TCIRG1 Research Articles

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

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. 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. 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. 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.

Autosomal Dominant Osteopetrosis (ADO) Caused by a Missense Variant in the TCIRG1 Gene.

Autosomal dominant osteopetrosis (ADO) is a rare genetic disorder resulting from impaired osteoclastic bone resorption. Clinical manifestations frequently include fractures, osteonecrosis (particularly of the jaw or maxilla), osteomyelitis, blindness, and/or bone marrow failure. ADO usually results from heterozygous missense variants in the Chloride Channel 7 gene (CLCN7) that cause disease by a dominant negative mechanism. Variants in the T-cell immune regulator 1 gene (TCIRG1) are commonly identified in autosomal recessive osteopetrosis but have only been reported in 1 patient with ADO. Here, we report 3 family members with a single heterozygous missense variant (p.Gly579Arg) in TCIRG1 who have a phenotype consistent with ADO. Three of 5 protein prediction programs suggest this variant likely inhibits the function of TCIRG1. This is the first description of adult presentation of ADO caused by a TCIRG1 variant. Similar to families with ADO from CLCN7 mutations, this variant in TCIRG1 results in marked phenotype variability, with 2 subjects having severe disease and the third having very mild disease. This family report implicates TCIRG1 missense mutations as a cause of ADO and demonstrates that the marked phenotypic variability in ADO may extend to disease caused by TCIRG1 missense mutations.

Modeling TCIRG1 Neutropenia By Utilizing Patient Derived Induced Pluripotent Stem Cells

Background: Severe congenital neutropenia (SCN) is a hereditary hematopoietic disorder characterized by recurrent infections and leukemic transformation. We previously identified a novel heterozygous missense mutation (R736S) in TCIRG1 causing SCN in a large multigenerational family (PMID: 24753205). We also identified a statistically significant correlation in individuals with lower ANC with rare missense variants in TCIRG1 in a large cohort of population-based genome screening (PMID: 27229898). Recently, we have discovered another TCIRG1 heterozygousvariant: R736C in two unrelated families with congenital neutropenia. Independently, Shinwari et al identified a novel TCIRG1 mutation, V52L, which was correlated with the neutropenic phenotype (PMID: 35573728). Homozygous mutations of TCIRG1 are causing autosomal recessive osteopetrosis (ARO), which is characterized by osteopetrosis and secondary hematological and neurological abnormalities. The Genome Aggregation Database (gnomAD) has no data about TCIRG1 R736S and R736C variants accruing in the healthy population. This is in contrast of numerous healthy individuals with a various heterozygous variants throughout the TCIRG1 gene. Aims: We aim to generate in-vitro model of TCIRG1 neutropenia by utilizing patient derived induced pluripotent stem cells (iPSCs) and characterize the hematological abnormalities caused by mutant TCIRG1 expression. We also aim to characterize the incidence of TCIRG1-associated neutropenia and the prevalence of TCIRG1 variants by sharing this new information within the scientific community through identifying more cases/families with this disease. Methods: Medical history, differential WBC counts and whole exome sequencing (WES) was used to identify the genetic basis of neutropenia in the family with R736S mutation. The R736C mutation in the new families also were identified by WES of the index cases. Medical history, blood counts and Sanger sequencing identified their affected relatives. iPSC lines of 4 affected individuals from 2 families harboring TCIRG1 R736S and R736C mutations and healthy volunteer were generated using standard methodology. S732F benign heterozygous TCIRG1 variant was generated by utilizing CRISPR/Cas9 knock-In gene editing using a healthy volunteer iPSC line. iPSCs hematopoietic differentiation was achieved by using STEMdiff Hematopoietic Kit. Resultant CD34+ cells were differentiated towards neutrophils using a published protocol (PMID: 35795780). Cell proliferation, survival and differentiation characteristics were measured by automated cell counter and flow cytometry, cell viability staining and surface markers analysis for granulocytic differentiation. Results: The discovery of these families came through the reputation and work of the Severe Congenital Neutropenia International Registry (SCNIR) and the expanding use of WES to identify the causes of hereditary disorders. With the identification of the index cases, we have identified several affected family members in each family. In all three families the severity of neutropenia is variable and correlates with the incidence of recurrent bacterial infections. Myeloid differentiation of CD34+ cells derived from iPSCs of 4 patients from two families with R736S and R736C mutations revealed maturation, survival and proliferation abnormalities compared to volunteer cells and cell line harboring benign S732F heterozygous TCIRG1 mutation. All patient cells had from 2 to 3-fold drop in cell proliferation and cell viability drop by over 70% for the patients with R736S and up to 90% with R736C mutation compared to the controls. The myeloid differentiation in the patients' cells, measured by flow cytometry and assessed by CD66b+/CD14+ and CD11b+/CD15+ markers was impaired by 38% and 23% respectively for the patients with R736S and 43% and 24% for the patients harboring R736C mutation. Conclusions: The pathogenesis of TCIRG1-associated neutropenia is currently unknown. Carriers of pathogenic variants from these families appear to have neutropenia with variable severity. Our preliminary studies show that patient iPSC derived hematopoietic cells closely recapitulate the neutropenic phenotype. Population based genomic studies; careful family histories and WES will identify new patients with this disorder.

