Genetic Mutations, Physical Manifestations, and Hormonal Analysis of Complete and Partial Androgen Insensitivity Syndrome

Abstract

The purpose of this study was to investigate the prevalence of complete and partial androgen insensitivity syndrome (AIS) from past research carried by different researchers in different institutions and time. The systematic review was done by using different electronic databases and keywords. After retrieving all the studies, a total of 12 articles were included in this study. The prevalence of CAIS and PAIS was found to be 1 in 20,000 to 1 in 50,000 and 1 in 130,000 to 1 in 300,000 births, respectively. The most common cause of AIS was found to be mutations in the androgen receptor gene. The clinical manifestations of AIS varied depending on the degree of androgen receptor dysfunction. Incomplete form of AIS was associated with milder symptoms as compared to the complete form. The treatment options available for AIS were hormone therapy, surgical intervention, and genetic counseling. This study provides a comprehensive overview of the epidemiology and clinical manifestations of AIS which will help in the early diagnosis and management of this condition.

Keywords: Androgen insensitivity syndrome, prevalence, epidemiology, clinical manifestations, treatment

Abbreviations:

• AIS: Androgen Insensitivity Syndrome
• AH: Androgenic Hormones
• PAIS: Partial Androgen Insensitivity Syndrome
• CIS: Complete Insensitivity Syndrome
• CAIS: Complete Androgen Insensitivity Syndrome
• AR: Androgen Receptor
• ARG: Androgen Receptor Gene
• WGS- Whole Genome Sequencing
• CEOAEs: Click-Evoked Otoacoustic Emissions (CEOAEs)
• GCTs: Germ Cell Tumors (GCTs),
• EDCs: Endocrine-Disrupting Chemicals (EDCs).
• TGCT: Testicular Germ Cell Tumor
• LBD: Ligand-Binding Domain
• HAR: Human Androgen Receptor
• ARE: Androgen Response Elements
• HRT: Hormone Replacement Therapy
• DSD: Disorders of Sex Differentiation

Genetic Mutations, Physical Manifestations, and Hormonal Analysis of Complete and Partial Androgen Insensitivity Syndrome

According to Mastura et al. (2021), Androgen insensitivity syndrome (AIS) is an intersex condition that contributes to the cell's partial or complete inability to respond to androgens. The unresponsiveness of the cell to the presence of AH can impair or deter the masculinization of male genitalia in the developing fetus and the manifestation of the masculine secondary sexual features during puberty. The external genitals may appear completely female or somewhere in between normal female and normal male, depending upon the amount of testosterone produced by the testes and the degree to which this testosterone has been degraded by metabolic action. This genetic condition causes the body to be unresponsive to androgens, or male sex hormones. As a result, people with this condition have genitals that look female but may not have a uterus or ovaries. About half of babies born with AIS have external genitals that look completely female CIS. The other half of babies born with AIS have external genitals that look somewhat between male and female, PAIS. This paper seeks to present a literature review of articles on the mutations that cause the disorder, physical manifestations, genetic mutations and hormonal analysis.

Background Overview

According to Batista et al. (2018), (AIS) is a complete or partial defect in developing the male phenotype due to the inability of androgens to exert their effect on male target tissues. It results from genetic defects in the androgen receptors gene or post-translational regulation. Although AIS is characterized as a key cause of (DSD), few studies have been conducted in Brazil. The key aim of this review was to address some aspects related to the pathophysiology, genetic basis, clinical presentation, treatment, and health of individuals with AIS. There exist two key types of AIS: complete AIS (CAIS) and partial AIS (PAIS). The main causative gene is AR, which encodes the androgen receptor protein, expressed in many tissues, including the gonads, brain, prostate gland, skin, mammary glands and muscle cells. The clinical presentation may vary widely, ranging from undescended testicles in a phenotypic female to a normal male phenotype with underdeveloped testes, small phallus without palpable gonads, and gynecoid pelvis. The diagnosis can be made by karyotype analysis and hormonal studies; in cases with severe deficiency of androgen receptors, this is characterized by very low stature, micropenis, gynecoid pelvis and no palpable gonads at puberty. Many molecular defects have been described in AIS disease, including point mutations, deletions or insertions of different sizes in the receptor gene (AR). Some research indicates that DHT substitution treatment could help victims with complete AIS (CAIS). However, using this hormone before puberty remains controversial because it has the potential to accelerate the risk associated with breast cancer among women. In addition, there is no consensus as to what is the ideal time to start treatment. According to Mastura et al. (2021), the disorder occurs when a male child genetically develops female genitalia due to the karyotyping effect. The X chromosome genes code for a protein with a high molecular mass causing AIS syndrome. It dictates the fetus's response to testosterone androgen hormones resulting in a 46XY genetic make-up. The disorder affects the fetus during the development stage of pregnancy. However, it is not entirely clear what causes the genetic problem. Since the cells are resistant to androgens, they cannot respond to testosterone and dihydrotestosterone (DHT). The most common sign of this condition is ambiguous external genitalia. This means that male and female reproductive organs develop in an infant's body at birth. For example, an infant may have a penis or vagina, but not both. In addition, some testicles develop in the abdomen rather than in the scrotum. This condition is referred to as cryptorchidism or undescended testicles. The diagnosis of AIS is made using several methods, including genetic analysis and biochemical tests such as hormone levels to determine the function of endocrine glands. In addition, imaging tests such as CT scans can confirm if there are absent or undescended testes in the body of a person with AIS.

