Six novel Mutation analysis of the androgen receptor gene in 17 Chinese patients with androgen insensitivity syndrome

Clinica Chimica Acta 506 (2020) 180–186

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Six novel Mutation analysis of the androgen receptor gene in 17 Chinese patients with androgen insensitivity syndrome

T

⁎

Xuanyu Jianga, Yanling Tengb, Xin Chena, Nana Lianga, Zhuo Lia, Desheng Lianga,b, , ⁎ Lingqian Wua,b, a b

Center for Medical Genetics & Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan 410078, China Hunan Jiahui Genetics Hospital, Changsha, Hunan 410078, China

A R T I C LE I N FO

A B S T R A C T

Keyword: Androgen receptor Androgen insensitivity syndrome Molecular genetics

Background: Androgen insensitivity syndrome (AIS) is the most common type of 46, XY disorders of sex development (DSD), with a wide range of clinical heterogeneity, from male infertility, hypospadias to completely normal female external genitalia. Mutation of the androgen receptor (AR) gene on the X chromosome (Xq11.2q12) is the main cause of AIS. Methods: By phenotype evaluation, hormone test, ultrasound scan and G-banding karyotype, 17 unrelated Chinese patients were clinical diagnosed with AIS. Sanger sequencing of the AR was performed in these 17 patients. Functional studies were carried out for the novel mutations. Results: We identified 16 mutations in all patients, including six novel mutations (Q59*, F171Sfs*4, E204*, G209E, I870T, *921R). It is the first time that a stop codon mutation (*921R) in AR has been identified. Expression and nuclear localization analysis showed the *921R mutation caused an elongated abnormal polypeptide chain of the AR protein, and the abnormal protein could not be transported to the nucleus to stimulate the expression of downstream genes after androgenic treatment. Expression analysis showed the protein level of G209E mutation was obviously decreased. Conclusion: Our study expands the spectrum of AR mutations and could provide evidence for the genetic and reproductive counseling of families with AIS. All of these findings broadened the mutation spectrum of AR, which were significantly valuable for patient gender assignment, genetic counseling and the clinical and psychological management.

1. Introduction Androgen insensitivity syndrome (AIS) [OMIM 300068] is an Xlinked recessive genetic disease that causes male infertility and male hermaphroditism. 30% of patients have no family history [1]. According to a 10-year study conducted by Boehmer et al, the incidence of AIS is probably between 1:40,800 and 1:99,000 [2]. The disease was first described by Morris in 1953 and was named testicular feminization syndrome [3]. The name change from testicular feminization syndrome to AIS is due to the lack of therapeutic effect of methyltestosterone treatment in patients, which shows that the characteristics of the syndrome is that the androgen receptor (AR) of 46, XY individuals is completely or partially resistant to androgen [4]. According to the AR sensitivity to androgen, AIS can be divided into three types. Patients with complete androgenic insensitivity (CAIS) exhibit

typical female external genitalia, slightly or well developed breasts, secondary sexual characteristics in puberty, but primary amenorrhea. Partial androgen insensitivity (PAIS) is characterized by small penis and hypospadias. Mild androgen insensitivity (MAIS) is generally a completely normal male phenotype, with adolescent breast development or infertility in adulthood [5,6]. AIS is mainly caused by mutation of the AR on chromosome Xq1112. It is a member of the nuclear receptor superfamily. Like other nuclear receptors, it is composed of four functional domains: a large Nterminal transcriptional activation domain (NTD), a DNA-binding domain (DBD), a hinge domain and a C-terminal ligand binding domain (LBD). The AR mutation of the gene leads to the abnormal structure and function of AR protein, causing insensitivity of the target tissues to androgens that are related to male sexual development [7,8]. To date, more than 600 AIS-related AR mutations have been

⁎ Corresponding authors at: Center for Medical Genetics & Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan 410078, China. E-mail addresses: [email protected] (D. Liang), [email protected] (L. Wu).

https://doi.org/10.1016/j.cca.2020.03.036 Received 24 December 2019; Accepted 25 March 2020 Available online 27 March 2020 0009-8981/ © 2020 Elsevier B.V. All rights reserved.

