Vindication for proteomics in research on ageing

The April publication in Science of the identity of the faulty gene involved in the devastating premature-ageing disorder, Hutchison-Gilford progeria syndrome (HGPS), was a triumph for geneticists at the National Human Genome Research Institute in Washington.

But it was only half the story -- a collaboration between teams at the University of Sydney and Sydney-based company Proteome Systems had already independently identified the Lamin A (LMNA) gene as the culprit in progeria, using proteomics.

The US study, led by Francis Collins, director of the National Human Genome Research Institute (NHGRI), identified only the gene, and the mutation that leads to progeria.

Collins described his team's discovery as "the first piece in solving the tragic puzzle of progeria. Without such information, we in the medical community were at loss about where to focus our efforts to help these children and their families. Now, we finally know where to begin."

The Sydney project, led by Dr Nicolle Packer, at Proteome Systems, and Prof Tony Weiss, head of molecular biotechnology at Sydney University, had already found a dozen more pieces to the puzzle, that are far more informative about what goes wrong in progeria -- and, by extension, what happens in the normal processes of ageing.

Not only has the Sydney team identified the mutant gene, it has catalogued the disastrous, ripple-down effects of the Lamin A mutation on downstream genes that collectively cause progeria.

The Packer/Weiss collaboration, which will publish its findings later this year, has provided a tour-de-force demonstration of the advantages of proteomics over genetics in rapidly identifying genes involved in severe genetic disorders, and then linking cause with consequences.

Weiss's team obtained skin and blood-cell samples from US progeria patients and their healthy siblings from the Coriell Research Institute in the US, which maintains cell lines from individuals with genetic disorders and their relatives.

Rare disease model for ageing

Progeria, according to the NHGRI team, is a spontaneous, not inherited, genetic disorder -- it occurs only once in every 8 million births, so cases in Australia are extremely rare.

Children with progeria usually appear normal at birth. However, within a year, their growth rate slows and their appearance begins to change. They typically become bald, and have aged-looking skin that has lost its elasticity and pinched noses. They often suffer from symptoms typically seen in elderly people, especially severe cardiovascular disease, and the average age of death is around 13, usually from heart attack or stroke.

Packer said that while the disorder was a potential model for normal ageing, it had been difficult to study because children with progeria do not live long enough.

To identify the gene involved, Weiss's team at Sydney University prepared protein extracts from the affected and healthy cells, and sent them to Proteome Systems. "Obviously they were enriched in proteins we thought would be interesting," he said.

Packer said an honours student working in both their laboratories, Leanne Robinson, did most of the work, running the extracts on 2D electrophoresis gels and doing mass spectrometry analyses on the purified proteins.

They were able to identify proteins in the progeria cells that had changed in abundance, or had been abnormally spliced or modified by processes such as glycosylation or phosphorylation.

Converging lines

Weiss said converging lines of evidence pointed towards the Lamin A protein being the primary cause of progeria. Researchers had previously found chromosomal abnormalities, including translocations, in regions lying close to the Lamin A gene.

Proteome Systems' analysis showed that Lamin A was abnormal in progeria cells. This revealed that the Lamin A gene was likely to be mutated in the progeria cells.

The Lamin A protein is a component of the nuclear membrane; the mutation identified by the US team involves a single DNA base, but it has dramatic effects on the way the protein is spliced -- the protein molecule itself ends up with a large amino acid sequence missing from its C-terminus.

Packer said the missing region appeared to be involved in activating other proteins by phosphorylation. These changes cause cumulative errors in the biochemical pathways leading from the Lamin A protein, with disastrous effects on normal cell function.

After implicating Lamin A as the source of progeria, Weiss and Packer emailed Collins in the US, who told them in confidence that his team had the same gene using conventional molecular genetic techniques.

"We had big smiles on our faces when he told us -- it came at just the right time," Weiss said. "We now had verification from two sides -- proteomics and genetics -- that the Lamin A gene was involved."

Collins' team beat them into print with the discovery, but Weiss said the proteomics approach has provided a much better picture of changes occurring inside the cell in progeria.

"Not only do we see changes in the Lamin A protein, we see a range of additional protein changes that we are confident are direct consequences of the mutation involved," he said.

Sydney University and Proteome Systems plan to spin off a new private company, Nucleos, to commercialise discoveries from the progeria study related to ageing; it will also use a proteomics-based approach to identify genes involved in other genetic disorders.