Ritscher-Schinzel syndrome can be characterized as an endosomal recyclinopathy

It may state two separate claims as one.

LRP4 binds SNX17, and this binding is lost in the LRP4(Y1769A) mutant.

“Expression alongside mCherry-SNX17 in HEK293T cells followed by mCherry-trap immuno-isolation revealed LRP4 binding to SNX17 that was lost in a LRP4(Y1769A) mutant (Fig. 4A and fig.S5A).”

It may state two separate claims as one.

Cell surface abundance of FGFR2 is decreased in Snx17- and Vps35l-suppressed MC3T3E1 cells, resulting in attenuated FGF-mediated signaling through the MAPK pathway.

“The cell surface abundance of this receptor was decreased in both Snx17-and Vps35l-suppressed MC3T3E1 cells that resulted in attenuated FGF-mediated signaling through the MAPK pathway (Fig. 4E and F).”

It may state two separate claims as one.

Knock-out of VPS35L, CCDC22, and WASHC5 destabilizes the corresponding Retriever, CCC and/or WASH complexes and causes loss of endosomal localization of COMMD1.

“Western blotting suggested knock-out of VPS35L, CCDC22, and WASHC5 destabilized the corresponding Retriever, CCC and/or WASH complexes and the loss of endosomal localization of the CCC complex subunit COMMD1 was observed in immunofluorescence (fig.S2A to C).”

It may state two separate claims as one.

LRP8 and VLDLR associate with SNX17, and this association is reduced in LRP8(Y866A) and VLDLR(Y837A) mutants.

“Immunoprecipitation of wild-type human-LRP8 and VLDLR revealed association with mCherry-SNX17, that was reduced in LRP8(Y866A) and VLDLR(Y837A) mutants (Fig. 5A).”

It may state two separate claims as one.

In H4 neuroglioma cells knocked out for SNX17, VPS35L, CCDC22, or WASHC5, cell surface amount of LRP4 is reduced and rescued by re-expression of wild-type proteins.

“In human H4 neuroglioma cells knocked-out for the expression of SNX17, VPS35L, CCDC22, or WASHC5, the cell surface amount of LRP4 was clearly reduced and rescued upon re-expression of the wild-type proteins (Fig. 4B).”

It may state two separate claims as one.

Patients harbouring the COMMD4-L41R mutation were born to consanguineous parents and all three patients passed away in early infancy.

“patients harbouring the COMMD4-L41R mutation were born to consanguineous parents and all three patients passed away in early infancy.”

It may state two separate claims as one.

Cell surface abundance of endogenous LRP2 is decreased in HEK293T knock-out cells targeting SNX17, VPS35L, CCDC22, or WASHC5, and is recovered by re-expression of wild-type proteins.

“Western blotting revealed that the cell surface abundance of endogenous LRP2 was clearly decreased in knock-out HEK293T cell lines targeting SNX17 as well as VPS35L, CCDC22, and WASHC5(4) compared to parental cells, which was recovered by re-expression of wild-type proteins (Fig. 3D).”

• cites [4]Not matched

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

It may state two separate claims as one.

Exogenous overexpression of wild-type DPYSL5, DPYSL5-E41K, or DPYSL5-G47R does not alter expression of SNX17-Retriever/CCC/WASH pathway proteins or cargo proteins, nor does it affect endogenous COMMD1 localization to endosomes.

“Exogenous overexpression of wild-type GFP-DPYSL5, and GFP-DPYSL5-E41K or GFP-DPYSL5-G47R in HEK293T cells did not alter the expression of proteins in the SNX17-Retriever/CCC/WASH pathway or their cargo proteins, nor did it affect the localization of endogenous COMMD1 to endosomes, suggesting that these mutants do not exhibit a dominant negative effect on the recycling pathway (fig.S3H and I).”

It may state two separate claims as one.

FGFR2 associates with SNX17, and this association is reduced in FGFR2-(Y656A) and FGFR2-(Y733A) mutants.

“Immunoprecipitation of the GFP-tag transmembrane and cytoplasmic facing tail of Human-FGFR2 co-expressed with FLAG-tagged SNX17 identified an association with wild-type FGFR2 that was clearly reduced in FGFR2-(Y656A) and -(Y733A) mutants -FGFR2 contains INNIDY 656 , CTNELY 733 and TTNEEY 769 motifs (Fig. 4D).”

The quote only partly states this claim.

