Carriers Of Duchenne Muscular Dystrophy Research Articles

Carrier detection of Duchenne dystrophy by frequency analysis of the electromyogram

A new method for Duchenne muscular dystrophy (DMD) carrier detection based on frequency analysis of electromyograms (EMG) taken from relative force contractions shows great promise in classifying possible carriers. Nine carriers were examined and compared to nineteen normals in an attempt to define a discriminant function that would be effective in classification of possible carriers of the gene responsible for DMD. EMG data were taken at 20%, 40%, and 60% of maximum effort from the biceps brachii using a specially designed dynamometer apparatus. The apparatus was able to isolate the biceps muscle group and allowed for adjustment to accommodate different body sizes. Three signal processing methods were used to find variables in the frequency domain that would provide the best discriminant function based on the jackknife classification method. Of these methods, the most promising appears to be the high-to-low ratio method at 20% maximum contraction. When combining the high and low values found with this test in conjunction with the peak cepstrum values also found at 20% maximum contraction, discrimination was found to be 83.8% accurate. Because this classification is not based solely on definite carriers, and appears to be independent of serum creatine phosphokinase (CPK) values, it would seem that classification based on a definite carrier population could be performed with greater accuracy, especially with the addition of CPK values.

GENE STUDIES IN NEWBORN MALES WITH DUCHENNE MUSCULAR DYSTROPHY DETECTED BY NEONATAL SCREENING

18 000 newborn males were screened for Duchenne muscular dystrophy (DMD) by creatine kinase (CK) analysis of filter paper blood spots between Jan 1, 1986, and Dec 31, 1987. 5 affected boys have been identified, and in 3 of 5 probands molecular deletions or duplications have been found. 3 of 5 mothers were judged highly likely to be carriers of DMD because of repeatedly raised CK levels, identified gene rearrangements, or both abnormalities. 1 mother has a very low probability of being a carrier and 1 is at an intermediate risk. The use of DNA analyses in new DMD probands identified by neonatal screening has allowed confirmation of the diagnosis and accurate assignment of carrier status in mothers and female relatives in over half the cases studied, and may help to reduce the population incidence of DMD by avoiding delay before clinical diagnosis.

A grandpaternally derived de novo deletion within Xp21 initially presenting in carrier females diagnosed as Kugelberg-Welander syndrome.

We report on two sisters with a history of muscle weakness and an electromyogram (EMG) diagnosis of Kugelberg-Welander syndrome (KWS) or juvenile spinal muscular atrophy. A half-brother to these women was diagnosed to have Duchenne muscular dystrophy (DMD). Using molecular probes, we identified a deletion within Xp21 in this isolated case of DMD. Sequences detected by pXJ1.1 are deleted, while fragments detected by pERT87 are intact. Both of these probes are derived from the DMD locus. We have shown that the affected sisters share with their half-brother DNA markers that are linked to the DMD gene and inherited from their maternal grandfather. Dosage analysis of Southern blots show monosomy for pXJ1.1, which has allowed us to determine carrier status within this family and to show that the half-sisters are manifesting DMD carriers.

Service experience using DNA analysis for genetic prediction in Duchenne muscular dystrophy.

In August 1985 we instituted a carrier and prenatal testing service for Duchenne muscular dystrophy (DMD) using direct DNA analysis. The experience over the first nine months is described. We...

Duchenne muscular dystrophy: high frequency of deletions.

DNA probes are available for Duchenne muscular dystrophy (DMD) carrier detection and prenatal diagnosis. With probes for about 25% of the proximal portion of the gene, we found the proximal probes detected deletions in 23% of nonselected DMD boys, while a single distal probe detected 17% more as deletions. The combined percentage was 39% for all probes tested. Prenatal diagnosis and carrier detection are more accurate if deletions are mapped rather than by use of restriction fragment length polymorphism analysis. The effort involved in screening all affected boys for deletions is considerably less, and provides an accurate genetic marker for subsequent prenatal diagnosis in the family and prospective counseling for female relatives. It seems likely that, once the entire gene (cDNA) is available for screening, most DMD boys will show deletions.

Germline mosaicism and Duchenne muscular dystrophy mutations.

