A case of mild partial androgen insensitivity syndrome in a juvenile boy

Abstract

Androgen insensitivity syndrome (AIS) is a rare disorder with X-linked recessive inheritance in 46 XY patients. The clinical manifestations vary between patients, especially regarding external genitalia development. Herein, the case of AIS in a 13-year-old male, who was born with hypospadias and presented to the hospital with gynaecomastia that had developed from 8 years of age, is reported. No micropenis, cryptorchidism or bifid scrotum were found. Testis volume was 12 ml on both sides. His testosterone and luteinizing hormone levels were normal compared with sex- and age-adjusted reference range. His bone age was approximately 13 years according to Greulich-Pyle assessment. Sequence analysis of the androgen receptor (AR) gene revealed a mutation (c.2041A>G) in exon 4, a novel mutation site in the AR gene. Prediction analysis suggested this to be a disease-causing variant. A milder clinical presentation and normal hormone levels in cases of partial AIS might differ from the usually reported signs and symptoms. A diagnosis of AIS should not be ignored in teenage patients who present with gynaecomastia and hypospadias, but normal hormone levels.

Introduction

Androgen insensitivity syndrome (AIS) is a rare disease. Precise figures for the prevalence of AIS are unavailable due to a large number of mild AIS and partial AIS being misdiagnosed or underreported, however, the incidence of complete AIS is thought to be between 1 in 20 400 and 1 in 99 100. ¹ As a heterogeneous disease, the clinical manifestations of AIS range between varying levels of impaired virilism, gynaecomastia and infertility. Besides AIS, similar presentations may result from androgen deficiency, excess oestrogens, decreased androgen action, and 5α-reductase type 2 deficiency.^2,3 In particular, 5α-reductase type 2 deficiency shares similar clinical and biochemical manifestations with AIS, such as androgen concentration and luteinizing hormone (LH) concentration. As a result, the differential diagnosis of AIS and 5α-reductase type 2 deficiency remains challenging, particularly in prepubertal teenagers without gynaecomastia. Here, we report the case of a teenager with mild partial AIS with normal hormonal change and a novel mutation site.

The study was performed in accordance with the Declaration of Helsinki and was approved by Tongji Hospital Ethics Committee (TJ-IRB20210746). Written informed consent to publish the case was provided by the legal guardian (father) of this patient.

Case report

A 13-year-old male patient presented to the hospital with gynaecomastia. Bilateral, symmetrical breast enlargement had been noted over the previous 5 years without breast pain. His past medical history was unremarkable, except for simple hypospadias at birth. His family history revealed that his two sisters had normal puberty, but his maternal uncle had hypospadias and gynaecomastia (Figure 1). Physical examination revealed height of 169 cm, a body mass index (BMI) of 16.8 kg/m², normal blood pressure, and an apparently normal male phenotype with testicular volume of 12 ml in both testes (normal testis volume for a 13–14-year-old male, 8.33 ± 4.87 ml) with bilateral gonads having already descended into the scrotum, and a penis length of 6.5 cm (normal penis length for a 13–14-year-old male, 4.52 ± 1.21 cm). ⁴ The patient exhibited abnormal tanner stage III breast development. A blood sample was collected into a tube with coagulant and separator gel, processed to obtain serum, and analysed immediately. Laboratory tests showed that his progesterone level was 0.43 ng/ml (sex- and age-adjusted reference range, 0.06–6.09 ng/ml), follicle-stimulating hormone (FSH) was 2.83 mIU/ml (sex- and age-adjusted reference range, 1.52–10.30 mIU/ml), LH was 7.23 mIU/ml (sex- and age-adjusted reference range, 0.79–7.47 mIU/ml), testosterone was 5.21 ng/ml (sex- and age-adjusted reference range, 0.20–6.60 ng/ml), ⁵ and sex hormone-binding globulin was 27.3 nmol/L (sex- and age-adjusted reference range, 8.6–64.2 nmol/L). According to the laboratory centre, the patient’s calculated free testosterone was 424.0 pmol/L (reference range is shown below under ‘Interpretation of hormone reference interval’), and calculated bioactive testosterone was 9.950 nmol/L (reference range is shown below under ‘Interpretation of hormone reference interval’). Thyroid function, β-human chorionic gonadotropin (β-HCG) level, insulin-like growth factor 1, insulin-like growth factor-binding protein 3, prolactin and oestradiol were within normal range. The patient’s bone age was approximately 13 years, according to Greulich-Pyle assessment. No genital organ was found in the pelvic cavity through pelvic ultrasound. No abnormal findings were identified via pituitary magnetic resonance imaging, adrenal computed tomography or pelvic ultrasound scans. Chromosome analysis of peripheral blood revealed a 46, XY karyotype.

