In this study we reported 19 unrelated patients with androgen insensitivity syndrome whose phenotype was in relation to the nature of the specific AR variant. A total of 19 AR variants were identified. Eight variants were firstly reported. Six novel AR variants showed seriously impaired transcription activity approximately of 1%. One AR mutant protein (V819G) presented mild AR-deficient activity of 59%. Interestingly, another AR mutant protein (K718X) showed increased transcription activity but impaired nuclear localization. The current study broadened the genetic spectrum of androgen insensitivity syndrome which may facilitate in the real clinical-setting for individualized medical approach.
During embryonic development, sex differentiation of male fetus required androgen secreted by Leydig cells to act on normal androgen receptor to exert its biological effects. Subsequently, the wolffian duct developed, male external genitalia differentiated and testes gradually descended to the scrotum. Phenotype spectrum of AIS patients is broad and closely related to the severity of AR impairment, ranging from typical female genitalia at birth to male hypospadias, micropenis, cryptorchidism, oligospermia and gynecomastia. In our study, half of the CAIS patients (50%) came for consultation due to inguinal swelling before puberty which is easily overlooked but an important clue for early diagnosis of AIS. Like Costagliola’s results, the median age of genetic diagnosis for CAIS patients in our study was delayed compared with the age of first symptom occurrence (18.5 [16–22] vs 14 [IQR 1.5–22]). It is reported that CAIS patients accounted for 1–2% in infancies presented with inguinal hernia which is rare in normal female infants, and nearly 57% of the CAIS population presented with an inguinal hernia in the U.K[11, 12]. Therefore, further examination in females with inguinal hernia, especially karyotype analysis, is needed for timely diagnosis of CAIS. Two female patients had regular sexual life without vagina creation and dilation. Although the vagina varies from dimple in the perineum to normal length, most CAIS women were satisfied with their psychosexual development and sexual function. Another two CAIS patients presented pubic hair (Tanner P2-P3) which developed in sparse CAIS patients while axillary hair was reported generally absent[1, 14].
Partial androgen insensitivity syndrome should be excluded in infants presented with hypospadias. All five male patients in the current study showed varying degrees of severities of hypospadias. Urethral rupture was repaired and testicular descent was performed in three patients. The prevalence of DSD is estimated to be 5‰ in births, with 73% of them being boys with hypospadias. Although success in reaching a molecular diagnosis in 46,XY DSD was relatively low, as Eggers et al reported in a large international patient cohort, AR presented to be the largest percent of variants identified in 46,XY DSD patients [16, 17]. For male patients with gynecomastia at puberty, the possibility of AIS should be considered. 80% (4/5) patients complained about gynecomastia in our study. Retrospective studies have reported gynecomastia often occurred in young adulthood of PAIS and MAIS patients and it was usually associated with signs of under virilization, such as hypospadias and cryptorchidism[18, 19]. To be noted, 22.2% (4/18) patients in our cohort had a sibling with AIS. Molecular studies in Touzon’s study revealed other affected or carrier relatives in 87% of the index cases. Therefore, genetic counseling for the individual and family should be strongly encouraged.
More than 1000 AR mutations have been reported so far, and up to 30% were sporadic de novo mutations. Most AR mutations were identified in androgen insensitivity syndrome, and a small number of variants were detected in prostate cancer, spinal and bulbar muscular atrophy. Missense variants in the current study accounted for the most 63.2% (12/19), followed by small insertions and deletion 21.1% (4/19) and nonsense mutations 15.8% (3/19). Seven out of 19 variants (36.8%) result in premature stop codon of the AR protein. The variant pattern was similar with the previous studies that most AR variants in all AIS phenotypes are non-synonymous point mutations and frameshift leading to a premature stop codon are more frequently reported in CAIS patients. However, different from the previous studies which found that exon 5 and exon 7 were commonly involved exons in AR, most variants in our study located on exon 1, exon 2 and exon 5, each accounting for 21.1% (4/19). This may be due to the high proportion of CAIS enrolled, as Batista et al said, defects in NTD (mainly encoded by exon 1) are more frequent in CAIS patients. Four variants (A749D, A844E, G569W, A597T) have been reported to be associated with partial AIS which is consistent with phenotype presentation in our study[23–26]. A novel AR variant V819G on ligand-binding domain was identified in case 18 with PAIS. It is reported that most missense mutations involved in LBD are related to partial AIS. Ten variants (52.6%) resided in ligand-binding domain which has been reported to be the most frequently involved domain.
AR belongs to the nuclear receptor superfamily which needs to combine with androgen to form a complex and enter to nucleus, then activates transcription factors and promotes the expression of downstream genes. It is widely expressed in the body, such as liver, adipose tissue, endometrium, ovary, prostate, testis, skin, etc., and is closely related to the occurrence and development of many diseases. The residual activity of the affected AR underlies the phenotype of AIS and could be analyzed based on reporter assay in vitro. In our study, the transcription activity analysis demonstrated six out of eight AR mutant proteins (P15Afs*69, S258Efs*47, W435Gfs*44, C560F, C577W, C580Afs*46) have severely impaired residual activity in vitro (about 1%) which showed a strong correlation between genotype and phenotype. In terms of the two variants C560F and C577W which located in DNA-binding domain, different nucleotide substitution at the same position has been reported and all variants were associated with the phenotype of CAIS[28, 29]. The four frameshift variants resulting in premature stop codon have never been reported and the transcriptional activity was severely impaired. In contrast to decreased transcription activity, the AR mutant protein (K718X) on ligand-binding domain showed activated transcription compared with wildtype under DHT stimulation at different concentrations. The similar phenomenon has been reported in Bevan et al study. The two variants D864N and L907F were identified in complete androgen insensitivity and presented with considerable binding and transactivation activity. Considering the mechanism of nuclear receptor action, we further studied nuclear localization function of the novel AR variant K718X. Under 100nM DHT, the AR variant K718X showed impaired transport function from the cytoplasm to nucleus in COS-1 cells whose normality is vital to exert androgen biological function. In fact, in terms of the correlation between phenotype and genotype of AR variant, it is heterogenous even with the same substitution. A small number of mutations, as L581R, R608Q, R609K, Q641X, L723F, R727L, W742C, W752X, Y764C, R787X, Q799E, V867L, were identified in patients with androgen insensitivity syndrome but also found to be gain of transcription activity in prostate cancer which may imply the complex mechanism of AR action . The AR mutant protein (V819G) showed residual activity of 59%. The patient with the AR variant V819G presented with mild hypospadias and gynaecomastia at puberty. Reporter assay showed increased transcription activity with gradually elevated DHT concentration which further confirmed the proof of pathogenicity and severity. As Hellmann et al reported gynaecomastia may be ameliorated by androgen therapy, definite molecular diagnosis of AIS may reveal the severity of the AR variant and contribute to the individual’s clinical management.
Our findings suggested the necessity of genetic test and functional study in patients with AIS, especially in PAIS patients. For female patients presented with inguinal swelling before puberty, karyotype analysis is greatly encouraged. Functional analysis of the AR variant may be in favor of assessment of the responsiveness to androgen treatment. For those variants with increased transcription activity, AR function may be severely affected through abnormal nuclear localization in androgen insensitivity syndrome.