A new murine Rpl5 (uL18) mutation provides a unique model of variably penetrant Diamond-Blackfan anemia.

Ribosome dysfunction is implicated in multiple abnormal developmental and disease states in humans. Heterozygous germline mutations in genes encoding ribosomal proteins are found in most individuals with Diamond-Blackfan anemia (DBA), whereas somatic mutations have been implicated in a variety of cancers and other disorders. Ribosomal protein-deficient animal models show variable phenotypes and penetrance, similar to human patients with DBA. In this study, we characterized a novel ENU mouse mutant (Skax23m1Jus) with growth and skeletal defects, cardiac malformations, and increased mortality. After genetic mapping and whole-exome sequencing, we identified an intronic Rpl5 mutation, which segregated with all affected mice. This mutation was associated with decreased ribosome generation, consistent with Rpl5 haploinsufficiency. Rpl5Skax23-Jus/+ animals had a profound delay in erythroid maturation and increased mortality at embryonic day (E) 12.5, which improved by E14.5. Surviving mutant animals had macrocytic anemia at birth, as well as evidence of ventricular septal defect (VSD). Surviving adult and aged mice exhibited no hematopoietic defect or VSD. We propose that this novel Rpl5Skax23-Jus/+ mutant mouse will be useful in studying the factors influencing the variable penetrance that is observed in DBA.

Identification and characterization of a novel chemically induced allele at the planar cell polarity gene Vangl2.

Planar cell polarity (PCP) signaling controls a number of morphogenetic processes including convergent extension during gastrulation and neural tube formation. Defects in this pathway cause neural tube defects (NTD), the most common malformations of the central nervous system. The Looptail (Lp) mutant mouse was the first mammalian mutant implicating a PCP gene (Vangl2) in the pathogenesis of NTD. We report on a novel chemically induced mutant allele at Vangl2 called Curly Bob that causes a missense mutation p.Ile268Asn (I268N) in the Vangl2 protein. This mutant segregates in a semi-dominant fashion with heterozygote mice displaying a looped tail appearance, bobbing head, and a circling behavior. Homozygote mutant embryos suffer from a severe form of NTD called craniorachischisis, severe PCP defects in the inner hair cells of the cochlea and posterior cristae, and display a distinct defect in retinal axon guidance. This mutant genetically interacts with the Lp allele (Vangl2 S464N ) in neural tube development and inner ear hair cell polarity. The Vangl2I268N protein variant is expressed at very low levels in affected neural and retinal tissues of mutant homozygote embryos. Biochemical studies show that Vangl2I268N exhibits impaired targeting to the plasma membrane and accumulates in the endoplasmic reticulum. The Vangl2I268N variant no longer physically interacts with its PCP partner DVL3 and has a reduced protein half-life. This mutant provides an important model for dissecting the role of Vangl2 in the development of the neural tube, establishment of polarity of sensory cells of the auditory and vestibular systems, and retinal axon guidance.

Scribble1 plays an important role in the pathogenesis of neural tube defects through its mediating effect of Par-3 and Vangl1/2 localization.

Scribble1 (Scrib1) is a tumor suppressor gene that has long been established as an essential component of apicobasal polarity (ABP). In mouse models, mutations in Scrib1 cause a severe form of neural tube defects (NTDs) as a result of a defective planar cell polarity (PCP) signaling. In this study, we dissected the role of Scrib1 in the pathogenesis of NTDs in its mouse mutant Circletail (Crc), in cell lines and in a human NTD cohort. While there were no obvious defects in ABP in the Scrib1Crc/Crc neuroepihelial cells, we identified an abnormal localization of the apical protein Par-3 and of the PCP protein Vangl2. These results were concordant with those obtained following a partial knockdown of Scrib1 in MDCK II cells. Par-3 was able to rescue the localization defect of Vangl1 (paralog of Vangl2) caused by partial knockdown of Scrib1 suggesting that Scrib1 exerts its effect on Vangl1 localization indirectly through Par-3. This conclusion is supported by our findings of an apical enrichment of Vangl1 following a partial knockdown of Par-3. Re-sequencing analysis of SCRIB1 in 473 NTD patients led to the identification of 5 rare heterozygous missense mutations that were predicted to be pathogenic. Two of these mutations, p.Gly263Ser and p.Gln808His, and 2 mouse NTD mutations, p.Ile285Lys and p.Glu814Gly, affected Scrib1 membrane localization and its modulating role of Par-3 and Vangl1 localization. Our study demonstrates an important role of Scrib1 in the pathogenesis of NTDs through its mediating effect of Par-3 and Vangl1/2 localization and most likely independently of ABP.

Loss-of-function de novo mutations play an important role in severe human neural tube defects.

