June 08, 2007
Sensitive Genetic Analysis Reveals Vast Changes Associated with Hypertrophic Cardiomyopathy
The one-gene, one-disease concept is elegant, but incomplete. A single gene mutation can cause many other genes to start—or stop—working, and it may be these changes that ultimately cause clinical symptoms. Identifying the complete set of affected genes used to appear impossible. Not anymore.
Studying genetically modified mice, researchers led by Christine E. Seidman, a Howard Hughes Medical Institute investigator at Brigham and Women's Hospital, and her husband Jonathan G. Seidman, who is at Harvard Medical School, have identified hundreds of genes with altered expression in preclinical hypertrophic cardiomyopathy. The study, which is coauthored by colleagues at Harvard Medical School, is published in the June 9, 2007, issue of the journal Science. The discovery could help scientists define the pathways that lead to the disease and lead to the discovery of targets for early detection, prevention, and treatment.
“Some of these low-abundant molecules may be very important in altering cell biology in ways that may be part of the root cause, or a compensatory response to very early manifestations of disease.”
Christine E. Seidman
To obtain a complete picture of the genetic changes associated with the disease, the researchers developed a new gene sequencing technique called polony multiplex analysis of gene expression, or PMAGE. The technique can find messenger RNA transcripts—the directions for making a protein, spun out from the DNA of an active gene—that occur as rarely as one copy for every three cells.
To use PMAGE, researchers attach short sequences (called tags) cut from mRNAs to tiny beads. This tag is amplified, so that each bead contains millions of copies of the same mRNA tag sticking out from it like a minuscule Koosh ball. All of the beads — now called polonies (short for polymerase chain reaction of colonies) — are placed in one layer onto glass, and all of the tags are sequenced simultaneously. A computer program then matches the tags to known genes. The more tags associated with a gene, the higher the expression of that gene.
The industry standard for gene sequencing is serial active gene expression, or SAGE. "There are a couple of labs that have been dedicated to developing this technology," Seidman said, including HHMI investigator Bert Vogelstein at Johns Hopkins and George Church at Harvard. But PMAGE analysis costs between 1/20 and 1/9 of a comparable SAGE analysis, making it more appropriate for the kind of large-scale expression profiling undertaken in this study, she explained. "With SAGE, you can't afford to sequence 4 million transcripts."
Using PMAGE, the researchers compared a healthy group of mice to a group with a genetic mutation that causes hypertrophic cardiomyopathy (HCM) after about 25 weeks of age. In people with HCM, the heart muscle thickens and fails to relax normally after contraction. HCM is the most common cause of sudden death in athletes.
Seidman's group used cardiac tissue from 8-week-old mice to create two PMAGE libraries totaling 4.4 million mRNA tags. They found 706 genes that were overactive or underactive in HCM mice, compared with normal mice. Some genes already have been linked with HCM or heart development. Others are new to the scene.
Overactive genes included:
Nppa (natriuretic peptide precursor a), which encodes atrial natriuretic peptide, or ANP. This protein is an important marker for HCM.
Ctgf (connective tissue growth factor), Tgfß1 (transforming growth factor beta-1), and Postn (periostin), powerful regulators of fibrosis and collagen deposition. The early activation of these genes indicates that fibrosis is probably a primary contributor to heart dysfunction, not a reaction to other changes.
Vgll2 (vestigial-like 2 homolog) and Egr3 (early growth response-3), transcriptional regulators involved in the fetal development of the heart muscle
Nr1h3 (nuclear receptor subfamily 1, group h, member 3) and Nfkbie (nuclear factor kappa light polypeptide gene enhancer in B cells inhibitor epsilon), which have never before been linked with HCM
Underactive genes included:
Hod (homeobox-only protein) and Hand2 (hand and neural crest derivatives 2), transcriptional regulators involved in the fetal development of heart muscle
Abcc9 (adenosine triphosphate cassette subfamily C member 9), which encodes part of the cardiac potassium channel. This channel helps to regulate calcium balance. Mice who lack Abcc9 develop arrhythmias and myocardial calcium overload.
Sln (sarcolipin) and Pln (phospholamban), which regulate calcium uptake into muscle cells
"It's important that we could statistically quantify changes even in genes with very low expression levels," Seidman said. "Some of these low- abundant molecules may be very important in altering cell biology in ways that may be part of the root cause, or a compensatory response to very early manifestations of disease."
Seidman is now repeating the PMAGE sequencing using tissue from younger mice. "We want to get at the drivers of the pathology," Seidman said. "HCM largely affects structural proteins, and we don't really understand how a change in one protein affects the cascade that ultimately affects physiology. If we do this sequencing early enough, we'd like to think we'll see signals that point us to something that is fundamentally changing the whole downstream cascade."