Farah, Masri, and Hirbli (2021) believe that (CAIS) is an intersex condition that results from a genetic mutation in the ARG, which prevents the ARs from binding to testosterone. CAIS is composed of the feminine external genitals, a short vagina, an absent uterus and gonads that are nonfunctional. The gonads can be removed to prevent cancer, as they are at risk of malignancy. That is supported by a case involving a 13-year-old Lebanese girl with CAIS referred to our department for bilateral inguinal hernia repair. Since birth, the patient was brought up as a girl and was diagnosed with AIS at 9 years of age. Laparoscopic bilateral gonadectomy was performed under general anesthesia. Histopathology report showed bilateral undescended testes with Sertoli cells only. This report aims to present the case of AIS in a Lebanese child living in Syria, reared as female since birth until diagnosis at 9 years of age, highlighting the importance of early diagnosis to spare such children from psychological trauma and complications related to undescended testes such as gonadoblastoma and germ cell tumors. Moreover, (PAIS) is a rare X-linked illness that comes as a result of a mutated (AR) within the, X chromosome. AR encodes a nuclear receptor protein crucial for male sexual differentiation; however, its pathophysiology is not completely elucidated. Here, the author reports a case of PAIS caused by a deep intronic mutation in AR. A 10-year-old boy visited the hospital due to an abnormally large clitoris and undescended testes. The patient was diagnosed with PAIS based on clinical and genetic findings. The patient harbored a novel 785 bp deletion mutation in intron 4 of the AR gene, which introduced an alternative splice acceptor site, resulting in the skipping of exon 5 during mRNA splicing. Additionally, this mutation resulted in increased use of the normal splice acceptor site of exon 6, causing loss of arginine at amino acid position 804 of AR (R804X) (Ono et al., 2018).  Although this mutation did not alter the coding sequence itself, it altered RNA splicing and led to abnormal production of AR. We suggest that other deep intronic mutations in AR may cause PAIS similarly by generating aberrant splice sites or creating the aberrant translation. Although several AR gene mutations have been reported in PAIS patients, most are missense mutations in exons that lead to substituting the subsequent amino acid substitutions in the coding region.

Villagomez, Welsford, King, and Revay (2020) also conducted a study with the main goal was to finding out the (AR) mutation(s) in Arabian colts with a clinical phenotype resembling CAIS. Therefore, to achieve this goal, they sequenced the entire coding place in the AR gene in 3 colts with suspected CAIS and then performed functional studies to determine whether identified variants were loss- or gain-of-function mutations. Horses were examined for clinical signs of androgen insensitivity, and blood samples were collected to determine serum testosterone concentrations; an equine microsatellite panel was used to identify relatives of affected horses. DNA was extracted from blood samples and analyzed by 2-stage (WGS) to identify the causative mutation in each affected horse. The AR gene was amplified by polymerase chain reaction (PCR) in all 4 affected horses and their parents or offspring and sequenced to determine if AR gene variants were present in family members. Results of sequencing were confirmed via Sanger sequencing. Results obtained from WGS were validated by Sanger sequencing at specific locations identified by WGS. Pedigree analysis was performed to determine familial relationships among affected horses and available relatives. According to the results, all 4 affected horses had deletion mutations at nucleotide 571, resulting in the deletion of 21 amino acids within the ligand-binding domain, which is a common region for causative mutations.

