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.

This file has methods used in the study in addition to methods described in the manuscript.

This file has detail figure legends for supplementary figures.

Supplemental Figures contains supplementary figures S1-S13. Supplementary Figure S1. The effect of MSH2 deficiency on cell sensitivity to baicalein. Supplementary Figure S2. Baicalein-treated HEC59 cells are deficient in CHK2-regulated S phase checkpoint arrest. Supplementary Figure S3. Baicalein disrupts the interaction between MutSα and CHK2-ATM. Supplementary Figure S4. Baicalein binds to MutSα. Supplementary Figure S5. Baicalein intercalates DNA but does not form crosslinks. Supplementary Figure S6. XPF deficiency reduces baicalein-induced γ-H2AX and rescues baicalein-induced cell death in HT29 MSH2-deficient cells. Supplementary Figure S7. Baicalein pellets reduce the size of MSH2-deficient xenograft tumors but have little effect on MSH2-proficient tumors. Supplementary Figure S8. Baicalein shrinks MSH2-deficient xenograft tumors in a time-dependent manner. Supplementary Figure S9. Baicalein shrinks xenograft tumors formed by MSH2-deficient LoVo cells. Supplementary Figure S10. Baicalein inhibits the growth of chronic-AOM-DSS-induced colon tumors in Msh2LoxP/LoxPVilCre mice. Supplementary Figure S11. Baicalein inhibits the growth of acute-AOM-DSS-induced colon tumors in Msh2LoxP/LoxPVilCre mice. Supplementary Figure S12. Baicalein have little effect on the growth of chronic-AOM-DSS-induced colon tumors in WT mice. Supplementary Figure S13. Model describing baicalein's mechanism of action.

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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.

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.

Significance Understanding molecular and cellular mechanisms of rare genetic diseases provides invaluable insights into the human biology and pathology of both rare and related common diseases. Fibrous dysplasia (FD) is a mosaic disease resulting from postzygotic activating mutations of GNAS . The mouse models we created allowed us to precisely model FD by expressing the FD Gα s mutation under the control of its endogenous genetic locus. We found in our FD mouse models that up-regulated Wnt/β-catenin signaling resulted in impaired differentiation and proliferation of bone marrow stem cells, which in turn caused marrow fibrosis. Our work provides a solid new foundation for therapeutic development of FD and understanding the principles whereby Gα s signaling governs bone formation and maintenance and bone marrow stromal cell differentiation.

Isolated Methylmalonic Acidemia (MMA) comprises a relatively common and heterogeneous group of inborn errors of metabolism. Most affected individuals display severe multisystemic disease with episodes of metabolic instability, chronic renal disease, and neurological complications. The most severe clinical presentations are typically associated with complete loss of methylmalonyl-CoA mutase (MUT) enzymatic activity. MUT converts methylmalonyl-CoA to succinyl-CoA within mitochondria but defects in the transport or synthesis of its cobalamin co-factor also variably impair MUT activity. Previous mouse models of Mut MMA generated by our group have been critical to developing gene therapy for MMA but have either displayed neonatal lethality or required concomitant transgenesis for viability. Thus, the need for ameliorated models to further investigate disease-associated pathophysiology and facilitate gene therapy studies exists. We have therefore constructed a mouse model of an attenuated form of isolated MMA, the cblA subtype, by using homologous recombination to create a deletion allele of Mmaa, the enzyme that performs the gated transfer of adenosylcobalamin to Mut and protects Mut from oxidative inactivation. Patients with the cblA subtype of MMA can have variable presentations, spanning the full spectrum of MMA associated symptoms and pathology, yet always harbor an element of clinical and biochemical responsiveness to injectable B12. Mmaa-/- mice were born in mendelian proportions, exhibited decreased survival after weaning (P<0.0001), weighed 50% less than littermates at 6 months (P<0.0001), lacked immunoreactive Mmaa and Mmaa mRNA in multiple tissues, including the liver, and manifested severe metabolic perturbations. Plasma methylmalonic acid concentrations ranged between 63.88-1641.91 µM, representing a 62-fold or more elevation over age matched littermates. Mmaa-/- mice also display diminished 1-C-13 propionate oxidative capacity (P<0.0001) and hepatic complex IV activity (P=0.002). Electron microscopy confirmed the presence of hepatic megamitochondria and abnormal proximal renal tubular mitochondria. The glomerular filtration rate, measured using FITC-sinistrin decay, showed that Mmaa-/- mice have diminished kidney function compared to heterozygous littermates. Following hydroxycobalamin administration, Mmaa-/- mice had improved survival and increased weight (P=0.0003), which was associated with improved 1-13C-propionate oxidative capacity. This new mouse model fully recapitulates the clinical and biochemical features seen in cblA patients, and provides a robust platform for testing gene and cell therapies for MMA because affected mice are relatively severe but survive until weaning. These animals also offer the opportunity to longitudinally examine disease biomarkers and more easily assay vectors across a wide age range.

