A mutation in the ZNF687 gene that is responsible for the severe form of Paget's disease of bone causes severely altered bone remodeling and promotes hepatocellular carcinoma onset in a knock-in mouse model.

Paget's disease (PDB) is a late-onset bone remodeling disorder with a broad spectrum of symptoms and complications. One of the most aggressive forms is caused by the P937R mutation in the ZNF687 gene. Although the genetic involvement of ZNF687 in PDB has been extensively studied, the molecular mechanisms underlying this association remain unclear. Here, we describe the first Zfp687 knock-in mouse model and demonstrate that the mutation recapitulates the PDB phenotype, resulting in severely altered bone remodeling. Through microcomputed tomography analysis, we observed that 8-month-old mutant mice showed a mainly osteolytic phase, with a significant decrease in the trabecular bone volume affecting the femurs and the vertebrae. Conversely, osteoblast activity was deregulated, producing disorganized bone. Notably, this phenotype became pervasive in 16-month-old mice, where osteoblast function overtook bone resorption, as highlighted by the presence of woven bone in histological analyses, consistent with the PDB phenotype. Furthermore, we detected osteophytes and intervertebral disc degeneration, outlining for the first time the link between osteoarthritis and PDB in a PDB mouse model. RNA sequencing of wild-type and Zfp687 knockout RAW264.7 cells identified a set of genes involved in osteoclastogenesis potentially regulated by Zfp687, e.g., Tspan7, Cpe, Vegfc, and Ggt1, confirming its role in this process. Strikingly, in this mouse model, the mutation was also associated with a high penetrance of hepatocellular carcinomas. Thus, this study established an essential role of Zfp687 in the regulation of bone remodeling, offering the potential to therapeutically treat PDB, and underlines the oncogenic potential of ZNF687.

Profilin 1 deficiency drives mitotic defects and reduces genome stability.

Profilin 1-encoded by PFN1-is a small actin-binding protein with a tumour suppressive role in various adenocarcinomas and pagetic osteosarcomas. However, its contribution to tumour development is not fully understood. Using fix and live cell imaging, we report that Profilin 1 inactivation results in multiple mitotic defects, manifested prominently by anaphase bridges, multipolar spindles, misaligned and lagging chromosomes, and cytokinesis failures. Accordingly, next-generation sequencing technologies highlighted that Profilin 1 knock-out cells display extensive copy-number alterations, which are associated with complex genome rearrangements and chromothripsis events in primary pagetic osteosarcomas with Profilin 1 inactivation. Mechanistically, we show that Profilin 1 is recruited to the spindle midzone at anaphase, and its deficiency reduces the supply of actin filaments to the cleavage furrow during cytokinesis. The mitotic defects are also observed in mouse embryonic fibroblasts and mesenchymal cells deriving from a newly generated knock-in mouse model harbouring a Pfn1 loss-of-function mutation. Furthermore, nuclear atypia is also detected in histological sections of mutant femurs. Thus, our results indicate that Profilin 1 has a role in regulating cell division, and its inactivation triggers mitotic defects, one of the major mechanisms through which tumour cells acquire chromosomal instability.

The Osteoclast Traces the Route to Bone Tumors and Metastases.

Osteoclasts are highly specialized cells of the bone, with a unique apparatus responsible for resorption in the process of bone remodeling. They are derived from differentiation and fusion of hematopoietic precursors, committed to form mature osteoclasts in response to finely regulated stimuli produced by bone marrow-derived cells belonging to the stromal lineage. Despite a highly specific function confined to bone degradation, emerging evidence supports their relevant implication in bone tumors and metastases. In this review, we summarize the physiological role of osteoclasts and then focus our attention on their involvement in skeletal tumors, both primary and metastatic. We highlight how osteoclast-mediated bone erosion confers increased aggressiveness to primary tumors, even those with benign features. We also outline how breast and pancreas cancer cells promote osteoclastogenesis to fuel their metastatic process to the bone. Furthermore, we emphasize the role of osteoclasts in reactivating dormant cancer cells within the bone marrow niches for manifestation of overt metastases, even decades after homing of latent disseminated cells. Finally, we point out the importance of counteracting tumor progression and dissemination through pharmacological treatments based on a better understanding of molecular mechanisms underlying osteoclast lytic activity and their recruitment from cancer cells.

