Tissue samples of seven affected calves were first examined for bovine viral diarrhea virus, a known infectious cause of osteopetrosis, but the results were negative. However, pedigree analysis of the seven calves revealed both maternal and paternal common ancestry, suggesting an association between their genes and the disease.

Scientists compared DNA from affected Red Angus calves and their carrier parents to DNA from unaffected animals. They then searched the entire genomes of all calves for chromosomal segments common to the affected animals, but different from the normal animals.

The Illumina Bovine SNP50 BeadChip, developed earlier by ARS researchers, along with industry and university partners, was used to identify suspect genes. The BeadChip, a glass slide containing thousands of DNA markers, identifies relationships between markers simultaneously.

“We had just the right tool to genotype DNA samples from animals with this mutation,” Sonstegard says. “Once samples were assembled and extracted, running the chip didn’t take very long.”

A single chip generates more than 50,000 genotypes for each animal. DNA samples were applied to the BeadChip, chemically labeled, and scanned to produce genotypes.

“We were looking for those regions from the affected calves where the chromosome was similar on both the mother’s copy and the father’s copy,” Sonstegard says.

“We were able to detect through an analysis program when it was homozygous — having two copies of the same gene — only in a specific region of chromosome 4.”

A segment on cattle chromosome 4 contains SLC4A2, a gene necessary for proper osteoclast maintenance and function, McDaneld says. Osteoclasts are types of cells responsible for breaking down old bone during bone development and remodeling.

In the osteopetrosis-affected calves, researchers found that some of the SLC4A2 genetic material had been deleted. The discovery of the deletion in this gene was a first for cattle, McDaneld says.

While the cattle study was being conducted, scientists elsewhere were studying bone development in mice. They intentionally created a similar mutation in SLC4A2 to determine the function of the gene, and it was observed to cause the same marble bone disease phenotype, McDaneld says.

The fact the exact same gene in mice was responsible for the osteopetrosis mutation confirmed the findings in Red Angus.