SLIDES

OMIM ENTRY for DMD an official site with complete, up to date information

A. Nature of the Disease

- a neuromuscular dystrophy-there are several types, but DMD is the most common (G.B.A Duchenne, a French neurologist 1868)
- a degenerative muscle disease - often associated with mental retardation
- affects about 1/3300 males - one of the most common genetic diseases
- BMD (Becker) occurs in 1 out of 30,000 live male births
- onset of the disease at 3-4 years of age; wheelchair dependency at ~ 10 yrs.
- death generally in the second decade

no known cure at the moment, although cell therapy with muscle stem cells has been proposed and the first attempt in humans was described in the LA Times ten years ago (~Feb.14, 1990)
- (most recently it was shown that transplanted bone marrow cells can populate muscle in mice and potentially become stem cells for myogenesis)

B. Genetics of the disease

- a sex-linked recessive disease, i.e. the gene maps to the X chromosome
- 2/3 of the cases are familial, i.e. there are pedigrees suggesting that the mother is a carrier, i.e. other affected male sons exist
- 1/3 of the cases are sporadic, i.e. they appear to be due to new mutations; this is unusual, because the mutation rate at this locus must be exceptionally high to account for the large absolute number of sporadic cases (explanation to follow)

- occasionally females are affected: they are found to have X/autosome translocations (explanation to follow)

C. Cloning of the Gene for DMD

A. several motivations for cloning the gene (pre 1987):

1. because of the high incidence, carrier identification would be highly desirable, but in the absence of a biochemical test (enzyme assay) this was not possible
- can one find a closely linked RFLP or even a probe for the gene itself to do the testing?

2. cloning of the gene would lead to the sequence, hence to a protein sequence, or a protein product made in bacteria: a) one can make an antibody to localize the protein and perhaps study it in carriers b) the aa sequence may reveal the nature and function of the protein
- a cloned gene would also allow the use of probes and other approaches for a direct identification of mutated genes in potential carrier females

3. mapping
   a. females with the disease had X/autosome translocations with variable breakpoints on different autosomes, but there was always a breakpoint within band Xp21
note that in such translocations the normal X chromosome is preferentially inactivated, i.e. the normal X chromosome is the late replicating X, and the normal DMD gene is silenced
- hypothesis: the break has been made right in the middle of the gene; therefore, heterozygous females express the DMD phenotye

   b. males with X chromosome deletions
- a male patient was described and cytologically characterized in 1985 who had the following problems:
                    chronic granulomatous disease associated with cytochrome b245 deficiency
                    McLeod red cell phenotype - associated with an abnormal antigen
                    Duchenne muscular dystrophy
                    retinitis pigmentosa - a diffuse atrophy of pigment epithelium of the retina
                    slight mental retardation

all these diseases had been mapped to the X chromosome
- cytological investigations showed a reduced size of the central dark band on the short arm (Xp21); question: was the material from this band elsewhere in the genome of this patient?
- the child had been adopted, and the parents were unknown

i) investigators made somatic cell hybrids in which the deleted chromosome was the only human X chromosome present on a Chinese hamster background

ii) investigators made a lymphoblastoid cell line from the patient by transformation with EBV
- hybridized to 20 different anonymous fragments previously isolated from human Xp.
- Luck (!!!!!!): one of the probes (#754) was missing from the patient's X chromosome and from total DNA from his    lymphoblast line

CONCLUSION:   there is a deletion which is very precisely mapped, AND one has a cloned fragment of DNA from this region
the region still contains potentially many genes, and the fragment may be part of any one of them;
assumption: the DMD locus is within this region;

B. Two independent strategies for cloning the gene

1. Kunkel et al. "subtraction hybridization"

i) 49,XXXXY DNA from "normal" individual digested with MboI
ii) DNA from patient with DMD, RP, CGD and the deletion was sheared
iii) a 200 fold excess of sheared DNA was mixed with MboI-cleaved DNA from 49,XXXXY "normal" individual
iv) use of "PERT technique" to increase the reassociation reaction....
only DNA fragments from the region of the deletion will form clonable restriction fragments with MboI sites at both ends; all others will have staggered nonrecognizable ends

cloning into the BamHI site of pBR322 yields a library which can be screened for DNA segments which are derived from the region deleted in the patient