Modulation of Endocannabinoid Tone in Osteoblastic Differentiation of MC3T3-E1 Cells and in Mouse Bone Tissue over Time.

Bone is a highly complex and metabolically active tissue undergoing a continuous remodeling process, which endures throughout life. A complex cell-signaling system that plays role in regulating different physiological processes, including bone remodeling, is the endocannabinoid system (ECS). Bone mass expresses CB1 and CB2 cannabinoid receptors and enzymatic machinery responsible for the metabolism of their endogenous ligands, endocannabinoids (AEA and 2-AG). Exogenous AEA is reported to increase the early phase of human osteoblast differentiation in vitro. However, regarding this cell context little is known about how endocannabinoids and endocannabinoid-related N-acylethanolamines like PEA and OEA are modulated, in vitro, during cell differentiation and, in vivo, over time up to adulthood. Here we characterized the endocannabinoid tone during the different phases of the osteoblast differentiation process in MC3T3-E1 cells, and we measured endocannabinoid levels in mouse femurs at life cycle stages characterized by highly active bone growth (i.e., of juvenile, young adult, and mature adult bone). Endocannabinoid tone was significantly altered during osteoblast differentiation, with substantial OEA increment, decline in 2-AG and AEA, and consistent modulation of their metabolic enzymes in maturing and mineralized MC3T3-E1 cells. Similarly, in femurs, we found substantial, age-related, decline in 2-AG, OEA, and PEA. These findings can expand existing knowledge underlying physiological bone cell function and contribute to therapeutic strategies for preventing bone-related metabolic changes accruing through lifespan.

Identification of sixteen novel candidate genes for late onset Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative movement disorder affecting 1-5% of the general population for which neither effective cure nor early diagnostic tools are available that could tackle the pathology in the early phase. Here we report a multi-stage procedure to identify candidate genes likely involved in the etiopathogenesis of PD. METHODS: The study includes a discovery stage based on the analysis of whole exome data from 26 dominant late onset PD families, a validation analysis performed on 1542 independent PD patients and 706 controls from different cohorts and the assessment of polygenic variants load in the Italian cohort (394 unrelated patients and 203 controls). RESULTS: Family-based approach identified 28 disrupting variants in 26 candidate genes for PD including PARK2, PINK1, DJ-1(PARK7), LRRK2, HTRA2, FBXO7, EIF4G1, DNAJC6, DNAJC13, SNCAIP, AIMP2, CHMP1A, GIPC1, HMOX2, HSPA8, IMMT, KIF21B, KIF24, MAN2C1, RHOT2, SLC25A39, SPTBN1, TMEM175, TOMM22, TVP23A and ZSCAN21. Sixteen of them have not been associated to PD before, were expressed in mesencephalon and were involved in pathways potentially deregulated in PD. Mutation analysis in independent cohorts disclosed a significant excess of highly deleterious variants in cases (p = 0.0001), supporting their role in PD. Moreover, we demonstrated that the co-inheritance of multiple rare variants (≥ 2) in the 26 genes may predict PD occurrence in about 20% of patients, both familial and sporadic cases, with high specificity (> 93%; p = 4.4 × 10- 5). Moreover, our data highlight the fact that the genetic landmarks of late onset PD does not systematically differ between sporadic and familial forms, especially in the case of small nuclear families and underline the importance of rare variants in the genetics of sporadic PD. Furthermore, patients carrying multiple rare variants showed higher risk of manifesting dyskinesia induced by levodopa treatment. CONCLUSIONS: Besides confirming the extreme genetic heterogeneity of PD, these data provide novel insights into the genetic of the disease and may be relevant for its prediction, diagnosis and treatment.

Early Alpine occupation backdates westward human migration in Late Glacial Europe.

