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Previous Volume 329:915-920 September 23, 1993 Number 13 Next

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ABSTRACT

Background DNA analysis of peripheral-blood leukocytes is routinely used to demonstrate mutations in the dystrophin gene in patients with Duchenne's or Becker's muscular dystrophy. In approximately 35 percent of patients, DNA studies are not informative; in these patients immunochemical analysis of a muscle-biopsy specimen can determine whether dystrophin, the protein product of the gene for Duchenne's dystrophy, is present at reduced levels or absent. DNA analysis can be performed in amniocytes or chorionic-villus cells to identify mutations of the dystrophic gene prenatally, but immunochemical testing for dystrophin cannot be performed because the protein is not expressed in these cells.

Methods To circumvent this limitation in prenatal diagnosis, we induced myogenesis in 21 cultures of skin fibroblasts, 49 amniocyte cultures, and 6 chorionic-villus cell cultures by infecting the cells with a retrovirus vector containing MyoD, a gene regulating myogenesis. Transfection of MyoD into cells that do not normally develop into muscle cells results in the production of a protein that switches on myogenesis. We performed immunocytochemical analysis for dystrophin in the MyoD-converted muscle cells.

Results We found that 60 of 61 myotube cultures from subjects with no family history of Duchenne's dystrophy expressed dystrophin. Both myotube cultures from the two patients with Becker's dystrophy also expressed dystrophin, but all cultures from nine patients and two fetuses with Duchenne's dystrophy were dystrophin-deficient.

Conclusions Immunocytochemical analysis of dystrophin in genetically altered non-muscle cells is feasible and may be applicable to the prenatal and postnatal diagnosis of Duchenne's muscular dystrophy when conventional DNA analysis is not informative.

When myoblasts from fetal skeletal muscle or satellite cells from muscle specimens are grown in culture, the progeny fuse to form multinucleated myotubes that express muscle-specific proteins,⁷ including dystrophin⁸. Cultures derived from muscle satellite cells of patients with Duchenne's muscular dystrophy are deficient in dystrophin according to immunocytochemical⁹ or Western blot¹⁰ analysis. In cultured myotubes from one patient with Becker's muscular dystrophy, the expected abnormal dystrophin was found on Western blotting¹⁰. Prenatal study of dystrophin may be possible if amniocytes and chorionic-villus cells can be induced to express muscle-specific genes.

Davis et al.¹¹ isolated a gene, MyoD, which is expressed exclusively in skeletal muscle. Transfection of MyoD into cells that do not normally develop into muscle cells generates a protein that switches on the program of myogenesis. Other structurally related myogenic regulatory genes have similar actions¹². We used a MyoD retrovirus vector¹³ to convert cultured human fibroblasts, amniocytes, and chorionic-villus cells into cells expressing skeletal-muscle proteins, and performed immunocytochemical analysis for dystrophin to determine whether this technique could be used to diagnose Duchenne's muscular dystrophy in patients and fetuses at risk.

Methods

Patients

Duchenne's muscular dystrophy was diagnosed in nine boys and Becker's muscular dystrophy in two on the basis of their clinical and family history, serum creatine kinase measurement, and histologic examination of muscle. Five patients (four with Duchenne's dystrophy and one with Becker's dystrophy) had a deletion in the gene for Duchenne's dystrophy as determined by multiple-exon polymerase chain reaction,^14,15 and three patients with Duchenne's dystrophy had no detectable mutation. Three patients with Duchenne's dystrophy (two with a deletion and one not studied genetically) had no detectable dystrophin in muscle. Muscle specimens from the other eight boys were no longer available. We initiated cultures of skin fibroblasts from all 11 patients as described elsewhere⁷. Some cultures had been stored frozen for eight years or longer. As controls, cultures of fibroblasts from 10 patients who underwent biopsies to evaluate other neurologic or neuromuscular diseases were performed.

