CASE REPORT
Nagoya J. Med. Sci. 85. 362–368, 2023
doi:10.18999/nagjms.85.2.362

An extremely rare missense mutation of the androgen
receptor gene in a Vietnamese family with complete
androgen insensitivity syndrome
Thi Minh Thi Ha1,2, Phan Tuong Quynh Le1,2,Thanh Nha Uyen Le3 and Thi Thuy Yen Hoang4
1
Department of Medical Genetics, University of Medicine and Pharmacy, Hue University, Hue, Vietnam
Center of Prenatal and Neonatal Screening - Diagnosis, University of Medicine and Pharmacy Hospital,
Hue University, Hue, Vietnam
3
Center for Endocrinology and Metabolism, Hudson Institute of Medical Research, Melbourne, Australia
4
Department of Pediatrics, University of Medicine and Pharmacy, Hue University, Hue, Vietnam

2

ABSTRACTWe report a Vietnamese family with complete androgen insensitivity syndrome that included several
phenotypic females who have a 46,XY karyotype with an extremely rare mutation of the androgen receptor
gene. The proband was a 27-year-old phenotypic adult female referred to our department for karyotyping
due to primary amenorrhea. Ultrasound examination revealed a small uterus. Chromosomal analysis showeda 46,XY karyotype. A polymerase chain reaction assay revealed the presence of the sex-determining region
Y gene. Next-generation sequencing detected the NM_000044.6(AR):c.2170C>T(p.Pro274Ser) mutation,
which was confirmed by Sanger sequencing. There is only one previous report of this mutation in a childwith complete androgen insensitivity syndrome. In the family presented in this study, there were four
more phenotypic adult females with primary amenorrhea and a phenotypic female infant with testes in
the inguinal canals. The infant (first cousin once removed of the proband) presented with inguinal hernia/swelling in a phenotypic female and one of the four abovementioned adults had similar genetic analysis
results. This is the second report of a missense mutation NM 000044.6(AR):c.2170C>T in the world andthe first study to document a pedigree consisting of several individuals with CAIS as a result of thismutation. The presence of a tiny uterus in the proband, which is a rare occurrence in complete androgen
insensitivity syndrome, is a unique clinical indicator of the disorder’s variable expressivity.
Keywords: androgen insensitivity syndrome, androgen receptor gene, c.2170C>T (p.Pro274Ser), primaryamenorrhea, testes in the inguinal canals
Abbreviations:
CAIS: complete androgen insensitivity syndrome
AR: androgen receptor gene
SRY: sex-determining region Y gene
AMH: anti-Müllerian hormone

Received: April 6, 2022; accepted: June 1, 2022
Corresponding Author: Thi Minh Thi Ha, MD, PhD
Department of Medical Genetics, University of Medicine and Pharmacy, Hue University, 6, Ngo Quyen
Street, Hue 49100, Vietnam
Center of Prenatal and Neonatal Screening - Diagnosis, University of Medicine and Pharmacy Hospital, Hue
University, 51, Nguyen Hue Street, Hue 49100, Vietnam
Tel: +84-91-404-6350, E-mail: htmthi@hueuni.edu.vn, htmthi@huemed-univ.edu.vn
362

Rare AR mutation in a family with CAISThis is an Open Access article distributed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International
License. To view the details of this license, please visit (http://creativecommons.org/licenses/by-nc-nd/4.0/).

