessive neurovisceral lysosomal storage disorder that results in defective intracellular transport of cholesterol. The major form of human NPC (NPC1) has been mapped to chromosome 18, the NPC1 gene (NPC1) has been sequenced and several mutations have been identified in NPC1 patients. A feline model of NPC has been characterized and is phenotypically, morphologically, and biochemically similar to human NPC1. Complementation studies using cultured fibroblasts from NPC affected cats and NPC1 affected humans support that the gene responsible for the NPC phenotype in this colony of cats is orthologous to human NPC1. Using human-based PCR primers, initial fragments of the feline NPC cDNA were amplified and sequenced. From these sequences, feline-specific PCR primers were generated and designed to amplify six overlapping bands that span the entire feline NPC1 open reading frame. A single base substitution (2864G-C) was identified in NPC1 affected cats. Obligate carriers are heterozygous at the same allele and a PCR-based assay was developed to identify the geneotype of all cats in the colony. The mutation results in an amino acid change from cysteine to serine (C955S). Several of the mutations identified in people occur in the same region. Marked similarity exists between the human and feline NPC1 cDNA sequences, and is greater than that between the human and murine NPC1 sequences. The human cDNA sequence predicts a 1278aa protein with a lysosomal targeting sequence, several trans-membrane domains and extensive homology with other known mediators of cholesterol homeostasis.     

Rapid DNA haplotyping using a multiplex heteroduplex approach: Application to duchenne muscular dystrophy carrier testing

A new strategy has been developed for rapid haplotype analysis based on an initial multiplex amplification of several polymorphic sites, followed by heteroduplex detection. Heteroduplexes formed between two different alleles are detected because they migrate differently than the corresponding homoduplexes in Hydrolink-MDE gel. This simple, rapid method does not depend on specific sequences such as restriction enzyme sites or CA boxes and does not require the use of isotope. This approach has been tested using commonly occurring polymorphisms spanning the dystrophin gene as a model. We describe the use of the method to assign the carrier status of females in Duchenne muscular dystrophy (DMD) pedigrees. The method may be used for other genetic diseases when mutations are unknown or there are few dinucleotide markers in the gene proximity, and for the identification of haplotype backgrounds of mutant alleles. © 1995 Wiley-Liss, Inc.     

Sister chromatid exchange and chromosome breakage in complete hydatidiform moles.

Women with complete hydatidiform moles (CHM) are at a 10% risk for developing persistent trophoblastic disease or choriocarcinoma. We studied sister chromatid exchange (SCE) as a prognostic indicator for malignancy in peripheral blood lymphocytes (PBL) from women with CHM and their husbands, but found no differences from normal control couples. SCE levels in cultured tissue derived from 11 CHM (avg. 7.9) and 2 choriocarcinomas (avg. 6.8) were not significantly different from those of 8 normal skin fibroblast cultures (avg. 7.8). These same tissues were then examined for chromosome breakage which was significantly higher for CHM (0.48/cell) and choriocarcinoma (0.87/cell) than normal fibroblasts (0.33/cell). Chromosome breaks occurred at 50-60% known fragile sites and at 50-55% of cancer breakpoints. Whereas SCE was only associated with 13% of breaks in the three tissues, half of these were at known fragile sites. Our results suggest that SCE is not an indicator of malignancy in PBL or cultured cells from CHM or choriocarcinoma and that the level of SCE is not elevated in CHM or choriocarcinoma. However, our results confirm the increased breakage seen in the latter two tissues which may represent general DNA instability predisposing to choriocarcinoma and its accompanying chromosomal rearrangements.     

