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Updated 12 March 2008 E-mail discussion
Printable version

Live Discussion: Gene Expression Profiling of Postmortem Human Brain


Akira Sawa

Marquis Vawter

The venerable medical tradition of postmortem research would seem to be poised for a quantum leap in this era of molecular science, but old provisos and emergent issues must clearly be taken into account.

On 9 January 2008, Akira Sawa of Johns Hopkins University and Marquis Vawter of the University of California, Irvine, led an SRF online discussion of the fine art of generating and interpreting reliable gene expression data in psychiatric research. We invite you to read their background text below, along with an extensive comment by Vawter on several recent papers, particularly one in the Journal of Neuroscience Methods by Christine Miller and colleagues at Johns Hopkins University and the Stanley Medical Research Institute. We then ask that you share your ideas, warnings, horror stories, and other useful information in the form of a comment.

View Transcript of Live Discussion — Posted 12 March 2008

View Comments By:
Karoly Mirnics, Fuller Torrey — Posted 30 December 2007
Christine Miller — Posted 7 January 2008
Alberto Arregui — Posted 7 January 2008
Beth Thomas — Posted 7 January 2008
Sherry Leonard — Posted 7 January 2008
Kenji Hashimoto — Posted 7 January 2008
Tadafumi Kato, Kazuya Iwamoto — Posted 8 January 2008
Sinthuja Sivagnanasundaram, Duncan Sinclair, Cynthia Shannon Weickert — Posted 8 January 2008
Christine Miller — Posted 9 January 2008
Paul Harrison — Posted 9 January 2008
Christine Miller — Posted 9 January 2008
Barbara K. Lipska, Joel Kleinman — Posted 9 January 2008
Sinthuja Sivagnanasundaram — Posted 17 January 2008


Background Text
By Akira Sawa and Marquis Vawter

The purpose of this forum is to discuss factors that impact gene expression profiles in postmortem human brain in the context of microarray, quantitative PCR, in-situ hybridization, and other expression assessment methods.

The pathophysiology of several psychiatric disorders has been extensively studied using postmortem human brains. Enormous molecular information has been obtained from autopsied brains from schizophrenia patients through microarray and proteomic approaches. However, there are caveats to the use of this tissue as well: schizophrenia appears to be a disorder of neurodevelopmental origin; thus, major pathological events that affect neurodevelopment may be compensated for at later stages, such that they would not be seen or detected in autopsied brains. In addition, mechanisms and processes of brain maturation may be different between patients with schizophrenia and controls. More practically, confounding factors such as medication, smoking, and diet must be considered in studying autopsied brains. Gene expression profiles are affected by RNA quality, fixation, agonal factors, postmortem interval, as well as freeze-thaw effects of autopsied brains.

Thus, we plan to discuss methodological approaches for quality control in postmortem brain gene expression studies, as well as data interpretation, alternative approaches, and subject selection or matching. Our discussion will mainly focus on studies of gene expression profiles, since whole genome studies have been conducted. Similar strategies may also refer to studies targeting protein and peptide profiling.

Some provisional questions for discussion will be

1. What are the reliable parameter(s) for quality control of gene expression profiling with postmortem brains?

2. What are the appropriate strategy(ies) in data analysis and interpretation, considering possible confounding factors?

3. How can we address molecular changes associated with neurodevelopment in the autopsied brains? How can we address the question of possible differences in brain maturation and aging between patients and controls?

4. What types of tissues are to be considered as alternatives to autopsied brains?

5. What types of brain banks or collections are to be developed in the research community?

6. What kinds of data sharing systems are expected to promote research in the field?

Read Mark Vawter’s comment on several recent postmortem methods papers.


Comments on Online Discussion
Comment by:  Karoly Mirnics, SRF AdvisorFuller Torrey
Submitted 30 December 2007 Posted 30 December 2007

Summary of Workshop Regarding Future Use of Stanley Brain Collection Tissue
(30 November 2007, Bethesda, Maryland)

There appears to be good consensus among researchers working with Stanley Brain Collection tissue on what is needed for future brain collections. Much has been learned from the existing collections and so there is a lot of expertise available. Thanks to the postmortem research, we have identified processes and molecular mechanisms that are critically involved in schizophrenia, and there is an emerging consensus about some of the most important findings. However much more is to be done.

The complexity of the anatomical brain structures must be respected. The cellular phenotypes making up the brain are different, and the disease process will affect them differently. Thus, we must find ways to respect the microanatomy in our experiments. There appears to be a consensus that laser-capture techniques using single cells is the most promising wave of the future. The problems are that it is difficult, labor intensive, and expensive. Other...  Read more


View all comments by Karoly Mirnics
View all comments by Fuller Torrey

Comment by:  Christine Miller
Submitted 7 January 2008 Posted 7 January 2008

My view is that postmortem studies of the affected organ (the brain, in this case) will always be important and that the focus should be on expanding and improving the repertoire of available techniques. Data derived from a variety of methods help to form the most complete picture, and the significance attributed to any given set of results must be evaluated and re-evaluated based on current knowledge about the limitations of the methods used.

