Essential Oils for the Conservation of Paper Items.

Archival documents and artworks stored in libraries frequently undergo degradative processes promoted by the so-called "biodeteriogens" that inhabit these places. A renewed interest in plant-derived products has arisen in those research groups focusing on cultural heritage preservation and looking for new and safe disinfection techniques. In this view, essential oils (EOs) and their volatile organic constituents are very appealing thanks to their versatility of action. A literature survey of the scientific publications involving EOs and/or their major constituents related to the conservation of paper items of cultural heritage interest is presented here, aiming to reveal benefits and limitations of such peculiar plant-derived compounds.

Expanded circulating hematopoietic stem/progenitor cells as novel cell source for the treatment of TCIRG1 osteopetrosis.

Allogeneic hematopoietic stem cell transplantation is the treatment of choice for autosomal recessive osteopetrosis caused by defects in the TCIRG1 gene. Despite recent progress in conditioning, a relevant number of patients are not eligible for allogeneic stem cell transplantation because of the severity of the disease and significant transplant-related morbidity. We exploited peripheral CD34+ cells, known to circulate at high frequency in the peripheral blood of TCIRG1-deficient patients, as a novel cell source for autologous transplantation of gene corrected cells. Detailed phenotypical analysis showed that circulating CD34+ cells have a cellular composition that resembles bone marrow, supporting their use in gene therapy protocols. Transcriptomic profile revealed enrichment in genes expressed by hematopoietic stem and progenitor cells (HSPCs). To overcome the limit of bone marrow harvest/ HSPC mobilization and serial blood drawings in TCIRG1 patients, we applied UM171-based ex-vivo expansion of HSPCs coupled with lentiviral gene transfer. Circulating CD34+ cells from TCIRG1-defective patients were transduced with a clinically-optimized lentiviral vector (LV) expressing TCIRG1 under the control of phosphoglycerate promoter and expanded ex vivo. Expanded cells maintained long-term engraftment capacity and multi-lineage repopulating potential when transplanted in vivo both in primary and secondary NSG recipients. Moreover, when CD34+ cells were differentiated in vitro, genetically corrected osteoclasts resorbed the bone efficiently. Overall, we provide evidence that expansion of circulating HSPCs coupled to gene therapy can overcome the limit of stem cell harvest in osteopetrotic patients, thus opening the way to future gene-based treatment of skeletal diseases caused by bone marrow fibrosis.

B lymphocytes limit senescence-driven fibrosis resolution and favor hepatocarcinogenesis in mouse liver injury.

Hepatocellular carcinoma (HCC) is a frequent neoplasia and a leading cause of inflammation-related cancer mortality. Despite that most HCCs arise from persistent inflammatory conditions, pathways linking chronic inflammation to cancer development are still incompletely elucidated. We dissected the role of adaptive immunity in the Mdr2 knockout (Mdr2-/- ) mouse, a model of inflammation-associated cancer, in which ablation of adaptive immunity has been induced genetically (Rag2-/- Mdr2-/- and µMt-Mdr2-/- mice) or with in vivo treatments using lymphocyte-specific depleting antibodies (anti-CD20 or anti-CD4/CD8). We found that activated B and T lymphocytes, secreting fibrogenic tumor necrosis factor alpha (TNFα) and other proinflammatory cytokines, infiltrated liver of the Mdr2-/- mice during chronic fibrosing cholangitis. Lymphocyte ablation, in the Rag2-/- Mdr2-/- and µMt-Mdr2-/- mice, strongly suppressed hepatic stellate cell (HSC) activation and extracellular matrix deposition, enhancing HSC transition to cellular senescence. Moreover, lack of lymphocytes changed the intrahepatic metabolic/oxidative state, resulting in skewed macrophage polarization toward an anti-inflammatory M2 phenotype. Remarkably, hepatocarcinogenesis was significantly suppressed in the Rag2-/- Mdr2-/- mice, correlating with reduced TNFα/NF-κB (nuclear factor kappa B) pathway activation. Ablation of CD20+ B cells, but not of CD4+ /CD8+ T cells, in Mdr2-/- mice, promoted senescence-mediated fibrosis resolution and inhibited the protumorigenic TNFα/NF-κB pathway. Interestingly, presence of infiltrating B cells correlated with increased tumor aggressiveness and reduced disease-free survival in human HCC. CONCLUSION: Adaptive immunity sustains liver fibrosis (LF) and favors HCC growth in chronic injury, by modulating innate components of inflammation and limiting the extent of HSC senescence. Therapies designed for B-cell targeting may be an effective strategy in LF. (Hepatology 2018;67:1970-1985).

