Identification of an AR Mutation-Negative Class of Androgen Insensitivity by Determining Endogenous AR Activity

Abstract

Context: Only approximately 85% of patients with a clinical diagnosis complete androgen insensitivity syndrome and less than 30% with partial androgen insensitivity syndrome can be explained by inactivating mutations in the androgen receptor (AR) gene.

Objective: The objective of the study was to clarify this discrepancy by in vitro determination of AR transcriptional activity in individuals with disorders of sex development (DSD) and male controls.

Design: Quantification of DHT-dependent transcriptional induction of the AR target gene apolipoprotein D (APOD) in cultured genital fibroblasts (GFs) (APOD assay) and next-generation sequencing of the complete coding and noncoding AR locus.

Setting: The study was conducted at a university hospital endocrine research laboratory.

Patients: GFs from 169 individuals were studied encompassing control males (n = 68), molecular defined DSD other than androgen insensitivity syndrome (AIS; n = 18), AR mutation-positive AIS (n = 37), and previously undiagnosed DSD including patients with a clinical suspicion of AIS (n = 46).

Intervention(s): There were no interventions.

Main outcome measure(s): DHT-dependent APOD expression in cultured GF and AR mutation status in 169 individuals was measured.

Results: The APOD assay clearly separated control individuals (healthy males and molecular defined DSD patients other than AIS) from genetically proven AIS (cutoff < 2.3-fold APOD-induction; 100% sensitivity, 93.3% specificity, P < .0001). Of 46 DSD individuals with no AR mutation, 17 (37%) fell below the cutoff, indicating disrupted androgen signaling.

Conclusions: AR mutation-positive AIS can be reliably identified by the APOD assay. Its combination with next-generation sequencing of the AR locus uncovered an AR mutation-negative, new class of androgen resistance, which we propose to name AIS type II. Our data support the existence of cellular components outside the AR affecting androgen signaling during sexual differentiation with high clinical relevance.

Figure 1.

NGS of the AR locus. A, Graphic representation of the AR locus and the regions amplified by the haloplex design (chrX: 66,754,874–66955461 [hg19]) (shown in green). Highly repetitive sequences were excluded from the design (shown in brown). For comparison, repetitive elements present in this locus are shown in black. B, Division of the four patient groups from whom cultured GFs were analyzed. When mutations were found in the AR-CDS of GFs from group 4 (clinically suspected androgen resistance), they were reallocated to group 3 (genetically proven AIS). Therefore, two of the initially 41 samples from group 4 with clinically suspected PAIS and seven of the initially eight samples with clinically suspected CAIS were reallocated to group 3, resulting in 15 GF samples with genetically proven PAIS and 22 samples with genetically proven CAIS, respectively. C, Distribution of mutations found in the CDS of the AR. Red dots represent nonsense mutations and green dots missense mutations. Synonymous mutations in the coding region were not considered as CDS mutations. The graph was designed using the Mutation Mapper software from cBioPortal. D, Distribution of nonannotated SNPs along the sequenced region (green bars). DNTD, N-terminal transactivation domain; DBD, DNA-binding domain; HR, hinge region; LBD, ligand-binding domain.

Figure 2.

DHT-dependent, AR-induced APOD mRNA expression represented as the ratio between ethanol (EtOH)- and DHT-treated GFs. A, Scrotum-derived male controls (vasectomy, orchidopexy [group 1]), labioscrotal derived molecular defined DSDs (other DSDs [group 2]), and AR-CDS mutation-positive AIS (CAIS, PAIS [group 3]). B, Depiction of cutoff values between male controls (vasectomy and orchidopexy) and AR-CDS mutation-positive AIS (CAIS and PAIS) of 2.29 (100% sensitivity, 97.7% specificity, P < .0001) and between the same AR-CDS mutation-positive AIS and molecular defined DSDs (other DSDs) of 2.36 (100% sensitivity, 93,3% specificity, P < .0001). C, Foreskin-derived male controls (circumcision [group 1]) and molecular-defined DSDs (other DSDs [group 2[) as well as AR-CDS mutation-positive AIS (CAIS, PAIS [group 3]). D, Depiction of the cutoff value between male controls (circumcision) and AR-CDS mutation-positive AIS (CAIS and PAIS) of 2.36 (100% sensitivity, 100% specificity, P < .0001). Means and SDs are included as error bars. Values of P < .001 are denoted by three stars, and those values of P < .01 are denoted by two stars. Among the DSD diagnoses other than AIS, empty squares represent SRD5A2, horizontally half-filled squares represent HSD17B3, vertically half-filled squares represent CYP17A1, crossed squares represent CYP21A2, and dotted squares represent NR5A1 mutations.

Figure 3.

DHT-dependent, AR-induced APOD induction in response to different DHT concentrations in the culture media. A, GF-11, GF-16, and GF-35 are scrotum-derived male control GFs (Supplemental Table 1). GFs derived from a CAIS patient carrying the p.Ser310fs mutation served as negative control. The p.Val867Met mutation is shown in black. B, GF-120, GF-123, and GF-124 are foreskin-derived male control GFs (Supplemental Table 2). GFs derived from a CAIS patient carrying the p.Ser220fs mutation served as the negative control. The p.Tyr782Asp mutation is shown in black.

Figure 4.

DHT-dependent, AR-induced APOD expression in AR-CDS-negative individuals with clinically suspected androgen resistance (group 4) derived from scrotum/labia majora (A) and from foreskin/labia minora (B). Suspected androgen-resistant GFs of group 4 are divided into mild androgen resistance (MAIS, micropenis), partial androgen resistance (PAIS, ambiguous external genitalia), and complete androgen resistance (CAIS, completely female external genitalia). Included are means and SDs. For comparison, the tissue-specific controls and AR-CDS mutation-positive GFs are shown as well. The calculated cutoffs are drawn as dotted lines.

Figure 5.

Analysis of AR-CDS mutation-negative but functionally androgen-insensitive GF. A, Distribution of potentially damaging mutations in the sequenced region outside the AR-CDS. B, AR protein expression in male scrotum-derived controls and AR-CDS mutation-negative but functionally androgen-insensitive labioscrotal GFs. C, AR protein expression in male foreskin-derived controls and AR-CDS mutation-negative but functionally androgen-insensitive GFs. Included are means and SDs.