H2AFY promoter deletion causes PITX1 endoactivation and Liebenberg syndrome

Abstract

BackgroundStructural variants (SVs) affecting non-coding cis-regulatory elements are a common cause of congenital limb malformation. Yet, the functional interpretation of these non-coding variants remains challenging. The human Liebenberg syndrome is characterised by a partial transformation of the arms into legs and has been shown to be caused by SVs at the PITX1 locus leading to its misregulation in the forelimb by its native enhancer element Pen. This study aims to elucidate the genetic cause of an unsolved family with a mild form of Liebenberg syndrome and investigate the role of promoters in long-range gene regulation.MethodsHere, we identify SVs by whole genome sequencing (WGS) and use CRISPR-Cas9 genome editing in transgenic mice to assign pathogenicity to the SVs.ResultsIn this study, we used WGS in a family with three mildly affected individuals with Liebenberg syndrome and identified the smallest deletion described so far including the first non-coding exon of H2AFY. To functionally characterise the variant, we re-engineered the 8.5 kb deletion using CRISPR-Cas9 technology in the mouse and showed that the promoter of the housekeeping gene H2afy insulates the Pen enhancer from Pitx1 in forelimbs; its loss leads to misexpression of Pitx1 by the pan-limb activity of the Pen enhancer causing Liebenberg syndrome.ConclusionOur data indicate that housekeeping promoters may titrate promiscuous enhancer activity to ensure normal morphogenesis. The deletion of the H2AFY promoter as a cause of Liebenberg syndrome highlights this new mutational mechanism and its role in congenital disease.

Successful application of genome sequencing in a diagnostic setting: 1007 index cases from a clinically heterogeneous cohort

“…Recently, Cassini et al reported a deep intronic variant in IGHMBP2 shown to lead to nonsense mediated decay via activation of a cryptic splicing site in a patient with Charcot-Marie-Tooth [34]. As part of a more complex mechanism, Kragesteen et al described a deletion in the first noncoding exon of H2AFY that leads to abnormal expression of Pitx1 and Liebenberg syndrome [35].…”

Enhancer dependence of cell-type–specific gene expression increases with developmental age

“…3 H and I ), suggesting that Myb is essential for EryD-specific distal enhancer activation. Disruption, inversion, or insertion of enhancers can perturb tissue-specific chromatin architecture and lead to inappropriate expression of target gene ( 43 – 47 ). Here, CRISPR/Cas9-mediated deletion of selected enhancers resulted in a striking decrease in target gene expression in adult stage MEL cells, but not in mESC-derived erythroblasts ( Fig.…”

The regulatory landscapes of developmental genes

“…This is because in forelimbs the Pen enhancer is sequestered by other contacts or titrated out by the closely located H2afy (Macroh2a1) gene promoter, which prevents interactions with Pitx1. In the hindlimbs, the enhancer escapes its local 3D domain to interact with the Pitx1 promoter, thus allowing transcription and the conferral of hindlimb identity (Kragesteen et al, 2018(Kragesteen et al, , 2019.…”