A Novel Role for HAPLN1 in Skeletal Development

Adolescent idiopathic scoliosis (AIS) is a potentially progressive spinal curvature that occurs during rapid growth and has a multi-factorial genetic etiology. Prior genetic studies in humans and animal models support differences in cartilage development in the pathogenesis of AIS. Here, we performed genome sequencing in families with inherited AIS and identified a co-segregating rare variant in the HAPLN1 gene. HAPLN1 encodes Hyaluronan and Proteoglycan Link Protein 1, also known as Cartilage Link Protein, that is reported to stabilize the extracellular matrix (ECM) by binding hyaluronic acid (HA) and proteoglycans in multiple tissues. One variant, p.C304S, was predicted to disrupt a critical disulfide bridge of the HAPLN1 C-terminal loop domain. In transient over-expression experiments, we detected wild type mouse Hapln1 protein in the cell lysate, media, and ECM, while Hapln1 p.C306S (C304S in human) was detected in cell lysate and media, but not in the ECM. A truncated Hapln1 p.C306fs*5 (ET) lacking the C-terminal loop domain was detected only in the cell lysate. By mutagenesis experiments we found that replacing cysteines in Hapln1 primary disulfide bridges disrupted secretion and retention in the ECM, whereas replacing cysteines in secondary loops disrupted ECM retention only. Thus, all disulfide loops are required for ECM retention. Mice were engineered to harbor the orthologous mutations. While Hapln1ET/ET mice died shortly after birth, exhibiting a chondrodysplastic phenotype with shortened limbs and dome-shaped head, Hapln1C306S/C306S mice were viable, born at expected Mendelian ratios, and showed no significant skeletal malformations up to one year of age. Surprisingly, we were unable to detect Hapln1 protein in tissues of Hapln1C306S/C306S mice although they were phenotypically normal. Furthermore, we found that Hapln1 negatively regulates apoptosis in growth plate hypertrophic zones. Collectively, our data suggest an essential function for Hapln1 that is potentially independent of its role in the ECM.