Estimation of the male and female mutation rates in Duchenne muscular dystrophy (DMD)

Summary We present the results of an international collaborative study aimed at estimating the ratio of male to female mutation rates in Duchenne muscular dystrophy based on the method of C. Müller and T. Grimm. With a sample size of 295, this ratio is found to be very close to 1, thus giving evidence for equal mutation rates in males and females in Duchenne muscular dystrophy.     

On the power to detect differences between male and female mutation rates for Duchenne muscular dystrophy, using classical segregation analysis and restriction fragment length polymorphisms

The power to detect departures from the theoretical proportion of new mutants in X-linked lethal disorders has been analyzed for several types of segregation analysis, including methods based on completely linked restriction fragment length polymorphisms. It is shown that all methods require large sample sizes in order to detect even large differences between male and female mutation rates. Ascertainment bias is shown to have a great effect on the outcome of the segregation analysis. All reviewed studies concerning the proportion of new mutants in Duchenne muscular dystrophy, whether they claimed equality or inequality between the male and female mutation rates, give insufficient evidence because of ascertainment bias and a too low power. An ascertainment bias-free method is given, with the advantage that information from many studies can be combined. By doing so, in the long run, even moderate departures from equality in mutation rates (if present) can be detected.     

DNA analysis in Turkish Duchenne/Becker muscular dystrophy families

Summary The molecular genetics of Duchenne/Becker muscular dystrophy was investigated in 81 affected Turkish families. Deletions were detected by multiplex polymerase chain reaction assays and cDNA Southern analyses. The distribution of the deletions along the gene and their correlation to clinical phenotype were different from the studies reported on other populations. Moreover, DNA polymorphisms in mothers were determined using 8 DNA probes and three CA repeat sequences, and a high degree of informativeness was observed.     

Estimation of male to female ratio of mutation rates from carrier-detection tests in X-linked disorders.

A method is presented for the estimation of the ratio of male to female mutation rates from female carrier-detection test data from pedigrees containing an isolated male manifesting an X-linked necessive disorder. Pedigrees of any size and complexity (barring consanguinity) and containing any number of tested females can be utilized. The relative fitness of affected males and carrier females, and the segregation probability of the abnormal gamete in females, can be estimated simultaneously with the ratio of mutation rates in order to test specific hypotheses against given bodies of data. Here this method is applied to families containing isolated individuals affected with Lesch-Nyhan syndrome.     

Genetic linkage study between the loci for Duchenne and Becker muscular dystrophy and nine X-chromosomal DNA markers

Summary A set of nine polymorphic loci defined by DNA probes was studied for linkage with the disease locus in ten families with a history of Duchenne muscular dystrophy (DMD), and three families with a history of Becker muscular dystrophy (BMD). The results confirm DMD and BMD linkage to all marker loci and suggest closer linkage of several probes than hitherto detected. This will be of practical interest for risk calculations in affected families.     

Frameshift deletion mechanisms in Egyptian Duchenne and Becker muscular dystrophy families

Partial gene deletion is the major type of mutation leading to Duchenne muscular dystrophy (DMD) and its mild allelic form, Becker muscular dystrophy (BMD). Amplification of the genomic DNAs of 152 unrelated dystrophin patients using multiple primers detected 78 (51.3%) probands with deletion mutations. We predicted the translational reading frame for all the deletions in Egyptian dystrophin males. The frameshift rule was confirmed positively ranging for 50 to 67% of the cases depending on the type of disease. We discuss ways of accounting for some exceptions from the frameshift hypothesis in the central and proximal regions. These explanations may help in developing procedures for reducing the severity of dystrophin phenotypes to restore the correct frame by disrupting the translational fidelity. Great efforts have been put into the development of effective 'gene correction' procedures via such intrinsic mechanisms. In addition, we mapped regional difference in deletion mutation frequencies within the DMD gene locus between the different Egyptian governorates. There were no double deletions in the Egyptian dystrophin males.     

