Summed and Weighted Summary Scores for the Medsger Disease Severity Scale Compared with the Physician's Global Assessment of Disease Severity in Systemic Sclerosis.

OBJECTIVE: To develop a weighted summary score for the Medsger Disease Severity Scale (DSS) and to compare its measurement properties with those of a summed DSS score and a physician's global assessment (PGA) of severity score in systemic sclerosis (SSc). METHODS: Data from 875 patients with SSc enrolled in a multisite observational research cohort were extracted from a central database. Item response theory was used to estimate weights for the DSS weighted score. Intraclass correlation coefficients (ICC) and convergent, discriminative, and predictive validity of the 3 summary measures in relation to patient-reported outcomes (PRO) and mortality were compared. RESULTS: Mean PGA was 2.69 (SD 2.16, range 0-10), mean DSS summed score was 8.60 (SD 4.02, range 0-36), and mean DSS weighted score was 8.11 (SD 4.05, range 0-36). ICC were similar for all 3 measures [PGA 6.9%, 95% credible intervals (CrI) 2.1-16.2; DSS summed score 2.5%, 95% CrI 0.4-6.7; DSS weighted score 2.0%, 95% CrI 0.1-5.6]. Convergent and discriminative validity of the 3 measures for PRO were largely similar. In Cox proportional hazards models adjusting for age and sex, the 3 measures had similar predictive ability for mortality (adjusted R(2) 13.9% for PGA, 12.3% for DSS summed score, and 10.7% DSS weighted score). CONCLUSION: The 3 summary scores appear valid and perform similarly. However, there were some concerns with the weights computed for individual DSS scales, with unexpected low weights attributed to lung, heart, and kidney, leading the PGA to be the preferred measure at this time. Further work refining the DSS could improve the measurement properties of the DSS summary scores.

The planar cell polarity protein Vangl2 is required for retinal axon guidance.

Vangl2 plays a critical role in the establishment of planar cell polarity (PCP). Previously, we detected expression of Vangl2 in the developing retina during late embryogenesis, which led us to investigate the possible role of Vangl2-mediated PCP signaling in eye development. We have generated a Vangl2(BGeo) knock-in mouse allowing us to evaluate Vangl2 mRNA expression during retinal development, and used an isoform-specific antibody to examine Vangl2 protein expression in cryosections. To investigate the role of Vangl2 in retinal development, we examined eyes taken from embryos homozygous for independent alleles of Looptail (Lp, Lp(m1jus) ) mutant mice. We found that Vangl2 mRNA and protein are dynamically expressed in the developing embryonic and postnatal retina, with Vangl2 expression becoming progressively restricted to the ganglion cell layer and optic nerve as the retina matures. The expression pattern of Vangl2 transcript and protein is most prominent in retinal ganglion cells (RGC), and their axons. Additionally, we show that Vangl2 is required for retinal and optic nerve development as Vangl2 (Lp/Lp) mutant embryos display a significantly reduced eye size, marked thickening of the retina, and striking abnormalities in the morphology of the optic nerve (significant hypoplasia, and aberrant exit trajectory). Notably, we identified a salient intraretinal axon guidance defect in Vangl2 (Lp/Lp) mutant embryos through which axon bundles traverse the entire thickness of the retina and become trapped within the subretinal space. Our observations identify a new and essential role for Vangl2-dependent PCP signaling in the intraretinal path-finding of RGC axons.

Modulation of Malaria Phenotypes by Pyruvate Kinase (PKLR) Variants in a Thai Population.

Pyruvate kinase (PKLR) is a critical erythrocyte enzyme that is required for glycolysis and production of ATP. We have shown that Pklr deficiency in mice reduces the severity (reduced parasitemia, increased survival) of blood stage malaria induced by infection with Plasmodium chabaudi AS. Likewise, studies in human erythrocytes infected ex vivo with P. falciparum show that presence of host PK-deficiency alleles reduces infection phenotypes. We have characterized the genetic diversity of the PKLR gene, including haplotype structure and presence of rare coding variants in two populations from malaria endemic areas of Thailand and Senegal. We investigated the effect of PKLR genotypes on rich longitudinal datasets including haematological and malaria-associated phenotypes. A coding and possibly damaging variant (R41Q) was identified in the Thai population with a minor allele frequency of ~4.7%. Arginine 41 (R41) is highly conserved in the pyruvate kinase family and its substitution to Glutamine (R41Q) affects protein stability. Heterozygosity for R41Q is shown to be associated with a significant reduction in the number of attacks with Plasmodium falciparum, while correlating with an increased number of Plasmodium vivax infections. These results strongly suggest that PKLR protein variants may affect the frequency, and the intensity of malaria episodes induced by different Plasmodium parasites in humans living in areas of endemic malaria.

Independent mutations at Arg181 and Arg274 of Vangl proteins that are associated with neural tube defects in humans decrease protein stability and impair membrane targeting.

