The dystrophin associated protein complex in zebrafish.

Many cases of muscular dystrophy in humans are caused by mutations in members of the dystrophin associated protein complex (DAPC). Zebrafish are small vertebrates whose bodies are composed predominantly of skeletal muscle, making them attractive models for studying mammalian muscle disorders. Potential orthologs to most of the human DAPC proteins have been found in zebrafish by database screening. Expression of the sarcoglycans, dystroglycan and dystrophin has been confirmed by western blotting. Immunohistochemical and biochemical techniques localize these proteins to the muscle cell membrane in adult zebrafish. Morpholino (MO) experiments designed to inhibit the translation of dystrophin mRNA produce juvenile zebrafish that are less active than zebrafish injected with control morpholinos. Western blot analysis of the dystrophin morpholino-injected zebrafish shows concurrent reduction of dystrophin and the sarcoglycans, suggesting that these proteins, like those in mammals, are part of a complex whose integrity is dependent on dystrophin expression. These results indicate that the zebrafish is an excellent animal model in which to approach the study of dystrophin and its associated proteins.

Detection of mutations in the dystrophin gene via automated DHPLC screening and direct sequencing.

BACKGROUND: Currently molecular diagnostic laboratories focus only on the identification of large deletion and duplication mutations (spanning one exon or more) for Duchenne Muscular Dystrophy (DMD) yielding 65% of causative mutations. These mutations are detected by an existing set of multiplexed polymerase chain reaction (PCR) primer pairs. Due to the large size of the dystrophin gene (79 exons), finding point mutations (substitutions, deletions or insertions of one or several nucleotides) has been prohibitively expensive and laborious. The aim of this project was to develop an effective and convenient method of finding all, or most, mutations in the dystrophin gene with only a moderate increase in cost. RESULTS: Using denaturing high performance liquid chromatography (DHPLC) screening and direct sequencing, 86 PCR amplicons of genomic DNA from the dystrophin gene were screened for mutations in eight patients diagnosed with DMD who had tested negative for large DNA rearragements. Mutations likely to be disease-causative were found in six of the eight patients. All 86 amplicons from the two patients in whom no likely disease-causative mutations were found were completely sequenced and only polymorphisms were found. CONCLUSIONS: We have shown that it is now feasible for clinical laboratories to begin testing for both point mutations and large deletions/duplications in the dystrophin gene. The detection rate will rise from 65% to greater than 92% with only a moderate increase in cost.

Dystrophin and muscular dystrophy: past, present, and future.

Duchenne muscular dystrophy was described in the medical literature in the early 1850s but the molecular basis of the disease was not determined until the late 1980s. The cloning of dystrophin led to the identification of a large complex of proteins that plays an important, although not yet well understood, role in muscle biology. Concomitant with the elucidation of the function of dystrophin and its associated proteins has been the pursuit of therapeutic options for muscular dystrophy. Although there is still no cure for this disorder, great advances are being made in the areas of gene introduction and cell transplant therapy.

Genomic organization and single-nucleotide polymorphism map of desmuslin, a novel intermediate filament protein on chromosome 15q26.3.

BACKGROUND: Desmuslin is an alpha-dystrobrevin-interacting protein expressed primarily in heart and skeletal muscle. The desmuslin protein interacts with and is closely related to desmin, a protein encoded by a locus mutated in some forms of hereditary distal myopathy. As a muscle-specific intermediate filament protein, desmuslin is also a candidate for myopathies of unknown etiology. RESULTS: The desmuslin gene was localized to chromosome 15q26.3 by electronic screening of the human DNA sequence database. Primer pairs were designed to amplify the 5 exons of the desmuslin gene in 11 overlapping DNA segments. The desmuslin gene was screened for mutations in 71 patients with various forms of myopathy for which there was no known cause. In this analysis, 10 common and 2 rare amino acid altering single-nucleotide polymorphisms were identified, all of which were seen in a control population of individuals thus making these unlikely causes of the phenotype. Interestingly, one of the single-nucleotide polymorphisms found in a patient resulted in a premature stop codon in the first exon. The nonsense mutation was also detected in the patient's unaffected father and one unaffected control; it was detected in 0.44% (2/454) of unrelated chromosomes and is therefore predicted to have a homozygous frequency of 0.002%. CONCLUSION: No causative mutations were found in the desmuslin gene. However, the single-nucleotide polymorphisms mapped in this study represent a well-mapped group that can be used for disequilibrium studies of this region of chromosome 15q26.3.