Germline and somatic mosaicism in a female carrier of Duchenne muscular dystrophy

The family of a male with Duchenne muscular dystrophy (DMD) and a deletion within the dystrophin gene has been studied. Polymerase chain reaction analysis of ectopic mRNA from peripheral blood T+B lymphocytes and the use of (CA) _n repeat polymorphisms in and around the deleted region showed the proband's mother to be both a germline mosaic and a somatic mosaic for the deletion seen in her son. The mutation therefore occurred as a mitotic event early in embryogenesis.     

Utility of MLPA in mutation analysis and carrier detection for Duchenne muscular dystrophy

CONTEXT:

Multiplex ligation probe amplification (MLPA) is a new technique to identify deletions and duplications and can evaluate all 79 exons in dystrophin gene in patients with Duchenne muscular dystrophy (DMD). Being semi-quantitative, MLPA is also effective in detecting duplications and carrier testing of females; both of which cannot be done using multiplex PCR. It has found applications in diagnostics of many genetic disorders.

AIM:

To study the utility of MLPA in diagnosis and carrier detection for DMD.

MATERIALS AND METHODS:

Mutation analysis and carrier detection was done by multiplex PCR and MLPA and the results were compared.

RESULTS AND CONCLUSIONS:

We present data showing utility of MLPA in identifying mutations in cases with DMD/BMD. In the present study using MLPA, we identified mutations in additional 5.6% cases of DMD in whom multiplex PCR was not able to detect intragenic deletions. In addition, MLPA also correctly confirmed carrier status of two obligate carriers and revealed carrier status in 6 of 8 mothers of sporadic cases.     

Mapping DNA sequences in a human X-chromosome deletion which extends across the region of the Duchenne muscular dystrophy mutation.

A somatic cell hybrid has been constructed and characterized using fibroblasts from a phenotypically normal woman who possesses an X chromosome with an interstitial deletion of the short arm. High-resolution banding indicates that the deleted segment is either Xp22.13-p11.4 or Xp22.11-p11.23. Southern blot hybridization to previously mapped DNA sequences confirms that the missing segment of the X chromosome is a deletion and not an interstitial translocation and supports the cytogenetic interpretation that the deletion extends proximal of Xp11.3 and therefore probably comprises Xp22.11-p11.23. Three further DNA sequences have been localized to the region of the deleted segment. The following order has been assigned to the seven probes used: Xpter-RC8-pXUT22-(OA1,C7,M2C)-L1.28-RD6 -Xcen.     

Enhanced sensitivity to calcium in Duchenne muscular dystrophy.

The ionophore A23187 causes an increase in the Ca content of human erythrocytes and a Ca-dependent increase in K efflux (Gardos effect). These changes are associated with a reduction in osmotic fragility and cell size. Treatment of erythrocytes from patients with Duchenne muscular dystrophy with A23187 results in ⁴⁵Ca uptake comparable to that of erythrocytes from control subjects. However, the reduction in osmotic fragility and K content observed in dystrophic erythrocytes is twofold greater than in control erythrocytes. These results indicate that an alteration in the regulation of erythrocyte membrane function by Ca occurs in Duchenne muscular dystrophy. This alteration may be responsible for other changes in erythrocyte membrane properties observed in Duchenne muscular dystrophy.     

Uniparental disomy of the entire X chromosome in a female with Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe, progressive, X-linked muscle-wasting disorder with an incidence of approximately 1/3,500 male births. Females are also affected, in rare instances. The manifestation of mild to severe symptoms in female carriers of dystrophin mutations is often the result of the preferential inactivation of the X chromosome carrying the normal dystrophin gene. The severity of the symptoms is dependent on the proportion of cells that have inactivated the normal X chromosome. A skewed pattern of X inactivation is also responsible for the clinical manifestation of DMD in females carrying X;autosome translocations, which disrupt the dystrophin gene. DMD may also be observed in females with Turner syndrome (45,X), if the remaining X chromosome carries a DMD mutation. We report here the case of a karyotypically normal female affected with DMD as a result of homozygosity for a deletion of exon 50 of the dystrophin gene. PCR analysis of microsatellite markers spanning the length of the X chromosome demonstrated that homozygosity for the dystrophin gene mutation was caused by maternal isodisomy for the entire X chromosome. This finding demonstrates that uniparental isodisomy of the X chromosome is an additional mechanism for the expression of X-linked recessive disorders. The proband's clinical presentation is consistent with the absence of imprinted genes (i.e., genes that are selectively expressed based on the parent of origin) on the X chromosome.     