Figure 1.

Family tree (pedigree) of a 13-year-old male patient who presented with gynaecomastia and a history of simple hypospadias at birth, according to genealogical symptoms. The maternal uncle (I2) of the proband (II4, arrow) had gynaecomastia and hypospadias. Due to personal reasons, no blood sampling or genetic analysis of the uncle (I2) was performed. Other family members were normally developed.

Considering the patient was a teenage boy with hypospadias and gynaecomastia, AIS was suspected. A peripheral blood sample was obtained from the patient for Sanger sequencing of the androgen receptor (AR) gene, revealing a novel point mutation in exon 4. The variant was denoted as c.2041A>G at the cDNA level or as p.Ile681Val at the protein level (NM_000044; Figure 2). Sanger sequencing was also performed on samples obtained from his mother and both sisters. His maternal uncle declined genetic testing for personal reasons. Genetic test results showed that his mother and elder sister were heterozygous for the c.2041A>G mutation. The identified mutation site was predicted to be damaging in MutationTaster2 software (http://www.mutationtaster.org/) and Sorting Intolerant From Tolerant (SIFT, version 4.0.3) prediction software (http://provean.jcvi.org/index.php). In addition, the genetic variant was found to be absent in the normal Chinses population, according to the 1000 genomes database (https://www.internationalgenome.org/) and Exome Aggregation Consortium (ExAC) database (http://exac.broadinstitute.org). The Genomic Evolutionary Rate Profiling (GERP) score of the mutation site was found to be 5.03, suggesting an evolutionarily conservative site. With the permission of this patient and his legal guardian, treatment with 40 mg oral testosterone undecanoate, twice daily, was initiated, however, due to inconvenient access to medication, oral testosterone undecanoate treatment was discontinued by the patient and his legal guardian after 1 month, and no further treatment has been administered since. The discontinuation of testosterone undecanoate treatment was agreed with the clinical team, as the patient had normal penile length and testis volume. Two years later, he underwent reduction mammoplasty for enlarged breast. The patient is now studying in a vocational-technical school and declined to attend a follow-up assessment at the outpatient clinic, however, he reported no changes in penile length and testis volume. The patient reported that the illness has hurt him a bit right now, but he is generally fine.

Figure 2.

Results of sequencing of the androgen receptor (AR) gene in the proband (a) and his mother (b), showing nucleotide substitution.

