Complex Changes in the Efficiency of the Expression of Many Genes in Monogenic Diseases, Mucopolysaccharidoses, May Arise from Significant Disturbances in the Levels of Factors Involved in the Gene Expression Regulation Processes

Abstract

Monogenic diseases are primarily caused by mutations in a single gene; thus, they are commonly recognized as genetic disorders with the simplest mechanisms. However, recent studies have indicated that the molecular mechanisms of monogenic diseases can be unexpectedly complicated, and their understanding requires complex studies at the molecular level. Previously, we have demonstrated that in mucopolysaccharidoses (MPS), a group of monogenic lysosomal storage diseases, several hundreds of genes reveal significant changes in the expression of various genes. Although the secondary effects of the primary biochemical defect and the inefficient degradation of glycosaminoglycans (GAGs) might be considered, the scale of the changes in the expression of a large fraction of genes cannot be explained by a block in one biochemical pathway. Here, we demonstrate that in cellular models of 11 types of MPS, the expression of genes coding for proteins involved in the regulation of the expression of many other genes at various stages (such as signal transduction, transcription, splicing, RNA degradation, translation, and others) is significantly disturbed relative to the control cells. This conclusion was based on transcriptomic studies, supported by biochemical analyses of levels of selected proteins encoded by genes revealing an especially high level of dysregulation in MPS (EXOSC9, SRSF10, RPL23, and NOTCH3 proteins were investigated). Interestingly, the reduction in GAGs levels, through the inhibition of their synthesis normalized the amounts of EXOSC9, RPL23, and NOTCH3 in some (but not all) MPS types, while the levels of SRSF10 could not be corrected in this way. These results indicate that different mechanisms are involved in the dysregulation of the expression of various genes in MPS, pointing to a potential explanation for the inability of some therapies (such as enzyme replacement therapy or substrate reduction therapy) to fully correct the physiology of MPS patients. We suggest that the disturbed expression of some genes, which appears as secondary or tertiary effects of GAG storage, might not be reversible, even after a reduction in the amounts of the storage material.

Keywords: gene expression; glycosaminoglycans; mucopolysaccharidosis; transcriptomics.

Figure 1

Number of transcripts coding for proteins involved in gene expression (GO:0010467) with changed levels (at FDR < 0.1; p < 0.1) in different types of MPS relative to control cells (HDFa). Up-regulation (UP) and down-regulation (DOWN) in MPS relative to HDFa are marked.

Figure 2

Number of transcripts coding for proteins involved in the regulation of gene expression (GO:0010468) with changed levels (at FDR < 0.1; p < 0.1) in different types of MPS relative to control cells (HDFa). Down- and up-regulated transcripts are marked (DOWN and UP, respectively).

Figure 3

Number of transcripts of genes with changed levels (at FDR < 0.1; p < 0.1) in different types of MPS relative to control cells (HDFa) from the following sub-categories of the term “gene expression” (GO:0010467) (child terms): GO:0140053: mitochondrial gene expression, GO:0006396: RNA processing, GO:0010628: positive regulation of gene expression, GO:0010629: negative regulation of gene expression, GO:0097659: nucleic acid-templated transcription, GO:0006412: translation, GO:0006406: mRNA export from the nucleus, and GO:0051604: protein maturation. Down- and up-regulated transcripts are marked (DOWN and UP, respectively).

Figure 4

Levels of selected proteins (EXOSC9, panel (A); RPL23, panel (B); SRSF10, panel (C); NOTCH3, panel (D)) in fibroblasts of different types of MPS relative to control HDFa cells. The lower parts of each panel represent representative fragments of the blots (‘control’ represents loading control; in panel (C), short and long exposures are shown due to the low-level signal under standard conditions), and the upper parts demonstrate the quantification of the results. The results indicated by the columns are the mean values from three independent experiments with error bars representing SD. Statistically significant differences relative to the HDFa control (p < 0.05) are indicated by asterisks (*).

