Analysis of potential biomarkers and modifier genes affecting the clinical course of CLN3 disease

Abstract

Mutations in the CLN3 gene lead to juvenile neuronal ceroid lipofuscinosis, a pediatric neurodegenerative disorder characterized by visual loss, epilepsy and psychomotor deterioration. Although most CLN3 patients carry the same 1-kb deletion in the CLN3 gene, their disease phenotype can be variable. The aims of this study were to (i) study the clinical phenotype in CLN3 patients with identical genotype, (ii) identify genes that are dysregulated in CLN3 disease regardless of the clinical course that could be useful as biomarkers, and (iii) find modifier genes that affect the progression rate of the disease. A total of 25 CLN3 patients homozygous for the 1-kb deletion were classified into groups with rapid, average or slow disease progression using an established clinical scoring system. Genome-wide expression profiling was performed in eight CLN3 patients with different disease progression and matched controls. The study showed high phenotype variability in CLN3 patients. Five genes were dysregulated in all CLN3 patients and present candidate biomarkers of the disease. Of those, dual specificity phosphatase 2 (DUSP2) was also validated in acutely CLN3-depleted cell models and in CbCln3(Δex7/8) cerebellar precursor cells. A total of 13 genes were upregulated in patients with rapid disease progression and downregulated in patients with slow disease progression; one gene showed dysregulation in the opposite way. Among these potential modifier genes, guanine nucleotide exchange factor 1 for small GTPases of the Ras family (RAPGEF1) and transcription factor Spi-B (SPIB) were validated in an acutely CLN3-depleted cell model. These findings indicate that differential perturbations of distinct signaling pathways might alter disease progression and provide insight into the molecular alterations underlying neuronal dysfunction in CLN3 disease and neurodegeneration in general.

Figure 1

Clinical scoring of 25 CLN3 patients homozygous for the 1-kb deletion in the CLN3 gene. Scoring for each problem category was made as follows: normal function (3); problem slight, but readily recognized (2); problem severe (1); total loss of function (0). The range between the 10th and 90th percentile is represented by the shaded area. The black line represents the median.

Figure 2

Comparison of clinical scoring of CLN3 patients with rapid (n = 2) versus slow disease progression (n = 2). Clinical scoring is shown in red for patients with rapid disease progression (patient 1, solid line; patient 2, broken line) and in blue for patients with slow disease progression (patient 3, solid line; patient 4, broken line). The shaded area represents the range between the 10th and 90th percentile calculated from clinical scoring of 25 patients who are homozygous for the 1-kb deletion in the CLN3 gene (including the two patients with rapid and slow disease progression).

Figure 3

Relative mRNA expression of DUSP2. (A) The relative mRNA expression of DUSP2 in lymphocytes derived from CLN3 patients receiving anticonvulsive medication compared with age- and gender-matched controls was measured by RT-PCR and normalized to RPS18 expression. The patients were 14, 13 and 14 years old (P14M, male; P13F, female; P14F, female) and were all homozygous for the 1-kb deletion in the CLN3 gene. They were classified as having average (P14M), slow (P13F) and rapid disease progression (P14F). (B) The relative mRNA expression of Dusp2 in CbCln3^Δex7/8 cerebellar precursor cells compared with wild-type cells was measured by RT-PCR and normalized to Actb expression. Data are means ± SD of three independent RT-PCR experiments. *P < 0.05 compared with controls; **P < 0.01 compared with controls; ***P < 0.001 compared with controls.

Figure 4

Relative mRNA expression of candidate genes in siRNA-mediated acutely CLN3-depleted cells. HeLa cells or SH-SY5Y cells were transfected with CLN3 siRNA for 72 h in total. The relative mRNA expressions of candidate genes were measured by RT-PCR and normalized to ACTB expression. mRNA levels of scrambled siRNA-treated cells (white bar) were set as 1 and used as control. (A) Relative mRNA expression of CLN3, and DUSP2 in CLN3 siRNA-treated HeLa cells. (B) Relative mRNA expression of CLN3, and DUSP2 in CLN3 siRNA-treated SH-SY5Y cells. (C) Relative mRNA expression of CLN3, RAPGEF1 and SPIB in CLN3 siRNA-treated HeLa cells. Data are mean ± SD of three (B) or five (A, C) independent transfection experiments. **P < 0.01 compared with controls; ***P < 0.001 compared with controls.