Adult Osteosclerotic Metaphyseal Dysplasia With Progressive Osteonecrosis of the Jaws and Abnormal Bone Resorption Pattern Due to a LRRK1 Splice Site Mutation

Osteosclerotic metaphyseal dysplasia (OSMD) is a rare autosomal recessive sclerosing skeletal dysplasia. We report on a 34-year-old patient with sandwich vertebrae, platyspondyly, osteosclerosis of the tubular bones, pathologic fractures, and anemia. In the third decade, he developed osteonecrosis of the jaws, which was progressive in spite of repeated surgical treatment over a period of 11 years. An iliac crest bone biopsy revealed the presence of hypermineralized cartilage remnants, large multinucleated osteoclasts with abnormal morphology, and inadequate bone resorption typical for osteoclast-rich osteopetrosis. After exclusion of mutations in TCIRG1 and CLCN7 we performed trio-based exome sequencing. The novel homozygous splice-site mutation c.261G>A in the gene LRRK1 was found and co-segregated with the phenotype in the family. cDNA sequencing showed nearly complete skipping of exon 3 leading to a frameshift (p.Ala34Profs*33). Osteoclasts differentiated from the patient's peripheral blood monocytes were extremely large. Instead of resorption pits these cells were only capable of superficial erosion. Phosphorylation of L-plastin at position Ser5 was strongly reduced in patient-derived osteoclasts showing a loss of function of the mutated LRRK1 kinase protein. Our analysis indicates a strong overlap of LRRK1-related OSMD with other forms of intermediate osteopetrosis, but an exceptional abnormality of osteoclast resorption. Like in other osteoclast pathologies an increased risk for progressive osteonecrosis of the jaws should be considered in OSMD, an intermediate form of osteopetrosis

Efficient generation of osteoclasts from human induced pluripotent stem cells and functional investigations of lethal CLCN7‐related osteopetrosis

Human induced pluripotent stem cells (hiPSCs) hold great potential for modeling human diseases and the development of innovative therapeutic approaches. Here, we report on a novel, simplified differentiation method for forming functional osteoclasts from hiPSCs. The three-step protocol starts with embryoid body formation, followed by hematopoietic specification, and finally osteoclast differentiation. We observed continuous production of monocyte-like cells over a period of up to 9 weeks, generating sufficient material for several osteoclast differentiations. The analysis of stage-specific gene and surface marker expression proved mesodermal priming, the presence of monocyte-like cells, and of terminally differentiated multinucleated osteoclasts, able to form resorption pits and trenches on bone and dentine in vitro. In comparison to peripheral blood mononuclear cell (PBMC)-derived osteoclasts hiPSC-derived osteoclasts were larger and contained a higher number of nuclei. Detailed functional studies on the resorption behavior of hiPSC-osteoclasts indicated a trend towards forming more trenches than pits and an increase in pseudoresorption. We used hiPSCs from an autosomal recessive osteopetrosis (ARO) patient (BIHi002-A, ARO hiPSCs) with compound heterozygous missense mutations p.(G292E) and p.(R403Q) in CLCN7, coding for the Cl-/H+-exchanger ClC-7, for functional investigations. The patient's leading clinical feature was a brain malformation due to defective neuronal migration. Mutant ClC-7 displayed residual expression and retained lysosomal co-localization with OSTM1, the gene coding for the osteopetrosis-associated transmembrane protein 1, but only ClC-7 harboring the mutation p.(R403Q) gave strongly reduced ion currents. An increased autophagic flux in spite of unchanged lysosomal pH was evident in undifferentiated ARO hiPSCs. ARO hiPSC-derived osteoclasts showed an increased size compared to hiPSCs of healthy donors. They were not able to resorb bone, underlining a loss-of-function effect of the mutations. In summary, we developed a highly reproducible, straightforward hiPSC-osteoclast differentiation protocol. We demonstrated that osteoclasts differentiated from ARO hiPSCs can be used as a disease model for ARO and potentially also other osteoclast-related diseases. (c) 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR)

Development of a disease model for autosomal recessive osteopetrosis and novel CRISPR/Cas9-based gene therapeutic approaches in human stem cells

