Putative second hit rare genetic variants in families with seemingly GBA-associated Parkinson's disease

Abstract

Rare variants in the beta-glucocerebrosidase gene (GBA1) are common genetic risk factors for alpha synucleinopathy, which often manifests clinically as GBA-associated Parkinson's disease (GBA-PD). Clinically, GBA-PD closely mimics idiopathic PD, but it may present at a younger age and often aggregates in families. Most carriers of GBA variants are, however, asymptomatic. Moreover, symptomatic PD patients without GBA variant have been reported in families with seemingly GBA-PD. These observations obscure the link between GBA variants and PD pathogenesis and point towards a role for unidentified additional genetic and/or environmental risk factors or second hits in GBA-PD. In this study, we explored whether rare genetic variants may be additional risk factors for PD in two families segregating the PD-associated GBA1 variants c.115+1G>A (ClinVar ID: 93445) and p.L444P (ClinVar ID: 4288). Our analysis identified rare genetic variants of the HSP70 co-chaperone DnaJ homolog subfamily B member 6 (DNAJB6) and lysosomal protein prosaposin (PSAP) as additional factors possibly influencing PD risk in the two families. In comparison to the wild-type proteins, variant DNAJB6 and PSAP proteins show altered functions in the context of cellular alpha-synuclein homeostasis when expressed in reporter cells. Furthermore, the segregation pattern of the rare variants in the genes encoding DNAJB6 and PSAP indicated a possible association with PD in the respective families. The occurrence of second hits or additional PD cosegregating rare variants has important implications for genetic counseling in PD families with GBA1 variant carriers and for the selection of PD patients for GBA targeted treatments.

Fig. 1. Genetic and clinical characteristics of family A and B.

A The pedigree structure of families A and segregation of GBA: c.115+1G>A and DNAJB6; p.T193A (c.A577G) are shown. Affected individuals are shown as filled symbols and the arrow points to the index patient. The affection status of the family members of generation I and II could not be ascertained. The affection status of deceased individuals (diagonal lines) was reported by immediate family members and was confirmed by available medical records. Symbols with black lines (IV:6, V:4, and V:5) indicate asymptomatic GBA variant carriers. Asterisks mark the individuals for whom whole-genome sequencing and rare variant analysis was performed. B Representative Sanger sequence chromatograms showing GBA1: c.115+1G>A and DNAJB6; p.T193A (c.A577G) variant positions in subjects IV:4 and IV:7 of family A. Arrowhead points to heterozygous substitutions. C The pedigree structure of families B and segregation of GBA; p.L444P (c.1448T>C) and PSAP; p.N157S (c.A470G) variants is shown. Affected individuals are shown as filled symbols and the arrow points to the index patients. The symbol with a white circle indicates an individual with PD but without GBA variant (phenocopy PD patient, III:4). The PSAP; p.N157S (c.A470G) variant was found in all PD, and in addition in one healthy family member (IV:7). Asterisks mark the individuals for whom whole-genome sequencing and rare variant analysis was performed. D Representative sanger sequence chromatograms showing GBA; p.L444P (c.1448T>C) and PSAP; p.N157S (c.A470G) variant positions in subjects III:1 and III:2 of family B. Arrowhead points to heterozygous substitutions.

Fig. 2. Functional impact of the PD cosegregating DNAJB6 variant c.A577G:p.T193A identified in family A.

A Schematic diagram of the human DNAJB6 domain structure is shown. The approximate position of p.T193A is indicated. Alignment of a region of the human DNAJB6 protein sequence containing an S/T-rich motif with the corresponding polypeptide sequence from various species is shown below. Numbers indicate amino acid positions. Blue shading indicates the conservation status of the corresponding residues. Arrow points to the variant position, the S/T motif (SxSTST) is also highlighted. B The model structure of the DNAJB6 homodimer with the variant position (T193, red spheres) is shown. C Representative images showing the cellular distribution of alpha-synuclein-dsRed fusion protein in an alpha-synuclein aggregation reporter HEK293 cell line lacking endogenous DNAJB6 expression. Cells were transfected with plasmids expressing EGFP alone or as a fusion protein with wild type or the T193A variant containing DNAJB6. Focal and diffuse cytoplasmic distributions of alpha-synuclein-dsRed can be observed. D Quantification of focal cytoplasmic distribution of aggregated alpha-synuclein-dsRed (n = 3 transfections). Statistical analysis was performed by ANOVA. ***P < 0.001, *P < 0.05. Graphs represent mean and standard deviation.

Fig. 3. Functional impact of the PD cosegregating PSAP variant c.A470G:p.N157S identified in family B.

A The domain structure of human the PSAP protein is shown. The location of the p.N157S variant (red) and the predicted Cathepsin D site (L177–L179) in the intersaposin A–B region is indicated (green). The alignment of human PSAP with various species is shown. Numbers indicate the position of amino acid residues. Blue shading indicates the conservation status of the corresponding residues; arrow points to the variant position. The predicted Cathepsin D site, L177–L179, is indicated (underlined in green). B The structure of the human PSAP protein is shown. The variant positions (N157) in the intersaposin A–B region is marked in red and the predicted Cathepsin D site (L177–L179) is displayed in pink. C Representative images showing the distribution of LAMP1-mTurquoise2 and PSAP-EGFP fusion proteins in HEK293T cells. DAPI was used as a nuclear stain (blue). Arrowheads in the bottom right image point to the EGFP-PSAP puncta in the perinuclear region not overlapping with LAMP1-mTurquoise2. D Quantification of green (PSAP-EGFP fusion protein) signal intensity overlapping with turquoise signal (mTurquoise2-LAMP1 marking the perinuclear lysosomal compartment) is shown. Graphs represent mean and standard deviation ***P = 0.001. Also shown is the immunoblot image of wild type and N157S variant containing PSAP proteins from HEK293 cells (top panel). HEK293 cells were transfected with an equal amount of plasmids expressing the wild type or N157S variant PSAP proteins as Flag-tag conjugate. Full-length blots are presented in Supplementary Fig. 3C. All blots are derived from the same experiment and gels/blots were processed in parallel. E Representative images showing LAMP1-positive intracellular inclusions decorated with alpha-synuclein-EGFP. Cells were transfected with three plasmids: a plasmid-expressing EGFP-tagged aggregation-prone alpha-synuclein protein (see “Methods”), a plasmid-expressing mTurquoise2-LAMP1 expression protein as a lysosomal marker, and a plasmid-expressing wild type or p.PN157S variant containing PSAP. Arrowheads in the merged panel point to the EGFP-PSAP puncta in the perinuclear region overlapping with LAMP1-mTurquoise2 as well as alpha-syuclein-EGFP puncta indicating lysosomal aggregation. F The quantification of lysosomal alpha-synuclein-EGFP aggregates in transfected cells is shown (n = 3 transfections). Statistical analysis was performed by ANOVA. Graphs represent mean and standard deviation, **P < 0.01.