Murine Rankl-/- Mesenchymal Stromal Cells Display an Osteogenic Differentiation Defect Improved by a RANKL-Expressing Lentiviral Vector.

Autosomal recessive osteopetrosis (ARO) is a severe bone disease characterized by increased bone density due to impairment in osteoclast resorptive function or differentiation. Hematopoietic stem cell transplantation is the only available treatment; however, this therapy is not effective in RANKL-dependent ARO, since in bone this gene is mainly expressed by cells of mesenchymal origin. Of note, whether lack of RANKL production might cause a defect also in the bone marrow (BM) stromal compartment, possibly contributing to the pathology, is unknown. To verify this possibility, we generated and characterized BM mesenchymal stromal cell (BM-MSC) lines from wild type and Rankl-/- mice, and found that Rankl-/- BM-MSCs displayed reduced clonogenicity and osteogenic capacity. The differentiation defect was significantly improved by lentiviral transduction of Rankl-/- BM-MSCs with a vector stably expressing human soluble RANKL (hsRANKL). Expression of Rankl receptor, Rank, on the cytoplasmic membrane of BM-MSCs pointed to the existence of an autocrine loop possibly activated by the secreted cytokine. Based on the close resemblance of RANKL-defective osteopetrosis in humans and mice, we expect that our results are also relevant for RANKL-dependent ARO patients. Data obtained in vitro after transduction with a lentiviral vector expressing hsRANKL would suggest that restoration of RANKL production might not only rescue the defective osteoclastogenesis of this ARO form, but also improve a less obvious defect in the osteoblast lineage, thus possibly achieving higher benefit for the patients, when the approach is translated to clinics. Stem Cells 2017;35:1365-1377.

Genetics of Osteopetrosis.

PURPOSE OF REVIEW: The term osteopetrosis refers to a group of rare skeletal diseases sharing the hallmark of a generalized increase in bone density owing to a defect in bone resorption. Osteopetrosis is clinically and genetically heterogeneous, and a precise molecular classification is relevant for prognosis and treatment. Here, we review recent data on the pathogenesis of this disorder. RECENT FINDINGS: Novel mutations in known genes as well as defects in new genes have been recently reported, further expanding the spectrum of molecular defects leading to osteopetrosis. Exploitation of next-generation sequencing tools is ever spreading, facilitating differential diagnosis. Some complex phenotypes in which osteopetrosis is accompanied by additional clinical features have received a molecular classification, also involving new genes. Moreover, novel types of mutations have been recognized, which for their nature or genomic location are at high risk being neglected. Yet, the causative mutation is unknown in some patients, indicating that the genetics of osteopetrosis still deserves intense research efforts.

3D Bone Biomimetic Scaffolds for Basic and Translational Studies with Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are recognized as an attractive tool owing to their self-renewal and differentiation capacity, and their ability to secrete bioactive molecules and to regulate the behavior of neighboring cells within different tissues. Accumulating evidence demonstrates that cells prefer three-dimensional (3D) to 2D culture conditions, at least because the former are closer to their natural environment. Thus, for in vitro studies and in vivo utilization, great effort is being dedicated to the optimization of MSC 3D culture systems in view of achieving the intended performance. This implies understanding cell⁻biomaterial interactions and manipulating the physicochemical characteristics of biomimetic scaffolds to elicit a specific cell behavior. In the bone field, biomimetic scaffolds can be used as 3D structures, where MSCs can be seeded, expanded, and then implanted in vivo for bone repair or bioactive molecules release. Actually, the union of MSCs and biomaterial has been greatly improving the field of tissue regeneration. Here, we will provide some examples of recent advances in basic as well as translational research about MSC-seeded scaffold systems. Overall, the proliferation of tools for a range of applications witnesses a fruitful collaboration among different branches of the scientific community.

Correction of osteopetrosis in the neonate oc/oc murine model after lentiviral vector gene therapy and non-genotoxic conditioning.

