A novel variant in the 5’ UTR of the androgen receptor gene without coding region alterations in three patients with complete androgen insensitivity syndrome

A novel variant in the 5’ UTR of the androgen receptor gene without coding region alterations in three patients with complete androgen insensitivity syndrome | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A novel variant in the 5’ UTR of the androgen receptor gene without coding region alterations in three patients with complete androgen insensitivity syndrome Anne Bergougnoux, Guillaume Perez, Abdelhay Boulahtouf, Delphine Mallet, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8151404/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract A syndrome-causing androgen receptor ( AR) gene variant is identified in > 95% of 46,XY individuals with a female phenotype due to complete androgen insensitivity syndrome (CAIS). Here, we describe three patients (two adults, 37 and 32 years of age, and a 14-year-old teenager) with CAIS harboring a new 5’UTR variant of AR . Sanger sequencing of the AR coding region did not identify any known syndrome-causing variant. Massive parallel sequencing of genes, known to be involved in differences in sexual development, and their regulatory regions identified a novel c.-829C > T variant in the 5’UTR sequence of AR in all three patients. The ORF Finder software predicted the use of a new AUG codon located 296 bp downstream of the transcription start site (not confirmed by western blotting). Luciferase activity was slightly decreased in U2SO cells after transfection of the AR 5’UTR-c.-829C > T construct, but this could not explain the CAIS phenotype. Western blotting with an anti-AR antibody showed increased expression of a high molecular weight band and a decrease of the native AR protein. Aberrant splicing and mRNA level alterations were not detected. This study identified the c.-829C > T AR variant in three unrelated patients with CAIS. The functional analysis suggests that a posttranslational modification in AR may increase its molecular weight. The reduced bioavailability of the native AR protein could explain CAIS in these three patients. This second 5’UTR-coding sequence variant highlights the need to analyze AR exons and non-coding regions in all patients with CAIS. androgen receptor complete androgen insensitivity syndrome (CAIS) non-coding gene sequence 5’UTR variant differences of sexual development Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Androgen insensitivity syndrome is the most frequent cause of differences of sex development (DSD) in individuals with a 46,XY karyotype and normal/high testosterone concentration (Sultan et al. 2014). This syndrome is due to disease-causing variants in the androgen receptor ( AR ) gene, the cellular mediator of androgen activity that is crucial for masculinization of the external genitalia during fetal life and puberty. The phenotype ranges from complete androgen insensitivity syndrome (CAIS), where androgen-dependent tissues do not respond at all to the androgenic effects of testosterone and dihydrotestosterone, to partial and mild androgen insensitivity syndrome, where response is partial. The estimated prevalence of CAIS ranges from 1/20,000 to 1/64,000 male births (Mongan et al. 2015; Quigley et al. 1995). The timing of referral to clinicians extends from birth, or even before birth, to adulthood. During fetal life, referral is usually based on a discrepancy between the 46,XY karyotype and the presence of female external genitalia on prenatal ultrasonography (US). As individuals with CAIS are born with unambiguous external female genitals, a DSD may not be suspected until puberty onset. Primary amenorrhea is the most typical presentation in a phenotypic female adolescent, with normal breast development, but very sparse axillary and pubic hair. Height is typically normal or slightly increased. The genitourinary exam reveals normal external genitalia and a rudimentary, blind-ending vagina without uterus at US. In the neonatal period and infancy, CAIS should be suspected in patients with inguinal hernia and female phenotype. Various data indicate that CAIS