In the present study a splice

In the present study, a splice acceptor site mutation (c.1769-1G > C) was identified in AR gene from patients with CAIS. The c.1769-1G > C mutation results in replacement of the normal sequence (CAG/G) with a new sequence (CAC/G) at the splice acceptor site of intron 2. In the splice acceptor site recognition, U2AF35 interacts with the AG dinucleotides in a sequence-specific fashion, thus the new sequence was not recognized as a splice site signal, and this was predicted by all the three bioinformatics software. Furthermore, mRNA analysis proved that a cryptic splice acceptor site located at position −71/−70 bp upstream of the normal splice acceptor site was used, thus resulting in an aberrant AR transcript containing an insertion of 69 nucleotides between exon 2 and exon 3. Interestingly, the 69-nucleotide insertion in cDNA does not lead to a premature termination codon. This abnormal transcript would result in the replacement of Gly by Glu at amino pannexin-1 inhibitor 590 as well as an in-frame insertion of 23 amino acids between position 590 and 591 (p.[Gly590_Lys591insIleProGluGluArgAspSerGlyAsnSerLeuSerGlyLeuSerThrLeuValPheValLeuProArg; Gly590Glu]) in the AR protein. The 23 amino acids insertion occurs in a key region of the DBD, between the carboxyl-terminal (C-terminal) part of the first zinc finger, which contains the P-box (proximal box) responsible for specific DNA binding, and the second zinc finger, which contains the d-box (distal box) involved in DNA-dependent dimerization [19]. We did not investigate whether the patients aberrant mRNA could be translated or if the encoded protein possessed any activity. However, Jääskeläinen et al. [20] previously reported a c.1769-1G > A mutation at the same splice acceptor site that resulted in the same 23 amino acids insertion in AR protein from a CAIS patient. They found that the mutant AR protein retains ligand binding activity for the C-terminal part of the AR protein remains intact by in vitro binding assay. Brüggenwirth et al. [21] identified a c.1769–11 T > A mutation that resulted in the same 23 amino acids insertion in the AR protein as present study. They demonstrated that the mutant AR protein had no DNA-binding activity in a band shift assay, and unable to activate the transcription of androgen target genes by transcription activation assay. Consequently, The 23 amino acids in-frame inserted between the two zinc clusters, displaying defective DNA binding and defective transcription activation. Therefore, our identified mutation is analogous to those previously studied, and is expected to produce the same effect. Furthermore, we investigated whether the 23 amino acids insertion in the AR protein could influence its nuclear translocation by analysis the subcellular localization of this mutant AR in the COS-1 cells. We found that compared with the normal AR protein, the mutant AR was unable to be translocated into the nucleus after hormone stimulation, and formed perinuclear aggregates in the cytoplasm. Interestingly, Jagla et al. [22] reported a similar mutant AR to the present study, named AR23 p.[Glu589_Gly590insGluIleProGluAspSerGlyAsnSerLeuSerGlyLeuSerThrLeuValPheValLeuPro], containing an insertion of 23 amino acids at position 589 between the two zinc fingers of AR DBD, which was detected in a metastatic prostate cancer (PCa) specimen via a yeast functional assay. They also demonstrated that the nuclear entry of AR23 upon hormone stimulation was impaired. Thus, we can conclude that in the present study the additional 23 amino acids insertion affects AR intracellular trafficking by impairing its translocation from the cytoplasm to the nucleus after hormone stimulation. Subsequently, the c.1769-1G > C mutation leads to loss of the AR protein biological function which explains the pathogenicity of this mutation in the patients. According to the AR database, twenty-five splice site mutations have been reported in the AR gene, of which 21 splice mutations were associated with complete insensitivity syndrome. Most of the splice mutations are located at the donor site; only four mutations are located at the acceptor site, including one mutation in intron 3/exon 4 and three mutations in intron 2/exon 3. The c.1769-1G > C mutation identified in the present study also occurred in intron 2/exon 3 splice acceptor site, indicating that the intron 2/exon 3 splice site mutation maybe a hotspot mutation site in the AR gene.