In an additional, blinded study we cultured amniocytes or chorionic-villus cells from four women with a family history of Duchenne's muscular dystrophy, each of whom was pregnant with a male fetus. One woman had an 18 percent risk of being a carrier, and the fetus had a 17 percent risk of being affected according to linkage analysis. Two months after birth the child had an increased serum level of creatine kinase, and dystrophin was undetectable in his muscle. The second woman was a definite carrier with a partial duplication of the gene for Duchenne's dystrophy. DNA analysis of amniocytes indicated that the fetus had inherited the mutation. The pregnancy was terminated, and dystrophin was undetectable in fetal muscle. The third woman, also a definite carrier, was pregnant with a fetus who had a risk of less than 1 percent of being affected according to linkage analysis. The pregnancy was continued to term, and the child was normal. The fourth woman had a deletion of the gene that was not detected in a culture of chorionic-villus cells, and the child was also normal. (Cultures and follow-up information about the pregnancies were kindly provided by Drs. C.T. Caskey and P.A. Ward, Baylor College of Medicine, Houston).

As controls, 48 amniocyte and 3 chorionic-villus cell cultures were performed. The cells had been obtained during routine karyotype screening of women with no family history of muscular dystrophy.

The research protocol for these studies was reviewed and approved by the institutional review board of Columbia University.

Culture Conditions

Initially, cultures were grown at 37 °C in 5 percent carbon dioxide and air, in 75-cm² flasks or 100-mm petri dishes in Dulbecco's modified Eagle's medium supplemented with 15 percent fetal bovine serum and containing gentamicin and amphotericin B (GIBCO, Life Technologies, Grand Island, N.Y.). For infection and G418 selection (see below), fetal bovine serum was inactivated by heating at 56 °C for one hour. After G418 selection, 10,000 or 20,000 trypsinized cells were plated on coverslips in 35-mm dishes. At confluency the serum concentration was reduced to 2 percent in order to promote muscle-cell fusion and differentiation.

Retrovirus Infection

To convert the cells, we used an amphotropic retrovirus vector containing the MyoD coding region and the bacterial gene for neomycin phosphotransferase (neo), which allows selection of infected cells with G418^13,16. Subconfluent cultures were infected by exposure to medium containing the retrovirus (approximately 10⁷ G418-resistant colony-forming units per milliliter)¹³ for 24 or 48 hours, with 4 µg of hexadimethrine bromide (Polybrene) per milliliter (Sigma, St. Louis). Selection was carried out in medium containing G418 (400 µg of active compound per milliliter [Geneticin, an analogue of neomycin; GIBCO]) for three or four days. Surviving cells continued to grow well, most likely because adequate medium conditioning had occurred before the G418-sensitive cells were eliminated.

Immunocytochemical Procedure

            Antibodies

We used a 60-kd polyclonal antibody directed against a fusion protein containing an amino acid sequence near the N-terminal of dystrophin¹⁷ and a monoclonal antibody (NCL-Dys2) directed against a synthetic polypeptide at the C-terminal of dystrophin (Novocastra Laboratories, Newcastle-upon-Tyne, United Kingdom). The 60-kd antibody was purified by repeated exposure to methanol-fixed monolayers of human fibroblasts to reduce diffuse background staining caused by cross-reactivity with dystrophin homologues, such as dystrophin-related protein^18,19. NCL-Dys2 antibody did not immunostain undifferentiated myoblasts, and on dot-blot analysis it did not cross-react with a fusion protein containing the C-terminal of dystrophin-related protein.

An anti-myosin heavy-chain monoclonal antibody recognizing sarcomeric myosin and an anti-titin antibody produced in guinea pigs were used as muscle-specific markers.

            Immunostaining

The cultures were grown on coverslips, rinsed in phosphate-buffered saline (pH 7.3), fixed in acetone at -10 °C for one minute, and air-dried. After exposure to the 60-kd or NCL-Dys2 antibody for 1 hour, the cultures were rinsed five times with phosphate-buffered saline and 10 percent calf serum for 5 minutes, then incubated with antisheep biotinylated antibody (cultures exposed to the 60-kd antibody) or with antimouse biotinylated antibody (cultures exposed to NCL-Dys2) for 45 minutes. After rinsing, the cultures were exposed to fluorescein-labeled streptavidin for 45 minutes. The cultures were pair-labeled with anti-myosin heavy-chain antibody or anti-titin antibody and rhodamine-conjugated second antibodies. Staining specificity was confirmed by incubating some cultures with nonimmune serum. The preparations were studied with an oil-immersion objective (63 x) in a Zeiss ultraviolet microscope with appropriate filters to detect fluorescein and rhodamine fluorescence.

