Surprising findings from just five patients has led to the first proof of how the rare disorder Fanconi anemia causes chromosomal instability. A team of international researchers, led by scientists at Rockefeller University, reports the findings in the September issue of Nature Genetics.
The scientists found a gene mutation not previously known to be related to Fanconi anemia, and they say that BRIP1 is the first gene associated with the disease whose protein has a known function. That protein, known as BACH1, normally helps DNA unwind in order to be repaired, and if it cannot function, chromosomal damage accumulates, they say.
"We have known for decades that patients with Fanconi anemia have chromosomes that break easily, but none of the many genes previously found to be associated with the disease explained this phenomenon. This new link to BRIP1 mutations may have revealed a central player in development of the disease," says the study's principal investigator, Arleen Auerbach, Ph.D., who directs the Laboratory of Human Genetics and Hematology at Rockefeller. Working with her were researchers from two German universities and from Memorial Sloan-Kettering Cancer Center in New York.
"Given these new findings, we can now suggest that DNA double-strand breaks are the lesions that underlie the pathology of this disease," says Auerbach, who is internationally known for her work on the disorder and for the large Fanconi anemia registry she maintains at Rockefeller.
Fanconi anemia (FA) is an inherited disorder characterized by developmental abnormalities, life-threatening bone-marrow failure, and predisposition to a variety of cancers. Researchers have long known that patients with the disease have chromosomes that are not readily repaired when they break; in fact, a blood test created in 1981 by Auerbach, which uses a chemical that specifically increases that damage, is now used worldwide to diagnose FA.
Auerbach and others suspected this hallmark chromosomal instability is associated with defects in caretaker genes that help maintain the integrity of DNA. One reason for this hypothesis is that some already identified Fanconi anemia proteins accumulated in the nuclei of normal cells along with protein produced by the gene BRCA1, which is believed to help maintain DNA stability, but when mutated, is the major breast cancer susceptibility protein.
Researchers had theorized that the underlying fault in FA lies in the seven genes that need to work together to produce a protein "complex" that activates another existing cellular protein known as FANCD2. FANCD2 is then believed to work with BRCA1 protein to repair the constant DNA damage that results from excessive sunlight, radiation, exposure to carcinogenic chemicals and even from normal cell division.
"All of these seven Fanconi genes have to be normal -- if one isn't, then FANCD2 is not activated," says Auerbach. But she adds that no one knows what the proteins FANCD2, BRCA1 or even BRCA2 -- produced by another breast cancer susceptibility gene that has also been linked to FANCD2 -- are actually doing.
"No one knows the precise role of any of these genes and proteins, but we believe that if BRCA1 or BRCA2, or any of the Fanconi genes that activate D2 are defective, a sequence of events is disrupted and DNA repair is blocked," she says.
But Auerbach and her team of researchers were puzzled that about 20 patients in the 1,000-plus International Fanconi Anemia Registry (IFAR) had no mutations in any of the genes known to be associated with the disease, yet there was no question they had Fanconi anemia. "These patients had the disease, yet their FANCD2 was activated normally, and there were no problems with BRCA1 or BRCA2," she says.
So Auerbach and her colleagues selected four families for a detailed gene analysis, based on the suspicion that there was, in each of the families, a "founder effect" -- a change in the frequency of a gene mutation that occurs when a population is descended from only a few individuals. Two of these families were Inuit (aboriginal Canadians): one had two children with Fanconi anemia and the other family had a single child with the disease. "We suspected there was a single mutation in a single gene that affected these children," Auerbach says.
The researchers also selected two Hispanic families in which they knew the parents were first cousins, and each had an affected child.
The researchers first applied a test that could tell them whether the offending gene was "upstream" or "downstream" from activated FANCD2 -- that is, did action of the mutant gene fall in the molecular pathway before FANCD2 was activated, or after, respectively? The answer was that the problem was located downstream from a normally functioning FANCD2.
The researchers then mapped SNPs in the genome of those patients and families, looking for changes in which a single chemical building block in the DNA differs from the usual building block at that position. Because FA is a recessive genetic disease, an affected child needs to inherit two copies of an errant gene, each from a parent that carried a single mutation.
They were startled to find only one suspect location in the entire genome, on chromosome 17, that was present in all four families. Further research uncovered two candidate genes within that region, and none of the patients had an abnormality in one of them. But they all had mutations in the second gene, BRIP1.
"What was very surprisingly to us is that while all five patients were homozygous for a mutation in the gene, as expected, all had the same mutation in this gene," Auerbach says. In other words, the five patients each inherited two copies of the same mutation, one from each parent.
When the researchers looked at the other families in their registry with no known mutations in any of the genes associated with the disease, they found six more patients with this same BRIP1 mutation, three of whom were homozygous.
Now the story began to make sense to the researchers, since the protein, BACH1, produced by BRIP1, was known to be a DNA helicase, a class of enzymes which unwind the two strands of the DNA double helix so that DNA synthesis can take place. And they knew from the scientific literature that BACH1 interacts with BRCA1 protein.
"This is the first gene associated with Fanconi anemia that we have a defined function for," says Auerbach. "It interacts directly with BRCA1, and is known to play a role in the repair of DNA double-strand breaks."
BACH1 could be the link between FANCD2 and BRCA1, the researchers say.
"It may be that DNA can't be repaired without a normally functioning BACH1," says Auerbach. "So perhaps FANCD2 activation isn't the endpoint, as had been thought, but that it has to do something downstream that can't be accomplished if BACH1 is not present."