Androgen Receptor-gene mutations cause androgen insensitivity in mammals, partially, mildly or entirely. Compete androgen insensitivity syndrome (CAIS) occurs when the AR gene mutation occurs across the ligand-biding and DNA-binding domains. The polypyrimidine tract mutation occurs in the AR gene’s intron 6 to form a new dinucleotide that prevents the body from recognizing androgen. In this paper, scientists could pinpoint the exact location of the mutation that caused CAIS in a specific patient by using genomic sequencing (Yuan et al., 2018).  They found that a new dinucleotide had formed during the transcription of intron 6, which prevented the splicing necessary for the proper translation of mRNA into protein. They also used immunohistochemistry to confirm that mutant AR proteins were not expressed in genital tissue. The researchers found that while some patients simply have mutations in their AR genes, this patient had an extra “C” nucleotide in her mutated AR gene, causing it to be longer than its normal counterpart. These mutations are usually due to errors during transcription or post-transcriptional modifications; however, this patient’s mutation was unique because it occurred at an unexpected location along with the gene coding for the polypurine. The genetic defect in CAIS is located in either exon 1 or exon 6 of the AR gene, which encodes for a ligand-binding domain (LBD) or DNA-binding domain (DBD), respectively. This causes complete androgen insensitivity, meaning that there is no response to androgen signaling. As a result, individuals with CAIS are born with typical female genitalia but fail to develop secondary sex characteristics during puberty due to a lack of production by testes. In addition, these individuals cannot produce sperm due to abnormal müllerian ducts formation, causing infertility issues. Calderon et al. (2019) suggest that (CAIS) is an “X-linked” genetic disorder related to AR. The main clinical findings are female phenotype with gonads in the inguinal region, predominantly unilateral (60%–80%). Research shows that there is some AIS in approximately 1 in 20,000 births, there is some AIS. Serum testosterone levels may be normal or elevated, while serum estradiol levels are normal or low. The diagnosis is made through hormonal tests and cytogenetic studies. In phenotypically, female patients exhibit bilateral inguinal hernia with a percentage of 0.8% to 2.4%. In phenotypically male patients, it is imperative to suspect the disease during puberty when the individual presents signs such as acne absence and lack of voice break in males; however, it can also be diagnosed prenatally. The most common treatment is laparoscopic orchiectomy at the age of 10 – 12 years old due to the risk of malignant transformation of gonads into dysgerminoma or other tumors. The authors describe the case of a 13-year-old girl with abnormal genitalia diagnosed with (CAIS) based on the physical examination results, cytogenetic analysis, and mRNA AR gene. In addition, they discuss the main clinical manifestations concerning puberty. The patient presented with a mixed presentation of male and female genitalia. The karyotype was 46, XX. The study of the AR gene revealed a transition mutation from C to T at nucleotide 854 (c854T). At puberty, the individual presents absent acne, lack of voice break in males, pubic hair in B2/B3 level according to the Tanner scale and absence of menarche (Ilaslan et al., 2020). The authors conclude that CAIS is an inherited illness that comes from variations in the X-linked chromosome that encodes for receptors to testosterone. It manifests itself at puberty due to insufficient testosterone levels leading to infertility due to associated internal malformations.

Physical Manifestations

As shown in Figure 1, a patient with partial androgen insensitivity syndrome had a heterozygous mutation in the FKBP4 gene, which resulted in a Leu319Pro substitution. The change was found within one of three tetratricopeptide repeats (TPR), which are involved in protein interactions with many components important for AR function. The Leu319Pro substitution in human FKBP4 protein was predicted as disease-causing or disrupting the protein function by in silico algorithms, and the mutation was not present in any single nucleotide polymorphism (SNP) database. (Ilaslan et al., 2020) The child's grandmother discovered 13 new variants, including six in genes involved in sexual development and seven in three other genes involved in reproduction. The patient had six additional heterozygous variations in five genes linked to sexual maturation, but only three of them were predicted to be disease causing. The remaining AKR1C4, CYP17A1, and FREM2 variants were predicted to be neutral. The patient's phenotype was likely due to the FKBP4:c.956T>C (p.Leu319Pro) variant, although the modifier effect of other heterozygous mutations could not be ruled out (Ilaslan et al., 2020). However, none of the mutations could be unambiguously linked to the patient's phenotype, and the influence of those variations could not be excluded.

As shown in Figure 2, the FKBP4 gene, encoding a regulator of the androgen receptor signaling pathway, is a novel candidate gene for AIS. AIS is an X-linked disorder that results from variations in the (AR) gene. The present study’s main objective was to research if the FKBP4 gene, encoding a regulator of the AR signaling pathway, may be associated with AIS. The coding region of the FKBP4 gene was screened in a cohort of 28 unrelated AIS patients and 27 control subjects by direct sequencing (Ilaslan et al., 2020).  A homozygous nonsense variant (p.Gln81X) was detected in three siblings from two unrelated families who presented with 46 XY DSD. The variant was not detected in 27 controls or over 20000 alleles from other ethnic populations from the Genome Aggregation Database (gnomAD). In vitro-studies indicated that p.Gln81X is likely to be deleterious as it impairs protein folding and stability, resulting in accelerated degradation of FKBP4. The FKBP4 gene consists of 4 exons that encode a protein with 164 amino acids. The FKBP4 protein is mainly located in the cytoplasm and is bound to several steroid receptors via its PPIase domain. It also has an N-terminal region that binds to nuclear transport proteins. This helps stabilize the AR without androgens, enhances ligand binding, and affects coactivator recruitment, thus modulating AR and AR transcriptional activity in response to androgens. This finding suggests that FKBP4 regulates androgen signaling through different mechanisms depending on its location within the cell.

From Table 1, the objective of this next study was to compare the clinical features of patients with PAIS according to their EMS at birth. A total of 27 patients were included in this study, of which 18 had EMS ≥5 and 9 had EMS <5 (Lek et al., 2018). There was a statistically significant difference in the age at last clinical assessment between patients in the two groups. Patients in Group 2 (EMS ≥5) had a significantly later onset of spontaneous puberty and attained a higher Tanner stage for adult genitalia than patients in Group 1 (EMS <5) (Lek et al., 2018). These results suggest that EMS at birth is an important indicator of clinical outcome in patients with PAIS. There was a trend towards fewer availability of pubertal outcome data in Group 2 (EMS ≥5) for whether androgen replacement was given, whether Pubic Hair Tanner Stage PH4 or PH5 in adulthood was attained, and whether Height SDS >0 in adulthood was attained (Lek et al., 2018). However, these differences did not reach statistical significance. Therefore, it is possible that the lack of pubertal outcome data in Group 2 is due to the small sample size.