Duchenne muscular dystrophy (DMD) is a severe X-linked neuromuscular disease with an incidence of approximately 1 in 3,500 newborn boys. The DMD locus has a high mutation frequency: one third of the cases is thought to result from a new mutation. Linkage studies using probes to detect restriction fragment length polymorphisms and DNA deletion studies have greatly improved DMD carrier detection and prenatal diagnosis. Here we report on two families in which a pERT87 (DXS164) deletion was transmitted to more than one offspring by women who showed no evidence for the mutation in their own somatic (white blood) cells. We also show that the deletion in both siblings in one of the families is identical, indicating that the deletion must have occurred during mitosis in early germline proliferation, leading to a germline mosaicism. This phenomenon may turn out to be a major factor contributing to the induction of DMD mutations, and has important implications for the counselling of DMD families.

Making the most of multiple measurements in estimating carrier probability in Duchenne muscular dystrophy: the Bayesian incorporation of repeated measurements using logistic discrimination.

In carrier detection studies, females-at-risk are usually tested several times if the results are ambiguous, whereas subjects in the control and obligate carrier reference groups may not be tested as often. The question is how to incorporate the multiple measurements most effectively with information in the family pedigree into combined carrier risks. Sets of measurements on individuals are not independent, but are related with the correlation coefficient 0 less than rho less than 1. We have developed a procedure for incorporating repeated measurements on individuals and their a priori chance of having the disease into logistic models. This procedure utilizes the set of measurements and an estimate of rho. We describe application of this procedure to carrier detection in Duchenne muscular dystrophy (DMD) using serum creatine kinase (CK) measurements as the biochemical indicator of carrier status. Estimates of rho for controls and obligate DMD carriers did not differ significantly from 0.5. Repeated testing with use of rho = 0.5 significantly decreased the median logistic carrier probability for controls and increased it for carriers. In some cases four to six rather than the three CK tests conventionally used in genetic counseling were necessary to obtain a stable logistic carrier probability for a subject.

The application of linkage analysis to genetic counselling in families with Duchenne or Becker muscular dystrophy.

A total of 278 families of probands with Duchenne or Becker muscular dystrophy has been ascertained and offered genetic counselling. Linkage studies have been performed in these families using polymorphic...

Increased chloride efflux in fibroblasts from X-linked muscular dystrophies and clones from Duchenne carriers

Previous studies have suggested an increased chloride membrane permeability in Duchenne muscular dystrophy (DMD) fibroblasts. We report that an increased chloride efflux with respect to controls is present not only in fibroblasts from DMD, but also from two other X-linked muscular dystrophies, Becker and Emery-Dreifuss, as well as in clones from DMD carrier females. The latter observation suggests that, at least in DMD, the increased chloride efflux phenotype might be subject to lyonization.

Spreading behaviour of cultured fibroblasts from carriers of Duchenne muscular dystrophy.

Cultured skin fibroblasts from patients with Duchenne muscular dystrophy (DMD) are more sensitive than normal cells to prolonged exposure to the ionophore monensin. In a cell spreading assay in which cells were preincubated with monensin and subsequently allowed to adhere to and spread on a glass substratum in serum-free medium for 100 min, the mean transformed cell area of normal and DMD cells was 5.97 +/- 0.11 and 5.29 +/- 0.03, respectively. Cultured fibroblasts from carriers of DMD yielded a value of 5.59 +/- 0.03, which is intermediate between, and significantly different from, the values for both normal and DMD cultures. This result would be predicted on the basis of random X-chromosome inactivation in female carriers of this disorder. However, comparison of DMD carrier cell spreading data with data obtained from pooled and summated measurements taken from separate experiments using either normal or DMD fibroblasts suggest a more complex situation. Examination of the variance of the means of cell area for the true carrier population and the summated normal and DMD population provides evidence suggesting that some form of cellular interaction may occur between the two cell genotypes in culture.

Detection of Duchenne muscular dystrophy carriers: quantitative echography and creatine kinasemia.

Data obtained from simultaneous determinations of serum creatine-kinase levels and estimation of ultrasound attenuation values in muscles greatly improved the detection of obligate carriers of Duchenne muscular dystrophy than when only one of these methods was employed alone. Eleven carriers out of 19 had a high creatine-kinasemia level and nine carriers out of 19 had a high (abnormal) attenuation value. Because of the limited overlapping between the two parameters studied, we were able to recognize 17 obligate carriers out of the 19. This indicates that the parameters studied concern different features of the disease, and the practical and theoretical considerations are discussed. The techniques are discussed together with molecular genetic investigations.

