Novel GLI3 variant causing overlapped Greig cephalopolysyndactyly syndrome (GCPS) and Pallister-Hall syndrome (PHS) phenotype with agenesis of gallbladder and pancreas

Autopsy carried out 2 h after delivery of the 600 g stillborn revealed multiple systemic malformations. Macroscopic autopsy findings are summarized in Table 1. The stillborn demonstrated a proportionally unbalanced, large head with acrocephaly (Fig. 1a), postaxial polysyndactyly (Fig. 1b and c), gastrointestinal malformations including malrotation and atresia of the anus, agenesis of the gallbladder (Fig. 1d) and the pancreas, hypoplasia of both kidneys (Fig. 1e), and the endocrine organs. Except these malformations, histopathological alteration of other major organs such as heart, liver, and bone was not noted. In view of these macroscopic features suggestive of GCPS or PHS, mutation of the GLI3 gene was analyzed. Routinely formalin-fixed (10%) and paraffin-embedded archival samples of infant thymus tissue samples obtained from Kobe University Hospital (Kobe, Hyogo) deparaffinized with xylene, suspended in 5 μl of 1 × TE and then mixed with pre-warmed and liquefied low-melting agarose (3.2%) at 1:1. A total of 10 μl of agarose beads containing 1 × TE and tissue fragments was formed in 250 μl of pre-chilled mineral oil, and then incubated at 50 °C overnight in 1000 μl of 200 μg/ml proteinase K, 10 mM Tris-HCl (pH 8.0) and 25 mM ethylene diamine tetraacetic acid. Bead fragments were washed in 1 × TE, sliced into several pieces, and analyzed by PCR using sets of primers encompassing all known coding exons and exon–intron boundaries of the GLI3 gene. Primers were designed as described [6]. The PCR mixture contained Mighty AMP® DNA polymerase (Takara, Tokyo, Japan) and bead fragments in a final volume of 25 μl. The PCR products were electrophoresed in a 3% agarose gel and stained with ethidium bromide. Purified PCR products were cloned into TA-vector, and amplified plasmids were analyzed for the DNA sequence. Each PCR product was analyzed by sequencing, and the missense mutation (c.2155 C > T) leading to p.P719S was detected in 6 of 12 independent clones from PCR targeting exon 6, which altered the amino acid property from hydrophobic (Proline, P) to hydrophilic (Serine, S) within the proteolytic cleavage (PC) site of GLI3 (Fig. 2). Because this missense mutation was absent in both parents (data not shown), without family history of GCPS or PHS, and non-maternity factors like egg donation, surrogate motherhood, and errors in embryo transfer, the c.2155 C > T mutation probably occurred de novo in the fetus, albeit parental germline mosaicism cannot be ruled out.