Results

MyoD Conversion

First, we established that the MyoD gene could convert normal fibroblasts, amniocytes, and chorionic-villus cells (i.e., those unaffected by Duchenne's dystrophy) to muscle cells, and that differentiating myotubes produced detectable amounts of dystrophin on immunocytochemical examination. We exposed 10 fibroblast, 48 amniocyte, and 3 chorionic-villus cell cultures obtained for routine biochemical or genetic screening to the MyoD-encoding retrovirus, which also contained a neo gene (Figure 1A). Some multinucleated syncytia were noted as early as two to three days after selection in medium containing G418. Most cells, however, continued to proliferate and began to fuse as the cultures approached confluency (Figure 1B). Differentiation was further promoted by reducing the serum concentration of the medium.

View larger version (34K): [in this window] [in a new window]   Figure 1. Induction of Myogenesis in Cell Cultures to Permit Detection of Dystrophin Expression. Panel A shows the retrovirus vector used in these studies; it contains the MyoD coding region driven (direction indicated by arrows) by the viral long-terminal-repeat (LTR) promoter and a neomycin phosphotransferase gene (neo) driven by a simian virus 40 (SV) early promoter13. (A)n denotes the polyadenylation site. Panel B shows the steps leading to the detection of dystrophin expression (or its absence). Cultured amniocytes, skin fibroblasts, or chorionic-villus cells were infected with the MyoD retrovirus vector. The infected cells rendered resistant to G418 were transferred to 35-mm dishes with coverslip inserts. When the culture approached confluency, the cells were induced to fuse and differentiate in mitogen-poor medium. The cultures were fixed and evaluated for dystrophin expression as described in the Methods section.

 
Although G418-resistant cells were present in all cultures studied, the cell yield varied considerably. Cell counts performed in four fibroblast and nine amniocyte cultures indicated that at least 0.1 percent and up to 2 percent (mean, 1 percent) of fibroblasts survived G418 selection. The cell yield of amniocyte cultures was considerably higher (4 to 23 percent; mean, 13 percent). Clonal analysis of the G418-selected cells indicated that at least 5 percent and up to 90 percent (mean, 25 percent) of the cell colonies yielded multinucleated myotubes about two to three weeks after G418 selection. Similar results were obtained in cultures of cells from the patients with Duchenne's or Becker's muscular dystrophy. Despite the variable efficiency of MyoD conversion, sufficient numbers of myogenic cells for immunocytochemical analysis were generated from all cultures three to four weeks after exposure to the MyoD retrovirus.

Immunocytochemical Findings

G418-selected cells grown on coverslips were allowed to differentiate for three or seven days in mitogen-poor medium. The cultures were immunostained as described in the Methods section. Of the immunostained control cultures, 10 fibroblast, 47 amniocyte, and 3 chorionic-villus cell cultures expressed dystrophin in about 95 percent of multinucleated syncytia after three days (Table 1). At seven days all myotubes expressed dystrophin, as did some spindle-shaped cells (Figure 2A) or multipolar mononuclear cells. Staining was found predominantly at the cell surface and was either patchy or continuous (Figure 2A and Figure 2E). Some irregular areas of diffuse fluorescence were also commonly seen in the sarcoplasm (Figure 2A and Figure 2C). Paired labeling with anti-myosin heavy-chain antibody (Figure 2B, 2D, and 2F) or anti-titin antibody -- markers of muscle differentiation -- showed that these proteins were expressed concurrently with dystrophin. In one MyoD-converted control amniocyte culture, muscle cells that expressed myosin heavy chain did not stain for dystrophin, not even after two weeks in mitogen-poor medium (Table 1).

