Peripheral biomarkers in Autism: secreted amyloid precursor protein-alpha as a probable key player in early diagnosis

Abstract

Autism is a pervasive developmental disorder characterized by impairments in socialization and communication. There is currently no single molecular marker or laboratory tool capable of diagnosing autism at an early age. The purpose of this study is to explore the plausible use of peripheral biomarkers in the early diagnosis of autism via a sensitive ELISA. Here, we measured plasma secreted amyloid precursor protein alpha (sAPP-alpha) levels in autistic and aged-matched control blood samples and found a significantly increased level of sAPP-alpha in 60% of the known autistic children. We then tested 150 human umbilical cord blood (HUCB) samples and found significantly elevated levels of plasma sAPP-alpha in 10 of 150 samples. As an additional confirmatory measure, we performed Western blot analysis on these samples which consistently showed increased sAPP-alpha levels in autistic children and 10 of 150 HUCB samples; suggesting a group of autistic patients which could be identified in early childhood by levels of sAPP-alpha. While there is need for further studies of this concept, the measurement of sAPP-alpha levels in serum and human umbilical cord blood by ELISA is a potential tool for early diagnosis of autism.

Keywords: Autism; autism spectrum disorders (ASD); brain derived neurotrophic factor (BNDF); secreted amyloid precursor protein-α (sAPP-α).

Figure 1

Levels of sAPP-α are elevated in autistic children. (A and B) Plasma sAPP-α levels were measured by sAPP-α ELISA. Data are presented as mean ± SEM (n = 25 for autistic children, 15 ♂/10 ♀F; n = 25 for healthy age matched children, 15 ♂/10 ♀) of sAPP-α (ng/mg total plasma protein). (C) Western blotting analysis consistently shows increased sAPP-α levels in autistic children versus age-matched healthy controls as indicated. Blood plasma samples for both groups of children were randomly selected. The selected samples were then pooled and loaded in triplicate for electrophoresis. The lack of similarity between all three lanes for the autism samples is not due to differences in the individual samples. (D) As quantified in comparison to total protein (normalization), densitometry analysis shows significantly increased density in Western blotting band density as indicated. Data are presented as mean ± SEM [n = 15 (autistic children), 11 ♂/4 ♀; n = 15 (healthy controls), 8 ♂/7 ♀] of Western blotting band density.

Figure 2

A significantly increased level of sAPP-α is observed in approximately 7% of 150 human umbilical cord blood (HUCB) samples. (A) The plasma was isolated, prepared, and subjected to sAPP-α ELISA. Data indicated elevated levels (> 3 ng/mg total plasma protein) of plasma sAPP-α in 10 of 150 samples. Data are presented as mean ± SEM of sAPP-α (ng/mg total protein). (B) Western blotting analysis demonstrated increased sAPP-α levels in the same samples which demonstrated elevated sAPP-α by ELISA. (C) As quantified with total protein (normalization), densitometry analysis shows significantly increased density of Western blotting band as indicated. Likewise, samples demonstrating a < 3 ng/mg difference in sAPP-α by ELISA demonstrated the same changes upon Western blotting analysis. HUCB samples from both groups of infants were randomly selected. The selected samples were then pooled and loaded in triplicate for electrophoresis. Data are presented as mean ± SEM (n = 10, 8 M/7 F) of band density.