Russell Vanderboom, Ph.D.
Medical Scientific Writer, Lerner Research Institute
In a family beset with nine heart attacks among 21 individuals over three generations, the influence of genes on coronary disease seems inevitable.
For the Steffensen family from Des Moines, Iowa, the family medical history replete with infarctions, bypass surgeries, and angioplasties begged for answers. The Steffensens wanted to know why heart disease affected the lives of so many in their family. Six of nine Steffensen children had heart attacks, as had their father. Two grandchildren also suffered heart attacks in their early forties. Other family members required significant cardiac care. The family was well aware that their hearts were their Achilles’ heel.
The Steffensens looked to the medical community for care, but they also sought answers. They wanted to know whether the answer to the cause of their family heart disease could be helpful to future generations of Steffensens, as well as to other families who may share the genetic propensity for increased instances of coronary artery disease and myocardial infarctions (CAD/MI).
In the November 28 issue of Science,* genetic researchers from the Cleveland Clinic Foundation published an answer the Steffensen family had sought. Qing Wang, Ph.D., Eric Topol, M.D., and their CCF colleagues identified a specific pathogenic mutation in a human gene that is responsible for the overwhelming incidence of CAD/MI occurring within the family from mid-America. The CCF scientists determined that a 21- base-pair deletion in the MEF2A gene results in changes in the gene’s protein functions in cells within the heart. The altered gene causes defects resulting in CAD/MI. This is the first gene to be established as a direct cause for coronary heart disease within humans.
The discovery was possible because the Steffensens chose to participate in comprehensive genetic analysis. The critical step that the Steffensens took to resolve the quandary of their family cardiac health problem was to take part in genetic research on heart disease. After Donald Steffensen had a heart attack in 1992, he came to The Cleveland Clinic for care. Mr. Steffensen continues to consult with Bruce Wilkoff, M.D., CCF Pacemaker Clinic, regarding his pacemaker and routine cardiac care follow-up.
But in the mid-1990’s, he also took the key step toward resolving the mystery of why so many family members shared his problem with heart disease. In the course of his treatment and evaluation at the Clinic, Mr. Steffensen told Randall Starling, M.D., Cardiovascular Medicine, that eight of his ten siblings had heart attacks at about the same age. That information raised the red flag indicating a genetic link to the disease. Mr. Steffensen then enrolled in GeneQuest, the nation’s first and largest clinical genetic study directed at finding the genetic basis for heart disease. GeneQuest was conducted during the mid to late 1990’s by Eric Topol, M.D., CCF Chief Academic Officer and Provost, and Chairman, Department of Cardiovascular Medicine.
After Mr. Steffensen’s initial involvement in the heart disease studies, he encouraged many other members of the family to participate in the GeneQuest 1A study, an extension of the original GeneQuest project. To date, more than 100 family members across several generations have contributed DNA for genetic analysis.
The goal of GeneQuest 1A is to use single large families to map and find the gene(s) for coronary heart disease. The Cleveland Clinic researchers decided to focus on large families for the gene identification studies, a strategy used successfully in previous studies by Dr. Wang in finding other types of cardiac disease genes.
Qing Wang, Ph.D., M.B.A., Director of CCF’s Center for Cardiovascular Genetics
Dr. Topol and Dr. Wang, who is the Director of CCF’s Center for Cardiovascular Genetics, and their colleagues identified four families, including the Steffensens, as candidate carriers of genes that might cause heart disease. Procuring additional support funding from a Doris Duke Charitable Foundation grant for Innovations in Clinical Research, Drs. Topol and Wang started the hunt for a gene that caused heart disease leading to CAD and MI.