miRNome profiling in Duchenne muscular dystrophy; identification of asymptomatic and manifesting female carriers

Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disorder that occurs due to inactivating mutations in DMD gene, leading to muscular dystrophy. Prediction of pathological complications of DMD and the identification of female carriers are important research points that aim to reduce disease burden. Herein, we describe a case of a late DMD patient and his immediate female family members, who all carry same DMD mutation and exhibited varied degrees of symptoms. In our study, we sequenced the whole miRNome in leukocytes and plasma of the family members and results were validated using real-time PCR. Our results highlighted the role of miR-409-3p, miR-424-5p, miR-144-3p as microRNAs that show correlation with the extent of severity of muscular weakness and can be used for detection of asymptomatic carriers. Cellular and circulating levels of miR-494-3p had shown significant increase in symptomatic carriers, which may indicate significant roles played by this miRNA in the onset of muscular weakness. Interestingly, circulating levels of miR-206 and miR-410-3p were significantly increased only in the severely symptomatic carrier. In conclusion, our study highlighted several miRNA species, which could be used in predicting the onset of muscle and/or neurological complications in DMD carriers.     

Skewed X inactivation in a female MZ twin results in Duchenne muscular dystrophy.

One of female MZ twins presented with muscular dystrophy. Physical examination, creatine phosphokinase levels, and muscle biopsy were consistent with Duchenne muscular dystrophy (DMD). However, because of her sex she was diagnosed as having limb-girdle muscular dystrophy. With cDNA probes to the DMD gene, a gene deletion was detected in the twins and their mother. The de novo mutation which arose in the mother was shown by novel junction fragments generated by HindIII, PstI, or TaqI when probed with cDNA8. Additional evidence of a large gene deletion was given by novel SfiI junction fragments detected by probes p20, J-Bir, and J-66 on pulsed-field gel electrophoresis (PFGE). Immunoblot analysis of muscle from the affected twin showed dystrophin of normal size but of reduced amount. Immunofluorescent visualization of dystrophin revealed foci of dystrophin-positive fibers adjacent to foci of dystrophin-negative fibers. These data indicate that the affected twin is a manifesting carrier of an abnormal DMD gene, her myopathy being a direct result of underexpression of dystrophin. Cytogenetic analysis revealed normal karyotypes, eliminating the possibility of a translocation affecting DMD gene function. Both linkage analysis and DNA fingerprint analysis revealed that each twin has two different X chromosomes, eliminating the possibility of uniparental disomy as a mechanism for DMD expression. On the basis of methylation differences of the paternal and maternal X chromosomes in these MZ twins, we propose uneven lyonization (X chromosome inactivation) as the underlying mechanism for disease expression in the affected female.     

Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification.

The application of recombinant DNA technology to prenatal diagnosis of many recessively inherited X-linked diseases is complicated by a high frequency of heterogeneous, new mutations (1). Partial gene deletions account for more than 50% of Duchenne muscular dystrophy (DMD) lesions, and approximately one-third of all cases result from a new mutation (2-5). We report the isolation and DNA sequence of several deletion prone exons from the human DMD gene. We also describe a rapid method capable of detecting the majority of deletions in the DMD gene. This procedure utilizes simultaneous genomic DNA amplification of multiple widely separated sequences and should permit deletion scanning at any hemizygous locus. We demonstrate the application of this multiplex reaction for prenatal and postnatal diagnosis of DMD.     

Evidence for mutation by unequal sister chromatid exchange in the Duchenne muscular dystrophy gene.

We have studied three families each containing a male with Duchenne or Becker muscular dystrophy. Southern blot analysis using both genomic and cDNA probes revealed that an exon-containing segment of DNA within the gene is duplicated in the probands, their mothers, and, in two cases, their sisters. The grandpaternal origin of the duplication has been demonstrated in these families by RFLP and duplication analysis. The results suggest that unequal sister-chromatid exchange, which most likely occurred in the germ cell lineage of the proband's grandfather, is responsible for generating these duplications and that this type of intrachromosomal rearrangement, although rarely reported in humans, is not uncommon in the muscular dystrophy gene.