View this table: [in this window] [in a new window]   Table 1. Correlation of Clinical Phenotype with Dystrophin Expression in MyoD-Converted Cells.

 

View larger version (148K): [in this window] [in a new window]   Figure 2. Immunostaining of MyoD-Converted Cultures from Controls (Panels A through F) and a Patient with Duchenne's Muscular Dystrophy (Panel G and Panel H). Staining procedures are described in the Methods section. A postmitotic mononuclear muscle cell and multinucleate myotubes from controls express dystrophin at the cell surfaces (Panel A and Panel E) and in the cytoplasm (Panel A and Panel C). A myotube from the patient (Panel G) lacks immunostaining for dystrophin. The same muscle cells, pair-labeled with anti-myosin heavy-chain antibody used as a muscle-specific marker, show prominent immunostaining in all the cultures (Panels B, D, F, and H). The bar equals 20 microm.

 
            Patients

In contrast, myotubes from the nine patients with Duchenne's dystrophy did not show dystrophin fluorescence (Figure 2G and Table 1). Pair-labeling with anti-muscle myosin heavy-chain antibody or anti-titin antibody confirmed that the dystrophin-deficient multinucleated syncytia in the patients' cultures were differentiated muscle cells (Figure 2H). Dystrophin expression in myotubes from the two patients with Becker's dystrophy was similar to expression in control cell cultures.

            Prenatal Diagnosis

To assess the validity of the method as a tool for the prenatal diagnosis of Duchenne's dystrophy, we performed a blinded study in four coded cultures (three cultures of chorionic-villus cells and one of amniocytes). The cells had been obtained from four women (three of whom were definite carriers of mutations causing Duchenne's dystrophy, and one of whom had an 18 percent risk of being a carrier according to linkage analysis) who were pregnant with male fetuses, for whom adequate follow-up data on the outcome of their pregnancies were available (see the Methods section).

All four cultures were successfully converted by the MyoD retrovirus vector. The cultures were immunostained with NCL-Dys2 antibody and anti-titin antibody. The culture of chorionic-villus cells from the fetus with a 17 percent risk of being affected by Duchenne's dystrophy, as well as the culture of amniocytes from the fetus with a duplication in the gene for the disease, did not express dystrophin. The cultures of chorionic-villus cells from the two normal fetuses (one with a risk of less than 1 percent of being affected and one who did not inherit the deletion from the mother) showed normal staining for dystrophin (see the Methods section and Table 1).

Discussion

We have developed a method of prenatal diagnosis of Duchenne's muscular dystrophy that can be used when direct DNA analysis and linkage studies are inconclusive, obviating the invasive, technically difficult procedure of fetal muscle biopsy. We converted cells from easily accessible non-muscle tissues into myogenic cells and showed that normal amniocytes, chorionic-villus cells, and fibroblasts can be induced to express myogenic proteins, including dystrophin. Among the cell cultures that we studied, we correctly identified all patients and fetuses with Duchenne's dystrophy, demonstrated by the absence of immunostaining for dystrophin in MyoD-converted cells. With respect to the cell culture of the fetus in whom linkage analysis demonstrated only a 17 percent risk of Duchenne's dystrophy and in whom the diagnosis could be established only after birth, the MyoD-conversion method, showing dystrophin deficiency in chorionic-villus cells, would have aided in genetic counseling in this case if this method had been available during the pregnancy. The converted cells from the two patients with Becker's dystrophy showed normal staining for dystrophin, presumably because these patients expressed only a quantitatively reduced amount of dystrophin or a truncated protein that retained epitopes reacting with the antibody. Therefore, if the MyoD-conversion method were used to diagnose Becker's muscular dystrophy, a more specific analysis of dystrophin, by Western blotting¹⁰ or an enzyme-linked immunosorbent assay,²⁰ would be required.