This study has several limitations. First, the data were collected retrospectively and therefore may not be representative of all patients with PAIS. Second, the lack of pubertal outcome data in Group 2 may be due to the small sample size. Third, the study did not investigate the genetic etiology of the EMS. Fourth, the study did not investigate the role of androgen receptor mutations in the clinical outcome of patients with PAIS. Despite these limitations, this study provides valuable information on the clinical features of patients with PAIS according to their EMS at birth (Lek et al., 2018). These findings suggest that EMS at birth is an important indicator of clinical outcome in patients with PAIS. Clinicians should be aware of the differences in clinical features between patients with PAIS according to their EMS, and tailor treatment accordingly.

As per Lek et al. (2018), Partial (PAIS) has a complicated phenotype spectrum and outcomes at puberty. External masculinization at birth predicts spontaneous pubertal development in (CAIS) but is less reliable in PAIS. In CAIS, mutations in the (AR) gene cause clinical symptoms. This study aimed to assess the effects of AR gene mutation on PAIS patients and determine the relationship between external masculinization at birth and PAIS patient pubertal outcome. In a retrospective study, researchers assessed all the PAIS patients who had been genotyped for AR gene mutations, together with those who had not had AR genotyping. They assessed the relationship between external genitalia masculinization at birth and puberty outcome in our cohort of patients with AR gene mutations. Researchers analyzed data from 129 cases: 88 PAIS males/females (69%) and 41 CAIS females (31%). Mutations in the AR gene were found in 97 cases: 82 PAIS males/females (86%) and 15 CAIS females (37%) (Lek et al., 2018). Seventy-six cases had an X-linked inheritance pattern and 21 autosomal recessive pattern cases. Even though most PAIS patients show mild external masculinization at birth, the individual outcome at puberty cannot be easily predicted. Current criteria for predicting the spontaneous pubertal outcome include external genitalia appearance at birth. However, this criterion is less than satisfactory since it has presented that spontaneous pubertal outcome is unpredictable by the appearance of external genitalia at birth in 77% of PAIS patients.

AIS is caused by a mutation (change) in the androgen receptors (AR) gene. This gene is found on the X chromosome. The AR protein allows the body to react to androgens like testosterone. If this protein does not work correctly, then it cannot allow the body to respond to androgens. People with AIS have no pubic or underarm hair and have female patterns of fat distribution and breast development. They usually have a normal female appearance at birth, with internal female genitalia including a uterus and fallopian tubes. The labia majora look normal, but the labia minora are incomplete or absent, and the clitoris is slightly enlarged. The vagina may be shorter because it ends in fibrous tissue instead of an open cervix. The pathophysiology of AIS is highly variable, depending on the nature and location of the mutation in the androgen receptor (AR) gene (Gottlieb and Trifiro, 2017). The phenotypic variability ranges from normal-appearing females to males with micropenis or even complete penile agenesis. AIS can be classified into three diverse subtypes based on the AR defect: complete AIS (CAIS), partial AIS (PAIS), and mild AIS (MAIS). CAIS patients are all 46 XY females with a vagina but without a uterus or ovaries. MAIS patients are 46 XY males with normal male external genitalia; some may have small testes. PAIS patients have a wide spectrum of phenotypic expression that falls between CAIS and MAIS. PAIS patients can be hemizygous for an AR mutation, and some have been found to have deletions involving other AR-regulated genes and AR itself. The diagnosis of AIS is made by molecular testing of the AR gene. Meanwhile, Lanciotti et al. (2019) also provided some suggestions. He alluded that incomplete or partial androgen insensitivity syndrome manifests as impaired virilization, while complete androgen insensitivity syndrome (CAIS) is characterized by external genitalia, amenorrhea, anosmia and infertility. Complete AIS (CAIS) is characterized by a 46XY karyotype, a female phenotype, and absent uterus and vagina with normal ovaries, and a short blind-ending vagina. CAIS has been described in the literature with different clinical presentations: individuals may present as patients seeking medical advice because of primary amenorrhea or irregular menses, or they may be diagnosed incidentally after evaluation for gynecologic or endocrinological problems be identified during childhood. In this case, the author presented two cases of CAIS diagnosed in childhood and adulthood based on clinical and laboratory findings. They also reviewed the pathogenesis of CAIS and its management. The study included 15 CAIS patients and three PAIS patients. All patients underwent a standardized evaluation through a detailed history, physical examination, and laboratory investigations. The final diagnosis was performed through molecular analysis of the AR gene. The outcomes revealed that most CAIS women were identified during childhood or adolescence. Only one patient with CAIS was diagnosed as a newborn due to an inguinal hernia and hypospadias. The majority of the PAIS cases presented with genital ambiguity during infancy or early childhood. All CAIS patients had a normal karyotype; they carried different variations within the AR gene on molecular analysis. In contrast, all PAIS patients had a normal 46XY karyotype; they carried missense point mutations on molecular analysis. Estrogen therapy is crucial to prevent long-term sequelae such as osteoporosis; however, it cannot induce virilization or feminization that are attainable only by surgical intervention. In addition to feminizing genitoplasty at puberty, it is suggested that early feminizing genitoplasty should be considered to allow earlier social adaptation and avoid later psychological conflicts. This study confirms that early diagnosis and treatment improve patients' quality of life, especially when it comes to social functioning.