Before the end of the Last Glacial Maximum (LGM, ∼16.5 ka ago)1 set in motion major shifts in human culture and population structure,2 a consistent change in lithic technology, material culture, settlement pattern, and adaptive strategies is recorded in Southern Europe at ∼18-17 ka ago. In this time frame, the landscape of Northeastern Italy changed considerably, and the retreat of glaciers allowed hunter-gatherers to gradually recolonize the Alps.3-6 Change within this renewed cultural frame (i.e., during the Late Epigravettian phase) is currently associated with migrations favored by warmer climate linked to the Bølling-Allerød onset (14.7 ka ago),7-11 which replaced earlier genetic lineages with ancestry found in an individual who lived ∼14 ka ago at Riparo Villabruna, Italy, and shared among different contexts (Villabruna Cluster).9 Nevertheless, these dynamics and their chronology are still far from being disentangled due to fragmentary evidence for long-distance interactions across Europe.12 Here, we generate new genomic data from a human mandible uncovered at Riparo Tagliente (Veneto, Italy), which we directly dated to 16,980-16,510 cal BP (2σ). This individual, affected by focal osseous dysplasia, is genetically affine to the Villabruna Cluster. Our results therefore backdate by at least 3 ka the diffusion in Southern Europe of a genetic component linked to Balkan/Anatolian refugia, previously believed to have spread during the later Bølling/Allerød event. In light of the new genetic evidence, this population replacement chronologically coincides with the very emergence of major cultural transitions in Southern and Western Europe.

Novel autophagic vacuolar myopathies: Phenotype and genotype features.

BACKGROUND: Autophagic vacuolar myopathies (AVMs) are an emerging group of heterogeneous myopathies sharing histopathological features on muscle pathology, in which autophagic vacuoles are the pathognomonic morphologic hallmarks. Glycogen storage disease type II (GSDII) caused by lysosomal acid α-glucosidase (GAA) deficiency is the best-characterised AVM. AIMS: This study aimed to investigate the mutational profiling of seven neuromuscular outpatients sharing clinical, myopathological and biochemical findings with AVMs. METHODS: We applied a diagnostic protocol, recently published by our research group for suspected late-onset GSDII (LO-GSDII), including counting PAS-positive lymphocytes on blood smears, dried blood spot (DBS)-GAA, muscle biopsy histological and immunofluorescence studies, GAA activity assay and expression studies on muscle homogenate, GAA sequencing, GAA multiplex ligation-dependent probe amplification (MLPA) and whole exome sequencing (WES). RESULTS: The patients had a limb girdle-like muscular pattern with persistent hyperCKaemia; vacuolated PAS-positive lymphocytes, glycogen accumulation and impaired autophagy at muscle biopsy. Decreased GAA activity was also measured. While GAA sequencing identified no pathogenic mutations, WES approach allowed us to identify for each patient an unexpected mutational pattern in genes cooperating in lysosomal-autophagic machinery, some of which have never been linked to human diseases. CONCLUSIONS: Our data suggest that reduced GAA activity may occur in any condition of impaired autophagy and that WES approach is advisable in all genetically undefined cases of autophagic myopathy. Therefore, deficiency of GAA activity and PAS-positive lymphocytes should be considered as AVM markers together with LC3/p62-positive autophagic vacuoles.

The two faces of giant cell tumor of bone.

Giant cell tumor (GCT) is a bone-destructive benign neoplasm characterized by distinctive multinucleated osteoclast-like giant cells with osteolytic properties distributed among neoplastic stromal cells. GCT is locally aggressive with progressive invasion of adjacent tissues and occasionally displays malignant characteristics including lung metastasis. GCT is characterized genetically by highly recurrent somatic mutations at the G34 position of the H3F3A gene, encoding the histone variant H3.3, in stromal cells. This leads to deregulated gene expression and increased proliferation of mutation-bearing cells. However, when GCT complicates Paget disease of bone (GCT/PDB) it behaves differently, showing a more malignant phenotype with 5-year survival less than 50%. GCT/PDB is caused by a germline mutation in the ZNF687 gene, which encodes a transcription factor involved in the repression of genes surrounding DNA double-strand breaks to promote repair by homologous recombination. Identification of these driver mutations led to novel diagnostic tools for distinguishing between these two tumors and other osteoclast-rich neoplasms. Herein, we review the clinical, histological, and molecular features of GCT in different contexts focusing also on pharmacological treatments.

ZNF687 Mutations in an Extended Cohort of Neoplastic Transformations in Paget's Disease of Bone: Implications for Clinical Pathology.