Introduction: Autosomal recessive osteopetrosis (ARO) is a rare genetic disease, characterized by increased bone density due to defective osteoclast function. Most of the cases are due to TCIRG1 gene mutation, leading to severe bone phenotype and death in the first years of life. The standard therapy is the hematopoietic stem cell transplantation (HSCT), but its success is limited by several constraints. Conversely, gene therapy (GT) could minimize the immune-mediated complications of allogeneic HSCT and offer a prompt treatment to these patients. Methods: The Tcirg1-defective oc/oc mouse model displays a short lifespan and high bone density, closely mirroring the human condition. In this work, we exploited the oc/oc neonate mice to optimize the critical steps for a successful therapy. Results: First, we showed that lentiviral vector GT can revert the osteopetrotic bone phenotype, allowing long-term survival and reducing extramedullary haematopoiesis. Then, we demonstrated that plerixafor-induced mobilization can further increase the high number of HSPCs circulating in peripheral blood, facilitating the collection of adequate numbers of cells for therapeutic purposes. Finally, pre-transplant non-genotoxic conditioning allowed the stable engraftment of HSPCs, albeit at lower level than conventional total body irradiation, and led to long-term survival and correction of bone phenotype, in the absence of acute toxicity. Conclusion: These results will pave the way to the implementation of an effective GT protocol, reducing the transplant-related complication risks in the very young and severely affected ARO patients.

Plant Sampling for Production of Essential Oil and Evaluation of Its Antimicrobial Activity In Vitro.

Essential oils (EOs) and oleoresins are complex mixtures mainly made up of terpenes, synthesized by a wide variety of plants. Individual terpenes may show broad-spectrum activity against different plant pathogens, and their combination into EO and oleoresin mixtures enhances plant chemical defense. The interest in EOs has significantly increased due to the trend of using natural products as herbicides, insecticidal and antimicrobial agents. In addition, the use of plant mixtures is an emerging approach to face the problem of antimicrobial resistance in agriculture. This chapter reports guidelines about plant sample collection for the production of EOs and provides protocols to test their activity as antimicrobial agents against bacteria and fungi. It also describes a solvent-free method for the inclusion of EOs into ß-cyclodextrins. This type of formulate is prepared to turn liquid EOs into easily manageable water-soluble powders, and to control the release of volatile compounds, aiming to increase EOs' applications in agriculture.

A novel intronic variant in PIGB in Acrofrontofacionasal dysostosis type 1 patients expands the spectrum of phenotypes associated with GPI biosynthesis defects.

Acrofrontofacionasal dysostosis type 1 (AFFND1) is an extremely rare disorder characterized by several dysmorphic features, skeletal abnormalities and intellectual disability, and described only in seven patients in the literature. A biallelic variant in the Neuroblastoma Amplified Sequence (NBAS) gene was recently identified in two Indian patients with AFFND1. Here we report genetic investigation of AFFND1 in the originally described Brazilian families and the identification of an extremely rare, recessively-inherited, intronic variant in the Phosphatidylinositol Glycan class B (PIGB) gene NC_000015.10 (NM_004855.4): c.795-19T > G) in the affected individuals. The PIGB gene encodes an enzyme involved in the biosynthesis of the glycosylphosphatidylinositol (GPI) anchor, which is required for the post-translational modification of a large variety of proteins, enabling their correct cellular localization and function. Recessive variants in PIGB have previously been reported in individuals with a neurodevelopmental syndrome having partial overlap with AFFND1. In vitro assays demonstrated that the intronic variant leads to exon skipping, suggesting the Brazilian AFFND1 patients may be null for PIGB, in agreement with their severe clinical phenotype. These data increase the number of pathogenic variants in the PIGB gene, place AFFND1 among GPI deficiencies and extend the spectrum of phenotypes associated with GPI biosynthesis defects.

Pathobiologic Mechanisms of Neurodegeneration in Osteopetrosis Derived From Structural and Functional Analysis of 14 ClC-7 Mutants.

ClC-7 is a chloride-proton antiporter of the CLC protein family. In complex with its accessory protein Ostm-1, ClC-7 localizes to lysosomes and to the osteoclasts' ruffled border, where it plays a critical role in acidifying the resorption lacuna during bone resorption. Gene inactivation in mice causes severe osteopetrosis, neurodegeneration, and lysosomal storage disease. Mutations in the human CLCN7 gene are associated with diverse forms of osteopetrosis. The functional evaluation of ClC-7 variants might be informative with respect to their pathogenicity, but the cellular localization of the protein hampers this analysis. Here we investigated the functional effects of 13 CLCN7 mutations identified in 13 new patients with severe or mild osteopetrosis and a known ADO2 mutation. We mapped the mutated amino acid residues in the homology model of ClC-7 protein, assessed the lysosomal colocalization of ClC-7 mutants and Ostm1 through confocal microscopy, and performed patch-clamp recordings on plasma-membrane-targeted mutant ClC-7. Finally, we analyzed these results together with the patients' clinical features and suggested a correlation between the lack of ClC-7/Ostm1 in lysosomes and severe neurodegeneration. © 2020 American Society for Bone and Mineral Research (ASBMR).