Autosomal recessive osteopetrosis type I: description of pathogenic variant of TCIRG1 gene

Autosomal recessive osteopetrosis type I: description of pathogenic variant of TCIRG1 gene

Osteoclast rich osteopetrosis due to defects in the TCIRG1 gene.

Discovery that mutations in TCIRG1 (also known as Atp6i) gene are responsible for most instances of autosomal recessive osteopetrosis (ARO) heralded a new era for comprehension and treatment of this phenotypically heterogeneous rare bone disease. TCIRG1 encodes the a3 subunit, an essential isoform of the vacuolar ATPase proton pump involved in acidification of the osteoclast resorption lacuna and in secretory lysosome trafficking. TCIRG1 defects lead to inefficient bone resorption by nonfunctional osteoclasts seen in abundance on bone marrow biopsy, delineating this ARO as 'osteoclast-rich'. Presentation is usually in early childhood and features of extramedullary haematopoiesis (hepatosplenomegaly, anaemia, thrombocytopenia) due to bone marrow fibrosis, and cranial nerve impingement (blindness in particular). Impaired dietary calcium uptake due to high pH causes the co-occurrence of rickets, described as "osteopetrorickets". Osteoclast dysfunction leads to early death if untreated, and allogeneic haematopoietic stem cell transplantation is currently the treatment of choice. Studies of patients as well as of mouse models carrying spontaneous (the oc/oc mouse) or targeted disruption of Atp6i (TCIRG1) gene have been instrumental providing insight into disease pathogenesis and development of novel cellular therapies that exploit gene correction.

