- Authors: Xue JY. et al.
- Year: 2021
- Journal: J Bone Miner Res
- Applications: in vitro / mRNA / jetMESSENGER
- Cell type: RAW 264.7
Description: Mouse monocytes/macrophages
Known as: RAW
RAW264.7 were seeded at a density of 5300 cells/cm2 in chamber slides or bone resorption assay plates. On the next day, medium was replaced and the cells were incubated with 100 nb/mL of RANKL. After 48 hours of RANKL treatment, the cells were transfected with mRNA using jetMESSENGER (Polyplus) according to the manufacturer's instructions. The medium was replaced with the fresh medium containing 100 ng/mL RANKL after 4 hours of transfection, and cells were further cultured for 48 hours.
Osteopetrosis is a group of rare inherited skeletal disorders characterized by a marked increase in bone density due to deficient bone resorption. Pathogenic variants in several genes involved in osteoclast differentiation and/or function have been reported to cause osteopetrosis. Solute carrier family 4 member 2 (SLC4A2, encoding anion exchanger 2) plays an important role in osteoclast differentiation and function by exchange of Cl- with HCO3 - . Biallelic Slc4a2 loss-of-function mutations in mice and cattle lead to osteopetrosis with osteoclast deficiency; however, pathogenic SLC4A2 variants in humans have not been reported. In this study, we describe a patient with autosomal recessive osteopetrosis due to biallelic pathogenic variants in SLC4A2. We identified novel compound heterozygous variants in SLC4A2 (NM_003040.4: c.556G>A [p.A186T] and c.1658T>C [p.V553A]) by exome sequencing. The measurement of intracellular Cl- showed that the variants decrease the anion exchange activity of SLC4A2. The impact of the variants on osteoclast differentiation was assessed by a gene knockout-rescue system using a mouse macrophage cell line, RAW 264.7. The Slc4a2-knockout cells show impaired osteoclastogenesis, which was rescued by the wild-type SLC4A2, but not by the mutant SLC4A2s. Immunofluorescence and pit assay revealed that the mutant SLC4A2s leads to abnormal podosome belt formation with impaired bone absorption. This is the first report on an individual affected by SLC4A2-associated osteopetrosis (osteopetrosis, Ikegawa type). With functional studies, we prove that the variants lead to SLC4A2 dysfunction, which altogether supports the importance of SLC4A2 in human osteoclast differentiation. © 2021 American Society for Bone and Mineral Research (ASBMR).