Genome-first approach for the characterization of a complex phenotype with combined NBAS and CUL4B deficiency.

Biallelic variants in neuroblastoma-amplified sequence (NBAS) cause an extremely broad spectrum of phenotypes. Clinical features range from isolated recurrent episodes of liver failure to multisystemic syndrome including short stature, skeletal osteopenia and dysplasia, optic atrophy, and a variable immunological, cutaneous, muscular, and neurological abnormalities. Hemizygous variants in CUL4B cause syndromic X-linked intellectual disability characterized by limitations in intellectual functions, developmental delays in gait, cognitive, and speech functioning, and other features including short stature, dysmorphism, and cerebral malformations. In this study, we report on a 4.5-month-old preterm infant with a complex phenotype mainly characterized by placental-related severe intrauterine growth restriction, post-natal growth failure with spontaneous bone fractures, which led to a suspicion of osteogenesis imperfecta, and lethal bronchopulmonary dysplasia with pulmonary hypertension. Whole exome sequencing identified compound heterozygosity for a known frameshift and a novel missense variant in NBAS and hemizygosity for a known CUL4B nonsense mutation. In vitro functional studies on the novel NBAS missense substitution demonstrated altered Golgi-to-endoplasmic reticulum retrograde vesicular trafficking and reduced collagen secretion, likely explaining part of the patient's phenotype. We also provided a comprehensive overview of the phenotypic features of NBAS and CUL4B deficiency, thus updating the recently emerging NBAS genotype-phenotype correlations. Our findings highlight the power of a genome-first approach for an early diagnosis of complex phenotypes.

Generation of an immunodeficient mouse model of tcirg1-deficient autosomal recessive osteopetrosis.

BACKGROUND: Autosomal recessive osteopetrosis is a rare skeletal disorder with increased bone density due to a failure in osteoclast bone resorption. In most cases, the defect is cell-autonomous, and >50% of patients bear mutations in the TCIRG1 gene, encoding for a subunit of the vacuolar proton pump essential for osteoclast resorptive activity. The only cure is hematopoietic stem cell transplantation, which corrects the bone pathology by allowing the formation of donor-derived functional osteoclasts. Therapeutic approaches using patient-derived cells corrected ex vivo through viral transduction or gene editing can be considered, but to date functional rescue cannot be demonstrated in vivo because a relevant animal model for xenotransplant is missing. METHODS: We generated a new mouse model, which we named NSG oc/oc, presenting severe autosomal recessive osteopetrosis owing to the Tcirg1 oc mutation, and profound immunodeficiency caused by the NSG background. We performed neonatal murine bone marrow transplantation and xenotransplantation with human CD34+ cells. RESULTS: We demonstrated that neonatal murine bone marrow transplantation rescued NSG oc/oc mice, in line with previous findings in the oc/oc parental strain and with evidence from clinical practice in humans. Importantly, we also demonstrated human cell chimerism in the bone marrow of NSG oc/oc mice transplanted with human CD34+ cells. The severity and rapid progression of the disease in the mouse model prevented amelioration of the bone pathology; nevertheless, we cannot completely exclude that minor early modifications of the bone tissue might have occurred. CONCLUSION: Our work paves the way to generating an improved xenograft model for in vivo evaluation of functional rescue of patient-derived corrected cells. Further refinement of the newly generated mouse model will allow capitalizing on it for an optimized exploitation in the path to novel cell therapies.

One Disease, Many Genes: Implications for the Treatment of Osteopetroses.

Osteopetrosis is a condition characterized by increased bone mass due to defects in osteoclast function or formation. In the last decades, the molecular dissection of osteopetrosis has unveiled a plethora of molecular players responsible for different forms of the disease, some of which present also primary neurodegeneration that severely limits the therapy. Hematopoietic stem cell transplantation can cure the majority of them when performed in the first months of life, highlighting the relevance of an early molecular diagnosis. However, clinical management of these patients is constrained by the severity of the disease and lack of a bone marrow niche that may delay immune reconstitution. Based on osteopetrosis genetic heterogeneity and disease severity, personalized therapies are required for patients that are not candidate to bone marrow transplantation. This review briefly describes the genetics of osteopetrosis, its clinical heterogeneity, current therapy and innovative approaches undergoing preclinical evaluation.

