Molecular genetics of myotonic dystrophy

syndrome,

Molecular Genetics of Myotonic Dystrophy M. Benjamin Perryman, David L. Friedman, Y.-H. Fu, and C. Thomas Caskey

Myotonic muscular dystrophy (DM) has been shown to be caused by the expansion of an unstable triplet nucleotide repeat sequence located in the 3’ untranslated region of a gene coding for a putative serine-threonine protein kinase. Isolation of genomic and cDNA clones for the DM kinase have significantly simplified the genetic diagnosis of DM. The cellular localization, enzymatic activity, and role in the pathophysiology of DM of the kinase protein are as yet unknown. (Trends

Cardiovasc

Med

1993;3:82-84)

Myotonic

muscular

dystrophy

the most

prevalent

form

dystrophy

in adults

Duchenne-Becker in incidence

and

(1909), dominant

plete penetrance ity. The unique

disease pattern

other

is inherited

including

of muscular

(1989)].

cataracts,

many

gastrointesti-

[for a review, see usually

con-

a skeletal muscle

disease, DM, like Duchenne-Becker cular dystrophy,

ptosis,

may be involved,

Although

sidered to be primarily

by a

weakness

In addition,

cardiovascular,

nal, and reproductive Harper

expressiv-

is characterized

systems

as an

defect with incom-

and variable

balding.

organ

of inherited

DM, first described

and wasting, myotonia, and frontal

to

dystrophy

of new cases

by Steiner-t autosomal

is second

muscular

muscular dystrophies.

(DM) is

of muscular

mus-

has a high incidence

of

serious cardiac involvement.

Cardiac dys-

function

reported

in DM has been

many instances,

in

but its exact incidence

M. Benjamin Perxyman is at the Division of Cardiology, the University of Colorado Health Sciences Center, Denver, CO 80262; and David L. Friedman is at the Section of Cardiology, Y.-H. Fu is at the Institute of Molecular Genetics, and C. Thomas Caskey is at the Institute of Molecular Genetics and Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX 77030, USA.

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has not been established. The more common findings involving the heart in DM are conduction abnormalities, particularly heart block, atria1 arrhythmias, and cardiomyopathy (Prystowsky et al. 1979, Moorman et al. 1985, Hiromasa et al. 1987). The severity of the disease ranges from obligate heterozygotes with no detectable symptoms to profoundly disabled individuals. A particularly devastating form of the disease is congenital DM in which hypotonia, muscle weakness, mental retardation, respiratory distress, and feeding difficulty appear in children of affected mothers. In most instances, the mothers of congenital DM patients have mild or subclinical disease. The entire range of severity of presentation may be found within a single sibship. It has been reported that disease symptoms begin progressively earlier and more severe disease is present in successive generations of families with DM, but this phenomenon, termed anticipation, has been considered an artifact due to ascertainment bias (Penrose 1948). One reason for the reluctance to accept anticipation as a biological phenomenon was the difficulty in providing an explanation of the mechanism on the basis of Mendelian inheritance. The identification of an unstable CGG triplet repeat in the 5’ untranslated region of the FMR-1 gene as the molecular basis for fragile X

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another

disease

character-

ized by the anticipation phenomenon, provided both a molecular explanation for anticipation consistent with Mendelian inheritance and a potential strategy for cloning the defect responsible for DM [for a review of unstable triplet repeats as a cause of human disease, see Caskey et al. (1992)]. With oligonucleotide probes containing GC-rich triplets to identify repeat sequences (Fu et al. 1992) and positional cloning strategies (Buxton et al. 1992a, Harley et al. 1992a, Brooke et al. 1992, Aslanidis et al. 1992, Mahadevan et al. 1992), an unstable GCT nucleotide repeat was identified as the DM mutation. In children born with congenital DM, the expansion of the triplet repeat sequence was shown to be up to several kilobases in length. The DM GCT repeat sequence is highly polymorphic, with most unaffected individuals having 5-30 repeats, 13 repeats being the most frequent allele size. The identification of expansion of the GCT repeat sequence as the mutation responsible for DM has greatly simplified disease diagnosis and family counseling. cDNA or genomic probes can be used to detect the large expansion of the GCT repeat sequence found in congenital cases, and polymerase chain reaction (PCR) amplification using oligonucleotide primers flanking the repeats can be used to detect smaller expansions of the region. While it has been shown that the premutation expansion allele size for fragile X is 52-100 repeats, the same information for DM premutation allele size has not been rigorously established. It has been recently proposed that the size of the expansion in DM can be used to make predictions about disease severity (Harley et al. 1992b), particularly in congenital cases (Tsilfidis et al. 1992). While disease severity may correlate broadly with expansion size, caution must be used in applying predictions to individual patients, since disease severity may also increase with a concomitant decrease in expansion of the repeat sequence within a single family, suggesting a complex relationship between repeat stability and disease severity (Ashizawa et al. 1992). The complex nature of this relationship is further emphasized by the inheritence pattern of the congenital form of DM (see below). DNA probes derived from the region containing the CTG repeat sequence

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were used to isolate cDNAs confirming

AMINO ACID SEQUENCE

that the CTG repeat

1

sequence

is tran-

100

200

scribed (Fu et al. 1992, Brook et al. 1992, Mahadevan et al. 1992). With the cDNA as a probe, a 3.0- to 3.3-kb transcript

