Speaker
Description
Background: Sutures of the human cranium remain patent postnatally to enhance cranial growth to accommodate the growing brain. Craniosynostosis ensues from the premature fusion of cranial sutures, adversely impacting brain growth. The condition has a multifactorial aetiology, with ~30% of cases being syndromic. The current study deciphered the contribution of genetic risk factors to CS aetiology in a Ghanaian cohort.
Methods: Whole exome sequencing (WES) was carried out on DNA from 14 families employing Illumina HiSeq at 100X based on GRCh38. Quality control, read alignment and variant calling were conducted utilising Sentieon workflow. Normalised VCF files were annotated using Ensembl VEP. Variants having MAF >0.01 were filtered out, and pathogenicity of variants was ascertained using 12 dbNSFP tools, with variant prioritisation using VarElect ACMG guidelines. In zebrafish functional experiments, two sets of targets and fluorescent PCR primers were designed to target exons in EPHA8. sgRNA synthesis and its injection with Cas9 mRNA into 70 single-cell zebrafish embryos followed. Embryos were profiled for CRISPR activity using real-time PCR and Sanger sequencing. The best target sgRNA and Cas9 mRNA were injected into another set of 70 embryos. Positive and negative controls were set up as well. After 5 days post-fertilisation (dpf), embryos were phenotyped using a fluorescent microscope, and CRISPR activity was assessed as stated.
Results and Discussions: WES revealed novel and de novo variants in genes such as EPHA8, CNOT2, ATP2B3, DVL3, PIEZO1, TJP2, ZEB2, EPHB3, IGFBP6, BBS9, CENPM, BCL11A, EFNA2, and ARID1B. Zebrafish crispants exhibited abnormal craniofacial bones and jaw morphology, fused and single eyes, and cyclops with disrupted faces, with over 78% of embryos being severely malformed and failing to thrive by five dpf.
Conclusions: Novel genes and genetic variants contribute to craniosynostosis aetiology among Ghanaians. Our findings are crucial for elucidating pathophysiology, genetic counselling and personalised and precision medicine.