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.

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.

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 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.

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.

Paget disease of bone-associated UBA domain mutations of SQSTM1 exert distinct effects on protein structure and function.

SQSTM1 mutations are common in patients with Paget disease of bone (PDB), with most affecting the C-terminal ubiquitin-associated (UBA) domain of the SQSTM1 protein. We performed structural and functional analyses of two UBA domain mutations, an I424S mutation relatively common in UK PDB patients, and an A427D mutation associated with a severe phenotype in Southern Italian patients. Both impaired SQSTM1's ubiquitin-binding function in pull-down assays and resulted in activation of basal NF-κB signalling, compared to wild-type, in reporter assays. We found evidence for a relationship between the ability of different UBA domain mutants to activate NF-κB signalling in vitro and number of affected sites in vivo in 1152 PDB patients from the UK and Italy, with A427D-SQSTM1 producing the greatest level of activation (relative to wild-type) of all PDB mutants tested to date. NMR and isothermal titration calorimetry studies were able to demonstrate that I424S is associated with global structural changes in the UBA domain, resulting in 10-fold weaker UBA dimer stability than wild-type and reduced ubiquitin-binding affinity of the UBA monomer. Our observations provide insights into the role of SQSTM1-mediated NF-κB signalling in PDB aetiology, and demonstrate that different mutations in close proximity within loop 2/helix 3 of the SQSTM1 UBA domain exert distinct effects on protein structure and stability, including indirect effects at the UBA/ubiquitin-binding interface.

Unique X-linked familial FSGS with co-segregating heart block disorder is associated with a mutation in the NXF5 gene.

Focal segmental glomerulosclerosis (FSGS) is the consequence of a disease process that attacks the kidney's filtering system, causing serious scarring. More than half of FSGS patients develop chronic kidney failure within 10 years, ultimately requiring dialysis or renal transplantation. There are currently several genes known to cause the hereditary forms of FSGS (ACTN4, TRPC6, CD2AP, INF2, MYO1E and NPHS2). This study involves a large, unique, multigenerational Australian pedigree in which FSGS co-segregates with progressive heart block with apparent X-linked recessive inheritance. Through a classical combined approach of linkage and haplotype analysis, we identified a 21.19 cM interval implicated on the X chromosome. We then used a whole exome sequencing approach to identify two mutated genes, NXF5 and ALG13, which are located within this linkage interval. The two mutations NXF5-R113W and ALG13-T141L segregated perfectly with the disease phenotype in the pedigree and were not found in a large healthy control cohort. Analysis using bioinformatics tools predicted the R113W mutation in the NXF5 gene to be deleterious and cellular studies support a role in the stability and localization of the protein suggesting a causative role of this mutation in these co-morbid disorders. Further studies are now required to determine the functional consequence of these novel mutations to development of FSGS and heart block in this pedigree and to determine whether these mutations have implications for more common forms of these diseases in the general population.

Association of a GRIA3 gene polymorphism with migraine in an Australian case-control cohort.

BACKGROUND: The excitatory neurotransmitter glutamate has been implicated in both the hyperexcitability required for cortical spreading depression as well as activation of the trigeminovascular system required for the allodynia associated with migraine. Polymorphisms in the glutamate receptor ionotropic amino-3-hydroxy-5-methyl-4-isoxazole-propionin acid 1 (GRIA1) and GRIA3 genes that code for 2 of 4 subunits of the glutamate receptor have been previously associated with migraine in an Italian population. In addition, the GRIA3 gene is coded within a previously identified migraine susceptibility locus at Xq24. This study investigated the previously associated polymorphisms in both genes in an Australian case-control population. METHODS: Variants in GRIA1 and GRIA3 were genotyped in 472 unrelated migraine cases and matched controls, and data were analyzed for association. RESULTS: Analysis showed no association between migraine and the GRIA1 gene. However, association was observed with the GRIA3 single nucleotide polymorphism (SNP) rs3761555 (P=.008). CONCLUSION: The results of this study confirmed the previous report of association at the rs3761555 SNP within the migraine with aura subgroup of migraineurs. However, the study identified association with the inverse allele suggesting that rs3761555 may not be the causative SNP but is more likely in linkage disequilibrium with another causal variant in both populations. This study supports the plethora of evidence suggesting that glutamate dysfunction may contribute to migraine susceptibility, warranting further investigation of the glutamatergic system and particularly of the GRIA3 gene.

