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New Facioscapulohumeral Dystrophy Mutation Explains FSHD2, and More

Robinson, Richard

doi: 10.1097/01.NT.0000425717.04100.ef
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MUSCLES AFFECTED in facioscapulohumeral dystrophy

MUSCLES AFFECTED in facioscapulohumeral dystrophy

Investigators implicate mutations in a gene called structural maintenance of chromosomes flexible hinge domain-containing protein 1 (SMCHD1) as a cause of facioscapulohumeral dystrophy.

A new gene discovery in facioscapulohumeral dystrophy type 2 (FSHD2) not only explains the cause of a disease in a subset of patients, but also confirms the most likely cause of FSHD1, and suggests a new mechanism that may be responsible for diseases far beyond neurology.

The study, published in the Nov. 11 online edition of Nature Genetics, implicates mutations in a gene called structural maintenance of chromosomes flexible hinge domain-containing protein 1 (SMCHD1) as the cause of FSHD2. The normal gene is responsible for silencing large chromosomal regions, including the region that contains double homeobox 4 (DUX4), a transcription factor normally expressed widely only during development. Aberrant reactivation of DUX4 through other means is the leading hypothesis for causing FSHD1.

FSHD is characterized by progressive weakness and degeneration of the skeletal muscles that control movement. It usually begins in the teenage years, progresses slowly, and its symptoms vary from mild to disabling.

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“There has been a question for some time of what specific gene is responsible for the pathology of FSHD,” said Daniel G. Miller, MD, PhD, associate professor of pediatrics at the University of Washington in Seattle, and co-leader of the study, along with Stephen Tapscott, MD, PhD, of the Fred Hutchinson Cancer Research Center in Seattle, and Silvere van der Maarel, PhD, of Leiden University in the Netherlands.

The evidence pointing to DUX4 as the cause of FSHD began to accumulate in 1999, but it has only been in the past several years that the case for the gene became especially strong. The gene is located within an array of repeated genetic material called D4Z4 units, located at the tip of chromosome 4. Normally, the chromosome has between 20 and 50 of these units, Dr. Miller explained, but people with FSHD1 have fewer than 10.

With the normal number of repeats, the chromatin containing the D4Z4 array is condensed, and the genes within, including DUX4, are unavailable for transcription. “But for a long time, people have noted that when this array becomes shortened, chromatin structure in the region becomes relaxed, and the DUX4 gene within each D4Z4 repeat in the array starts to become expressed,” he said.

Because there are other genes in the array, there has been a lingering question about the role of DUX4, but, Dr. Miller said, that has recently been put to rest. “DUX4 is clearly the culprit in this disease.”

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But about 5 percent of FSHD patients, those with FSHD2, don't have shortened D4Z4 arrays. What accounts for their disease? That question led Dr. Miller and colleagues to investigate further the arrays in these patients.

“When we looked closely at the arrays in those individuals,” he said, “we found they were relaxed and hypomethylated, despite not being shortened.” Methylation of DNA is an important means of triggering chromatin condensation, and the lack of methylation in these arrays was likely responsible for their open structure.

Other normally condensed regions on other chromosomes were also affected, suggesting the cause lay beyond the D4Z4 region itself. So the team turned to whole-exome sequencing in multiple unrelated individuals who have FSHD2 to find the gene that might be responsible for this change. With the decreased cost of such large-scale sequencing techniques, and the increased power of the bioinformatic analysis that follows, this gene-hunting method is becoming increasingly common for rare genetic disorders.

They found that most individuals carried mutations in the SMCHD1 gene. The protein's role is to aid in methylation of large chromosomal regions, including the X chromosome and, apparently, the D4Z4 array. They also found some FSHD families with neither shortened D4Z4 arrays or SMCHD1 mutations, suggesting more genes remain to be discovered.

