The Dark Side of RNA

Strange as it seems, a new class of diseases is emerging that appears to be caused by mutations in untranslated regions of RNA. The protein-coding sequence of the relevant gene is uninterrupted and yet features of the disease flourish. Exactly how this happens is the subject of keen investigation that now promises to further intensify, following the publication in August of an important discovery. Researchers at the University of Minnesota showed that myotonic dystrophy, known formally as dystrophia myotonica (DM), derives primarily from a mutation in untranslated RNA.1 Their finding, which concerns DM type 2, corroborates evidence of a decade earlier that DM1 is similarly caused.2 The two mutations, which occur on chromosomes 19 and 3, respectively, both result in the multisystemic signs and symptoms that characterize myotonic dystrophy. Moreover, both mutations are repeat expansions-nucleotide sequences that recur adjacently many more times than normal. A third type of DM is thought to exist, but it has not been identified. "I think this will be considered the major pathogenic mechanism for DM," asserts Laura P.W. Ranum, the DM2 paper's senior author and a University of Minnesota associate professor of genetics, cell biology, and development. The paper points out that untranslated repeat expansions also are involved in two types of spinocerebellar ataxia, SCA8 and SCA10. However, the role that transcribed RNA plays in their pathogenesis is yet to be conclusively shown. Admits coauthor John W. Day, an associate professor in the university's department of neurology: "We're not sure where this will lead." He considers it likely that researchers will now focus relatively more on the mutant RNA and less on protein kinases thought to be involved in DM. First described almost a century ago, myotonic dystrophy is the most common form of muscular dystrophy that affects adults, with an estimated minimum incidence of one in 8,000. Dominantly inherited, it causes muscle contraction problems, weakness, and emaciation. Yet, the disease also produces abnormalities in other systems, including cardiac conduction defects, cataracts, abnormal glucose response, neuropsychiatric impairment, and, in males, premature balding and testicular atrophy. After the DM1 repeat expansion was identified, scientists focused on possible regulatory problems of the relevant gene, dystrophia myotonica protein kinase (DMPK). It codes for a protein of the same name that is important to muscle function. It had long been known that DM patients have reduced phosphorylation of membrane proteins in muscle tissue and red blood cells, suggesting the possibility of a protein kinase deficiency. In 1996, knockout mice were developed to see if the absence of DMPK would result in DM's constellation of clinical features. But instead of multisystemic effects, only problems of skeletal muscle structure and function were found.3 In 1999, electrophysiological analysis of mice lacking DMPK demonstrated cardiac conduction disturbances.4 In May 2000, two groups simultaneously showed that mice deficient in a gene called SIX5, homologous to an eye development gene in the fruit fly, would develop cataracts.5,6 (The DM1 mutation extends into SIX5, which is adjacent to DMPK.) A few months later, researchers demonstrated that even when the untranslated RNA repeat of DM1 is expressed in messenger RNA (mRNA) unrelated to DMPK, mice will still develop the disease's problems with muscle relaxation and weakness.7 Three Theories Prevail These and other investigations have kept alive three theories concerning how repeat expansions in a noncoding region of a gene can result in DM1. The first concerns disturbances at the DNA level, where the mutation begins as a CTG expansion. It somehow alters the regional chromatin or genetic material in the nucleus, suppressing expression of SIX5 and other nearby genes, together resulting in the various features of DM1. The second theory involves a loss of function at the DMPK protein level. This occurs after the mutation is transcribed from its CTG sequence on the DNA strand to a CUG sequence in the RNA (the thymine being replaced by uracil). The then-mutant RNA does not leave the nucleus to enter the cytoplasm, where translation of mRNA into protein normally takes place. As a result, less DMPK protein is produced. The third theory posits a "toxic gain" at the RNA level: once transcribed, the mutant RNA binds to other proteins and disrupts nuclear function. Researchers wondered which of the theories would be corroborated by the discovery of DM2's underlying cause. The University of Minnesota group's paper shows that the repeat expansion in RNA occurs in DM2, just as it does in DM1, thus identifying this mutation as responsible for the disease's multisystemic effects. The RNA toxic gain theory is ascendant, but how scientists will view contributing causes of DM, says Ranum, "almost depends on if you're a lumper or a splitter." To date, researchers have concentrated more on the likenesses than the differences between DM1 and DM2, observes Stephen J. Tapscott, a neurologist in the human biology division of the Fred Hutchinson Cancer Research Center in Seattle. Tapscott and colleagues showed that disruption of SIX5 in mice causes cataracts.5 He agrees that the Ranum group's findings "should get a lot of people interested" in studying the RNA mutations, but another question is how might other effects contribute to DM's clinical profile? The more severe of the two disease types is DM1, which is at its worst