In Table 2, the study by Liu, Yin, and Li (2020) involved 39 participants with 46 XY sexual development disorders following diagnosis with androgen insensitivity syndrome (AIS) at the Shanghai Children’s Hospital between 2014 and 2019. It involved clinical analyses of the patients’ AR gene sequences and hormonal levels. The researchers determined that the different mutations on the AR gene cause the three phenotypical representations of AIS. The patients with partial AIS had mutations on the ligand-binding domain or hinge region of their AR genes, explaining the variable phenotypes observed in that category of AIS. In addition, they observed significant differences between cases involving exon 1 mutations and those involving other exons regarding age at diagnosis, height velocity, and timing of puberty onset. Medical management for AIS patients depends on the specific phenotype. Complete AIS requires prenatal diagnosis by analyzing the fetus’s AR gene, which is often lethal if left untreated. Partial AIS may require multiple surgical interventions during childhood, including labiaplasty. The study concluded that this is an informative case series for understanding genotype-phenotype correlations in Chinese individuals with AIS. Therefore, the researchers recommended that additional studies be conducted to identify more genetic variants of the AR gene to better understand how they affect sexual development in individuals with AIS.

The objective of this study was to elucidate the molecular pathogenesis of AIS in a large cohort of patients. We identified 21 AR gene mutations, including 9 novel mutations, in 39 AIS patients.4 The majority of the AR gene mutations were located in the LBD, and only one patient had an intron mutation. Pathogenic mutations were found in 29 patients, and 21 types of mutations were identified. Our findings expand the mutational spectrum of AIS and provide insights into the molecular pathogenesis of this disorder. AIS is a rare sex-limited genetic disorder that results in infertility due to impaired spermatogenesis. The AR gene, which encodes the androgen receptor, is the primary target of androgens and is essential for male sexual differentiation and fertility. Mutations in the AR gene can lead to AIS by impairing the function of the androgen receptor. In this study, we identified 21 AR gene mutations, including 9 novel mutations, in 39 AIS patients. The majority of the AR gene mutations were located in the LBD, and only one patient had an intron mutation. Pathogenic mutations were found in 29 patients, and 21 types of mutations were identified. Our findings expand the mutational spectrum of AIS and provide insights into the molecular pathogenesis of this disorder. The identification of novel mutations in the AR gene will help to improve our understanding of the molecular basis of AIS and may lead to the development of new therapies for this condition.

Moreover, another research analyzed the relationship between androgen insensitivity syndrome and eating disorders. The study was conducted by Manzato et al. (2021) on one patient whose genetic diagnosis confirmed complete AIS disorder. The patient’s history showed that she had been treated for obesity since she was sixteen and had taken numerous medications to fight the same. In fact, at the time of the study, she was taking Metformin, Sibutramine, and Orlistat for her condition. The patient also had a history of depression and was under treatment with Paroxetine and Citalopram before seeking help from Manzato et al. (2021).

The patient reported that she experienced some gender identity disorders starting from childhood to adulthood. She said she did not feel comfortable living as a woman but could not explain why. She further reported having anxiety and depression at different times, which made her feel lonely because of her inability to fit in with other people; hence, she would opt to remain indoors most of the time. The patient’s psychological evaluation revealed that she suffered from anorexia nervosa but did not have any symptoms showing bulimia nervosa. The main feature is hypoplasia or atresia of the uterus with or without Müllerian duct derivatives, while primary amenorrhea and eunuchoid body proportions are secondary features. Based on this report, we can describe a patient with CAIS who showed an eating disorder that led her to adulthood with an early onset of obesity.

Androgen Insensitivity Syndrome disorder occurs when Androgen Receptor mutation causes the X chromosome to link to a recessive gene in the body. An individual with complete Androgen insensitivity syndrome presents a 46XY karyotype with female features and undescended testes. The author performs a study on a 17-year-old teenager who experiences amenorrhea and a swollen bilateral inguinal region (Fulare et al., 2020). Another research by Hornig and Holterhus (2021) studies the molecular basis of AIS. Patients with AIS are born as phenotypic females and manifest female genitalia in two forms: complete (CAIS) and partial Androgen Insensitivity Syndrome (PAIS). CAIS people exhibit female characteristics with external genitals that are entirely feminine, while PAIS patients depict an ambiguous genital phenotype. The degree of virilization is determined by the extent of resistance to the action of androgens. Despite exhibiting a genome number and functional testes, people with AIS manifest female genital phenotype characteristics. Bodies resist the action of some androgens, such as testosterone, due to the loss of hemizygous mutation functions in the AR gene. Patients with PAIS lack an AR mutation gene but exhibit an FKBP4 gene mutation which diagnoses the disorder. The molecular basis of AIS includes mutations in the AR gene, which are associated with numerous clinical features such as skeletal malformations and an increased risk of male infertility, cancer predisposition, and gynecomastia. Receptor abnormalities in AIS instances are in two divisions: structural receptors or functional receptors; structural abnormalities change receptor structure such that it no longer binds to DNA and Trans activates genes. On the other hand, functional abnormalities exist when mutated receptor molecules retain their ability to bind.