181

25 20 22 17 4 26 17 22 17 7

22 20

17

5 6 7 8 9 10 11 12 13 14

15 16

17

d

c

b

Female

Female Female

Female Female Female Female Male Female Female Female Female Female

Female Female Male Male

Social sex

Primary amenorrhea

Primary amenorrhea Primary amenorrhea Primary amenorrhea Primary amenorrhea Hypospadias Primary amenorrhea Primary amenorrhea Primary amenorrhea Inguinal hernia Absence of uterus and accessories Primary amenorrhea Abnormal external genitalia

Inguinal hernia Inguinal hernia Abnormal external genitalia Hypospadias

Reason for consultation

46,XY

46,XY 46,XY

46,XY 46,XY 46,XY 46,XY 46,XY 46,XY 46,XY 46,XY 46,XY 46,XY

46,XY 46,XY 46,XY 46,XY

Karyotypes

Absent Absent Absent Absent / Absent Absent Scant Absent / Absent Scant Absent

Ⅳ Ⅰ Ⅱ

/ / / Scant

Pubic-axillary hair

III nd Ⅱ Ⅳ / Ⅴ nd III nd /

/ / / Ⅰ

Tanner stage of breast

FSH: follicle‑stimulating hormone, reference range: 3.5–12.5 IU/L. LH: luteinizing hormone, reference range: 2.4–12.6 IU/L. E2: estradiol, reference range: 46.0–607 pmol/L. T: testosterone, reference range: 0.22–2.9 nmol/L; /: no data due to prepuberty; nd: no data.

12 2 1/12 19

1 2 3 4

a

Age (year)

Case

Table 1 Clinical phenotypes of subjects in this study.

None

None None

Rudimentary uterus None None None None None None None None None

None None None None

Vterus

Inguinal testis Unilateral inguinal testes and other side normal Abdominal testes

nd nd Bilateral testicles normal Unilateral inguinal testes and other side normal Abdominal testes Inguinal testes Inguinal testes Inguinal testis Bilateral testicles normal nd Abdominal testes Abdominal testes Inguinal testis Inguinal testis

Gonads

7.96

nd 13.69

nd 13.31 nd 4.83 1.03 6.67 12.38 70.28 14.6

10.97 nd nd nd

FSHa(IU/ L)

34.28

nd 37.1

nd 56.46 nd 48.98 0.19 11.89 45.54 40.58 33.96

2.38 nd nd nd

LHb(IU/L)

144.7

nd 35.55

nd 173.3 nd 14.76 nd 50.28 110.6 38.29 128.3

5 nd nd nd

E2c(pmol/ L)

14.67

nd 65.1

nd 30.48 nd 18.67 4.01 30.43 29.76 17.59 29.7

0.174 nd nd nd

Td(nmol/L)

X. Jiang, et al.

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Fig. 1. According to the inguinal ultrasound, the left and right inguinal area were found to have moderate echogenic nodules of 3.51 × 1.26 cm (a) and 312 × 1.43 cm (b), respectively, with regular morphology and clear boundaries, which were considered as testicles.

Fig. 2. Diagram of the AR cDNA with the locations of variations detected in patient. Reported variations (top) and unreported variations (bottom) are shown.

2.3. Plasmid construction and site-directed mutagenesis

recorded in HGMD (http://www.hgmd.cf.ac.uk/). In this study, 16 AR mutations, including 6 novel mutations, were detected in 17 unrelated Chinese AIS patients. We explored the consequences of all mutations through software prediction and functional studies.