I have two points to make: one about improving our understanding of postmortem effects and the other about variability in sectioning.

1. The first relates to animal models of postmortem factors that affect mRNA, protein, and metabolite measurements from brain tissue. To my knowledge, the animal studies that have been published do not mimic the postmortem conditions that are in effect for deceased humans. Specifically, the effects of postmortem interval have been modeled after the brain has been removed from the cranium, not before, whereas the PMI referred to in the human studies relates to the time between death and the removal...  Read more


View all comments by Christine Miller

Comment by:  Alberto Arregui
Submitted 7 January 2008 Posted 7 January 2008

In research projects involving postmortem material, extreme care needs to be applied in formulating conclusions. The long-term effects of neuroleptics, even in patients who have died without use of meds for many months, on brain substances is unknown. The known effect of "protracted" illnesses on brain transmitters is also present. I participated in studies done at the MRC Neurochemical Unit in Cambridge in the late 1970s (e.g., Arregui et al., 1979; Arregui et al., 1980; Mackay et al., 1982), and many findings of altered peptides were left unpublished because it was very difficult to find appropriate explanations for the findings. Good luck.

References:

Arregui A, Mackay AV, Iversen LL, Spokes EG. Reduction of angiotensin-converting enzyme in substantia nigra in early-onset schizophrenia. N Engl J Med. 1979 Mar 1;300(9):502-3. Abstract

Arregui A, Mackay AV, Spokes EG, Iversen LL. Reduced activity of angiotensin-converting enzyme in basal ganglia in early onset schizophrenia. Psychol Med. 1980 May 1;10(2):307-13. Abstract

Mackay AV, Iversen LL, Rossor M, Spokes E, Bird E, Arregui A, Creese I, Synder SH. Increased brain dopamine and dopamine receptors in schizophrenia. Arch Gen Psychiatry. 1982 Sep 1;39(9):991-7. Abstract

View all comments by Alberto Arregui


Comment by:  Beth Thomas
Submitted 7 January 2008 Posted 7 January 2008

I have a question about whether there is any consensus over how to best address the potential influences of lifetime antipsychotic drug exposure on gene expression findings. This is certainly an important confounding factor in any gene expression study using postmortem samples from schizophrenic subjects. Three approaches have been used in the literature, although each has its weakness.

1. Comparisons of gene expression levels from drug-treated schizophrenic subjects to those who had been “medication-free” for various periods of time. The doubt here is that, after a presumed lifetime of antipsychotic drug treatment, would a period of 3-6 months “drug-free” be long enough to reverse the chronic effects of drugs?

2. Correlations between gene expression levels and recorded drug doses. This seems logical; however, it is known that many patients are noncompliant with their reported medications, and sometimes drug traces or metabolites are found in subjects that are different from what is listed in the medical records.

3. Measurements of antipsychotic drug influences...  Read more


View all comments by Beth Thomas

Comment by:  Sherry Leonard
Submitted 7 January 2008 Posted 7 January 2008

The topics for discussion are all important! Would like to add one more: cause of death. Some years ago we did in vitro translation and PAGE from mRNA isolated from control subjects, matched for age, sex and ethnicity, who had died from disparate causes. The protein profiles were VERY different. Since that time we also match samples for cause of death if possible. Wondering if anyone else has thought about this issue.

What is really needed is a database with expression profiles from controls with different causes of death, but that is dreaming!

View all comments by Sherry Leonard


Comment by:  Kenji Hashimoto
Submitted 7 January 2008 Posted 7 January 2008

We believe that postmortem human brain samples are critical for examining molecular changes associated with the pathophysiology of schizophrenia. However, there are a number of confounding factors, including the postmortem interval (PMI). A number of studies have used autopsied brain samples for which there were no significant differences between the mean PMI values, but there were wide ranges (e.g., ~72 hrs for Stanley Brain Samples) of PMI. For example, levels of amino acids, including glutamate, in the brain are clearly affected by PMI. In a study of brain amino acids in postmortem samples from schizophrenia patients, we first normalized their time-course changes to simple equations in rodents, which were then applied to the studies of human autopsied brain samples (Hashimoto et al., 2007). Therefore, the use of simple equations using animals may be necessary to determine the substances affected by confounding factors.

Schizophrenia is a neurodevelopmental disorder. Therefore, we may not see or detect evidence of the...  Read more


View all comments by Kenji Hashimoto

Comment by:  Tadafumi KatoKazuya Iwamoto
Submitted 7 January 2008 Posted 8 January 2008

Thanks to the endeavors of Dr. Torrey and all the other contributors of the Stanley Medical Research Institute, postmortem brain study has been opened to the research community worldwide. Gene expression profiles have been analyzed in the Stanley brain bank samples by several laboratories including ours and the data are now open to the public. However, the description on the results shown in the database and the content of the papers from individual groups are not always consistent, partly because of the difference in the philosophy of how the confounding factors should be controlled for.

