ESPE Abstracts

Introduction: Variants in the N-terminal of the Indian-hedgehog gene (IHH) have been associated with Brachydactyly type-A1 (AD) and Acrocapitofemoral dysplasia (AR), only three of which have been functionally studied. However, heterozygous IHH variants, majority classified as variants of unknown significance (VUS) are being increasingly identified, not only in the N-terminal but also in the uncharacterized C-terminal, by NGS, in individuals with short stature and/or brachydactyly. IHH plays a crucial role in endochondral bone development. It is synthesized as a precursor which is translocated to the endoplasmic reticulum and auto-cleaved into the active-secreted N-terminal peptide (IHH-N) and a C-terminal peptide (IHH-C), critical for self-cleavage.

Aims: To design a functional assay to determine the pathogenicity of IHH VUS and to determine whether variants localized in the C-terminal are pathogenic or not.

Methods: Eight IHH variants (3 IHH-N, 5 IHH-C) observed in short stature/brachydactyly individuals, 4 coding variants listed in gnomAD (2 IHH-N, 2 IHH-C) and 2 synonymous variants were introduced into pCMV6-IHH vector by site-directed mutagenesis and transiently transfected into HEK293T cells. Cultured media and cell lysates were collected and the expression of the different IHH peptides were analyzed by western blot.

Results: All 8 variants showed reduced IHH-N secretion (<50%) and decreased intracellular stability of both, IHH-N and IHH-C peptides compared to wildtype. All four gnomAD variants also showed significantly reduced IHH-N secretion whilst the synonymous variants behave as wildtype.

Conclusions: 1) All studied IHH variants, regardless of their localization in the N- or C-terminal significantly reduced the active IHH-N secretion. 2) The IHH variants can be re-classified to pathogenic variants. 3) Surprisingly, variants present at frequencies 0.0092-0.1% in gnomAD also showed significant reductions. 4) This study highlights the importance of performing functional studies to confirm the pathogenicity of VUS in short stature genes. 5) However, can IHH-associated short stature be considered as a monogenic disorder or a contributing factor to height deficit?