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Original Article |

Convergent Evidence for 2′,3′-Cyclic Nucleotide 3′-Phosphodiesterase as a Possible Susceptibility Gene for Schizophrenia FREE

[+] Author Affiliations

Author Affiliations: Department of Psychological Medicine (Drs Peirce, Bray, Williams, Norton, Owen, and O’Donovan and Ms Preece) and Biostatistics and Bioinformatics Unit (Dr Moskvina), School of Medicine, Cardiff University, Cardiff, Wales; Department of Psychiatry, Mount Sinai School of Medicine, New York, NY (Drs Haroutunian and Buxbaum); Mental Illness Research, Education and Clinical Centers, Bronx Veterans Affairs Medical Center, New York (Dr Haroutunian).


Arch Gen Psychiatry. 2006;63(1):18-24. doi:10.1001/archpsyc.63.1.18.
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Context  Convergent data make 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) a candidate gene for schizophrenia. Reduced expression has been reported in the schizophrenic brain. The CNP gene maps to a region to which we have reported linkage to schizophrenia. Mice in which the CNP gene has been knocked out display central nervous system pathological characteristics reminiscent of some features observed in schizophrenia. 2′,3′-Cyclic nucleotide 3′-phosphodiesterase is used as a marker of myelin-forming cells and is detectable in cells of oligodendrocyte lineage throughout life. Because CNP is thought to be important for oligodendrocyte function, altered expression has been interpreted as supportive of the hypothesis that altered oligodendrocyte function may be an etiological factor in schizophrenia. However, it is unclear whether the observed changes in the schizophrenic brain are primary or secondary.

Objectives  To determine if CNP expression is influenced by DNA polymorphisms and to verify if these polymorphisms are associated with schizophrenia.

Design  Allele-specific messenger RNA expression assay and genetic association studies.

Setting  Unrelated subjects were ascertained from secondary psychiatric inpatient and outpatient services.

Participants  We used brain tissue from 60 anonymous individuals with no known psychiatric disorder; a case-control sample of 708 white individuals from the United Kingdom meeting DSM-IV criteria for schizophrenia matched for age, sex, and ethnicity to 711 blood donor controls; and a pedigree with DNA from 6 affected siblings and 1 parent, showing evidence for linkage to CNP.

Main Outcome Measures  Association between allele and gene expression. Association between allele and schizophrenia.

Results  The exonic single nucleotide polymorphism rs2070106 was associated with CNP expression (P<.001). Compatible with underexpression of CNP messenger RNA in schizophrenia, the lower-expressing A allele was significantly associated with schizophrenia (P = .04) in the case-control sample. All affected individuals in the linked pedigree were homozygous for the lower-expression allele, providing independent support for the association (P = .03).

Conclusions  Our data support the hypothesis that reduced CNP expression in the schizophrenic brain is relevant to disease etiology and therefore provide support for the general hypothesis that altered oligodendrocyte function is an etiological factor in schizophrenia.

Figures in this Article

Methods for testing genes using association designs in general offer more power to detect small effects than methods based on linkage, but until genomewide association scans become economically realistic, such analyses are dependent on prior functional and/or positional support for a disease locus. Unfortunately, our current knowledge of the functional pathological characteristics of schizophrenia so far only provides broad clues about the pathological mechanisms that might be involved, and therefore, it is difficult to define a group of candidate genes small enough to be comprehensively studied.

Global surveys of messenger RNA (mRNA) expression levels offer an alternative approach to linkage for selecting candidate genes and/or pathophysiological pathways for a disease in the absence of a prior hypothesis linking the function of that gene or pathway to the disease etiology.1 Although the large number of transcripts to be examined, as well as the numerous potential sources of experimental and sampling variance that accompany the use of complex tissues obtained post mortem, poses significant analytic and design problems, recent studies suggest that robust and etiologically relevant changes in gene expression can be detected even in tissues as complex as the human postmortem brain. Examples include the identification of altered expression of the neuregulin receptor erbB3 in the schizophrenic brain,2,3 a finding whose relevance is supported by the evidence implicating variation in the gene encoding its ligand, neuregulin 1, in susceptibility to schizophrenia.4 Others include the identification of the G protein receptor kinase 3 gene as a candidate gene for bipolar disorder5 and the regulator of G-protein signaling 4 as a candidate gene for schizophrenia,6 findings that have received supportive evidence from follow-up genetic studies.7,8 In the cases of regulator of G-protein signaling 4 and the G protein receptor kinase 3 gene, each was selected for genetic analysis because, as well as displaying altered expression, each mapped to a putative region of linkage for the relevant disorder. Thus, each candidate gene was supported by data from 2 non−hypothesis based methods, an approach that has been termed convergent functional genomics.5

Recently, microarray studies have reported the down-regulation of genes related to oligodendrocyte function and myelination in the schizophrenic brain compared with control subjects.2,3,9 Among the mRNAs down-regulated was that encoded by the 2′,3′-cyclic nucleotide 3′-phosphodiesterase gene (CNP), a finding confirmed at the protein level by Flynn and colleagues.10 These observations are of interest because CNP maps to 17q21.2, a region of the genome in which we have recently observed genomewide significant evidence for linkage (logarithm odds score, 8.32; genomewide empirical P value ≤ .02) to schizophrenia in a single pedigree.11 Moreover, given evidence for altered myelination and oligodendrocyte function in schizophrenia,12CNP is also a plausible functional candidate gene.

