Prediction of phenotypic severity in mucopolysaccharidosis type IIIA

Abstract

Objective: Mucopolysaccharidosis IIIA or Sanfilippo disease type A is a progressive neurodegenerative disorder presenting in early childhood, caused by an inherited deficiency of the lysosomal hydrolase sulfamidase. New missense mutations, for which genotype-phenotype correlations are currently unknown, are frequently reported, hampering early prediction of phenotypic severity and efficacy assessment of new disease-modifying treatments. We aimed to design a method to determine phenotypic severity early in the disease course.

Methods: Fifty-three patients were included for whom skin fibroblasts and data on disease course and mutation analysis were available. Patients were phenotypically characterized on clinical data as rapidly progressing or slowly progressing. Sulfamidase activity was measured in fibroblasts cultured at 37 °C and at 30 °C.

Results: Sulfamidase activity in fibroblasts from patients homozygous or compound heterozygous for a combination of known severe mutations remained below the limit of quantification under both culture conditions. In contrast, sulfamidase activity in fibroblasts from patients homozygous or compound heterozygous for a known mild mutation increased above the limit of quantification when cultured at 30 °C. With division on the basis of the patients' phenotype, fibroblasts from slowly progressing patients could be separated from rapidly progressing patients by increase in sulfamidase activity when cultured at 30 °C (p < 0.001, sensitivity = 96%, specificity = 93%).

Interpretation: Phenotypic severity strongly correlates with the potential to increase sulfamidase activity in fibroblasts cultured at 30 °C, allowing reliable distinction between patients with rapidly progressing or slowly progressing phenotypes. This method may provide an essential tool for assessment of treatment effects and for health care and life planning decisions. Ann Neurol 2017;82:686-696.

Figure 1

Linearity of (A) protein concentration and (B) incubation time in sulfamidase (SGSH) activity assay in cultured fibroblasts of control cell line.

Figure 2

Residual sulfamidase (SGSH) enzyme activity in fibroblasts at 37 °C and at 30 °C. Group 1 (n = 23) was homozygous or compound heterozygous for the common severe mutations p.R245H, p.Q380R, p.S66W, or c.1080delC. Group 2 (n = 18) was compound heterozygous for 1 of the severe mutations and the mild p.S298P mutation. Group 3 (n = 4) was homozygous for the mild p.S298P mutation. The limit of quantification is indicated as a dashed line.

Figure 3

Residual sulfamidase (SGSH) enzyme activity in fibroblasts obtained from 53 mucopolysaccharidosis type IIIA patients and cultured at 30 °C. The patients are divided into 2 phenotypic group: rapidly progressing (RP) and slowly progressing (SP). The numbers correspond to the patient numbers as listed in Tables 1 and 2. The limit of quantification is indicated as a dashed line.

Figure 4

Western blot of patient fibroblast cell lysates. An immunoreactive 56kDa band was observed in samples from control cells and from slowly progressing mucopolysaccharidosis type IIIA patient cells cultured at 30 °C. ß‐actin was used as a protein loading control. Culture temperatures in °C are indicated below the lanes. SGSH = sulfamidase.

Figure 5

Western blot (A) and sulfamidase (SGSH) enzyme activity (B) in fibroblast cell lysate with addition of bortezomib at 2 concentrations (0 and 10nM) for 48 hours. Mucopolysaccharidosis type IIIA (lane 2: c.1080delC/c.376insG; lanes 3–6: p.S298P/p.S298P) and control fibroblasts (lane 1) were cultured at 37 °C and 30 °C for 48 hours instead of 1 week. To show the location of the mature SGSH band more accurately a diluted control sample was loaded in lane 7. SGSH activity was measured in p.S298P/p.S298P cell lysates.

Figure 6

Heparan sulfate levels measured in control cells (C) and in patient cell lines cultured at 37 °C and at 30 °C. The samples were measured in duplicate. The data are represented as medians and ranges. The numbers correspond to patient numbers as listed in Tables 1 and 2. RP = rapidly progressing; SP = slowly progressing.

Figure 7

Immunofluorescence microscopy using a monoclonal antibody directed at sulfamidase (stained yellow). Upper panels: Fibroblasts cultured at 37 °C. Lower panels: Fibroblasts cultured at 30 °C. (A, B) Fibroblasts from a healthy control. (C–H) Fibroblasts from mucopolysaccharidosis type IIIA patients (rapidly progressing [RP], Patient 1; slowly progressing [SP], Patients 29 and 53). Scale bars indicate 20 μm.