Figure 5

Levels of the EXOSC9 protein in fibroblasts of different types of MPS with (+) and without (−) treatment with 50 μM genistein for 48 h (only MPS types which revealed significantly changed amounts of this protein relative to HDFa control cells (see Figure 4) are shown). Panel (A) shows representative blots and the quantification of the results by densitometry, where value 1 corresponds to the level of EXOSC9 determined in MPS cells not treated with genistein. Panel (B) shows the scheme of results where the column (−) indicates the levels of EXOSC9 in MPS cells relative to HDFa control while the column (+) indicates the levels of this protein in cells of the corresponding MPS types after 48 h of treatment with 50 μM genistein relative to untreated MPS cells. Up-headed arrows indicate increased levels, down-headed arrows indicate decreased levels, and dashes indicate no significant changes. In panel (A), the results indicated by the columns are mean values from three independent experiments with error bars representing SD. Statistically significant differences relative to the untreated MPS cells (p < 0.05) are indicated by asterisks (*).

Figure 6

Levels of the RPL23 protein in fibroblasts of different types of MPS with (+) and without (−) treatment with 50 μM genistein for 48 h (only MPS types which revealed significantly changed amounts of this protein relative to HDFa control cells (see Figure 4) are shown). Panel (A) shows representative blots and the quantification of the results by densitometry, where value 1 corresponds to the level of RPL23 determined in MPS cells not treated with genistein. Panel (B) shows the scheme of results where the column (−) indicates the levels of RPL23 in MPS cells relative to HDFa control while the column (+) indicates the levels of this protein in cells of the corresponding MPS types after 48 h of treatment with 50 μM genistein relative to untreated cells. Up-headed arrows indicate increased levels, down-headed arrows indicate decreased levels, and dashes indicate no significant changes. In panel (A), the results indicated by columns are mean values from three independent experiments with error bars representing SD. Statistically significant differences relative to the untreated MPS cells (p < 0.05) are indicated by asterisks (*).

Figure 7

Levels of the SRSF10 protein in fibroblasts of different types of MPS with (+) and without (−) treatment with 50 μM genistein for 48 h (only MPS types which revealed significantly changed amounts of this protein relative to HDFa control cells (see Figure 4) are shown). Panel (A) shows representative blots and the quantification of the results by densitometry, where value 1 corresponds to the level of SRSF10 determined in MPS cells not treated with genistein. Panel (B) shows the scheme of results where the column (−) indicates the levels of SRSF10 in MPS cells relative to HDFa control while the column (+) indicates the levels of this protein in cells of the corresponding MPS types after 48 h of treatment with 50 μM genistein relative to untreated MPS cells. Up-headed arrows indicate increased levels, down-headed arrows indicate decreased levels, and dashes indicate no significant changes. In panel (A), the results indicated by the columns are mean values from three independent experiments with error bars representing SD. Statistically significant differences relative to the untreated MPS cells (p < 0.05) are indicated by asterisks (*).

Figure 8

Levels of the NOTCH3 protein in fibroblasts of different types of MPS with (+) and without (−) treatment with 50 μM genistein for 48 h (only MPS types which revealed significantly changed amounts of this protein relative to HDFa control cells (see Figure 4) are shown). Panel (A) shows representative blots and the quantification of the results by densitometry, where value 1 corresponds to the level of NOTCH3 determined in MPS cells not treated with genistein. Panel (B) shows the scheme of results where the column (−) indicates the levels of NOTCH3 in MPS cells relative to HDFa control while the column (+) indicates the levels of this protein in cells of the corresponding MPS types after 48 h of treatment with 50 μM genistein relative to untreated MPS cells. Up-headed arrows indicate increased levels, down-headed arrows indicate decreased levels, and dashes indicate no significant changes. In panel (A), the results indicated by the columns are mean values from three independent experiments with error bars representing SD. Statistically significant differences relative to the untreated MPS cells (p < 0.05) are indicated by asterisks (*).