Analysis of human sarcospan as a candidate gene for CFEOM1.

BACKGROUND: Congenital fibrosis of the extraocular muscles type 1 (CFEOM1) is an autosomal dominant eye movement disorder linked to the pericentromere of chromosome 12 (12p11.2 - q12). Sarcospan is a member of the dystrophin associated protein complex in skeletal and extraocular muscle and maps to human chromosome 12p11.2. Mutations in the genes encoding each of the other components of the skeletal muscle sarcospan-sarcoglycan complex (alpha - delta sarcoglycan) have been shown to cause limb girdle muscular dystrophy (LGMD2C-F). To determine whether mutations in the sarcospan gene are responsible for CFEOM1 we: (1) attempted to map sarcospan to the CFEOM1 critical region; (2) developed a genomic primer set to directly sequence the sarcospan gene in CFEOM1 patients; and (3) generated an anti-sarcospan antibody to examine extraocular muscle biopsies from CFEOM1 patients. RESULTS: When tested by polymerase chain reaction, sarcospan sequence was not detected on yeast or bacterial artificial chromosomes from the CFEOM1 critical region. Sequencing of the sarcospan gene in CFEOM1 patients from 6 families revealed no mutations. Immunohistochemical studies of CFEOM1 extraocular muscles showed normal levels of sarcospan at the membrane. Finally, sarcospan was electronically mapped to bacterial artificial chromosomes that are considered to be outside of the CFEOM1 critical region. CONCLUSIONS: In this report we evaluate sarcospan as a candidate gene for CFEOM1. We have found that it is highly unlikely that sarcospan is involved in the pathogenesis of this disease. As of yet no sarcospan gene mutations have been found to cause muscular abnormalities.

Micronuclei in lymphocytes of prostate cancer patients undergoing radiation therapy.

To further verify the applicability of the micronucleus (MN) assay in biodosimetry, we measured the MN yield in cytokinesis-blocked (CB) peripheral blood lymphocytes (PBL) of eight prostate cancer (PC) patients. These patients had no previous chemotherapy or radiotherapy (xRT). They were treated with standardized schemes of fractionated pelvic xRT. Before xRT, and at one random time-point during the course of xRT, blood samples were collected from each patient for the following purposes: (1) to verify the relationship between the MN yield in PBL and the estimated equivalent (EQ) total-body absorbed dose; and (2) to evaluate the individual differences of ex vivo radiation dose-response (1-4 Gy) relationship of MN yield in PBL before xRT. The number of xRT fractions, cumulative tumor dose, and EQ total-body absorbed doses of these patients represented a wide range. We found in PBL of these patients that (1) MN yield (Y) increased linearly with the estimated EQ total-body absorbed dose as Y=14.6+9.2D (R(2)=0.7, p=0.007); the distributions of MN yield were overdispersed; the ratio of relative increment of MN yield per 1000 binucleated (BN) PBL ranged from 0.9 to 8.2 (median: 4.1) folds above that of the respective baseline levels; and (2) before xRT, the MN yields also increased linearly with the ex vivo radiation dose; at each radiation dose level, the distributions of MN yield were overdispersed in most patients. In two of the three patients with xRT-induced early side effects (cystitis, diarrhea), the MN yield in PBL induced by ex vivo irradiation before xRT was significantly higher than in the other patients without xRT-induced side effects. These findings suggest that MN yields in CB PBL can be used as an in vivo biodosimeter. Since the differences in individual ex vivo radiation dose-response relationship of MN yield in PBL before xRT appeared to be significant, our preliminary results also suggest that it may be possible to identify individual intrinsic radiosensitivity before the start of xRT.

Recent mortality patterns in California.

Mortality among people with developmental disabilities was reviewed using recent data obtained from the California Department of Developmental Services. The time interval for this report was 1991-1995. We defined two study cohorts: one beginning in January 1991 and a second in April 1993. The latter period represented the years of implementation of the Coffelt settlement. Our primary interest was in the Coffelt period cohort. Statistically significant association with increased rates of mortality was found for community residence. A trend of declining mortality was noted for the community facilities from 1991-1995, but not for the developmental centers.

Reliability of finger stick capillary blood for the lymphocyte micronucleus assay.

