Identification of reduced tryptophan metabolism in the ASD cells

We initiated an investigation of the metabolic profile of lymphoblastoid cell lines from patients with different neurobehavioral disorders and normal individuals, utilizing Biolog Phenotype MicroArray plates to see if patients with specific disorders had a characteristic profile. The assay, as carried out, analyzes the NADH generation in the presence of each substrate by measuring the redox color change of a tetrazolium dye after 24 h of incubation.

We first tested the ability of lymphoblastoid cells to utilize the 367 substrates in the PM plates in 18 controls and 55 patients. Seventeen of these patients were affected with ASDs and 38 had 15 different intellectual disability (ID) conditions and known pathogenic mutations in specific ID-associated genes. Of the 17 ASD patients, 10 had non-syndromal autistic disorder with known genetic alterations: two of these patients were monozygotic male twins with a chromosome 3p26.2 deletion, not involving any reported gene, one had a chromosome 2p21 duplication and a chromosome 20p21.1 deletion, three had SHANK3 mutations, two had NLGN4 mutations, and one had a balanced translocation involving chromosomes 14 and 15. The remaining seven cell lines were from patients with syndromal autism: four cases had Fragile X syndrome, autistic features and full methylation at the FRAXA locus; two cases had Rett syndrome and MECP2 mutations; one case had a ZNF711 mutation, X-linked intellectual disability and clinical findings consistent with ASDs (see Additional file 1: Table S1 for more detailed description).

The analysis of the Biolog PM plate data was conducted in a blind fashion so that one of us (CFC) was ignorant of the clinical information connected to each patient. The analysis of the assay data revealed significant differences in the utilization of multiple substrates as an energy source for most patient cell lines when they were compared to the controls. However, once the clinical information was attached to the data, if an ASD was present, it was noted that the 25 substrates with the greatest statistical significance (P value ≤0.001) contained a form of tryptophan, and all 27 wells containing tryptophan had P values ≤0.05 (Additional file 2: Table S2). This collective finding was independent of whether tryptophan was alone in the well or in the first or second position of a dipeptide. These observations suggested that lymphoblastoid cell lines from either syndromal or non-syndromal ASD patients exhibit reduced tryptophan metabolism as compared to normal individuals. Among the top 50 wells with a significant difference between patients without ASDs and controls, none contained tryptophan (Additional file 3: Table S3). Out of 222 substrates that met the minimal statistical threshold for significance (P <0.05), only two (0.9%) contained tryptophan (Lys-Trp and Pro-Trp) for patients without ASDs. The results suggest that tryptophan metabolism in lymphoblast cells from non-ASD patients with neurodevelopmental disorders is not significantly different as compared to normal individuals.

It is noteworthy that some of these patients were affected by conditions that sometimes may present with autistic traits: (1) two cell lines were from patients with Angelman syndrome, a condition often considered in the differential diagnosis of Rett syndrome, who did not meet criteria for ASDs; and (2) a patient with a ZNF711 mutation who did not exhibit ASD features.

In order to evaluate the specificity of our finding with regard to the behavioral phenotype associated with ASDs, we tested 10 patients with schizophrenia, a condition that shares some features with ASDs. We did not detect significant abnormalities in tryptophan metabolism, as compared to controls (data not shown).

In summary, these data suggested that the reduced tryptophan metabolism: (1) was a prominent feature common for the 17 ASD patient cell lines tested, as a group, independent of whether the autistic traits were the sole clinical findings in the patients or whether they were accompanied by other signs in a syndromal condition; (2) was extremely rare in patients with similar conditions but without features consistent with ASDs; and (3) was not observed in other neurodevelopmental conditions.

Validation studies and feature selection

In order to replicate our initial results, we tested an additional 20 patients with autistic disorders, according to the DSM IV-Revised criteria, 10 new controls and 10 of the previously tested controls randomly selected. Thirteen of the 20 ASD patients had genetic abnormalities: seven patients had the MET rs1858830 C/C genotype, four had chromosomal rearrangements found by array-CGH analysis, one had a balanced translocation involving chromosomes 13 and 15, and one had a mutation in the OCRL1 gene. To focus our analysis on tryptophan metabolism, we utilized only the PM-M4 plate, which contains the 12 wells with tryptophan as the first amino acid in various dipeptides. The PM-M4 plate also contains 17 wells with tyrosine, an amino acid that shares some common pathways with tryptophan and whose utilization was significantly reduced in 19/27 wells (70.4%) in our previous cohort. The Biolog results in these ASD cell lines, on average, indicated that the tryptophan metabolism was reduced when compared to the controls (Figure 1 and Additional file 4: Table S4), which was consistent with our findings for the previously tested 17 ASD cell lines. No significant changes were detected for the wells containing tyrosine, except for the one with the tryptophan-tyrosine dipeptide (Additional file 4: Table S4).

