Genotype-phenotype analysis of selective failure of tooth eruption-A systematic review.

Tooth eruption is an important and unique biological process during craniofacial development. Both the genetic and environmental factors can interfere with this process. Here we aimed to find the failure pattern of tooth eruption among five genetic diseases. Both systematic review and meta-analysis were used to identify the genotype-phenotype associations of unerupted teeth. The meta-analysis was based on the characteristics of abnormal tooth eruption in 223 patients with the mutations in PTH1R, RUNX2, COL1A1/2, CLCN7, and FAM20A respectively. We found all the patients presented selective failure of tooth eruption (SFTE). Primary failure of eruption patients with PTH1R mutations showed primary or isolated SFTE1 in the first and second molars (59.3% and 52% respectively). RUNX2 related cleidocranial dysplasia usually had SFTE2 in canines and premolars, while COL1A1/2 related osteogenesis imperfecta mostly caused SFTE3 in the maxillary second molars (22.9%). In CLCN7 related osteopetrosis, the second molars and mandibular first molars were the most affected. While FAM20A related enamel renal syndrome most caused SFTE5 in the second molars (86.2%) and maxillary canines. In conclusion, the SFTE was the common characteristics of most genetic diseases with abnormal isolated or syndromic tooth eruption. The selective pattern of unerupted teeth was gene-dependent. Here we recommend SFTE to classify those genetic unerupted teeth and guide for precise molecular diagnosis and treatment.

A Mild Case of Autosomal Recessive Osteopetrosis Masquerading as the Dominant Form Involving Homozygous Deep Intronic Variations in the CLCN7 Gene.

Osteopetrosis is a heterogeneous group of rare hereditary diseases characterized by increased bone mass of poor quality. Autosomal-dominant osteopetrosis type II (ADOII) is most often caused by mutation of the CLCN7 gene leading to impaired bone resorption. Autosomal recessive osteopetrosis (ARO) is a more severe form and is frequently accompanied by additional morbidities. We report an adult male presenting with classical clinical and radiological features of ADOII. Genetic analyses showed no amino-acid-converting mutation in CLCN7 but an apparent haploinsufficiency and suppression of CLCN7 mRNA levels in peripheral blood mononuclear cells. Next generation sequencing revealed low-frequency intronic homozygous variations in CLCN7, suggesting recessive inheritance. In silico analysis of an intronic duplication c.595-120_595-86dup revealed additional binding sites for Serine- and Arginine-rich Splicing Factors (SRSF), which is predicted to impair CLCN7 expression. Quantitative backscattered electron imaging and histomorphometric analyses revealed bone tissue and material abnormalities. Giant osteoclasts were present and additionally to lamellar bone, and abundant woven bone and mineralized cartilage were observed, together with increased frequency and thickness of cement lines. Bone mineralization density distribution (BMDD) analysis revealed markedly increased average mineral content of the dense bone (CaMean T-score + 10.1) and frequency of bone with highest mineral content (CaHigh T-score + 19.6), suggesting continued mineral accumulation and lack of bone remodelling. Osteocyte lacunae sections (OLS) characteristics were unremarkable except for an unusually circular shape. Together, our findings suggest that the reduced expression of CLCN7 mRNA in osteoclasts, and possibly also osteocytes, causes poorly remodelled bone with abnormal bone matrix with high mineral content. This together with the lack of adequate bone repair mechanisms makes the material brittle and prone to fracture. While the skeletal phenotype and medical history were suggestive of ADOII, genetic analysis revealed that this is a possible mild case of ARO due to deep intronic mutation.

Diagnosis and discovery: Insights from the NIH Undiagnosed Diseases Program.