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 melatonin receptor 1A (MTNR1A) gene is associated with recurrent and idiopathic calcium nephrolithiasis.

BACKGROUND: Experimental evidence indicate that melatonin regulates some renal tubular functions via specific melatonin receptors (MTNRs) located in the kidney of several avian and mammalian species, including humans. We hypothesized that single nucleotide polymorphisms (SNPs) in the melatonin receptor 1A gene (MTNR1A) might influence the risk of calcium nephrolithiasis. METHODS: We performed a systematic analysis of the MTNR1A gene in 246 recurrent calcium stone formers (136 men, 110 women; mean age 40.2 ± 12.0 years; body mass index 25.8 ± 4.5 kg/m2) and 269 healthy controls comparable for age and gender without a history of nephrolithiasis. RESULTS: Two SNPs in Intron 1 of MTNR1A were significantly associated with calcium nephrolithiasis: rs13140012 (P = 0.0004) and rs6553010 (P = 0.009). The haplotypes resulting from the two SNPs were also differently distributed between stone formers and controls, the haplotype A-T being more represented among stone formers (P = 0.00001) and the haplotype T-C being more common in healthy controls (P = 0.00001). Preliminary functional studies showed that the SNP rs13140012 could modify the binding sites for transcription factors. CONCLUSION: The results of this case-control study indicate a strong association between allelic variants of MTNR1A and recurrent calcium nephrolithiasis.

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.

Common variants in the regulative regions of GRIA1 and GRIA3 receptor genes are associated with migraine susceptibility.

BACKGROUND: Glutamate is the principal excitatory neurotransmitter in the central nervous system which acts by the activation of either ionotropic (AMPA, NMDA and kainate receptors) or G-protein coupled metabotropic receptors. Glutamate is widely accepted to play a major role in the path physiology of migraine as implicated by data from animal and human studies. Genes involved in synthesis, metabolism and regulation of both glutamate and its receptors could be, therefore, considered as potential candidates for causing/predisposing to migraine when mutated. METHODS: The association of polymorphic variants of GRIA1-GRIA4 genes which encode for the four subunits (GluR1-GluR4) of the alpha-amino-3- hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor for glutamate was tested in migraineurs with and without aura (MA and MO) and healthy controls. RESULTS: Two variants in the regulative regions of GRIA1 (rs2195450) and GRIA3 (rs3761555) genes resulted strongly associated with MA (P = 0.00002 and P = 0.0001, respectively), but not associated with MO, suggesting their role in cortical spreading depression. Whereas the rs548294 variant in GRIA1 gene showed association primarily with MO phenotype, supporting the hypothesis that MA and MO phenotypes could be genetically related. These variants modify binding sites for transcription factors altering the expression of GRIA1 and GRIA3 genes in different conditions. CONCLUSIONS: This study represents the first genetic evidence of a link between glutamate receptors and migraine.

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.

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.

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.

DDX11L: a novel transcript family emerging from human subtelomeric regions.

BACKGROUND: The subtelomeric regions of human chromosomes exhibit an extraordinary plasticity. To date, due to the high GC content and to the presence of telomeric repeats, the subtelomeric sequences are underrepresented in the genomic libraries and consequently their sequences are incomplete in the finished human genome sequence, and still much remains to be learned about subtelomere organization, evolution and function. Indeed, only in recent years, several studies have disclosed, within human subtelomeres, novel gene family members. RESULTS: During a project aimed to analyze genes located in the telomeric region of the long arm of the human X chromosome, we have identified a novel transcript family, DDX11L, members of which map to 1pter, 2q13/14.1, 2qter, 3qter, 6pter, 9pter/9qter, 11pter, 12pter, 15qter, 16pter, 17pter, 19pter, 20pter/20qter, Xpter/Xqter and Yqter. Furthermore, we partially sequenced the underrepresented subtelomeres of human chromosomes showing a common evolutionary origin. CONCLUSION: Our data indicate that an ancestral gene, originated as a rearranged portion of the primate DDX11 gene, and propagated along many subtelomeric locations, is emerging within subtelomeres of human chromosomes, defining a novel gene family. These findings support the possibility that the high plasticity of these regions, sites of DNA exchange among different chromosomes, could trigger the emergence of new genes.