We performed functional studies on unreported stop codon mutation (*921R) and missense mutation (G209E). The stop codon mutation is predicted to cause 95 amino acids extension of wild polypeptide chain by Mutationtaster (http://www.mutationtaster.org/). Therefore, we take normal human cDNA as a template and use primers AR-Ecor1F: 5′-CCGGAATTCCGGATGGAAGTGCAGTTAGGGCTG-3′, and ARXhor1-R: 5′-CCGCTCGAGCGGTTAAAGGCATACAACACCATTCAA AAC-3′ amplifies the human AR cDNA and predicted extended base length. The amplification products were linked to pCMV-N-HA by double enzyme digestion. Using wild type pCMV-N -HA-wt-AR plasmid as template, primers AR-2761-F: 5′-ACACCCAGcGAAGCATTGGAAAC CCTATTTCC-3′, AR-2761-R: 5′- AATGCTTCgCTGGGTGTGGAAATAGA TGGGCT-3′, AR-626-F: 5′- CAGCAGCGaGAGAGCGAGGGAGGCCTCGG GGG-3′, AR-626-R: 5′- TCGCTCTCtCGCTGCTGCTGCCTTCGGATACT and Mut Express II Fast Mutagenesis kit for site-directed mutagenesis (Vyzyme; Nanjing, China). All the above plasmids were identified by Sanger sequencing, and transiently transfected plasmids were extracted using Endo-Free Plasmid DNA Maxi Kit (OMEGA).

2. Materials and methods 2.1. Patients From 2005 to 2018, we enrolled 17 patients who were clinically diagnosed with AIS into this study. These patients showed varying degrees of abnormal external genitalia with high levels of testosterone. Patients were measured for breast growth examination, pubic and axillary hair examination, external genital differentiation examination, sex hormone level examination, and ultrasound examination. This study obtained the consent of the adult patient and the guardian of the minor patient, signed the informed consent form of the patient, and was also approved by the ethics committee of Hunan Jiahui Genetic Hospital.

2.4. Western blot

2.2. Sample extraction and Sanger sequencing

293T cell lines obtained from the Center for Medical Genetics & Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University (Changsha, China). 293T cell were inoculated in 6-well plates with 5 × 105 per well and cultured in 37 °C and 5%CO2 incubator. The cells were cultured overnight and transfected in strict accordance with the instructions of Lipofectamine 3000 (Invitrogen). Cells were scraped for western blot two days after transfection. HA-AR wild or mutant protein was diluted with mouse anti-HA antibody (Beyotime; Shanghai, China) for 1:1000, and internal reference was diluted with anti-actin antibody (Abcam) for 1:1500.

The peripheral blood of patients and their parents were collected into EDTA anticoagulation tubes by venipuncture, and genomic DNA was extracted by phenol-chloroform method. According to the AR (NM_000044) reference sequence provided by the UCSC database HumanGRCh37/hg19, primer design was performed using premier5. The designed primers were specifically analyzed by the premier blast on NCBI. All patients' AR coding regions and their flanking sequences were subjected to PCR amplification and sanger sequencing, and the available parental samples were verified. 182

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2.5. Immunofluorescence staining 293T cells were inoculated in 24-well plates with 5 × 104 cells per well and transfected 24 h later with 5 μmol/L DHT (dihydrotestosterone, Meilun) or vehicle (DMSO). After 24 h, it was fixed with 4% paraformaldehyde. The corresponding antibodies were incubated and fluorescence images were obtained by TCS SP5 laser confocal microscopy (Leica).

+: affected, ± : heterozygous for mutation; PolyPhen‑2: sensitivity, 0.00, specificity, 1.00; /: No data due to non-missense mutation; P: pathogenic, LP: likely pathogenic, VUS: uncertain significance.

P P P VUS P LP p P LP LP P P P P P P P This study This study This study This study Gottlieb [23] Zhou [14] Mowszowicz [13] Lee [25] Eggers [15] Jakubiczka [16], Matias [17] Yaegashi [26] Baldazzi [27] Batch JA [18], Heo YJ [19] Batch JA [18], Heo YJ [19] This study Batista [20] This study Disease_causing Disease_causing Disease_causing Polymorphism Disease_causing Disease_causing Disease_causing Disease_causing Disease_causing Disease_causing Disease_causing Disease_causing Disease_causing Disease_causing Disease_causing Disease_causing Disease_causing One sister(+) None None None One maternal aunt(+) Four maternal aunt(+) None One maternal aunt(+);four cousin(+) None One maternal sister( ± );one niece(+) One sister(+) None Two cousin(+) None One sister(+) None None 1/NTD 1/NTD 1/NTD 1/NTD 3/DBD 3/DBD 3/DBD 4/Hinge 5/LBD 5/LBD 5/LBD 5/LBD 6/LBD 6/LBD 8/LBD 8/LBD 8/LBD c.175_c.177insTAG c.510delT c.610G > T c.626G > A c.1822C > T c.1846C > T c.1847G > A c.2156G > A c.2191G > A c.2248A > G c.2257C > T c.2301delT c.2324G > A c.2324G > A c.2609T > C c.2667C > T c.2761T > C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Q59* F171Sfs*4 E204* G209E R608* R616C R616H W719* V731M M750V R753* D768Ifs*21 R775H R775H I870T S889S *921R

nd Mother De novo nd Mother nd nd Mother nd nd Mother nd Mother Mother nd nd De novo