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.

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.

Diamond-Blackfan Anemia (DBA) is associated with mutations in several ribosomal protein genes, including Ribosomal Protein S19 (RPS19), which is mutated in approximately 25% of patients. Most RPS19 mutations are deletions of all or part of the RPS19 gene and are predicted to cause DBA by a haploinsufficiency mechanism. However, approximately 30% of RPS19 mutations are missense mutations in the RPS19 coding sequence, which we hypothesize act through a dominant negative mechanism. To test for a dominant negative effect, we generated a transgenic mouse model expressing a common and penetrant mutation at codon 62 that replaces an Arginine with a Tryptophan (R62W). The constructs contain the ubiquitous actin promoter linked to the wild-type or R62W human RPS19 cDNA followed by the 3′ region of the Gamma globin gene to provide RNA stability and intron splicing to facilitate RNA transport to the cytoplasm. The constructs are flanked by chicken HS4 barrier elements to ensure transgene expression regardless of the location in the genome. Eight lines of wild-type RPS19 transgenic mice were fertile, expressed RPS19 in all tissues, and had normal hematology. Twelve RPS19R62W founder animals were generated, six of which died before they reached 2 months of age. Two of these animals were analyzed and found to have a macrocytic anemia. None of the other 6 founder animals transmitted the RPS19R62W transgene to F1 pups or d13.5 embryos, suggesting either that the RPS19R62W transgene was not present in the germ line and/or that expression of the RPS19R62W protein may cause early lethality. Supporting this hypothesis, embryonic stem cells (ES) expressing wild-type RPS19 were viable, while ES cells expressing RPS19R62W were not viable. To circumvent potential embryonic lethality, we generated conditional RPS19R62W transgenic mice with stop sequences flanked by lox P sites inserted between the promoter and the RPS19 gene. In the presence of Cre recombinase, lox P sites are combined, excising the sequences between them. Adult mice carrying the conditional RPS19R62W transgene and the interferon inducible Mx1-Cre gene were treated with poly (I:C) to induce excision of the stop sequence. Following poly (I:C) administration, hematocrits dropped significantly in RPS19R62W/Mx1-Cre animals compared to controls, but rebounded to normal within two weeks, due to incomplete stop sequence excision and expansion of unexcised cells in the bone marrow. Colony-forming cell assays indicate that RPS19R62W-expressing bone marrow contains 2 to 3 fold fewer BFU-E and CFU-E (p<0.05) and similar numbers of CFU-GM compared to wild-type animals. The decrease in erythroid progenitors was variable, indicating different levels of excision as well as penetrance. When RPS19R62W mice were crossed to Prion-Cre mice, which express Cre at the early embryonic stage, small, anemic d13.5 embryos and occasional small, adult animals with macrocytic anemia were observed. Day 13.5 RPS19R62W/Prion-Cre fetal livers had reduced overall numbers of erythroid cells, and reduced numbers of BFU-E and CFU-E. The decrease in erythroid progenitors was variable, especially in the line carrying 1 copy of the transgene compared to the line carrying 4 copies of the transgene. FACS analysis of d13.5 fetal liver and adult RPS19R62W/Prion-Cre erythroid cells revealed a relative accumulation of erythroid progenitor cells and a relative decrease in the number of terminally differentiating erythroid cells, suggesting that terminal erythroid differentiation is delayed. These findings are consistent with the reticulocytopenia observed in adult RPS19R62W/Prion-Cre mice. In summary we have successfully generated a mouse model of DBA caused by ectopic expression of mutant human RPS19R62W. The development of a severe anemia following conditional expression of mutant RPS19 suggests that the R62W missense mutation has a dominant negative effect that delays erythropoiesis causing an overall reduction in erythroid cells.