incidence in children with premenarchal inguinal hernia is 1.1% (Hurme et al. 2009; Sarpel et al. 2005), and that 90% of all girls with CAIS will develop inguinal hernia (Oakes et al. 2008; Sarpel et al. 2005; Sultan et al. 2014). The AR gene is located on the X chromosome (Xq11-12). This gene (90 kb in length) contains eight exons and encodes a 919-amino acid protein (Eisermann et al. 2013). The AR protein has four major functional domains: N-terminal domain (residues 1-556) encoded by exon 1, central DNA-binding domain (residues 557-627) encoded by exons 2 and 3, C-terminal ligand-binding domain (residues 670-919) encoded by exons 4 to 8, and hinge region (residues 628-669) that connects the DNA-binding and ligand-binding domains (Sultan et al. 2014). To date, more than 1,000 syndrome-causing AR variants have been reported (Gottlieb et al. 2012), and more than 480 likely pathogenic and pathogenic variants were listed in the ClinVar database on July 2025 (www.ncbi.nlm.nih.gov<clinvar). Most variants are located in exonic regions, mostly exons 4-8 (Georget et al. 2006; Poujol et al. 2002), followed by exons 2-3, and then exon 1 (Philibert et al. 2010). In more than 95% of CAIS cases, a syndrome-causing AR variant is identified in the coding sequence; however, in the other 5%, the coding sequence is normal, despite typical clinical and hormonal features of CAIS (Tadokoro-Cuccaro and Hughes 2014). Various hypotheses have been formulated to explain this observation, such as defects in AR protein expression (Mongan et al. 2015) or AR transcriptional activity, linked to a defect in a co-regulator protein (Adachi et al. 2000). Few studies have described deep intronic AR variants located relatively far from the exonic boundaries (Känsäkoski et al. 2016; Ono et al. 2018) and the only 5’UTR sequence variant in CAIS (c-547C>T) was reported by Holterhus’ team in 2016 (Hornig et al. 2016). Here, we describe three unrelated patients with CAIS harboring a new likely pathogenic Single Nucleotide Variant (SNV) in the 5’UTR of the AR gene. Patients and Methods Clinical presentation Patient #1 was Caucasian and was referred to the pediatric unit of Marseille University Hospital, France, for neonatal unilateral inguinal hernia. Clinical examination revealed a palpable gonad in the left inguinal position and moderate clitoromegaly. US confirmed the presence of a gonad in the left hernial sac, and the uterus absence. The basal plasma testosterone concentration was 0.3 ng/ml, and the luteinizing hormone (LH) and follicle-stimulating hormone (FSH) concentrations were 0.1 and 0.2 IU/L, respectively, AMH 106 ng/mL. The patient’s karyotype was 46,XY. The diagnosis of CAIS was considered on the basis of the clinical presentation, but could not be confirmed by Sanger sequencing of the AR gene. In line with the guideline practices at that time, gonadectomy was performed at the age of 10 years. The normal structure of the two testes was confirmed by a pathologist. Female puberty induction, based on 17β estradiol supplementation, was started at the age of 11 years. After 2 years of supplementation, breast development was B5 and estrogen replacement therapy was continued. The patient was lost to follow-up until the age of 37 years, when she was referred to the adult endocrine unit for medical follow-up. The patient’s height was 168 cm, weight was 58 kg, and pubertal status was B5P2. The patient reported low adherence to the prescribed hormone replacement therapy. The patient was married and reported normal intercourse. Patient #2 (North African origin) was referred to the endocrine unit of Montpellier University Hospital, France, at the age of 32 years because of primary amenorrhea. The patient was married, but had never been able to have normal intercourse and was initiating divorce proceedings. At the age of 32 years, her doctor encouraged the patient to seek medical advice for her gynecological problems. The patient’s height was 170 cm, and weight was 58 kg. The patient refused the gynecological examination, but breast development was B5, which had occurred spontaneously at puberty, and pubic hair was absent. The presence of bilateral inguinal