Novel TCIRG1 Mutations Causing Congenital Neutropenia

Background: Severe congenital neutropenia (SCN) is a bone marrow failure disorder leading to severe neutropenia related infections and leukemia. More than 30 different genes associated with this disorder have been identified. We previously identified a novel heterozygous missense mutation (R736S) of TCIRG1 as the probable cause for SCN in a large multigenerational family through exome sequencing (Makaryan et al; Hum Mutat. 2014 Jul;35(7):824-7). We also identified a statistically significant correlation of rare missense variants of TCIRG1 with lower ANC in three cohorts from the NHLBI Grand Opportunity Exome Sequencing Project (GO ESP; Rosenthal et al; Genet Epidemiol. 2016 Sep;40(6):470-4). Recently, we have discovered a novel TCIRG1 variant (R736C) at the same position in a new family with congenital neutropenia. TCIRG1 encodes subunit a3, a 116-kD component (OC116) of V-ATPases, vacuolar proton pumps which mediate H+ transport through membranes. Homozygous loss-of-function mutations of TCIRG1 are the most common cause of autosomal recessive osteopetrosis (ARO). Mutations R736S and R736C occur at an amino acid residue critical for proton translocation. There are no reports in the literature of individuals with heterozygous mutations at this locus. This is in contrast to the numerous healthy individuals with various heterozygous variants throughout the TCIRG1 gene. Parents of ARO patients harboring homozygous TCIRG1 mutations are considered healthy with no musculoskeletal or hematopoietic abnormalities. Pathogenesis of TCIRG1-associated neutropenia is largely unknown. Hypothesis: We hypothesize that certain heterozygous TCIRG1 variants, like R736S and R736C, may cause SCN through specific molecular pathways. Methods: Family and medical history, differential WBC counts and whole exome sequencing/Identity by Decent (IBD) analysis were used to identify the genetic basis of neutropenia in the previously reported family. Sanger sequencing was used to confirm the findings of exome sequencing and to identify other affected individuals. The R736C mutation was identified by whole exome sequencing with further site-specific analysis of 35 genes. Affected relatives were identified by Sanger sequencing. The Polyphen 2 online prediction tool was used to predict the impact of both substitutions on TCIRG1 function. Results: Discovery and details of the original family were described in our previous report: (Makaryan et al; Hum Mutat. 2014 Jul;35(7):824-7). The second family was discovered in Taiwan, where the primary physician contacted SCNIR about a young patient with neutropenia who had a novel TCIRG1 mutation. Shortly thereafter, we identified two more affected family members. In both families the severity of recurrent bacterial infections is variable, roughly correlating with the severity of the neutropenia. For example, the grandmother of the index patient in the second family was not aware that she had neutropenia. The novel R736C TCIRG1 variant is located in the same position as the previous R736S variant. Arginine in this position is evolutionary highly conserved and both these substitutions cause a major change in amino acid size and properties. Polyphen-2 analysis predicts the effects of both mutations "probably damaging” (score 1.0). Conclusions: Understanding the genetic, epidemiological and molecular basis of TCIRG1-associated neutropenia is important for improving the diagnosis and treatment of this form of congenital neutropenia. The pathogenesis of TCIRG1-associated neutropenia is unknown. It is very important to emphasize that carriers of pathogenic variants from both families have neutropenia with variable severity. The report of this new family suggests that there may be other patients and families with neutropenia caused by mutations in TCIRG1. Population based genome studies, careful family histories and whole genome/exome sequencing will be highly valuable for identifying and helping new patients with this disorder.

P1434: CORRECTION OF CONGENITAL NEUTROPENIA-RELATED GENES IN NAÏVE IPS CELLS DERIVED FROM PATIENTS USING VIRAL-FREE CRISPR/CAS9 GENE-EDITING SYSTEM

Background: Severe congenital neutropenia (SCN) is a rare haematological condition with a heterogeneous genetic background that often leads to severe impairment of the immune system. For patients who do not respond to standard treatment, the only possible therapy is allogeneic haematopoietic stem cell transplantation. SCN patients have an increased risk of the development of myelodysplastic syndrome or acute myelogenous leukaemia. Rare genetic disorders, such as SCN, require prioritising bone marrow failure diagnosis and therapy tailored to individual patients. Human induced pluripotent stem (iPS) cells are a promising prospect for future cell therapy in a wide range of diseases like congenital neutropenia for which there are currently no cures or effective therapies. Aims: The aim of this study was to develop potential treatment procedure based on the use of iPS cells generated from epithelial cells of patients with congenital neutropenia. Using mutation-specific gRNA and CRISPR-Cas9 system, we made an attempt to repair patients-derived iPS cells which may have potential for renewal of the corrected granulocyte population in the bone marrow. Methods: Patient-derived epithelial cells were reprogrammed with the EBV-derived oriP/EBNA-1 system carrying transcription factors. The transition of these cells into the naïve state was accomplished with the use of cell culture media supplemented with Leukaemia inhibitory factor, an inhibitor of Protein kinase C, small molecule activators of WNT, and inhibitors of ERK1/2 and JNK signaling pathways. CRISPR-Cas9 was used to repair mutations in patients-derived naïve iPS cells. Into the target cells, a specific sgRNA combined with eSpCas9 protein and HDR template was introduced via nucleofection with Lonza 4D Nucleofector, as a CRISPR enhancer, brefedrin A was used. To verify the efficiency of transfection and gene edition, the targeted region were analysed with Sanger sequencing (Fig. 1). Results: We have generated primed and naïve iPS cells from 7 patients with different severity of neutropenic phenotype and genetic background, 4 another samples are pending. The morphology of the iPSCs line (primed cells) displayed a typical ESC-like colony appearance comprised of tightly packed cells. Alkaline phosphatase and immunocytochemical staining confirmed that the reprogrammed cells express pluripotent stem cell markers and can differentiate into the three germ layers. We successfully repaired a mutation in ELANE gene in 4 out of 30 screened colonies as well as TCIRG1 gene in 3 out of 14 screened colonies. Additionally, for further functional studies, we generated a homozygotic ELANE p.Cys55Arg mutant, that might serve as a unique model of the genotype-phenotype gradient of elastase activity. Image:Summary/Conclusion: We have successfully generated and repaired iPS cells derived from patients with congenital neutropenia which might serve as a procedure with a great potential in the field of cell therapy tailored to individual patients.

