Gene-wise association of variants in four lysosomal storage disorder genes in neuropathologically confirmed Lewy body disease

Abstract

Objective: Variants in GBA are associated with Lewy Body (LB) pathology. We investigated whether variants in other lysosomal storage disorder (LSD) genes also contribute to disease pathogenesis.

Methods: We performed a genetic analysis of four LSD genes including GBA, HEXA, SMPD1, and MCOLN1 in 231 brain autopsies. Brain autopsies included neuropathologically defined LBD without Alzheimer Disease (AD) changes (n = 59), AD without significant LB pathology (n = 71), Alzheimer disease and lewy body variant (ADLBV) (n = 68), and control brains without LB or AD neuropathology (n = 33). Sequencing of HEXA, SMPD1, MCOLN1 and GBA followed by 'gene wise' genetic association analysis was performed. To determine the functional effect, a biochemical analysis of GBA in a subset of brains was also performed. GCase activity was measured in a subset of brain samples (n = 64) that included LBD brains, with or without GBA mutations, and control brains. A lipidomic analysis was also performed in brain autopsies (n = 67) which included LBD (n = 34), ADLBV (n = 3), AD (n = 4), PD (n = 9) and control brains (n = 17), comparing GBA mutation carriers to non-carriers.

Results: In a 'gene-wise' analysis, variants in GBA, SMPD1 and MCOLN1 were significantly associated with LB pathology (p range: 0.03-4.14 x10(-5)). Overall, the mean levels of GCase activity were significantly lower in GBA mutation carriers compared to non-carriers (p<0.001). A significant increase and accumulation of several species for the lipid classes, ceramides and sphingolipids, was observed in LBD brains carrying GBA mutations compared to controls (p range: p<0.05-p<0.01).

Interpretation: Our study indicates that variants in GBA, SMPD1 and MCOLN1 are associated with LB pathology. Biochemical data comparing GBA mutation carrier to non-carriers support these findings, which have important implications for biomarker development and therapeutic strategies.

Fig 1. GCase and HexA activity in autopsy brain tissue.

A) GCase activity was significantly reduced in LBD cases carrying GBA mutations (n = 16) compared to LBD non-GBA carriers (n = 18) and controls (n = 30). Differences in activity for HEXA were not significant in any group. B) GCase was significantly reduced in LBD cases with mutations classified as ‘severe’ type (L444P, 84insGG etc.) compared to controls, and to LBD cases with ‘mild’ mutations (N370S) or variants of unknown phenotypic effect (E326K, T369M). Differences in activity for HEXA were not significant. * p<0.05, ** p<0.01, *** p<0.001.

Fig 2. Heat Maps showing significant changes in lipid classes.

A) Heat map showing statistically significant changes in major lipid subclasses in LBD GBA mutation carriers compared to LBD wildtype, AD cases and controls and B) Heat map showing statistically significant changes in lipid classes in LBD GBA mutation carriers compared to LBD wildtype, AD cases and controls. The heat map columns reflect all significant lipid changes (q<0.05) in a diseased compared to control patients. The color bar represents the log2 value of the ratio of each lipid species. Statistical analysis for the AD and LBD Mutation samples was based on the one way analysis of variance followed by post hoc Fisher’s least significant difference test while the LBD (wildtype) samples was based on Student’s T-test. A false discovery rate control was used to correct for multiple comparisons.

Fig 3. Comparative lipid profile of post-mortem brain tissue obtained from patients diagnosed with various neurological conditions.

Comparative lipid profile of post-mortem brain tissue obtained from patients diagnosed with various neurological conditions. The individual lipid subclasses of each group of patients was expressed as relative to control group levels for 2 separate sets of experiments (i.e. AD and LBD GBA mutation carrier relative to Control S1, LBD non carrier (wildtype) relative to Control S2). Statistical analysis for the AD and LBD Mutation samples was based on the one way analysis of variance followed by post hoc Fisher’s least significant difference test while the LBD non carrier (wildtype) samples was based on Student’s T-test. A false discovery rate control was used to correct for multiple comparisons. * q<0.05, ** q<0.01, *** q<0.001. PC, phosphatidylcholine; ePC, ether phosphatidylcholine; PE, phosphatidylethanolamine; pPE, plasmalogen phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol; PA, phosphatidic acid; PG, phosphatidylglycerol; LBPA, lysobisphosphatidic acid; Cer, ceramide; SM, sphingomyelin; dhSM, dihydrosphingomyelin; GalCer, galactosylceramide; GluCer, glucosylceramide; Sulf, sulfatide; Sulf-h, hydroxylated sulfatide; GM3, monosialodihexosylganglioside