Introduction

Defects in the androgen receptor gene affects the androgen-dependent male sexual development in 46,XY individuals, leading to androgen insensitivity syndrome (AIS); the incidence being 1 in 20,000–64,000 male births (Ahmed et al. 2000). Clinically, based on the severity of the symptoms, AIS is categorized into complete, partial and mild. Complete androgen insensitivity syndrome (CAIS), the most severe manifestation of androgen insensitivity is characterized by female phenotypic appearance with 46,XY karyotype, typical female genitalia, normal breast development, absent/sparse pubic hair, absent uterus and bilateral intra-abdominal or inguinal testis, hypoplastic or absent wolffian structures associated with normal or elevated plasma testosterone (pl-T) and high luteinizing hormone (LH) levels (Sultan et al. 2001; Melo et al. 2011). Partial androgen insensitivity (PAIS) presents with either ambiguous genitalia or male phenotype with hypospadias and micropenis. Impaired spermatogenesis and/or pubertal undervirilization in otherwise normal men is considered as the mildest form of androgen insensitivity (MAIS) (Evans et al. 1997; Brinkmann 2001; Galani et al. 2008; Raicu et al. 2008). Other pathological conditions associated with defective androgen receptors are spinal bulbar muscular atrophy (La Spada et al. 1991), male breast cancer (Wooster et al. 1992) and prostate cancer (Tilley et al. 1996).

The two most important androgens are testosterone and 5 α-dihydrotestosterone, whose actions are mediated by functional androgen receptor, which upon receipt of signal activate transcription of specific genes in target tissues (Melo et al. 2011). The androgen receptor (AR) gene located at Xq11-12, has eight exons and encodes 920 amino acid residues. Akin to other members of the nuclear receptor superfamily, the AR gene contains four functional domains: an N-terminal domain (NTD) which serves transcriptional activation function encoded by exon 1; a central deoxyribonucleic acid (DNA) binding domain (DBD) rich in cysteine residues encoded by exons 2 and 3, a hinge region containing the nuclear targeting signal and a C-terminal ligand-binding domain (LBD) encoded by exons 4–8. LBD plays a critical role in nuclear localization, receptor dimerization and interaction with other proteins in addition to ligand binding (Brinkmann 2001; Raicu et al. 2008; Petroli et al. 2011).

SRY is another crucial gene that plays an important role in initiating male sexual development, mutations of which are known to cause XY sex reversal with gonadal dysgenesis and an increased risk of gonadal tumour. Mutations in SRY gene results in partial sex reversal implying its importance in male sex determination (McElreavy et al. 1992; Shahid et al. 2010). At birth, these patients are phenotypically normal female, however, during the pubertal age, they present with amenorrhoea and delayed secondary sexual characteristics. To date there are 60 different mutations identified within the open reading frame of SRY (Isidor et al. 2009).

In this study, we screened the SRY and AR genes in patients with gonadal dysgenesis and AIS and identified five sequence variations in AR gene.

Subjects and methods

Subjects and clinical history

The study adhered to the institutional ethical guidelines and was cleared by the same. Informed consent was obtained from the subjects, their family members and controls who participated in the study. Twenty-three cytogenetically confirmed XY females with mean age of 15.96 years were investigated. The clinical findings of the subjects are briefed in table 1. A total of 100 random healthy males were recruited in the study as normal controls.

Table 1 Details of clinical and hormonal profiles of patients.
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Cytogenetic and molecular analyses