In vertebrates, Vangl proteins play important roles during embryogenesis, including establishing planar polarity and coordinating convergent extension movements. In mice, homozygosity for mutations in the Vangl1 and Vangl2 genes or combined heterozygosity for Vangl1/Vangl2 mutations causes the very severe neural tube defect (NTD) craniorachischisis. Recently, a number of patient-specific VANGL1 and VANGL2 protein mutations have been identified in familial and sporadic cases of mild and severe forms of NTDs. The biochemical nature of pathological effects in these mutations remains unknown. Of interest are two arginine residues, R181 and R274, that are highly conserved in Vangl protein homologues and found to be independently mutated in VANGL1 (R181Q and R274Q) and VANGL2 (R177H and R270H) in human cases of NTDs. The cellular and biochemical properties of R181Q and R274Q were established in transfected MDCK kidney epithelial cells and compared to those of wild-type (WT) Vangl1. Compared to that of WT, these mutations displayed impaired targeting to the plasma membrane and were instead detected in an intracellular endomembrane compartment that was positive for the endoplasmic reticulum. R181Q and R274Q showed impaired stability with significant reductions in measured half-lives from >20 h for WT protein to 9 and 5 h, respectively. These mutations have a cellular and biochemical phenotype that is indistinguishable from that of Vangl mutations known to cause craniorachichisis in mice (Lp). These results strongly suggest that R181 and R274 play critical roles in Vangl protein function and that their mutations cause neural tube defects in humans.

Genetic control of susceptibility to Candida albicans in SM/J mice.

In the immunocompromised host, invasive infection with the fungal pathogen Candida albicans is associated with high morbidity and mortality. Sporadic cases in otherwise normal individuals are rare, and they are thought to be associated with genetic predisposition. Using a mouse model of systemic infection with C. albicans, we identified the SM/J mouse strain as unusually susceptible to infection. Genetic linkage studies in informative [C57BL/6JxSM/J]F2 mice identified a major locus on distal chromosome 15, given the appellation Carg5, that regulates C. albicans replication in SM/J mice. Cellular and molecular immunophenotyping experiments, as well as functional studies in purified cell populations from SM/J and C57BL/6J, and in [C57BL/6JxSM/J]F2 mice fixed for homozygous or heterozygous Carg5 alleles, indicate that Carg5-regulated susceptibility in SM/J is associated with a complex defect in the myeloid compartment of these mice. SM/J neutrophils express lower levels of Ly6G, and importantly, they show significantly reduced production of reactive oxygen species in response to stimulation with fMLF and PMA. Likewise, CD11b(+)Ly6G(-)Ly6C(hi) inflammatory monocytes were present at lower levels in the blood of infected SM/J, recruited less efficiently at the site of infection, and displayed blunted oxidative burst. Studies in F2 mice establish strong correlations between Carg5 alleles, Ly6G expression, production of serum CCL2 (MCP-1), and susceptibility to C. albicans. Genomic DNA sequencing of chromatin immunoprecipitated for myeloid proinflammatory transcription factors IRF1, IRF8, STAT1 and NF-κB, as well as RNA sequencing, were used to develop a "myeloid inflammatory score" and systematically analyze and prioritize potential candidate genes in the Carg5 interval.

The intracellular carboxyl terminal domain of Vangl proteins contains plasma membrane targeting signals.

Vangl1 and Vangl2 are integral membrane proteins that play a critical role in establishing planar cell polarity (PCP) in epithelial cells and are required for convergent extension (CE) movements during embryogenesis. Their proper targeting to the plasma membrane (PM) is required for function. We created discrete deletions at the amino and carboxy termini of Vangl1 and monitored the effect of the mutations on PM targeting in Madin-Darby canine kidney cells. Our results show that the Vangl1 amino terminus lacks PM targeting determinants, and these are restricted to the carboxy terminus, including the predicted PDZBM motif at the C-terminus.

An expanding role of Vangl proteins in embryonic development.

The mammalian Vangl1 and Vangl2 genes were discovered a decade ago through their association with neural tube defects, in particular the presence of Vangl2 mutations in independent alleles of the mouse mutant Loop-tail (Lp), a mouse model of the severe neural tube defect craniorachischisis. Vangl1 and Vangl2 variants have also been detected in familial and sporadic cases of spina bifida. Vangl proteins are highly conserved in evolution with relatives in flies, and distant invertebrates and vertebrates. In these organisms, they play a central role in planar cell polarity (PCP) and convergent extension (CE) movements. Over the past decade, these functional characteristics have also been established for mammalian Vangl genes. The careful analysis of mouse Vangl genes mutants has showed that these genes and the associated PCP pathway and CE movements are involved in many unexpected developmental processes, from morphogenesis of different tissues, left-right asymmetry, asymmetric cell division, and organization of many epithelial structures, as well as positioning and function of cellular appendages. Genetic studies in double mutants and biochemical studies of interacting proteins have started to elucidate the molecular pathways in which Vangl proteins participate and that regulate these complex events.

