Human stem cell cultures from cleft lip/palate patients show enrichment of transcripts involved in extracellular matrix modeling by comparison to controls

Abstract

Nonsyndromic cleft lip and palate (NSCL/P) is a complex disease resulting from failure of fusion of facial primordia, a complex developmental process that includes the epithelial-mesenchymal transition (EMT). Detection of differential gene transcription between NSCL/P patients and control individuals offers an interesting alternative for investigating pathways involved in disease manifestation. Here we compared the transcriptome of 6 dental pulp stem cell (DPSC) cultures from NSCL/P patients and 6 controls. Eighty-seven differentially expressed genes (DEGs) were identified. The most significant putative gene network comprised 13 out of 87 DEGs of which 8 encode extracellular proteins: ACAN, COL4A1, COL4A2, GDF15, IGF2, MMP1, MMP3 and PDGFa. Through clustering analyses we also observed that MMP3, ACAN, COL4A1 and COL4A2 exhibit co-regulated expression. Interestingly, it is known that MMP3 cleavages a wide range of extracellular proteins, including the collagens IV, V, IX, X, proteoglycans, fibronectin and laminin. It is also capable of activating other MMPs. Moreover, MMP3 had previously been associated with NSCL/P. The same general pattern was observed in a further sample, confirming involvement of synchronized gene expression patterns which differed between NSCL/P patients and controls. These results show the robustness of our methodology for the detection of differentially expressed genes using the RankProd method. In conclusion, DPSCs from NSCL/P patients exhibit gene expression signatures involving genes associated with mechanisms of extracellular matrix modeling and palate EMT processes which differ from those observed in controls. This comparative approach should lead to a more rapid identification of gene networks predisposing to this complex malformation syndrome than conventional gene mapping technologies.

Fig. 1

Clustering of 87 DEGs resulted from the comparison between 6 controls and 6 NSCL/P patients. Each GEDI map (or mosaic) represents a gene expression profile of a single individual. The blue color represents the lowest expression level and red color represents the highest expression level on a scale of −4.70 to 7.98, respectively. The black frame highlights four patients with similar gene expression profile

Fig. 2

Gene clusters 1, 4, 6 and 9 resulted from k-means method (k = 9). In both clusters it is possible to observe a similar gene expression profile among 4 out of 6 patients (F4280, F4281, F4282 and F4283), indicating that many of the 87 selected DEGs are co-regulated in these 4 NSCL/P patients

Fig. 3

Quantitative Real-Time PCR (qRT-PCR) initial analysis of NSCL/P patients and control samples for ERAP2, PPT2, COL15A1. E = primer amplification efficiency; CT = cycle threshold; delta_CT = sample’s average_CT normalized by pool’s average_CT; NF = normalization factor

Fig. 4

The most significant network built by IPA tool with the highest number of differentially expressed genes. The upregulated genes in NSCL/P patients are indicated in red and the downregulated genes in green. The blank symbols pertain to genes that were either not present in our array or not differentially expressed. Solid lines indicate a direct linkage among two genes. Lines with arrows indicate that one gene acts on the other, and lines without arrows indicate that the corresponding proteins interact with each other. The 6 genes circled in orange were used in clustering analysis of qRT-PCR and microarray

Fig. 5

Hierarchical clustering of all the patients analyzed by qRT-PCR and microarray, considering only the genes from IPA network 1 (Fig. 4). a Clustering of expression values obtained by qRT-PCR. b Clustering of expression values obtained by microarray. c Correlations (Spearman’s correlation test, r and p-values) between each co-regulated gene from qRT-PCR clustering. It is possible to observe that the gene MMP3 is significantly and inversely correlated to ACAN, COL4A1, COL4A2 genes in a subgroup of patients from qRT-PCR. This same pattern of co-regulation is also present in another group of patients analyzed by microarray, which includes two individuals (F4280 and F4281) from the mentioned qRT-PCR subgroup. In controls expression data of both assays, MMP3 is upregulated and ACAN, COL4A1 and COL4A2 downregulated