Chromosome analysis was performed on metaphases prepared from peripheral blood lymphocyte culture (Hungerford 1965), applying standard GTG banding technique (Seabright 1971). Karyotype analysis was done using Olympus microscope BX51 (Olympus Singapore Pte Ltd.) with applied spectral imaging systems karyotyping software (BandView ver. 6.0). Genomic DNA (100 ng) obtained from peripheral blood lymphocytes, using phenol–chloroform extraction was PCR amplified for the complete SRY (NM_003140.2) and AR (NM_000044.3) coding regions (exons 1 >8) in PTC 200 (DNA Engine, BioRad, USA) thermal cycler. Details of primers are listed in table 2. Two sets of overlapping primers were used to amplify the single exon of SRY with the PCR conditions of initial denaturation at 95∘C for 5 min, followed by 30 cycles of denaturation at 95∘C for 15 s, annealing at 58∘C for 30 s, extension at 68∘C for 1 min and final extension at 68∘C for 7 min. Five sets of overlapping primers were used to amplify exon 1 and a single set of primer was used to amplify each exons 2–8 of AR gene as initial denaturation at 94∘C for 3 min for 1 cycle, followed by 30 cycles of denaturation at 94∘C for 30 s, annealing for 30 s at variable temperatures (table 2) and extension at 72∘C for 45 s and a final extension at 72∘C for 5 min. PCR reactions for SRY and AR genes were performed using Accuprime Pfx mastermix (Invitrogen, Carlsbad, USA) and Jump start red taq (Sigma, St Louis, USA), respectively, as per the manufacturer’s instructions. The amplified products were visualized through 1.5% agarose gel. Purified amplicons (Qiagen PCR purification kit, Hilden, Germany) were commercially sequenced (Madras Diabetic Research Foundation, Chennai, India) and checked for variations through basic local alignment search test (BLAST) analysis.

Table 2 Details of primers used for amplification of SRY and AR genes (exons 1–8).
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Results

Chromosome analysis revealed the karyotype to be 46,XY in 22 of the patients studied and one subject (SA235) showed presence of an additional marker; 47,XY + mar (figure 1). All cases analysed (n= 23) were positive for SRY with no sequence variation in the coding region. Screening the complete coding region of AR gene (exons 1 >8) revealed the following variations: patient 1 (SA235) presented with C >T transition in exon 1 (n.1317C >T) substituting codon (CAG) for glutamine at 68 to stop codon (TAG) (p.Q68X) (figure 2). Patient 2 (SA125) carried a G–A substitution in exon 1 (n.1761G >A) resulting in a change from glycine (GGG) to arginine (AGG) at codon 216 (p.G216R) (figure 3). Patient 3 (SA35) harboured deletion of a single-nucleotide G at codon 266 (n.1911delG), which resulted in a frameshift and subsequently introduced a premature stop at codon 295 (D266fs295ter) (figure 4). Patient 4 (SA213) revealed a C > T substitution in exon 6 (n.3510C >T) (figure 5) that changes the codon for glutamine at 799 (CAA) to a stop codon (TAA) (p.Q799X). Patient 5 (SA263) showed deletion of nucleotide T in exon 7 at codon 853 (n.3672delT) resulting in a frameshift and introduction of premature stop at codon 882 (p.C853fs882ter) (figure 6). All the above variations in the respective AR domains are schematically shown in figure 7. The possibility of presence of the following mutations in normal population was ruled out as follows, mutation n.1911delG was analysed in 100 random male controls through sequencing analysis and mutations n.1761G >A, n.3510C >T, n.3672delT were studied in 100 male control samples through RFLP using the restriction enzyme XhoI, NlaIV and BsrBI, respectively (figure 8).

Figure 1
figure 1

GTG-banded karyogram of proband (SA235P) showing 47,XY + mar karyotype.

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Figure 2
figure 2

(a) Partial electrophoregram of AR gene (exon 1) of the proband (SA235) showing n.1317C >T leading to Q68X. (b) Normal control.

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Figure 3
figure 3

(a) Partial electrophoregram of AR gene (exon 1) of the proband (SA125) showing n.1761G >A leading to G216R. (b) Heterozygous mother. (c) Normal father.

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Figure 4
figure 4

(a) Partial electrophoregram of AR gene (exon 1) of the proband (SA35) with n.1911delG leading to frameshift mutation (D266fs295ter). (b) Normal control.

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Figure 5
figure 5

(a) Partial electrophoregram of AR gene (exon 6) of the proband (SA213) showing n.3510C >T leading to Q799X. (b) Same mutation in the sibling (SA213S). (c) Normal father.

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Figure 6
figure 6

(a) Partial electrophoregram of AR gene (exon 7) of the proband (SA263) showing deletion T (n.3672delT) leading to frame shift mutation. (b) Heterozygous mother showing normal and the mutated allele as overlapping sequences from the site of mutation indicated. (c) Normal control.

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Figure 7
figure 7

Gene structure of androgen receptor (AR) showing five mutations detected in the respective domains. Mutations p.Q68X, p.G216R and p.D266fs295ter are located in the transactivation domain (exon 1). Mutations p.Q799X and p.C853fs882ter are located in the ligand binding domain (exons 6 and 7, respectively).