2′,3′-Cyclic nucleotide 3′-phosphodiesterase is widely used as a marker protein of myelin-forming glial cells. In brain development, CNP is detectable in cells of oligodendrocyte lineage and is maintained in mature oligodendrocytes throughout life.13 2′,3′-Cyclic nucleotide 3′-phosphodiesterase is associated with noncompacted myelin regions, absent from compacted myelin, and also found in oligodendrocyte cytoplasm.10 Although the precise function of CNP in oligodendrocytes is unclear, recent evidence suggests that it interacts with mitochondria and cytoskeletal proteins and may act to promote microtubule assembly or act as a membrane anchor for tubulin.14 Intriguingly, a recent animal study showed that CNP-deficient mice display a reduction in overall brain size, enlarged ventricles, and corpus callosum atrophy, all of which are consistent with pathological features observed in schizophrenia.13

That altered CNP expression can be observed and replicated in small samples of cases and controls suggests a schizophrenia-related influence on CNP expression that is probably too large and homogeneous to attribute entirely to genetic variation at the CNP locus itself. If altered CNP expression is relevant to pathogenesis, it is probably as a final common pathway resulting from multiple trans-acting genetic and environmental risk factors. Alternatively, the replicated evidence for the association between altered CNP expression and schizophrenia in small samples might suggest that the former is a common consequence of the latter, for example, a result of its treatment.

In this study, we applied molecular genetic approaches in a bid to distinguish between the hypotheses mentioned earlier. The rationale underpinning our study was that if it is true that altered CNP expression influences schizophrenia susceptibility, any direct influences on CNP expression resulting from a cis-acting polymorphism at the CNP locus will have a similar effect, albeit probably with a small genetic effect size. Thus, we sought evidence that the expression of CNP is influenced by at least 1 cis-acting polymorphism using a highly quantitative allele-specific expression assay.15 Second, we sought direct and indirect evidence for association between CNP and schizophrenia in a large sample of schizophrenic cases and controls. Third, we examined the sequence of CNP in a family showing evidence for 17q linkage to schizophrenia for the presence of alleles that might represent rare alleles of large effect size. Fourth, we undertook de novo polymorphism discovery across the complete genomic sequence (including introns) of CNP and examined all variants for evidence of association with schizophrenia. The analyses presented show that CNP is indeed subject to cis-acting influences on gene expression, that an allele associated with lower CNP expression is also associated with schizophrenia, and that while all affected members of the pedigree showing evidence for linkage to schizophrenia were less identical by descent on 17q than suggested by our original linkage analysis, all were homozygous for the less common putative risk allele, a finding that is unlikely to occur simply by chance.

SAMPLE

Our case-control sample consisted of 708 white subjects with schizophrenia from the United Kingdom and Ireland (487 men and 221 women), matched for age, sex and ethnicity to 711 blood donor controls (478 men and 233 women). All patients had a consensus diagnosis of schizophrenia according to DSM-IV criteria made by 2 independent raters following a semistructured interview by trained psychiatrists or psychologists using the Schedules for Clinical Assessment in Neuropsychiatry interview16 and review of case records. High levels of reliability (κ >0.8) were achieved between raters for diagnoses. All cases were screened to exclude substance-induced psychotic disorder or psychosis due to a general medical condition. The mean (SD) age at first psychiatric contact was 23.6 (7.7) years and the mean (SD) age at ascertainment was 41.8 (13.5) years. The blood donor controls were not specifically screened for psychiatric illness but individuals were not taking regular prescribed medications. In neither country are blood donors remunerated even for expenses, and as a result, blood donors are not enriched for substance abusers or indigents who may have relatively high rates of psychosis. Ethics committee approval was obtained in all regions where patients were recruited, and informed written consent was obtained from all participants. The pedigree showing evidence for linkage to 17q11 consisted of a single sibship of 6 individuals with DSM-IV schizophrenia (diagnosed as described earlier) from whom DNA was available. DNA was also available from 1 parent of unknown diagnostic status but who was believed to be unaffected.

LABORATORY METHODS

Primers to amplify exons, promoters, flanking sequences, and introns were designed based on alignment of mRNA sequence (NM_033133) and the corresponding genomic sequence (NT_010755.14). Polymerase chain reaction amplification and mutation screening were performed using denaturing high-performance liquid chromatography and, where indicated, sequencing, using protocols we have described extensively.17,18

The sample for mutation screening consisted of 14 unrelated white subjects from the United Kingdom meeting DSM-IV criteria for schizophrenia, each of whom had at least 1 affected sibling. At a later stage, we also screened all available individuals from the linked pedigree.

