Van Buchem disease decoded

Study underscores the importance of non-coding DNA in disease determination

Today a team of scientists provides convincing evidence that the deletion of a large non-coding DNA segment on human chromosome 17 is responsible for Van Buchem disease. This genetic mutation is one of only a few disease-associated mutations discovered to date that alters a long-range transcriptional regulatory element. The study appears online in the journal Genome Research.

“Our study addresses a fundamental issue with regards to the majority of the human genome that is non-coding in nature, and its potential impact on human health,” explains Dr. Gabriela Loots, a scientist in the Department of Genome Biology at Lawrence Livermore National Laboratory who headed the study. “Non-coding regions located far away from the genes they regulate are critical for normal gene expression and are capable of leading to dramatic abnormal phenotypes if altered or deleted.”

Van Buchem disease is a rare hereditary disorder of the skeletal system that is characterized by progressive osteosclerosis, particularly in the skull and mandible, but also in the clavicles, ribs, and diaphyses of long bones. Consequences of this increased bone mass usually include facial distortions and pinching of cranial nerves, and the increased nerve pressure often leads to deafness and blindness. Onset of the disease generally occurs during childhood and is manifested only in individuals carrying two copies of the mutant allele.

The locus responsible for Van Buchem disease was previously mapped to the short arm of human chromosome 17 near the gene sclerostin (or SOST), whose protein product functions as a negative regulator of bone formation. Mutations in the protein-coding regions of SOST are known to be responsible for sclerosteosis, another genetic disorder with attributes similar to Van Buchem disease. Because SOST was therefore a strong causal candidate for Van Buchem disease, scientists screened the SOST coding sequence for associated mutations, but to no avail.

Recently, however, a large 52-kilobase deletion was identified at a significant distance – approximately 35 kilobases – from the SOST gene in humans. This deletion, although associated perfectly with Van Buchem disease, removed a large non-protein-coding region; thus, its function, if any, in the development of the disease remained unclear.

The current study, which was led by Dr. Loots, was designed to rigorously investigate the underlying molecular mechanism by which this non-coding sequence might ultimately give rise to Van Buchem disease. Her team included researchers from Lawrence Berkeley National Laboratory (Berkeley, CA), the Novartis Institutes for BioMedical Research (Basel, Switzerland), and the DOE Joint Genome Institute (Walnut Creek, CA).

Dr. Loots and her colleagues engineered a human bacterial artificial chromosome (BAC) and generated transgenic mice with and without the 52 kilobases of DNA that are absent in Van Buchem patients. Although SOST was expressed normally in the early mouse embryo from both lines, SOST was dramatically downregulated in adult mice carrying the deletion, when compared to wild-type transgenic mice. These results provided strong evidence that the lack of SOST expression in humans homozygous for the 52-kilobase deletion is caused by a regulatory element – most likely an enhancer – that is located within the 52-kilobase region and that functions in a bone- and age-specific manner.

The team then utilized comparative sequence analyses and transient transfection assays to identify the actual enhancer sequence within the 52-kilobase deletion region that is responsible for SOST regulation. They aligned 140-kilobases of the human SOST region with the orthologous mouse sequence and identified seven evolutionarily conserved regions (ECRs). The seven ECRs were subjected to in vitro transient transfection enhancer analyses, and one of them – a 250-base-pair region named ECR5 – was found to drive expression in osteoblast-like cells.

These results provide a strong causal link between the 52-kilobase deletion and Van Buchem disease in humans. Drs. Michaela Kneissel and Hansjoerg Keller, collaborators on the project from the Novartis Institutes for BioMedical Research in Basel, Switzerland, point out that, in addition to addressing a fundamental problem in genomics, this project has clinical relevance. “Human genetic diseases of the skeleton, such as sclerosteosis and Van Buchem disease, provide a starting point for understanding the modulation of anabolic bone formation, and ultimately have the potential to identify key molecular components that can be used as new therapeutic agents to treat individuals suffering from bone loss disorders,” say the researchers.

On an important scientific note, the methodological approaches employed by these researchers in elucidating the basis for Van Buchem disease will be widely applicable and very powerful in the characterization of other distant, cis-acting regulatory elements.