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Keywords: Alternative splicing; Intellectual and developmental disability; Mendelian disorder; Splicing mechanisms; Genotype-phenotype correlations

E. Siavriene 1; V. Mikstiene 1; D. Radzevicius 2; Z. Maldziene 1; T. Rancelis 1; G. Petraityte 1; I. Kavaliauskiene 1; L. Sarkinas 2; A. Utkus 1; V. Kucinskas 1; E. Preiksaitiene 1

1) Department of Human and Medical Genetics, Institute of Biomedical Sciences, Faculty of Medicine, Vilnius University, Vilnius, Lithuania; 2) The Children’s Hospital, affiliate of Vilnius University Hospital Santaros Klinikos, Vilnius, Lithuania

BACKGROUND: Preaxial polydactyly type IV (MIM 174700; ORPHA 93338), also referred as polysyndactyly, has been described in a few syndromes including Greig cephalopolysyndactyly syndrome (GCPS; MIM 175700; ORPHA 380). GCPS is caused by heterozygous mutation in the GLI3 gene (MIM 165240), which is located in the cytoband 7p14. In this study, we present the functional analysis of a novel GLI3 splice site variant, which cosegregates in three generations of a family with preaxial polydactyly type IV and other clinical features of GCPS.
METHODS & RESULTS: Sequencing analysis of the GLI3 coding region identified a novel donor splice site variant NC_000007.14(NM_000168.6):c.473+3A>T in the proband and the same variant was subsequently identified in other affected family members. In order to elucidate the pathogenicity of the detected variant in GLI3 gene, proband’s RNA was isolated from fibroblast culture and template cDNA was synthesized. Further Sanger sequencing of the proband’s cDNA sample revealed that the splice site variant disrupts the original donor splice site, thus leading to exon 4 skipping. Based on in silico analysis, the pathogenic splice site variant consequently results in a truncated protein NP_000159.3:p.(His123Argfs*57), which lacks functionally important domains: zinc finger domain, proteolytic cleavage site, transactivation domain 2, transactivation domain 1 as well as part of repressor domain.
CONCLUSION: The functional cDNA analysis revealed that NC_000007.14(NM_000168.6):c.473+3A>T led to the haploinsufficiency of GLI3 that causes GCPS in the affected family members. This analysis provides a unique possibility to identify the molecular basis of GCPS as well as many other hereditary diseases and conditions.
The work was funded by the Research Council of Lithuania (No. S-MIP-17-19/LSS-150000-1179, Ingenes project).