Discovering those fundamental drivers of change could result in targeted therapies to halt HCM in its tracks, or even prevent it altogether.
"We're doing this in a mouse right now, but we have access to tissue from human patients," Seidman said. "With the deep, rich analysis we obtain from a biopsy or resection, we can jump into understanding the human biology of heart disease quite quickly."
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February 27, 2005
Tailing the Cause of a Rare Heart Disease
Using genetic analyses and the translucent tail of a fish, researchers have pinpointed the underlying cause of a rare, mysterious heart disease that is preceded by hearing loss. Discovering the genetic cause of this disease provides researchers with a wealth of new ideas about the molecules involved in building the developing heart, as well as how diseases weaken heart muscle.
In an advance online publication on February 27, 2005, in the journal Nature Genetics , Howard Hughes Medical Institute investigators Christine E. Seidman and Jonathan G. Seidman and their colleagues identified the mutation that causes the disorder, dilated cardiomyopathy preceded by sensorineural hearing loss. The Seidmans and their colleagues at Harvard Medical School collaborated with researchers at University Hospital Würzberg in Germany, Massachusetts General Hospital, Children's Hospital Boston and The Wellcome Trust Sanger Institute in Britain.
“The finding that transcriptional regulation goes awry in this disorder gives us a top-down look at the molecules that must be appropriately expressed and regulated for normal function of the heart throughout life.”Christine E. Seidman
In dilated cardiomyopathy, muscle weakness causes the left ventricle to stretch. As a result, the heart becomes enlarged to the point where it can no longer pump blood efficiently. In earlier studies of patients with cardiomyopathy preceded by hearing loss, the Seidmans and their colleagues identified a region of chromosome 6 as the location of the culprit gene.
In the latest study, they sought to pinpoint the gene and identify the mutation that was responsible. A search of the human genome database identified candidate genes in the region, and subsequent tissue analysis revealed that one gene, called EYA4 , was expressed in both the heart and the cochlea of the ear. The researchers confirmed that affected people possess a characteristic mutation of the EYA4 gene, a finding which offered surprises, said Christine Seidman.
"The EYA4 gene had been implicated in hearing loss before, but in none of the patients where mutations had been characterized had there ever been an abnormality of the heart reported," she said. The Seidmans also found that the gene was expressed in adult heart tissues, which is unusual because other EYA gene family members are thought to function primarily during development.
Curiously, the gene does not code for a structural protein involved in known myocyte functions such as contraction, as is the case with other mutations that cause cardiomyopathies. Rather, EYA4 codes for a protein involved in activating other genes—controlling the copying, or transcription, of genetic information to messenger RNA that carries that genetic blueprint to the cell's protein-making machinery.
To confirm that the mutant EYA4 does indeed cause cardiomyopathy, the researchers turned to the zebrafish, whose genetic machinery for cardiovascular development closely resembles that of mammals. In their experiments with the fish, they used antisense genetic techniques to reduce the fish's production of eya4 protein and measured the resulting changes to the heart. The treated fish developed swelling of the heart ventricle that suggested cardiac dysfunction. High-speed video of the fish's beating heart—visible because the fish are translucent—indicated that the pumping function of the heart was dramatically reduced.
However, the researchers turned to the fish's translucent tail to confirm independently that blood flow was, indeed, reduced in the treated fish. "Since the cardiac imaging of zebrafish remains technically challenging," said Seidman, "we devised a means of tracking the movement of a single red blood corpuscle through the tail to assess flow. That analysis revealed a dramatically lower pumping velocity in the treated fish and provided a functional readout of heart performance," she said.
To understand the molecular mechanism by which the mutant eya4 protein might compromise cardiac function in humans, the researchers compared its function with that of other mutant forms that only caused hearing loss. They found that the mutant protein that caused both cardiomyopathy and hearing loss lacks a region necessary for the protein to attach to other proteins that help it enter the cell nucleus. Only if the eya4 protein enters the nucleus can it play the appropriate role in gene regulation, said Seidman.
"That finding suggests that this mutation causes a dramatic reduction in the amount of eya4 within the nucleus, and we presume that is what accounts for heart disease," said Seidman. "The implication of this finding is that the function of eya4 in the heart may be different than its functions in other tissues, such as the ear.”
Although the researchers' findings will not aid treatment of the cardiomyopathy immediately, said Seidman, they do offer the potential for important basic insights into the mechanisms of cardiomyopathies.
"Whenever a new disorder affecting the human population is identified, it adds new basic knowledge to the field of medicine," she said. "And sometimes rare disorders such as this one can provide very powerful insights into the biology of more common problems. In this case, the finding that transcriptional regulation goes awry in this disorder gives us a top-down look at the molecules that must be appropriately expressed and regulated for normal function of the heart throughout life."
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