Mesenchymal Stromal Cell-Seeded Biomimetic Scaffolds as a Factory of Soluble RANKL in Rankl-Deficient Osteopetrosis.

Biomimetic scaffolds are extremely versatile in terms of chemical composition and physical properties, which can be defined to accomplish specific applications. One property that can be added is the production/release of bioactive soluble factors, either directly from the biomaterial, or from cells embedded within the biomaterial. We reasoned that pursuing this strategy would be appropriate to setup a cell-based therapy for RANKL-deficient autosomal recessive osteopetrosis, a very rare skeletal genetic disease in which lack of the essential osteoclastogenic factor RANKL impedes osteoclast formation. The exogenously administered RANKL cytokine is effective in achieving osteoclast formation and function in vitro and in vivo, thus, we produced murine Rankl-/- mesenchymal stromal cells (MSCs) overexpressing human soluble RANKL (hsRL) following lentiviral transduction (LVhsRL). Here, we described a three-dimensional (3D) culture system based on a magnesium-doped hydroxyapatite/collagen I (MgHA/Col) biocompatible scaffold closely reproducing bone physicochemical properties. MgHA/Col-seeded murine MSCs showed improved properties, as compared to two-dimensional (2D) culture, in terms of proliferation and hsRL production, with respect to LVhsRL-transduced cells. When implanted subcutaneously in Rankl-/- mice, these cell constructs were well tolerated, colonized by host cells, and intensely vascularized. Of note, in the bone of Rankl-/- mice that carried scaffolds with either WT or LVhsRL-transduced Rankl-/- MSCs, we specifically observed formation of TRAP+ cells, likely due to sRL released from the scaffolds into circulation. Thus, our strategy proved to have the potential to elicit an effect on the bone; further work is required to maximize these benefits and achieve improvements of the skeletal pathology in the treated Rankl-/- mice. Stem Cells Translational Medicine 2019;8:22-34.

Absence of Dipeptidyl Peptidase 3 Increases Oxidative Stress and Causes Bone Loss.

Controlling oxidative stress through the activation of antioxidant pathways is crucial in bone homeostasis, and impairments of the cellular defense systems involved contribute to the pathogenesis of common skeletal diseases. In this work we focused on the dipeptidyl peptidase 3 (DPP3), a poorly investigated ubiquitous zinc-dependent exopeptidase activating the Keap1-Nrf2 antioxidant pathway. We showed Dpp3 expression in bone and, to understand its role in this compartment, we generated a Dpp3 knockout (KO) mouse model and specifically investigated the skeletal phenotype. Adult Dpp3 KO mice showed a mild growth defect, a significant increase in bone marrow cellularity, and bone loss mainly caused by increased osteoclast activity. Overall, in the mouse model, lack of DPP3 resulted in sustained oxidative stress and in alterations of bone microenvironment favoring the osteoclast compared to the osteoblast lineage. Accordingly, in vitro studies revealed that Dpp3 KO osteoclasts had an inherent increased resorptive activity and ROS production, which on the other hand made them prone to apoptosis. Moreover, absence of DPP3 augmented bone loss after estrogen withdrawal in female mice, further supporting its relevance in the framework of bone pathophysiology. Overall, we show a nonredundant role for DPP3 in the maintenance of bone homeostasis and propose that DPP3 might represent a possible new osteoimmunological player and a marker of human bone loss pathology. © 2019 American Society for Bone and Mineral Research.

Mutations in the Neuroblastoma Amplified Sequence gene in a family affected by Acrofrontofacionasal Dysostosis type 1.

Acrofrontofacionasal Dysostosis type 1 (AFFND1) is an extremely rare, autosomal recessive syndrome, comprising facial and skeletal abnormalities, short stature and intellectual disability. We analyzed an Indian family with two affected siblings by exome sequencing and identified a novel homozygous truncating mutation in the Neuroblastoma-Amplified Sequence (NBAS) gene in the patients' genome. Mutations in the NBAS gene have recently been associated with different phenotypes mainly involving skeletal formation, liver and cognitive development. The NBAS protein has been implicated in two key cellular processes, namely the non-sense mediated decay and the Golgi-to-Endoplasmic Reticulum retrograde traffic. Both functions were impaired in HEK293T cells overexpressing the truncated NBAS protein, as assessed by Real-Time PCR, Western blot analysis, co-immunoprecipitation, and immunofluorescence analysis. We examined the expression of NBAS protein in mouse embryos at various developmental stages by immunohistochemistry, and detected expression in developing chondrogenic and osteogenic structures of the skeleton as well as in the cortex, hippocampus and cerebellum, which is compatible with a role in bone and brain development. Functional genetics in the zebrafish model showed that depletion of endogenous z-nbas in fish embryos results in defective morphogenesis of chondrogenic cranial skeletal elements. Overall, our data point to a conserved function of NBAS in skeletal morphogenesis during development, support the hypothesis of a causative role of the mutated NBAS gene in the pathogenesis of AFFND1 and extend the spectrum of phenotypes associated with defects in this gene.

