The molecular characterization of seven novel GLI family zinc finger 3 (GLI3) variants in Chinese families with limb malformations

Siyuan Tao^1,2,3Xuyu Gu¹Xiaodong Wang¹Xiaofang Shen^1*Xiuli Zhao^1,2,3*

• ¹Children's Hospital of Soochow University, Suzhou, China
• ²Department of Laboratory Medicine, Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
• ³Chinese Academy of Medical Sciences and Peking Union Medical College Institute of Basic Medical Sciences, Beijing, China

Background: GLI family zinc finger 3 (GLI3) is a transcription factor involved in limb development. GLI3 gene variants have been shown to be associated with several human congenital limb malformations, including Greig cephalopolysyndactyly, Pallister–Hall syndrome, non-syndromic postaxial polydactyly (PAP-A/B), and preaxial polydactyly type IV (PPD-IV). The aim of this study was to identify GLI3 gene variants in ten Chinese families with limb malformations. Methods: Ten Chinese families with limb malformations were recruited. Variants screening in probands was then performed using NGS, with candidate pathogenic variants verified by polymerase chain reaction (PCR) combined with Sanger DNA sequencing. Variant pathogenicity was evaluated using bioinformatics, evolutionary conservation, and disease and mutant allele co-segregation approaches. The biological effects of missense variants were predicted by three-dimensional protein conformation analysis. Results: Ten GLI3 variants were identified, including two missense variants: c.1063G>A (p.Val355Ile) and c.1489C>A (p.Leu497Ile), four nonsense variants: c.2374C>T (p.Arg792*), c.2008C>T (p.Gln670*), c.1096 C>T (p.Arg366*), and c.2029C>T (p.Gln677*), three frameshift variants: c.600delC (p.Tyr200*), c.1880_1881del (p.His627Argfs*48), and c.811_812delCT (p.Leu271Serfs*5), and a large fragment deletion of NC_000007.14: g.42061081_42069739. Seven of these ten variants have never been recorded in the Human Gene Variant Database. Conclusions: Ten GLI3 variants were successfully identified in families with different limb malformations, and indicate significant clinical and allelic heterogeneity of GLI3 related limb malformations. The present study expands the spectra of pathogenic variants and clinical manifestation for GLI3-related morphological disorder, and provides solid evidence for genetic counseling and prenatal gene diagnosis in the affected families.