Neoplastic transformation is a rare but serious complication of Paget's disease of bone (PDB), occurring in fewer than 1% of individuals with polyostotic disease. Their prognosis is poor, with less than 50% surviving 5 years. In 2016, the genetic alteration of giant cell tumor (GCT) complicating PDB was identified as a founder germline mutation (P937R) in the ZNF687 gene. However, the study population was exclusively of Italian descent, and patients of different ethnic origins were not studied. To fill this gap, herein we performed mutation analysis of ZNF687 in a GCT in the pelvis of a 45-year-old black American woman with polyostotic PDB. The P937R mutation in ZNF687 was found in her tumor but, as expected, the ancestral haplotype that characterizes the Italian GCT/PDB patients was not found. Furthermore, we identified two additional Italian GCT/PDB patients with this ZNF687 mutation, now constituting a cohort of 18 GCT/PDB cases, all harboring the identical mutation. We also searched for ZNF687 mutations in a unique collection of tumor tissues derived from Italian PDB patients, including 28 osteosarcomas (OS/PDB), 8 undifferentiated sarcomas (SRC/PDB), 1 fibrosarcoma (FS/PDB), and 1 chondrosarcoma (CS/PDB). We identified the P937R mutation in one SRC/PDB and a different ZNF687 mutation (R331W) in 1 of 28 pagetic osteosarcomas. Thus, whereas GCT/PDB pathogenesis globally seems to involve the P937R mutation in ZNF687, other neoplasms associated with PDB seem to be less related to mutations in this gene. Finally, we identified the G34W mutation in the H3F3A gene in the maxillary tumor masses of two PDB patients, defining them as conventional GCT rather than GCT/PDB. Thus, combined molecular analysis of H3F3A and ZNF687 is essential to clarify the origin and diagnosis of tumors in PDB. © 2020 American Society for Bone and Mineral Research.

The Loss of Profilin 1 Causes Early Onset Paget's Disease of Bone.

Paget's disease of bone (PDB) is a late-onset disorder frequently caused by mutations in the SQSTM1 gene, leading to hyperactive osteoclasts and resulting in bone pain, deformities, and fractures. However, some more severe forms of PDB-negative for SQSTM1 mutations-have been described, in which the disease degenerates into bone cancers and shows a poor prognosis. Osteosarcoma is the most frequent and aggressive tumor arising in PDB (OS/PDB), with a 5-year survival rate almost nil, but the underlying molecular mechanism is unknown. Here, we investigated an extended pedigree with 11 individuals affected by early onset and polyostotic PDB, mainly interesting the appendicular skeleton. Interestingly, three members also developed secondary osteosarcoma. We performed exome sequencing and identified a 4-bp deletion in the PFN1 gene, resulting in the degradation of the mutant protein. Copy number screening on 218 PDB individuals of our biobank disclosed that four of them (~2%) carry a germline heterozygous deletion of PFN1. The identification of these subjects, who exhibit a particularly severe form of disease, emphasizes the diagnostic value of this genetic screening to identify PDB individuals predisposed to develop osteosarcoma. In fact, we detected allelic imbalance at PFN1 locus also in 8 of 14 (57%) sporadic OS/PDB, further proving its causative role. in vitro experiments also confirmed PFN1 involvement in this form of PDB. Indeed, CRISPR-Cas9-mediated Pfn1 knockout in pre-osteoclasts resulted into enhanced osteoclast differentiation and resorption, with the formation of large osteoclasts never described before in PDB. In addition, Pfn1 lacking pre-osteoblasts lost their differentiation capability and failed to efficiently mineralize bone. Moreover, they acquired features of malignant transformation, including loss of focal adhesions and increased invasion ability. In conclusion, these findings disclose PFN1 haploinsufficiency as the pathological mechanism in OS/PDB. © 2020 American Society for Bone and Mineral Research.

Early posterior vitreous detachment is associated with LAMA5 dominant mutation.