400

500

600

I

is

CONSENSUS SEQUENCES

identified in skeletal muscle, heart, and brain. Rather surprisingly, nucleotide sequence analysis of the DM cDNA revealed that the GCT repeat sequence was in the 3’ untranslated portion of the mRNA. In the other two human diseases

Protein Kinase Domain

known to be caused by expansion of unstable triplet repeats, Kennedy’s syndrome and fragile X syndrome, the expanded triplet repeats are found in the coding sequence and the 5’ untranslated sequence, respectively [for a review, see Caskey et al. (1992)]. We have used the Baylor College of Medicine Molecular Biology Information Resource to analyze the predicted domain structure of the DM protein deduced from the cDNA nucleotide sequence. The myotonic dystrophy gene codes for a protein of M, = 55,000. Three regions within the myotonic dystrophy gene product have significant homology with other known proteins. The first is an N-terminal protein kinase domain. The sequence is homologous with several different kinases, especially the CAMPand cGMP-dependent protein kinases, protein kinase C, calcium-calmodulindependent protein kinase, l3-adrenergic receptor kinase, and myosin light-chain kinase. This region also includes a sequence that is similar to an ATP-binding domain present in the myosin head. In the carboxy-terminal region of the DM gene product, there is a stretch of 60 amino acids that is similar to the coiledcoil domain characteristic of a-helical fibrous proteins. The coiled-coil sequence forms the basis for the side-to-side associations of subunits to form filaments and is characterized by a 28-amino-acid stretch that contains four heptad units. Each heptad unit contains a regular array of hydrophobic and charged amino acids, which segregate within the coiled structure to form nonpolar surfaces and positively and negatively charged surfaces, and align in a complementary fashion to stabilize filament formation (McLachlan and Karn 1982). A putative transmembrane domain is located near the C-terminal end of the protein and has a high degree of sequence similarity to the 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMG-COA reductase)

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300

Coiled-Coil Domain

TransDomain Membrane

Figure 1. Diagram of the consensus domain structure of myotonic dystrophy kinase.

transmembrane domain (Jingami et al. 1987). The transmembrane domain of HMG-CoA reductase spans the membrane seven times while the DM kinase domain contains a single membranespanning helix. A schematic summary of the predicted DM kinase domain structure is shown in Figure 1. Additional cDNAs isolated from mouse and human brain and heart libraries have detected multiple splice forms of the kinase mRNA (Jansen et al. 1992b). All of the splice forms in mouse and human revealed a complex pattern that both differed in length and resulted in a change in reading frame. These splice forms result in differences in the protein, which would be expected to alter the transmembrane domain, perhaps resulting in proteins that differ in cellular location and enzymatic activity. To facilitate the studies of alternative splicing and transcriptional control of DM kinase gene expression, we have recently determined the nucleotide sequence for the entire human DM kinase gene. The sequence may be accessed through Gene Bank accession number VO0727. Using a synthetic peptide antigen synthesized from the deduced amino acid sequence of the DM kinase, we have produced antisera that recognize proteins of molecular weights predicted from the alternative splicing pattern reported by Jansen et al. (1992b). It is not yet known whether the alternative splice forms are biologically relevant for tissue or developmental regulation or if they play any role in the pathogenesis of the disease. The major DM kinase gene detected in myocardium is the fulllength M, = 55,000 protein. A complete physical map of the region surrounding the DM region on human chromosome 19 has been developed, and a combination of cosmid and yeast

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artificial chromosome clones that span the region has been isolated (Jansen et al. 1992a, Buxton et al. 1992b, Shutler et al. 1992). These studies indicate that several other genes are located in the DM region of human chromosome 19. Whether expansion of the unstable GCT repeat sequence has any effect on transcription of these genes is currently unknown. Despite the remarkable progress in understanding the molecular genetics of DM, much remains to be learned. It is not yet known how the mutation affects the expression of the kinase protein to produce the disease in a dominant manner. It is possible to speculate that expansion of the repeat sequence in the 3’ untranslated region might either stabilize mRNA transcripts, resulting in increased protein expression and therefore protein kinase activity, or alternatively decrease message stability, resulting in underexpression of the protein and reduced kinase activity. Expansion of the repeat sequence might also affect expression of other genes in the DM region of chromosome 19. An especially intriguing aspect of the repeat is the question of the role of mitotic versus meiotic repeat expansion. The CGG repeat sequences in fragile X syndrome exhibit mitotic instability resulting in somatic mosaicism [for a review, see Caskey et al. (1992)]. Mitotic instability in the DM repeat region could play a role in generation of extremely variable symptomology observed in individuals with the disease. It would also appear that the repeat region may be more unstable in meiotic events in females, since the extremely large expansions seen in the congenital disease are the result of maternal transmission of the mutant allele. Finally, there are many questions concerning the cellular localization, enzymatic activity,

a3

developmental and tissue-specific regulation, and role of the kinase protein in the pathophysiology of DM that remain to be answered. Unraveling the answer to these questions will provide insight into a number of pathophysiologic processes, including muscle weakness and wasting, cardiac conduction abnorrnalities, and cataract formation.

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