Proteome Analysis of Lymphoblastoid Cell Lines of Patients with Autism Spectrum Disorder Carrying a Mutation In the Ribosomal Protein Gene RPL10

Background: Autism spectrum disorder (ASD) has a strong genetic background with a higher frequency of affected males suggesting involvement of X-linked genes. The complex genetic architecture points towards the contribution of rare genomic variants distributed over the complete genome targeting genes in pathways for synaptic signaling, cell adhesion, secretion or scaffolding. These are crucial processes in the establishment of synaptic plasticity (SP).

Objectives: We previously identified two rare missense mutations, L206M and H213Q, in the ribosomal protein L10 (RPL10) gene on Xq28 in three independent families. Functional analyses in yeast showed that both mutant alleles alter translation, a modulating mechanism of SP. Gene expression studies of RPL10 itself in lymphoblastoid cell lines (LCL) of male patients harboring the H213Q mutation showed no significant difference at the transcriptional level. Therefore, we aim to analyze the impact of RPL10 mutations on differential translation applying 2D gel/mass spectrometry (MS) methods using these mutant LCLs.

Methods: Protein extracts of two biological replicates for DIGE were prepared from LCLs established from the two male index patients of each family, their heterozygous carrier mothers, an unaffected brother and a heterozygous carrier sister, respectively, and a male and female control. Each of the samples was stained with Cy3 and compared versus the Cy5 stained internal control, which was an isogenic mixture of each of the samples. Data acquisition was performed on the Typhoon Scanner System and data were subsequently analyzed using DeCyder Software. Data were normalized against the internal standard and relative volumes of spots were calculated using normalized intensity values. Three different types of statistical analyses (two-tailed t-tests) were performed: patients versus mothers, patients versus non-patients and mutant allele carriers versus wild-type allele carriers.

Results: Following data analysis ten significantly up- or down-regulated spots in the samples of the mutation carriers were selected and picked from a preparative, Coomassie-stained 2D-PAGE. Proteins were digested with trypsin and analyzed by MALDI-MS. Interestingly three of the ten spots could be identified as posttranslational modifications of a single protein. This down-regulated protein is a central regulator in gluconeogenesis. One other up-regulated protein was identified as a peroxisomal member of the lipid metabolism. Validation via RT-PCR and Western Blot is in progress.

Conclusions: There is evidence from the literature that RPL10 is not only involved in the translation processes as a component of the large ribosomal subunit, but also in the establishment of the cytoskeleton, mRNA turnover and ageing. We will use information of differential protein expression caused by these autism specific RPL10 mutations to search for underlying pathways specifically relevant for synaptogenesis in neuronal development and therefore causative for the autism phenotype.