Lysosomal Storage and Albinism Due to Effects of a De Novo CLCN7 Variant on Lysosomal Acidification.

Optimal lysosome function requires maintenance of an acidic pH maintained by proton pumps in combination with a counterion transporter such as the Cl-/H+ exchanger, CLCN7 (ClC-7), encoded by CLCN7. The role of ClC-7 in maintaining lysosomal pH has been controversial. In this paper, we performed clinical and genetic evaluations of two children of different ethnicities. Both children had delayed myelination and development, organomegaly, and hypopigmentation, but neither had osteopetrosis. Whole-exome and -genome sequencing revealed a de novo c.2144A>G variant in CLCN7 in both affected children. This p.Tyr715Cys variant, located in the C-terminal domain of ClC-7, resulted in increased outward currents when it was heterologously expressed in Xenopus oocytes. Fibroblasts from probands displayed a lysosomal pH approximately 0.2 units lower than that of control cells, and treatment with chloroquine normalized the pH. Primary fibroblasts from both probands also exhibited markedly enlarged intracellular vacuoles; this finding was recapitulated by the overexpression of human p.Tyr715Cys CLCN7 in control fibroblasts, reflecting the dominant, gain-of-function nature of the variant. A mouse harboring the knock-in Clcn7 variant exhibited hypopigmentation, hepatomegaly resulting from abnormal storage, and enlarged vacuoles in cultured fibroblasts. Our results show that p.Tyr715Cys is a gain-of-function CLCN7 variant associated with developmental delay, organomegaly, and hypopigmentation resulting from lysosomal hyperacidity, abnormal storage, and enlarged intracellular vacuoles. Our data supports the hypothesis that the ClC-7 antiporter plays a critical role in maintaining lysosomal pH.

Induced GnasR201H expression from the endogenous Gnas locus causes fibrous dysplasia by up-regulating Wnt/ß-catenin signaling.

Fibrous dysplasia (FD; Online Mendelian Inheritance in Man no. 174800) is a crippling skeletal disease caused by activating mutations of the GNAS gene, which encodes the stimulatory G protein Gαs FD can lead to severe adverse conditions such as bone deformity, fracture, and severe pain, leading to functional impairment and wheelchair confinement. So far there is no cure, as the underlying molecular and cellular mechanisms remain largely unknown and the lack of appropriate animal models has severely hampered FD research. Here we have investigated the cellular and molecular mechanisms underlying FD and tested its potential treatment by establishing a mouse model in which the human FD mutation (R201H) has been conditionally knocked into the corresponding mouse Gnas locus. We found that the germ-line FD mutant was embryonic lethal, and Cre-induced Gnas FD mutant expression in early osteochondral progenitors, osteoblast cells, or bone marrow stromal cells (BMSCs) recapitulated FD features. In addition, mosaic expression of FD mutant Gαs in BMSCs induced bone marrow fibrosis both cell autonomously and non-cell autonomously. Furthermore, Wnt/ß-catenin signaling was up-regulated in FD mutant mouse bone and BMSCs undergoing osteogenic differentiation, as we have found in FD human tissue previously. Reduction of Wnt/ß-catenin signaling by removing one Lrp6 copy in an FD mutant line significantly rescued the phenotypes. We demonstrate that induced expression of the FD Gαs mutant from the mouse endogenous Gnas locus exhibits human FD phenotypes in vivo, and that inhibitors of Wnt/ß-catenin signaling may be repurposed for treating FD and other bone diseases caused by Gαs activation.

Planar cell polarity breaks bilateral symmetry by controlling ciliary positioning.

Defining the three body axes is a central event of vertebrate morphogenesis. Establishment of left-right (L-R) asymmetry in development follows the determination of dorsal-ventral and anterior-posterior (A-P) body axes, although the molecular mechanism underlying precise L-R symmetry breaking in reference to the other two axes is still poorly understood. Here, by removing both Vangl1 and Vangl2, the two mouse homologues of a Drosophila core planar cell polarity (PCP) gene Van Gogh (Vang), we reveal a previously unrecognized function of PCP in the initial breaking of lateral symmetry. The leftward nodal flow across the posterior notochord (PNC) has been identified as the earliest event in the de novo formation of L-R asymmetry. We show that PCP is essential in interpreting the A-P patterning information and linking it to L-R asymmetry. In the absence of Vangl1 and Vangl2, cilia are positioned randomly around the centre of the PNC cells and nodal flow is turbulent, which results in disrupted L-R asymmetry. PCP in mouse, unlike what has been implicated in other vertebrate species, is not required for ciliogenesis, cilium motility, Sonic hedgehog (Shh) signalling or apical docking of basal bodies in ciliated tracheal epithelial cells. Our data suggest that PCP acts earlier than the unidirectional nodal flow during bilateral symmetry breaking in vertebrates and provide insight into the functional mechanism of PCP in organizing the vertebrate tissues in development.

The MFSD12 p.Tyr182His common variant is sufficient to alter mouse agouti coat color.

MFSD12 functions as a transmembrane protein required for import of cysteine into melanosomes and lysosomes. The MFSD12 locus has been associated with phenotypic variation in skin color across African, Latin American, and East Asian populations. The frequency of a particular MFSD12 coding variant, rs2240751 (MAF = 0.08), has been reported to correlate with solar radiation and occur at highest frequency in Peruvian (PEL MAF = 0.48) and Han Chinese (CHB MAF = 0.40) populations, suggesting it could be causative for associated phenotypic variation in skin color. We have generated a mouse knock-in allele, Mfsd12Y182H , to model the human missense p.Tyr182His human variant. We demonstrate that the variant transcript is stably expressed and that agouti mice homozygote for the variant allele are viable with an altered coat color. This in vivo data confirms that the MFSD12 p.Tyr182His variant functions as a hypomorphic allele sufficient to alter mammalian pigmentation.

