Genome-wide transcription analysis of histidine-related cataract in Atlantic salmon (Salmo salar L)

Abstract

Purpose: Elevated levels of dietary histidine have previously been shown to prevent or mitigate cataract formation in farmed Atlantic salmon (Salmo salar L). The aim of this study was to shed light on the mechanisms by which histidine acts. Applying microarray analysis to the lens transcriptome, we screened for differentially expressed genes in search for a model explaining cataract development in Atlantic salmon and possible markers for early cataract diagnosis.

Methods: Adult Atlantic salmon (1.7 kg) were fed three standard commercial salmon diets only differing in the histidine content (9, 13, and 17 g histidine/kg diet) for four months. Individual cataract scores for both eyes were assessed by slit-lamp biomicroscopy. Lens N-acetyl histidine contents were measured by high performance liquid chromatography (HPLC). Total RNA extracted from whole lenses was analyzed using the GRASP 16K salmonid microarray. The microarray data were analyzed using J-Express Pro 2.7 and validated by quantitative real-time polymerase chain reaction (qRT-PCR).

Results: Fish developed cataracts with different severity in response to dietary histidine levels. Lens N-acetyl histidine contents reflected the dietary histidine levels and were negatively correlated to cataract scores. Significance analysis of microarrays (SAM) revealed 248 significantly up-regulated transcripts and 266 significantly down-regulated transcripts in fish that were fed a low level of histidine compared to fish fed a higher histidine level. Among the differentially expressed transcripts were metallothionein A and B as well as transcripts involved in lipid metabolism, carbohydrate metabolism, regulation of ion homeostasis, and protein degradation. Hierarchical clustering and correspondence analysis plot confirmed differences in gene expression between the feeding groups. The differentially expressed genes could be categorized as "early" and "late" responsive according to their expression pattern relative to progression in cataract formation.

Conclusions: Dietary histidine regimes affected cataract formation and lens gene expression in adult Atlantic salmon. Regulated transcripts selected from the results of this genome-wide transcription analysis might be used as possible biological markers for cataract development in Atlantic salmon.

Figure 1

Cataract scores in selected dietary groups throughout the experimental period. The cataract score for each dietary group is given as the mean of the sums of the scores for both eyes, resulting in a possible maximum score of 8 (4 for each lens). Error bars show the standard error of the mean (SEM). The number of fish per group (n) varied from 31 to 113.

Figure 2

Individual cataract scores and N-acetyl histidine (NAH) concentrations in lenses of the fish used for microarray analysis. The right lens of the fish was used for microarray analysis, and thus the cataract scores (on a scale from 0 to 4) of the right lens are presented in the graphs. The NAH concentrations were determined in the left lens of the same fish. A: Cataract scores and NAH concentrations for the individual samples are shown in this graph. Under the sample names, the sample clustering (obtained by hierarchical clustering of genes and samples, see Figure 4) is shown to relate individual cataract scores and NAH concentrations to gene expression patterns (the closer the samples are in the cluster tree, the more similar is the lens transcriptome). B: Lens NAH concentrations were significantly negatively correlated to the cataract scores of the right lens (Spearman rank test; r=−0.63, p<0.002, n=22).

Figure 3

Correspondence analysis plot. The principal components 1 and 2, which explain the highest amounts of variance in the data set, are shown on the x- and y-axis of the plot, respectively. The samples are colored according to the dietary groups. The low-His samples (LLL) are blue, and the medium-His samples (MMM) are dark red. The dark red and blue lines are plotted from the point of origin through the respective group medians, which are marked by an equally colored dot. The total variance retained in the plot is 16.349%, the x-axis component variance is 10.623%, and the y-axis component variance is 5.726%.

Figure 4

Hierarchical clustering of samples and transcripts. The samples are arranged in columns, and the transcripts are arranged in rows. Only the transcripts with a q-value of 0% in the SAM list were clustered. Negative log intensity ratios are shown in green and positive log intensity ratios are shown in red in the heat map as indicated by the color bar. The blue color represents missing values. The transcripts divide into two distinct clusters. The first cluster contains the transcripts that are up-regulated in the low-His group compared to the medium-His group and is marked by a red bar at the right side of the heat map. The second cluster contains the down-regulated transcripts and is marked by a green bar at the right side of the heat map. The samples divide into three main clusters, reflecting the His feeding regimes. Low-His samples are clearly separated from medium-His samples.

Figure 5

Examples of transcripts with different expression patterns related to cataract score. For four selected significantly differentially expressed transcripts, the log intensity ratios are plotted against the cataract score of the respective sample, not taking into account which dietary group the samples belong to. For a certain transcript, if the difference between the mean log intensity ratios of the lenses with a score of 0 and the lenses with a score of 1 was 0.2 or greater, this transcript was classified as “early” regulated. If this difference was less than 0.2, the transcript was classified as “late” regulated. A: SPARC precursor (SPARC; CA052160) was chosen as an example for “early” up-regulated transcripts. B: Metallothionein B (MT-B; CK990996) was chosen as an example of “late” up-regulated transcripts. C: Ependymin (EPN; CA042089) was chosen as an example of “early” down-regulated transcripts. D: Fatty acid binding protein 2 (FABP2; CA054659) was chosen as an example of “late” down-regulated transcripts.

Figure 6

Correlation between fold change values obtained by microarray analysis and qRT–PCR for 16 selected transcripts. Fold change (FC) values obtained by microarray analysis were significantly correlated to those obtained by qRT–PCR (Spearman rank test; r=0.89, p<0.0001, n=16).