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Blood test development by UK scientists may provide an early diagnosis for Alzheimer’s disease.It is one hundred years since the German neuroscientist, Alois Alzheimer, first described a case of this particularly devastating form of dementia. Alzheimer’s patient was a woman in her 50s known as Auguste D, who died in 1906. He commented of her condition “she suffered from constant restlessness and anxious confusion”.

A century on and scientists know more about the condition; yet there is still no treatment that goes beyond the symptomatic treatments that are currently causing such controversy through NICE. Alzheimer’s disease (AD) is the most common cause of dementia in the elderly and is the fourth most common cause of death in the Western world. It is estimated that there are over half a million suffers in the UK alone, representing massive demands on health systems.

At the present time, diagnosis is primarily achieved through clinical assessment involving informant interviews and supplementary investigations to rule out other causes of confusion. This form of assessment, although high in sensitivity, has a low specificity when discriminating from other forms of dementia. Thus there is high demand for a way of increasing the accuracy of clinical diagnosis for the disease. Many studies are underway to try to find a biomarker, or test, that would aid diagnosis, especially in early stages, and could be used to monitor disease progression. The Alzheimer’s Association and the National Institute of Ageing recognised the need for a marker of disease and laid down criteria for a successful biomarker. Ideally it would be ‘reliable, reproducible, non-invasive, simple to perform and inexpensive’ whilst having levels of sensitivity and specificity of over 80%.

As yet there are no suitable markers available that fit these criteria. However, progress has been made in trying to find changes at the protein level using proteomics. Proteomics is the study of proteins, especially their structure and function, and is becoming an important technique in the search for biomarkers. Many proteomic and candidate protein studies have analysed cerebral spinal fluid (CSF) to look at changes between disease and control cases. The most reproduced findings of these studies have been an increased level of total tau and phospho-tau and a decreased level of amyloid beta (Ab 1-42) in AD patients relative to normal ageing.

Although sensitive detection of both of these proteins is now established, the specific discrimination between different forms of dementia is still low. Another drawback to this analysis is that lumbar puncture, the way of obtaining the sample, is very invasive, and repeated collection from elderly patients in large groups is very challenging. Blood, on the other hand, is much easier to obtain and the added fact that about 500mls of CSF are absorbed into the blood each day makes it a rich source for biomarkers. Our group, based at King’s College London, recently published work in the journal Brain (Hye et al., 2006). This five-year study, led by Professor Simon Lovestone and funded by the Alzheimer’s Research Trust is, to the best of our knowledge, the largest proteomic study of its kind.

Our analysis involved the use of two-dimensional gel electrophoresis (2-DGE) and liquid chromatography, coupled with quadruple time-of-flight mass spectrometry (MS/MS) to seek possible biomarkers in peripheral venous blood from a large patient sample set.

The initial discovery phase involved a comparison of plasma from 50 normal elderly control subjects directly compared with 50 AD subjects. After separation by 2-DGE, image analysis and statistical analysis, 15 spots were found to have significant case-control differences. Analysis of these spots by MS/MS identified a number of proteins, many previously implicated with disease pathology, including complement factor H (CFH) and alpha-2-macroglobulin (a-2M). These two proteins also showed the greatest fold-change differences. As the 2-DGE data was reproducible, we performed a class prediction analysis to determine whether the pattern of peptide spots could predict clinical caseness.

The outcome of this prediction was a sensitivity of 56% and a specificity of 80% and, although not meeting the ideal criteria for a biomarker, these data demonstrate that there are protein differences between AD cases and normal elderly controls.

To confirm our proteomic data, we analysed CFH and a-2M by western blotting (Figure 1). This validation involved separate sets of patients from those used in the proteomic analysis and in all included over 500 subjects. This cohort of samples comprised AD patients and patients with other neurodegenerative diseases including Huntington’s disease (HD), motor neurone disease (MND), vascular dementia (VaD) and progressive supranuclear palsy (PSP). Quantitative densitometry showed significant elevation of both CFH and a-2M in AD compared to controls and other neurodegenerative diseases, thus confirming our initial discovery (Figure 2). This elevation also seems to correlate with disease severity but levels of specificity and sensitivity would need to be improved with alternative assays.

The role of a-2M is of considerable interest in AD. This proteinase inhibitor has been shown to be present in amyloid plaques and also has a role in regulating immune response, being induced by inflammatory cytokines. CFH is an inhibitor involved in the alternative complement pathway and has, like a-2M, also previously been detected in amyloid plaques, although studies have shown there to be no increase in levels in brain tissue or CSF.

Our study, on the other hand, has shown that levels of CFH are related to disease severity, with inverse correlation to cognitive decline. The results of our study suggest that blood-based biomarkers are a possibility for the early diagnosis of Alzheimer’s disease. However, in order to translate these findings to patient benefits we need first to design a more suitable assay and then test this in a large sample of patients.

We hope that these findings pave the way for future biomarkers in Alzheimer’s.

Steve Lynham
MRC Centre for Neurodegenerative Research
Institute of Psychiatry
King’s College London.

Hye A, Lynham S, Thambisetty M, et al, 2006. Proteome-based plasma biomarkers for Alzheimer’s disease, Brain, 129: 3042-3050.