RNA sequencing of chondrocytes from E16.5 Vps35l-cKO Prx1 tibiae reveals downregulated genes enriched for 'regulation of canonical Wnt signaling pathway' and 'MAPK cascade'.

“mRNA extraction from chondrocytes in E16.5 mouse tibiae and RNA sequencing revealed downregulated genes in Vps35l-cKO Prx1 (Fold change < -0.585, q < 0.1) with gene ontology (GO) enrichment for gene sets”

It may state two separate claims as one.

Loss of DPYSL5 expression has no detectable effect on cell surface amounts of ITGB1 and LRP1, nor on steady-state expression of Retriever, CCC, or WASH complexes.

“In stark contrast to the loss of cell surface ITGB1 and LRP1 observed in VPS35L, VPS26C and WASHC4 knock-out H4 cells, the loss of DPYSL5 expression had no detectable effect on surface amounts of these cargo, nor did it alter the steady-state expression of Retriever, the CCC complex or the WASH complex assessed by western blotting (fig.S3G).”

It may state two separate claims as one.

DPYSL5-E41K displays disrupted interaction with TPM3, whereas DPYSL5-G47R does not.

“Although the proteins retain their ability to form DPYSL5 homo-and hetero-tetramers, both amino-and carboxy-terminal GFP-tagged DPYSL5-E41K (a mutation identified in eight patients with overlapping phenotypes) displayed a disrupted interaction with tropomyosin alpha-3 (TPM3), which associates with muscle and non-muscle actin.This loss in TPM3 binding was not observed with the DPYSL5-G47R mutant”

It may state two separate claims as one.

Vps35l-cKO Prx1 mice show smaller body weight and shorter tibia length.

“Consistent with patients with RSS displaying growth impairment and short stature, Vps35l-cKO Prx1 mice showed a smaller body weight and shorter tibia length (Fig. 4G and H).”

The quote only partly states this claim.

In ex vivo tibiae culture, gene sets related to the MAPK cascade are the top hit among upregulated genes in littermate control mice but not in Vps35l-cKO Prx1 mice.

“Gene enrichment analysis of upregulated genes (Fold change > 0.585, q < 0.05) revealed that gene sets related to the”

It may state two separate claims as one.

Vps35l-cKO Nestin mice show reduced LRP2 expression especially in the S1 segment of proximal tubules compared to littermate controls, with no structural abnormality on PAS staining or ultrastructural analysis.

“Immunohistochemistry of renal sections showed reduced LRP2 expression especially in the S1 segment of proximal tubules of Vps35l-cKO Nestin mice compared to littermate controls, while no structural abnormality were observed based on PAS staining and ultrastructural analysis (Fig. 3G, fig.S4F to H).”

It may state two separate claims as one.

CCDC93-S631A retains CCC complex assembly but displays enhanced degradative turnover compared to wild-type CCDC93.

“Finally, Serine-631 of CCDC93 is conserved across species and although CCDC93-S631A retained CCC complex assembly as assessed by GFP-immunoprecipitation, it also displayed an enhanced degradative turnover compared to wild-type CCDC93 in CHX chase assay (Fig. 1G, fig.S1F and G).”

It may state two separate claims as one.

Human SLITRK2, SLITRK3, and SLITRK5 each contain an NPxY motif required for mCherry-SNX17 association.

“Human SLITRK2, SLITRK3, and SLITRK5, each contain an NPxY motif, CQNPIY 701 , CNNPIY 756 , and CKNPIY 765 respectively, that are required for mCherry-SNX17 association (Fig. 5G).”

It may state two separate claims as one.

Thickness of brain cortex and migration and number of neurons appear not significantly affected in Vps35l-cKO Nestin mice lacking profound hydrocephaly.

“Thickness of the brain cortex, and the migration and number of neurons appeared not to be significantly affected in the brains of mice lacking profound hydrocephaly (Fig. 6D and fig.S7A and B).”

It may state two separate claims as one.

HEK293T cells carrying a COMMD4-L41R knock-in exhibit decreased steady-state abundance of COMMD4 compared to parental cells.

“Indeed, western blot analysis showed that HEK293T cells carrying a COMMD4-L41R knock-in exhibited a decrease in the steady-state abundance of COMMD4 compared to the parental cells (Fig. 1F).”

It may state two separate claims as one.

All Vps35l-cKO Nestin mice show growth impairment, behavioral problems, and high mortality rates compared to littermate controls.

