Abstract
Acheiropodia, congenital limb truncation, is associated with homozygous deletions in the LMBR1 gene around ZRS, an enhancer regulating SHH during limb development. How these deletions lead to this phenotype is unknown. Using whole-genome sequencing, we fine-mapped the acheiropodia-associated region to 12 kb and show that it does not function as an enhancer. CTCF and RAD21 ChIP-seq together with 4C-seq and DNA FISH identify three CTCF sites within the acheiropodia-deleted region that mediate the interaction between the ZRS and the SHH promoter. This interaction is substituted with other CTCF sites centromeric to the ZRS in the disease state. Mouse knockouts of the orthologous 12 kb sequence have no apparent abnormalities, showcasing the challenges in modelling CTCF alterations in animal models due to inherent motif differences between species. Our results show that alterations in CTCF motifs can lead to a Mendelian condition due to altered enhancer–promoter interactions.
Highlights
Acheiropodia, congenital limb truncation, is associated with homozygous deletions in the limb development membrane protein 1 (LMBR1) gene around zone of polarizing activity regulatory sequence (ZRS), an enhancer regulating Sonic Hedgehog (SHH) during limb development
The ZRS is completely intact in the Brazilian individuals with acheiropodia who are homozygous for the LMBR1 exon 4 deletion, suggesting that other functional units associated with SHH limb expression may be disrupted by this deletion
We identified a 12,041 base pair homozygous deletion that overlaps LMBR1 exon 4 along with two base pairs (CA) that were inserted at the breakpoint (Fig. 1b–d, Supplementary Fig. 1)
Summary
Acheiropodia, congenital limb truncation, is associated with homozygous deletions in the LMBR1 gene around ZRS, an enhancer regulating SHH during limb development. How these deletions lead to this phenotype is unknown. Homozygous deletions encompassing the ZRS lead to acheiropodia in humans and mice[15,16] These results indicate that acheiropodia is likely caused by reduced SHH expression during limb development. Previous studies in mice deleted individual and combinations of CTCF sites in the Shh locus, some of them affecting interactions between the ZRS and Shh promoter and leading to a reduction of up to ~52% of Shh expression in the limb, but none of which led to an observable limb phenotype[4,21]. Ectopic CTCF sites appeared in these CTCF motif knockout mice likely supporting compensatory interactions[21]
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