in a congenital form transmitted maternally. Generally, DM is transmitted paternally. "What's special about DM1, that it's more severe?" he asks. "It's going to be important to look at electrocardiac conduction abnormalities and other discrete features." Baylor College of Medicine neurologist Tetsuo Ashizawa notes that the DM2 mutation occurs in a gene that codes for an RNA-binding protein. The gene, zinc finger protein 9, may interact with RNA and DNA, including the promoter regions of some genes. This raises a possibility that the expansion affects the expression of certain genes by changing the ZNF9 expression. Ashizawa believes that more research concerning the RNA expansion's toxic gain of function is now justified, but he cautions that suppressed expression of neighboring genes by the mutant DNA cannot be ignored, either. Last year, Ashizawa and colleagues identified a repeat expansion in an intron, a noncoding region of the gene that is responsible for SCA10.8 Ashizawa thinks chances are good that SCA10 will prove to be a third RNA disease. However, he warns, "there isn't really compelling evidence yet that this expansion is transcribed into RNA." No changes in mRNA levels were found in the lymphoblast cell lines studied, but he points out that SCA10 does not affect these tissues. His group is now working on the transcription question. Unstable Expansions The mutations that cause DM and SCA10 are unstable. There is evidence of genetic anticipation (decreasing age of onset and increasing severity through the generations), but the correlation between age of onset and repeat size is not consistent. The expansions can vary widely in size between families and individuals, and even within different tissues of one person. In the case of DM2, the pathogenic range can extend from 50 repeats to more than 11,000. The picture is even murkier for SCA8. For example, its expansion shows up in people who don't have the disease, in families with no history of ataxia. Consequently, concerns exist over whether the expansion of the untranslated CTG repeat in SCA8 is the true, disease-causing mutation. Ashizawa says incomplete penetrance could explain these false positives. Penetrance, the probability of expressing a phenotype given a genotype, may be regulated by modifier genes. A transgenic mouse model whose CTG repeat mutation is expressed at the RNA level should provide critical data to settle this controversy. Other mysteries concerning RNA diseases also remain. One concerns periodic interruption of the primary expanded motif in both DM2 and SCA8 by another repeat mutation. John B. Vincent, a neurogeneticist at the Hospital for Sick Children in Toronto, has closely studied SCA8 mutations, including the CTG repeat and its CTA interrupts. "The CTA repeat length and/or repeat interrupts may be important for the pathogenicity or penetrance of the expanded alleles," he says. However, work by Ranum's group suggests no such evidence. Vincent's team has found expansions at the SCA8 locus in psychiatric patients who don't have ataxia, but not frequently enough to prove a connection. "I don't have a strong conviction that we will find a clear link between the SCA8 expansion and major psychoses," he acknowledges. "However, as a genetic model for psychiatric disorders, unstable DNA mutations, particularly working via pathogenic RNAs, could explain many of the inconsistencies with Mendelian inheritance such as low penetrance, genetic anticipation, parent-of-origin effect and discordance between monozygotic twins." Ranum, whose group first identified the SCA8 mutation in 1999,9 says, "the CTG expansion at the SCA8 locus can cause ataxia but it can also not develop, and we don't know why." She adds, "To me, the thing that makes the most sense is there's another, unlinked genetic factor." In any case, she believes that RNA disease might not be limited to DM and SCA. "I think it could very well be something that's found more and more." Steve Bunk (sbunk@the-scientist.com) is a contributing editor for The Scientist. References 1. C.L. Liquori et al., "Myotonic dystrophy type 2 caused by a CCTG expansion in intron 1 of ZNF9," Science, 293:864-7, Aug. 3, 2001. 2. J.D. Brook et al., "Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3' end of a transcript encoding a protein kinase family member," Cell, 68:799-808, 1992. 3. S. Reddy et al., "Mice lacking the myotonic dystrophy protein kinase develop a late onset progressive myopathy," Nature Genetics, 13:325-35, 1996. 4. C.I. Berul et al., "DMPK dosage alterations result in atrioventricular conduction abnormalities in a mouse myotonic dystrophy model," The Journal of Clinical Investigation, 103[4]:R1-7, 1999. 5. T.R. Klesert et al., "Mice deficient in Six5 develop cataracts: implications for myotonic dystrophy," Nature Genetics, 25:105-9, 2000. 6. P.S. Sarkar et al., "Heterozygous loss of Six5 in mice is sufficient to cause ocular cataracts," Nature Genetics, 25:110-4, 2000. 7. A. Mankodi et al., "Myotonic dystrophy in transgenic mice expressing an expanded CUG repeat," Science, 289:1701-2, 2000. 8. T. Matsuura et al., "Large expansion of the ATTCT pentanucleotide repeat in spinocerebellar ataxia type 10," Nature Genetics, 26:191-4, 2000. 9. M.D. Koob et al., "An untranslated CTG expansion causes a novel form of spinocerebellar ataxia (SCA8)," Nature Genetics, 21:379-84, 1999. See also, S. Bunk, "Ataxia discoveries open window to neurodegeneration," The Scientist, 13[9]:14, April 26, 1999.

The Dark Side of RNA

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