----> pERT clones - still not definitely identified as being from the DMD locus, but potentially close

verified by Southern blots to DNA from human cells with X chromosome deletions, and hybrid cells with abnormal X chromosomes

2. Worton et al. used female with DMD with a translocation (X;21)(p21;p11) chromosome 21 is an acrocentric chromosome with a very small short arm which encodes 30-40 repeats of 43 kb containing the 18S, 5.8S, and 28S ribosomal genes

                                              2121rrrrrrrrrrrrrrrrrrrrrr..........CEN-21......q
                                                                            X
xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxDMDxxxxxxxxxxxxxxxxxxxxxxxCEN-X....q

recombinant chromosomes: der 21 and der X  (der - derivative; named after centromere)

der21 xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxDMrrrrrrrrrrrrrr..........CEN-21......q

                                                         derX    2121rrrrrrrrDxxxxxxxxxxxxxxxxxxxxxxxCEN-X

the ribosomal DNA clones were available, but there are ribosomal gene clusters on several acrocentric human chromosomes: 13, 14, 15, 21, 22

the translocation chromosomes were separated from other human chromosomes in somatic cell hybrids with mouse A9 cells.

Mouse and human ribosomal DNA gene sequences must be distinguished: they vary in the spacer region

...........|----------***|----------***|----------***|----------***|----------***|..................

13,000 nt transcript (45S) is processed in the nucleolus to
18S rRNA (~2000 nt) + 5.8S rRNA (~160 nt) + 28S rRNA (~5000 nt)
- small ribosome, large ribosome

three useful hybrids were generated: two with the derivative X, and one with the derivative 21.

the junction creates a new restriction fragment
- use of several probes from the 43 kb repeat to look at restriction fragments from hybrid DNA cut with a variety of restriction enzymes to find a unique restriction fragment derived from the X;21 junction region (one copy per cell)
- such a unique restriction fragment must differ from restriction fragments from normal human DNA detected with the same probe

Note: one could not use just 18S or 28S rRNA probes, because these would also hybridize to the mouse rRNA genes

- a probe identifying uniquely human rDNA and such a junction region was found
- use of the same probe to isolate the corresponding region from phage or plasmid libraries made from the hybrids

spacer...18S...5.8S.....28S.J.Dxxxxxxxxx...

a probe (100-3) was isolated and shown to be specific for human 28S rDNA which was even closer to the junction

after lots of work: junction region cloned, and hence X chromosome-derived human DNA clones close to or within DMD locus (the female is affected-the locus is likely to be disrupted at the junction)
clone XJ-1.1
    linkage to TaqI RFLP that tracks DMD through pedigrees
    mapping of pERT clones near junction fragment XJ-1.1
    mapping junction fragment XJ-1.1 in Xp21 deletion

3. cloning of full length cDNA and identification of mRNA (~ 16,000 nt) in muscle
                                       - abnormal transcripts in muscle of some DMD patients

D. Physical Mapping of the DMD Locus

- the DMD locus includes >2.3 Mb of DNA
- analysis by pulsed field electrophoresis (CHEF gels)

E. Identification of the Gene Product and Its Possible Function

- probes on Northern blots detect 16 kb transcript (long!!!) in muscle but not in fibroblasts
- cDNA clones identified by Worton et al and Kunkel et al: none of the cDNAs were complete: W and K had different cDNAs which however detected the same transcript on a Northern blot
- suggestion:      very long transcript very large gene: lots of exons (79) and introns

lots of work to get the whole cDNA sequence: obviously, none of the labs rested until this was accomplished

1. nature of the protein
        multiple domains in the protein

        repeat domains in the central portion
        relationship of domains to spectrin and a-actinin
        antiparallel homodimer is most likely quaternary structure

- possibility of unequal crossing over within the gene generating duplications and deletions
- explanation for milder form in BMD patients: the reading frame is preserved
- in the more severe DMD patients there is also a reading frame shift and hence no useful proteinis made

