Scientists have used next-generation DNA sequencing tools to identify a mutation in a gene that underlies one of the most common forms of severe hypertension.

More than one billion people worldwide suffer from hypertension, putting them at increased risk of heart attack or stroke. Scientists know that some individuals are genetically predisposed to developing high blood pressure, but the complexity of both the condition and the human genome makes identifying risk factors extremely challenging. Howard Hughes Medical Institute (HHMI) scientists have used next-generation DNA sequencing tools to identify a mutation in a gene that underlies one of the most common forms of severe hypertension.

HHMI investigator Richard P. Lifton and colleagues report in the February 11, 2011, issue of Science that mutations in the gene encoding the KCNJ5 potassium channel drive development of aldosterone-producing adenomas, a tumor of the adrenal gland. The hormone aldosterone is normally only made by the adrenal gland in response to dehydration or high levels of potassium in the blood potassium. Excessive levels of aldersterone produce hypertension.

Because of the high impact of these tumors among the severely hypertensive population, these findings are of substantial clinical importance.

Richard P. Lifton

“These tumors are clinically important; they are found in five to ten percent of patients with severe hypertension. We also thought they might be biologically interesting because unlike many other cancers they rarely become invasive or lead to distant metastases,” says Lifton, professor and chair of genetics at Yale University School of Medicine.

Lifton, who worked with a team of researchers from Yale, Uppsala University, New York Medical College, and Henry Ford Hospital in Detroit, says their analysis of the tumors was a problem particularly well-suited to new capabilities to efficiently sequence all the genes in the genome.

Using a DNA sequencing strategy called whole exome sequencing, which was developed in the lab by the paper’s first author, Murim Choi, the team sequenced all 23,000 or so human genes in tumor DNA from four patients. They compared those sequences to each patient’s own blood-cell DNA, searching for mutations that had occurred somatically, meaning after normal development, in the tumors.

They found very few protein-altering somatic mutations, only about two per tumor. Remarkably, however, a gene encoding a potassium channel named KCNJ5 was mutated twice. When they studied additional aldosterone-producing adenomas they found either of two mutations in KCNJ5 present in eight of the 22 tumors they studied. “These findings were exceptionally unlikely to be a chance event and established these mutations in the causation of these tumors,” says Lifton.

Potassium channels are cellular pores that normally allow only potassium ions to diffuse in or out of cells. Structural analysis suggested that the types of KCNJ5 mutations seen in adenoma tumors introduced changes in the protein that could decrease its preference for potassium ions. Electrophysiology analysis confirmed that by showing that when the mutant channels were expressed in cultured cells, they allowed normally prohibited sodium ions to pass through the mutant channels.

“In the 1990s Rod MacKinnon’s lab demonstrated that the basis for channel specificity is a selectivity filter defined by specific amino acids that permit potassium but not sodium ions to pass through the channel,” says Lifton, referring to fellow HHMI investigator Rod MacKinnon of The Rockfeller University, who received the Nobel Prize in Chemistry in 2003 for work on ion channel structure and function. “One of the two mutations we found in tumors was a mutation altering one of the exact same amino acids that MacKinnon defined.”

The team then showed that sodium influx into tumor cells caused by KCNJ5 leakiness sets off a chain of events resulting in increased intracellular calcium, turning on both aldosterone production and cell proliferation. The latter is a “first” for ion channels. “To our knowledge this is the first report of an ion channel mutation with a role in neoplasia,” says Lifton.

While the novel mutations identified in the Science paper are occurred somatically, they raised the question of what would happen if such a mutation was present in every cell in the body. This prompted Lifton to revisit a study his lab published in 2008. In it, his team described a family with an inherited condition in which the father and two daughters had severe hypertension as children due to uncontrolled aldosterone secretion and massive growth of all the cells of the adrenal gland that normally make aldosterone. In that study, the researchers could not identify the precise gene responsible for condition.

Now, armed with a suspect, they re-examined blood DNA samples from that family and found that the father had a similar mutation in KCNJ5 and had transmitted this mutation to both of his affected daughters. This discovery, also reported in the Science paper, links mutations altering critical regions of the KCNJ5 protein—whether inherited or somatically acquired—to excessive aldosterone production by adrenal tumors and accompanying high blood pressure.

The team’s analysis would have been impossible without today’s powerful DNA sequencing technology. Rather than applying that technology to all of the DNA in the tumor samples in their study, Lifton’s team narrowed their search using a method called whole exome sequencing. Conceptually, the method is straightforward. Only about one percent of a cell’s DNA accounts for genes that encode proteins, but those genes (the “exome”) are sites of about 85 percent of disease-causing mutations. Whole exome methodology enables researchers to ignore the extraneous sequences and focus on the mere 30 million or so base pairs that make up the one percent. “Whole exome sequencing is enabling geneticists to study both rare and common complex diseases,” says Choi, referring to conditions like hypertension or diabetes that emerge from a constellation of factors.

Choi taught himself the computational techniques needed to carry out the study. “When I started my project in 2007, I needed to develop skills to analyze huge sets of data, so I bought a book to learn PERL,” he says, referring to a programming language used in bioinformatics analysis. “I did bench work in the daytime and read the book at night. I finished it in two weeks.” Clearly, that was two weeks well spent: By 2009, Choi was first author on a Lifton lab “whole exome” proof-of-concept study published in Proceedings of the National Academy of Sciences. And for the current paper in Science, Choi analyzed all the genomic data.

“Because of the high impact of these tumors among the severely hypertensive population, these findings are of substantial clinical importance,” Lifton says. He says his team’s findings reveal surprisingly simple biology for these adrenal tumors and raise the possibility of developing of a screening test to identify patients with these tumors by finding one of these two mutations in cells or DNA shed from the tumors and circulating in the blood. “A simple screening test for these tumors would have important impact for the treatment of this disease since this form of hypertension can be cured by removal of the tumor,” he notes.

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