The ANKH gene is located on chromosome 5p15.2 within the genomic interval 5:14,704,799-14,871,777 (GRCh38). ANKH (ANK in mouse) is a multipass transmembrane protein. The solute carrier is involved in regulating inorganic pyrophosphate (PPi) levels in virtually all tissues. PPi regulates mineralization in bone and prevents unwanted mineralization in soft tissues.
The ANKH gene was first identified as the gene responsible for the joint ankylosis phenotype in the ank/ank mouse model. Mutations in the C-terminal end of the ANKH protein were identified in patients with craniometaphyseal dysplasia. Mutations include in-frame deletions, insertions or point mutations. Mutations for CCAL2 are located in the N-terminal domain. Known mutations consist of point mutations, an in-frame deletion and a change in translation start site.
Functionally, ANKH was thought to be a PPi transporter. Recent findings suggest that ANKH is a transporter of small molecules such as citrate, nucleotides. The role of ANKH/ANK in pathologic mineralization has been reviewed by Williams, 2016.
Previous work on CMD showed that a knock-in mouse model where a phenylalanine deletion (Phe377del) was introduced in ANK reproduces the CMD phenotype. Homozygous mice show a strong phenotype early while heterozygous mice show a progressively developing intermediate phenotype throughout life. Mutant ANKH in patients and mutant ANK in the knock-in mouse model is rapidly degraded. However, the major contribution to the CMD phenotype appears to be due to gain of functions of the mutant protein and not to the reduced amounts of protein as the characteristic CMD features of the knock-in mouse are not expressed in the knockout mouse. Extracellular PPi concentrations in bone is reduced in the CMD knock-in mouse model but the mechanism for this has not been fully explored.
In all cases of CPP disease, whether familial or idiopathic, the formation of CPP crystals in the pericellular matrix of cartilage is essential in the disease process. In order for this to occur, there must be excessive metabolic elaboration of PPi. It was not until the discovery of ANKH that a potential route for this proposed pathogenesis was explored. ANKH mutations in CCAL2 families appear to cause excessive PPi and ATP efflux from articular chondrocytes. However, identifying the exact function of ANKH in CCAL2 patients has been challenging. It appears that, under normal conditions, ANKH functions to transport ATP and perhaps other important small molecule metabolites. When mutated, ANKH may alter its functional profile to regulate the activity and transport of intracellular PPi (PPi is a by-product of many metabolic reactions in cells) to the extracellular space, or alternatively, alter its interactions with other proteins that are critical to the generation of extracellular PPi from ATP. Ultimately, the result is a gain of function with respect to elaboration of extracellular PPi, such that pericellular conditions in the articular cartilage of CCAL2 patients become amenable to CPP crystal formation.