Psychiatry 1997;41:759-761

Frances Yee, Robert H. Yolken

With recent advances in molecular biology techniques and
methodologies, it is now possible to study the etiology and
pathogenesis of many neuropsychiatric illnesses in which little
is currently known about the disease process. Cloning of the
genes involved in these disorders may serve as genetic and
diagnostic markers, as well as provide insights into the
underlying mechanisms of the disease so that more effective
treatments can be developed. This editorial will present an
overview of various strategies used to identify candidate clones,
and applications of these screening techniques to the study of
neuropsychiatric illnesses, specifically schizophrenia and
bipolar disorder.

There are several differential screening methods, in which
comparisons are made between two sample populations, i.e.
affected and normal. The most widely used technique is
differential hybridization, where two separate complementary DNA
(cDNA) libraries are constructed from the affected and normal
samples, and these are then screened for various cDNAs that have
altered expression levels in only one population (reviewed in
Calvet 1991; Hoog 1991). The selection of nucleic acid to be
examined is an important consideration; genomic DNA is not ideal
for studying differential gene expression, because of its
complexity (~3 x 109 base pairs in human genome) and
the fact that much of the genome contains noncoding sequences. A
better choice is RNA, which is transcribed from expressed
portions of the DNA genome, and this also reduces the amount of
screening required. Since RNA is readily degraded and difficult
to work with, the RNA is converted by an enzyme called reverse
transcriptase to its cDNA form, and the resulting cDNA is used in
constructing libraries, which are representative of the starting
cell/tissue RNA. These libraries consist of plasmids (circular
DNA) into which the newly synthesized cDNA has been inserted, and
the plasmid DNA can be propagated by bacteriophage hosts. This
differential hybridization screening method has been used
successfully to identify affected genes (reviewed in May et al
1989); Watson and Margulies 1993); however, this technique has
several drawbacks. A major limitation is that is only allows
comparisons to be made between two sample sets at a time, and
this may pose a problem in the screening of complex tissues such
as the brain, where a great deal of individual variation exists.
Another issue is that rare RNA transcripts tend to be
underrepresented in cDNA libraries, since their expression is at
very low levels, and their corresponding cDNAs may be detected in
the screening process.

The development of PCR techniques has overcome many of the
limitations of differential hybridization screening. Two similar
PCR-based methods were recently introduced to study differential
expression of cDNAs; one method is called differential display
reverse transcription-PCR (DDRT-PCR; Liang and Pardee 1992), and
the second technique is called RNA fingerprinting by arbitrarily
primed-PCR (RAP-PCR; Welsh et al 1992). As before, RNA is reverse
transcribed to it cDNA form and then used as templates for the
PCR along with PCR primers of arbitrary sequence. The resulting
DNA products are then electrophoresed on acrylamide gels and
visualized by either radiolabeling or fluorescent staining of
these PCR products. The patterns of DNA bands generated by the
PCR are then compared between the cases and controls. There are
several advantages of these two reverse transcription
(RT)-PCR-based screening methods; they allow for the simultaneous
analysis of two or more samples, and PCR is also more sensitive
than traditional cDNA library screening, so that it is possible
to detect rare cDNA sequences. In addition, due to the greater
sensitivity of PCR-based methods, smaller amounts of starting
material are needed compared with constructing libraries, which
could be a crucial consideration in cases where availability of
tissue samples is limited. Furthermore, it is possible to
determine if the expression level is increased or decreased in
the affected sample relative to the normal control. This is also
an unbiased approach, since the PCR primers used are not specific
for a particular target cDNA, and as a result they can amplify
any RNA that is differentially expressed. It should be pointed
out that this approach can detect RNA transcripts from human
genes, as well as exogenous agents such as viruses, bacteria, or

The main difference between the RAP-PCR and DDRT-PCR methods
is the reverse transcription step; the RAP-PCR uses an arbitrary
primer for this reaction, whereas the DDRT-PCR uses a modified
olio deoxythymidine (dT) primer (detailed in McClelland et al
1995), which binds to poly A tails of messenger RNA (mRNA)
transcripts and synthesizes the cDNA from the
3’-untranslated region (3’-UTR). The main disadvantage
of the latter procedure is that not all mRNA transcripts are
polyadenylated; for example, many virally encoded RNAs lack the
poly A tail. Furthermore, if degradation has occurred the poly A
tail may not be intact, since ribonucleases (enzymes that digest
RNA) degrade the transcript from the 3’ terminus, which
means that these transcripts would be missed in the screening.
Another disadvantage of the DDRT-PCR method is that it detects
sequences mostly in the 3’-UTR, and not in the coding region
of the sequence, which can make the identification of the
sequence difficult, since the 3’-UTR of mammalian genes are
highly divergent. In contrast, the RAP-PCR method uses the same
arbitrary primer for the initial reverse transcription and the
subsequent PCR reaction, and such palindromic sequences are more
likely to be present in coding regions of mRNAs (McClelland and
Welsh 1994). These coding regions, or open reading frames (orfs),
are more informative, since the amino acid sequence of the
translated protein an often be deduced, which enables sequence
searches of both the nucleotide and protein databases, and this
could facilitate identification of a candidate clone. The main
drawback of the RAP-PCR techniques is that there is no selection
for the mRNA subpopulation of total RNA by using oligo dT primers
for cDNA synthesis as with DDRT-PCR, and consequently there is a
greater likelihood of detecting ribosomal RNAs due to their
greater abundance (85% of total RNA versus 5% for mRNAs).