2. localization in muscle cells
    association with plasma membrane glycoprotein
    link between PM (sarcolemma) and cytoskeleton (discussion in Cell Biology Lectures)

Selected References

- Boyce, F.M., Beggs, A.H., Feener, C., and Kunkel, L.M. (1991). Dystrophin is transcribed in brain from a distant upstream promoter. PNAS 88, 1276-1280.
- Den Dunnen, J.T., Grootscholten, P.M., Bakker, E., Blonden, L.A.J., Ginjaar, H.B., Wapenaar, M.C., Van Paassen, H.M.B., Van Broeckhoven, C., Pearson, P.L., and van Ommen, G.J.B. (1989). Topography of the Duchenne muscular dystrophy (DMD) gene: FIGE and cDNA analysis of 194 cases reveals 115 deletions and 13 duplications. Am. J. Hum. Genet. 45, 835-847.
- Ervasti, J.M., and Campbell, K.P. (1991). Membrane organization of the dystrophin-glycoprotein complex. Cell 66, 1121-1131.
- Francke, U., et.al (1985). Minor Xp21 chromosome deletion in a male associated with expression of Duchenne muscular dystrophy, Chronic Granulaomatous Disease, retinitis pigmentosa, and McLeod syndrome. Am. J. Hum. Genet. 37, 250-267.
- Koenig, M., Beggs, A.H., Moyer, M., Scherpf, S., Heindrich, K., Bettecken, T., Meng, G., Müller, C.R., Lindlöf, M., Kaariainen, H., De la Chapelle, A., Kiuru, A., Savontaus, M.-L., Gilgenkrantz, H., Récan, D., Chelly, J., Kaplan, J.-C., Covone, A.E., Archidiacono, N., Romeo, G., Liechti-Gallati, S., Schneider, V., Braga, S., and Moser, H. (1989). The molecular basis for Duchenne versus Becker muscular dystrophy: Correlation of severity with type of deletion. Am. J. Hum. Genet. 45, 498-506.
- Koenig, M., Beggs, A.H., Moyer, M., Scherpf, S., Heindrich, K.,
- Koenig, M., Hoffman, E.P., Bertelson, C.J., Monaco, A.P., Feener, C., and Kunkel, L.M. (1987). Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell 50, 509-517.
- Koenig, M., Monaco, A.P., and Kunkel, L.M. (1988). The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein. Cell 53, 219-226.
- Kunkel, L.M., Monaco, A.P., Middlesworth, W., Ochs, H.D., and Latt, S.A. (1985). Specific cloning of DNA fragments absent from the DNA of a male patient with an X chromosome deletion. Proc. Natl. Acad. Sci. USA 82, 4778-4782.
- Mandel, J.L. (1989). Dystrophin: The gene and its product. Nature 339, 584-586.
- Monaco, A.P. (1989). Dystrophin, the protein product of the Duchenne/Becker muscular dystrophy gene. TIBS 14, 412-415.
- Ray, P.N., Belfall, B., Duff, C., Logan, C., Kean, V., Thompson, M.W., Sylvester, J.E., Gorski, J.L., Scjmickel, R.D., and Worton, R.G. (1985). Cloning of the breakpoint of an X;21 translocation associated with Duchenne muscular dystrophy. Nature 318, 672-675.
- Rowland, L.P. (1988). Dystrophin: a triumph of reverse genetics and the end of the beginning. New Engl. J. of Med. 318, 1392-1394.
- van Ommen, G.J.B., Verkerk, J.M.H., Hofker, M.H., Monaco, A.P., Kunkel, L.M., Ray, P., Worton, R., Wieringa, B., Bakker, E., and Pearson, P.L. (1986). A physical map of 4 million bp around the Duchenne muscula dystrophy gene on the human X chromosome. Cell 47, 499-504.
-Anderson, M.S. and Kunkel, L.M. (1992). The molecular and biochemical basis of Duchenne muscular dystrophy. Trends Biochem. Sci. 17, 289-292.
- Tennyson, C.N., Klamut, H.J., and Worton, R.G. (1995). The human dystrophin gene requires 16 hours to be transcribed and is cotranscriptionally spliced. Nature Genet. 9, 184-190.