BACKGROUND: Extracellular matrix molecular components, previously linked to multisystem syndromes include collagens, fibrillins and laminins. Recently, we described a novel multisystem syndrome caused by the c.9418G>A p.(V3140M) mutation in the laminin alpha-5 (LAMA5) gene, which affects connective tissues of all organs and apparatus in a three generation family. In the same family, we have also reported a myopic trait, which, however, was linked to the Prolyl 4-hydroxylase subunit alpha-2 (P4HA2) gene. Results of investigation on vitreous changes and their pathogenesis are reported in the present study. MATERIALS AND METHODS: Nineteen family individuals underwent complete ophthalmic examination including best-corrected visual acuity (BCVA), fundus examination, fundus photography, intraocular pressure measurement, axial length measurement using ocular biometry, Goldmann visual field examination, standard electroretinogram, SD-OCT. Segregation analysis of LAMA5 and P4HA2 mutations was performed in enrolled members. RESULTS: The vitreous alterations fully segregated with LAMA5 mutation in both young and adult family members. Slight reduction of retinal thickness and peripheral retinal degeneration in only two patients were reported. CONCLUSIONS: In this work we showed that PVD is a common trait of LAMA5 multisystem syndrome, therefore occurring as an age-unrelated trait. We hypothesize that the p.(V3140M) mutation results in a reduction of retinal inner limiting membrane (ILM) stability, leading to a derangement in the macromolecular structure of the vitreous gel, and PVD. Further investigations will be necessary to elucidate the role of wild type and mutated LAMA5 in the pathogenesis of PVD.

The identification of H3F3A mutation in giant cell tumour of the clivus and the histological diagnostic algorithm of other clival lesions permit the differential diagnosis in this location.

BACKGROUND: Giant Cell Tumour of Bone (GCT) is a locally aggressive primary bone tumour that usually occurs at the epiphyses of the long bones of the appendicular skeleton with a tendency to recurrence. Recurrent somatic H3F3A mutations have been described in 92% of GCT cases. GCTs involving the Clivus are extremely rare lesions and less than 15 cases are described in the literature. They represent a surgery challenge and are easily misdiagnosed. Our aim was to reveal if the genetic bases underlying Clival GCTs were the same of GCTs of long bones to improve the diagnosis and treatment. METHODS: The targeted somatic sequencing of GCT-related genes (H3F3A, H3F3B, IDH1, IDH2 and ZNF687) was performed on Clival GCT biopsies of two different cases. Histological analyses on the same tissues were used to detect the neoplastic population and its expression profile. RESULTS: Sanger sequencing revealed that both patients were positive for the p.Gly34Trp mutation in the H3F3A gene. Immunofluorescence assay using monoclonal antibody, specifically detecting the mutant H3.3, highlighted that the mutation only involved the mononuclear cell population and not the multinucleated giant cells. Moreover, immunohistochemistry assay showed that RANKL was highly expressed by the stromal cells within Clival GCT, mimicking what happens in GCT of the long bones. In addition, systematic literature review allowed us to generate a histology-based diagnostic algorithm of the most common clival lesions. CONCLUSIONS: We conclude that the Clival GCT is genetically defined by somatic mutation in the H3F3A gene, linking it to the GCT of long bones. The similarity with GCTs of long bones let us to hypothesize the utility of Denosumab therapy (already effective for GCTs) in these surgically challenging cases. Moreover, H3F3A genetic screening can be combined to the histological analysis to differentiate GCTs from morphologically similar giant cell-rich sarcomas, while the histological diagnostic algorithm could help the differential diagnosis of other clival lesions.

Effect of genetic variants of OPTN in the pathophysiology of Paget's disease of bone.

Paget's disease of bone (PDB) is the second most frequent metabolic bone disease after osteoporosis. Genetic factors play an important role in PDB, but to date PDB causing mutations were identified only in the Sequestosome 1 gene at the PDB3 locus. OPTN has been recently associated with PDB, however little is known about the effect of genetic variants in this gene in PDB pathophysiology. By sequencing OPTN in SQSTM1 non-carriers PDB patients we found 16 SNPs in regulatory, coding and non-coding regions. One of those was found to be associated with PDB in our cohort - rs2234968. Our results show that rs2238968 effect may be explained by a change in OPTN splicing that give rise to a predicted truncated protein. We also performed functional studies on the variants located in OPTN promoter - rs3829923 and the rare variant -9906 - to investigate putative regulators of OPTN. Our results show that OPTN expression seems to be regulated by SP1, RXR, E47, and the E2F family. In conclusion, our work suggests a potential pathophysiological role of SNPs in OPTN, giving a new perspective about the regulatory mechanisms of this gene. Ultimately we discovered a new variant associated with PDB in OPTN, reinforcing the relevance of this gene for the development of this bone disease.

