In this report we have used cDNA microarray technology to analyze the expression pattern of 7,700 genes in LCLs from six non-related individuals with idiopathic autism. Each patient was selected from familial forms of AD (sib-pairs), in which at least one chromosome 7q-region segregated with the disorder. The analysis resulted in the identification of nine genes, all of which were down-regulated. None of these genes have been reported previously as associated with autism. Notably, one of the nine genes encodes semaphorin 5A, which is involved in axonal guidance [21
]. The SEMA5A
gene is positioned at chromosome 5p15, approximately 6 Mb from a marker indicating increased allele sharing in our previous genome-wide scan [7
]. Rodent expression of Sema5A has been reported in cerebral cortex, basal ganglia, thalamus and hippocampus in embryonic and postnatal rat brains [22
]. Semaphorin 5A is a bifunctional molecule, acting both as attracting and repulsing guidance cues [23
]. Failure of Sema5A expression leads to abnormal development of the axonal connections in the forebrain of mice [25
], which may affect the formation of functional synapses. Furthermore, it has been suggested that haploinsufficiency for SEMA5A
is responsible for mental retardation in Cri-du-chat, a syndrome resulting from deletions of chromosome 5p [26
]. Synaptic dysfunction of specific neurons is likely to be one etiologic mechanism in AD and this was supported by the identification of mutations in the NLGN3
genes in a subgroup of patients with autism [13
]. The NLGN3
genes encode synaptic cell-adhesion molecules. A down-regulation of SEMASA,
with an effect on axonal guidance, may therefore be an analogous candidate mechanism behind AD in some patients.
The HSPA1L, CCND2
genes were also found to be down-regulated in our microarray study. These genes were of interest due to their involvement in the immune system or brain development [27
], but differences in mRNA expression levels could not be confirmed by qPCR. This indicates that these genes may be false positives in our microarray analysis.
To our knowledge, no previous findings suggest association between the remaining five genes down-regulated in this study (CSDA, SARCOSIN, MGST1, RPN2 and SNX2) and mechanisms related to brain development or brain function.
Several genes in the 7q31 region, such as RAY1/ST7, WNT2, CORTB2
have been proposed as candidate genes for autism [35
]. In addition, linkage and association to the β3 subunit of the GABAA
gene on chromosome 15q have been reported by several groups [39
]. Unfortunately, the WNT2, RELN
genes were not expressed at levels high enough to detect true differences in our LCL samples. The results from a previous microarray analysis of brain specimens suggest that variation in the expression of EAAT1
, two members of the glutamate system, are associated with autism [19
]. We could not confirm changes in expression of these two candidate genes in our study. The RAY1/ST7
genes were not present on the microarrays used in this study.
Idiopathic autism is a genetically complex disorder, manifested in the brain. Since idiopathic autism is constitutional, altered transcript levels may be detected also in non-neuronal tissues, such as lymphoblastoid cells, which are used as a model in this study. Consequently, the significance of our findings in the etiology of AD depends on the presence of constitutional changes in mRNA levels from genes expressed in both brain and lymphocytes. The small number of individuals included renders generalized conclusions hazardous, and the risk of separate false positives should be considered. Another limitation is the sensitivity of the microarray system. Small variations in expression levels may be difficult to detect and is further complicated for genes with low constitutional expression levels.
The significance of the down-regulated genes in this study is yet unclear. Further analyses, including a larger number of patient samples and preferably on brain tissue from AD patients, are needed to confirm a possible involvement of these transcripts in the disease process. The microarray technique still provides a useful tool for the investigation of mechanisms behind complex psychiatric disorders, and the method may contribute to clarify both primary and secondary events in these disorders.