ANK1 and EPB41 Variants and The Risk of Glucocorticoid-Induced Osteonecrosis

Steroid-induced osteonecrosis of the femoral head (SONFH) is a refractory skeletal disorder influenced by genetic and environmental factors. However, conclusive pathogenic genetic evidence remains elusive due to the limited exploration of rare damaging variants. In this study, we aimed to identify rare variants associated with SONFH.

We conducted whole-exome sequencing in an SONFH case-control study comprising 174 patients with systemic lupus erythematosus (SLE) taking steroids, followed by comparing the cases with an ethnically matched healthy population and validation in an additional SONFH cohort of 246 patients without SLE. Rare damaging variants were identified via genetic burden analysis and confirmed by Sanger sequencing and pedigree studies. The functional assessments of the erythrocytes on target variants were performed.

We identified 10 heterozygous ANK1 and EPB41 rare variants in 9 (10.8%) of 83 patients with SONFH compared with 0 of the 91 non-SONFH controls (0%). The burden effect of them remained robust after adjusted analysis (Firth's logistic regression-adjusted odds ratio [aOR]_ANK1 11.3; aOR_EPB41 32.3; both P < 0.05). The variants were detected in 10 of the validated SONFH cohort (4.1%) with 246 non-SLE conditions. Functional analyses revealed that the variants result in membrane dysfunctions in the patients' erythrocytes, characterized by reduced ANK1 expression, abnormal morphology, and increased hypotonic hemolysis.

These findings suggest that the rare variants in ANK1 and EPB41 are novel SONFH disease risk factors that compromise erythrocyte membrane integrity, exacerbating microcirculatory damage in the presence of glucocorticoid as a second hit.