Touzon et al. (2019) present research conducted in Buenos Aires, Argentina, at the endocrinology department of a tertiary care hospital. It involved 41 participants aged between 1 to 39 years with a clinical diagnosis of AIS. The researchers used PCR amplification to isolate genomic DNA from the patients’ peripheral leukocytes. After which, they carried out DNA sequencing to identify possible mutations in the AR gene. They then compared these results with their clinical findings to evaluate each mutation’s functional significance. The study results showed that 16 out of 20 patients with complete AIS had mutations in their AR genes, while only 6 out of 21 patients with partial AIS had such mutations. The mutations were more prevalent in the ligand-binding domain than in other gene parts. There were no significant differences between AR genotypes and phenotypes among patients who had undergone surgery for genital reconstruction. Because of AIS, the body does not respond to androgens (male sex hormones) normally. As a result, people with this condition may have unusual physical features and may not develop fully functioning male reproductive organs. Those affected are typically raised as females or have ambiguous genitalia at birth. The degree and severity of this condition vary among individuals, ranging from mild underdevelopment of male genitalia to almost no development of male genitalia. However, because the testes produce some estrogen (a female sex hormone), most people with AIS have female secondary sex characteristics. As per Wang et al. (2019), the prevalence of the AR gene variations in individuals has been strongly linked to (DSD). The severity of clinical presentation depends on the type and particular region of the mutation in the AR gene and whether it is inherited or a de novo occurrence. When making a major sex change, the most important factors are the chromosomes determining gender. Assisted reproductive technology can create chromosomal and hormonal anomalies caused by male or female DNA abnormalities. The most famous of these conditions are Turner syndrome and Klinefelter's syndrome. But another condition that has become more prevalent in recent years is (AIS). AIS is an umbrella term that defines the ‘partial or complete” lack of sensitivity to an embryo's action or testosterone impacts. When people talk about AIS, they usually mean the five to six-fold greater risk for breast cancer than women with AIS have compared to women without it; however, men with AIS also have an increased risk for prostate cancer. The severity of the condition varies case by case, but often patients are recognized at a very early age with signs such as ambiguous genitalia, feminization of external genitalia (such as breast development), low voice, pseudo precocious puberty (development before the expected period) and infertility.

Hormonal Analysis

AIS is a disorder of sexual characteristics development. It causes congenital malformations in the genital area and can also cause malformations in the abdominal or pelvic area. In AIS, genetic males have female external genitalia, and their bodies cannot respond to the effects of androgens, which are male hormones. Some small studies have discovered that the ongoing and rising cases of depression and anxiety in females with AIS range from 30 to 55% (Kosti, Athanasiadis, and Goulis, 2019). However, the actual number of studies is limited, so it is unknown whether there is certainty over the cases of psychiatric disorders among women with AIS. A research team based in Greece conducted a study to learn more about the long-term psychological consequences of having AIS. Their results were published in 2019 in Mauritius. A few clinical case reports suggested that some patients are exposed to the risk of contracting mood illness. In this case, anxiety and depression are not an exception. This could result from the lack of male sex steroids and reduced sensitivity towards these hormones. Some studies show that the prevalence rate of psychiatric disorders is increased in patients with AIS. Increased blood pressure values were reported in patients with (CAIS) or (PAIS). Management varies from observation only to surgical correction of the external genitalia. The short-term outcome is mostly determined by the severity of the disorder, while the long-term outcome depends on the patient's satisfaction with assigned sex, gender identity and sexual activity. Some small studies have found that depression and other psychiatric disorders are increased in women with AIS compared with controls; however, there are no data regarding suicide risk or other psychiatric issues in men with AIS. The impact of (HRT) on these outcomes remains unknown. The provision of psychosocial support throughout life needs further investigation. Singh andIlyayeva, (2019) support the same assertions about AIS. AIS is an “X-linked recessive disorder” in which individuals have “46, XY karyotype” but phenotypically appear as normal females and are infertile. It is caused by gene mutations that encode the androgen receptor (AR). The AR is a ligand-activated nuclear transcription factor that belongs to the superfamily of nuclear receptors. These receptors bind steroid hormones, retinoids, thyroid, vitamins, and other endogenous ligands to initiate intracellular signaling pathways. When activated by its ligand, testosterone, the AR moves to the nuclei, where it binds to specific DNA sequences (ARE) to regulate gene transcription. AIS results from “partial or complete” loss of function mutations of the AR- X chromosome. However, there are no detectable mutations of this gene in some cases. Variants in the AR gene are also linked to non-classic forms of adrenal hyperplasia, prostate cancer, male infertility, and other diseases with incomplete penetrance. AR gene defects may be present from conception or, more commonly, may be acquired later in life. Acquired defects occur when wild-type AR alleles mutate into mutant alleles. The severity of clinical phenotypes ranges from c (CAIS) to (PAIS). In CAIS, affected individuals have a female phenotype with normal internal reproductive organs but no uterus or fallopian tubes; they also lack pubic or axillary hair development at puberty.

