ALS Gene Expression and Biomarkers: A Better Way to Microarray

Microarrays tantalize investigators with the promise of a new smorgasbord of genes to explore and proteins to target with therapeutics. But these studies are so variable, say researchers from the University of California, Los Angeles, David Geffen School of Medicine, that findings rarely overlap even when scientists are looking for genes involved in the same disease. For example, among 11 recent microarray studies for amyotrophic lateral sclerosis, 1,140 known genes have popped up. Yet only 95 were identified in more than one study, and of those, only 61 genes moved in the same direction (up- or downregulated with disease) in multiple studies.

The UCLA researchers, led by Stanislav Karsten and Martina Wiedau-Pazos, set out to conduct an ALS microarray study that would yield more reliable results. They shored up their study with multiple cell types from mice as well as human tissue, and considered different time points in disease progression. Four years later—this kind of careful validation takes time—they report their results in the June 18 Human Molecular Genetics online. Joint first authors Lili Kudo and Liubov Parfenova and their colleagues identified a number of potentially useful genes. Among the list are a baker’s dozen that could serve as blood biomarkers, which are sorely needed in ALS research.

There are several reasons previous studies did not corroborate each others’ results, the authors say. For one, when different scientists use different microarrays, and analyze their data differently, it is no surprise that one study does not match the next, Karsten said. And shortcomings in study design can also confuse results. One of the main problems is that the brain is a very complex tissue, Karsten said. Unfortunately, most of the previous studies neglected this fact and used whole tissue. If we really want to understand the mechanisms of the disease, we have to look at specific cells. Furthermore, in cases where researchers screen postmortem tissue, they only see gene changes at the end stage of disease, when neurons are already dead or dying.

The UCLA researchers designed their study to overcome some of these challenges. They compared a gene expression in wild-type mice with expression in two mouse models of ALS,. One was a classic model, expressing human mutant superoxide dismutase 1 (SOD1), a common cause for familial ALS. The other was a tau mutant mouse, which is relevant to frontotemporal dementia, a disease that shares much in common with ALS. The researchers collected tissue from these animals in the presymptomatic stage, hoping to identify common genes associated with the cause of the disease, not simply the effects of neural degeneration. And instead of whole tissue, they used laser capture microdissection to collect motor neurons as well as glia, which have been repeatedly linked to ALS pathogenesis.

From their initial screen, the researchers identified 251 genes, including 65 unknowns, that were differentially expressed in one of the ALS models compared to control animals. We hoped that we would find a lot of genes in common, Karsten said. But in fact, these two disease processes are almost entirely different, at least at the level of global transcripts. Only 12 genes—eight known—were differentially regulated in both the SOD1 and tau models.

To validate the microarray results, the researchers used RT-PCR and immunohistochemistry to confirm the changes in a few select genes. Even for genes that had small, 1.5-fold changes, there was noticeable change in protein expression under the microscope, they report.

Because a genetic mouse model can be a poor substitute for true human disease, the scientists sought to confirm the importance of these genes in human ALS samples. They used postmortem material from people who had sporadic ALS and from normal controls. They tested 10 top candidates, based on their mouse data and the availability of antibodies, and performed immunohistochemistry on the human samples. Among the 10, three genes were upregulated in both SOD1 and tau mouse models and people with ALS: CRB1, CNGA3, and OTUB2. Another, MMP14, was consistently downregulated. Because these genes were regulated similarly in two genetic models and sporadic cases, Wiedau-Pazos said, they may be relevant to sporadic as well as inherited ALS, Expression of the other six genes was inconsistent between mice and people, but Wiedau-Pazos noted this is not too surprising in a comparison of early mouse disease to late human disease.

Finally, the researchers went back to the mice to analyze gene changes in the peripheral blood. For clinical researchers to properly diagnose, assess disease progression, and evaluate drug efficacy, they need easily accessible biomarkers. The UCLA team designed a custom microarray with 1,449 potentially ALS-related genes from their earlier screen, and used it to test blood samples from mSOD1 mice. They were able to detect 13 genes that reliably went up or down in mSOD1 blood, compared to control wild-type blood. Among these were three genes already of interest in ALS. Neurofilament heavy chain and peripherin, both involved in motor neuron cytoskeletal structure, were upregulated in mSOD1 mice. Monoglyceride lipase, which inactivates endogenous cannabinoids and may be protective in the disease, was consistently downregulated in blood from the mSOD1 mice, (Micale et al., 2007).

The next step, the authors said, will be to validate these blood markers in human samples. If it works, Karsten noted, it would be an incredibly cheap test—just 13 probes on a chip, plus a drop of blood.

The authors say their approach, with multi-step validation, is the way to get solid results from microarray analysis. Because of that, Wiedau-Pazos thinks the candidates they have identified are likely to be confirmed in future work.

Kudo LC, Parfenova L, Vi N, Lau K, Pomakian J, Valdmanis P, Rouleau GA, Vinters HV, Wiedau-Pazos M, Karsten SL. Integrative gene-tissue microarray-based approach for identification of human disease biomarkers: application to amyotrophic lateral sclerosis. Hum Mol Genet. 2010 June 18. Abstract

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