Compared with peripheral blood sampling, capillary blood collecting by finger stick is less traumatic and more convenient. To assess the sensitivity and reliability of capillary blood for the lymphocyte micronucleus (MN) assay, this study was performed in three sample groups, i.e. healthy donors (n = 3), cancer patients before treatment (n = 7), and cancer patients who were undergoing fractionated partial-body radiotherapy (n = 9). For each group, we measured three intra-individual variables, i.e. micronucleus (MN) frequency, binucleate (BN) index, and micronucleated BN index of lymphocytes obtained from capillary blood and the corresponding peripheral blood. Our results indicated that in all three sample groups, the differences in these variables between capillary blood and peripheral blood either before or after ex vivo 137Cs irradiation (2 Gy) were insignificant. Since capillary blood is more accessible than peripheral blood, we believe that it is a reliable source for the lymphocyte MN assay especially when venipuncture is not convenient.

beta-dystrobrevin, a new member of the dystrophin family. Identification, cloning, and protein associations.

Dystrophin, the protein disrupted in Duchenne muscular dystrophy, is one of several related proteins that are key components of the submembrane cytoskeleton. Three dystrophin-related proteins (utrophin, dystrophin-related protein-2 (DRP2), and dystrobrevin) have been described. Here, we identify a human gene on chromosome 2p22-23 that encodes a novel protein, beta-dystrobrevin, with significant homology to the other known dystrobrevin (now termed alpha-dystrobrevin). Sequence alignments including this second dystrobrevin strongly support the concept that two distinct subfamilies exist within the dystrophin family, one composed of dystrophin, utrophin, and DRP2 and the other composed of alpha- and beta-dystrobrevin. The possibility that members of each subfamily form distinct protein complexes was examined by immunopurifying dystrobrevins and dystrophin. A beta-dystrobrevin antibody recognized a protein of the predicted size (71 kDa) that copurified with the dystrophin short form, Dp71. Thus, like alpha-dystrobrevin, beta-dystrobrevin is likely to associate directly with dystrophin. alpha- and beta-dystrobrevins failed to copurify with each other, however. These results suggest that members of the dystrobrevin subfamily form heterotypic associations with dystrophin and raise the possibility that pairing of a particular dystrobrevin with dystrophin may be regulated, thereby providing a mechanism for assembly of distinct submembrane protein complexes.

Concerning the analysis of 2 x 2 tables.

Recent papers (R. M. Engeman and G. D. Swanson, 1991, Comput. Biomed. Res. 24, 509; M. R. Massai and P. Barbini, 1993, Comput. Biomed Res. 26, 98) have expressed concern regarding differences in results among statistical methods of hypothesis testing for 2 x 2 contingency tables. These papers did not present current statistical thought on this topic, and they did not discuss measures of effect for such analyses. In this paper statistical literature on the 2 x 2 contingency table is presented and philosophical issues underlying the methods discussed. Measures of effect appropriate for several data origins or designs which result in 2 x 2 arrays are also presented. The paper presents an empirical adjustment to the Fisher's exact test known as the mid-P approach. The information should help provide the user of statistics a better understanding of the analysis of 2 x 2 tables. An example using data from Engeman and Swanson (1991) is provided, and some recommendations for the analysis of 2 x 2 tables are given.

Gender differences in laterality patterns for speaking and singing.

This study examined behaviors reflecting cerebral organization of speaking and singing in normal college students. The investigation focused on whether differences existed in the laterality patterns of two singing tasks and one speaking task in males and females. Performance was measured on a verbal/manual time-sharing paradigm, coupling finger tapping with three vocal tasks (speaking, singing a rote song, singing up and down a diatonic five note scale). Females exhibited less variation than males in mean tapping rates and laterality scores across all three vocal tasks, thus indicating that gender most likely influences lateralization of vocal tasks. Bilateral integration was indicated for both males and females during singing up/down the aforementioned scale. These findings suggest differential involvement of both hemispheres in processing musical functions.

Sensitivity and specificity of the Quetelet index to assess obesity in men and women.