0 0 0 0 0 0 0 0 0.00006 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0.00002 0 0 0 0 0 0 0 0

1 / 1 0.999 1 1 1 1 1 0.937 1 / 1 1 0.79 1 /

ACMG Mutation taster Family history Source Exon/functional domain AA change Nucleotide change Case

Table 2 Genetic variants identified in the affected subjects.

ExAC

1000 g

Polyhen-2

Reference

X. Jiang, et al.

3. Results 3.1. Clinical presentation A total of 17 unrelated patients were involved in this study. All patients had a karyotype of 46, XY. The patients came to hospital mainly because of an abnormal external genitalia after birth or primary amenorrhea in adulthood. Most patients present with typical female genitalia, cryptorchidism, hormone changes, and ultrasound abnormalities (Table 1). Except for patients 3, 4, 9 and 16 who are suspected of PAIS, all patients are intended to be diagnosed with CAIS. Patient 13 was typical CAIS, presenting as female external genitalia, but with primary amenorrhea, sex hormones suggesting higher than normal testosterone levels, and ultrasound suggesting bilateral inguinal substantial nodules, considering testicles (Fig. 1). 3.2. Molecular genetic analysis of the AR Sanger sequencing of the AR of 17 patients detected 16 variations (Fig. 2, Table 2). These different mutations include seven missense, four nonsense, two deletion, one insertion, one synonymous, and one stop codon variations. These variations are located in different domains of AR (Fig. 4). Among these 16 detected variations, Q59*, F171Sfs*4, E204*, G209E, I870T and *921R have not been described previously in the literature. Family pedigrees and Sanger sequence traces of six novel variations are shown in Figs. 2 and 3. All variations were not found or at extremely low frequency in EXAC (http://exac.broadinstitute.org) and 1000 Genome project (http://www.internationalgenome.org). Except for case 4, all variations were evaluated as pathogenic by Mutationtaster (http://www. mutationtaster.org/). Polyphen-2 (http://genetics.bwh.harvard.edu/ pph2/) predict that these variations are possibly damaging or probably damaging. These variations were interpreted to be pathogenic or likely pathogenic by American College of Medical Genetics (ACMG) standards and guidelines (table 2). 3.3. AR expression analysis We found a total of 6 AIS related AR novel variations, three of which would produce truncated proteins, resulting in the loss of key functional domains, and I870T was the change of different amino acids at the same site reported in previous studies. Therefore, we only conducted protein expression analysis on G209E and *921R. Through the Western Blot experiment, Compared with the wild-type AR protein, the molecular weight of HA-AR-*921R protein increased significantly, and the protein level of HA-AR-G209E was clearly lower than that of wild-type AR protein (Fig. 5). 3.4. Subcellular localization of AR in 293T cells Western blot showed *921R mutation express a larger protein, but the protein level has not changed significantly, so we conducted further experiments. Since AR protein functions by binding with androgen and being transported to the nucleus to stimulate the expression of target genes, we conducted immunofluorescence experiments. Immunofluorescence analysis showed that wild-type and mutant (*921R) AR were located in cytoplasm without DHT stimulation. After 183

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Fig. 3. Sanger sequence traces of the six novel AR gene variations are depicted.

Fig. 4. Partial pedigrees of the family 1, 2, 3, 4, 15 and 17. The arrows indicate the proband; black symbols indicate patients; white symbols indicate unaffected individuals; black spots indicate carriers.

DHT treatment, wild-type AR entered the nucleus, while mutant AR remained in the cytoplasm (Fig. 6).

