A large worldwide collaborative effort to identify genetic mutations in patients with multiple-system atrophy (MSA) has finally paid off — and offers a surprising hint about the molecular targets at work and how neurologists might think about treating patients. Right now, there are no treatments for the complex and disabling condition.
The scientists, led by Shoji Tsuji, MD, PhD, professor and chairman of the department of neurology and director of the Medical Genome Center at the University of Tokyo Hospital, identified two genetic mutations in two MSA families in Japan. They also conducted studies on MSA patients with no family history and reported that 10 percent of them also had evidence of a gene mutation or a common genetic variant that increased the risk of developing the disease. The gene, COQ2, encodes the enzyme parahydroxybenzoate-polyprenyl transferase, which is essential for the biosynthesis of coenzyme Q10.
The findings were published July 18 in the New England Journal of Medicine (NEJM).
MSA has long been thought to be a sporadic non-genetic condition. This is the first gene to be identified and Dr. Tsuji believes that it will lead to a better understanding of the disease and, for some, a potential treatment. The Japanese researchers are now trying to figure out how much coenzyme Q10 must be delivered into the brain to reverse or slow the disease process.
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This was a worldwide collaborative effort. Dr. Tsuji cases from all over Japan, Europe, and North America. The analysis received included 363 Japanese patients, 223 European patients, and 174 from the United States. Thousands of patients with Alzheimer's and Parkinson's diseases, as well as amyotrophic lateral sclerosis, were used as a control sample in the study.
The investigators first performed linkage analysis in the six MSA families and then when they detected several interesting loci, particularly two regions on chromosome 4, they selected one family with the strongest LOD [logarithm of odds] score for whole genome sequencing. They identified four single nucleotide variants, of which one variant in COQ2 gene was found to be the causative mutation for this family. They further identified compound heterozygous mutations in COQ2 gene in another family.
They then expanded mutational analysis in the COQ2 gene to a large series of sporadic MSA patients. They identified a common variant (V343A) and found multiple rare variants in the COQ2 gene are associated with increased risk for sporadic MSA.
The gene variant (V343A) identified as a risk factor in MSA is common among Japanese populations (1.6 to 2.2 percent allele frequency) but not in Europeans and people in the US. The allele frequency in MSA patients is 4.8 percent, suggesting the allele is a modest risk factor.
“We should consider that this variant in COQ2 is not necessarily causal but rather [it] confers a susceptibility to sporadic MSA,” the authors wrote in the NEJM paper. They also found multiple rare variants in COQ2 not only in Japanese sporadic MSA patients but also in European MSA patients.
Having a mutant copy of the gene is not guaranteed to trigger symptoms of MSA, they pointed out. Parents of two of the MSA families had variants in the heterozygous state and did not have signs of MSA. “The penetrance of the mutation is not high,” said Dr. Tsuji. “There must be something else going on.”
The investigators sequenced almost a dozen other genes that work on the coenzyme Q10 biosynthesis pathway but there were no genetic variants that congregated with the disease, said Dr. Tsuji.
MSA has a complicated phenotype that includes autonomic dysfunction, parkinsonism, cerebellar ataxia and pyramidal dysfunction, he pointed out. Scientists have identified accumulations of alpha-synuclein in oligodendroglia. Some patients have a form of MSA that is characterized by more severe cerebellar ataxia (MSA-c) and another by a more dominant parkinsonism (MSA-p). Interestingly, Japanese patients seem to have more of the cerebellar ataxia form and those in Europe and the US patients have more parkinsonism symptoms.
The investigators wanted to better understand the contribution of changes in COQ2 and its relationship to the pathophysiology of the disease. They designed a study to determine the functional effect of each variant (R337Q/V343A, R337X/V343A, and V343A/V343A) on mitochondria. They measured the enzyme activities with these mutations in lymphoblastoid cell lines and demonstrated that these mutations mildly but significantly decrease COQ2 enzyme activities.
For multiple rare variants of COQ2 identified in Japanese, European, and MSA patients and controls, where lymphoblastoid cell lines were unavailable, they measured the COQ2 enzymes activities using yeast. They prepared COQ2 complementary DNAs carrying these mutations and transformed COQ2-null mutant yeast and measured the growth rate of yeast under an aerobic condition.
They observed markedly decreased growth rates for some of these mutations (deleterious mutations). They also found that these deleterious mutations are strongly associated with increased risk (odds ratio = 11.97) for combined MSA patients (Japanese, European, and American MSA patients), although the carrier frequencies of these deleterious variants are substantially low (MSA cases; 1.06 percent and controls; 0.09 percent).