scars was observed, and the patient reported surgery for inguinal hernia at the age of ~ 10 years. The basal plasma testosterone concentration was 17 ng/ml, LH and FSH concentrations were 36 and 5.4 IU/L, respectively, and basal estradiol and anti-Müllerian hormone (AMH) concentrations were 44 pg/mL and 114 ng/mL, respectively. The karyotype was 46,XY. Magnetic resonance imaging revealed a very short vagina with a vaginal wall and two intraabdominal gonads without cysts or adenoma, whereas Müllerian structures were not identified. Sanger sequencing of the AR , SRD5A2 , and NR5A1 gene coding regions did not lead to the detection of any pathogenic variant. Patient #3 (Caucasian origin) was referred to the pediatric endocrine unit of Civil Hospices of Lyon during the neonatal period for bilateral inguinal hernia. US confirmed the presence of inguinal gonads and no uterus. The karyotype was 46,XY. Testosterone concentration was 0.4 ng/mL, LH and FSH concentrations were 0.6 and 1.1 IU/L, respectively, basal estradiol concentration was 4.6 pg/mL and AMH concentration was 118 ng/mL. Sanger sequencing of the AR , SRD5A2 and NR5A1 gene coding regions did not detect any pathogenic variant. The patient presented spontaneous puberty at the age of 14 years when pubertal status was B4P2 according to the Tanner stages. The patients’ clinical features are summarized in Table 1. All three patients (or the parents) gave their informed consent for genetic testing and publication. Genetic analyses The massively parallel sequencing (MPS) design included a panel of genes (coding sequences and known regulatory regions, such as enhancers) (list of genes available on request) implicated in DSD, hypogonadotropic hypogonadism, steroidogenesis, or infertility (either isolated or as part of a syndromic disorder) based on studies in humans or knockout and/or knockdown studies in animal models showing consequences on sex determination or differentiation. MPS libraries from blood samples of patients #1 and #2 were prepared following the manufacturer’s instructions, as briefly described below. The DNA libraries were prepared using SeqCap EZ probes and the KAPA HyperPrep Kits and HyperCap Target Enrichment Kits (Roche Diagnostics®, Basel, Switzerland). The libraries were sequenced in independent runs using a MiniSeq High Output Reagent Cartridge and Flowcell on a MiniSeq (Illumina, San Diego, CA, USA). Read alignment was performed using Local Run Manager (Illumina, San Diego, CA, USA) or the MobiDL workflow developed by the MoBiDiC team ( https://github.com/mobidic/MobiDL ). This workflow is dedicated to next generation sequencing data based on capture libraries and focuses on gene panels/exomes. It uses the Genome Analysis ToolKit (GATK) 4 HaplotypeCaller and Google DeepVariant for variant calling. Copy number variants (CNV) were detected using the MobiCNV tool ( https://github.com/mobidic/MobiCNV ). The Captain Achab workflow and the MoBiDiC Prioritization Algorithm (Yauy et al. 2018 ) were used for SNV filtering and prioritization. The pathogenicity of rare variants was then predicted using the online MobiDetails DNA Variant Interpretation tool ( https://mobidetails.iurc.montp.inserm.fr/MD/ ) For patient #3, MPS was performed as previously described (Mazel et al. 2022 ) using a custom design based on a SeqCap EZ Solution-Based Enrichment strategy (Roche NimbleGen) and paired-end sequencing 2 × 150 bp on a Nextseq 500 (Illumina). Targeted regions included coding exons ± 50 bp and the 5′ and 3′ UTRs (list of genes available on request). Genomic alignment against the hg19/GRCh37 assembly and variant calling were done using BWA-MEM v.0.7.12 and GATK HaplotypeCaller v.3.4 (Broad Institute), respectively. Variants were classified according to the American College of Medical Genetics (ACMG) guidelines (Richards et al. 2015 ). The suspected syndrome-causing variant was confirmed by Sanger sequencing with the following primers: forward 5’-GTGCTGTACAGGAGCCGAAG-3’; reverse 5’-GGTAACTCCCTTTGGCTGC-3’. In silico predictions of upstream Open Reading Frames (uORF) The Open Reading Frame Finder from NCBI ( https://www.ncbi.nlm.nih.gov/orffinder/ ) predicts