The distinct clinical features of giant cell tumor of bone in pagetic and non-pagetic patients are associated with genetic, biochemical and histological differences.

Giant Cell Tumor of Bone (GCT) is a tumor characterized by neoplastic mesenchymal stromal cells and a high number of osteoclast-like multinucleated giant cells. Rarely, GCT could arise in bones affected by Paget's disease of bone (GCT/PDB). Although it is already known that GCT/PDB and GCT show a different clinical profile regarding the age-onset and skeletal localization, our deep clinical comparison between the two GCT/PDB and GCT cohorts, permitted us to identify additional differences (e.g. focality, ALP serum levels, the 5-year survival rate and the familial recurrence), strongly suggesting a different molecular basis. Accordingly, driver somatic mutations in H3F3A and IDH2 were described in GCT patients, while we recently identified a germline mutation in ZNF687 as the genetic defect of GCT/PDB patients. Here, we detected H3F3A mutations in our GCT cohort, confirming its molecular screening as the elected diagnostic tool, and then we excluded the two-hit in H3F3A and IDH2 as the trigger event for the GCT/PDB development. Importantly, we also identified an alternative biochemical profile with GCT/PDB not exhibiting the up-regulation of the GCT marker FGFR2IIIc. Finally, our histological analysis also showed a different appearance of the two forms of the tumor, with GCT/PDB showing a higher number of osteoclast-like giant cells (twice), with an abnormal number of nuclei per cell, corroborating its different behaviour in terms of neoplastic properties. We demonstrated that the distinct clinical features of pagetic and conventional GCT are associated with different genetic background, resulting in a specific biochemical and histological behaviour of the tumour.

Identification of the first dominant mutation of LAMA5 gene causing a complex multisystem syndrome due to dysfunction of the extracellular matrix.

BACKGROUND: The laminin alpha 5 gene (LAMA5) plays a master role in the maintenance and function of the extracellular matrix (ECM) in mammalian tissues, which is critical in developmental patterning, stem cell niches, cancer and genetic diseases. Its mutations have never been reported in human disease so far. The aim of this study was to associate the first mutation in LAMA5 gene to a novel multisystem syndrome. METHODS: A detailed characterisation of a three-generation family, including clinical, biochemical, instrumental and morphological analysis, together with genetics and expression (WES and RNAseq) studies, was performed. RESULTS: The heterozygous LAMA5 mutation c.9418G>A (p.V3140M) was associated with skin anomalies, impaired scarring, night blindness, muscle weakness, osteoarthritis, joint and internal organs ligaments laxity, malabsorption syndrome and hypothyroidism. We demonstrated that the mutation alters the amount of LAMA5 peptides likely derived from protein cleavage and perturbs the activation of the epithelial-mesenchymal signalling, producing an unbalanced expression of Sonic hedgehog and GLI1, which are upregulated in cells from affected individuals, and of ECM proteins (COL1A1, MMP1 and MMP3), which are strongly inhibited. Studies carried out using human skin biopsies showed alteration of dermal papilla with a reduction of the germinative layer and an early arrest of hair follicle downgrowth. The knock-in mouse model, generated in our laboratory, shows similar changes in the tissues studied so far. CONCLUSIONS: This is the first report of a disease phenotype associated with LAMA5 mutation in humans.

Dysregulation of the Expression of Asparagine-Linked Glycosylation 13 Short Isoform 2 Affects Nephrin Function by Altering Its N-Linked Glycosylation.