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Figure 8
figure 8

Representative RFLP gel pictures of mutation screening in normal males (a) showing n.1761G mutation screening by XhoI RFLP. (b) showing n.3510C >T mutation screening by NlaIV RFLP. (c) showing n.3672delT mutation screening by BsrBI RFLP. (d) Table describing the primers, PCR product size, product size after RFLP in the context of WT and mutation with appropriate mutated sample as positive control.

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Discussion

AIS and complete gonadal dysgenesis (CGD) associated with 46,XY karyotype in females are often diagnosed at puberty due to the absence of menarche. Unlike CGD, mixed and partial gonadal dysgenesis often present with karyotypes including mosaicism-like 45,X/46,XY (Layman et al. 2009; Jorgensen et al. 2010; Ocal et al. 2012). Literature suggests the incidence of SRY mutation to be about 10–15% in cases of XY CGD and sex reversal including point mutations, frameshifts and deletions (Shahid et al. 2004; Rocha et al. 2011). Majority of the other cases are known to carry mutations in either SRY regulatory elements or other genes like AR, NR5A1, DHH, FGF9, M33, DMRT1, AMH (antimullerian hormone) that are involved in sex differentiation pathway (Katoh-Fukui et al. 1998; Boyer et al. 2002; Canto et al. 2005; DiNapoli et al. 2006; Jorgensen et al. 2010) or other unknown genes (Rocha et al. 2011).

Molecular analysis of the subjects that are clinically diagnosed and cytogenetically validated for AIS, revealed no alterations in SRY, but showed five variations in the AR gene. The human AR is a member of the nuclear receptor superfamily, which facilitates the development of male secondary sexual characteristics as well as the growth of prostate gland (Brinkmann 2001; McEwan 2009). Among the steroid receptors, the AR records the highest density, witnessing more than 600 different mutations leading to AIS (Gottlieb et al. 2012). Two thirds of mutation reports are in the LBD, 20% of the mutations occur in the DBD and the NTD is said to cover less than 15% of the mutations (Gottlieb et al. 2004). Mutations detected in individuals with AIS range from single-base variations, nucleotide insertions or deletions, complete or partial gene deletion to intronic mutations (Brinkmann 2001). Among genetic lesions, gene deletion (either partial or complete) and intronic mutations are at low frequency, whereas missense mutations leading to variable phenotypes account for the most common molecular lesion.

The present study reports three variations in exon 1 of AR gene. This exon constitutes a major portion of the AR gene, encoding about 50% of the AR protein with two repeat regions: a CAG repeat normally varying from 11 to 31 and a CGN repeat ranging from 10 to 25. The variable length of these polymorphic regions are likely to be involved in regulation of AR activity and is implicated with AR functional defects (La Spada et al. 1991; Dowsing et al. 1999; Liu et al. 2008). Patient 1 harboured a C >T transition within the CAG repeat region at codon 68 converting glutamine (CAG) to stop codon (TAG) (n.1317C >T; p.Q68X) (figure 2). Mutation at this site has not been reported previously but similar premature terminating mutations within this polyglutamine stretch has been shown elsewhere (Zoppi et al. 1993; Philibert et al. 2010). Family members of patient 1 could not be studied due to unavailability of sample.

Patient 3 was identified with a novel frameshift mutation D266fs295ter that resulted from a single-nucleotide deletion (n.1911delG) at codon 266 (figure 4). This deletion leads to frameshift and introduces premature stop codon at position 295. The patient’s father, mother and brother carried the normal allele indicating the mutation to be of de novo origin. Exon 1 encodes the NTD and it mediates the transactivation function, hence, the disruption of this region might often compromise the biological function of AR. Supporting this, Zoppi et al. (1993) demonstrated the possibility of translation initiation downstream of the premature termination at NTD. However, such a translation reinitiated protein containing the portions downstream of NTD still remained to cause CAIS, which might be due to the loss of regions necessary for transactivation or the loss of specific23 FXXLF 27 region that is involved in the C-terminal interaction (Thompson et al. 2001; He and Wilson2003). In both the cases of premature termination within exon 1 showing typical features of CAIS, the protein would remain nonfunctional even if translation reinitiation had occurred.

