Background: NLRP7 is responsible for recurrent hydatidiform moles, but its functional role is unknown. Results: NLRP7 mutations impair IL-1␤ and TNF secretion but do not affect IL-1␤ processing. NLRP7 co-localizes with the microtubule organizing center, and mutations impair cytokine trafficking. Conclusion: Patients with NLRP7 mutations have abnormal tolerance to aberrant pregnancies. Significance: We unravel a new mechanism for reproductive wastage.

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset autosomal dominant muscular dystrophy that results from small expansions of a polyalanine tract in the PABPN1 gene. Intranuclear inclusions are the pathological hallmark of OPMD. The mechanism by which protein aggregation in OPMD might relate to a toxic gain-of-function has so far remained elusive. Whether protein aggregates themselves are pathogenic or are the consequence of an unidentified underlying molecular mechanism is still unclear. Here, we report that protein aggregation in a cell model of OPMD directly impaires the function of the ubiquitin-proteasome pathway (UPP) as well as molecular chaperone functions. The proteasome inhibitor lactacystin causes significant increase of protein aggregation and toxicity. Moreover, overexpression of molecular chaperones (HSP40 and HSP70) suppressed protein aggregation and toxicity. We also provide evidence that mPABPN1-ala17 protein aggregation proportionally correlates with toxicity. Furthermore, we show that co-expression of chaperones in our OPMD cell model increases the solubility of mPABPN1-ala17 and transfected cell survival rate. Our studies suggest that molecular regulators of polyalanine protein solubility and degradation may provide insights into new mechanisms in OPMD pathogenesis. Further analysis of the cellular and molecular mechanisms by which UPP and molecular chaperones influence the degradation of misfolded proteins could provide novel concepts and targets for the treatment and understanding of the pathogenesis of OPMD and neurodegenerative diseases.

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Background NLRP7 mutations are responsible for recurrent molar pregnancies and associated reproductive wastage. To investigate the role of NLRP7 in sporadic moles and other forms of reproductive wastage, the authors sequenced this gene in a cohort of 135 patients with at least one hydatidiform mole or three spontaneous abortions; 115 of these were new patients. Methods/Results All mutations were reviewed and their number, nature and locations correlated with the reproductive outcomes of the patients and histopathology of their products of conception. The presence of NLRP7 mutations was demonstrated in two patients with recurrent spontaneous abortions, and some rare non-synonymous variants (NSVs), present in the general population, were found to be associated with recurrent reproductive wastage. These rare NSVs were shown to be associated with lower secretion of interleukin 1b and tumour necrosis factor and therefore to have functional consequences similar to those seen in cells from patients with NLRP7 mutations. The authors also attempted to elucidate the cause of stillbirths observed in 13% of the patients with NLRP7 mutations by examining available placentas of the stillborn babies and live births from patients with mutations or rare NSVs. A number of severe to mild placental abnormalities were found, all of which are known risk factors for perinatal morbidity. Conclusions The authors recommend close follow-up of patients with NLRP7 mutations and rare NSVs to prevent the death of the rare or reduced number of babies that reach term.

Autosomal dominant oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disease that presents in the fifth or sixth decade. The disease is characterized by progressive eyelid drooping (ptosis), swallowing difficulties (dysphagia) and proximal limb weakness. [1][2][3][4][5][6] Pathological studies showed the presence of unique intranuclear filamentous inclusions in skeletal muscle fibers of OPMD patients. 7,8 The OPMD locus was mapped by linkage analysis to chromosome 14q11.1 9-11 and the gene was identified as PABPN1, encoding the poly(A) binding protein nuclear 1 (PABPN1, PABP2, PAB II). 12 Dominant OPMD is caused by expansion of a short GCG ABSTRACT: Background: Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by progressive ptosis, dysphagia and proximal limb weakness. The autosomal dominant form of this disease is caused by short expansions of a (GCG) 6 repeat to (GCG) [8][9][10][11][12][13] in the PABPN1 gene. The mutations lead to the expansion of a polyalanine stretch from 10 to 12-17 alanines in the N-terminus of PABPN1. The mutated PABPN1 (mPABPN1) induces the formation of intranuclear filamentous inclusions that sequester poly(A) RNA and are associated with cell death. Methods: Human fetal brain cDNA library was used to look for PABPN1 binding proteins using yeast two-hybrid screen. The protein interaction was confirmed by GST pull-down and co-immunoprecipitation assays. Oculopharyngeal muscular dystrophy cellular model and OPMD patient muscle tissue were used to check whether the PABPN1 binding proteins were involved in the formation of OPMD intranuclear inclusions. Results: We identify two PABPN1 interacting proteins, hnRNP A1 and hnRNP A/B. When co-expressed with mPABPN1 in COS-7 cells, predominantly nuclear protein hnRNP A1 and A/B colocalize with mPABPN1 in the insoluble intranuclear aggregates. Patient studies showed that hnRNP A1 is sequestered in OPMD nuclear inclusions. Conclusions: The hnRNP proteins are involved in mRNA processing and mRNA nucleocytoplasmic export, sequestering of hnRNPs in OPMD intranuclear aggregates supports the view that OPMD intranuclear inclusions are "poly(A) RNA traps", which would interfere with RNA export, and cause muscle cell death. RÉSUMÉ: Interaction de hnRNP A1 et A/B avec PABPN1 dans la dystrophie musculaire oculopharyngée. Introduction: La dystrophie musculaire oculopharyngée (DMOP) est une maladie de l'âge adulte caractérisée par une ptose progressive des paupières, une dysphagie et une faiblesse musculaire proximale. La forme autosomique dominante est causée par de courtes expansions d'une répétition (GCG)6 à (GCG)8-13 dans le gène PABPN1. Les mutations donnent lieu à une expansion d'un tractus de polyalanine de 10 à 12-17 alanines dans la partie N-terminale de PABPN1. Le gène PABPN1 muté (PABPN1m) induit la formation d'inclusions filamenteuses intranucléaires qui séquestrent l'ARN poly(A) et entraînent la mort cellulaire. Méthodes: Une librairie d'ADNc provenant de cerveau foetal humain a été utilisée pour chercher...

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Mutations in NLRP7 (NOD-like-receptor family, pyrin domain containing 7) are responsible for a type of recurrent pregnancy loss known as recurrent hydatidiform mole (HYDM1). This condition is characterized by abnormal growth of the placenta, a lack of proper embryonic development and abnormal methylation patterns at multiple imprinted loci in diploid biparental molar tissues. The role of NLRP7 protein in the disease manifestation is currently not clear. In order to better understand how the effects of HYDM1 are associated with mutations on the structure of NLRP7, we performed an inter-domain interaction screen using a yeast two-hybrid system. Additionally, we generated in silico structural models of NLRP7 in its non-activated and activated forms. Our observations from the yeast two-hybrid screen and modeling suggest that the NACHT-associated domain (NAD) of the NLRP7 protein is central to its oligomeric assembly. Upon activation, the NAD and a small part of the leucine rich repeat (LRR) of one molecule emerged out of the protective LRR domain and interact with the NACHT domain of the second molecule to form an oligomer. Furthermore, we investigated the molecular basis for the pathophysiological effect of four missense mutations, three HYDM1-causing and one rare non-synonymous variant, on the protein using confocal microscopy of transiently transfected NLRP7 in HEK293T cells and in silico structural analysis. We found that with the two clinically severe missense mutations, L398R and R693W, the normal molecule to molecule interaction was apparently affected thus decreasing their oligomerization potential while aggresome formation was increased; these changes could disturb the normal downstream functions of NLRP7 and therefore be a possible molecular effect underlying their pathophysiological impact.