NEW YORK (GenomeWeb) – The combination of three rare genetic variants is responsible for a version of a congenital heart disease, a new study that was enabled by a CRISPR-Cas9 mouse model and patient-derived induced pluripotent stem cells has shown.
On their own, the three inherited missense single-nucleotide variants were not associated with a disease phenotype. But in two childrem who inherited all three – two from the father and one from the mother – the genetic interactions led to a cardiac anomaly called left ventricular noncompaction, where heart cells are thought to fail to mature.
The research, published today in Science, involved the use of multiple genomic technologies, including clinical whole-exome sequencing, CRISPR gene editing in mice, RNA sequencing, and iPS cells. Each of the technologies was crucial to parsing the complex genetic machinery underlying the patient's condition, said Deepak Srivastava, president of the Gladstone Institute of Cardiovascular Disease and the paper's senior author.
"Going back and forth between human and mouse to validate findings from human sequencing, it's the first study to put all that together to directly address a combination of variants that occur in the human population," he said.
Sequencing the parents and affected children revealed the variants, while CRISPR helped recapitulate features of the disease in mice. RNA-seq also helped show that the variants led to differences in gene expression in mouse heart tissues.
While many human diseases have been shown to be either monogenic or polygenic, establishing that diseases can also be oligogenic, or due to just a few genes in combination, has been difficult, Srivastava said.
He first started caring for the family affected by the heart condition almost a decade ago, he said. Prior to the two patients at the center of the new study, the parents had lost a fetus due to heart failure. "When we first sequenced their exomes, it was clear it was familial," he said.
With CRISPR, the research team was able to try to recapitulate the individual candidate mutations they found in the father and mother with sequencing. As they wrote in the study, the missense variants in MKL2, MYH7, and NKX2-5 were "sufficient to mimic the pathology […] in a mouse model," including differences in heart wall thickness and gene expression. In iPS cells generated from the patient, RNA-seq showed gene expression patterns similar to the mice with all three gene variants.
Srivastava estimated that the study cost approximately $1 million.
In the paper, the authors said that the NKX2-5 variant, inherited from the mother, acted as a genetic modifier. While rare, with an allele frequency of 0.0012, Srivastava stressed that the variant is out there in the human population. "When people have thought about genetic causes, we've largely, in order to deal with the thousands of variants everyone has, ignored those that occur in some frequency." His team was successful, though, because they considered variants that were more frequent and predicted to be less detrimental, he said.
"There are things that can push [a disease] over the edge so it manifests," Srivastava said, and the paper shows that NKX2-5 is one of them. But the fact that it can take so little to change a phenotype is actually encouraging, he said. "The corollary is that you can push it the other way. The hurdle may not be so great, and you may only have to tweak it a little bit."
"Now that we know the genetic mechanism [underlying the disease], we can actually screen for drugs in the cells we have, to try to correct the problems we identified," he said. "We're doing that now to try to figure out a potential treatment with these sorts of conditions."