OBJECTIVE AND METHODS: In a cross-sectional design, the sensitivity and specificity of the Quetelet Index (QI) was determined in relation to percent body fat standards. Subjects were 1280 men and 365 women asymptomatic for coronary heart disease. Contingency tables by QI quartiles were created using two sets of criteria of obesity: body fat > or = 25% and QI > or = 28 kg.m-2 for men and 30% and 27 kg.m-2 for women. Percent body fat was determined with hydrodensitometry in all subjects. RESULTS: In men, sensitivity, positive, and negative predictive value of QI in relation to percent body fat was 54.5%, 91.8%, 82.7% and 73.8%, respectively. The corresponding values for women were 26.9%, 98.2%, 90.7% and 67.1%. Sensitivity of QI was 2.03 times greater for men than for women (P < 0.001). Receiver operator characteristic (ROC) curve analysis suggested that an acceptable trade-off between sensitivity (91%) and specificity (47%) occurred at a QI of 24.5 kg.m-2 for men and at a QI of 22 kg.m-2 for women. CONCLUSIONS: The data suggest that in an asymptomatic population, percent body fat-based QI cut-off values may misclassify the obese. A fat mass-based reference system could perhaps lead to an improved classification outcome.

Comparison of four methods to assess body composition in women.

The purpose of this study was to compare the accuracy of four methods to assess body composition of women. Seventy-seven Caucasian women [mean (+/- SD) age: 31.8 +/- 8.6 years; mass: 59.5 +/- 9.1 kg; stature: 162.4 +/- 6.9 cm; Quetelet Index: 22.5 +/- 3.1 kg/m2] were tested for percent body fat (%BF) with hydrostatic weighing (HW), near-infrared spectrophotometry (NIR), bioelectrical impedance analysis (BIA), and seven-site skinfolds (7-SFs). Compared to %BF with HW (24.9 +/- 6.5%), an analysis of variance revealed no mean differences (P > or = 0.05) among %BF with NIR (26.0 +/- 5.5%), BIA (25.7 +/- 5.8%) and 7-SFs (24.0 +/- 6.0%). The correlations between %BF with HW and NIR, BIA and 7-SF were r = 0.47, r = 0.77, and r = 0.79, respectively (P < or = 0.05), and prediction errors (SEE) were 5.8%, 4.2%, and 4.1%. Comparisons of %BF obtained from machine readings versus those computed from the manufacturer's equation indicated significant differences for the BIA (machine 25.7%, equation 27.8%) and NIR (machine 26.0%, equation 21.5%) methods. We concluded that although the mean %BF differences were small among the four methods, the large SEE values may allow the use of BIA and 7-SFs but not NIR. Inaccuracies of machine readings versus equation-computed %BF indicate that BIA and NIR variables and/or constants in the equations supplied are not identical to those used in machine-generated calculations.

Role of industry representatives in providing assistance to certified diabetes educators.

OBJECTIVE: As diabetes educators have become more frequently involved in decisions regarding medications, equipment, and supplies, industry representatives have increasingly provided various types of assistance. The major objectives of this survey were to determine 1) the types of assistance being provided to certified diabetes educators (CDEs) by industry representatives, 2) whether product recommendations are based on assistance provided, and 3) the types of assistance that CDEs consider appropriate. RESEARCH DESIGN AND METHODS: We developed the survey instrument and it was mailed to every fifth person listed in the 1989 directory of CDEs. Three hundred twenty-five (51%) of the active surveys were returned. RESULTS: Those responding indicated that 1) the types of support provided most frequently by industry representatives include instructional materials, samples, supplies, and equipment for patient care and education; 2) the number of types of support provided is directly related to the number of industry representatives seen on a regular basis and the frequency of interaction; 3) 22% of the educators state that they make product recommendations based on support provided; and 4) the role of the industry reps should be to continue support for patient care and education, to provide more support for public and professional education, and to minimize support for personal items, e.g., gifts or meals. CONCLUSIONS: The survey indicates that CDEs are receiving many types of assistance from industry representatives and that they prefer that such assistance be targeted toward patient and professional education.

Comparison of four methods to assess body composition in black and white athletes.

Four methods of assessing body composition were compared in 55 black and 35 white, Division I, American football players. Percent body fat (%BF) was estimated with hydrostatic weighing at residual volume, corrected for race; seven-site skinfolds (7 SF), corrected for race; bioelectrical impedance analysis (BIA); and near-infrared spectrophotometry (NIR). Percent body fat with HW in blacks (mean = 14.7%) and whites (19.7%) did not differ (P > .05) from %BF with 7 SF (blacks, 14.7%; whites, 19.0%). In relation to HW, BIA significantly (P < .05) overpredicted (blacks: 20.1%, SEE = 3.2%; whites: 22.3%, SEE = 4.3%) and NIR underpredicted %BF (blacks: 12.6%, SEE = 3.9%; whites: 17.7%, SEE = 3.6%). The contribution of BIA variables (resistance, phase angle, conductance) and NIR optical density to predict %BF was trivial compared to body mass index. It appears that race may not substantially influence %BF prediction by NIR and BIA. It was concluded that when considering the cost and expertise required with NIR and BIA, SF measurements appear to be a superior alternative for rapid and accurate body composition assessment of athletes, independent of race.