Most of the mutations they found were predicted to reduce production of the protein, which they confirmed in patient fibroblasts. They also showed that less SMCHD1 protein accumulated on D4Z4 arrays in patient tissue. Finally, they showed that DUX4 expression could be artificially increased by reducing SMCHD1 expression with RNA interference, or by using antisense oligonucleotides to recreate the same splicing errors in the SMCHD1 messenger RNA that are caused by the gene mutations.

Taken together, these results showed that the loss of SMCHD1 activity in FSHD2 patients reduced methylation of the D4Z4 array, opening it up and allowing the genetic transcription machinery to gain access to the DUX4 gene. “That results in increased DUX4 expression, but through a different mechanism from FSHD1,” Dr. Miller said.

Not everyone with a SMCHD1 mutation is likely to develop FSHD, Dr. Miller said, because they must also meet one other condition. Transcribed DUX4 RNA can only be translated if, like every other RNA transcript, it is stabilized by the addition of a polyadenine tail. About half the population in the United States lacks the necessary sequences in the D4Z4 array to accomplish that, meaning they would not develop FSHD from a SMCHD1 mutation, even if they carried one.

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There is much left to be learned about the disease. One intriguing and still unexplained point is that not every affected nucleus expresses DUX4. The SMCHD1 mutation causes transcriptional instability, Dr. Miller said, “but there is something stochastic, or some other trigger, that results in DUX4 expression.” But even those nuclei not making the protein may take it up from adjacent ones that are. “It may be that that is why this is a muscular dystrophy,” he said, since muscle cells are syncytia, with one cytoplasm shared among multiple nuclei. “If one nucleus makes DUX4, the other nuclei provide targets for that transcription factor. It amplifies the signal dramatically.”

It is also still unknown how elevation of DUX4 expression ultimately causes disease, although apoptosis of affected muscle cells is the leading hypothesis. The effects of SMCHD1 mutations on other tissues, if any, await further exploration. “We haven't found other genes, but we think the mutation is unlikely to only act at FSHD D4Z4 repeats,” Dr. Miller said. The obvious candidates would be cancers, he noted, since epigenetic deregulation is a common mechanism in cancer.

“We'd like to understand all these pathways better, so we can design treatments for repression of DUX4 expression,” Dr. Miller continued. He noted the disease may be an ideal target for intervention, since it progresses very slowly, with first symptoms typically appearing in the teenage years, but loss of mobility not occurring for another 20 years. “There is a lot of time to apply a therapy,” he said.



The study was supported and funded by the NIH, including the NINDS, the University of Washington Center for Mendelian Genomics, and various FSHD patient advocacy groups and foundations.

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“This paper is important for at least three reasons,” according to Thomas Bird, MD, professor of medicine, neurology, and medical genetics at the University of Washington, who also serves on the editorial advisory board of Neurology Today. “It confirms a recent hypothesis of FSHD, that it is due to reactivation of DUX4. Just a couple of years ago, when it was proposed, this was controversial. Second, it now describes a whole different genetic variant of FSHD, a whole new way to get that disease.” And third, it suggests a “new model” for other diseases, in which pathogenesis requires two genetic events, in this case the presence of a functional polyadenylation signal within normally contracted chromatin, and a mutation that changes the condensation status of that chromatin. That type of “two-hit” requirement may underlie the variable penetrance seen in other genetic disorders as well.

Dr. Bird, who was not involved in the study, also suggested there are likely to be other effects of SMCHD1 mutation, acting at other sites. “I would think it is likely to have another effect. There is no reason why it should be specific to this one region on chromosome 4. This could be the basis of a number of diseases we don't even know about.”

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• Lemmers RJ, Tawil R, van der Maarel SM, et al. Digenic inheritance of an SMCHD1 mutation and an FSHD-permissive D4Z4 allele causes facioscapulohumeral muscular dystrophy type 2. Nat Genet 2012: E-pub 2012 Nov. 12.
    Neurology Today archive on muscular dystrophies:
      • NIH archive on genetics of facioscapulohumeral muscular dystrophy:
        © 2012 American Academy of Neurology