Living with an undiagnosed medical condition places a tremendous burden on patients, their families, and their healthcare providers. The Undiagnosed Diseases Program (UDP) was established at the National Institutes of Health (NIH) in 2008 with the primary goals of providing a diagnosis for patients with mysterious conditions and advancing medical knowledge about rare and common diseases. The program reviews applications from referring clinicians for cases that are considered undiagnosed despite a thorough evaluation. Those that are accepted receive clinical evaluations involving deep phenotyping and genetic testing that includes exome and genomic sequencing. Selected candidate gene variants are evaluated by collaborators using functional assays. Since its inception, the UDP has received more than 4500 applications and has completed evaluations on nearly 1300 individuals. Here we present six cases that exemplify the discovery of novel disease mechanisms, the importance of deep phenotyping for rare diseases, and how genetic diagnoses have led to appropriate treatment. The creation of the Undiagnosed Diseases Network (UDN) in 2014 has substantially increased the number of patients evaluated and allowed for greater opportunities for data sharing. Expansion to the Undiagnosed Diseases Network International (UDNI) has the possibility to extend this reach even farther. Together, networks of undiagnosed diseases programs are powerful tools to advance our knowledge of pathophysiology, accelerate accurate diagnoses, and improve patient care for patients with rare conditions.

Anterior cruciate ligament rupture in a patient with Albers-Schonberg disease.

BACKGROUND: Osteopetrosis is an uncommon inherited disease marked with elevated bone density and frequent bone fractures owing to flawed osteoclast activity. Autosomal dominant osteopetrosis type 2 (ADO-2), a benign form of osteopetrosis, is also known as Albers-Schonberg disease. CASE PRESENTATION: We report the first successful anterior cruciate ligament (ACL) reconstruction surgery for ACL rupture treatment in a 30-year-old female with ADO-2, who carried a heterozygous missense mutation c.2227C > T (p.Arg743Trp) in exon 23 of the chloride channel 7 (CLCN7) gene. Histopathological analysis of the ruptured ACL sample revealed massive calcium salt deposition in the ligament tissue. A ligament advanced reinforcement system (LARS) artificial ligament was employed in her ACL reconstruction surgery. At her final 16 month's follow-up, she reported no knee instability symptoms and other complications. The range of motion of the affected knee was good. The side-to-side difference in knee laxity, as evidenced by a KT-1000 arthrometer was 0.9 mm. The Lysholm score improved from 45 before operation to 83 after operation. The Tegner activity score improved from 1 before operation to 4 after operation. CONCLUSIONS: Our findings further confirmed that the newly identified mutated locus (p.Arg743Trp) may lead to acid secretion disorders at different sites (including calcified ACL in our case). In terms of clinical treatment, ligament reconstruction surgery in patients with Albers-Schonberg disease presents a unique challenge to orthopedic surgeons and requires further preparation and time.

Novel CLCN7 mutation identified in a Han Chinese family with autosomal dominant osteopetrosis-2.

Osteopetrosis is a heritable bone condition featuring increased bone density due to defective osteoclastic bone resorption. Exome sequencing and Sanger sequencing were conducted in Han Chinese family members, some of whom had typical osteopetrosis, and a novel missense variant c.2350A>T (p.R784W) in the chloride channel 7 gene (CLCN7) was identified. This variant cosegregated with the disorder in the family but was not observed in 800 controls. The data indicate that exome sequencing is a powerful and effective molecular diagnostic tool for detecting mutations in osteopetrosis, which is a genetically and clinically heterogeneous disorder. This discovery broadens the CLCN7 gene mutation spectrum and has important implications for clinical therapeutic regimen decisions, prognosis evaluations, and antenatal diagnoses.

Novel mutations of CLCN7 cause autosomal dominant osteopetrosis type II (ADO-II) and intermediate autosomal recessive osteopetrosis (IARO) in Chinese patients.