“All mice showed growth impairment, behavioral problems, and high mortality rates compared to their littermate controls (Fig. 6E andF, and movies S1 to S4), phenotypes consistent with our Vps35l-cKO Nestin mouse model recapitulating many of the neurological phenotypes observed in patients with RSS.”

It may state two separate claims as one.

In primary neurons, Snx17 or Vps35l suppression reduces Reelin uptake and Reelin-mediated phosphorylation of DAB1.

“In primary neurons Snx17 or Vps35l suppression reduced Reelin uptake (Fig. 5C), and reduced Reelin-mediated phosphorylation of DAB1, a downstream phosphorylation target of Reelin signalling (Fig. 5D).”

It may state two separate claims as one.

Knock-out of COMMD4, COMMD9, and CCDC93 reduces cell surface amounts of ITGB1 and LRP1, destabilizes the Commander complex, and alters COMMD1 localization.

“Also, in the knock-out of COMMD4, COMMD9, and CCDC93, western blotting revealed reduced cell surface amount of ITGB1 and LRP1, destabilization of the Commander complex, and altered localization of COMMD1n (Fig. 2A and B).”

It may state two separate claims as one.

COMMD4-L41R knock-in cells display reduced cell surface abundance of ITGB1 and LRP1 compared to wild-type cells, suggesting a defect in Commander-mediated cargo recycling.

“Finally, the COMMD4-L41R knock-in cells displayed reduced cell surface abundance of ITGB1 and LRP1 compared to wild-type cell in western blotting suggesting a defect in Commander-mediated cargo recycling (fig.S2F).”

It may state two separate claims as one.

In the CCDC93(Y457*/S631A) patient cell line, CCDC22 and VPS35L amounts are significantly decreased (P < 0.001), whereas SNX27, VPS35, and WASHC5 expression remains unchanged.

“The amount of CCDC22 (P < 0.001) and VPS35L (P < 0.001) were also significantly decreased in the patient cell line whereas the expression of SNX27, VPS35, and WASHC5, endosomal proteins that are not part of Commander complex, remained unchanged (Fig. 1H and fig.S1H).”

It may state two separate claims as one.

Vps35l-cKO Nestin mice show loss of VPS35L expression and reduced LRP1 expression in brain homogenates.

“Loss of VPS35L expression and reduced LRP1 expression was confirmed by western blot analysis of mouse brain homogenates (fig.S4E).”

It may state two separate claims as one.

Five patients from three families with RSS carry biallelic mutations in CCC complex genes: homozygous COMMD4 L41R, homozygous COMMD9 L70Gfs*5, or compound heterozygous CCDC93 Y457* and S631A.

“Through international collaboration fostered by GeneMatcher (30), we identified five patients from three families, each of whom carried biallelic mutations in genes of the CCC complex, either a homozygous missense variant in COMMD4 [NM_017828.5:c.122T>G;p.Leu41Arg (L41R)]; a homozygous frameshift variant in COMMD9 [NM_014186.3:c.208_209del;p.Leu70GlyfsTer5 (L70Gfs*5)]; or compound heterozygous”

• cites [30]Not matched

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

It may state two separate claims as one.

VPS26C or WASHC4 knock-out H4 cells show loss of COMMD1 endosomal localization and reduced cell surface abundance of ITGB1 and LRP1.

“Consistent with this, VPS26C or WASHC4 knock-out H4 cells displayed a loss in COMMD1 endosomal localization in immunofluorescence and reduced cell surface abundance of ITGB1 and LRP1 in western blotting (Fig. 2C and D).”

It may state two separate claims as one.

Dysregulation of cargo recycling through the SNX17-Retriever/CCC/WASH pathway is causative for RSS, and perturbed sorting of tissue-specific cargo proteins through this pathway is the principal root cause of the RSS recyclinopathy.

“The multi-organ phenotypes found in Ritscher-Schinzel syndrome stem from defective endosomal protein recycling, leading to a proposed recyclinopathy.”

• Also stated in: § DISCUSSION · boosted

  “By combining clinical and genetic analyses with cellular and biochemical approaches, we have defined how dysregulation of cargo recycling through the SNX17-Retriever/CCC/WASH pathway is causative for RSS and have identified a spectrum of integral membrane proteins whose cell surface expression is reduced in key tissues affected in RSS.”