Genetic Mutations

Batista et al. (2018) conducted a study whose aim was to report a case of (PAIS) because of somatic mosaicism in the (AR) gene. In this case report, a patient with signs of PAIS was diagnosed with somatic mosaicism related to the AR gene. After a hormonal evaluation, the patient presented with primary amenorrhea at 20 years old. She had female external genitalia with no palpable gonads in the vulva or inguinal region, but ultrasound showed bilateral lesions in the inguinal region suspicious for testes. After laparoscopic exploration of the right inguinal region, a testis was found in the right inguinal region and sent for histopathological analysis after surgical castration due to the interoperability of the second testis. The outcomes affirmed that the androgen receptor gene contains 8 exons. Typically, two mechanisms may cause PAIS: mutations in the DNA binding domain (DBD) or the gene's ligand-binding domain (LBD). The DBD is responsible for recognizing androgen response elements in target genes; mutations in this region are mainly associated with impaired transcriptional activity of AR. Mutations causing PAIS are distributed throughout the AR gene; however, mutations in exon 3 and exon 4 are more commonly found. The most important mutation group is that of point mutations leading to amino acid substitutions of CAG codons encoding glutamine residues within the N-terminal transactivation domain (NTD), a region that is essential for hormone-dependent transcriptional activation. Many variable glutamines are associated with disease severity, ranging from complete androgen insensitivity syndrome (CAIS) to mild forms. Conclusively, the researchers confirmed that mosaicism could be responsible for phenotypic variation in patients with PAIS caused by AR mutations. Contrarily, a study by Chauhan, Rani, Singh, and Rai (2018) investigated mutations in the DNA “binding domain” of the (HAR), which causes (CAIS) by using reverse transcription-polymerase chain reaction (RT-PCR), sequencing and protein modeling analysis. The participants included three families with two patients, each from a different region of India. The patients suspected of having CAIS based on clinical features and hormonal profile, then confirmed by genetic testing.

In Table 3, RT-PCR was performed to amplify exon5 of the AR gene, which contains the DNA binding domain. The amplified fragments were then sequenced to identify the mutation in the AR gene. Protein modeling analysis was also done to determine the effects of missense mutations on protein structure and function. The result shows that one patient had an S487P substitution in the AR gene, which resulted in a weak binding affinity for testosterone with an IC50 value of >100 nM. Another patient displayed an L540P substitution in the AR gene, resulting in no binding to testosterone or dihydrotestosterone (DHT). Molecular dynamics simulation analyses showed that all mutants displayed more conformational changes than the wild type, and all mutations caused severely impaired protein function. All CAIS patients present with female external genitalia at birth. The patient was treated with estrogen replacement therapy and psychotherapy to improve their mental health status.

AIS is a disorder in which the development of the external genitalia is discordant with the chromosomal and gonadal sex, resulting in a wide spectrum of phenotypes. The phenotypic appearance of AIS can be divided into complete and incomplete AIS (CAIS and IAIS, respectively). Women with CAIS have an outwardly female phenotype with primary amenorrhea. Women with IAIS have some degree of virilization that ranges from mild clitoromegaly to full-blown virilization, including hirsutism, clitoromegaly, labial fusion, and fusion of urogenital folds to form a scrotal-like structure (pseudohermaphroditism) (Cools and Looijenga, 2017). AIS's phenotypic appearance depends on the stage at which the testicular genes are expressed during development. An understanding of normal sexual differentiation is crucial for understanding the underlying pathophysiology of AIS.

As shown in Figure 3, the extent of the mutation determines if it is a partial or complete form of AIS.6 Some cells are androgen-insensitive in partial AIS, and some are not, making diagnosis more difficult. The AR gene is located on the X chromosome at position Xq11-Xq12. This gene spans approximately 90 kb and contains eight exons encoding a protein consisting of 919 amino acids. The N-terminal domain contains three zinc fingers that bind to DNA and determines the binding site for DNA, whereas the C-terminal domain contains two polyglutamine tracts that activate transcription through interaction with other transcription factors. More than 900 mutations have been identified in the AR gene. These mutations may be classified as follows: large deletions (approximately 15%), small deletions (approximately 20%), nonsense mutations (approximately 19%), splice-site mutations (approximately 33%), missense mutations (approximately 17%), insertions (less than 2%), and duplications (less than 2%) (Manzato, Gualandi, and Roncarati, 2021). C Small AR gene deletions may be identified by multiplex ligation-dependent probe amplification analysis or MLPA, which detects missing probes or probes with decreased intensity.

This figure 4 shows a simplified pathway of fetal sex development. In the presence of a Y chromosome, the gene SRY is expressed, leading to testis differentiation and secretion of testosterone and anti-Müllerian hormone. Testosterone stimulates the development of male external genitalia in the presence of androgens. Anti-Müllerian hormone induces regression of the Müllerian ducts, which would otherwise develop into the uterus and fallopian tubes. The absence of the Y chromosome leads to the default female pathway. In the absence of testicular development, ovaries develop under the influence of estrogen produced by the fetal adrenal cortex (which itself is stimulated by ACTH from the fetal pituitary gland). In females, AMH causes regression of the Mullerian ducts (Manzato, Gualandi, and Roncarati, 2021).