SUMMARY: Osteopetrosis is a group of genetic bone disorders. Mutations in the chloride channel 7 gene (CLCN7) lead to chloride channel defect, which results in autosomal dominant osteopetrosis type II (ADO-II), autosomal recessive osteopetrosis (ARO), and intermediate autosomal recessive osteopetrosis (IARO). In the present study, we identified seven novel mutations of the CLCN7 gene and reported the first case of IARO with compound heterozygous mutation in Chinese population. INTRODUCTION: Osteopetrosis is a heritable bone disorder due to the deficiency of or function defect in osteoclasts. Mutations in the CLCN7 lead to chloride channel defects, which result in osteopetrosis with diverse severity ranging from asymptomatic or relatively mild symptoms in ADO-II to the very severe phenotype in ARO. Heterozygous mutations in CLCN7 are associated to ADO-II, while homozygous and compound heterozygous mutations in CLCN7 may result in ARO and IARO. To date, a total of 24 mutations in CLCN7 were identified in ADO-II, and only 3 mutations were identified in IARO. In the present study, we reported seven unrelated ADO-II patients and one IARO patient from Chinese population and elucidated the characteristics of CLCN7 gene mutations in these patients. METHODS: All 25 CLCN7 exons and exon-intron boundaries from genomic DNA were amplified and sequenced in eight affected individuals suffering from ADO-II/IARO. The clinical, biochemical, and radiographic analysis were evaluated to compare the differences between ADO-II and IARO both in genotype and phenotype. RESULTS: The results showed that there were seven novel CLCN7 mutations identified in these ADO-II/IARO patients, including six heterozygous missense mutations (p.L224R, p.S290Y, p.R326G, p.G347R, p.S473N, and p.L564P) and a novel splice mutation (p.K691FS). CONCLUSIONS: The compound heterozygous mutations (p.L224R and p.K691FS) were firstly observed in one IARO patient. The present study would enrich the database of CLCN7 mutations and improve our understanding of this heritable bone disorder.

Two novel mutations of CLCN7 gene in Chinese families with autosomal dominant osteopetrosis (type II).

Autosomal dominant osteopetrosis type II (ADO-II) is a heritable bone disorder characterized by osteosclerosis, predominantly involving the spine (vertebral end-plate thickening, or rugger-jersey spine), the pelvis ("bone-within-bone" structures) and the skull base. Chloride channel 7 (CLCN7) has been reported to be the causative gene. In this study, we aimed to identify the pathogenic mutation in four Chinese families with ADO-II. All 25 exons of the CLCN7 gene, including the exon-intron boundaries, were amplified and sequenced directly in four probands from the Chinese families with ADO-II. The mutation site was then identified in other family members and 250 healthy controls. In family 1, a known missense mutation c.296A>G in exon 4 of CLCN7 was identified in the proband, resulting in a tyrosine (UAU) to cysteine (UGU) substitution at p.99 (Y99C); the mutation was also identified in his affected father. In family 2, a novel missense mutation c.865G>C in exon 10 was identified in the proband, resulting in a valine (GUC) to leucine (CUC) substitution at p.289 (V289L); the mutation was also identified in her healthy mother and sister. In family 3, a novel missense mutation c.1625C>T in exon 17 of CLCN7 was identified in the proband, resulting in an alanine (GCG) to valine (GUG) substitution at p.542 (A542V); the mutation was also identified in her father. In family 4, a hot spot, R767W (c.2299C>T, CGG>TGG), in exon 24 was found in the proband which once again proved the susceptibility of the site or the similar genetic background in different races. Moreover, two novel mutations, V289L and A542V, occurred at a highly conserved position, found by a comparison of the protein sequences from eight vertebrates, and were predicted to have a pathogenic effect by PolyPhen-2 software, which showed "probably damaging" with a score of approximately 1. These mutation sites were not identified in 250 healthy controls. Our present findings suggest that the novel missense mutations V289L and A542V in the CLCN7 gene were responsible for ADO-II in the two Chinese families.

Multisystem disorder associated with a pathogenic variant in CLCN7 in the absence of osteopetrosis.