Novel Disease-Associated Missense Single-Nucleotide Polymorphisms Variants Predication by Algorithms Tools and Molecular Dynamics Simulation of Human TCIRG1 Gene Causing Congenital Neutropenia and Osteopetrosis.

T Cell Immune Regulator 1, ATPase H + Transporting V0 Subunit A3 (TCIRG1 gene provides instructions for making one part, the a3 subunit, of a large protein complex known as a vacuolar H + -ATPase (V-ATPase). V-ATPases are a group of similar complexes that act as pumps to move positively charged hydrogen atoms (protons) across membranes. Single amino acid changes in highly conserved areas of the TCIRG1 protein have been linked to autosomal recessive osteopetrosis and severe congenital neutropenia. We used multiple computational approaches to classify disease-prone single nucleotide polymorphisms (SNPs) in TCIRG1. We used molecular dynamics analysis to identify the deleterious nsSNPs, build mutant protein structures, and assess the impact of mutation. Our results show that fifteen nsSNPs (rs199902030, rs200149541, rs372499913, rs267605221, rs374941368, rs375717418, rs80008675, rs149792489, rs116675104, rs121908250, rs121908251, rs121908251, rs149792489 and rs116675104) variants are likely to be highly deleterious mutations as by incorporating them into wild protein they destabilize the wild protein structure and function. They are also located in the V-ATPase I domain, which may destabilize the structure and impair TCIRG1 protein activation, as well as reduce its ATPase effectiveness. These mutants have not yet been identified in patients suffering from CN and osteopetrosis while (G405R, R444L, and D517N) reported in our study are already associated with osteopetrosis. Mutation V52L reported in our study was identified in a patient suspected for CN. Finally, these mutants can help to further understand the broad pool of illness susceptibilities associated with TCIRG1 catalytic kinase domain activation and aid in the development of an effective treatment for associated diseases.

Infantile malignant osteopetrosis caused by a new mutation of TCIRG1 gene： a case report

Infantile malignant osteopetrosis caused by a new mutation of TCIRG1 gene： a case report

Clinical presentation and analysis of genotype-phenotype correlations in patients with malignant infantile osteopetrosis.

Malignant infantile osteopetrosis (MIOP) is the autosomal recessive, severe form of osteopetrosis. This rare genetic syndrome usually presents soon after birth and is often fatal if left untreated. Early diagnosis is key for proper management but clinical presentation is diverse, and oftentimes diagnosis may be challenging. In this study, we retrospectively collected data of genetic mutations and phenotypic characteristics at the initial presentation of 81 MIOP patients and analyzed genotype-phenotype correlations. The most common genetic mutation was in the TCIRG1 gene (n=46, 56.8%), followed by SNX10 (n=20, 25%). Other genetic mutations included RANK (n=7, 8.7%), CLCN7 (n=5, 6.2%) and CA2 (n=3, 3.7%). More than half of the patients presented with growth retardation (n=46, 56.8%). Twenty-one of the patients were blind (26%) and thirty-seven patients had other neurological deficits (45.7%) at the time of initial presentation. Most patients presented with hematological signs of bone marrow failure including anemia (n=69, 85.2%) and thrombocytopenia (n=33, 40.7%). Thrombocytopenia at initial presentation was significantly more prevalent in patients with mutations in the TCIRG1 gene (p=0.036). Other phenotypic presenting features were not found to be significantly correlated to specific gene mutations. In conclusion, the initial presentation of MIOP is variable, but some features are common such as growth retardation, visual impairment, and cytopenias. High awareness of MIOP presenting signs is essential for prompt diagnosis of this challenging disease.