Figure 1 Histogram of energy metabolism in the ASD cells vs . control cells. The bar height of the histogram indicates the mean of measurements of 20 cell lines for each group; vertical bars are standard errors of the means. Star* symbol denotes level of statistical significance determined by a t test: **P <0.01; *P <0.05. The substrates were ordered according to their individual P values with the lowest on the left. Full size image

We investigated whether each tryptophan dipeptide possesses the same statistical power in distinguishing patients from controls. Ten feature selection methods were utilized to analyze the data obtained from the experiments of 20 controls vs. 20 patients. The analysis revealed that the top tryptophan dipeptides in order of informative power were: tryptophan-glycine (Trp-Gly), tryptophan-lysine (Trp-Lys), tryptophan-alanine (Trp-Ala), tryptophan-arginine (Trp-Arg), and tryptophan-leucine (Trp-Leu).

The results allowed us to design a customized 96-well plate with 12 columns of eight wells. These customized plates were used to test lymphoblastoid cell lines from new, unrelated cohorts of 50 ASD patients and 50 controls. This experiment, using five tryptophan dipeptides along with a positive control (α-D-glucose), a negative control (empty well), and L-tryptophan, replicated our previous observations of reduced tryptophan metabolism in lymphoblastoid cell lines from patients with ASDs (Figure 2). We also noticed that the positive control well containing glucose showed a difference between controls and patients with ASDs, even if not as significant as the one observed in the wells containing tryptophan. This observation suggested that there might exist some fundamental differences in the general energetic metabolism between ASD cases and controls, which were not observed in our previous data. To determine if the contribution of this possible general effect of energy utilization affected our results, we re-analyzed the data by ‘normalizing’ the tryptophan values with the positive control well. For each individual we have tested, we subtracted the value of each well containing tryptophan from the corresponding value of the well containing glucose. The result of this ‘normalization’ was that data still supported the previous observation that the lymphoblastoid cell lines of the patients with ASDs, on average, utilize tryptophan less effectively than those of controls (data not shown).

Figure 2 Histogram of energy metabolism in the ASD cells vs . control cells. The bar height of the histogram indicates the mean of measurements of 50 cell lines for each group; vertical bars are standard errors of the means. Star symbol (*) denotes level of statistical significance determined by a t test: ***P <0.001. The substrates were ordered according to their individual P values with the lowest on the left. Full size image

Since the experiment exploring the detected five tryptophan dipeptides utilized a larger cohort and more diverse group of samples, we tested whether age was a co-factor for the difference observed. A Spearman correlation analysis failed to find any age-related effect on the tryptophan utilization either in the patients with ASDs or in the controls (results not shown).

Gene expression in the tryptophan metabolic pathway

It is reasonable to hypothesize that the observed reduction of tryptophan metabolism in ASD cell lines may stem from abnormal functions along tryptophan metabolic pathways in the cells. We therefore mined data generated from a small gene expression profiling study we had conducted previously to examine this possibility. The study employed the Agilent Whole Human Genome Oligo Microarrays and RNA extracted from the initial 10 ASD cell lines and 10 controls (Figure 3 and online). The data indicated that two genes, SLC7A5 and SLC7A8, coding for tryptophan transporter subunits, expressed in both blood and brain, had reduced expression in all patients (Figures 3 and 4). The mitochondrial isoform of tryptophanyl tRNA synthetase (WARS2) had significantly reduced expression (P <0.001) in a majority (6/10) of the ASD cell lines while the cytoplasmic isoform (WARS) showed no difference in expression levels (Figures 3 and 4, Additional file 5: Table S5).

Figure 3 Histogram of gene expression of selected genes in tryptophan metabolic pathway in the ASD cells vs . control cells. The bar height of the histogram indicates the mean of measurements of 10 cell lines for the controls and 10 cell lines of patients with ASDs. Vertical bars are standard errors of the means. Symbol denotes level of statistical significance determined by a t test: *P <0.05; **P <0.01. ns, non-significant. Full size image

Figure 4 Tryptophan pathways. The figure illustrates the main intracellular pathways involving tryptophan. Genes with reduced expression in our microarray dataset are in blue, genes with increased expression are in red. Genes with statistically significant reduction of expression are underlined. Reactions generating NADH are indicated in the top section of the figure. FAD, flavin adenine dinucleotide. Full size image

The two main pathways of tryptophan metabolism lead to the synthesis of serotonin and kynurenine [18]. Tryptophan hydroxylase is the rate-limiting enzyme in the biosynthesis of serotonin and the gene encoding the isoform 2 of this enzyme (TPH2) showed significantly reduced expression levels (Figures 3 and 4, Additional file 5: Table S5). Also, several genes coding for enzymes involved in the kynurenine pathway showed significant differences (P <0.05) between ASD cell lines and controls. The expression levels of AADAT, HAAO, and MAOA were reduced in patients with ASDs, while QPRT showed a non-significant trend towards over-expression (Figure 3 and Additional file 5: Table S5).

Not all genes in tryptophan related pathways exhibited significant expression differences between ASD patients and controls, and each patient exhibited a different profile for the group of genes examined. However, each of the 10 ASD patients showed significant differences (P <0.05) from controls in the expression levels of at least 9/15 genes involved in tryptophan metabolism (Additional file 5: Table S5).