Characterisation of molecular mechanisms for PLCγ2 disease-linked variants

May combine two claims.

PLCγ1 shares 52% sequence identity with PLCγ2, and their overall domain organisation is conserved; therefore, homology models are plausible.

“PLCγ1 shares 52% sequence identity with PLCγ2, and their overall domain organisation is conserved; therefore, homology models are plausible.”

• cites Hajicek et al., 2019 sourcePartly supported

  The Results section, “Structure of full-length PLC-γ1” (page unavailable), describes domains shared by the PLC-γ isozymes. The Materials and methods section, “Homology modeling” (page unavailable), reports building a PLC-γ2 model from a PLC-γ1 template. Our copy does not substantiate the specific figure of 52% sequence identity. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites Le Huray et al., 2022 sourcePartly supported

  The Discussion (page not provided) says PLCγ1 and PLCγ2 share a distinctive regulatory array, supporting conserved domain organisation. Our copy does not give 52% sequence identity between them or explicitly assess the plausibility of homology models; the 98% identity reported in Materials and Methods concerns rat and human PLCγ1. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites Liu et al., 2020 sourcePartly supported

  The Results section on PLCγ1 architecture (page unavailable) describes PLCγ-family domains and reports using conserved PLC-core structures to make a homology model of PLCγ1. It does not give 52% sequence identity between PLCγ1 and PLCγ2 or explicitly establish that their overall domain organisation is conserved. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

May combine two claims.

In B-cells, phosphorylation of Tyr759 in the regulatory array correlates with PLCγ2 activation and is largely mediated by Btk tyrosine kinase.

“For example, in B-cells, phosphorylation of the Tyr759 in the regulatory array (cSH2-SH3 linker) correlates with the PLCγ2 activation and is largely mediated by Btk tyrosine kinase.”

• cites Kim et al., 2004 sourceSupported

May combine two claims.

The rare PLCγ2 P522R variant is associated with protection against Alzheimer's disease and with a supporting role in healthy ageing.

“Importantly, it has also been found that a rare variant of PLCγ2 (P522R) has a protective role in Alzheimer's disease (AD).”

• Also stated in: § Introduction · plain

  “Notably, genetic evidence related to neurodegeneration, including genome-wide association studies (GWASs), linked the rare PLCγ2 P522R (rs72824905) variant with protection against Alzheimer's disease (AD) and with a supporting role in healthy ageing (de Rojas and al., 2020;Sims et al., 2017;van der Lee et al., 2019).”

• cites de Rojas and al., 2020Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites Sims et al., 2017 sourceSupported
• cites van der Lee et al., 2019 sourceSupported

Classified as probably not a substantial claim.

Several studies have suggested that the protective role of the P522R PLCγ2 variant is linked to functional effects on microglia.

“Several studies have suggested that the protective role of the P522R PLCγ2 variant is linked to functional effects on microglia (Maguire et al., 2021;Takalo et al., 2020;Tsai et al., 2023).”

• cites Maguire et al., 2021 sourceSupported
• cites Takalo et al., 2020 sourceSupported
• cites Tsai et al., 2023 sourceSupported

Classified as probably not a substantial claim. May combine two claims.

Many mutations discovered in PLCγ2 have been linked to the development of complex immune disorders as well as resistance to ibrutinib treatment in chronic lymphocytic leukaemia.

“Furthermore, many mutations discovered in PLCγ2 have been linked to the development of complex immune disorders as well as resistance to ibrutinib treatment in chronic lymphocytic leukaemia.”

• cites Baysac et al., 2024 sourceSupported
• cites Burger et al., 2016 sourcePartly supported

  The Results report four PLCG2 mutations emerging during ibrutinib treatment in a patient with CLL, supporting the resistance link. The References cite a study of one PLCG2 mutation causing an autoinflammatory disease with immunodeficiency, but this paper does not establish that many PLCG2 mutations are linked to complex immune disorders. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites Woyach et al., 2014 sourcePartly supported

  The Results and Discussion link three PLCγ2 mutations to acquired ibrutinib resistance in patients with chronic lymphocytic leukaemia. The Results also describe one of them, S707Y, as previously reported in an inherited inflammatory disease, but the paper does not link many PLCγ2 mutations to complex immune disorders. Page numbers are unavailable in our copy. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

A link between PLCγ enzymes, cytoskeletal changes and cell motility has been previously documented.