Comparison of body composition assessment by hydrodensitometry, skinfolds, and multiple site near-infrared spectrophotometry.

Percent body fat (%BF) was assessed in 171 men with underwater weighing (UWW), seven-site skinfolds (7 SF), and near-infrared spectrophotometry (NIR). NIR was determined at eight sites, including biceps, triceps, axilla, chest, abdomen, suprailium, subscapula and thigh. Analysis of variance and Dunnett's post-hoc procedure revealed that NIR significantly (P less than 0.05) underpredicted %BF when using the biceps (12.9%), chest (11.3%), abdomen (10.2%), subscapula (11.3%) and thigh (9.9%) sites compared to the criterion %BF measured with UWW (13.4% +/- SD = 6.49). %BF with 7 SF was only 0.3% higher than %BF with UWW (r = 0.94, standard error of estimate = 2.9%). Correlation coefficients between SF thickness and NIR optical density readings at 940 nm (OD1) and 950 nm (OD2) wavelengths ranged from r = -0.30 (subscapula) to r = -0.67 (biceps) for OD1 and r = -0.39 (axilla) to r = -0.68 (biceps) for OD2. Multiple linear regression showed that the OD readings were not significant predictors of UWW %BF when using body mass, stature, activity level, and frame size as predictors, except for the OD2 reading at the biceps site. Stepwise regression revealed that NIR OD readings did not predict additional variance in %BF beyond mass, stature, activity level, frame size, or 7 SF. It was concluded that 7 SF are better predictors of %BF than NIR, and that NIR at multiple sites is inconsistent in assessing body composition.

Peak running velocity, submaximal energy expenditure, VO2max, and 8 km distance running performance.

The purposes of this study were to examine (1) if the peak running velocity achieved during maximal treadmill testing is related to 8 km distance running performance (DRP) and (2) if the relationship between selected physiological determinants are consistant for two 8 km races within a competitive season. Seven members of a University cross-country team (mean +/- SE) (age, 19.4 +/- 0.5 years, VO2max 67.0 +/- 1.0 ml/kg/min were tested 2-3 days following a mid-season race (Race M) and the season-ending conference championship (Race C). Despite similar weather and terrain, Race C (29:22 +/- 1:22 min:sec) was significantly (p less than 0.05) slower than Race M (28:31 +/- 0:58 min:sec). However, no significant differences (p greater than 0.05) were observed between testing sessions for calculated energy expenditure heart rate oxygen consumption and RER during submaximal running (248 and 268 m/min), postsubmaximal run lactate, peak running velocity, and VO2max. A significant correlation (p less than 0.05) was observed for Race M with with calculated energy expenditure during submaximal running at both speeds (r = 0.85) and with peak running velocity (r = -0.76). Regression analysis revealed that 92% of the total variance for Race M was accounted for by calculated energy expenditure during submaximal running and VO2max. No significant correlation or regression relationship was observed for any variables with Race C. These results suggest that peak running velocity, calculated energy expenditure during submaximal running, and VO2max can be associated with 8 km running performance. However, the relationship between 8 km DRP and the variables measured can differ for two races over a competitive season.

Mortality in a large southeastern facility for persons with mental retardation.

Mortality rates for institutionalized persons with mental retardation were presented. Rates were provided for two time intervals, 1974 through 1979 and 1980 through 1985, and by age, race, and gender. Consistent differences between black and white residents or by gender were not indicated. However, significant improvement in mortality did occur between the two time periods. Persons with profound retardation were found to have higher mortality than those whose retardation was mild to severe. Respiratory disease was the most prevalent cause of death among the individuals with profound mental retardation, whereas heart disease and cancer were the most common causes of death among persons with mild, moderate, or severe retardation. Aging of the population was noted over the period of the study, indicative of the increasing frailty of the institutionalized population.

Trend analysis for repeated measures designs.

This paper outlines a method of analysis that is well-suited to repeated measures designs. This method uses polynomial functions of time to characterize an outcome variable measured at several distinct time points on the same subjects. Profiles for different treatment groups may be compared using the appropriate interaction terms in an analysis of variance table. The method is illustrated with data from two recent studies.