👤 Philippe Pals

To broaden our understanding of bradyarrhythmias and conduction disease, we performed common variant genome-wide association analyses in up to 1.3 million individuals and rare variant burden testing in 460,000 individuals for sinus node dysfunction (SND), distal conduction disease (DCD) and pacemaker (PM) implantation. We identified 13, 31 and 21 common variant loci for SND, DCD and PM, respectively. Four well-known loci (SCN5A/SCN10A, CCDC141, TBX20 and CAMK2D) were shared for SND and DCD, while others were more specific for SND or DCD. SND and DCD showed a moderate genetic correlation (r Show less

The survival and proliferation of multiple myeloma (MM) cells in the bone marrow (BM) critically depend on interaction with stromal cells expressing the chemokine CXCL12. CXCL12 regulates the homing to the BM niche by mediating the transendothelial migration and adhesion/retention of the MM cells. The gamma isoform of CXCL12 (CXCL12γ) has been reported to be highly expressed in mouse BM and to show enhanced biological activity compared to the 'common' CXCL12α isoform, mediated by its unique extended C-terminal domain, which binds heparan sulfate proteoglycans (HSPGs) with an extraordinary high affinity. Here, we investigated the expression of CXCL12γ in human BM and studied its functional role in the interaction of MM cells with BM stromal cells (BMSCs). We assessed CXCL12γ mRNA and protein expression by human BMSCs using qPCR, flow cytometry, and immunohistochemistry. CRISPR-Cas9 was employed to delete CXCL12γ and the heparan sulfate (HS) co-polymerase EXT1 in BMSCs. To study the functional roles of BMSC-derived CXCL12γ and HSPGs in the interaction of MM cells with BMSCs cells, MM cell lines and primary MM cells were co-cultured with BMSCs. We observed that CXCL12γ is expressed in situ by reticular stromal cells in both normal and MM BM, as well as by primary BMSC isolates and BMSC lines. Importantly, upon secretion, CXCL12γ, unlike the CXCL12α isoform, was retained on the surface of BMSCs. This membrane retention of CXCL12γ is HSPG mediated, since it was completely annulated by CRISPR-Cas9-mediated deletion of the HS co-polymerase EXT1. CXCL12γ expressed by BMSCs and membrane-retained by HSPGs supported robust adhesion of MM cells to the BMSCs. Specific genetic deletion of either CXCL12γ or EXT1 significantly attenuated the ability of BMSCs to support MM cell adhesion and, in addition, impaired their capacity to protect MM cells from bortezomib-induced cell death. We show that CXCL12γ is expressed by human BMSCs and upon secretion is retained on their cell surface by HSPGs. The membrane-bound CXCL12γ controls adhesion of MM cells to the stromal niche and mediates drug resistance. These findings designate CXCL12γ and associated HSPGs as partners in mediating MM-niche interaction and as potential therapeutic targets in MM. Show less

Dementia with Lewy bodies (DLB) and Parkinson's disease (PD) are clinically, pathologically and etiologically disorders embedded in the Lewy body disease (LBD) continuum, characterized by neuronal α-synuclein pathology. Rare homozygous and compound heterozygous premature termination codon (PTC) mutations in the Vacuolar Protein Sorting 13 homolog C gene (VPS13C) are associated with early-onset recessive PD. We observed in two siblings with early-onset age (< 45) and autopsy confirmed DLB, compound heterozygous missense mutations in VPS13C, p.Trp395Cys and p.Ala444Pro, inherited from their healthy parents in a recessive manner. In lymphoblast cells of the index patient, the missense mutations reduced VPS13C expression by 90% (p = 0.0002). Subsequent, we performed targeted resequencing of VPS13C in 844 LBD patients and 664 control persons. Using the optimized sequence kernel association test, we obtained a significant association (p = 0.0233) of rare VPS13C genetic variants (minor allele frequency ≤ 1%) with LBD. Among the LBD patients, we identified one patient with homozygous missense mutations and three with compound heterozygous missense mutations in trans position, indicative for recessive inheritance. In four patients with compound heterozygous mutations, we were unable to determine trans position. The frequency of LBD patient carriers of proven recessive compound heterozygous missense mutations is 0.59% (5/844). In autopsy brain tissue of two unrelated LBD patients, the recessive compound heterozygous missense mutations reduced VPS13C expression. Overexpressing of wild type or mutant VPS13C in HeLa or SH-SY5Y cells, demonstrated that the mutations p.Trp395Cys or p.Ala444Pro, abolish the endosomal/lysosomal localization of VPS13C. Overall, our data indicate that rare missense mutations in VPS13C are associated with LBD and recessive compound heterozygous missense mutations might have variable effects on the expression and functioning of VPS13C. We conclude that comparable to the recessive inherited PTC mutations in VPS13C, combinations of rare recessive compound heterozygous missense mutations reduce VPS13C expression and contribute to increased risk of LBD. Show less

Multiple myeloma (MM) is characterized by the expansion of malignant plasma cells in the bone marrow (BM). Most MMs display aberrant Wnt/β-catenin signaling, which drives proliferation; however, they lack oncogenic Wnt pathway mutations, suggesting activation by autocrine Wnt ligands and/or paracrine Wnts from the BM microenvironment. Expression of the heparan sulfate (HS) proteoglycan syndecan-1 is a hallmark of MM. Syndecan-1 is a critical player in the complex reciprocal interaction between MM cells and their BM niche, mediating growth factor/cytokine binding and signaling by its HS chains. Here, by means of CRISPR/Cas9-mediated knockout and doxycycline-inducible short hairpin RNA-mediated knockdown of EXT1, a critical enzyme for HS polymerization, we demonstrate that the HS chains decorating syndecan-1 mediate aberrant Wnt pathway activation in MM. HS-deficient MM cells exhibited strongly decreased autocrine Wnt/β-catenin pathway activity and reduced Wnt pathway-dependent proliferation. In addition, we demonstrate that Wnts bind to the HS side chains of syndecan-1 and that this binding contributes to paracrine Wnt pathway activation through the Wnt receptor Frizzled (Fzd). Furthermore, in an HS-dependent fashion, syndecan-1 also binds osteoblast-produced R-spondin, which represses Fzd degradation by activation of LGR4, an R-spondin receptor aberrantly expressed on MM cells. Costimulation with R-spondin and its binding to HS chains decorating syndecan-1 are indispensable for optimal stimulation of Wnt signaling in MM. Taken together, our results identify syndecan-1 as a crucial component of the Wnt signalosome in MM cells, binding Wnts and R-spondins to promote aberrant Wnt/β-catenin signaling and cell growth, and suggest HS and its biosynthetic enzymes as potential targets in the treatment of MM. Show less

Expression of the heparan sulfate proteoglycan syndecan-1 is a hallmark of both normal and multiple myeloma (MM) plasma cells. Syndecan-1 could affect plasma cell fate by strengthening integrin-mediated adhesion via its core protein and/or by accommodating and presenting soluble factors via its HS side chains. Here, we show that inducible RNAi-mediated knockdown of syndecan-1 in human MM cells leads to reduced growth rates and a strong increase of apoptosis. Importantly, knockdown of EXT1, a copolymerase critical for HS chain biosynthesis, had similar effects. Using an innovative myeloma xenotransplantation model in Rag-2(-/-)gamma(c)(-/-) mice, we demonstrate that induction of EXT1 knockdown in vivo dramatically suppresses the growth of bone marrow localized myeloma. Our findings provide direct evidence that the HS chains of syndecan-1 are crucial for the growth and survival of MM cells within the bone marrow environment, and indicate the HS biosynthesis machinery as a potential treatment target in MM. Show less