s at a single DNA position. "So in each case among the 84, we know there is a patient who had this exact change, accompanied by mild to severe disease," Mayfield explains.
Next, the researchers transferred each of the variants into a yeast cell, yielding 84 yeast strains that differed at only a single place in the CBS gene. A control strain containing the fully functional major allele of the CBS gene was constructed for comparison.
"Once we put an allele into yeast we can conduct experiments to ask whether it's functional or not by growing the colonies, both in dishes and in liquid culture," the geneticist explains. A mildly mutated allele may allow yeast cells to grow normally, but severely mutated cells fail to grow at all. Many variants function somewhere in between. "When you know where two sequences differ, you can start to identify changes that affect biological function from those that don't," he says.
But "the really interesting and exciting part of this project is what we learned about treating the disease," Mayfield adds. Some homocystinuria patients respond to simple vitamin B6 supplementation, while others require severe dietary restriction to avoid disease symptoms. In a series of experiments with yeast in liquid culture, the researchers added different levels of supplemental vitamin B6 to the 84 disease alleles and found that 37 percent of the alleles were functional or could be rescued by vitamin B6. "In the clinic, then, you can expect the patients with these alleles to respond to B6 treatment," the geneticist explains.
"This method gives us information about allele function and is applicable to other disease genes. Metabolic diseases and proteins that need vitamin co-factors are amenable to similar studies. Our findings indicate clearly that sequences alone can't tell the full story. This is a first step in gaining information from the genome to move toward a treatment. We can't claim to cure the disease, but it is good news."
This study was funded by the Howard Hughes Medical Institute as part of a larger project at UC Berkeley to design an entry-level biology curriculum that included discovery-based learning. It was performed in part by undergraduates mentored by Mayfield, who feels it is extremely important that university-level science courses begin to teach the next generation techniques they will need to make sense of natural variation in gene sequences and the difference between natural variation and disease.
"Right now, our ability to generate DNA sequences is far ahead of our ability to understand it," he notes. "We're going to see in our personal genome sequences that each of us has variations in disease genes. We must not panic. In this study, even in the extreme case of sequences from patients with a disease, we saw that some variants were coincidental, not causal, while others were treatable. We need to develop a suite of tools like this to help us investigate which alleles are functional and which are not, before we can comprehend the human genome."
|Contact: Janet Lathrop|
University of Massachusetts at Amherst