Elucidating the Antigenic Epitopes in Maternal Autoantibody Related Autism Spectrum Disorder (ASD)
Numerous researchers have described the presence of maternal autoantibodies reactive to fetal brain proteins in a subset of mothers of children with autism spectrum disorder (ASD). Our laboratory recently determined the identity of the autoantigens targeted by autoantibodies in maternal autoantibody related (MAR) ASD; we identified seven autoantigens: lactate dehydrogenase A and B (LDH-A, LDH-B), stress-induced phosphoprotein 1 (STIP1), collapsin response mediator proteins 1 and 2 (CRMP1, CRMP2), cypin, and Y-box binding protein 1(YBX1). A detailed understanding of the targeted epitopes on each autoantigen will be critical in dissecting the pathology associated with maternal autoantibodies.
The specific objective of this study was to elucidate and verify the immunodominant epitope sequences targeted by maternal autoantibodies for each of the MAR ASD autoantigens.
The amino acid sequences of the candidate autoantigens were obtained from the NCBI protein database. Working in conjunction with PEPperPRINT© microarray solutions, each protein was translated into a peptide array composed of duplicate, overlapping peptide sequences, which were 15 amino acids long with a 14 amino acid peptide-peptide overlap. Two microarray schemes were synthesized: one microarray containing LDH-A, STIP1, and CRMP1 sequences, and the other comprising LDH-B, CRMP2, Cypin, and YBX1 sequences. Each array was incubated with maternal plasma samples obtained from the Childhood Autism Risk from Genetics and Environment (CHARGE) Study at UC-Davis, which were predetermined via western blot and ELISA to be highly reactive to the target proteins. Immunodominant epitopes were consequently verified using microarrays, which contained peptide sequences identified in the previous arrays. We ran 24 plasma samples from mothers of children with severe ASD status, 12 plasma samples from mothers of children with mild ASD, and 12 samples from mothers of TD controls. Diagnosis of all enrolled children was confirmed at the UC Davis M.I.N.D. Institute.
We successfully determined that the MAR ASD autoantibodies recognized unique, discrete peptide sequences on each target autoantigen. We further determined that there are 3 epitopes for LDH-A, 3 epitopes for LDH-B, 3 epitopes for STIP1, 1 epitope for cypin, 3 epitopes of YBX1, 4 epitopes for CRMP1, and 9 epitopes for CRMP 2. Overall, we have determined that autoantibodies from mothers of children with ASD reacted to more autoantigens and more epitopes per autoantigen than autoantibodies from mothers of typically developing children.
The results of this project will enable the development of robust biomarkers for ASD risk assessment and subphenotype stratification. Additionally, we have used these peptide epitopes to break tolerance in a mouse model, which will help us to better understand how the maternal autoantibodies are interacting with the candidate autoantigens, thus providing insight into the pathologic mechanisms through which these antibodies alter developmental trajectory, and the critical pathways that may serve as a template for therapeutic intervention. Future experiments using a larger sample population will be performed to determine if maternal autoantibody reactivity to individual or combinations of peptides correlate with increased aberrant behaviors and/or increased cognitive and social deficits.