INTRODUCTION
Androgen insensitivity syndrome, OMIM# 300068, was first described as testicular feminization by Morris.1 It is characterized by resistance to androgen in 46,XY individuals.2 The
external genitalia of patients with androgen insensitivity syndrome vary from a normal female
form in complete androgen insensitivity syndrome (CAIS) to an undervirilized male form inpartial androgen insensitivity syndrome and a normal male form in mild androgen insensitivity
syndrome.3 The prevalence of CAIS is estimated to range from one in 20,400 to one in 99,100
individuals with a 46,XY karyotype.2 Primary amenorrhea in a phenotypic female adolescentand inguinal hernia/swelling in a phenotypic female infant are typical scenarios in which CAIS
is diagnosed.4 Although CAIS patients have a female appearance with breast development andcomplete female external genitalia, they have a 46,XY karyotype with the presence of the
SRY gene. The clinical presentation of CAIS results from a lack of masculinization caused byineffective androgen action due to receptor function loss.5 The androgen receptor is encoded by
the androgen receptor (AR) gene (OMIM# 313700), which is located on the X chromosome at
Xq12.2 Approximately 95% of CAIS patients have AR gene mutation.6 CAIS is thus an X-linked
recessive genetic disorder in which 70% of AR mutations are inherited and 30% are de novo.2
Several hundred AR mutations have been identified, and the number is steadily increasing. Since
2012, the Androgen Receptor Gene Mutations Database has added over 800 entries of mutations
causing androgen insensitivity syndrome, including over 500 different AR mutations. 7 To date,the Human Gene Mutation Database includes more than 700 AR mutations, the vast majority ofwhich are missense and nonsense mutations.8 Thus, it is critical to detect AR gene mutations,particularly rare or novel ones in diverse populations, and determine their association with the
CAIS phenotype. We present a Vietnamese family with several phenotypic females who have a
46,XY karyotype with an extremely rare AR gene mutation.

CASE REPORTThe proband was a 27-year-old phenotypic female adult referred to the Department of Medical
Genetics of University of Medicine and Pharmacy, Hue University (Vietnam) for karyotyping
because of primary amenorrhea. The physical examination revealed normal female genitalia,
sparse pubic and axillary hair, developed breasts, and a height of 158 cm (the average heightof Vietnamese women is 153.4 cm). The patient reported no sexual intercourse problems.
Biochemistry tests showed serum hormone levels as follows: luteinizing hormone 34.17 mIU/
mL, follicle-stimulating hormone 9.13 mIU/mL, estradiol 49.81 pg/mL, testosterone 15.00 ng/
mL, and anti-Müllerian hormone (AMH) 23.00 ng/mL. Ultrasound examination showed a small
uterus with a longitudinal dimension of 43 mm, an anteroposterior dimension of 12.5 mm, andno endometrium (Fig. 1). No gonad structure was found via ultrasound. A second specialist’s
ultrasound examination also revealed a small uterus and no gonad structure. Laparoscopy was
not available.
Conventional karyotyping of a peripheral blood sample showed a 46,XY karyotype. A
polymerase chain reaction assay detected the presence of the sex-determining region Y (SRY)gene. Genomic DNA samples was subjected to next-generation sequencing targeting coding
region (exome sequencing) using a NextSeq system (Illumina, USA) at Gene Solutions (HoNagoya J. Med. Sci. 85. 362–368, 2023

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Thi Minh Thi Ha et al

Chi Minh City, Vietnam) for a panel of 45 genes associated with disorders of sex development (DSD). The next-generation sequencing results revealed a missense mutation of the AR
gene, NM_000044.6(AR):c.2170C>T p.(Pro724Ser). This substitution was confirmed by Sanger
sequencing (Applied Biosystems 3730 Genetic Analyzer; Thermo Fisher Scientific, USA) (Fig.
2). We have submitted this variant to the ClinVar database under the accession SCV001593154.1

Fig. 1

Ultrasound image of the proband’s pelvic area, showing a small uterus with a longitudinal dimensionof 43 mm and an anteroposterior dimension of 12.5 mm

Fig. 2 Sequencing chromatogram of the proband’s AR gene with NM_000044.6(AR):c.2170C>T mutation
The arrow indicates the c.2170C>T mutation on the sense strand of the AR gene.
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Rare AR mutation in a family with CAIS

Fig. 3 The pedigree of the proband’s family
Members of II-7, III-2, III-9, III-14 (proband), and III-18 were phenotypic adult females with primary amenorrhea;
IV-5 was a phenotypic female infant with testes in the inguinal canals.