One of the 61 control cultures showed no staining for dystrophin even though myoconversion was successful, as demonstrated by the expression of myosin heavy chain. This represents a possible error rate of less than 2 percent. The reason for the error is unclear. It is most likely that the level of expression of dystrophin was below the level of detection of the anti-dystrophin antibody that we used. Alternatively, since the fetal cells were those of a boy, this false negative result could have represented an unsuspected or spontaneous mutation of the dystrophin gene; however, follow-up was not possible because the cultures had been coded to maintain anonymity. It is unlikely that insertion of the retrovirus in genomic DNA could interfere with dystrophin expression, because the cultures were not derived from single clones.

Analysis of DNA in peripheral-blood lymphocytes, amniocytes, or chorionic-villus cells by Southern blotting identifies approximately two thirds of patients and fetuses with deletion mutations in the gene for Duchenne's dystrophy^1,2,3. Screening DNA for deletions by Southern blotting has been largely replaced by a simpler and more rapid method, multiple-exon polymerase chain reaction,^14,15 but duplication mutations, microdeletions, and point mutations (which occur in approximately one third of patients) may go undetected. A new powerful and rapid diagnostic technique has been developed by which trace amounts of dystrophin messenger RNA expressed in peripheral-blood lymphocytes and other non-muscle cells are reverse transcribed and amplified by nested polymerase chain reaction. This direct method can unambiguously identify deletions or insertions in the Duchenne's dystrophy gene²¹. The same technique can also identify point mutations through chemical mismatch analysis and direct sequencing to distinguish deleterious mutations from neutral polymorphisms²². Because chemical mismatch analysis and sequencing are not routine procedures in laboratories performing genetic diagnosis, this technique is not used at present.

When direct DNA analysis cannot detect a mutation, the current approach to prenatal diagnosis relies on linkage analysis²³. Southern blot-based linkage analysis is now being replaced by a more rapid and accurate polymerase-chain-reaction assay, which uses newly discovered highly polymorphic markers and can reduce the number of unresolved cases^24,25.

Obviously, direct methods to detect the mutations of Duchenne's dystrophy are preferred. Because the protein product (dystrophin) of the mutated gene in Duchenne's dystrophy and Becker's dystrophy is known, reaching an accurate direct diagnosis is also feasible in most cases by study of the abnormal protein qualitatively or quantitatively in muscle-biopsy specimens^4,20. However, dystrophin testing for prenatal diagnosis is only feasible by study of a fetal muscle specimen^5,6.

The MyoD-conversion method should permit Duchenne's muscular dystrophy to be diagnosed by a simple skin-punch biopsy. This method may even be useful in producing an entirely new source of donor myoblasts to be implanted into the muscle of patients with genetic defects such as Duchenne's muscular dystrophy²⁶. Most important, however, this technique should be applicable to the identification of dystrophin deficiency in fetuses with this disorder whenever conventional DNA analysis is uninformative, and it could be performed in diagnostic laboratories skilled in tissue culture and immunocytochemical examination.

Supported by grants from the National Institutes of Health and the Muscular Dystrophy Association and by an award from Telethon-Italy (to Dr. Mongini). Dr. Sancho was the recipient of a Muscular Dystrophy Association Sammy Davis, Jr., Neuromuscular Disease Research Fellowship.

We are indebted to Drs. L.P. Rowland, S. DiMauro, E. Bonilla, S. Shanske, and P.A. Ward for their advice and criticism; to Drs. N. Kardon and D. Warburton for providing us with amniocyte cultures; to Dr. E. Hoffman (University of Pittsburgh) for the 60-kd antibody; to Drs. D. Fischman and D. Bader (Cornell University) for anti-myosin heavy-chain antibody; and to Dr. G. Salviati (University of Padua, Italy) for anti-titin antibody.

Source Information

From the Departments of Pathology (S.S., A.F.M.) and Neurology (K.T., W.F.W.), Columbia University College of Physicians and Surgeons, New York; the Department of Neurology, University of Turin, Clinica Neurologica II, Turin, Italy (T.M.); and the Departments of Genetics (H.W.) and Molecular Medicine (S.J.T., A.D.M.), Fred Hutchinson Cancer Research Center, Seattle.

Address reprint requests to Dr. Miranda at the Department of Pathology, Columbia University College of Physicians and Surgeons, 630 W. 168th St., New York, NY 10032.

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