Osteoclast Rich Osteopetrosis Due to Defects in TCIRG1 Gene

Osteoclast Rich Osteopetrosis Due to Defects in TCIRG1 Gene

Two novel mutations in TCIRG1 induced infantile malignant osteopetrosis: a case report

BackgroundInfantile malignant osteopetrosis (IMO) is a rare autosomal recessive disease characterized by a higher bone density in bone marrow caused by the dysfunction of bone resorption. Clinically, IMO can be diagnosed with medical examination, bone mineral density test and whole genome sequencing.Case presentationWe present the case of a 4-month-old male infant with abnormal skull development, hypocalcemia and premature closure of the cranial sutures. Due to the hyper bone density showed by his radiographic examination, which are characteristic patterns of IMO, we speculated that he might be an IMO patient. In order to confirm this diagnosis, a high-precision whole exome sequencing of the infant and his parents was performed. The analysis of high-precision whole exome sequencing results lead to the identification of two novel heterozygous mutations c.504-1G > C (a splicing site mutation) and c.1371delC (p.G458Afs*70, a frameshift mutation) in gene TCIRG1 derived from his parents. Therefore, we propose that there is a close association between these two mutations and the onset of IMO.ConclusionsTo date, these two novel mutations in gene TCIRG1 have not been reported in the reference gene database of Chinese population. These variants have likewise not been reported outside of China in the Genome Aggregation Database (gnomAD). Our case suggests that the use of whole exome sequencing to detect these two mutations will improve the identification and early diagnosis of IMO, and more specifically, the identification of homozygous individuals with TCIRG1 gene mutation. We propose that these mutations in gene TCIRG1 could be a novel therapeutic target for the IMO in the future.

Knockdown of Tcirg1 inhibits large-osteoclast generation by down-regulating NFATc1 and IP3R2 expression.

The TCIRG1 gene encodes the a3 isoform of vacuolar H+-ATPase (V-ATPase), which forms a proton transport channel in osteoclasts. Defects in this gene lead to functional impairment of osteoclasts and increased bone mass; however, the molecular mechanisms of TCIRG1 loss have not been fully elucidated. In the current study, we transfected mouse bone marrow-derived monocytes with control or Tcirg1-knockdown lentiviruses to further investigate the mechanisms of TCIRG1. Our results demonstrate that knockdown of Tcirg1 inhibits large-osteoclast (>100 μm) generation by decreasing the expression of nuclear factor of activated T-cells 1 (NFATc1) and inositol-1,4,5-trisphosphate receptor 2 (IP3R2). The decreased IP3R2 reduces intracellular calcium levels, which limits the nuclear translocation of NFATc1 in RANKL-induced mouse bone marrow-derived monocytes. These findings provide a mechanism to explain the effects of TCIRG1 impairment, with potential implications for the development of therapies for osteopetrosis.

Osteopetrosis in a six-month-old infant

Osteopetrosis is a severe and frequently fatal condition characterized by the formation of thick, dense bone, with a concomitant loss of bone marrow spaces secondary to defective osteoclast function or development. Clinical symptoms include pancytopenia, hepatosplenomegaly, fractures, cranial nerve impingement, and increased cranial pressure, among others. The condition requires rapid diagnosis and clinical intervention, as hematopoietic stem cell transplantation performed early in life can be curative in the majority of cases and can prevent the irreversible neurological impairment associated with the disease. We report a six-month-old male with osteopetrosis where bone marrow examination and molecular studies allowed a definitive diagnosis. Microscopic examination of a bone marrow biopsy demonstrated increased bone formation at various stages of ossification, decreased marrow spaces, and increased osteoclasts rimming the bone. Next-generation sequencing (NGS) demonstrated that the patient was homozygous for mutations in the TCIRG1 gene, which encodes an osteoclast vacuolar proton pump and is the most common mutation associated with the autosomal recessive or infantile malignant form of osteopetrosis. The patient underwent a cord blood stem cell transplant, but unfortunately expired four months after diagnosis. This case highlights the importance of bone marrow biopsy evaluation as part of the integrated approach to diagnosing osteopetrosis.

