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Genome of Rat Model Reveals 60 Deleted Genes that May be Involved in Hypertension

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By Monica Heger

Animal studies have always been important for understanding and characterizing human disease. Now, researchers have demonstrated that sequencing the genome of an animal model could yield even more insight by identifying causative mutations, genes, and pathways involved in complex genetic human disease.

In a study published in Genome Research last week, an international team sequenced the genome of a spontaneously hypertensive rat, the most widely studied animal model of hypertension. The study, which marks the first whole-genome sequencing study of an animal model of disease, identified 60 genes that had been completely deleted and an additional 47 that had deletions of one or more coding exon.

The researchers said the affected genes offered insight into potential driver mutations and pathways involved in the disease. Additionally, they reported that because animal models are homozygous, it is easier to search for interesting variants than it would be in human subjects. Furthermore, sequencing animal models would not require sequencing to as great a depth as human genomes would, which reduces the cost.

Senior author Tim Aitman, a chair in clinical and molecular genetics at Imperial College London, said that this approach will be a good way to study other complex human diseases. He added that his lab plans to sequence five additional rat genomes and, together with the other laboratories involved in this paper, eventually plans to sequence around 100 rat genomes representing disease models of hypertension, kidney failure, arthritis, autoimmune diseases, a number of different cancer types, and neurological disorders like hyperactivity.

"There are a whole range of disorders that are in some ways best studied in the rat because there has been extensive work done in those types of diseases," Aitman said. "Models in which detailed genetic studies have been done are the ones that will benefit the most."

Peter Harris, a professor of biochemistry and molecular biology and medicine at the Mayo Clinic who has studied kidney failure in animal models and who was not involved with the study, said the work is an important step forward. "I think it could really help identify the causative genes that underlie these [quantitative trait loci] that have been mapped. It gives a list of candidates that can be followed up very quickly … Overall, I was very impressed."

Aitman and a team of international researchers sequenced the spontaneously hypertensive rat to 10.7-fold coverage using a paired-end sequencing strategy on the Illumina Genome Analyzer with average read lengths of 41 base pairs. They mapped about 681.8 million reads to the reference Black Norway rat genome, and identified 3.6 million high quality SNPs; 343,243 short indels; more than 13,000 large deletions; and 588 copy number variants.

They then used Ensembl build 54 for the reference rat genome to determine the effects of the variations. They found that 60 genes were completely deleted from the genome sequence compared to the reference, and 47 additional genes were partially deleted.

Some mutations of note include a copy number variation in a gene that has previously been shown to cause insulin resistance and hypertension in the rat strain. The team found SNPs and indels in a gene that regulates left ventricular mass, and also a deletion in a gene that codes for a protein that regulates calcium homeostasis, electrical activity, and normal heart rhythm. The authors reported that the deletion appeared to "reduce greatly or even abolish the function of the encoded gene product."

They also found single base deletions in a gene, whose human ortholog has been associated with systolic or diastolic blood pressure in human genome-wide association studies.

"We thought there would be a large number of genes where there were subtle changes," said Aitman. "But, we found an extraordinary number of genes [whose function] was totally depleted. That was unexpected."

He said that at least some of those gene deletions will explain why the animal gets hypertension, and could provide insights into human disease.

Aitman said that some of the genes implicated in the rat hypertension model will probably be conserved in humans, but more importantly, he said that it is likely the pathway will be conserved. "If we can find the entire pathway in rats, we think it will be the same that you find in humans."

In particular, many of the genes that were mutated in the hypertensive rat were genes that affect ion transport, and previous studies have implicated that pathway in hypertension as well as kidney failure, said Aitman.

"In some cases, the transport of ions in the kidney leads to hypertension. So we think that may represent the tip of the iceberg for ion transport in general as a cause of hypertension," said Aitman.

Harris noted that while he was surprised by the number of deleted genes the researchers found, the "full significance" of these genes isn't immediately clear. He added, however, that they would be good candidates for further testing.

Kim Worley, an associate professor at the human genome sequencing center at Baylor College of Medicine, who worked on the sequencing of the rat reference genome, said the study showed "clear confirmations of known mutations that are of interest to the hypertensive phenotype." She added that she thought there was "a lot of intriguing information and a lot of new research that can be done."

As the first study to sequence the genome of an animal model of disease, the project also demonstrated the utility of the technique, highlighting its advantages over whole-genome sequencing of humans to study complex diseases.

"In model organisms, you can specify how the animals will breed, and by controlling the breeding, you can set up an experiment that is statistically much more powerful than you could do in humans," said Aitman. "You can create inbred strains, and the inbred strains are essentially homozygous at every base pair in the genome, and this means that all animals from a particular strain are genetically identical."

Harris agreed that sequencing animal models is a more straight-forward approach than sequencing humans. Because the animal models are homozygous, the variants are easier to find at a lower sequence density, said Harris. "And you can be more certain about their possible physical effect as well. The gene is either completely missing or has a stop codon mutation."

Being able to sequence animal genomes at a lower fold coverage also reduces the cost of the sequencing as compared to human genomes. Aitman said that the cost of sequencing reagents for the study was less than $50,000 when they did sequencing but that today, it would probably cost around $5,000.

Aitman said his team is now looking at the genes implicated in this study, and is trying to see if correcting the mutation and restoring the function of the genes affect hypertension. If so, then those genes could be potential targets for drug development, he said.

Aitman said his lab will continue to use the Illumina GA and that his lab plans to get an Illumina HiSeq 2000 instrument. "We anticipate that will greatly accelerate progress," he said.

In the future, Aitman predicted that many more animal disease models will be sequenced and made available by other research groups. "Because of these genome sequences that will be available, the rate of progress in understanding the genetic basis of disease will increase exponentially. There will be an explosion of knowledge, and some [of the genome sequences] will have quite rapid implications for understanding human disease."

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