NEW YORK (GenomeWeb News) – A combination of rare and dominant negative common genetic variants contribute to a condition called Bardet-Biedl syndrome, according to a new study that relied on a zebrafish model system and cell culture experiments to sift through more than 100 potential disease alleles.
Using assays that relied on known physiological features of Bardet-Biedl syndrome and zebrafish development, researchers from the US and Uruguay systematically tested 125 alleles affecting 14 genes in order to learn more about the functional consequences of these genetic changes. The study, which appeared online this week in the Proceedings of the National Academy of Sciences, suggests the condition is a consequence of both strong, rare variants as well as a subset of common variants that exacerbate the condition.
"Our work demonstrates that it is possible to develop functional bioassays using a vertebrate model that predicts whether a mutation has a role in a complex disease, like Bardet-Biedl syndrome," senior author Nicholos Katsanis, a cell biology researcher at Duke University and director of its human disease modeling center, said in a statement.
"[T]his might help settle a 100-year-old argument about common versus rare mutations and how they might underlie human genetic disorders … the answer is both, in a context-dependent fashion," he added.
Bardet-Biedl syndrome is an autosomal recessive, inherited disease that belongs to a group of conditions affecting the cilia structures projecting from some cells' surfaces. A range of symptoms, including mental retardation, obesity, retinal degeneration, and kidney problems, characterize the condition. And past studies have linked Bardet-Biedl syndrome to at least 14 genes that appear capable of interacting with one another epistatically.
But while research is progressing in terms of characterizing normal genetic variation and finding alleles linked to the disease risk, severity, and progression, the researchers noted, there is still a way to go before disease-related functions can be established for such variants.
"Knowing all of the disease-related variants in a genome is only a starting point, because our work suggests that there is complexity that many do not yet appreciate in disease architecture," Katsanis said.
In an effort to get a better handle on the mutations that contribute to disease and their effects, Katsanis and his team exploited a zebrafish embryo system that let them look for features resembling known features of Bardet-Biedl syndrome.
The researchers injected zebrafish embryos with morpholinos — antisense oligonucleotides used to curb the translation of specific transcripts — targeting a dozen potential Bardet-Biedl syndrome genes called bbs1-12.
They then looked for phenotypes reminiscent of BBS, such as shortened bodies or notochord problems, showing that such phenotypes occurred when they interfered with the translation of each of the 12 bbs genes. When the team tossed in human versions of the genes, though, they were able to rescue these unusual phenotypes with the mRNA.
The researchers took advantage of this rescue in their subsequent assays, looking at the effects of adding back human genes that had a range of BBS-related single nucleotide changes.
Of the mutants tested, the team found 14 that rescued the morpholino-treated embryos similar to full length human mRNA, along with 15 mutants that rescued the phenotypes to a lesser extent, and 45 that did not improve the phenotype.
Interestingly though, adding back another 35 mutant human genes actually made the morpholino-induced effects more severe — a finding consistent with a dominant negative effect for these mutations.
"It is possible that such interactions of mutant proteins exacerbate the disease phenotype but that interaction of mutant and [wild type] protein, although detrimental to [wild type] protein function, does not by itself result in disease," Katsanis and his co-authors wrote.
After testing the sensitivity and specificity of the zebrafish assay, the team also verified the patterns they'd seen in zebrafish through a series of experiments in mouse and human cell lines.
And, they reported, at least some of the dominant negative alleles identified in the assay also turn up in individuals with Bardet-Biedl syndrome and their family members: about 18 percent of the BBS-related alleles found in 218 individuals tested belonged to the dominant-negative allele set. That number was even higher when the researchers focused on alleles that appear to epistatically modify the effects of other variants.
Based on their results so far for Bardet-Biedl syndrome, the team argued that a similar approach may also prove useful for studying other human diseases. And, they say, such approaches will be increasingly necessary for evaluating variants coming out of large-scale genetic studies of disease.
"[T]he study of BBS mutations has benefited from a combination of strong evolutionary conservation and a priori understanding of some of the pathways involved in disease pathogenesis. Nonetheless, it is likely that this approach is conceptually feasible for a broad range of other clinical phenotypes," the researchers concluded.
"The availability of large-scale resequencing will necessitate the development and application of bioassays such as those described here to assess the functional relevance of variants identified in individuals with phenotypes of interest."