BACKGROUND: N-linked glycosylation, which is a post-translational modification process, plays an important role in protein folding, intracellular trafficking and membrane targeting, as well as in regulating the protein function. Recently, we identified a missense variant (p.T141L) in the short isoform 2 of the X-linked gene asparagine-linked glycosylation 13 (ALG13-is2), which segregated with focal segmental glomerulosclerosis and PCCD in a large Australian pedigree; however, any evidence of its pathogenicity was demonstrated. ALG13 gene encodes, through alternative splicing, 2 glycosyltransferase isoforms, which catalyse the second sugar addition of the highly conserved oligosaccharide precursor in the endoplasmic reticulum (ER). Mutations in the long isoform 1 were associated with epilepsy. METHODS AND RESULTS: Here, we show a different expression of the 2 isoforms depending on the tissue. Specifically, the long isoform is highly expressed in lungs, ovaries, testes, cerebellum, cortex, retina, pituitary gland, and olfactory bulbs, while the short isoform is highly expressed in mouse podocytes and in human podocyte cell lines, at both mRNA and protein levels. The silencing of ALG13-is2 by specific siRNAs induces an altered N-linked glycosylation pattern of nephrin, as demonstrated by the presence of an additional immunostaining band of about 130 kD. In knock-down cells, immunofluorescence analysis shows perturbed organization of the cytoskeleton and altered localization of nephrin on the cellular membrane. We also demonstrated that the altered pattern of N-linked glycosylation induces an over-expression of binding immunoglobulin protein and calreticulin, suggesting ER stress. CONCLUSIONS: These results provide preliminary evidence that ALG13-is2 could be an important modifier of renal filtration defects.

Evidence for epistatic interaction between VDR and SLC13A2 genes in the pathogenesis of hypocitraturia in recurrent calcium oxalate stone formers.

BACKGROUND: Genetic factors play a key role in the pathogenesis of hypocitraturia, a common risk factor for nephrolithiasis. The Na+-dicarboxylate cotransporter NaDC1, encoded by the sodium-dicarboxylate cotransporter (SLC13A2) gene, is a major determinant of urinary citrate excretion and its biological functions are regulated also by the vitamin D/Vitamin D receptor (VDR) biological system. The aim of this case-control study was to evaluate the possible epistatic interaction between VDR rs731236and SLC13A2 rs11567842 allelic variants in the pathogenesis of hypocitraturia. METHODS: Recurrent calcium-oxalate stone formers (SF) with or without hypocitraturia and healthy controls (C) were genotyped. Gene-gene interactions were estimated by the 1.0 software package of multifactor dimensionality reduction (MDR). RESULTS: The prevalence of VDR TT and SLC13A2 GG genotypes was higher in hypocitraturic SF compared to C (odds ratio [OR] 3.24, 95 % confidence interval [CI] 1.38-7.60 for VDR TT vs. VDR tt and OR 4.06, 95 % CI 1.75-9.42 for SLC13A2 GG vs. SLC13A2 AA ). MDR analysis indicated a significant interaction between VDR TT and SLC13A2 GG in hypocitraturic SF compared to C [OR 3.81 (2.11-6.88)]. These data are compatible with an epistatic interaction between the VDR TT and SLC13A2 GG genotypes with a significant impact on the magnitude of the effect (suppressive effect). CONCLUSIONS: These results point to an epistatic interaction between the VDR and the SLC13A2 alleles in the pathogenesis of idiopathic hypocitraturia in calcium-oxalate SF.

ZNF687 Mutations in Severe Paget Disease of Bone Associated with Giant Cell Tumor.

Paget disease of bone (PDB) is a skeletal disorder characterized by focal abnormalities of bone remodeling, which result in enlarged and deformed bones in one or more regions of the skeleton. In some cases, the pagetic tissue undergoes neoplastic transformation, resulting in osteosarcoma and, less frequently, in giant cell tumor of bone (GCT). We performed whole-exome sequencing in a large family with 14 PDB-affected members, four of whom developed GCT at multiple pagetic skeletal sites, and we identified the c.2810C>G (p.Pro937Arg) missense mutation in the zinc finger protein 687 gene (ZNF687). The mutation precisely co-segregated with the clinical phenotype in all affected family members. The sequencing of seven unrelated individuals with GCT associated with PDB (GCT/PDB) identified the same mutation in all individuals, unravelling a founder effect. ZNF687 is highly expressed during osteoclastogenesis and osteoblastogenesis and is dramatically upregulated in the tumor tissue of individuals with GCT/PDB. Interestingly, our preliminary findings showed that ZNF687, indicated as a target gene of the NFkB transcription factor by ChIP-seq analysis, is also upregulated in the peripheral blood of PDB-affected individuals with (n = 5) or without (n = 6) mutations in SQSTM1, encouraging additional studies to investigate its potential role as a biomarker of PDB risk.

