pp. 53-56 in Intractable Neurological Disorders, Human Genome Research and Society. Proceedings of the Third International Bioethics Seminar in Fukui, 19-21 November, 1993.

Editors: Norio Fujiki, M.D. & Darryl R.J. Macer, Ph.D.

Copyright 1994, Eubios Ethics Institute All commercial rights reserved. This publication may be reproduced for limited educational or academic use, however please enquire with Eubios Ethics Institute.

Molecular genetic diagnosis of muscular dystrophies

Kiichi Arahata & Hideo Sugita
National Institute of Neuroscience, NCNP, Tokyo 187, Japan

Fifty percent of the weight of the human body consists of muscle tissue. The skeletal, cardiac and smooth muscles endow humans with the power of movement. Progressive muscular dystrophy (PMD) is a group of inherited diseases marked by wasting and progressive weakness of the skeletal muscles. The involvement of other organs such as cardiac insufficiency and dilation of stomach can also be demonstrated by a careful examinations. The genetic cause may be inherited by three modes of inheritance pattern (dominant, recessive, X-linked), or the gene may also be defective due to a new mutation.

There have been great advances in the molecular genetic diagnosis of muscular dystrophies in recent years, which in turn have valuable implications for both genetic counseling and the elucidation of the etiology of the disease (1-4). Since molecular diagnoses are independent of the patient's age, they provide useful information before any clinical symptoms appear. At the genetic level, DNA and RNA-based molecular diagnosis and linkage analysis can be performed as shown in Figures 1 and 2 (4-6) (Fig. 1, 2). Isolation of the gene(s) permitted the direct identification of mutations using standard Southern or Northern blotting, PCR or RT-PCR techniques. We also use a PCR assay to study X inactivation. At the biochemical level, protein-based molecular diagnosis of the diseases can also be done by both immunoblotting (Fig, 1A) and immunocytochemistry (7, 8) (Fig. 3).

X-linked dystrophinopathy is the most common cause of muscular dystrophy. The high frequency of isolated male Duchenne patients (~30%) implies that there are also isolated female dystrophinopathy patients including Duchenne muscular dystrophy (DMD) carriers (9). Of the 3,048 diagnostic biopsies processed over 12 years at our Institute, 41 cases had the clinical diagnosis of limb-girdle muscular dystrophy (LGD) and 4 cases were quadriceps myopathy (QM). We found 17% of our LGD patients to be suffering from dystrophinopathy, indicating that they in fact had a disorder related to DMD/BMD (10) (Table 1). 31% (4/13) of isolated male LGD patients and 13% (2/15) of isolated female LGD patients were misclassified. All 4 patients with QM had clear abnormalities of dystrophin (11). Our study stresses the clinical overlap between LGD, QM and dystrophinopathy, and reinforces the necessity of dystrophin protein and gene studies for the accurate diagnosis of isolated cases of muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) and myotonic dystrophy (MD) are autosomal dominant PMD, and both show highly variable clinical phenotypes from almost normal (abortive) to severe forms. We analyzed nine FSHD families (7 familiar, and 2 sporadic patients), using a p13E-11 probe, specific to the most teromeric locus of chromosome 4q (distal to the D4S139 locus: (12). We found that p13E-11 probe detected EcoRI fragments of smaller size (13~27kb) in Japanese FSHD patients similar to those observed in Dutch patients, which thus co-segregated with Japanese FSHD (Fig.4) (13). The two point LOD score for linkage between D4S810 and FSHD was calculated with the LIPED program and was shown to have a maximum LOD score (Zmax) of 3.31 at a recombinant fraction () 0.00. Thus, so far neither patients with genetic heterogeneity nor recombinant FSHD have been found among the Japanese with FSHD examined. This study emphasizes the clinical importance of the p13E-11 probe for the diagnosis and genetic counseling of FSHD patients and their family. However, the overlap in the distribution of fragment size with controls and FSHD affected patients imply that p13E-11 could be considered as a tightly linked polymorphic marker of FSHD.

Figure 1: Dystrophin gene and protein abnormalities in a patient with muscular dystrophy (see ref. 5). A: Western blot with anti-30kD dystrophin antisera showing no detectable dystrophin in lane 2 from the patient 9. D=dystrophin, M=myosin. B: PCR multiplex analysis of genomic DNA, showing lane 2 and 5, patient 9; lane 1 and 4 normal controls; lane 3 and 6 are a negative control, a patient with complete deletion of his dystrophin gene. C: Southern blot to define the 5' end of patient 9's deletion (lane 2), showing exon numbers on the right.

Figure 2: DNA Analysis of Duchenne (DMD) and Becker (BMD) muscular dystrophy

Figure 3: Immunofluoresent analysis of dystrophin expression in skeletal muscles from other neurological diseases (a,b,c, as controls); BMD (d,e,f) and DMD (g,h,i). (See ref. 8). Frozen sections were stained with three region specific antibodies against dystrophin.

Figure 4: Southern blot analysis of EcoRI digested leucocyte genomic DNA from a FSHD family with the p13E-11 probe. Both the proband and his affected sister have a small (24kb) fragment which is not detected in the other family members (Ref. 13). Figure 5: Southern blot analysis of EcoRI digested leucocyte genomic DNA from a myotonic muscular dystrophy (lanes 3-6) family and two healthy controls (lanes 1,2) with the pM10M6 probe. The most severely affected patient (lane 4) had a larger 12kb fragment than his brother (lane 3; 10kb) or father (lane 6; 9.4kb).

Table 1: Summary of 41 limb-girdle patients included in the study (From Ref. 10)

Application of a chromosome 19q13 marker, pM10M6 (14), for the detection of the unstable triplet (CTG) repeat in MD has also been accomplished and was discussed (Fig.5).

We have recently reported a marked reduction of laminin subunits (particularly laminin M or merosin) in most muscle fibres of FCMD patients (15). The immunocytochemical pattern and intensity of laminin subunits were virtually normal in dystrophin-deficient, DAG-reduced, DMD muscle and other controls. Whether these findings in FCMD muscle represent a primary defect or an epiphenomenon remains unclear. One may infer that further studies are needed to identify the FCMD gene product that may interact directly or indirectly with dystrophin (16). More recently, we found a laminin M defect in dystrophic dy mice (17). This observation suggests that laminin M defect is primarily responsible for the pathogenesis of muscle fibre damage of the dy mice.

Accurate genetic counseling based on molecular genetic diagnosis is one of the most important targets of our research work. Thus we are trying to improve the molecular genetic techniques which are feasible on these families. However, we need to carefully preserve the privacy of the patients, even from her (or his) spouse unless she (or he) has told them.

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17. Arahata, K., et al. Laminin in animal models for muscular dystrophy: Defect of laminin M in skeletal and cardiac muscles and peripheral nerve of the homozygous dystrophic dy/dy mice. Proc. Japan Acad. 1993 (In Press).
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