ORFs from a pasted sequence and provides the predicted protein sequences from newly detected ORFs. Plasmids and transient transactivation experiments The AR 5’UTRwt-HIS-GFP plasmid that contains the 5’UTR of AR was a gift from PL Holtertus (Hornig et al. 2016 ). The AR 5’UTRwt-luciferase plasmid was created by inserting by PCR the 5’UTR of AR (-1117; +3) from the AR 5’UTRwt-HIS-GFP plasmid in the Kpn1 and Nco1 sites of the pGL4 luciferase reporter vector (Promega, Charbonnières-les Bains, France). The AR 5’UTRc-829C > T-luciferase and AR 5’UTRc-547C > T-luciferase plasmids were constructed by overlap extension PCR. The used primers are listed in supplementary Table 1. U2OS cells (Merck, France) were grown in DMEM-F12 medium (Merck, France) supplemented with 10% fetal bovine serum (Sigma-Aldrich/Merck, France). Typically, 100,000 cells/well were plated in 12-well plates and transfected the day after using JetPEI (Dutscher, Bernolsheim, France) and 1.9 µg of AR 5’UTR-AR-luciferase plasmid with 0.1µg Renilla-luciferase reporter vector (Thermo Fisher Scientific, USA). After 48h, Firefly/Renilla luciferase activity was measured following the Dual-Luciferase Reporter Assay protocol (Promega) and a MicroBeta Wallac luminometer (PerkinElmer). Quantitative RT-PCR analysis Total RNA was extracted from foreskin fibroblasts of a man who underwent posthectomy (control #1), from skin fibroblasts of another man (control #2) and from genital skin (labia majora) fibroblasts of patient #3 using a standard TRIzol/propanol protocol. Then, RNA was reverse transcribed with MMLV reverse transcriptase (Invitrogen, 28025013). Quantitative PCR was carried out with a LightCycler® 480 System from Roche, using the LightCycler 480 SYBR Green I Master (Roche, 04887352001). The primers used were in exon 5 of AR : F/5’-CATCCCTAAGGATACCCAGGGACCATC-3’, R/ 5’-GGATGGTCCCTGGGTATCCTTAGGGGATG-3’. A written consent for medical research was obtained for the two healthy donors and patient #3. Ex vivo AR mRNA quantification Total RNA from foreskin fibroblasts of control #1, from skin fibroblasts of control #2 and from genital skin fibroblasts of patient #3 was isolated using the RNA Now kit (Biogentex Laboratories, Inc., League City, TX, USA). RNA was reverse transcribed using random hexamers with the GeneAmp® RNA PCR kit (Thermo Fisher Scientific) following the manufacturer’s instructions. Then, AR cDNA was PCR amplified using primers in exon 1 (A9 Fw 5‘-GACTTCACCGCACCTGATGTGTGG-3’) and exon 8 (H2 Rv 5’- TTCCCCAAGGCACTGCAGAGGA-3’), with the Taq CORE Kit (MP Biomedicals) and the following touch-down program: initial denaturation at 94°C for 3 minutes; 14 cycles with denaturation at 94°C for 20 seconds, hybridization at 63°C for 40 seconds (-0.5°C at each cycle), extension at 72°C for 2 minutes; 26 cycles with denaturation at 94°C for 20 seconds, hybridization at 56°C for 40 seconds, extension at 72°C for 2 minutes; final extension at 72°C for 7 minutes. PCR products (patient #3 and two controls) were analyzed and compared with the LabChip® 90 system (Caliper Life Science, Hopkinton, MA, USA). Direct Sanger sequencing was performed using the same primers and the BigDye 1.1 Terminator sequencing kit on a ABI 3130 automated sequencer (Applied Biosystems, Foster City, CA, USA). Sequences were aligned using SeqScape V2.5. Western blot analysis Protein extracts (30 µg) from genital skin (labia majora) fibroblasts from patient #3 and of preputial skin (posthectomy) from control #1 were separated by SDS-PAGE on NuPAGE 7% gels (Thermo Fisher Scientific France, Illkirch-Graffenstaden). To identify the predicted shorter AR protein, NuPAGE 15% gels (Thermo Fisher Scientific France, Illkirch-Graffenstaden) were used. After transfer, PVDF membranes (Thermo Fisher Scientific France, Illkirch-Graffenstaden) were incubated with antibodies against native AR (D6F11, Cell Signaling Technology, USA; 1/2000) and the short AR form pAR-62AA (custom synthesis by Eurogentec, Belgium, H-CQSATLSQPPSPPFS-NH2; 1/1000, 1/10 and 1/2). RESULTS Identification of the same 5’UTR mutation in the AR gene in all three patients Genetic analysis of the three 46,XY patients with DSD revealed a novel c.