We have selected the RAP-PCR technique to compare cDNA
expression in cortical areas obtained postmortem from individuals
with schizophrenia or bipolar disorder and normal controls with
no history of mental illness. These brain tissues were collected
as part of the Stanley Neuropathology Consortium. An example of
one of our RAP-PCR gels is shown in Figure 1, in which the cDNA
pattern from three cortical regions from an individual with
schizophrenia is compared with a normal control. The bands of
interest were excised from the gel, subcloned into a
PCR-compatible vector (e.g., Stratagene’s PCR-Script SK+),
and sequenced. The sequences of these candidate clones are then
compared to the DNA databases to identify them; however, novel
transcripts not present in the databases are often found, e.g.,
10-20% of our candidate clones are unknown genes, and then it
becomes necessary to screen a cDNA library to obtain a
full-length clone. The most common problems with these PCR-based
screening techniques are that the extracted gel bands may contain
multiple PCR fragments, which makes identifying the clones of
interest more difficult, and false positives can also be
generated by spurious PCR products. Therefore, it is necessary to
confirm the differential expression of the candidate clones by
either Northern blotting (described in Liang and Pardee 1995), or
by more sensitive techniques, e.g., ribonuclease protection
assays and RT-PCR (with specific PCR primers), which are more
capable of detecting low-abundance messages. A variety of
differentially expressed transcripts can be detected with
RAP-PCR; our candidate clones include genes with known function,
e.g., retroviral sequences, transcription factors, and tyrosine
kinases, and clones with homologies to expressed sequence tags
(ESTs), which are previously described genes that are isolated
from cDNA libraries but their function is unknown. We have used
RT-PCR to screen larger sample sets to confirm differential
expression of these candidate clones, and have found that many of
the isolated clones exhibit a wide range of expression in the
cases and normals; however, a few of these clones show intergroup
differences (affecteds vs normals), and we are in the process of
further characterizing these candidate clones (to be reported in
a future publication). Our findings indicate that brain RNAs
suitable for performing these PCR screening techniques can be
isolated postmortem from individuals with schizophrenia and
bipolar disorder (detailed in Johnston et al, manuscript in
preparation), and that the analysis of these samples can identify
interesting target transcripts in terms of disease etiology.

Figure 1:
Differential amplification of case and control cortical brain
regions. A comparison of RAP-PCR patterns from frontal (Fr),
occipital (Oc) and cerebellar (Cb) cortical areas obtained from
an individual with schizophrenia (S) and a normal (N) control is
shown. The arrows indicate two products that were expressed at
higher levels in the case than the controls. The DNA size markers
(lanes 1, 5, and 9) are 200 and 300 base pairs.

Despite the fact
that the RAP-PCR and DDRT-PCR techniques are relatively new, they
have been successfully used by several groups in the
neurosciences to study development in the rat brain (Dalal et al
1994); Joseph et al 1994), learning and memory in rats (Inokuchi
et al 1996), as well as the effects on gene expression following
treatment with cocaine and amphetamine in rats (Douglass et al
1995), and lithium treatment of C6 glioma cells (Wang and Young
1996), and they should be invaluable for the study of human brain
disorders. Furthermore, the availability of several commercial
kits for both of these PCR procedures should enable researchers
to obtain reproducible findings by standardizing these
techniques, and facilitate their widespread usage. An important
consideration in using these PCR screening methods to study
complex disorders, such as schizophrenia, is to determine whether
the differential expressed transcript is disease-associated and
not merely due to normal individual variation or due to the
effects of medication; therefore, it is crucial to screen larger
sample sets than the small sample sizes initially examined.
Nevertheless, the application of these powerful PCR-based methods
should provide many contributions to our understanding of
neuropsychiatric disorders.

This research
was supported by the Theodore and Vada Stanley Foundation.
We thank Christopher Ross for helpful discussions, and Fuller
Torrey for suggestions on the manuscript.

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