Die autosomal-rezessive Osteopetrose (ARO) ist eine schwere, erblich bedingte Knochenerkrankung, die auf den Verlust der osteoklastenvermittelten Knochenresorption zurückzuführen ist. Eine Ursache dieses Osteoklastendefekts sind Mutationen im CLCN7-Gen, das für den Cl-/H+-Austauscher ClC-7 codiert. Da Osteoklasten von hämatopoetischen Stamm- und Progenitorzellen (HSPCs) abstammen, kann ARO mittels allogener Stammzelltransplantation geheilt werden. Jedoch ist diese Therapie mit einer hohen Mortalitätsrate assoziiert und oftmals sind keine passenden Spender verfügbar. Die autologe Transplantation von mittels Genomeditierung korrigierten HSPCs könnte die Therapie der ARO verbessern. Für die Etablierung eines solchen Behandlungsansatzes wurde hier zunächst ein ARO-Krankheitsmodell entwickelt, da aufgrund der Seltenheit der Erkrankung und des jungen Alters der Patienten die HSPCs nicht in ausreichendem Maß für Forschungszwecke zur Verfügung stehen. Hierfür wurden mononukläre Zellen des peripheren Blutes eines Patienten mit CLCN7-bedingter ARO zu induzierten pluripotenten Stammzellen (iPS) reprogrammiert, die anschließend zu Osteoklasten differenziert wurden. Diese waren nicht in der Lage Knochen zu resorbieren und spiegelten so den zellulären Phänotyp der Osteopetrose wider. Mithilfe dieses iPS-basierten Krankheitsmodells als auch mit HSPCs gesunder Kontrollspender wurden neue CRISPR/Cas9-basierte gentherapeutische Strategien für ARO entwickelt und getestet. Durch die Anwendung optimierter Protokolle zur CRISPR/Cas9-Genomeditierung konnten sowohl iPS als auch HSPCs selektionsfrei hocheffizient gezielt genetisch verändert werden. Darüber hinaus konnte durch die heterozygote Korrektur der pathogenen Mutation der Patienten-iPS die Knochenresorptionsfunktion der Osteoklasten partiell wiederhergestellt werden. In dieser Arbeit wurden die Grundlagen für eine somatische Gentherapie der CLCN7-bedingten ARO geschaffen, die es in Zukunft weiterzuentwickeln gilt.Autosomal recessive osteopetrosis (ARO) is a severe hereditary bone disease caused by lacking osteoclast-mediated bone resorption. One reason for this osteoclast defect are mutations in the CLCN7 gene coding for the Cl-/H+-exchanger ClC-7. Since osteoclasts develop from hematopoietic stem and progenitor cells (HSPCs), allogeneic HSPC transplantation is a curative treatment for ARO. However, HSPC transplantation is associated with high mortality and often no suitable donors are available. Autologous transplantation of HSPCs corrected by genome editing could improve the treatment of ARO. Due to the rarity of the disease and the patient’s young age, HSPCs are not sufficiently available for research. Therefore, an ARO disease model was developed. Peripheral blood mononuclear cells of a patient with CLCN7-related ARO were reprogrammed into induced-pluripotent stem cells (iPSCs) and differentiated into osteoclasts. These patient osteoclasts lost their bone resorption capacity, thus recapitulating the cellular ARO phenotype. Patient-derived iPSCs and HSPCs from healthy control donors were used to develop and test novel CRISPR/Cas9-based gene therapeutic approaches for ARO. Highly efficient targeted genome editing was achieved in both iPSCs and HSPCs using optimized protocols for CRISPR/Cas9-based genome editing. Heterozygous correction of the pathogenic mutation in patient-derived iPSCs partially restored bone resorption of the iPSC-derived osteoclasts. Taken together, this work lays the foundation for a somatic gene therapy for CLCN7-related ARO, which is to be developed further in future

Generation of a human induced pluripotent stem cell line (BIHi002-A) from a patient with CLCN7-related infantile malignant autosomal recessive osteopetrosis

Autosomal recessive osteopetrosis (ARO) is a genetic bone disease that can be caused by mutations in the CLCN7 gene preventing osteoclast-mediated bone resorption. We generated a human induced pluripotent stem cell (hiPSC) line, BIHi002-A, from peripheral blood mononuclear cells of an ARO patient carrying the CLCN7 mutations c.875G>A and c.1208G>A using Sendai viral vectors. The pluripotent identity of the BIHi002-A line was confirmed by their expression of typical markers for undifferentiated hiPSCs, their capacity to differentiate into cells of the three germ layers and by PluriTest analysis. The BIHi002-A line provides a tool for disease modelling and therapy development