BACKGROUND: Neural tube defects (NTDs) are very common and severe birth defects that are caused by failure of neural tube closure and that have a complex aetiology. Anencephaly and spina bifida are severe NTDs that affect reproductive fitness and suggest a role for de novo mutations (DNMs) in their aetiology. METHODS: We used whole-exome sequencing in 43 sporadic cases affected with myelomeningocele or anencephaly and their unaffected parents to identify DNMs in their exomes. RESULTS: We identified 42 coding DNMs in 25 cases, of which 6 were loss of function (LoF) showing a higher rate of LoF DNM in our cohort compared with control cohorts. Notably, we identified two protein-truncating DNMs in two independent cases in SHROOM3, previously associated with NTDs only in animal models. We have demonstrated a significant enrichment of LoF DNMs in this gene in NTDs compared with the gene specific DNM rate and to the DNM rate estimated from control cohorts. We also identified one nonsense DNM in PAX3 and two potentially causative missense DNMs in GRHL3 and PTPRS. CONCLUSIONS: Our study demonstrates an important role of LoF DNMs in the development of NTDs and strongly implicates SHROOM3 in its aetiology.

Novel mutations in Lrp6 orthologs in mouse and human neural tube defects affect a highly dosage-sensitive Wnt non-canonical planar cell polarity pathway.

Wnt signaling has been classified as canonical Wnt/ß-catenin-dependent or non-canonical planar cell polarity (PCP) pathway. Misregulation of either pathway is linked mainly to cancer or neural tube defects (NTDs), respectively. Both pathways seem to antagonize each other, and recent studies have implicated a number of molecular switches that activate one pathway while simultaneously inhibiting the other thereby partially mediating this antagonism. The lipoprotein receptor-related protein Lrp6 is crucial for the activation of the Wnt/ß-catenin pathway, but its function in Wnt/PCP signaling remains largely unknown. In this study, we investigate the role of Lrp6 as a molecular switch between both Wnt pathways in a novel ENU mouse mutant of Lrp6 (Skax26(m1Jus)) and in human NTDs. We demonstrate that Skax26(m1Jus) represents a hypermorphic allele of Lrp6 with increased Wnt canonical and abolished PCP-induced JNK activities. We also show that Lrp6(Skax26-Jus) genetically interacts with a PCP mutant (Vangl2(Lp)) where double heterozygotes showed an increased frequency of NTDs and defects in cochlear hair cells' polarity. Importantly, our study also demonstrates the association of rare and novel missense mutations in LRP6 that is an inhibitor rather than an activator of the PCP pathway with human NTDs. We show that three LRP6 mutations in NTDs led to a reduced Wnt canonical activity and enhanced PCP signaling. Our data confirm an inhibitory role of Lrp6 in PCP signaling in neurulation and indicate the importance of a tightly regulated and highly dosage-sensitive antagonism between both Wnt pathways in this process.

A novel hypomorphic Looptail allele at the planar cell polarity Vangl2 gene.

Vangl2 forms part of the planar cell polarity signalling pathway and is the gene defective in the Looptail (Lp) mouse mutant. Two previously described alleles, Lp and Lp(m1Jus) , segregate in a semi-dominant fashion, with heterozygotes displaying the looped-tail appearance, while homozygotes show the neural tube defect called craniorachischisis. Here, we report a novel experimentally induced allele, Lp(m2Jus) , that carries a missense mutation, R259L, in Vangl2. This mutation was specific to the Lp phenotype and absent from both parental strains and 28 other inbred strains. Notably, this mutation segregates in a recessive manner with all heterozygotes appearing normal and 47% of homozygotes showing a looped-tail. Homozygous Lp(m2Jus) embryos showed spina bifida in 12%. Lp(m2Jus) genetically interacts with Lp with 77% of compound heterozygotes displaying craniorachischisis. Vangl2(R259L) behaved like the wild-type allele in overexpression and morpholino knockdown/rescue assays in zebrafish embryos. These data suggest that Lp(m2Jus) represents a new hypomorphic allele of Lp.

An apoptotic molecular network identified by microarray: on the TRAIL to new insights in epithelial ovarian cancer.

BACKGROUND: In a previous microarray expression analysis, the authors identified candidate genes that were expressed differentially between ovarian tumors with low malignant potential and invasive serous epithelial ovarian tumors. Among them, the apoptosis-related candidate genes tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), caspase 8 (CASP8), FLICE-inhibitory protein (FLIP), and cytochrome C (CYC) were identified. METHODS: For the current study, the authors conducted immunohistochemical analyses of a tissue array comprised of 235 serous tumors of different grades and stages to evaluate whether there was differential protein expression for these candidates and for the 4 death cell receptors of Trail: Dr4, Dr5, DcR1, and DcR2. RESULTS: All proteins except DcR1 and DcR2 had significantly differential expression levels between grade 0 tumors (low malignant potential) and grade 2 and 3 tumors. Trail also showed differential expression between grade 0 tumors and grade 1 tumors. When all tumors were compared, the expression levels of Trail, Dr4, Dr5, DcR1, and Flip differed significantly between early-stage and advanced-stage disease. High Dr5 expression was associated with a poor prognosis in patients who had invasive tumors and in the subgroup of patients who had grade 3 tumors. Furthermore, the combinations of 2 proteins (Trail and Dr5, DcR2 and Cyc, Flip and Dr5, Flip and DcR2, DcR1 and Dr5 or Dr4 and Flip) revealed an association with patient prognosis. CONCLUSIONS: The identification of new proteins in the initial diagnosis and prognosis of patients with epithelial ovarian cancer may lead to a better understanding of the disease, highlighting new potential therapeutic targets, and may be useful in patient management.