-829C > T point mutation in the 5’UTR sequence of the AR gene (RefSeq NM_000044.6) (Fig. 1A). According to gnomAD V3, this variant has not been reported in the general population, whatever the ethnicity. No other variant detected in the patients’ DNA was considered pathogenic (data available on request). According to the ACMG classification criteria, the new c.-829C > T variant could be considered as likely pathogenic (class 4, PS4, PM2, PS3 and PP4). The ORF Finder tool detected a new uORF due to the creation of an initiation codon in the presence of the c.-829C > T variant (Fig. 1B). This new AUG was located 296 bp downstream of the transcription start site (TSS) of the AR gene. The new AUG is embedded in a vertebrate Kozak sequence (i.e., cXXATGG), with an expected A/GXXATGG consensus sequence. This could allow the recognition of the newly identified AUG by the translation machinery, theoretically leading to the production of a short protein of 62 amino acids, called AR-p62AA. Functional analysis of the AR -5’UTR mutation in vitro To functionally characterize the impact of the c-829C > T point mutation, the wild type (wt) or mutated (c-547C > T and c-829C > T) AR 5’UTR was cloned directly upstream of the luciferase reporter gene and the obtained plasmids were transfected in U2OS cells (Fig. 2A-B). Luciferase activity was decreased in cells that express the AR 5’UTRc-829C > T compared with the AR 5‘UTRwt (Fig. 2C) and also compared with the previously described disease-causing AR 5’UTR variant (AR 5’UTRc-547C > T), used as positive control. AR mRNA quantity and quality Then, RT-qPCR was used to quantify AR mRNA quantity in foreskin and skin fibroblasts from controls #1 and #2, respectively, and genital skin fibroblasts of patient #3. This analysis did not show any significant difference in AR mRNA quantity (Fig. 3A). To determine whether any additional syndrome-causing variant located in intronic regions in linkage disequilibrium with the c.-829C > T mutation was present, AR transcripts in genital fibroblasts from patient #3 and in foreskin and skin fibroblasts from controls #1 and #2, respectively, were quantitatively analyzed by touch-down PCR to cover all exons (Fig. 3B). No additional peak was detected compared to the AR transcript pattern in the two controls, confirming that only the full AR transcript was transcribed, without any detectable aberrant spliced form (Fig. 3C). AR protein analysis Given the discrepancy between the severe phenotype with complete feminization and the low effect of the mutation on AR transcriptional activity and mRNA quality and quantity, AR protein expression was assessed by western blotting. AR protein level (native form; 140 kDa) was decreased in genital skin fibroblasts from patient #3 compared with control #1 (Fig. 4A). In addition, the intensity of a high molecular weight (~ 170 kDa) band was significantly increased in patient #3. However, as the average weight of an amino acid is \(\:\sim110\:\text{D}\text{a},\) the putative AR-p62AA protein should approximatively weight \(\:62\cdot\:110\:\text{D}\text{a}\:=\:6.82\:\text{k}\text{D}\text{a}\) . Western blot analysis using an antibody against the AR-p62AA protein did not detect any band between 5 kDa and 10 kDa (Fig. 4B). DISCUSSION This study describes a new 5’UTR variant in the AR (c.-829C > T) detected in three patients with CAIS without mutations in AR coding region. The three patients had inguinal hernia: in patients #1 and #3 it appeared in the neonatal period and in patient #2 at the age of 10 years. In the presence of inguinal hernia, CAIS was only considered in patients #1 and #3. Patient #2 did not seek medical attention until much later, although she had primary amenorrhea. The molecular diagnosis of CAIS was only confirmed in adulthood in patients #1 and #2 (37 and 32 years, respectively), because patient #1 was lost to follow-up for several years and patient #2 was too embarrassed to consult earlier about her sexual difficulties. The association between inguinal hernia and CAIS in girls is well known (Sultan et al. 2014 ) and was a major reason for early referral in several case reports (Konar et al. 2015 ; Listyasari et al. 2019 ; Nair and Bhavana 2012 ; Sharma et al. 2011 ) and in large series (Hurme et al. 2009 ; Sarpel et al. 2005 ). For instance, Deeb found that 57% of 120 patients with CAIS presented with inguinal hernia, thus the most frequent mode of clinical presentation of CAIS in childhood (Deeb and Hughes 2005 ). Inguinal hernia in CAIS is explained by the role of testosterone and its receptor AR in the second phase of testicular descent (Hutson et al. 2015 ). Therefore, irrespective of the age at which the inguinal hernia appears, the diagnosis of CAIS should be considered. For the three patients reported here, AR coding sequence was first analyzed using the Sanger method, but no syndrome-causing variant was identified. However, the clinical, radiological and hormonal phenotype clearly indicated resistance to androgens. Thus, MPS was used. This approach allowed identifying the c.-829C > T variant in the 5’UTR of AR . The absence of other potentially causative variants and of any aberrant splicing pattern was confirmed by MPS and touch-down PCR. Variants in the non-coding sequences of the AR gene have been rarely detected in patients with androgen insensitivity syndrome. The McGill database (Gottlieb et al. 2012 ) contains only few DNA variants in the 5’UTR of AR involved in prostate cancer. A pathogenic insertion of a LINE-1 retrotransposon in the 5’UTR of AR has been associated with partial androgen insensitivity syndrome in nine XY individuals from the same family (Batista et al. 2019 ). To our knowledge, a syndrome-causing SNV in this region has been reported only once in two unrelated patients with CAIS (Hornig et al. 2016 ). The authors showed that this upstream variant (c.-547C > T) creates a novel translation start site (AUG) located downstream the TSS, thus limiting the translation of the native AR form. Here, in silico prediction tools suggested that the novel c.-829C > T variant induces the recognition of another alternative AUG (located upstream the native AUG) and the production of a short protein of 62 amino acids, called AR-p62AA. However, this short protein could not be detected by western blotting. Yet, the three patients had a markedly severe CAIS phenotype. Therefore, it seems evident that AR biology was strongly affected. Western blot analysis of genital skin fibroblasts from patient #3 showed the presence of a band of the expected molecular weight for AR, the intensity of which was strongly decreased compared with the control. This suggests a decrease of the AR protein amount in patient #3, which could partly explain the observed phenotype. In addition, a high molecular weight band (~ 170 kDa) was over-represented compared with the control. This high-molecular-weight band may indicate the presence of polyubiquitinated AR. Ubiquitination is a post-translational modification that involves the conjugation of ubiquitin, a highly conserved peptide of 76 residues, alone or in a polymerized chain (Komander and Rape 2012 ). Ubiquitination has many functions, ranging from labeling proteins to be degraded to modifying the interaction pattern of the ubiquitinylated protein. Considering the observed phenotype, this finding could suggest AR polyubiquitination, resulting in increased AR degradation. Furthermore, polyubiquitination has other effects that could influence AR functionality. These include confinement of proteins to the cytosol and impairment of the interaction with some partners. This could affect AR binding to HSP90, which is required to regulate AR binding affinity. Additionally, if due to confinement in the cytosol, AR could not enter the nucleus and dimerize, it would lose its transcription-activating function. These effects would reinforce the particularly low bioavailability of AR, which could be consistent with the patients' severe phenotype. The presence of this high-molecular weight band suggests that other post-translational modifications (e.g. acetylation or phosphorylation) are not implicated, but does not exclude sumoylation. This modification is nuclear and is mostly initiated by DNA damage and stress (Hendriks and Vertegaal 2016 ). As sumoylation modifies the interacting