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, an important 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 (BM), supporting their use in gene therapy protocols. Transcriptomic profile revealed enrichment in genes expressed by hematopoietic stem and progenitor cells (HSPC). To overcome the limit of BM harvest/HSPC mobilization and serial blood drawings in TCIRG1 patients, we applied UM171-based ex vivo expansion of HSPC coupled with lentiviral gene transfer. Circulating CD34+ cells from TCIRG1- defective patients were transduced with a clinically-optimized lentiviral vector 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 NOD scid gamma common chain (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 HSPC 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 BM fibrosis.

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

BackgroundAutosomal 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. MethodsWe generated a new mouse model, which we named NSG oc/oc, presenting severe autosomal recessive osteopetrosis owing to the Tcirg1oc mutation, and profound immunodeficiency caused by the NSG background. We performed neonatal murine bone marrow transplantation and xenotransplantation with human CD34+ cells. ResultsWe 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. ConclusionOur 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.

Stone bone – Is osteopetrosis as hard as it can be???

Bone is a vibrant tissue comprising of osteoblasts and osteoclasts. Osteoblasts synthesise bone matrix, whereas osteoclasts help in bone resorption. Osteopetrosis is a heritable disorder that leads to failure of osteoclasts to resorb bone. This leads to impairment in bone modelling and remodelling as changes occur in the shape and structure of the bone. Nearly 70% of osteopetrosis is caused by genetic mutations of TCIRG1, CLCN7, OSTM1, RANKL, RANK and PLEKHM1 genes. Clinical manifestations range from increased bone density, skeletal fragility, disturbed tooth eruptions to haematopoietic insufficiency, nervous degeneration, mental retardation and developmental delay. Diagnosis is mainly by clinical presentation and radiological findings. Treatment is primarily supportive to stimulate host osteoclasts by administering calcium and Vitamin D supplementation, high-dose calcitriol, joint arthroplasty, interferon gamma 1b and gene therapy. Haematopoietic stem cell transplantation is indicated for severe forms of autosomal recessive osteopetrosis. Infantile and intermediate forms of autosomal recessive osteopetrosis can be fatal if left untreated. This article includes a case report of osteopetrosis with focused nursing care using nursing process approach.

Hematopoietic Stem Cell-Targeted Neonatal Gene Therapy with a Clinically Applicable Lentiviral Vector Corrects Osteopetrosis in oc/oc Mice

Infantile malignant osteopetrosis (IMO) is an autosomal recessive disorder characterized by nonfunctional osteoclasts. Approximately 50% of the patients have mutations in the TCIRG1 gene, encoding for a subunit of the osteoclast proton pump. Gene therapy represents a potential alternative treatment to allogeneic stem cell transplantation for IMO. The oc/oc mouse is a model of IMO characterized by a 1,500 bp deletion in the TCIRG1 gene, severe osteopetrosis, and a life span of only 3 weeks. Here we show that the osteopetrotic phenotype in oc/oc mice can be reversed by hematopoietic stem cell-targeted gene therapy with a clinically applicable lentiviral vector expressing a wild-type form of human TCIRG1 under the mammalian promoter elongation factor 1α short (EFS-hT). oc/oc c-kit+ fetal liver cells transduced with EFS-hT were transplanted into sublethally irradiated oc/oc mice by temporal vein injection 1 day after birth. A total of 9 of 12 mice survived long term (19-25 weeks) with evidence of tooth eruption, uncharacteristic of oc/oc mice. Splenocytes were harvested 19-25 weeks after transplantation and differentiated into osteoclasts on bone slices to assess resorption and on plastic to assess TCIRG1 protein expression. Vector-corrected osteoclasts showed human TCIRG1 expression by Western blot. CTX-I release relative to that mediated by oc/oc-derived osteoclasts increased 8-239-fold. Resorption pits on bone slices were observed for osteoclasts derived from 7/9 surviving transplanted oc/oc mice. Histopathology of the bones of surviving animals showed varying degrees of rescued phenotype, the majority with almost full correction. The average vector copy number per cell in the bone marrow was 1.8 ± 0.5. Overall, 75% of transplanted mice exhibited long-term survival and marked reversal of the osteopetrotic bone phenotype. These findings represent a significant step toward the clinical application of gene therapy for IMO.