Clinical, pathological, and biochemical studies on an infantile case of sulfatide/GM1 activator protein deficiency

A 28-month-old black male died with severe complications of mental and motor deterioration, seizures, and aspiration. Autopsy demonstrated moderate liver enlargement, normal spleen and kidneys, small testes, and a grossly normal brain. Further examination showed irregular macrogyrae with evidence of a storage or sclerotic process. Thin layer chromatography of the lipids in formalin-fixed tissue demonstrated elevated levels of ceramide trihexoside and possibly sulfatides in liver and a decrease in the ratio of galactosylceramide to sulfatide in brain. Examination of the gangliosides in formalin-fixed brain indicated a slight increase in the percentage of GM1 ganglioside and a clear elevation in GM2 and GM3 gangliosides. Cultured skin fibroblasts had a normal activity for a large number of lysosomal enzymes including arylsulfatase A and galactocerebrosidase. When the cells were loaded with [14C]sulfatide only about 12% of the sulfatide was metabolized after 3 days. Extracts of the cells were subjected to SDS-PAGE and immunoblotting with antisphingolipid activator protein-1 (SAP-1) rabbit antiserum, and no cross-reacting material was detected confirming the diagnosis of metachromatic leukodystrophy caused by SAP-1 deficiency. This patient was clinically more severe than the other patients described previously with this deficiency. Further studies are underway to define the nature of the mutation in this patient.     

The effects of resistance training on aerobic and anaerobic power of young boys

A 4-wk interval-type training program incorporating omni-kinetic equipment and stationary cycling elicited an increase in the absolute and relative VO2max of an active group of young boys. The improvement in aerobic function was independent of the training protocols of high velocity-low resistance and low velocity-high resistance. However, the training programs failed to increase anaerobic function as measured by an "all-out" cycle test in which power output was calculated in watts and watts per kilogram for 0- to 15-s and 15- to 30-s work periods. Changes in aerobic and anaerobic functions were independent of physiological maturity as determined by serum testosterone level (ng X dl-1).     

A field test for the assessment of abdominal muscular endurance in professional ice hockey players

The purpose of this study was to develop a test protocol and produce normative data for measuring abdominal muscular endurance in professional ice hockey players. The protocol was developed using elite speed skaters and professional ice hockey players. The test involves the measurement of total number of curl-ups that can be completed at a prescribed rate of 25 repetitions per minute. The test protocol maximizes the isolation of the abdominal muscle group and the control of the movement. Normative data is presented from tests on 11 7 professional hockey players. This protocol provides discriminatory power and has high face validity in its application.J Orthop Sports Phys Ther 1984;6(1):30-33.     

Working conditions and health behavior as causes of educational inequalities in self-rated health: an inverse odds weighting approach

Objective:Using a novel mediation method that presents unbiased results even in the presence of exposure–mediator interactions, this study estimated the extent to which working conditions and health behaviors contribute to educational inequalities in self-rated health in the workforce.Methods:Respondents of the longitudinal Survey of Health, Ageing, and Retirement in Europe (SHARE) in 16 countries were selected, aged 50–64 years, in paid employment at baseline and with information on education and self-rated health (N=15 028). Education, health behaviors [including body mass index (BMI)] and working conditions were measured at baseline and self-rated health at baseline and two-year follow-up. Causal mediation analysis with inverse odds weighting was used to estimate the total effect of education on self-rated health, decomposed into a natural direct effect (NDE) and natural indirect effect (NIE).Results:Lower educated workers were more likely to perceive their health as poor than higher educated workers [relative risk (RR) 1.48, 95% confidence interval (CI) 1.37–1.60]. They were also more likely to have unfavorable working conditions and unhealthy behaviors, except for alcohol consumption. When all working conditions were included, the remaining NDE was RR 1.30 (95% CI 1.15–1.44). When BMI and health behaviors were included, the remaining NDE was RR 1.40 (95% CI 1.27–1.54). Working conditions explained 38% and health behaviors and BMI explained 16% of educational inequalities in health. Including all mediators explained 64% of educational inequalities in self-rated health.Conclusions:Working conditions and health behaviors explain over half of the educational inequalities in self-rated health. To reduce health inequalities, improving working conditions seems to be more important than introducing health promotion programs in the workforce.