Soluble Factors on Stage to Direct Mesenchymal Stem Cells Fate.

Mesenchymal stem cells (MSCs) are multipotent stromal cells that are identified by in vitro plastic adherence, colony-forming capacity, expression of a panel of surface molecules, and ability to differentiate at least toward osteogenic, adipogenic, and chondrogenic lineages. They also produce trophic factors with immunomodulatory, proangiogenic, and antiapoptotic functions influencing the behavior of neighboring cells. On the other hand, a reciprocal regulation takes place; in fact, MSCs can be isolated from several tissues, and depending on the original microenvironment and the range of stimuli received from there, they can display differences in their essential characteristics. Here, we focus mainly on the bone tissue and how soluble factors, such as growth factors, cytokines, and hormones, present in this microenvironment can orchestrate bone marrow-derived MSCs fate. We also briefly describe the alteration of MSCs behavior in pathological settings such as hematological cancer, bone metastasis, and bone marrow failure syndromes. Overall, the possibility to modulate MSCs plasticity makes them an attractive tool for diverse applications of tissue regeneration in cell therapy. Therefore, the comprehensive understanding of the microenvironment characteristics and components better suited to obtain a specific MSCs response can be extremely useful for clinical use.

Hematopoietic stem cell transplantation corrects osteopetrosis in a child carrying a novel homozygous mutation in the FERMT3 gene.

Osteopetrosis (OPT) is a rare skeletal disorder with phenotypic and genotypic heterogeneity: a variety of clinical features besides the bony defect may be present, and at least ten different genes are known to be involved in the disease pathogenesis. In the framework of this heterogeneity, we report the clinical description of a neonate, first child of consanguineous parents, who had osteoclast-rich osteopetrosis and bone marrow failure in early life, but no other usual classical features of infantile malignant OPT, such as visual or hearing impairments. Because of the severe presentation at birth, the patient received Hematopoietic Stem Cell Transplantation (HSCT) at 2months of age with successful outcome. Post-HSCT genetic investigation by means of exome sequencing identified a novel homozygous mutation in the Fermitin Family Member 3 (FERMT3) gene, which was predicted to disrupt the functionality of its protein product kindlin 3. Our report provides information relevant to physicians for recognizing patients with one of the rarest forms of infantile malignant OPT, and clearly demonstrates that HSCT cures kindlin 3 deficiency with severe phenotype.

Synonymous Mutations Add a Layer of Complexity in the Diagnosis of Human Osteopetrosis.

Autosomal recessive osteopetroses (AROs) are rare, genetically heterogeneous skeletal diseases with increased bone density that are often lethal if left untreated. A precise molecular classification is relevant for the patient's management, because in some subgroups hematopoietic stem cell transplantation (HSCT), which is the only curative therapy, is contraindicated. In two unrelated ARO patients, the molecular analysis revealed the presence of a synonymous variant in known ARO genes, namely in the TCIRG1 gene in one patient and in the CLCN7 in the other patient, predicted to impact on the splicing process. In the latter case, sequencing of the transcript confirmed the splicing defect, whereas in the former, for whom an RNA sample was not available, the defect was reconstructed in vitro by the minigene technology. These results strongly suggest that these synonymous changes were responsible for the disease in our patients. Our findings are novel with respect to ARO and add to the few reports in literature dealing with different diseases, underlining the importance of cDNA analysis for the correct assessment of exonic changes, even when exome sequencing is performed. In particular, we highlight the possibility that at least in some cases ARO is due to synonymous changes, erroneously considered clinically silent, in the genes already described in literature, and suggest carefully reevaluating the sequencing results of these genes when mutations are not found at a first analysis. In addition, with respect to the CLCN7 gene, we suggest that synonymous variants might also contribute to the large spectrum of severity typical of CLCN7-dependent osteopetrosis through more subtle, but not negligible, effects on protein availability and functionality. © 2016 American Society for Bone and Mineral Research.