G216R is a single-base alteration (n.1761G >A) that was identified in patient 2 (figure 3), which results in substitution of nonpolar (G) by a strong basic (R) residue at position 216. Molecular analysis of the patient’s mother revealed her to be heterozygous for the variation, while her father carried the normal allele, thus explaining maternal inheritance. The same variation has been previously reported by Deeb et al. (2005) in three unrelated patients; two females with ambiguous genitalia and a male with oligozoospermia. Another study by Wang et al. (1998) in a male with severe oligospermia revealed this mutation to be the cause for reduced AR activity, however the same mutation was also reported in a normal male suggesting that the presence of this variation alone cannot exclude fertility. Actually, this mutation has been reported to be present with variable phenotypes (PAIS, MAIS and normal) according to the AR mutation database (Gottlieb et al. 2012) and also associated with differences in sex of rearing (Deeb et al. 2005). The proband in our study diagnosed for PAIS, presented ambiguous genitalia with cliteromegaly, her gonads were of testicular origin and raised as a female.

The above discussed three mutations are clustered in the NTD of AR gene. The NTD encoded by exon 1 so far was considered to carry least number of mutations, probably because this exon may not have been analysed in most series. Missense mutations occurring in exon 1 are known to have a mild effect on AR function causing MAIS (Gottlieb et al. 2012). Single amino acid changes are considered to be rather infrequent in exon 1 of AR gene than stop codons, insertions or deletions as the unstructured NTD might tolerate single amino acid changes without affecting AR function (Audi et al. 2010). Truncation mutations in exon 1 is said to have increased by 25% (Audi et al. 2010; Philibert et al. 2010), which is reflected in this study.

Present study revealed two truncation mutations in the LBD, which is the hot spot for loss or defective functional mutations in AR. Patient 4 (SA213) harboured a truncation mutation (n.3510C >T) in exon 6 changing the codon 799 for glutamine to a stop codon (CAA >TAA) (p.Q799X) (figure 5). Even though the mother was deceased and father was found to carry normal allele, the elder sibling of the proband also harboured the same mutation suggestive of maternal inheritance. Notably, the elder sibling presented with amenorrhoea and poor secondary sexual characters like the proband, suggesting a strong correlation between the phenotype and this mutation. Moreover, Ignacak et al. (2004) reported the same mutation in a female with typical features of CAIS and abdominal tumour. Patient 5 (SA263) affected by CAIS, had a p.C853fs882ter mutation, where there is deletion of nucleotide T at codon 853 in exon 7 leading to frameshift and stop codon at exon 882 (figure 6).This n.3672delT novel mutation located in exon 7 was also found in her heterozygous mother demonstrating the maternal inheritance. There are similar reports of truncation mutations in exon 7 at different sites (Ahmed et al. 2000; Goulis et al. 2006; Cheikhelard et al. 2008) and all of these are reported to be present with CAIS. Truncation mutations in the highly conserved LBD of C-terminal results in the formation of receptor proteins, which are either incapable of binding to androgen or binding with high affinity (Imasaki et al. 1995; Ignacak et al. 2002; Goulis et al. 2006; Sun et al. 2010).

Truncation mutations are known to occur in all the exons of the AR gene, while missense mutations are said to be more common in the LBD. Here, we report four truncation mutations leading to truncated AR protein, two mutations in the NTD; and the other two in the LBD. Irrespective of the domain involved in mutation, it was noted that all four patients carrying truncation mutation presented features of complete androgen insensitivity, suggesting the truncated AR protein to be functionally compromised. This could be due to the loss of essential functional domain (LBD or DBD) in the truncated receptor protein (Bagatell and Bremner 2003). The fifth variation reported here is a missense mutation occurring in the NTD. A ligand-mediated N and C terminal interaction plays a critical role in stabilization and transcriptional activation of AR (Li et al. 2006; Centenera et al. 2008). Presence of a second nuclear localization signal in the LBD has also been reported (Poukka et al. 2000), suggesting that the substantial loss of AR protein in our subjects might lead to a defective protein stabilization, transcriptional activation or localization of AR. Truncated receptors could easily be predicted for their fully compromised biological function or incapable of binding to androgen with high affinity leading to insufficient activity. These alterations are uniformly said to be associated with phenotype of CAIS as seen in all four patients.

In conclusion, our study has identified three novel mutations and two known variations in the AR gene that might add more insights into the molecular mechanisms of AIS. Moreover, this study emphasizes the importance of mutation screening in the NTD, in addition to the LBD of AR. Further, our current result strengthens the direct role of AR gene as the most susceptible target in association with AIS.