Transmembrane topology of mammalian planar cell polarity protein Vangl1.

Vangl1 and Vangl2 are membrane proteins that play an important role in neurogenesis, and Vangl1/Vangl2 mutations cause neural tube defects in mice and humans. At the cellular level, Vangl proteins regulate the establishment of planar cell polarity (PCP), a process requiring membrane assembly of asymmetrically distributed multiprotein complexes that transmit polarity information to neighboring cells. The membrane topology of Vangl proteins and the protein segments required for structural and functional aspects of multiprotein membrane PCP complexes is unknown. We have used epitope tagging and immunofluorescence to establish the secondary structure of Vangl proteins, including the number, position, and polarity of transmembrane domains. Antigenic hemagglutinin A (HA) peptides (YYDVPDYS) were inserted in predicted intra- or extracellular loops of Vangl1 at positions 18, 64, 139, 178, 213, and 314, and individual mutant variants were stably expressed at the membrane of MDCK polarized cells. The membrane topology of the exofacial HA tag was determined by a combination of immunofluorescence in intact (extracellular epitopes) and permeabilized (intracellular epitopes) cells and by surface labeling. Results indicate that Vangl proteins have a four-transmembrane domain structure with the N-terminal portion (HA18 and HA64) and the large C-terminal portion (HA314) of the protein being intracellular. Topology mapping and hydropathy profiling show that the loop delineated by TMD1-2 (HA139) and TMD3-4 (HA213) is extracellular while the segment separating predicted TMD2-3 (HA178) is intracellular. This secondary structure reveals a compact membrane-associated portion with very short predicted extra- and intracellular loops, while the protein harbors a large intracellular domain.

Loss of membrane targeting of Vangl proteins causes neural tube defects.

In the mouse, the loop-tail mutation (Lp) causes a very severe neural tube defect, which is caused by mutations in the Vangl2 gene. In mammals, Vangl1 and Vangl2 code for integral membrane proteins that assemble into asymmetrically distributed membrane complexes that establish planar cell polarity in epithelial cells and that regulate convergent extension movements during embryogenesis. To date, VANGL are the only genes in which mutations cause neural tube defects in humans. Three independently arising Lp alleles have been described for Vangl2: D255E, S464N, and R259L. Here we report a common mechanism for both the naturally occurring Lp (S464N) and a novel ENU-induced mutation Lp(m2Jus)(R259L). We show that the S464N and R259L variants stably expressed in polarized MDCK kidney cells fail to reach the plasma membrane, their site for biological function. The mutant variants are retained intracellularly in the endoplasmic reticulum, colocalizing with ER chaperone calreticulin. Furthermore, the mutants also show a dramatically reduced half-life of ∼3 h, compared to ∼22 h for the wild-type protein, and are rapidly degraded in a proteasome-dependent and MG132-sensitive fashion. Coexpressing individually the three known allelic Lp variants with the wild-type protein does not influence the localization of the WT at the plasma membrane, suggesting that the codominant nature of the Lp trait in vivo is due to haploid insufficiency caused by a partial loss of function in a gene dosage-dependent pathway, as opposed to a dominant negative phenotype. Our study provides a biochemical framework for the study of recently identified mutations in hVANGL1 and hVANGL2 in sporadic or familial cases of neural tube defects.

Molecular and cellular mechanisms underlying neural tube defects in the loop-tail mutant mouse.

Loop-tail (Lp) mice show a very severe neural tube defect (craniorachischisis) caused by mutations in the Vangl2 gene (D255E, S464N). Mammalian Vangl1 and Vangl2 are membrane proteins that play critical roles in development such as establishment of planar cell polarity (PCP) in epithelial layers and convergent extension movements during neurogenesis and cardiogenesis. Vangl proteins are thought to assemble with other PCP proteins (Dvl, Pk) to form membrane-bound PCP signaling complexes that provide polarity information to the cell. In the present study, we show that Vangl1 is expressed exclusively at the plasma membrane of transfected MDCK cells, where it is targeted to the basolateral membrane. Experiments with an inserted exofacial HA epitope indicate that the segment delimited by the predicted transmembrane domains 1 and 2 is exposed to the extracellular milieu. Comparative studies of the Lp-associated pathogenic mutation D255E indicate that the targeting of the mutant variant at the plasma membrane is greatly reduced; the mutant variant is predominantly retained intracellularly in endoplasmic reticulum (ER) vesicles colocalizing with the ER marker calreticulin. In addition, the D255E variant shows drastically reduced stability with a half-life of approximately 2 h, compared to >9 h for its wild type counterpart and is rapidly degraded in a proteasome-dependent and MG132 sensitive pathway. These findings highlight a critical role for D255 for normal folding and processing of Vangl proteins, with highly conservative substitutions not tolerated at that site. Our study provide an experimental framework for the analysis of human VANGL mutations recently identified in familial and sporadic cases of spina bifida.