ALLELE-SPECIFIC EXPRESSION ASSAY

The principle of the assay is that in heterozygous carriers of a cis-acting polymorphism that affects the transcription or stability of a species of mRNA, the quantity of mRNA originating from each gene copy will be unequal. The simplest method for distinguishing between mRNA molecules originating from each copy of a pair of autosomal genes is to use a polymorphism within the mRNA sequence as a copy-specific tag.15 It is then possible to apply quantitative methods of allele discrimination to mRNA samples originating from individuals who are known to be heterozygous for the marker polymorphism in order to measure relative copy expression.15 Factors that can influence the measured amount of total expression of a specific gene (eg, tissue preparation, mRNA quality, drug exposure, preagonal state, hormones, effects secondary to regulatory polymorphisms in other genes) will, in the absence of a cis-acting allele-specific interaction, influence the amount of mRNA originating from each chromosome equally and are therefore controlled for by this assay. While not allowing the measurement of total RNA abundance, the allelic expression assay does have the major advantage of allowing the detection of small polymorphic cis-acting influences even in the face of large trans-acting influences.

Postmortem brain tissue was derived from the frontal, parietal, or temporal cortex of 60 white, European, unrelated, anonymized human adults of whom 50 were from the United Kingdom (the Medical Research Council London Neurodegenerative Diseases Brain Bank, London, England) and 10 were from Sweden (Department of Clinical Neuroscience, Karolinska Institute, Stockholm). All were free from psychiatric or neurological disorder at the time of death. Genomic DNA and RNA were extracted from each individual tissue sample, with subsequent deoxyribonuclease treatment of RNA as described.15 Heterozygotes for the transcribed marker single-nucleotide polymorphism (SNP) were identified by genotyping genomic DNA from all subjects. Allelic expression was estimated as described by Bray et al.15,19 Briefly, deoxyribonuclease-treated RNA samples were subject to reverse-transcription polymerase chain reaction and primer extension with allele-specific dye-terminator incorporation using the SNaPshot kit (Applied Biosystems, Foster City, Calif), and the relative levels of the products representing each transcribed allele were measured on a capillary sequencer. Samples were assayed using primers based on single exonic sequence, capable of amplifying either genomic DNA or complementary DNA (cDNA). The cDNA samples were assayed alongside the corresponding heterozygote genomic DNA, which represents a perfect 1:1 ratio of the 2 alleles. The ratio obtained from genomic DNA thus provided a correction factor for any inequalities in the efficiency of allelic representation specific to each cDNA assay.20 Absence of genomic DNA in the RNA extracts was confirmed by including the RNA samples that had not been reverse transcribed. Analysis of heterozygous samples was performed as 2 separate experiments. In each experiment, 2 cDNA samples were assayed for each heterozygous individual (as 2 separate reverse-transcription reactions), alongside the corresponding genomic DNA sample.

Individual genotyping was performed using AcycloPrime reagents (PerkinElmer Life Science Products, Boston, Mass). Genotypes were scored blind to affection status, and each plate of samples contained a mixture of equal numbers of cases and controls.

The DNA pools from 691 of the cases and 710 of the controls were also genotyped using the SNaPshot primer extension (Applied Biosystems) protocol described by us in detail by Norton et al.21

INDIRECT ASSOCIATION ANALYSIS

A grid of SNPs (minimum density of 1 SNP every 5 kilobases [kb]) across CNP, spanning the genomic sequence from 4 kb 5′ to the transcription start site through to 1 kb 3′ to the end of transcription was identified from the CHIP bioinformatics database.22 A linkage disequilibrium (LD) approach was used to supplement our direct gene analyses because genetic variation altering expression may occur in genomic sequences whose locations are unpredictable with reference to the exonic sequence.

STATISTICAL ANALYSIS

Contingency tables were used to test genotypes and alleles for association with schizophrenia and to calculate odds ratios. All analyses were 2-tailed. Power estimations were calculated using the Web-based program provided by the Department of Statistics, University of California, Los Angeles.23 The D′ and r2 estimates of LD were calculated using the Web program Haploview.24 Haplotype frequency analysis was performed using EH plus25 with a permutation test.26 A low-redundancy set of SNPs for haplotype analysis was selected using an entropy method developed in house (V.M., unpublished data, 2003). Entropy as a measure of haplotypic diversity is calculated as:

Figures

Place holder to copy figure label and caption
Figure.

Corrected genomic and complementary DNA (cDNA) ratios for rs2070106. Comparison between the observed corrected genomic ratios and the corrected cDNA ratios assayed for 2′,3′-cyclic nucleotide 3′-phosphodiesterase (n = 25). Data are expressed as the mean of the ratio of A:G for 2 measurements of each genomic DNA sample and 4 measurements of each cDNA sample.

Graphic Jump Location

Tables

Table Graphic Jump LocationTable 1. Individual Genotyping Results for rs2070106
Table Graphic Jump LocationTable 2. Pooled and Individual Genotyping Results for CNP SNPs
Table Graphic Jump LocationTable 3. Results of Linkage Disequilibrium Analysis*

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