Hypovitaminosis D and organ damage in patients with arterial hypertension: a multicenter double blind randomised controlled trial of cholecalciferol supplementation (HYPODD) : study design, clinical procedures and treatment protocol.

INTRODUCTION: At this time, good quality randomized clinical trials assessing the effects of vitamin D supplementation on cardiometabolic outcomes are lacking in the international literature. AIM: To fill this gap, the Working Group on Vitamin D and Cardiorenal Disorders established jointly by the Italian Society of Hypertension (SIIA) and the Forum in Bone and Mineral Research conceived the HYPODD study (HYPOvitaminosis D and organ Damage). METHODS: HYPODD is a no-profit multicenter 12-month parallel-group double-blind placebo controlled randomized trial aiming to assess the effects of cholecalciferol supplementation on blood pressure control, antihypertensive drugs consumption and progression of target organ damage in patients with essential hypertension and 25-hydroxyvitamin D serum level lower than 20 ng/ml (vitamin D deficiency). HYPODD is coordinated by the European Society Excellence Center of Hypertension of Federico II University, Naples, and involves 12 academic institutions in Italy (Ancona, Milan, Padua, Perugia, Rome, Siena, Trieste, Turin, Udine, Varese, and Verona). RESULTS AND CONCLUSION: The HYPODD study has been registered at the Agenzia Italiana del Farmaco-Osservatorio sulla Sperimentazione Clinica del Farmaco (AIFA-OsSC) and EUDRACT sites (n° 2012-003514-14) and has been approved by the Ethical Committees of all the Centers involved in the study. The patients' recruitment is currently underway.

Clinical characteristics and evolution of giant cell tumor occurring in Paget's disease of bone.

Patients with Paget's bone disease (PDB) have an increased risk of developing giant cell tumor (GCT). This study was performed to evaluate the clinical characteristics and evolution of GCT complicating PDB and to compare these clinical characteristics to those observed in two large PDB cohorts, the PDB Italian Registry and the United Kingdom's Multi-Centre Randomised Controlled Trial of Symptomatic Versus Intensive Bisphosphonate Therapy for Paget's Disease (PRISM) study. A systematic literature review identified 117 cases of PDB complicated by GCT (PDB-GCT), which involved the skeletal sites affected by PDB (110 patients) or the extraskeletal tissues adjacent to affected bones (7 patients). In contrast to what previously reported for GCT patients without GCT patients (83.2%) were white and one-fourth of them (24.8%) had multifocal GCTs. Compared to PDB patients without GCT, PDB-GCT patients showed a higher male/female ratio (2.1 versus 1.2) and more severe disease (age at PDB onset 52.1 ± 12.1 versus 63.3 ± 10.6 years; number of affected sites 6.1 ± 2.9 versus 2.34 ± 1.6; prevalence of polyostotic PDB 93.3% versus 60.6%). The mortality rate of PDB-GCT patients was higher than those occurring in GCT patients without PDB (about 50% versus 0% to 5% at 5 years) or in PDB patients without GCT (log rank = 29.002). Moreover, up to 98% of PDB-GCT cases had elevated total alkaline phosphatase levels at neoplasm diagnosis, suggestive of active PDB. Importantly, PDB-GCT patients from Southern Italy (45.6% of all GCT patients) showed a higher prevalence of multifocal GCT (51.7%) and of positive familial history for PDB (70.8%) and GCT (65.0%). Finally, indirect evidence suggests a decline in the incidence of GCT in PDB patients. The occurrence of GCT in PDB patients is associated with severe disease and reduced life expectancy of affected patients. The increased prevalence of familial diseases in PDB-GCT patients from Southern Italy suggests a founder effect. The observed changes over time in the incidence of GCT in PDB patients could be related to improved clinical management and/or living conditions of patients.