“A link between PLCγ enzymes, cytoskeletal changes and cell motility has been previously documented [for example (Obst et al., 2021;Weddell et al., 2018)] and in the context of microglia could be important for other functions that are relevant for AD, for example, for phagocytosis and removal of amyloid fibres.”

• cites Obst et al., 2021 sourceSupported
• cites Weddell et al., 2018 sourcePartly supported

  The Discussion reports that VEGFR1-induced PLCγ activation is required for macrophage migration, supporting the PLCγ–cell motility link. It does not directly document cytoskeletal changes as part of that link; cytoskeletal reorganization appears in the title of a work listed in the References, not as a finding reported for PLCγ in this paper. The later suggestion about microglial phagocytosis is the citing authors’ own framing. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

May combine two claims.

Initial cellular studies of individual mutations, as well as their comparison in a standard cell-based assay, have shown a clear increase in basal PLC activity of these variants and further enhancement after stimulation.

“Initial cellular studies of individual mutations, as well as their comparison in a standard cell-based assay, have shown a clear increase in basal PLC activity of these variants and further enhancement after stimulation [(Liu et al., 2020); Fig S1].”

• cites Liu et al., 2020 sourcePartly supported

  The Results section (‘Distribution and functional impact of mutations in PLCγ enzymes’; page unavailable) reports increased activity for the majority of variants tested in a standard cellular assay, and says most were further stimulated by EGF. It also identifies several deletions that lost responsiveness to EGF, so further enhancement does not apply to every tested variant. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

iPSC-derived microglia expressing another highly active APLAID variant display diminished functionality for key microglial processes.

“This possibility is further strengthened by our recent findings that the iPSC-derived microglia expressing another highly active APLAID variant display diminished functionality for key microglial processes (Bull et al., 2024).”

• cites Bull et al., 2024 sourceSupported

May combine two claims.

The DSA construct and M1141K PLCγ2 variant were clearly able to interact with liposomes, whereas interactions for WT and P522R were not detectable.

“In this setup, the DSA construct, with the membrane binding surfaces exposed, and the M1141K PLCγ2 variant were clearly able to interact with the liposomes.In contrast, for the autoinhibited PLCγ2 WT and the P522R variant such interactions were not detectable (Fig. 4A).”

May combine two claims.

Residue Met1141 is in the C2 domain at the C2/cSH2 interface and is mutated to Lys in both APLAID and CLL.

“For example, residue Met1141 is in the C2 domain at the C2/cSH2 interface and is mutated to Lys in both pathologies.”

May combine two claims.

The heparin-Sepharose chromatography elution profile of M1141K differs from those of WT and P522R, which are comparable.

“Consistent with this, a heparin-Sepharose chromatography elution profile of the M1141K PLCγ2 variant differs from those of the WT and the P522R PLCγ2 variants that are comparable (Fig. S2A).”

May combine two claims.

Several interdomain linkers are not visible in any crystal structures and their predicted positions vary between the two PLCγ2 models.

“Several interdomain linkers are not visible in any of the crystal structures and their predicted positions vary between the two PLCγ2 models (Fig. 1B).”

May combine two claims.

In in vitro PLC activity assays, PLCγ2 M1141K had significantly higher activity than WT but not as high as the PLC-core DSA construct.

“In both assays, PLC activity of the M1141K variant was significantly higher compared to the PLCγ2 WT but not as high as the activity of the PLC-core where autoinhibition has been completely eliminated by the deletion of the γSA (DSA construct) (Fig. 3A and B).”

May combine two claims.

In serum-starved, unstimulated cells, PLC activity of P522R was comparable to WT, contrasting a higher activity of M1141K.

“Consistent with the studies in vitro (Fig. 3), in serum-starved, unstimulated cells, the PLC activity of the P522R PLCγ2 variant was comparable to the WT, contrasting a higher activity of M1141K PLCγ2 variant (Fig. S4).”

May combine two claims.

Expression levels of P522R and WT PLCγ2 were similar and comparable to endogenous PLCγ2 in microglia.

“The assessment of expression levels for different proteins revealed similar expression of the P522R and WT PLCγ2, comparable to expression levels of endogenous PLCγ2 in microglia (Fig. 5A).”

