Objectives: The focus of this project was to identify the molecular targets of ASD associated, fetal-brain reactive maternal IgG antibodies.
Methods: A protein extract derived from fetal Rhesus macaque (152 day gestation) was fractionated by molecular weight and individual fractions were probed with plasma from mothers of children with ASD. Fractions containing antigenic proteins were subjected to duplicate 2-dimensional gel electrophoresis, with one gel being transferred to nitrocellulose and probed with maternal plasma to identify antigen location, and the other used for spot picking and tandem MS/MS analysis. Verification of MS results was carried out using commercially available purified or recombinant proteins, and further confirmed using blocking studies. Reactivity to the identified protein antigens was determined in a sample of 246 mothers of children with ASD and 149 mothers of typically developing children.
Results: Seven proteins were identified and confirmed to be the antigenic targets of ASD-associated maternal IgG. The 37kDa antigen is an essential metabolic enzyme with well characterized functions in neurogenesis. The 44kDa antigen is an enzyme known to regulate post-synaptic targeting. The 39kDa protein has an important role in late embryogenesis. Two proteins were identified as 73kDa antigens – one that is critical for neuronal growth cone collapse upon and the other that functions as a chaperone for several heat-shock proteins and mediates neuritogenesis. Confirmed reactivity to the 37kDa and both of the 73kDa proteins is observed exclusively among mothers of children with ASD with a prevalence of approximately 8%, yielding an odds ratio of 24.2 (95% CI: 45-405). Their children displayed significantly elevated stereotypical behaviors compared to ASD children from mothers lacking these antibodies.
Conclusions: Maternal IgG reactivity to the protein antigens identified in this study constitutes the most significant biomarker of ASD risk identified to date. In our study sample, reactivity to the 37kDa and 73kDa proteins was observed in approximately 8% of mothers of children with ASD and absent in mothers of typically developing children yielding a highly significant association with ASD (p=0.00001). Furthermore, previous findings from our group and others indicate that such maternal antibodies are often present during pregnancy, supporting the hypothesis that they could play a causal role in precipitating the behavioral outcomes noted in some cases of ASD.