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Tuesday, November 11, 2008

Mapping CJD: Human Mad Cow Disease

Creutzfeldt-Jakob disease, commonly known as CJD, is a brain disease that progresses rapidly and generally results in the death of the patient. It probably came to the public notice following the outbreaks of mad cow disease in Europe in the 1990s because the agent that causes bovine spongiform encephalopathy (BSE) in cows is thought by some to be responsible for the onset of variant CJD in humans. Yet, despite the swathe of publicity, CJD is a rare disease.

Of the various types, sporadic CJD is the most common, accounting for about 85% of cases in the US. The symptoms only show their faces around the age of 60 and most patients die within a year. Hereditary CJD, accounting for 5-10% of US cases, and acquired CJD, which is transmitted by exposure and accounts for 1% of cases, are the other major categories. The rapid progression and fatal outcome of the disease point to the fact that there is no cure for CJD.

The conventional way to confirm CJD in humans is to carry out a brain biopsy. This occurs in post-mortem examinations but biopsies on suspected patients are generally discouraged because, even if the disease is diagnosed, there is no treatment. So, the medical world needs a reliable test for live patients. Currently, one available test looks for the 14-3-3 proteins in cerebrospinal fluid (CSF). These are indicative of CJD but the immunoblot procedure gives a false positive rate of 5-10%, so is unsuitable for screening. In addition, a high proportion of patients with variant CJD do not produce these proteins. A second test, developed by Amorfix Life Sciences Ltd. and currently undergoing validation in the UK, detects prion proteins associated with vCJD in human blood and has given promising results.

A team of scientists in Germany has adopted a different approach, looking for a panel of proteins in CSF which are indicative of CJD and can differentiate it from other neurodegenerative diseases in the largest study of this type. Markus Otto from the University of Ulm with colleagues from six other institutions analysed CSF from a total of 72 patients: 36 diagnosed as having probable CJD (according to WHO criteria), 24 with Alzheimer's disease, 6 with dementia with Lewy bodies and 6 non-demented controls.

The samples were subjected to an optimised sample preparation procedure for analysis by 2D differential gel electrophoresis using the CyDyes 2, 3, and 5. This method has the potential to separate thousands of proteins on a single gel and is a popular choice for comparative, quantitative proteomics studies.

Following 20-fold concentration, CSF was depleted of the abundant proteins albumin and immunoglobulin G, which tend to mask less abundant proteins. There is always a risk that some less abundant proteins will be co-depleted, or reduced in volume so that there is insufficient quantity remaining on the gels for subsequent identification. However, the advantage gained by removing albumin and IgG were considered to outweigh these disadvantages.

The depleted CSF was mixed with acetone to induce protein precipitation and the dried proteins were lysed in buffer to get at the soluble proteins. Then, proteins from the different disease groups were labelled with the respective dyes and subjected to gel electrophoresis. An initial separation at pH 4-10 showed that few proteins were resolved in the pH 7-10 region, so the samples were run at pH 4-7. In this way, about 2200 spots were resolved.

The use of an internal standard allowed normalisation and comparison between the various gels. Using stringent criteria, five protein spots were found to be statistically different in the CJD samples compared with the others. These were cut from the gel to identify the proteins by mass spectrometry, although one protein remained a mystery because there was insufficient sample for identification. The team regarded this protein as one with high diagnostic potential for CJD and will aim to identify it in the future.

Two of the other four proteins were members of the 14-3-3 family, which was not unexpected. The remaining two were L-lactate dehydrogenase B-chain and gamma-enolase. Their suitability as biomarkers has yet to be validated but their discovery presents more possibilities for developing a diagnostic test that is more reliable than the current 14-3-3 immunoblotting test.

Other proteins that have been proposed previously as candidate markers for CJD, such as transthyretin, serotransferrin and gelsolin, were regulated on some gels but did not fulfil the stringent criteria for selection.

Otto and the team have demonstrated the validity of their approach for differentiating CJD from other neurodegenerative diseases and have presented several new candidates for biomarkers. The technique should also be applicable to other brain diseases, especially those in which brain proteins are transferred into CSF but the limitations with regard to low abundance proteins should be taken into account.

Related links:

Proteomics 2008, 8, 4357-4366: "Cerebrospinal fluid-optimized two-dimensional difference gel electrophoresis (2-D DIGE) facilitates the differential diagnosis of Creutzfeldt-Jakob disease"
Article by Steve Down

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