Disease Mutation Tracked Down, Ending ‘Curse’ for Colombian Families

Discovery of their gene mutation was cause for celebration among two extended families who had been cursed with migraines, early strokes, and dementia for generations.

In their insular world in an isolated Colombian valley, the affliction had become synonymous with one of the family names, invoking a social stigma with darker overtones of punishment for ancestral misdeeds. Then, suddenly, their misfortune had a different label—cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, or CADASIL.

“For them, the mutation was a relief,” said Joseph Arboleda-Velasquez, a graduate student at Harvard Medical School (HMS) who first organized the family trees and then led the scientific team that identified the mutations. “It was a disease caused by a mutation in a gene. It happens to other families in the world and has nothing to do with something they’ve done wrong.”

Joseph Arboleda-Velasquez tracked down more than 1,000 members of two extended families that have passed down a rare disease-causing mutation for generations. Photo by Graham Ramsay

Now, three years later, Arboleda-Velasquez and his collaborators have worked out an early step in the cascade of events leading from the mutated gene to the disease phenotype. The findings were published online April 27 in Human Molecular Genetics.The molecular investigation eventually may have an impact beyond the genetically affected families.

“Just as rare inherited mutations tell us a lot about the more common sporadic Alzheimer’s disease, I hope these CADASIL mutations will teach us about the genetic basis of strokes,” said senior author Kenneth Kosik, HMS professor of neurology at Brigham and Women’s Hospital.

Molecular Miscues
The clinical phenotype of migraine attacks, recurrent strokes, and cognitive difficulties first described in European families came to be called CADASIL about 10 years ago. Around 600 families with the disease have been identified worldwide. There is no treatment.

The inherited syndrome springs from various mutations in the Notch3 gene, which are specific to each family. Somehow, each familial mutation interferes in a common way with the receptor that helps define smooth muscle cells encircling blood vessels and coordinate the roles of neighboring tissues. In CADASIL, the smooth muscle cells in the brain’s vessels appear to degenerate early.

Children in families with CADASIL often suffer from migraines. Adults in their 30s and 40s can accumulate small strokes deep in the brain that eventually disable their speech and fine movements, and lead to dependence on others for daily activities.

At the suggestion of his adviser, neurologist Francisco Lopera, Arboleda-Velasquez began studying CADASIL during his neurology internship in the last year of medical school at the University of Antioquia in Medellin, Colombia. By the end of the year, he had tracked down 1,000 members of one extended pedigree—the largest known CADASIL family in the world—and found a second, smaller family with a more aggressive form of the disease.

This genealogy trace was not a computer exercise. Arboleda-Velasquez trekked repeatedly into remote corners of the region between two mountain chains in northwest Colombia.

“Just as rare inherited mutations tell us a lot about the more common sporadic Alzheimer’s disease, I hope these CADASIL mutations will teach us about the genetic basis of strokes.”

His queries took him across the invisible lines of the violent civil conflict among the government army, left-wing guerrilla groups, and right-wing paramilitary troops. On one such quest, a man denied another man was his brother (to whom he had not spoken in 15 years due to sharply divided loyalties). “You belong to this family,” Arboleda-Velasquez told him. “We know that because you happen to have this particular disease. But in addition, you share many other things that are good and valuable, and you can help each other.” The man escorted Arboleda-Velasquez through dangerous territory to his brother’s farm. He did not go onto the farm, but a tentative connection through intermediaries was established when the brother inquired about the man’s health and welfare.

In this way, the research project serves a greater good than biomedical science, said Arboleda-Velasquez. “For Colombia and this particular generation of Antioquians, it is important to create unity by any means,” he said.

Family members have become a key part of the team that recruits blood relatives—now numbering more than 2,000 individuals—to the neurology clinic for exams and blood samples. “These people are very committed,” Arboleda-Velasquez said. “They give their time, their blood, their skin biopsies, more blood, and when they die, we want their brains for pathological studies. They are very generous and want to help.”

Back to the Community
Arboleda-Velasquez came to HMS on a six-month scholarship in an exchange program between the two universities. The program was established by Lopera and Kosik, longtime collaborators on a research project investigating an inherited form of Alzheimer’s disease. He opted out of the planned clinical rotations and joined Kosik’s lab, applying to graduate school at HMS to continue his research.

Arboleda-Velasquez, Lopera, and Kosik jointly won Colombia’s top science award for their collaborative discovery of the two family mutations in Notch3. They used their prize as seed money for a nonprofit foundation to provide sustained genetic counseling and other support to people in Antioquia with genetic disorders.

The new paper may help explain how the various familial mutations in Notch3 all create a similar clinical phenotype. Arboleda-Velasquez and his co-first author, Raajit Rampal, an MD-PhD student at the State University of New York at Stony Brook, found that Notch3 mutations impair proper placement of the sugar groups necessary to make the receptor more or less sensitive to other molecules.

Specifically, the process goes askew at the second step, when an enzyme named Lunatic Fringe cannot extend a chain of sugars.

Surprisingly, CADASIL mutations usually do not directly affect the site where Fringe places its sugar. “When you study diseases caused by mutations in a particular gene, the paradigm clearly says that mutations should be related to the area of the particular defect,” said Arboleda-Velasquez. “In this case, mutations don’t affect the glycosylation sites directly.”

The culprit is the misshapen end of the receptor, the researchers found. Most CADASIL mutations result in a missing or extra cysteine somewhere at the end. Normally, the cysteines at the end of the receptor give the protein its accordion bends by pulling close to another cysteine. Instead, in CADASIL, an extra unpaired cysteine on one Notch3 receptor reaches out to bond with a lone cysteine on a neighboring receptor, forming an aberrant double receptor that effectively closes out Fringe.

The researchers also found evidence that a cysteine in Fringe may also aberrantly bond with a mutant Notch3 receptor. The researchers intend to examine postmortem brain tissue to determine if either of the dysfunctional duos are found in the extracellular aggregates common to people with CADASIL.

This week, Arboleda-Velasquez heads to Colombia for another CADASIL family reunion. He will introduce two mouse models he is developing in the lab of Spyridon Artavanis-Tsakonas, the Kurt J. Isselbacher/Peter D. Schwartz professor of cell biology at HMS and Massachusetts General Hospital, which carry the family mutations.

(Originally published in Focus, the twice monthly newsletter for the Harvard medical community)


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