A transgenic mouse model demonstrates a dominant negative effect of a point mutation in the RPS19 gene associated with Diamond-Blackfan anemia.

Diamond Blackfan anemia (DBA) is an inherited erythroblastopenia associated with mutations in at least 8 different ribosomal protein genes. Mutations in the gene encoding ribosomal protein S19 (RPS19) have been identified in approximately 25% of DBA families. Most of these mutations disrupt either the translation or stability of the RPS19 protein and are predicted to cause DBA by haploinsufficiency. However, approximately 30% of RPS19 mutations are missense mutations that do not alter the stability of the RPS19 protein and are hypothesized to act by a dominant negative mechanism. To formally test this hypothesis, we generated a transgenic mouse model expressing an RPS19 mutation in which an arginine residue is replaced with a tryptophan residue at codon 62 (RPS19R62W). Constitutive expression of RPS19R62W in developing mice was lethal. Conditional expression of RPS19R62W resulted in growth retardation, a mild anemia with reduced numbers of erythroid progenitors, and significant inhibition of terminal erythroid maturation, similar to DBA. RNA profiling demonstrated more than 700 dysregulated genes belonging to the same pathways that are disrupted in RNA profiles of DBA patient cells. We conclude that RPS19R62W is a dominant negative DBA mutation.

Author Correction: Mutations in COMP cause familial carpal tunnel syndrome.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Mutations in COMP cause familial carpal tunnel syndrome.

Carpal tunnel syndrome (CTS) is the most common peripheral nerve entrapment syndrome, affecting a large proportion of the general population. Genetic susceptibility has been implicated in CTS, but the causative genes remain elusive. Here, we report the identification of two mutations in cartilage oligomeric matrix protein (COMP) that segregate with CTS in two large families with or without multiple epiphyseal dysplasia (MED). Both mutations impair the secretion of COMP by tenocytes, but the mutation associated with MED also perturbs its secretion in chondrocytes. Further functional characterization of the CTS-specific mutation reveals similar histological and molecular changes of tendons/ligaments in patients' biopsies and the mouse models. The mutant COMP fails to oligomerize properly and is trapped in the ER, resulting in ER stress-induced unfolded protein response and cell death, leading to inflammation, progressive fibrosis and cell composition change in tendons/ligaments. The extracellular matrix (ECM) organization is also altered. Our studies uncover a previously unrecognized mechanism in CTS pathogenesis.

Wnt-induced Vangl2 phosphorylation is dose-dependently required for planar cell polarity in mammalian development.

Planar cell polarity (PCP) is an evolutionarily conserved essential mechanism that provides directional information to control and coordinate polarized cellular and tissue behavior during embryonic development. Disruption of PCP leads to severe morphological defects in vertebrates and its dysregulation results in a variety of human diseases such as neural tube defects and skeletal dysplasia. PCP is governed by a set of highly conserved core proteins that are asymmetrically localized at the cell surface throughout the polarized tissues. The uniform directionality of PCP is established by global cues, such as Wg/Wnt signaling gradients that break the original symmetrical localization of core PCP proteins including Vang/Vangl and Fz/Fzd. However, the exact mechanism remains elusive. In this study, we found that Vangl2 phosphorylation, which was previously identified to be induced by Wnt5a signaling, is required for Vangl2 functions in mammalian PCP in multiple tissues. The in vivo activities of Vangl2 are determined by its phosphorylation level. Phospho-mutant Vangl2 exhibits dominant negative effects, whereas Vangl2 with reduced phosphorylation is hypomorphic. We show that Vangl2 phosphorylation is essential for its uniform polarization pattern. Moreover, serine/threonine kinases CK1ɛ and CK1δ are redundantly required for Wnt5a-induced Vangl2 phosphorylation. Dvl family members are also required for Wnt5a-induced Vangl2 phosphorylation by enhancing the interaction of CK1 and Vangl2. These findings demonstrate that induction of Vangl protein phosphorylation plays an essential role in transducing Wnt5a signaling to establish PCP in mammalian development, suggesting a phosphorylation-regulated "Vangl activity gradient" model in addition to the well-documented "Fz activity gradient" model in Wnt/PCP signaling.

A Novel Chemotherapeutic Agent to Treat Tumors with DNA Mismatch Repair Deficiencies.

Impairing the division of cancer cells with genotoxic small molecules has been a primary goal to develop chemotherapeutic agents. However, DNA mismatch repair (MMR)-deficient cancer cells are resistant to most conventional chemotherapeutic agents. Here we have identified baicalein as a small molecule that selectively kills MutSα-deficient cancer cells. Baicalein binds preferentially to mismatched DNA and induces a DNA damage response in a MMR-dependent manner. In MutSα-proficient cells, baicalein binds to MutSα to dissociate CHK2 from MutSα leading to S-phase arrest and cell survival. In contrast, continued replication in the presence of baicalein in MutSα-deficient cells results in a high number of DNA double-strand breaks and ultimately leads to apoptosis. Consistently, baicalein specifically shrinks MutSα-deficient xenograft tumors and inhibits the growth of AOM-DSS-induced colon tumors in colon-specific MSH2 knockout mice. Collectively, baicalein offers the potential of an improved treatment option for patients with tumors with a DNA MMR deficiency. Cancer Res; 76(14); 4183-91. ©2016 AACR.