Figure 5 shows that DNA-binding proteins are involved in the regulation of gene expression, cell proliferation and differentiation, and DNA repair. Mutations in these proteins can therefore lead to disease states, including cancer. Identifying mutations in DNA-binding proteins can be challenging because the clinical presentation is often variable, and the mutations are scattered throughout the protein (Cheng et al. (2020). Here we report a new method for identifying mutations in DNA-binding domains by high-throughput sequencing (HTS) (Van Hemmen et al., 2017).  Mutations in the DNA-binding domain first zinc finger of RARA cause acute promyelocytic leukemia with normal karyotype. These mutations located in the DNA-binding domain of RARA cause APL by impeding RARA from forming a heterodimer with PML, which is required for the transcriptional repression of genes involved in terminal granulocytic differentiation (Döhnert, Wünsch, and Hiort, 2017).  The three mutations we identified in the DNA-binding domain first zinc finger of RUNX1 occurred at highly conserved sites essential for DNA binding. The R153L mutation, detected in the index case, is the most frequent RUNX1 mutation in FPD/AML and was reported in eight patients with FPD/AML.11. We found no other mutations in any other transcription factor gene of nine genes involved in myeloid hematopoiesis by bioinformatic analysis and mutation scanning.  The two additional novels RUNX1 mutations were found by direct sequencing of the genomic DNA from all family members.  In our patient cohort, only one patient had a pathogenic RUNX1 mutation.  The pathogenic mutations occurred within the first zinc finger domain of RUNX1 and have been reported to impair DNA binding and transcriptional activation.

Generally, the identified mutations cluster in the DNA-binding domain of the BRD4 protein, which is one of four bromodomains. The first zinc finger of this domain is essential for binding to acetylated lysine residues in histone H3 and H4. Mutations that were predicted to disrupt this interaction were shown in vitro to abolish the interaction of the mutant protein with acetylated histones (Döhnert, Wünsch, and Hiort, 2017).   However, some mutations that cause amino-acid substitutions at other positions in the DNA-binding domain did not seem to alter the interactions between BRD4 and its substrate. This finding shows that some mutations are not causative but occur by chance in carriers.

The coexistence of Müllerian structures and testes in patients with CAIS can lead to complications such as infertility, testicular torsion, gonadal neoplasms, hematogenous spread of germ cells or virilization due to ectopic testosterone secretion by gonads or adrenal tumors.9 An abdominal ultrasound scan revealed a right inguinal mass of 2 × 6 cm in diameter. A laparoscopic exploration was performed through an infraumbilical incision with two working ports on both sides of the abdomen. A right inguinal hernia was found with two testes inside it. The labial folds were joined together in the middle line with no opening for the urethra or vagina. The laparoscopic findings confirmed our suspicion of CAIS with persistent Müllerian structures. CAIS is a rare disorder of sex development caused by mutations in the androgen receptor gene located on the X chromosome. Phenotypic females characterize it with female external genitalia, who have an XY karyotype.1-3 In most cases, it manifests at puberty; however, it can be detected. Here is a report on a case of CAIS diagnosed in a young adult with persistent müllerian ducts removed at the age of 15 (Van Hemmen et al., 2017). The patient had primary amenorrhea and did not develop virilization at puberty. Bilateral gonadectomy was performed because she was diagnosed with CAIS based on histopathologic findings. The patient was subsequently hormonally maintained on estrogen and progesterone after the operation. Based on these findings, CAIS should be considered in patients with bilateral gonads containing testicular tissue even after extensive evaluation for other causes. The removal of internal organs of the female reproductive tract is essential because they are prone to malignancy in CAIS patients with testis persistence.

Conclusion

AIS or androgen resistance syndrome is a rare genetic condition caused by a mutation in the AR, resulting in the partial or complete inability of the cell to react to androgens. The result is that medically and surgically, individuals with AIS present as female (or non-mated), but phenotypically, anatomically, genetically and chromosomally, they are 46XY males. AIS presents a wide clinical spectrum involving complete or partial insensitivity to androgen hormones. The complete form (CAIS), characterized by ambiguous genitalia, causes karyotype 46XY women with primary amenorrhea, non-descending testes and a uterus absent or rudimentary. In the partial form (PAIS), the genital phenotype may resemble 46XY men or 46XY women. Reproduction is difficult in individuals with CAIS because of lacking a uterus and the low quality of ovaries. Fertility may be preserved in PAIS patients because of a milder insensitivity to androgens. The main molecular diagnosis methods are direct sequencing and deletion/ duplication tests in cases with a strong suspicion of AIS. Other methods like methylation analysis have been used, but they are not widely available. Since it is an inherited disease, genetic counseling should be performed to prevent transmission to future generations. A multidisciplinary team that includes endocrinologists, gynecologists, urologists and psychologist is essential for treating patients with AIS.

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