Probably not a substantial claim: may not pass S3 (load-bearing).

The corresponding sPH/nSH2 linker is not visible in crystal structures of PLCγ1 (PDB IDs: 6PBC and 7Z3J).

“The sPH/nSH2 linker is not visible in this PLCγ2 crystal structure; similarly, the corresponding linker is not visible in crystal structures of PLCγ1 (PDB IDs: 6PBC and 7Z3J).”

May combine two claims.

The structural features of the autoinhibitory interfaces are conserved between the two PLCγ2 models and consistent with the data describing PLCγ2 crystal structures.

“The structural features of these regions are conserved between the two PLCγ2 models (shown in Fig.1B) and consistent with the data describing PLCγ2 crystal structures.”

May combine two claims.

Several properties of P522R characterised in vitro are comparable with WT, including low basal PLC activity, absence of membrane interactions and good protein stability.

“Several properties of this variant characterised in vitro are comparable with the PLCγ2 WT, including a low basal PLC activity, absence of membrane interactions and good protein stability (Fig. 3, Fig. 4 and Fig. S2).”

May combine two claims.

The PLCγ2 P522R substitution uniquely occurs within the flexible sPH/nSH2 linker in the regulatory γSA array, a linker without any other defined functional role.

“The PLCγ2 P522R substitution uniquely occurs within the flexible sPH/nSH2 linker in the regulatory γSA array, the linker without any other defined functional role (Fig. 1).”

Probably not a substantial claim: may not pass S3 (load-bearing).

The sPH/nSH2 linker is not visible in the PLCγ2 crystal structure (PDB ID: 8T7C).

“The sPH/nSH2 linker is not visible in this PLCγ2 crystal structure; similarly, the corresponding linker is not visible in crystal structures of PLCγ1 (PDB IDs: 6PBC and 7Z3J).”

May combine two claims.

The M1141K mutation causes substantial conformational changes facilitating membrane interactions; such large conformational changes are not taking place in P522R.

“These data suggest that the M1141K mutation in PLCγ2 causes substantial conformational changes, facilitating membrane interactions; such large conformational changes are not taking place in the P522R PLCγ2 protein.”

May combine two claims.

In contrast to M1141K, the functional impact of the P522R substitution completely depends on further interactions with upstream kinases and other regulatory proteins in a relevant cellular context, where changes in P522R could facilitate processes such as phosphorylation and protein-protein interactions.

“It is also likely that in contrast to the PLCγ2 M1141K, the functional impact of the P522R substitution completely depends on further interactions with upstream kinases and other regulatory proteins in a relevant cellular context, where changes in the PLCγ2 P522R variant could facilitate processes such as phosphorylation and protein-protein interactions.”

May combine two claims.

The PLCγ2 P522R variant has subtle, localised structural changes that do not directly affect PLC activity by compromising autoinhibition, as determined for M1141K.

“These data, combined with the structural insights, suggest that the PLCγ2 P522R variant has subtle, localised structural changes that do not directly affect the PLC activity by compromising autoinhibition, as determined for PLCγ2 M1141K.”

• Also stated in: § Discussion · hedged

  “It is unlikely that in the PLCγ2 P522R variant this linker affects autoinhibitory interfaces.”

May combine two claims.

Although P522R substitution doesn't result in a large conformational change, subtle changes in the regulatory γSA facilitate Btk phosphorylation of the functionally important Tyr759.

“Therefore, although the P522R substitution doesn't result in a large conformational change, as observed for the M1141K PLCγ2, subtle changes in the regulatory γSA facilitate Btk phosphorylation of the functionally important Tyr759.”

May combine two claims.

These morphological differences suggest changes in actin cytoskeleton, supported by Phalloidin staining.

“These morphological differences suggest changes in actin cytoskeleton; this is further supported by Phalloidin staining (Fig. S5).”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

Other properties of the γSA are affected by the P522R substitution (e.g. regulatory protein-protein interactions) that can contribute to the AD protective functions of this variant.

“It is also likely that other properties of the γSA are affected by the P522R substitution (e.g. regulatory protein-protein interactions) that can contribute to the AD protective functions of this variant.”

Classified as probably not a substantial claim. May combine two claims.

The presence of the mutation of interest was confirmed with sequencing around sites of the mutation followed by full sequencing of the complete ORF to confirm sequence fidelity.