• Also stated in: § DISCUSSION · plain

  “Collectively, perturbed sorting of tissue-specific cargo proteins through SNX17-Retriever/CCC/WASH endosomal recycling is the principal root cause of the RSS recyclinopathy.”

It may state two separate claims as one.

Loss of the SNX17-Retriever/CCC/WASH-recycling pathway results in LRP2 dysfunction, leading to proteinuria.

“Loss of the SNX17-Retriever/CCC/WASH-recycling pathway results in LRP2 dysfunction, leading to proteinuria.”

It may state two separate claims as one.

Loss-of-function mutations in VPS35L, CCDC22, and WASHC5, subunits of the SNX17-Retriever/CCC/WASH pathway, cause Ritscher-Schinzel syndrome, a congenital malformation syndrome characterized by cerebellar, cardiac, and craniofacial malformations.

“Loss-of-function mutations in VPS35L, CCDC22, and WASHC5 subunits of the SNX17-Retriever/CCC/WASH pathway are responsible for Ritscher-Schinzel syndrome (RSS), a congenital malformation syndrome characterized by cerebellar, cardiac, and craniofacial malformations (hence its alternative name, 3C syndrome) (18)(19)(20)(21)(22)(23)(24)(25).”

• cites (18)(19)(20)(21)(22)(23)(24)(25)Not matched

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

It may state two separate claims as one.

Loss of SNX17-Retriever/CCC/WASH pathway function leads to LRP1 and LDLR mis-sorting into the lysosomal degradative pathway and reduced cell surface amounts of these lipoprotein receptors.

“Loss of pathway function leads to LRP1 and LDLR mis-sorting into the lysosomal degradative pathway and reduced cell surface amounts of these lipoprotein receptors (18,26).”

• cites [18]Not matched

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

• cites [26]Not matched

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

It may state two separate claims as one.

Impairment of endosomal recycling of LRP2 and resulting reduction of its cell surface abundance contributes to proteinuria in RSS, with low molecular weight LRP2 ligands such as β2-microglobulin leaking into urine.

“Cellular and mouse histopathological analyses revealed that impairment of endosomal recycling of LRP2, and the resulting reduction of its cell surface abundance, contributes to the development of proteinuria observed in RSS, wherein low molecular weight LRP2 ligands such as β2microglobulin leak into the urine.”

Probably not a substantial claim. The paper’s conclusions may not depend on it.

To ensure compliance with the 3Rs in animal experimentation, the authors calculated the required sample size prior to conducting experiments.

“To ensure compliance with the 3Rs (Replacement, Reduction, and Refinement) in animal experimentation, we calculated the required sample size prior to conducting experiments.”

It may state two separate claims as one.

Hypercholesterolemia in RSS arises from reduced endosomal recycling of hepatocyte LRP1 and LDLR, decreasing their cell surface expression and reducing uptake of circulating LDL cholesterol.

“Other phenotypes include lipid metabolism disorders and renal disorders (18), with the observed hypercholesterolemia arising from reduced endosomal recycling of hepatocyte lipoprotein receptors, low-density lipoprotein receptor-related protein 1 (LRP1) and low-density lipoprotein receptor (LDLR), leading to a decrease in their cell surface expression and a reduction in the uptake of circulating LD”

• cites [18]Not matched

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

• cites [26]Not matched

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

Probably not a substantial claim. The paper’s conclusions may not depend on it.

For all biochemical assays using cultured cells, three to four different experiments were performed and analyzed as stated in figure legends and methods.

“For all biochemical assays using cultured cells, three to four different experiments were performed and analyzed as stated in figure legends and methods.”

Probably not a substantial claim. The paper’s conclusions may not depend on it.

There is no randomization or blinding in this study.

“There is no randomization or blinding in this study.”

It may state two separate claims as one.

The SNX17-Retriever/CCC/WASH recycling pathway regulates a variety of cargos in the neuronal system, including integrins, SLITRK and SEZ6 protein families, Reelin receptors, and APP proteins, and dysregulation in their cell surface expression likely explains the complex neurodevelopmental pathology in RSS.

“These data establish that the SNX17-Retriever/CCC/WASH recycling pathway regulates a variety of cargos in the neuronal system, including integrins, SLITRK and SEZ6 protein families, Reelin receptors and APP proteins, and that dysregulation in their cell surface expression likely explains the highly complex neurodevelopmental pathology observed in RSS.”