Family-based whole-exome sequencing implicates a variant in lysyl oxidase like 4 in atypical femur fractures

Abstract

Atypical femur fractures (AFFs) are rare adverse events associated with bisphosphonate use, having unclear pathophysiology. AFFs also cluster in families and have occurred in patients with monogenetic bone diseases sometimes without bisphosphonate use, suggesting an underlying genetic susceptibility. Our aim was to identify a genetic cause for AFF in a Caucasian family with 7 members affected by osteoporosis, including 3 siblings with bisphosphonate-associated AFFs. Using whole-exome sequencing, we identified a rare pathogenic variant c.G1063A (p.Gly355Ser) in lysyl oxidase like 4 (LOXL4) among 64 heterozygous rare, protein-altering variants shared by the 3 siblings with AFFs. The same variant was also found in a fourth sibling with a low-trauma femur fracture above the knee, not fulfilling all the ASBMR criteria of AFF and in 1 of 73 unrelated European AFF patients. LOXL4 is involved in collagen cross-linking and may be relevant for microcrack formation and bone repair mechanisms. Preliminary functional analysis showed that skin fibroblast-derived osteoblasts from the unrelated patient with the LOXL4 variant expressed less collagen type I and elastin, while osteogenic differentiation and mineralization were enhanced compared with 2 controls. In conclusion, this LOXL4 variant may underlie AFF susceptibility possibly due to abnormal collagen metabolism, leading to increased formation of microdamage or compromised healing of microcracks in the femur.

Keywords: LOXL4; atypical femur fractures; bisphosphonates; collagen; family study; osteoporosis; whole-exome sequencing.

Figure 1

Family pedigree.

Figure 2

Polygenic risk scores for low BMD in the family members and the association of LOXL4 with BMD and fracture. A-B, association between standardized PGS for BMD and BMD T-scores of the subjects (descending trend line). The dots forming the clouds represent Rotterdam study subjects; the triangle dots represent the family members; the vertical lines represent 1 SD and 2 SD from the mean in the Rotterdam study population. C-D, associations of LOXL4 and BMD (C) and all types of fractures (D) in UK Biobank. E, association of rs4919173 with LOXL4 by expression quantitative trait loci in GTEX. NES, normalized effect size, defined as the slope of the linear regression, and it represents the effect of the alternative allele relative to the reference allele in the human genome reference. PGS, polygenic scores. BMD, bone mineral density. GTEX, Genotype-Tissue Expression.

Figure 3

Functional changes in patient-derived fibroblasts with LOXL4 variant. A, LOXL4 mRNA expression during differentiation of bone marrow mesenchymal stromal cell-derived osteoblasts and adipocytes. Gene expression was normalized by correcting for the expression of the housekeeping gene 36B4. B, ALP/protein in fibroblast-derived osteoblast culture on day 7; samples from patient and 2 healthy controls. Calcium deposition by fibroblast-derived osteoblast cultures on day 24; samples from patient and 2 healthy controls. C, collagen type I immunocytochemistry in fibroblast-derived osteoblast cultures on day 7 and day 14, from the patient and 2 healthy controls (representative for 5 images of 3 replicates per individual). Green: collagen type I; red: actin cytoskeleton; blue: nuclei (DAPI). NC = negative control. Data are presented as mean ± SEM and analyzed by unpaired two sample T-test. SEM, Standard Error of the Mean.