Functional Analyses of Four CYP1A1 Missense Mutations Present in Patients with Atypical Femoral Fractures

Abstract

Osteoporosis is the most common metabolic bone disorder and nitrogen-containing bisphosphonates (BP) are a first line treatment for it. Yet, atypical femoral fractures (AFF), a rare adverse effect, may appear after prolonged BP administration. Given the low incidence of AFF, an underlying genetic cause that increases the susceptibility to these fractures is suspected. Previous studies uncovered rare CYP1A1 mutations in osteoporosis patients who suffered AFF after long-term BP treatment. CYP1A1 is involved in drug metabolism and steroid catabolism, making it an interesting candidate. However, a functional validation for the AFF-associated CYP1A1 mutations was lacking. Here we tested the enzymatic activity of four such CYP1A1 variants, by transfecting them into Saos-2 cells. We also tested the effect of commonly used BPs on the enzymatic activity of the CYP1A1 forms. We demonstrated that the p.Arg98Trp and p.Arg136His CYP1A1 variants have a significant negative effect on enzymatic activity. Moreover, all the BP treatments decreased CYP1A1 activity, although no specific interaction with CYP1A1 variants was found. Our results provide functional support to the hypothesis that an additive effect between CYP1A1 heterozygous mutations p.Arg98Trp and p.Arg136His, other rare mutations and long-term BP exposure might generate susceptibility to AFF.

Are secondary effects of bisphosphonates on the vascular system of bone contributing to increased risk for atypical femoral fractures in osteoporosis?

“…[ 27 ] Thus, any proposed mechanism must account for both the location and the time frame for development. The bilateral nature of the potential risk for an AFF may also indicate that a genetic component is involved. [ 28–30 ] This conclusion has been supported by recent reports indicating the potential involvement of missense mutations in the CYP1A1 gene [ 31 ] and the ENPP1 gene, [ 32 ] as well as osteomalacia causing genes. [ 32 ] The potential role of the indicated genes and others in AFF [ 33 ] has recently been reviewed by Serra‐Vinardell et al.…”

“…This may create a state of functional avascular necrosis for the bone associated with the endothelium of the nutrient canal. This compromise of the sub‐endothelial bone integrity could also be influenced regarding the extent and rate of loss of integrity consistent with the known association of some of the genetic risk factors for AFF, [ 23,31,32 ] particularly those genes associated with osteomalacia. [ 32 ] Thus, a continual loss of the integrity of the sub‐EC/microvasculature bone in a nutrient canal could progress until a critical “tipping point” was reached and the risk for an AFF had increased until other secondary variables associated with Asian ethnicity and femur geometry became critical.…”

Whole Exome Sequencing in Two Southeast Asian Families With Atypical Femur Fractures

“…Except these two variants, no other variants were identified in the candidate gene analysis, including the analysis of the GGPS1 and CYP1A1 genes, which were implicated to be associated with BP-associated AFF by a genetic study of three sisters affected with BP-associated AFFs and subsequent functional studies. (17,37) The unbiased variant filtering approach assuming a dominant inheritance model on the biallelic variants where both sisters (2-a and 2-b) share the same heterozygous variants resulted in 132 variants with a frequency <0.005 in both the overall population and the East Asian subpopulation in public databases. Potential causal variant selection in WES data in only two samples leaves many variants to select from; hence, we prioritized variants predicted to be most damaging (truncating, or CADD >30) as potential candidates, because these often disrupt the function of the protein and are known to cause Mendelian disorders.…”

“…Except these two variants, no other variants were identified in the candidate gene analysis, including the analysis of the GGPS1 and CYP1A1 genes, which were implicated to be associated with BP‐associated AFF by a genetic study of three sisters affected with BP‐associated AFFs and subsequent functional studies. ( 17,37 )…”

Association between single nucleotide variants and severe chronic pain in older adult patients after lower extremity arthroplasty

“…Variants in these genes alter enzyme activity, preventing drug metabolism or transformation into active forms in the body, leading to great differences in the demand for analgesic drugs in different patients. For example, AMACR, encoding α-methylacyl-CoA racemase, catalyzes key steps in ibuprofen metabolism (Lloyd et al, 2013), while AOX1 encodes xanthine dehydrogenase, which metabolizes aza-and oxo-heterocycles representing the scaffold of many drugs like morphine and fentanyl (Garattini & Terao, 2012) and CYP1A1 encodes cytochrome oxidase and participates in steroid catabolism (Ugartondo et al, 2021). In addition to exogenous drugs, some active molecules in the human body also have analgesic effects, such as endogenous palmitoylethanolamide (PEA), which reduces pain by activating PPAR-α.…”