(VCV001077100.1).
The proband’s family included several more phenotypic adult females with primary amenorrhea.
They were identified as II-7, III-2, III-9, and III-18 in the pedigree (Fig. 3), with III-9 having
palpable masses in the groin bilaterally. Moreover, a phenotypic female infant who was a firstcousin once removed (IV-5) of the proband (III-14) had testes in the inguinal canals, suspected
due to inguinal swelling and confirmed by ultrasound examination at the age of one month.
Karyotyping, polymerase chain reaction assays for the SRY gene, and Sanger sequencing forthe aforementioned substitution were also performed on III-9 and IV-5. The results showed a
46,XY karyotype, the presence of SRY gene, and the NM_000044.6(AR):c.2170C>T mutation inboth. There was no genetic testing available for other family members with primary amenorrhea.
This research was approved by the Institutional Review Board of the University of Medicine
and Pharmacy Hospital, Hue University (approval number 48BV/21).

DISCUSSION
The proband (III-14) in this study was a completely external phenotypic adult female withprimary amenorrhea. Karyotyping revealed a 46,XY pattern. Further genetic analysis revealed
SRY gene positivity. Taken together, these findings led to the diagnosis of a 46,XY DSD. CAIS
is the most common disorder that results in a female phenotype in 46,XY individuals. 9 The
proband’s breast development was consistent with CAIS. While Müllerian structures are notfound in most CAIS cases,2,10 an ultrasound examination revealed that our patient had a small
uterus measuring 43 × 12.5 mm. Therefore, Swyer syndrome (complete gonadal dysgenesis)
was another possibility in this case. However, the patient’s hormonal profile, which included
elevated levels of luteinizing hormone, estradiol, testosterone, and AMH, appeared to be moreconsistent with CAIS than with Swyer syndrome.11 In general, females with CAIS have normal
or higher testosterone levels than males, due to intact testes. Their elevated luteinizing hormone
concentrations indicate androgen resistance at the hypothalamic-pituitary level. When testosterone
is aromatized, it produces estradiol levels that are higher than those in men but lower thanthose in women.3,12 Females with CAIS also have an elevated AMH levels due to the secretionof Sertoli cells.13 On the other hand, Swyer syndrome has a hormonal profile that includes low
testosterone and AMH levels.11
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Using next-generation sequencing, we detected the patient’s AR gene variant (NM_000044.6:
c.2170C>T), which was located on exon 4 of the AR gene. Sanger sequencing confirmed the mutation. The AR gene encodes for the androgen receptor protein of 919 amino acids, including threefunctional domains: N-terminal transactivation domain (residues 1–555), DNA-binding domain(residues 556–623), C-terminal ligand binding domain (residues 666–919), and a hinge region
between DNA-binding domain and C-terminal ligand binding domain (residues 624–665).2,14 The
AR mutation in this study was a substitution of serine for proline at codon 724 in C-terminal ligand binding domain, which has been recorded on UniProt with an identifier code “VAR_009786”and has been identified as a pathogenic variant in androgen insensitivity syndrome.15 The proline
amino acid is nonpolar and hydrophobic, while serine is polar and hydrophilic. Therefore, thissubstitution might affect the androgen receptor protein structure and the ligand-binding abilityof C-terminal ligand binding domain. The AR gene c.2170C>T mutation was also predicted
as a damaging variant in bioinformatics tools. It has been reported as a deleterious variant in
PROVEAN (score: −6.29; cut-off: −2.5), as a probably damaging effect in Polyphen-2 (score:
0.999; sensitivity: 0.09; specificity: 0.99), and as disease causing in MutationTaster (probability
value: 0.999, score: 74, range: 0–215). Furthermore, Hannema16 reported this mutation as C2529T(P723S) in a 3-month-old child with CAIS as additional evidence of its pathogenic potential.
According to the Androgen Receptor Gene Mutation Database, since 2012, there has been a+1 shift in mutation numbering in the DNA-binding domain and the C-terminal ligand-binding
domain compared to most previously documented mutations.7 Consequently, the mutation in ourcase (Pro724Ser) is identical to the C2529T (P723S) mutation in the Hannema’s case.
All evidence considered, our patient was diagnosed with CAIS. Furthermore, the phenotypic
female infant (IV-5) with testes in the inguinal canals and one of the females with primaryamenorrhea in this family (III-9) had the same genetic analysis results, including a 46,XY
karyotype, SRY gene positivity, and the AR gene c.2170C>T mutation. Unfortunately, IV-5’s
mother postponed her genetic testing due to a psychological issue. Nonetheless, the fact that
IV-5, her maternal aunt (III-9) and her maternal aunt once removed (the proband) had thesame CAIS-causing AR mutation suggests that the mutation was inherited in the family andIV-5’s mother was an obligate carrier of AR mutation. It can be speculated that the other threephenotypic adult females with primary amenorrhea (II-7, III-2, and III-18) may also have CAIS
due to the same AR gene mutation in a 46,XY karyotype. To our knowledge, Hannema was thefirst to report the c.2170C>T mutation in a child with CAIS. This is the second report of thec.2170C>T mutation and the first study to document a pedigree that includes several individuals
with CAIS due to this mutation.
In terms of clinical presentation, most CAIS patients show regression of the Müllerian structures. Surprisingly, our proband still had a small uterus, which was examined by two specialists
using ultrasound. The patient did not appear to perceive her vagina to be small or tight anddid not report any problems with sexual intercourse. Persistent Müllerian remnants in patientswith CAIS were first described by Oka et al.17 Similar cases have subsequently been reported,albeit very rarely. Some studies have suggested that the presence of Müllerian structures does
not exclude a CAIS diagnosis.18,19 The presence of Müllerian remnants in some CAIS patientsdemonstrates the variable expressivity of this disorder. The etiology of this phenomenon in
CAIS remains unclear. In the first report, Oka et al17 suggested that it may be attributed to
impaired AMH synthesis and/or function. The proband in our study had elevated serum AMH,
suggesting that, although Sertoli cells secreted AMH,13 the hormone could not act on the target
organs. Dodge et al20 also reported a case of CAIS with incomplete Müllerian regression. They
hypothesized that the testes descended early, before AMH could affect the Müllerian structures.
Both Oka et al’s and Dodge et al’s explanations might be applicable to our case. We also
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Rare AR mutation in a family with CAIS