HSP90, and localizes in the cytoplasm. When testosterone, especially dihydrotestosterone, enters the cell, the LBD domain of the AR binds to androgen, and then induces a change in the androgen conformation to form a dimer, which induces it to change from an inactive state to a target DNA active state. The formation of this dimerization mainly depends on the interaction of DBD functional domains of two AR proteins and the interaction of N/C of AR. The dimerization AR is further

4. Discussion In the absence of androgens, the AR mainly binds to molecular chaperones, such as members of the heat shock protein family HSP70, 184

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independent transactivation function 1 (AF1), which contains two important transcription activation units: Tau-1 (amino acids 100_370) and Tau-5 (amino acids 360_485) [5]. In this study, the novel mutation (G209E) in Tau-1 of the AR in case 4 was tested via expression analysis, resulted in a lower level of AR protein than that of the wild type, indicating the effect of the mutation on the AR protein. The DBD domain is encoded by exon 2 and exon 3, and contains two zinc finger structures. The first zinc finger structure contains a P-box sequence, which is involved in the recognition of DNA and binding to specific response elements. The second zinc finger structure includes a D-box sequence, which is involved in the formation of androgen receptor dimer and can stabilize the binding of androgen receptor and DNA [12]. In the present study, two patients (case 6 and case 7) with DBD mutations (R616C, R616H) in the same amino acid position detected in this region. Mowszowicz et al. study constructed a R616H mutant plasmid, co-transfected it with reporter chloramphenicol acetyltransferase gene, and treated it with DHT. It was found that it could not induce chloramphenicol acetyltransferase activity, indicating that the mutation made the androgen receptor nonfunctional [13]. R616C has also been discovered in Zhou et al studies [14]. LBD is a functional domain that binds to androgens. According to HGMD database records, about 40% of AIS-related AR mutations are missense mutations in the LBD region. A total of 4 missense mutations in LBD were detected in this study. V731M, M750V, and R775H were among patients with these mutations in different studies [15–19]. Among them, R775H was detected in two of our patients, Batch and Heo et al. reports suggest that the mutation may be a hotspot mutation. I870T was detected in case 15. Although there was no functional study of the mutation, there were three reports of substitution of isoleucine with methionine at this 870 position. The phenotypes of the patients caused by I870M in these three reports are PAIS phenotypes such as hypospadias. Our patient caused CAIS, which may be caused by the non-polar isoleucine changed to the polar threonine, which led to the disorder of the protein structure. The replacement of isoleucine with methionine in the previous report, because these are non-polar amino acids, does not have such a large impact on the protein, which only causes PAIS with mild symptoms. Due to the degeneracy of codons, a synonymous mutation is a

Fig. 5. Western blot analysis of AR protein product and β-actin (reference protein).

transferred to the nucleus by microtubule dependent transport. The AR in the nucleus combines with the specific androgen recognition element in DNA to collect regulatory factors and activate the expression of downstream genes [9,10]. In this study, we detected 16 different mutations from 17 unrelated AIS patients. These mutations are distributed in different positions of the AR. In total, we detected seven truncated mutations in different functional domains. Truncation mutations can lead to the complete or partial loss of different functional domains of AR protein, making AR protein incapable of functioning normally. Such mutations are considered intolerable. Except for case 3, all of the truncation mutations in this study resulted in a relatively severe CAIS. Interestingly, case 3 carries a c.610G > A heterozygous mutation, whereas the patient has a karyotype of 46, XY. This indicates that case 3 is a chimera, and there is still partially well functioning AR protein to ensure part of its physiological function. Therefore, the clinical symptoms of case 3 are relatively mild, showing only division of scrotum and short penis, belonging to partial androgenic insensitivity syndrome. Case3 is an infant, and gender distribution after diagnosis is particularly important. Considering that there may still be some normal AR proteins, the possibility of future virilization should be considered in gender assignment [11]. The NTD of AR protein is an important functional domain that guarantees the correct folding of AR, recruits molecular chaperones, and then regulates AR activity [5]. The NTD contains a ligand-

Fig. 6. Confocal image of 293T cells transfected with AR wild or mutant. Representative pictures of the variants pCMV-N-HA-AR- *921R (MU) in cultured cells treated with DHT or DMSO. Plasmids containing the pCMV-N-HA-AR (WT) served as a control. Bar = 10 μm. 185

