Fetal alcohol syndrome (FAS) in C57BL/6 mice detected through proteomics screening of the amniotic fluid

Abstract

Background: Fetal Alcohol Syndrome (FAS), a severe consequence of the Fetal Alcohol Spectrum Disorders, is associated with craniofacial defects, mental retardation, and stunted growth. Previous studies in C57BL/6J and C57BL/6N mice provide evidence that alcohol-induced pathogenesis follows early changes in gene expression within specific molecular pathways in the embryonic headfold. Whereas the former (B6J) pregnancies carry a high-risk for dysmorphogenesis following maternal exposure to 2.9 g/kg alcohol (two injections spaced 4.0 h apart on gestation day 8), the latter (B6N) pregnancies carry a low-risk for malformations. The present study used this murine model to screen amniotic fluid for biomarkers that could potentially discriminate between FAS-positive and FAS-negative pregnancies.

Methods: B6J and B6N litters were treated with alcohol (exposed) or saline (control) on day 8 of gestation. Amniotic fluid aspirated on day 17 (n = 6 replicate litters per group) was subjected to trypsin digestion for analysis by matrix-assisted laser desorption-time of flight mass spectrometry with the aid of denoising algorithms, statistical testing, and classification methods.

Results: We identified several peaks in the proteomics screen that were reduced consistently and specifically in exposed B6J litters. Preliminary characterization by liquid chromatography tandem mass spectrometry and multidimensional protein identification mapped the reduced peaks to alpha fetoprotein (AFP). The predictive strength of AFP deficiency as a biomarker for FAS-positive litters was confirmed by area under the receiver operating characteristic curve.

Conclusions: : These findings in genetically susceptible mice support clinical observations in maternal serum that implicate a decrease in AFP levels following prenatal alcohol damage.

Figure 1

Sample MALDI-TOF spectrum of murine amniotic fluid samples collected on GD17. Samples from control (A) and alcoholic (B) B6J mouse fetuses: 50 μL aliquots were digested with methylated trypsin and 1 μL aliquots were prepared in an alpha-cyano matrix for linear and reflected MALDI-TOF.

Figure 2

Preprocessing effects. Example of raw (left panel) and preprocessed (right panel) mass spectra (1,000–1,500 m/z region).

Figure 3

Specificity-sensitivity analysis of the five top significant classifier peaks in the FAS-positive diagnostic profile. ROC curves were drawn for ROC characteristics of the top five peaks from MALDI-TOF based on linear discriminant classifier of alcohol-exposed and control B6J samples (named peaks 1–5, based on p values). Bootstrapped samples were randomly divided into training and test sets for linear discriminant analysis with varying classification cut-offs. ROC curves plotted 1-specificity versus sensitivity for peaks 1–4 (left panel) and peaks 4–5 (right panel). Maximum area under the ROC curve (1.0) was achieved using peaks 1–4, and near-unity (0.92) with peaks 4–5.