“The presence of the mutation of interest was confirmed with sequencing around sites of the mutation followed by full sequencing of the complete ORF to confirm sequence fidelity.”

Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

The phosphorylation time course data are representative for two independent experiments.

“The data are representative for two independent experiments.”

The quote only partly states this claim.

The differences in PLC activity between WT PLCγ2 and the P522R or M1141K variant are representative for 3 independent experiments.

“The differences are representative for 3 independent experiments.”

May combine two claims.

A small change in the γSA of the PLCγ2 P522R variant results in a higher rate of phosphorylation after stimulation and causes a mild increase in PLC activity.

“It is suggested that a small change in the γSA results in higher rate of phosphorylation after stimulation and causes a mild increase in PLC activity.”

May combine two claims.

The P522R PLCγ2 variant, in the context of growing microglia, has an increased PLC activity that is compatible with cell viability at its physiologically relevant expression levels.

“These data show that the P522R PLCγ2 variant, in the context of growing microglia, has an increased PLC activity that is compatible with cell viability at its physiologically relevant expression levels.”

May combine two claims.

Our data show clear differences, as well as some similarities, between the two proteins with respect to changes in PLC activity, membrane interactions, protein stability and the rate of phosphorylation.

“Our data show clear differences, as well as some similarities, between the two proteins with respect to changes in PLC activity, membrane interactions, protein stability and the rate of phosphorylation.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The protein samples from the same WES experiments allowed direct comparison, while the same negative and positive controls were prepared alongside with the different sample sets to allow cross comparison.

“The protein samples from the same WES experiments allowed direct comparison, while the same negative and positive controls were prepared alongside with the different sample sets to allow cross comparison.”

May combine two claims.

Our data show that the enhanced PLC activity of PLCγ2 P522R variant in BV2 cells, expressed at physiologically relevant levels, is well-tolerated in these cells, also showing changes in morphology towards higher motility.

“Furthermore, our data show that the enhanced PLC activity of PLCγ2 P522R variant in BV2 cells, expressed at physiologically relevant levels, is well-tolerated in these cells, also showing changes in morphology towards higher motility.”

May combine two claims.

A small structural change in the γSA of the PLCγ2 P522R variant may affect the rate of phosphorylation and the nature and dynamics of regulatory protein-protein interactions mediated by this region.

“A small structural change in the γSA of this variant may not only affect rate of phosphorylation but also nature and dynamics of regulatory protein-protein interactions mediated by this region (Fig. 6).”

Classified as probably not a substantial claim. May combine two claims.

All PLCγ2 mutations linked to APLAID affect residues that are close to the autoinhibitory interfaces; consequently, it has been considered that they could have a direct impact on PLC activity by overcoming or weakening the autoinhibition.

“All PLCγ2 mutations linked to APLAID (including S707Y/P, L848P, M1141K, L845-L484del, A708P, R742C/H and L845S), many of which also occur in drug-resistant CLL, affect residues that are in the PLCγ2 3D-structure close to the autoinhibitory interfaces; consequently, it has been considered that they could have a direct impact on PLC activity by overcoming or weakening the autoinhibition.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The altered regulatory protein-protein interactions of the PLCγ2 P522R variant could influence signalling connectivity to favour links to receptors promoting beneficial microglia functions (e.g. TREM2) instead of receptors involved in harmful inflammatory responses.

“In turn, this could influence signalling connectivity to favour links to receptors promoting beneficial microglia functions (e.g.TREM2) instead of receptors involved in harmful inflammatory responses.”

May combine two claims.

Based on these findings, supported by structural insights and further cell-based analyses, we propose mechanistic models that distinguish these PLCγ2 proteins and determine functional impact leading to different roles in two pathologies.

“Based on these findings, supported by the structural insights and further cell-based analyses, we propose mechanistic models that distinguish these PLCγ2 proteins and determine functional impact leading to different roles in two pathologies (Fig. 6).”

May combine two claims.

By reducing autoinhibition, the M1141K mutation affects an overall structure and increases basal PLC activity, which is further enhanced following stimulation, leading to aberrant signalling and APLAID.

“By reducing autoinhibition, this mutation affects an overall structure and increases basal PLC activity, that is further enhanced following stimulation, leading to aberrant signalling and APLAID.”