performed an NGS assay for the proband with the panel of 45 genes associated with DSD
including AMH and AMHR2 genes. No mutations were found in either gene. Further studies areneeded to clarify the pathogenesis of this substitution, particularly the association between the
AR c.2170C>T mutation and the presence of a small uterus, as well as the genetic modifiers
that lead to variable CAIS expressivity.
Although the three CAIS patients in this study had the same mutation, their gonad locations
were different: inguinal canals in the infant, groins in the III-9, and no location detected using ultrasound in the proband. The risk of gonadal tumors in CAIS patient and the timing of
gonadectomy have long been debated. The risk of gonadal malignancy has been estimated to be
14% (0–22%).21 However, most CAIS patient prefer to postpone gonadectomy due to the risksof the procedure and the subsequent need for hormone replacement therapy. The adult CAIS
patient in the current study had not undergone gonadectomy, growing into individuals with acompletely female appearance but with no signs of malignancy. Our findings support Hannema’s
recommendation that gonadectomy be postponed until puberty occurs naturally.22 Therefore, theinfant with CAIS in this study should be advised to keep her gonads until she reaches puberty.
In conclusion, this is the second report of a missense mutation NM 000044.6(AR):c.2170C>Tin the world and the first study to document a pedigree consisting of several individuals withCAIS as a result of this mutation. The presence of a tiny uterus in the proband, which is arare occurrence in CAIS, is a unique clinical indicator of the disorder’s variable expressivity.

PATIENT CONSENT
Written informed consent was obtained from the patient’s parents for the publication of thiscase report.

ACKNOWLEDGMENT
This research was supported by scientific grant from University of Medicine and Pharmacy
Hospital, Hue University, Vietnam. We sincerely thank the patient and her family for their
willingness to participate in this research. We would like to thank Gene Solutions, Ho Chi Minh
City, Vietnam, for helping us to perform next-generation sequencing.

DISCLOSURE STATEMENT
The authors have no conflicts of interest to declare.

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