Their strategy proved valid with the Steffensen family, in whom they identified the exact location of the trigger gene for heart disease on a specific region q26 of chromosome 15. To locate the errant gene, Dr. Wang’s laboratory scanned the entire genome of all participating members of the Steffensen family using 382 markers. Each genetic marker served as a landmark on human chromosomes 1 through 22. Dr. Wang’s approach spanned the entire human genome at an average of every 10 centiMorgans, or 10,000,000 base pairs apart. A specific marker, D15S120, was found to be associated with the disease occurring in the Steffensen family. Statistical analysis yielded the LOD score of 4.19, which is higher than 3.00, a threshold for indication of finding a genetic locus. As D15S120 is located on chromosome 15q26, and this marker is linked to the disease in the Steffensens, Drs. Wang and Topol concluded that the gene triggering the pathogenesis of CAD/MI is on chromosome 15q26. The research team then started the second phase of the project, to identify the exact gene.
The region within the Steffensen’s genome that contained the linked genetic coding resided on chromosome 15q26. That region contained approximately 93 genes. Forty-three of those genes were already identified when Dr. Wang examined the linkage. He noticed that MEF2A, a member of the myocyte enhancer factor-2 family of transcription factors, was located within the region of interest. MEF2A protein was known to function in vascular development and angiogenesis during fetal development in mice. Dr. Wang’s laboratory also detected MEF2A in the endothelial cell layer of coronary arteries. RNA message for the gene was localized within human endothelial cells as well.
Further analysis revealed the 21 deleted base pairs missing from the gene in family members who suffered heart disease, compared to the complete gene sequence for MEF2A present in one daughter and several grandsons who remained disease-free. The deletion was not found in 119 non-family members who had normal angiograms.
The 21 missing nucleotides in the altered MEF2A gene translated to a protein with seven fewer amino acids than the normal MEF2A protein. The deletion in the Steffensen’s MEF2A variant changed the protein near its carboxy terminus. That region of MEF2A has been shown to have dual functions. It is essential for trafficking the protein to the nucleus and driving the transcription of target genes.
To test whether the altered version of the gene found in the Steffensen family functioned normally, Dr. Wang used immunofluorescent staining of MEF2A to see where the protein localized within cells. Normal, wild-type MEF2A tracked to the nucleus of cells, while the variant with the missing seven amino acids appeared to be completely blocked from entering the nucleus. Transcription assays also indicated that the mutant protein lost functional control over gene transcription activity. Indeed, in cells in which both the wild-type and the mutant MEF2A gene variant were expressed, transcriptional activity was lost, indicating that the altered gene exerted dominant-negative control over normal MEF2A function.
Dr. Wang noted that the MEF2A mutations found in the Steffensen family were not observed in 50 randomly tested CAD/MI patients, suggesting that this particular MEF2A mutation may be rare within the general population. The penetration of the altered gene throughout the human gene pool will only be detected through a large clinical genetic study, he noted.
The discovery of the altered gene explained the genetic basis for the high incidence of heart disease in the Steffensen family. The Cleveland Clinic research also opens avenues for development of therapeutic targets that may control or rectify MEF2A induced heart disease.
“Finding the gene opens a new avenue through which scientists can track the pathway that leads to coronary heart disease and heart attack,” Dr. Wang said. “Genes in that pathway then can be used as targets to design new drugs intended to prevent or treat heart disease. Finding the gene also will make it easier to definitively diagnose patients with highly significant family histories of heart disease.”
Future research will focus on pinpointing the roles of the missing nucleotide pairs and amino acids, as well as searching for ways to counteract the negative impact on artery walls. This research is among the most exciting ever undertaken by The Cleveland Clinic, Dr. Topol said, because it can ultimately lead to refined preventive strategies for the disease with the highest fatality and disability burden.
Other researchers who worked on the MEF2A project include postdoctoral fellows Lejin Wang and Chun Fan, and cardiology study coordinator Sarah Topol, all of The Cleveland Clinic.
* Wang, L., Fan, C., Topol, S.E., Topol, E.J., and Q.Wang (2003) Mutation of MEF2A in an inherited disorder with features of coronary artery disease. Science 302:1578-1581.