The reporting of this study conforms to CARE guidelines. ⁶

Interpretation of hormone reference interval

When interpreting hormone levels, patient's age and sex are well-known to play a major role in making a clinical decision, however, there is no uniform sex- and age-adjusted reference range for hormone levels. Different detection methods, reagents and puberty stages have a great influence on the reference range. In the present case, the reference range recommended by the testing laboratory was initially adopted, followed by the reference range recommended by another Chinese hospital that uses the same detection method. In the current study, levels of LH, FSH, testosterone, and sex hormone-binding globulin were detected using a Beckman DxI 800 Immunoassay Analyser and corresponding chemiluminescent immunoassays (Beckman Coulter; Brea, CA, USA), according to the manufacturer’s instructions. The sex- and age-adjusted reference ranges were provided by Beckman Coulter Life Sciences and the Children’s Hospital of Shenzhen. ⁵ Free testosterone and bioactive testosterone were calculated through albumin, sex hormone-binding globulin and testosterone concentration via the Vermeulen equation, ⁷ using the online Free & Bioavailable Testosterone calculator from the International Society for the Study of the Aging Male (www.issam.ch/freetesto.htm). Although the values of free testosterone and bioactive testosterone in this patient were within the normal range for male adults, the sex- and age-adjusted reference ranges are required when diagnosing paediatric endocrine abnormalities, and were not provided by the local laboratory. The reference ranges of calculated free testosterone and bioactive testosterone in males aged 9–14 years have been reported as 1.45–250.62 pmol/L and 0.02–5.62 nmol/L, respectively, ⁸ where testosterone was detected by chemiluminescent immunoassay using an Abbott Architect i2000 analyser, which differed from the analyser used in the present study. Another previously published study reported reference ranges of calculated free testosterone and bioactive testosterone in males aged 13–14 years of 6.25–404.93 pmol/L and 0.15–9.49 nmol/L, respectively, ⁹ in which testosterone was measured by liquid chromatography and tandem mass spectrometry, unlike the present study. No matter which reference range was applied, the values of free testosterone and bioactive testosterone in the present patient were higher than those expected for teenagers of the same age and sex.

Discussion

Gynaecomastia, the benign proliferation of glandular breast tissue in males, is a common disorder among the male population. ² Physiologic gynaecomastia is the most common type, and is seen in infants, adolescents and the elderly. In the present case, gynaecomastia began at the age of 8 years and worsened during the subsequent 5 years. These characteristics were inconsistent with physiologic gynaecomastia. Obese teenagers have a tendency towards gynaecomastia during puberty, however the patient in the present case appeared slim (indicating normal BMI). Other less common causes of gynaecomastia include drugs, LH-producing tumours, β-HCG-producing tumours, congenital adrenal hyperplasia, aromatase excess syndrome, AIS, and systemic disease. ² A medical history, laboratory results, familial history and genetic sequencing support the differential diagnosis of AIS.

Hypospadias is a congenital anomaly of the male urethra that results in the abnormal ventral placement of the urethral opening. ¹⁰ The location of the displaced urethral meatus may range anywhere within the glans, the shaft of penis, the scrotum, or perineum. Severe hypospadias, and isolated simple hypospadias accompanied by micropenis with or without cryptorchidism, suggest a disorder of sex development. ¹¹ The differential diagnoses associated with hypospadias include AIS, 5α-reductase type 2 deficiency and 17-hydroxylase deficiency. ¹² 17-hydroxylase deficiency may be excluded by normal androgen level and normal blood pressure, and gynaecomastia is rarely seen in 5α-reductase type 2 deficiency. ¹³ Therefore, AIS was the most likely diagnosis in the present case, and was confirmed by genetic analysis.

Androgen insensitivity syndrome is an X-linked recessive disorder caused by impaired function of the androgen receptor. ¹² Varying degrees of androgen resistance result in different phenotypes, such as complete, partial or mild AIS. Complete AIS is characterized by female external genitals, primary amenorrhoea, absence of uterus and oviducts, gynaecomastia and absence of pubic and axillary hair. The clinical features of partial AIS depend on the degree of responsiveness of the external genitalia to androgen. Typical manifestations of partial AIS include micropenis, hypospadias and a bifid scrotum, which may contain descended testes. Mild AIS features gynaecomastia and infertility without genital anomalies. ¹²