The investigators also measured coenzyme Q10 levels in autopsied cerebellar tissue from three MSA patients and three controls. The coenzyme Q10 level from the patients with homozygous for M78V-V343A was substantially lower than that in brain tissue from the control samples.
They also measured intracellular coenzyme Q10 levels in lymphoblastoid cell lines from patients with MSA and controls. They reported that the intracellular levels in the cell lines in those MSA patients with two variant alleles were much lower than levels in cell lines from controls. Those with one variant V343A allele, however, did not significantly differ in the CoQ10 levels than the cell lines from controls.
“We are now convinced that these mutations have a role in MSA,” said Dr. Tsuji. He theorized that impaired COQ2 activity inhibits the production of coenzyme Q10 that is needed by mitochondria to produce ATP.
Low levels of coenzyme Q10 may also increase “vulnerability to oxidative stress,” said Dr. Tsuji. This isn't the first time that coenzyme Q10 deficiencies have been linked to a severe brain disease. COQ2 mutations have been found in infantile-onset multi-system disorder and nephropathy in some families. The effects of COQ2 mutations in MSA appear to be milder than the mutations found in these sick infants.
“That we also found that functionally impaired variants in COQ2 are associated with an increased risk for sporadic disease is exciting. We hope this will lead to a treatment for this devastating disease,” he added.
Dr. Tsuji's group is now measuring coenzyme Q10 levels in cerebrospinal fluid to see whether they can estimate how much supplementation would work. Clinical trials would begin in Japan, where the COQ2 mutations are more common. He estimates that there are more than 10,000 MSA patients in Japan. He added that it is possible that coenzyme Q10 supplementation might be effective in those with the variants they linked to MSA.
“We would like to conduct clinical trials with international [partners] to increase the size of the clinical trials, which is important to obtain conclusive evidence for this kind of disease modifying therapies,” Dr. Tsuji said. “Furthermore, the majority of patients with neurodegenerative diseases have sporadic illness, and we opened a new avenue to identify genes involved in development of sporadic neurodegenerative diseases,” said Dr. Tsuji.
Mitochondrial abnormalities are thought to trigger several neurodegenerative diseases, including Parkinson's and Huntington's. coenzyme Q10 is essential for energy production in mitochondria. Deficits decrease ATP production. Coenzyme Q10 is also a strong antioxidant. CoQ10 has been tried and failed in several clinical trials for Parkinson's.
The Japanese researchers are continuing to search for other genetic mutations in the other families. They have several candidate genes. There are a number of clues coming together about the pathogenesis of MSA. Scientists have found accumulation of alpha-synuclein in oligodendroglia.
EXPERTS WEIGH IN
“The finding gives us clues on a new pathway,” said Robert C. Griggs, MD, professor of neurology, medicine, pathology and laboratory medicine at the University of Rochester School of Medicine and Dentistry. “This was surprising. Chances are there will be other genes associated with MSA.”
“The finding establishes a new potential contributor to pathogenicity in a subgroup of MSA patients that is theoretically targetable with currently available agents,” added Peter K. Todd, MD, PhD, the Bucky and Patti Harris professor and assistant professor of neurology at the University of Michigan. However “the majority of the mutations associated with variants in CoQ2 are isolated to Japan, preventing the generalizability of these findings to all MSA cases. This may reflect the bias towards MSA-C in Japanese populations, but other reasons for this association need to be explored.
“Although some of these variants have clear impacts on COQ2 function and CoQ10 levels, we will have to wait until they are tested in animal models to see how these mutations contribute to pathogenicity, especially how they might impact alpha synuclein pathology. Also, the vast majority of MSA patients do not have variants in either COQ2 or alpha-synuclein, which was previously implicated with MSA in genome-wide association studies.”
“Most of the weight of this finding comes from Japanese patients and it will be interesting to see how this translates to patients in North America and Europe,” he added.
Stefan M. Pulst, MD, professor and chair of the department of neurology at the University of Utah, said the “finding makes a lot of sense. We have always looked for mitochondrial dysfunctions in neurodegenerative diseases.” Still, he added, “the geneticist in me says that we have to be careful in interpreting rare variants without a large enough control sample, which could mean as many as 10,000 people. But the researchers did what they could. There are complex issues. Now, scientists need to go back and mine the entire genetic region to see if there are other gene variants that modulate risk.”
The North American MSA Study Group shared their patient data for this study. Phillip Low, MD, a professor of neurology at the Mayo Clinic in Rochester, one of the U.S. study co-authors, said that “an abnormality of CoQ10 biosynthesis is more often relevant in Asian MSA but not in the MSA seen in the U.S.” He added that the “study is an important step forward but has little implications for North American MSA management.”
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