ESPE Abstracts

Background: Expression of SHOX, a transcription factor implicated in skeletal development, is regulated by the interaction of two promoters, weak, P₁ (exon 1) and strong, P₂ (exon 2), with at least, seven enhancers. SHOX haploinsufficiency, due to mutations in SHOX or its enhancers, explains ~70% of Leri-Weill dyschondrosteosis (LWD) and ~2.5% idiopathic short stature (ISS) cases whilst the underlying molecular mechanism in the remaining is unknown.

Objective and hypotheses: Several SHOX promoter variants have been reported but their pathogenicity is unknown. We set out: i) To determine the pathogenicity of the SHOX P₂ promoter variants detected in LWD/ISS patients (LOVD SHOX database); ii) To screen the SHOX P₂ promoter in our LWD/ISS cohorts with no known SHOX defect.

Method: i) Functional characterization of six SHOX P₂ promoter variants reported in LWD/ISS patients, by dual-promoter-luciferase experiments in U2OS cells. ii) Mutation screening of P₂ in two LWD/ISS cohorts: Cohort-A: 124 patients with suspected LWD without a known SHOX defect; Cohort-B: 1031 LWD/IISS referrals for SHOX testing.

Results: Functional assays showed a significant decrease in luciferase expression for variant, c.-243G>A, compared to wild-type. No P₂ variants were observed in cohort A, whilst in cohort B, 10 c.-65C>A and six c.-55C>T variants were detected.

Conclusion: The observation that the c.-243G>A variant reduced promoter activity and that it cosegregates with the LWD phenotype in a family, suggests that it is a pathogenic mutation. The two variants observed in cohort-B, have been shown by us and others to also cosegregate with LWD in two/three generations, but as with the other three variants, did not reduce promoter activity. Despite this, they still may affect SHOX expression by an alternative mechanism, such as affecting the interaction with SHOX enhancers. In conclusion we have confirmed the pathogenicity of one variant but further experiments are required for the remaining five variants.

Funding: This work was supported by a MINECO grant (SAF2012-30871) and the EndoScreen project (IdiPaz and UAM).