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Sequencing Study of Titin in 5,200 Patients May Help Improve Tests, Tailor Treatments for Dilated Cardiomyopathy

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NEW YORK (GenomeWeb) – A new sequencing study of titin, a major gene involved in dilated cardiomyopathy, in more than 5,200 individuals has allowed researchers to distinguish pathogenic variants from benign ones, pointing toward improved diagnostic genetic testing. The results also shed light on the mechanism by which mutations in titin cause DCM, suggesting new treatment avenues.

The study, from an international team led by researchers in the US, the UK, and Singapore, was published online in Science Translational Medicine today. In a conference call earlier this week, several of the authors spoke about their findings.

The study "solidifies the importance of titin in dilated cardiomyopathy and helps to identify those variants that we have strong evidence … are not pathogenic," said Christine Seidman, a professor of medicine and genetics at Harvard Medical School and one of the senior authors of the paper.

Non-ischemic dilated cardiomyopathy, which occurs in about 1 in 250 people, is an important cause of heart arrhythmia and heart failure, and is the most frequent indication for heart transplantation.

Genetic forms of DCM can be caused by mutations in a variety of genes, the most important being titin, a massive gene that encodes 364 exons and 34,350 amino acids – the largest human protein. Titin is expressed in various isoforms in different types of muscle cells and the titin protein is part of the sarcomere, where it functions in muscle contraction.

Two years ago, researchers showed that mutations that truncate the titin protein are the most common genetic cause of severe and familial DCM, accounting for about a quarter of all cases. However, that study also found truncating titin mutations in about 2 percent of healthy individuals, making it difficult to interpret titin variants in genetic tests.

In their current project, the researchers studied whether titin truncations play a role in less severe cases of DCM, so-called ambulatory patients. They also looked at differences between titin truncations in healthy controls and DCM patients, and how those differences could help clinicians interpret genetic test results and tailor patient treatment or follow-up. In addition, they sought to better understand the disease mechanism, based on the location of the mutations in the gene and their effect on protein expression.

For their study, they sequenced the titin gene – sometimes along with other genes – in five cohorts: 374 patients diagnosed with DCM, recruited through the Royal Brompton and Harefield Hospitals NHS foundation Trust; 155 end-stage DCM patients who were on a heart transplant list; 163 patients with familial DCM recruited by St. Vincent's Hospital and the Victor Chang Cardiac Research Institute; 308 healthy adult volunteers, with cardiovascular evaluations, recruited by the MRC Clinical Sciences Centre at Imperial College London; 1,623 participants in the Framingham Heart Study; 1,980 participants in the Jackson Heart Study; and 667 participants in the Women's Health Initiative.

To explore the expression of titin, they also performed RNA sequencing in 84 heart tissue samples from DCM patients undergoing heart surgery, and protein gel electrophoresis for a subset of these. This allowed them to gauge which titin exons are particularly important in the heart.

The analysis demonstrated that truncating titin mutations play a significant role in ambulatory DCM patients – 13 percent of patients in that group had such a mutation, compared to 20 percent in the group of end-stage DCM patients.

Similar to the previous study, they found that about 2 percent of the healthy controls carried truncating titin mutations.

However, mutations in DCM patients tended to occur in titin exons that are highly expressed in the heart, and were often located near the distal end of the gene, whereas many of the titin mutations in the control populations tended to fall into exons that are not expressed in the heart, and were often located at the proximal end of the gene.

"These findings help us to interpret the variants that we pick up during diagnostic genetic testing in patients with dilated cardiomyopathy, as well as variants found incidentally when sequencing for other reasons," said James Ware, a clinical lecturer at Imperial College London and postdoc at Harvard Medical School, and one of the lead authors of the paper.

For example, about half the titin variants found in healthy individuals could be excluded "off the bat" as non-pathogenic, he said, because they occur in exons not expressed in the heart.

The results could be particularly important for families affected by DCM. At the moment, first-degree relatives of DCM patients are evaluated by a cardiologist and monitored throughout their lives, even though fewer than half of them are expected to carry a mutation that puts them at risk for DCM. Such screening is costly, time-consuming, and stressful, and "anything to reduce this burden would be a great benefit," said Angharad Roberts, a researcher at Imperial College London and another lead author of the study.

If a truncating titin mutation in an exon expressed in the heart is found in a patient with familial DCM, it is highly likely to be the causative mutation, and other family members could be ruled out by testing for this mutation. "Those without the causative mutation can be reassured and discharged, while clinical follow-up is targeted to those who are at high risk," Roberts said. Initially, screening for titin truncations will likely be used in conjunction with clinical screening, she said.

The interpretation of titin truncations identified as secondary findings in genetic tests unrelated to cardiomyopathy could also benefit from the study's findings, although there are many mutations that cannot be categorized as benign at the moment. "Accurate interpretation of these results is vital to avoid unnecessary clinical follow-up and anxiety for these individuals," Roberts said.

The study also revealed that DCM patients with truncating titin mutations generally fared worse than DCM patients without such mutations, having more incidents of life-threatening heart arrhythmias and poorer survival. Therefore, "titin mutations may define a group of patients who are going to benefit from a tailored therapeutic approach," Ware said.

For example, DCM patients with titin truncations could receive targeted screening for heart rhythm problems and could preferentially receive internal cardiac defibrillators. "This therapy is not suitable for all patients and needs to be carefully targeted," Roberts said. "Genetic testing can help identify patients who would benefit most from this therapy."

In terms of the disease mechanism, the study data suggest that the truncated titin in patients somehow poisons the heart muscle cells. This points to new treatment strategies that try to eliminate the mutant protein rather than boost levels of the normal protein, Ware said.

The result was somewhat surprising, according to Seidman. "In general, we think that [a truncating mutation] is likely to be pretty damaging to the function of that peptide," she said. "The amazing result from this study is that this is not necessarily true."

One question the study did not answer is whether titin truncations also play a role in other DCM patient populations, for example in children or in pregnant women, and future studies will address this, Seidman said.

Another aspect the researchers would like to pursue is titin variants that don't truncate the protein, for example missense mutations. Seidman said everyone they have studied to date carries such a missense mutation in titin, and a subset of these mutations may contribute to disease.

Further understanding the disease mechanism of titin truncations is another focus of future studies. "We'd like to understand the signals that the abnormal titin molecule evokes" and find ways to stop those signals, Seidman said. "That clearly would allow for directed therapeutics that would provide great benefit to patients with these titin truncations."

In terms of basic science, she said, the study underscored "the incredible importance of alternative splicing" for the function of the titin gene. "We'd very much like to understand what the incorporation of modular units [into the heart] does to the contractile apparatus, the sarcomere, where titin performs most of its activities, and whether modulating the inclusion or exclusion of different domains will change heart function in ways that might benefit not just patients with dilated cardiomyopathy but perhaps other heart conditions as well," Seidman said.

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