BACKGROUND: We clinically and genetically evaluated a Taiwanese boy presenting with developmental delay, organomegaly, hypogammaglobulinemia and hypopigmentation without osteopetrosis. Whole-exome sequencing revealed a de novo gain-of-function variant, p.Tyr715Cys, in the C-terminal domain of ClC-7 encoded by CLCN7. METHODS: Nicoli et al. (2019) assessed the functional impact of p.Tyr715Cys by heterologous expression in Xenopus oocytes and evaluating resulting currents. RESULTS: The variant led to increased outward currents, indicating it underlies the patient's phenotype of lysosomal hyperacidity, storage defects and vacuolization. This demonstrates the crucial physiological role of ClC-7 antiporter activity in maintaining appropriate lysosomal pH. CONCLUSION: Elucidating mechanisms by which CLCN7 variants lead to lysosomal dysfunction will advance understanding of genotype-phenotype correlations. Identifying modifier genes and compensatory pathways may reveal therapeutic targets. Ongoing functional characterization of variants along with longitudinal clinical evaluations will continue advancing knowledge of ClC-7's critical roles and disease mechanisms resulting from its dysfunction. Expanded cohort studies are warranted to delineate the full spectrum of associated phenotypes.

Molecular Heterogeneity of Osteopetrosis in India: Report of 17 Novel Variants.

Osteopetrosis is a clinically and genetically heterogeneous group of inherited bone disorders that is caused by defects in osteoclast formation or function. Treatment options vary with the disease severity and an accurate molecular diagnosis helps in prognostication and treatment decisions. We investigated the genetic causes of osteopetrosis in 31 unrelated patients of Indian origin. Screening for the genetic variants was done by Sanger sequencing or next generation sequencing in 48 samples that included 31 samples from index patients, 16 from parents' and 1 chorionic villus sample. A total of 30 variants, including 29 unique variants, were identified in 26 of the 31 patients in the study. TCIRG1 was the most involved gene (n = 14) followed by TNFRSF11A (n = 4) and CLCN7 (n = 3). A total of 17 novel variants were identified. Prenatal diagnosis was done in one family and the foetus showed homozygous c.807 + 2T > G variant in TCIRG1. Molecular diagnosis of osteopetrosis aids in therapeutic decisions including the need for a stem cell transplantation and gives a possible option of performing prenatal diagnosis in affected families. Further studies would help in understanding the genetic etiology in patients where no variants were identified. Supplementary Information: The online version contains supplementary material available at 10.1007/s12288-023-01732-4.

Osteopetrosis complicated by multilevel spondylolysis.

Osteopetrosis is a heterogenous group of inheritable disorders which manifests as increased bone density and brittleness. The most common and mildest variant typically presents in adulthood with bone pain and pathologic fractures, including spondylolysis. We present the case of an otherwise healthy, active 17-year-old male with a history of osteopetrosis and 1 year of chronic back pain, found to have multilevel (L1-L4) spondylolysis in the setting of severe diffuse bony sclerosis consistent with osteopetrosis. While single-level spondylolysis is an uncommon complication of osteopetrosis, multilevel spondylolysis in the pediatric population is extremely rare and the genetics of prior cases studies have not been reported. Spondylolysis should be considered as one of the types of fractures that may occur in patients with osteopetrosis.

Case report: Gene mutations and clinical characteristics of four patients with osteopetrosis.

Osteopetrosis is characterized by increased bone density caused by decreased osteoclasts or dysfunction of their differentiation and absorption properties, usually caused by biallelic variants of the TCIRG1(OMIM:604592)and CLCN7(OMIM:602727) genes. Herein, the clinical, biochemical, and radiological manifestations of osteopetrosis in four Chinese children are described. Whole-exome sequencing identified compound heterozygous variants of the CLCN7 and TCIRG1 genes in these patients. In Patient 1, two novel variants were identified in CLCN7:c.880T > G(p.F294V) and c.686C > G(p.S229X). Patient 2 harbored previously reported a single gene variant c.643G > A(p.G215R) in CLCN7. Patient 3 had a novel variant c.569A > G(p.N190S) and a novel frameshift variant c.1113dupG(p.N372fs) in CLCN7. Patient 4 had a frameshift variant c.43delA(p.K15fs) and variant c.C1360T in TCIRG1, resulting in the formation of a premature termination codon (p.R454X), both of which were reported previously. Our results expand the spectrum of identified genetic variation in osteopetrosis and provide a deeper understanding of the relations between genotype and clinical characteristics of this disorder.