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their participation and support in this study.

mutation that does not change the amino acid sequence. In mutation’s prediction tools, synonymous mutations are often considered neutral and are completely ignored in studies. However, accumulating experiments show that synonymous mutations affect gene function through mRNA splicing, protein folding and translation fidelity. At present, more than 1000 cases of harmful synonymous mutations related to human diseases have been found. Synonymous mutations in AIS are very rare, and there are only three such reports. We also detected a synonymous mutation (S889S) of AR in case 16. In a report from patients with the same mutation as ours, Batista et al. found that the mutation resulted in partial deletion of AR cDNA 8 and 3′ untranslated region in the patient's genital skin fibroblasts. In addition, two other transcripts, including a complete coding region and a shortened 3′ untranslated region, were found in the genital skin fibroblasts of patients and normal people. It is believed that a small part of the complete AR protein can be produced, which can explain part of the androgenesis of this patient. Our patients are consistent with the patients in the report. They all have the PAIS phenotype, mainly manifesting the small penis and incomplete testicular decline [20]. In general, stop codon mutation is the mutation of one stop codon in the DNA molecule into the codon encoding amino acid, which causes the synthesis of the polypeptide chain to continue until the next stop codon, forming an abnormal extra-long polypeptide chain. In addition, when there is no substitutive stop codon in the 3 'UTR region after the stop codon into another codon, the mRNA without stop codon will be recognized and degraded through a mechanism called nonstop mRNA decay [21,22]. We identified a stop code mutation (*921R) in case 17 that was not found in previous studies. That mutant AR protein predict containing a C-terminal 95-amino acid extension, consistent with the results of protein expression. Immunofluorescence analysis showed that the mutant protein could not enter the nucleus and could not perform normal functions after DHT treatment. The mutation disabled the activation of genes associated with male development, allowing the patient to exhibit cryptorchidism and a completely normal appearance of female genitalia. At present, there are 20 stop codon mutations of genes related to human diseases. However, it is not clear how the extended peptide affects the function of the protein [22]. Our study demonstrated that this stop codon mutation can produce a stable protein with no significant change in expression. However, this abnormal protein eventually affects the nuclear transport ability. It is helpful to further study the function of the extended peptides. In summary, our study identified the cause of these 17 patients with AIS. 16 AR mutations were detected in these patients, including 6 novel mutations, which expanded the range of AR mutations. Among of 6 novel mutations, G209E mutant protein showed the reduced level of AR protein. Another one is a stop codon mutation (*921R), which is the first time that a stop codon mutation is identified in AR. This mutation causes the AR protein to become abnormally large and unable to function enter the nucleus under the stimulation of androgens. Our study provides a further understanding of the AIS pathogenesis. All of these findings provided a basis for their genetic and clinical management counseling. Especially for CAIS patients, timely gonadectomy is very necessary to avoid malignancies. For other AIS patients, surgical correction and hormone replacement therapy are the key to ensure their virilization.

Funding This work was supported by grants from the National Key R&D Program of China (2017YFC1001802, 2018YFC1002201), the Science and Technology Major Project of Hunan Province (2019SK1014) and the National Natural Science Foundation of China (81771599). References [1] Kaprova-Pleskacova Jana, Stoop Hans, Brüggenwirth Hennie, et al., Complete androgen insensitivity syndrome: factors influencing gonadal histology including germ cell pathology, Mod. Pathol. 27 (2014) 721–730. [2] A.L. Boehmer, O. Brinkmann, H. Brüggenwirth, et al., Genotype versus phenotype in families with androgen insensitivity syndrome, J. Clin. Endocrinol. Metab. 86 (2001) 4151–4160. [3] J.M. Morris, The syndrome of testicular feminization in male pseudohermaphrodites, Am. J. Obstet. Gynecol. 65 (1953) 1192–1211. [4] J.D. Wilson, M.J. Harrod, J.L. Goldstein, et al., Familial incomplete male pseudohermaphroditism, type 1. 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Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements We thank all the healthy individuals and the family members for 186