SET complex in serous epithelial ovarian cancer.

With low cure rates but increasing diverse treatment options that provide variable remission times, ovarian cancer is increasingly being recognized as a chronic disease. This reality indicates the need for a better understanding of factors influencing disease progression. In a previous global analysis of gene expression, we identified genes differentially expressed when comparing serous epithelial ovarian tumors of low and high malignant potential (grade 0 vs grade 3). In this analysis, 4 out of 5 members of the SET complex, SET, APE1, NM23 and HMGB2, were highly expressed in invasive grade 3 tumors. To further investigate the expression of these genes and the fifth member of the SET complex (pp32), we performed immunohistochemistry, on a tissue array composed of 235 serous tumors of different grades and disease stages. A significant correlation between expression of all SET complex proteins and the tumor differentiation was observed (p < 0.05). When combining all tumors, overexpression of Nm23 (p = 0.04), Set (p = 0.004) and Ape1 (p = 0.004) was associated with the clinical stage of the disease. No marker by itself was associated with prognosis. The combination of a high level of Nm23 in the context of a low level of Set compared to all other combinations of these markers did confer a better prognosis (p = 0.03). When combined, high expression of Hmgb2 and low expression of Ape1 was also associated with patient prognosis (p = 0.05). These findings suggest that a strategy that sums the activities of different partners within a pathway may be more appropriate in designing nomograms for patient stratification.

Tissue array analysis of expression microarray candidates identifies markers associated with tumor grade and outcome in serous epithelial ovarian cancer.

Molecular profiling is a powerful approach to identify potential clinical markers for diagnosis and prognosis as well as providing a better understanding of the biology of epithelial ovarian cancer. On the basis of the analysis of HuFL expression data, we have previously identified genes that distinguish low malignant potential and invasive serous epithelial ovarian tumors. In this study, we used immunohistochemistry to monitor a subset of differently expressed candidates (Ahr, Paep, Madh3, Ran, Met, Mek1, Ccne1, Ccd20, Cks1 and Cas). A tissue array composed of 244 serous tumors of different grades (0-3) and stages (I-IV) was used in this analysis. All markers assayed presented differential protein expression between serous tumors of low and high grade. Significant differences in Ccne1 and Ran expression were observed in a comparison of low malignant potential and grade 1 tumor samples (p<0.01). In addition, irrespective of the grade, Ccne1, Ran, Cdc20 and Cks1 showed significant differences of expression in association with the clinical stage of disease. While high level of Ccne1 have previously been associated with poor outcomes, here we found that high level of either Ran or Cdc20 appear to be more tightly associated with a poor prognosis (p<0.001, 0.03, respectively). The application of these biomarkers in both the initial diagnosis and prognostic attributes of patients with epithelial ovarian tumors should prove to be useful in patient management.

Endothelin-1 (ET-1) promotes MMP-2 and MMP-9 induction involving the transcription factor NF-kappaB in human osteosarcoma.

In the present study, we have investigated the effect of (i) ET-1 (endothelin-1) and its precursor, big ET-1, on MMP (matrix metalloproteinase)-2 and MMP-9 synthesis and activity in osteosarcoma tissue, and (ii) ET-1 receptor antagonists on cell invasion. Using Western blotting, zymography, RT-PCR (reverse transcription-PCR), immunohistochemistry, immunofluorescence and Northern blotting, we have shown that ET-1 and ET-1 receptors (ET(A) and ET(B)) were expressed in these cells. Additionally, we have demonstrated that ET-1 markedly induced the synthesis and activity of MMP-2, which was significantly increased when compared with MMP-9. Furthermore, inhibition of NF-kappaB (nuclear factor kappaB) activation blocked MMP-2 production and activity, indicating the involvement of NF-kappaB, a ubiquitous transcription factor playing a central role in the differentiation, proliferation and malignant transformation. Since ET-1 acts as an autocrine mediator through gelatinase induction and because inhibition of ET(A) receptor is beneficial for reducing both basal and ET-1-induced osteosarcoma cell invasion, targeting this receptor could be an attractive therapeutic alternative for the successful treatment of osteosarcoma.