Optimal precision in ultrasound attenuation estimation and application to the detection of Duchenne muscular dystrophy carriers.

This paper deals with the measurement of the attenuation of ultrasound in muscle and its application to the detection of Duchenne Muscular Dystrophy (DMD) carriers. The precision obtained when measuring the attenuation is an important parameter to be considered. A statistical approach is taken on simulated data and compared to in vivo results. The results allow discussion for the minimum tissue volume needed for the estimation. Variations in muscle attenuation between normals were obtained from studies on 27 volunteers. These attenuation values were compared to those obtained from 19 carriers of DMD. Attenuation appears to be a potential clinical indicator of DMD carriers.

Optimal precision in ultrasound attenuation estimation and application to the detection of Duchenne Muscular Dystrophy carriers

This paper deals with the measurement of the attenuation of ultrasound in muscle and its application to the detection of Duchenne Muscular Dystrophy (DMD) carriers. The precision obtained when measuring the attenuation is an important parameter to be considered. A statistical approach is taken on simulated data and compared to in vivo results. The results allow discussion for the minimum tissue volume needed for the estimation. Variations in muscle attenuation between normals were obtained from studies on 27 volunteers. These attenuation values were compared to those obtained from 19 carriers of DMD. Attenuation appears to be a potential clinical indicator of DMD carriers.

Use of serum creatine kinase, pyruvate kinase, and genetic linkage for carrier detection in Duchenne and Becker dystrophy.

Carrier detection in Duchenne dystrophy (DD) and Becker dystrophy (BD) can be achieved with DNA probes that recognize restriction fragment length polymorphisms (RFLPs). In 22 families, we found that 16 of 23 females at risk for being DD or BD carriers could be provided with more definitive indications of carrier status beyond the use of creatine kinase/pyruvate kinase and pedigree analysis. RFLP analysis was not possible for six individuals despite potentially informative probes, because family members critical to the analysis were unavailable. In only one instance were all eight probes uninformative.

The effect of aerobic exercise on serum creatine kinase activities.

Aerobic exercise is now a common form of recreational exercise among young women. In a previous study, more than a third of a group of young mothers volunteering blood samples to establish a creatine kinase (CK) reference range for Duchenne muscular dystrophy (DMD) carrier detection regularly participated in aerobic exercise programs. Aerobic exercise programs include eccentric exercises. As eccentric exercise is known to produce a delayed CK peak, this study was carried out to determine the effect of aerobic exercise on serum CK activities. The postexercise serum CK activity peak was monitored in 15 young women (age range 20-23 years) following aerobic exercise classes (45 minutes on 3 consecutive days). Peak values at 24-48 hours following the last class ranged from 90 to 3473 U/liter, or 1.55 to 34.71 times resting values. It is concluded that aerobic exercise programs should be excluded in order to obtain accurate resting serum CK values for muscle disease diagnosis.

Evaluation of carrier detection rates for Duchenne and Becker muscular dystrophies using serum creatine-kinase (CK) and pyruvate-kinase (PK) through discriminant analysis.

Serum pyruvate-kinase (PK) and creatine-kinase (CK) determinations have been carried out in a sample of 100 obligate carriers for the Duchenne muscular dystrophy (DMD) gene, 23 obligate carriers for the Becker muscular dystrophy (BMD) gene, and 50 normal adult control women. Blood samples were collected from all subjects three times on three independent occasions and the means of these three determinations were considered for both PK and CK activities in the statistical analysis. Discriminant analysis has shown that, in the group of carriers for the DMD gene, the estimated misclassification frequencies (M.F.) using either serum CK, PK, or both enzymes were: 26.5% for CK alone, 19.5% for PK alone, and 19% for both enzymes. In the group of carriers for the BMD gene, the estimated proportions of M.F. were: 31.7% for CK alone, 23.8% for PK alone, and 20.4% for both enzymes. It is concluded that, although a proportion of carries still remains undetected, the use of serum PK determinations enhances the capability of detecting carriers of both DMD and BMD mainly when compared with serum CK alone.