characteristics of the protein, AR could no longer undergo dimerization, resulting in impaired transcriptional activity, which may partly explain the observed phenotype. However, the decrease in AR protein amount clearly observed in patient #3 tends to exclude sumoylation. The western blot analysis and the luciferase activity assays gave contradictory results. However, the luciferase assays were obtained in U2OS cells that like other cell lines (e.g., HEK 293 cells) generally used for this kind of assay, are immortalized or cancer cells with karyotypic and/or genetic alterations and are considered “protein-producing machines” (Pontén 1967 ; Stepanenko and Dmitrenko 2015 ). The quality-controls for RNAs and proteins, as well as the regulatory mechanisms for transcription and translation, may be modified in U2OS cells, which makes it challenging to extrapolate the results to in vivo conditions. Although ubiquitination is the most attractive hypothesis, the precise relationship between ubiquitination and the mutation remains unclear because the in silico methods predicted the emergence of a short protein variant that was not identified by western blotting. The in silico analysis, which was not exhaustive, may have overlooked alternative translation initiation sequences, apart from the Kozak sequences, which may be dependent on the cell type or cellular conditions. Moreover, putative new motifs that can act as recognition motifs for degradation-associated enzymes and accessory proteins were not actively searched. On the other hand, it could be hypothesized that the mutation does not affect the protein but the mRNA. For example, it would be conceivable that the emergence of novel secondary structures within the 5'UTR of mRNA may destabilize ribosomes and activate RNA quality control systems, leading to increased mRNA degradation. Although the eIF4A factor displays RNA helicase activity that can undo secondary structures on RNA (Merrick and Pavitt 2018 ), a new structure may be interpreted as an anomaly and linked by factors to mRNA delivery and confinement in P-bodies. However, RT-qPCR did not find any difference in AR mRNA levels between patient #3 and the healthy controls. Furthermore, cDNA sequencing did not reveal any mRNA sequence abnormality or aberrant splicing pattern. Collectively, these results tend to demonstrate that AR transcription and mRNA are not affected by the c.-829C > T mutation. This reinforces the hypothesis that the problem concerns the AR protein metabolism/degradation. A significant decrease in AR protein bioavailability due to an increased degradation is biologically consistent with the CAIS phenotype. Conclusion This study reports the discovery of a new point mutation, c.-829C > T, in the 5’ UTR of the AR gene in three unrelated patients with similar CAIS phenotype. Although the in silico tools predicted that c.-829C > T induces the recognition of an alternative AUG and translation of a short protein of 62 amino acids called AR-p62AA, this new isoform could not be detected by western blotting. The presence of a high molecular AR form associated with a marked decrease in the native AR protein led to the hypothesis that AR ubiquitination is responsible for its increased degradation. However, the link between the c.-829C > T variant and ubiquitination was not demonstrated. As far as we know, this is the second report of patients with CAIS harboring a 5’UTR SNV of the AR gene. This work contributes to increase awareness of CAIS with a normal AR coding sequence and underlines the need to analyze also non-coding regions of the AR gene in typical CAIS. The presence of the c.