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Abstract

Human height is a complex trait with a high heritability. Mutations in the homeobox gene SHOX cause SHOX deficiency, the most frequent monogenic cause of short stature. SHOX deficiency has high penetrance. However, the clinical severity of SHOX deficiency varies widely, ranging from short stature without dysmorphic signs to mesomelic (disproportionate) skeletal dysplasia (Léri-Weill dyschondrosteosis, LWD) and different independent studies have reported rare SHOX deficiency individuals presenting with normal height and no dysmorphic signs. To shed light on the factors that modify disease severity/penetrance, we studied a three-generation family with five affected individuals presenting with LWD using whole genome linkage and whole exome sequencing analyses. By combining the data obtained with these two independent methods, we found that the variant allele p.Phe508Cys of the retinoic acid catabolizing enzyme CYP26C1 co-segregated with the SHOX variant allele p.Val161Ala in the 5 affected individuals, while the SHOX mutant alone was also present in 3 asymptomatic family members. Screening of a cohort of 68 LWD individuals led to the identification of two unrelated families with SHOX deficiency bearing also additional damaging CYP26C1 variants in the more severely affected family members. These results support a role for CYP26C1 in influencing the course of disease in SHOX deficiency patients. CYP26C1, similar to SHOX, is expressed in human primary chondrocytes and in zebrafish pectoral fins. Luciferase assays performed to functionally characterize the variants identified in SHOX and CYP26C1 demonstrated their damaging effects on their activity: SHOX mutants were not able to transactivate the reporter gene expression, whereas damaging variants in CYP26C1 affect its catabolic activity leading to increased levels of retinoic acid. High levels of retinoic acid significantly decreased SHOX expression in human primary chondrocytes and zebrafish embryos. Analysis of SHOX promoter unravelled an indirect effect of retinoic acid on SHOX expression. Individual morpholino knockdown of either gene resulted in shortened pectoral fins in zebrafish embryos, which was more pronounced in SHOX. Depletion of both genes simultaneously, aggravated the fin phenotype. Together our findings demonstrate that SHOX and CYP26C1 act in a common molecular pathway (retinoic acid signaling) controlling limb growth and describe CYP26C1 as the first genetic modifier for SHOX-associated disease.