CLCN7, a gene shared by autosomal recessive and autosomal dominant osteopetrosis.

After the discovery of abundant v-ATPase complexes in the osteoclast ruffled membrane it was obvious that in parallel a negative counter-ion needs to be transported across this membrane to allow for efficient transport of protons into the resorption lacuna. While different candidate proteins were discussed the osteopetrosis phenotype of Clcn7 knockout mice suggested that the chloride/proton-exchanger ClC-7 might be responsible for transporting the negative charge. In the following, individuals with autosomal recessive osteopetrosis (ARO) were found to carry biallelic CLCN7 pathogenic variants. Shortly thereafter, heterozygous pathogenic variants were identified as the exclusive cause of autosomal dominant osteopetrosis type 2 (ADO2). Since in most cell types other than osteoclasts ClC-7 resides in late endosomes and lysosomes, it took some time until the electrophysiological properties of ClC-7 were elucidated. Whereas most missense variants lead to reduced chloride currents, several variants with accelerated kinetics have been identified. Evidence for folding problems is also known for several missense variants. Paradoxically, a heterozygous activating variant in ClC-7 was described to cause lysosomal alteration, pigmentation defects, and intellectual disability without osteopetrosis. The counter-intuitive 2 Cl-/H+ exchange function of ClC-7 was shown to be physiologically important for intravesicular ion homeostasis. The lysosomal function of ClC-7 is also the reason why individuals with CLCN7-ARO can develop a storage disorder and neurodegeneration, a feature that is variable and difficult to predict. Furthermore, the low penetrance of heterozygous pathogenic CLCN7 variants and the clinical variability of ADO2 are incompletely understood. We aim to give an overview not only of the current knowledge about ClC-7 and its related pathologies, but also of the scientists and clinicians that paved the way for these discoveries.

A Mendelian Randomization-Based Causal Investigation on Bacterial Infection-Related Genes and Neurodegenerative Diseases.

Neurodegenerative diseases remain the most prevalent and unsolved health problems in human society, especially Alzheimer's disease (AD) and Parkinson's disease (PD). The pathogenesis, pathology, and potential clinical treatments of neurodegenerative diseases still require in-depth research. In the wake of the association between pandemics and a growing number of neurodegeneration patients, there has been growing speculation that infections are linked to AD and PD. The Aß peptide is an important causal-related biomarker of AD and is reported to share structural and functional similarities with certain antimicrobial peptides, suggesting that it has a role in eliciting an immune response against microbes. But how neurodegeneration is related to bacterial chronic infection has not been thoroughly investigated. Using the data from genome-wide association studies (GWAS), we performed Mendelian Randomization (MR) and map 7 genes in multiple bacterial infection pathways as exposure, which show a significant association with the outcome of AD or PD. As co-verification, we perform Gene Set Enrichment Analysis (GSEA) on selected genetic variants incorporating their perturb-seq gene list (combining single-cell RNA-seq and CRISPR-based perturbations). We observed clustering of the differentially expressed genes (DEGs) in the upstream and downstream of AD and PD-related KEGG pathways, hence confirming their causal association with AD and PD and providing new perspectives on the true cause of neurodegeneration.

CRISPR/Cas9-Mediated Gene Correction in Osteopetrosis Patient-Derived iPSCs.

