Increased glycolipid storage produced by the inheritance of a complex intronic haplotype in the α-galactosidase A (GLA) gene

Abstract

Background: Accumulation of galactosphingolipids is a general characteristic of Fabry disease, a lysosomal storage disorder caused by the deficient activity of α-galactosidase A encoded by the GLA gene. Although many polymorphic GLA haplotypes have been described, it is still unclear whether some of these variants are causative of disease symptoms. We report the study of an inheritance of a complex intronic haplotype (CIH) (c.-10C > T, c.369 + 990C > A, c.370-81_370-77delCAGCC, c.640-16A > G, c.1000-22C > T) within the GLA gene associated with Fabry-like symptoms and galactosphingolipid accumulation. We analysed α-Gal A activity in plasma, leukocytes and skin fibroblasts in patients, and measured accumulation of galactosphingolipids by enzymatic methods and immunofluorescence techniques. Additionally, we evaluated GLA expression using quantitative PCR, EMSA, and cDNA cloning.

Results: CIH carriers had an altered GLA expression pattern, although most of the carriers had high residual enzyme activity in plasma, leukocytes and in skin fibroblasts. Nonetheless, CIH carriers had significant galactosphingolipid accumulation in fibroblasts in comparison with controls, and also glycolipid deposits in renal tubules and glomeruli. EMSA assays indicated that the c.-10C > T variant in the promoter affected a nuclear protein binding site.

Conclusions: Thus, inheritance of the CIH caused an mRNA deregulation altering the GLA expression pattern, producing a tissue glycolipid storage.

Fig. 1

Pedigree of the family and complex intronic haplotype (c.-10C > T, c.369 + 990C > A, c.370-81_370-77delCAGCC, c.640-16A > G, c.1000-22C > T) carriers. Index case is indicated with an arrow

Fig. 2

Cytoplasmic vacuolation observed in a podocyte by light microscopy with a Masson’s trichrome and b Haematoxylin and eosin staining. c Myelin-like structures in a podocyte, with concentric lamellated ultra-structural appearance. (Electron Microscopy). d Focal areas of podocyte effacement; amorphous myelin-like structures are visible in glomerular parietal epithelial cells and in endothelial cells (Electron Microscopy). e Myelin-like structures parallel with zebra-like body appearance (Electron Microscopy)

Fig. 3

α-Gal enzyme activity. a Enzymatic activity in lysed leukocytes (nmol/mg protein/hour) and plasma (nmol/mL/hour), represented as mean ± SEM. b Enzymatic activity in lysed fibroblasts (nmol/mg protein/hour) represented as mean ± SEM, n = 3 *p ≤ 0.05

Fig. 4

Model of FD substrate accumulation in vitro. a Galactosphingolipid variation rate in wild-type fibroblasts under different conditions. Galactosphingolipids concentration was measured as described (n = 3) and changes in galactosphingolipids levels are represented. b Representative images of in vitro FD model. Control fibroblasts were stained with CD77 (Gb₃), green and LAMP1, red. CD77 and LAMP1 merged appear as orange/yellow. Nuclei were stained with DAPI, blue. b.1) Control 24 h; b.2) Control + TNFα 24 h; b.3) Control + TNFα 24 h + DGJ; b.4) Control + TNFα 24 h + agalsidase alfa. c Quantification of CD77/LAMP1 (orange/yellow) fluorescence rate (n = 3). d Quantification of CD17/LAMP1 (orange/yellow) fluorescence rate (n = 3)

Fig. 5

Accumulation of galactosphingolipids in fibroblast lysates. a Biochemical quantification after 5 days of culture represented as mean ± SEM. (n = 3) *p ≤ 0.05 **p ≤ 0.001. b Quantification of Gb₃ (CD77) confocal images represented as mean ± SD (n = 3) b.1) patient F1.10.*p ≤ 0.05; (+)TNFα vs. (−)TNFα and ^#p ≤ 0.05; Control vs. F1.10., b.2) Patient F1.4. c Immunocytochemistry of CD77 expression in fibroblasts from c.1) control, c.2) patient F1.4 and c.3) patient F1.10 after 16 h TNFα activation. Nuclei were stained with DAPI, blue

Fig. 6

EMSA analysis. a EMSA carried out with probes containing the C or T allele for the IVSO-10C > T variant in the GLA gene promoter. b The inverse of band densities from the EMSA plotted against the excess of cold allele T oligonucleotides, showing that allele T (slope = 0.003) was more easily displaced from the complex than allele C (slope = 0.001)