The myoblast defect identified in Duchenne muscular dystrophy is not a primary expression of the DMD mutation. Clonal analysis of myoblasts from five double heterozygotes for two X-linked loci: DMD and G6PD.

We previously proposed the hypothesis that the primary expression of the defect in X-linked Duchenne muscular dystrophy (DMD) occurred in the myoblast, or muscle precursor cell. This was based on the observation that the number of viable myoblasts obtained per gram DMD muscle tissue was greatly reduced and those that grew in culture had decreased proliferative capacity and an aberrant distended flat morphology. Here we test that hypothesis by determining whether the expression of the myoblast defect is X-linked. Muscle cells were obtained from five doubly heterozygous carriers of two X-linked loci, DMD and glucose-6-phosphate dehydrogenase (G6PD), and compared with those from five sex- and age-matched controls heterozygous for G6PD only. A total of 1,355 individual clones were determined to be muscle and evaluated at the single cell level for proliferative capacity, morphology, and G6PD isozyme expression. The results demonstrate that the proportion of defective myoblast clones is significantly increased in DMD carriers. However, since this cellular defect does not consistently segregate with a single G6PD phenotype in the myoblast clones derived from any of the carriers, it is unlikely to be the primary expression of the DMD mutant allele.

Unspecific reactions of HLA-B antisera on fibroblasts from patients and carriers of Duchenne muscular dystrophy.

Skin fibroblasts of patients with Duchenne muscular dystrophy were analysed for HLA tissue antigens. For the examination of the antigens two modified methods were used: the microdroplet cytotoxicity assay on fibroblast suspension and the cytotoxicity assay on fibroblast monolayer cultures using fluorochromasia. The HLA determination on the fibroblast suspensions of the patients showed a variety of unspecific HLA expressions which were not found in fibroblasts of healthy controls. These reactions were not apparent in the test in monolayer cultures. The exact cause has not been elucidated yet, but it appears to have a strong relation to some membrane abnormality in cells of DMD patients. The reproducible lack of unspecific reactions on monolayer cultures lead us to propose that these expressions arise from artificial injuries to membranes of these patient cells during trypsination or centrifugation. The validity of this test has been confirmed by a blind study.

Detection of carriers of human Duchenne muscular dystrophy by freeze-fracture analysis of erythrocyte plasmalemmal intramembrane particles.

Critical examination of plasma membrane particles on fracture faces of erythrocyte plasma membranes of human obligate carriers of Duchenne muscular dystrophy, control patients, and a patient afflicted with Duchenne muscular dystrophy revealed a 32% decrease in the number of intramembrane particles in erythrocytes of carriers compared with erythrocytes sampled from control patients. These results support the notion that quantitative analysis of intramembrane particles in freeze-fractured erythrocyte plasma membranes represents a new, rapid, simple, and highly accurate diagnostic tool for detection of carriers of human Duchenne muscular dystrophy.

Quantitative analysis of quadriceps muscle biopsy: Results in 7 definite and 45 possible carriers of duchenne muscular dystrophy

Quadriceps muscle biopsy was performed in 7 definite and 45 possible carriers of Duchenne muscular dystrophy. Unequivocal morphological changes were observed in 8 women; all but one of them were already detected as carriers by increased serum CK. Histometric analysis of muscle biopsy was performed on two fibre types (type 1 and type 2) in all 52 women; in 38 of them the two subgroups of type 2 fibres (type 2a and type 2b) were also quantified. Results of histometric analysis were matched with control values obtained from 30 healthy females in the same age as the carriers (20–50 yr). Histometric analysis on two fibre types detected a percentage of carriers (27%) much lower than that detected by serum CK determination (44%). The detection rate of combined serum CK determination and histometric analysis of muscle biopsy was: 100% in definite carriers, 65% in mothers possible carriers, 38% in sisters and 25% in the group of cousins and aunts. The histometric parameters most frequently altered were atrophy factor and variability coefficient of mean diameter. Histometric analysis carried out on three fibre types in 38 women detected 2 more carriers showing atrophy of type 2b fibres. The results indicate that quantitative analysis of muscle biopsy may be used to detect some possible carriers with normal serum CK. However, an adequate number of controls is mandatory; in fact the detection rate of histometric analysis of muscle biopsy decreased by increasing the number of controls, resulting much lower than previously reported.