BACKGROUND: Osteopetrosis represents a rare genetic disease with a wide range of clinical and genetic heterogeneity, which results from osteoclast failure. Although up to 10 genes have been identified to be related with osteopetrosis, the pathogenesis of osteopetrosis remains foggy. Disease-specific induced pluripotent stem cells (iPSCs) and gene-corrected disease specific iPSCs provide a platform to generate attractive in vitro disease cell models and isogenic control cellular models respectively. The purpose of this study is to rescue the disease causative mutation in osteopetrosis specific induced pluripotent stem cells and provide isogenic control cellular models. METHODS: Based on our previously established osteopetrosis-specific iPSCs (ADO2-iPSCs), we repaired the point mutation R286W of the CLCN7 gene in ADO2-iPSCs by the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) mediated homologous recombination. RESULTS: The obtained gene corrected ADO2-iPSCs (GC-ADO2-iPSCs) were characterized in terms of hESC-like morphology, a normal karyotype, expression of pluripotency markers, homozygous repaired sequence of CLCN7 gene, and the ability to differentiate into cells of three germ layers. CONCLUSIONS: We successfully corrected the point mutation R286W of the CLCN7 gene in ADO2-iPSCs. This isogenic iPSC line is an ideal control cell model for deciphering the pathogenesis of osteopetrosis in future studies.

A novel compound heterozygous mutation of the CLCN7 gene is associated with autosomal recessive osteopetrosis.

Osteopetrosis is a genetic condition of the skeleton characterized by increased bone density caused by osteoclast formation and function defects. Osteopetrosis is inherited in the form of autosomal dominant and autosomal recessive manner. We report autosomal recessive osteopetrosis (ARO; OMIM 611490) in a Chinese case with a history of scarce leukocytosis, vision and hearing loss, frequent seizures, and severe intellectual and motor disability. Whole-exome sequencing (WES) followed by Sanger sequencing revealed novel compound heterozygous mutations in the chloride channel 7 (CLCN7) gene [c.982-1G > C and c.1208G > A (p. Arg403Gln)] in the affected individual, and subsequent familial segregation showed that each parent had transmitted a mutation. Our results confirmed that mutations in the CLCN7 gene caused ARO in a Chinese family. Additionally, our study expanded the clinical and allelic spectrum of the CLCN7 gene and enhanced the applications of WES technology in determining the etiology of prenatal diagnoses in fetuses with ultrasound anomalies.

Natural History of Type II Autosomal Dominant Osteopetrosis: A Single Center Retrospective Study.

Background: Autosomal dominant osteopetrosis II (ADO II, MIM166600) is a sclerosing bone disorder caused by CLCN7 mutation. The main clinical characteristics include minor trauma-related fracture and hip osteoarthritis, whereas cranial nerve palsy and bone marrow failure rarely develop. Although it is generally believed that ADO II has a relatively benign course, the natural course of the disease in Chinese patients remains unclear. Materials and Methods: Thirty-six patients diagnosed with ADO II in Shanghai Jiao Tong University Affiliated Sixth People's Hospital from 2008 to 2021 were studied retrospectively. Among them, 15 patients were followed for an average of 6.3 years (1-14 years). Results: In this study, minor trauma-related fractures of the limb were the most typical clinical manifestations. Visual loss (1/36) and bone marrow failure (2/36), was rare in this study. The condition of ADO II seems to be stable in most patients. There were no correlations between markedly elevated bone mineral density (BMD) and minor trauma-related fractures. In total, 21 diseases causing mutations were detected. Among them, the mutation c.2299C>T (p.Arg767Trp) was the most common (16.67%), and mutation c.937G>A [p.(Glu313Lys)] was associated with severe fractures, haematological defects and cranial palsy. Conclusions: Minor trauma-related fracture is the most typical clinical manifestation of ADO II and always occurs in. The mutation c.2299C>T (p.Arg767Trp) is in general a relatively common variant, while the mutation c.937G>A [p.(Glu313Lys)] seems to be associated with severe phenotype. In our study, ADO II seems to remain stable over time.

Puerarin specifically disrupts osteoclast activation via blocking integrin-ß3 Pyk2/Src/Cbl signaling pathway.

