Phenotypic and biochemical characteristics and molecular basis in 36 Chinese patients with androgen receptor variants

Abstract

Background: Androgen insensitive syndrome (AIS) is a rare genetic disease resulting from androgen receptor (AR) mutations and one of the causes of 46, XY disorder of sexual development (DSD). This study aimed to describe the clinical features and molecular defects of 36 Chinese patients with AR variants and investigate the functional alterations of novel variants in vitro.

Material and methods: Subjects with AR variants were identified from 150 Chinese 46, XY DSD patients using targeted next-generation sequencing. In-silico and functional assays were performed to evaluate the transcriptional activity and nuclear localization of novel AR variants.

Results: Eight novel and fifteen reported AR variants were identified. 30.6% (11/36) of patients harbored additional variants other than AR. Mutations in the Arg841 residue were found in 7 unrelated patients. Postpubertal serum gonadotropin levels were significantly elevated in patients with complete AIS (CAIS) compared with those in patients with partial AIS (PAIS) (P < 0.05). All the novel variants initially predicted to be uncertain significance by in-silico analyses were reclassified as likely pathogenic for defective AR transcriptional activity in vitro, except p.L295P, which was found in an atypical patient with oligogenic mutations and reclassified as likely benign. c.368_369 ins T was observed to interfere with nuclear translocation.

Conclusions: Compared with PAIS patients, postpubertal CAIS patients had higher gonadotropin levels. Arg841 was disclosed as the location of recurrent mutations in Chinese AIS patients. Functional assays are important for reclassifying the novel AR variants and re-examining the diagnosis of AIS in specific patients with oligogenic mutations, instead of in-silico analysis.

Keywords: Androgen insensitive syndrome (AIS); Androgen receptor (AR) mutation; Disorder/differences of sex development (DSD); Functional assay.

Fig. 1

Distribution of eight novel AR mutations. a Novel AR mutations were distributed throughout the entire gene. b Amino acids L295, A588, G590, R761, L812, F879, and I907 are highly conserved through evolution. NTD N-terminal domain, DBD DNA-binding domain, HR hinge region, LBD ligand-binding domain

Fig. 2

The scatterplot of the serum hormones for postpubertal patients with CAIS and PAIS except for patient 5, 9 and 12. a The scatterplot of follicle-stimulating hormone (FSH). b The scatterplot of luteinizing hormone (LH). c The scatterplot of testosterone (T). CAIS (n = 11); PAIS (n = 24); *P < 0.05

Fig. 3

The transcriptional activities of WT and mutant AR were investigated using the androgen response element (ARE) promoter. The relative luminescence activity was expressed as a ratio of firefly to Renilla luciferase. a WT and all of the novel mutant AR in LNCaP cells. b WT AR, p.L295P, and p.A588P (as positive control) in 293T cells cultured with variant concentrations of DHT. The error bars represent the standard error. *versus WT P < 0.05; # versus Vector P > 0.05

Fig. 4

Nuclear translocation of GFP-tagged wild-type and mutant ARs. The upper panel from left to right: Empty vector and GFP-tagged WT AR (cultured without DHT) exhibited diffused localization in the cytoplasm. GFP-tagged WT AR (cultured with DHT) exhibited strong nuclear localization. The middle panel and lower pannel: Nuclear translocation of GFP-tagged eight mutant ARs in DHT treated cells. A similar nuclear localization pattern was observed in 7 novel missense mutants, while, the c.368_369 ins T mutant exhibited nuclear localization similar to empty vector. The scale bar represents 10 μm