Resumo

Different molecular approaches have been used trying to find genetic factors associated to Autism Spectrum Disorders (ASD). A rewarding strategy is the study of chromosomal abnormalities detected by karyotype, which is estimated to be present in around 3% of ASD cases. Functional analyses of genes identified by these approaches are also important, once the understanding of the pathways related to them can point to new candidate genes. Recently, we have identified an autistic male patient who has a de novo balanced translocation involving chromosomes 3 and 11 [t(3;11)(p21;q22)]. Using Fluorescent In Situ Hibridization (FISH), it was possible to confirm the disruption of VPRBP gene in chromosome 3. VPRBP is involved in DNA replication and, consequently, cellular proliferation and its silencing is lethal in early embryonic stages in mice. FISH analysis places the breakpoint in chromosome 11 in an area containing part of the TRPC6 gene. Quantification of some TRPC6 exons showed a reduction of 50% in the levels of expressions of exons 12 and 13 compared to exon 6, suggesting that the gene is transcribed until the breakpoint in the translocated chromosome. Indeed, sequencing the DNA sample, we found that the patient is heterozygous for a polymorphism in exon 6 and 13. However, when his cDNA is genotyped, we could only identify heterozygosity for the polymorphism in exon 6, but not for exon 13, which does not happen for a control sample also heterozygous for both polymorphisms. Since this gene has just one isoform, these results confirmed TRPC6 disruption. It was shown that Ca+2 influx through the nonselective channel TRPC6 promotes dendritic growth and synapse formation, processes previously implicated to ASD. This is also interesting, since a lot of genes involved in Ca+2 signaling controlled pathways are ASD candidates. Based on the potential involvement of this gene in ASD etiology, we screened 700 ASD patients for mutations in TRPC6, but any alteration was found. We also derived induced pluripotent stem cells (iPSCs) from the dental pulp stem cells (DPSC) of this patient and a control, and we are exploiting possible functional alterations associated to TRPC6 disruption in neural progenitor cells (NPCs) and neurons obtained from iPSCs. Our study is an example that cytogenetic analyses are very useful for the identification of potential ASD candidate genes. We are focused on TRPC6 due to its clear role in neuronal development, but we can not discard the possible involvement of vVPRBP in the etiology of ASD in this patient. Although we did not identify mutations in TRPC6 in any other patient, we believe that the study of the implications of TRPC6 disruption in NPCs and neurons functionality and development can help us to identify pathways and biological process related to ASD etiology.