OBJECTIVE: Given the limitations of current anti-resorption agents for postmenopausal osteoporosis, there is a need for alternatives without impairing coupling crosstalk between bone resorption and bone formation ie. osteoclastogenesis. Puerarin, a unique C-glycoside isoflavonoid, was found to be able to prevent bone loss by inhibiting bone resorption, but the underlying mechanism was controversial. In this study, we investigated the effects of puerarin on osteoclastic differentiation, activation and bone resorption and its underlying molecular mechanism in vitro, and then evaluated the effects of puerarin on bone metabolism using an ovariectomized (OVX) rat model. METHODS: In vitro, the effect of puerarin on osteoclastic cytotoxicity, differentiation, apoptosis, activation and function were studied in raw 264.7 â€‹cells and mouse BMMs. Mechanistically, osteoclast-related makers were determined by RT-PCR, western blot, immunofluorescence, and kinase activity assay. In vivo, Micro-CT, histology, serum bone biomarker, and mechanical testing were used to evaluate the effects of puerarin on preventing osteoporosis. RESULTS: Puerarin significantly inhibited osteoclast activation and bone resorption, without affecting osteoclastogenesis or apoptosis. In terms of mechanism, the expressions of protein of integrin-ß3 and phosphorylations of Src, Pyk2 and Cbl were lower in puerarin group than those in the control group. Oral administration of puerarin prevented OVX-induced trabecular bone loss and significantly improved bone strength in rats. Moreover, puerarin significantly decreased trap positive osteoclast numbers and serum TRAP-5b, CTx1, without affecting bone formation rate. CONCLUSIONS: Collectively, puerarin prevented the bone loss in OVX rat through suppression of osteoclast activation and bone resorption, by inhibiting integrin-ß3-Pyk2/Cbl/Src signaling pathway, without affecting osteoclasts formation or apoptosis. TRANSLATIONAL POTENTIAL OF THIS ARTICLE: These results demonstrate the unique mechanism of puerarin on bone metabolism and provide a novel agent for prevention of postmenopausal osteoporosis.

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. © 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).

Clinical and molecular characterization of five Chinese patients with autosomal recessive osteopetrosis.

BACKGROUND: Osteopetrosis is characterized by increased bone density and bone marrow cavity stenosis due to a decrease in the number of osteoclasts or the dysfunction of their differentiation and absorption properties usually caused by biallelic variants of the TCIRG1 and CLCN7 genes. METHODS: In this study, we describe five Chinese children who presented with anemia, thrombocytopenia, hepatosplenomegaly, repeated infections, and increased bone density. Whole-exome sequencing identified five compound heterozygous variants of the CLCN7 and TCIRG1 genes in these patients. RESULTS: Patient 1 had a novel variant c.1555C>T (p.L519F) and a previously reported pathogenic variant c.2299C>T (p.R767W) in CLCN7. Patient 2 harbored a novel missense variant (c.1025T>C; p.L342P) and a novel splicing variant (c.286-9G>A) in CLCN7. Patients 3A and 3B from one family displayed the same compound heterozygous TCIRG1 variant, including a novel frameshift variant (c.1370del; p.T457Tfs*71) and a novel splicing variant (c.1554+2T>C). In Patient 4, two novel variants were identified in the TCIRG1 gene: c.676G>T; p.E226* and c.1191del; p.P398Sfs*5. Patient 5 harbored two known pathogenic variants, c.909C>A (p.Y303*) and c.2008C>T (p.R670*), in TCIRG1. Analysis of the products obtained from the reverse transcription-polymerase chain reaction revealed that the c.286-9G>A variant in CLCN7 of patient 2 leads to intron 3 retention, resulting in the formation of a premature termination codon (p.E95Vfs*8). These five patients were eventually diagnosed with autosomal recessive osteopetrosis, and the three children with TCIRG1 variants received hematopoietic stem cell transplantation. CONCLUSIONS: Our results expand the spectrum of variation of genes related to osteopetrosis and deepen the understanding of the relationship between the genotype and clinical characteristics of osteopetrosis.