The paper by Dr. Miller and colleagues dealt with this important issue of confounding factors affecting gene expression profiles in postmortem brains using the largest numbers of brain samples to date (Weis et al., 2006). They reported that sample pH did not affect the RNA quality. This is well in accordance with our previous report showing that there was only a weak relationship between pH and call rate by GeneChip (  Read more


View all comments by Tadafumi Kato
View all comments by Kazuya Iwamoto

Comment by:  Sinthuja SivagnanasundaramDuncan SinclairCynthia Shannon Weickert (SRF Advisor)
Submitted 8 January 2008 Posted 8 January 2008

A Few Technical Comments About Evaluating RNA Quality From Human Brain

Since the measurement of RNA quality using several outputs from the Agilent 2100 Bioanalyser is considered a key tool in evaluating RNA quality from the human brain, we would like to point out just a few experimental factors that may impact the determination of RNA quality using the Bioanalyser: 1) the effect of heating the RNA samples prior to loading on the RNA 6000 Nano Chip, 2) the effect of drying down the samples, and 3) the effect of running the RNA through a purification column.

We will comment on each of these points based on our experience in the Schizophrenia Research Laboratory in Sydney Australia.

1) Although heating the total RNA sample before running the RNA 6000 Nano Chip is recommended by the manufacturer, it can result in lower RIN values (note that in both Lipska et al., 2006 and in Weis et al., 2007 the RNA...  Read more


View all comments by Sinthuja Sivagnanasundaram
View all comments by Duncan Sinclair
View all comments by Cynthia Shannon Weickert

Comment by:  Christine Miller
Submitted 9 January 2008 Posted 9 January 2008

Reply to Cynthia Shannon Weickert and colleagues:

Thanks for sharing your experience with concentrating RNA. It is important to clarify that our paper (Weis et al., 2007) did not assert that concentrating the RNA actually improved its quality for microarray analysis (i.e. degraded RNA was not restored to its original intact state). Rather, the point was that this was another example of RNA handling procedures that can affect the electropherogram without actually improving RNA quality.

I am curious as to the starting RNA used to test the effect of concentrating via drying down. Was it solvent extracted RNA (eg. with Trizol)? This RNA likely exists in a highly folded state due to the alcohol precipitation step during extraction, and much less additional folding would be expected to occur when concentrated by drying. The extent of folding should affect migration of the 28S and 18S bands in the nanochip. Column-extracted RNA, in contrast, has been denatured during the column binding...  Read more


View all comments by Christine Miller

Comment by:  Paul Harrison
Submitted 9 January 2008 Posted 9 January 2008

Several of the above comments pertain to the use of pH and/or RIN to predict/control for variation in mRNA quality in postmortem brains. Could I also raise the issue of utility and reliability: the pH of a given brain is pretty consistent between one brain area and another, and has a high test-retest reproducibility (and temporal stability). By contrast, we find RIN shows more variation in all these respects (as also shown by Lipksa et al., 2006, and other recent papers), presumably due to the technical factors discussed by Miller and Weickert, as well as to biological factors. Probably as a consequence, our experience is that (depending on the experimental dataset) pH sometimes does better than RIN in explaining variance in mRNA signals between brains, sometimes RIN does better, and sometimes but not always the combination is better than either alone. I think there are two implications of this:

1. Although RIN is a valuable index, it has its limitations and it should not be viewed uncritically or automatically as the gold standard in the field—or by implication...  Read more


View all comments by Paul Harrison

Comment by:  Christine Miller
Submitted 9 January 2008 Posted 9 January 2008

Clarification to my last commment: a higher 260/280 corresponds to less folding of the mRNA.

View all comments by Christine Miller

Comment by:  Barbara K. LipskaJoel Kleinman
Submitted 9 January 2008 Posted 9 January 2008

We have previously suggested that accurate assessment of multiple confounding factors and their inclusion as regressors in the analysis is critical for obtaining reliable and accurate quantification of mRNA expression (Lipska et al., 2006). One of these confounding factors is pH. Insofar as lower pH has been associated with decreased mRNA expression in postmortem human brain, decreased pH in schizophrenia may represent an important potential confound in comparisons between patients and controls. We are now showing that decreased pH is related to increased concentration of lactic acid (Halim et al., in press).

However, in contrast to the previous notion that an increase in lactic acid represents evidence for primary metabolic abnormalities in schizophrenia, we hypothesized that this increase is secondary to prior antipsychotic treatment. We have tested this by first demonstrating that lactate levels in the cerebellum of patients with schizophrenia are increased relative to control subjects. Second, we have shown that there is an...  Read more


View all comments by Barbara K. Lipska
View all comments by Joel Kleinman

Comment by:  Sinthuja Sivagnanasundaram
Submitted 17 January 2008 Posted 17 January 2008

Reply to Comment by Christine Miller
Thanks for the clarification. We did use Trizol for our RNA extraction and we plan to use this total RNA primarily for RT-PCR. We agree that different extraction protocols will result in different measures of RNA quality, and we suggest that details of the RNA isolation, purification, and amplification methods should be clearly stated in publications. Our measure of the A260/280 ratio for Trizol extracted RNA before and after purification was on average 1.89 and 2.05, respectively, which suggests that column purification improves the A260/280 ratio.

View all comments by Sinthuja Sivagnanasundaram

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