In the present case, the patient’s virilized genitalia and genital anomaly of hypospadias excluded the diagnosis of complete AIS and mild AIS. Partial AIS was diagnosed according to the clinical manifestations. The patient had an external masculinization (EMS) score of 10 (3 points for scrotal fusion, 3 points for no micropenis, 1 point for penile urethral meatus, and 3 points for bilateral scrotal gonad), which was much higher than reported for most patients with partial AIS (a median EMS score of 3). ¹⁴ His flaccid penis length was 6.5 cm, equal to the mean length reported for Chinese adult males, ¹⁵ and both testes were descended into the scrotum. Hence, the genital abnormality was mild. ¹⁶ The patient’s LH and testosterone concentrations were within normal range, and while normal LH and testosterone levels may be seen in patients with partial AIS, higher concentrations have been reported. For example, Vaidyanathan et al. ¹⁷ reported a similar case of a 17-year-old boy with gynaecomastia, a testosterone level of 1660 ng/dl (normal value, <1000 ng/dl), an LH level of 14 µ/ml (normal value, <3.6), and stretched penile length of 8 cm (<−2.5 SD of normal US values), while another case of partial AIS with prominent manifestation of gynaecomastia also had a higher level of LH and testosterone. ¹⁸ This suggests that the disease severity in the present case may be milder than in most patients with partial AIS.

More than 1000 variants have been identified in the AR gene, ¹⁹ with novel mutation sites frequently reported. In a study by Yuan et al., ²⁰ five novel mutation sites were found among 10 patients with AIS. In another study, ²¹ 12 previously unreported mutations were detected in 24 patients with AIS. Most of the mutation sites were localized in exons encoding DNA binding domains (exon 2 and 3) and ligand-binding domains (exon 4–8). ²⁰ The c.2041A>G mutation in the present case was located in exon 4, encoding the ligand binding domains. The various clinical manifestations of AIS are associated with varying molecular defects in the AR gene. In the study by Yuan et al., ²⁰ most missense mutations located in the ligand-binding domains led to complete AIS. A significant number of missense mutations between amino acids 214 and 511 have been shown to result in mild AIS, suggesting that missense mutations in this part of exon 1 have a relatively small effect on AR function. ²² However, the relationship between phenotype and genotype is difficult to determine. Phenotypic variation has been noted within affected families with the same mutation site, ²³ and in sporadic cases, phenotypic variation related to mutant c.2444G>A and c.2464C>G has been frequently reported to lead to either partial or mild AIS. ²² Many factors influence phenotypic variability, including different degrees of 5-α reductase activity, masculinization programming window and oligogenic effect. ²⁴ Some patients with AIS have mosaic mutations, in which the phenotypic manifestation depends on the time of occurrence of the mosaic, as well as its distribution in androgen dependent tissues. ²⁴ Androgen receptor coregulators and interacting proteins also influence phenotypic variability in patients with AIS. ²² The c.2041A>G mutation in the present case might involve a less severe responsiveness of the androgen receptor to its ligands due to unclear underlying aetiology.

Management of partial AIS with male predominance includes hormone replacement, puberty development, gonadectomy, hypospadias repair, mastectomy, fertility treatment and psychotherapy. Iatrogenic testosterone may improve penile length in male patients to some extent. Conversely, iatrogenic testosterone may aggravate gynaecomastia and trigger central precocious puberty, however, topical dihydrotestosterone might counteract this effect. Unfortunately, dihydrotestosterone is currently unavailable in China, and pharmacotherapy of AIS in China needs improvement. Surgery is important for hypospadias and gynaecomastia, which needs cooperation of the surgeon, and intra-cytoplasmatic sperm injection is an option for meeting the demands of fertility. ²⁵ Psychotherapy is extremely important, especially as the patient grows into adulthood.

Learning points

Androgen insensitivity syndrome is a rare disease demanding attention.

Novel mutation sites in the androgen receptor gene are common. The c.2041A>G mutation might involve a less severe responsiveness of the androgen receptor to its ligands due to unclear underlying aetiology.

The phenotype of partial AIS in the present case might be milder than usually reported. A diagnosis of partial AIS should be considered in patients who present with gynaecomastia and hypospadias, even if hormone levels are within the normal range of an adult. Age- and sex-adjusted reference range is of crucial importance for diagnosing such diseases in children.

Pharmacotherapy of AIS requires improvement in China.