HDAC9structural variants disruptingTWIST1transcriptional regulation lead to craniofacial and limb malformations

Abstract

Structural variants (SVs) can affect protein-coding sequences as well as gene regulatory elements. However, SVs disrupting protein-coding sequences that also function as cis-regulatory elements remain largely uncharacterized. Here, we show that craniosynostosis patients with SVs containing the Histone deacetylase 9 (HDAC9) protein-coding sequence are associated with disruption of TWIST1 regulatory elements that reside within HDAC9 sequence. Based on SVs within the HDAC9-TWIST1 locus, we defined the 3'-HDAC9 sequence as a critical TWIST1 regulatory region, encompassing craniofacial TWIST1 enhancers and CTCF sites. Deletions of either Twist1 enhancers (eTw5-7Δ/Δ) or Ctcf site (CtcfΔ/Δ) within the Hdac9 protein-coding sequence led to decreased Twist1 expression and altered anterior\posterior limb expression patterns of Shh pathway genes. This decreased Twist1 expression results in a smaller sized and asymmetric skull and polydactyly that resembles Twist1+/− mouse phenotype. Chromatin conformation analysis revealed that the Twist1 promoter interacts with Hdac9 sequences that encompass Twist1 enhancers and a Ctcf site and that interactions depended on the presence of both regulatory regions. Finally, a large inversion of the entire Hdac9 sequence (Hdac9INV/+) in mice that does not disrupt HDAC9 expression but repositions Twist1 regulatory elements showed decreased Twist1 expression and led to a craniosynostosis-like phenotype and polydactyly. Thus, our study elucidated essential components of TWIST1 transcriptional machinery that reside within the HDAC9 sequence It suggests that SVs, encompassing protein-coding sequence could lead to a phenotype that is not attributed to its protein function but rather to a disruption of the transcriptional regulation of a nearby gene.

The value of genome-wide analysis in craniosynostosis

“…We report the fourth case of CS and deletion involving HDAC9 without disrupting the TWIST1 -coding region. Recently, Hirsch et al (2022) showed that SVs involving HDAC9 disrupt TWIST1 -regulatory elements within HDAC9 in patients with CS. Our patient had coronal synostosis that was also described in two of the previously reported patients with deletion and one with a translocation breakpoint disrupting the HDAC9–TWIST1 locus ( Hirsch et al, 2022 ).…”

Copy Number Variation and Osteoporosis

“…CNVs residing in regulatory regions (enhancers and promoters) and long non-coding RNA can modulate gene expression of OP-related genes. In a comprehensive study by Hirsch et al, structural variations in HDAC9 gene that influenced the transcription of the neighbouring TWIST1 gene were discovered in craniosynostosis patients [ 40 ]. Deletions in HDAC9 gene, but not in the TWIST1 protein-coding sequence, caused development of craniosynostosis.…”

“…Deletions in HDAC9 gene, but not in the TWIST1 protein-coding sequence, caused development of craniosynostosis. Regulatory elements that reside in the HDAC9 gene contributed to the transcriptional regulation of the neighbouring craniofacial gene TWIST1 [ 40 ]. Deletion of TWIST1 enhancers within the HDAC9 gene induced a small size skull in their mouse model, confirming the functional role of the regulatory region of the TWIST1 gene.…”

“…Deletion of TWIST1 enhancers within the HDAC9 gene induced a small size skull in their mouse model, confirming the functional role of the regulatory region of the TWIST1 gene. TWIST1 is a transcription factor important for mesodermal development [ 40 ].…”

Case report: A third variant in the 5′ UTR of TWIST1 creates a novel upstream translation initiation site in a child with Saethre-Chotzen syndrome

“…The major underlying pathogenic mechanism responsible for the craniofacial and limb anomalies observed in SCS patients is haploinsufficiency, usually caused by heterozygous variants affecting the coding region of the gene or structural variants, such as inversions or translocations encompassing TWIST1 regulatory elements, leading to functional loss of one gene copy ( Hyder et al, 2021 ; Hirsch et al, 2022 ).…”