TCIRG1 and SNX10 gene mutations in the patients with autosomal recessive osteopetrosis

Autosomal recessive osteopetrosis (ARO) is a rare genetic bone disease characterized by dense and fragile bone, caused by a defect in osteoclasts responsible for the bone destruction. In this study, we aimed to investigate the mutations in TCIRG1 and SNX10 that are responsible for 50% and 4% of the cases, respectively. All amplicons were sequenced by Sanger sequencing following PCR amplification. As a result, six different mutations of the TCIRG1 gene were found in five of the twelve unrelated cases. These include two novel mutations, namely c.630 + 1G > T mutation and c.1778_1779delTG mutation of the gene which are identified as homozygous. A compound heterozygosity of known mutations c.649_674del26 and c.1372G > A and homozygous presence of the known c.2235 + 1G > A mutation were also observed in different patients. In addition, as a result of the prenatal testing in a family with osteopetrosis infant, the c.1674-1G > A mutation was detected as homozygous for the fetus. In TCIRG1, c.166C > T change, which is indicated as likely benign according to ClinVar database, was heterozygous. Several known polymorphisms; c.117 + 83 T > C, c.417 + 11A > G and c.714-19C > A in TCIRG1 gene; c.24 + 36 T > A and c.112-84G > A in SNX10 gene were also detected. In conclusion, our study revealed that five of the twelve cases carry at least one mutation of TCIRG1 gene. Further studies with more patients and other genes would help better understanding of genetic etiology of the disease.

Histopathological changes of bone marrow in patients with autosomal recessive osteopetrosis and mutation in TCIRG1 gene

Osteopetrosis is a group of rare hereditary diseases, the general structural manifestation of which is the excessive volume of bone tissue due to violation of osteoclastic resorption. The only way to treat this group of patients so far remains transplantation of hematopoietic stem cells, but the degree of its effectiveness largely depends on the severity of morphological changes in the hematopoietic microenvironment in the bone marrow. In this regard, a comprehensive clinical and morphological analysis, in conjunction with the results of transplantation can help in determining the prognosis of the disease depending on the genetic type of osteopetrosis. The material for the studies were biopsies and smears of bone marrow of patients with osteopetrosis, who were received at the R. Gorbacheva Memorial Research Institute for Pediatric Oncology, Hematology and Transplantation from the republics of Chuvashia and Mari El for carrying out transplantation of hematopoietic stem cells. The histological preparations were stained with hematoxylin and eosin, azur by Romanovsky and by Van Gieson. Bone marrow smears were stained by Romanovsky-Giemsa. The presence of the mutation c.807 + 5G>A in the gene TCIRG1 in patients with autosomal recessive osteopetrosis causes a complete loss of osteoclastic resorption, which is accompanied by pronounced early structural changes in the hematopoietic microenvironment already at the time of diagnosis. This is manifested by an excessive amount of lamellar bone at the same time as the almost complete obliteration of the medullar lacunae, as well as fibrosis of the bone marrow stroma. A common morphological phenomenon among this group of patients is the presence in the bone marrow of an excessive number of osteoclast differentiation cells without the formation of resorption lacunae. This causes a complete suppression of hemopoiesis, which clinically manifests cytopenia and the formation of foci of extramedullary hematopoiesis. Such pronounced structural changes in the hematopoietic microenvironment result in a high risk of primary graft failure during after transplantation of bone marrow hematopoietic stem cells, the effectiveness of which decreases as the child's age increases. Early development of pronounced structural changes in the hematopoietic microenvironment in patients with TCIRG1-mediated osteopetrosis determines the need for diagnosis and transplantation as soon as possible after birth.