-829C > T variant in the AR gene should orient the diagnosis toward CAIS. We declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. We would like to warmly thank Professor Holterhus for help with the primer design as well as Professor Didier Bessis and Doctor Boris Delaunay for collecting the skin/foreskin samples from the healthy male controls. This research did not receive any specific grant from any funding agency in the public, commercial or non-profit sector. Declarations Author Contribution Anne Bergougnoux and Guillaume Perez are co-first authorsAll the authors contributed to writing the manuscript.Anne Bergougnoux, Guillaume Perez, Françoise Paris, Delphine Mallet, Aurelie Gennetier, Abdlhay Boulahtof, Patrick Balaguer and Nadège Servant prepared the figures.Françoise Paris prepared the Table 1All authors reviewed the manuscript Acknowledgement We would like to warmly thank Professor Holterhus for help with the primer design as well as Professor Didier Bessis and Doctor Boris Delaunay for collecting the skin/foreskin samples from the healthy male controls. References Adachi M, Takayanagi R, Tomura A, Imasaki K, Kato S, Goto K, Yanase T, Ikuyama S, Nawata H (2000) Androgen-insensitivity syndrome as a possible coactivator disease. N Engl J Med 343: 856-62. doi: 10.1056/nejm200009213431205 Batista RL, Yamaguchi K, Rodrigues ADS, Nishi MY, Goodier JL, Carvalho LR, Domenice S, Costa EMF, Kazazian HH, Mendonca BB (2019) Mobile DNA in Endocrinology: LINE-1 Retrotransposon Causing Partial Androgen Insensitivity Syndrome. J Clin Endocrinol Metab 104: 6385-6390. doi: 10.1210/jc.2019-00144 Deeb A, Hughes IA (2005) Inguinal hernia in female infants: a cue to check the sex chromosomes? BJU Int 96: 401-3. doi: 10.1111/j.1464-410X.2005.05639.x Eisermann K, Wang D, Jing Y, Pascal LE, Wang Z (2013) Androgen receptor gene mutation, rearrangement, polymorphism. Transl Androl Urol 2: 137-147. doi: 10.3978/j.issn.2223-4683.2013.09.15 Georget V, Bourguet W, Lumbroso S, Makni S, Sultan C, Nicolas JC (2006) Glutamic acid 709 substitutions highlight the importance of the interaction between androgen receptor helices H3 and H12 for androgen and antiandrogen actions. Mol Endocrinol 20(4): 724-34. Gottlieb B, Beitel L, Nadarajah A, Paliouras M, Trifiro M (2012) The androgen Receptotr gene mutations database: 2012 update. http://www.androgendb.mcgill.ca, last update 2013. Hum Mutat 33: 887-94. Hendriks IA, Vertegaal AC (2016) A comprehensive compilation of SUMO proteomics. Nat Rev Mol Cell Biol 17: 581-95. doi: 10.1038/nrm.2016.81 Hornig NC, de Beaufort C, Denzer F, Cools M, Wabitsch M, Ukat M, Kulle AE, Schweikert HU, Werner R, Hiort O, Audi L, Siebert R, Ammerpohl O, Holterhus PM (2016) A Recurrent Germline Mutation in the 5'UTR of the Androgen Receptor Causes Complete Androgen Insensitivity by Activating Aberrant uORF Translation. PLoS One 11: e0154158. doi: 10.1371/journal.pone.0154158 Hurme T, Lahdes-Vasama T, Makela E, Iber T, Toppari J (2009) Clinical findings in prepubertal girls with inguinal hernia with special reference to the diagnosis of androgen insensitivity syndrome. Scand J Urol Nephrol 43: 42-6. doi: 10.1080/00365590802299247 Hutson JM, Li R, Southwell BR, Newgreen D, Cousinery M (2015) Regulation of testicular descent. Pediatr Surg Int 31: 317-25. doi: 10.1007/s00383-015-3673-4 Känsäkoski J, Jääskeläinen J, Jääskeläinen T, Tommiska J, Saarinen L, Lehtonen R, Hautaniemi S, Frilander MJ, Palvimo JJ, Toppari J, Raivio T (2016) Complete androgen insensitivity syndrome caused by a deep intronic pseudoexon-activating mutation in the androgen receptor gene. Sci Rep 6: 32819. doi: 10.1038/srep32819 Komander D, Rape M (2012) The ubiquitin code. Annu Rev Biochem 81: 203-29. doi: 10.1146/annurev-biochem-060310-170328 Konar S, Dasgupta D, Patra DK, De A, Mallick B (2015) Chromosomal Study is Must for Prepubertal Girl with Inguinal Hernia: Opportunity to Diagnose Complete Androgen Insensitivity Syndrome. J Clin Diagn Res 9: GD01-3. doi: 10.7860/jcdr/2015/11411.5750 Listyasari NA, Robevska G, Santosa A, Bouty A, Juniarto AZ, van den Bergen J, Ayers KL, Sinclair AH, Faradz SM (2019) Genetic Analysis Reveals Complete Androgen Insensitivity Syndrome in Female Children Surgically Treated for Inguinal Hernia. J Invest Surg: 1-7. doi: 10.1080/08941939.2019.1602690 Mazel B, Mallet D, Roucher-Boulez F, Signor CB, Bournez M, Darmency V, Bourgeois V, Poe C, El Khabbaz F, Vitobello A, Philippe C, Duffourd Y, Thauvin-Robinet C, Faivre L, Nambot S (2022) Epileptic encephalopathy as a new feature of the sudden infant death with dysgenesis of the testes syndrome caused by TSPYL1 variants. Am J Med Genet A 188: 3540-3545. doi: 10.1002/ajmg.a.62966 Merrick WC, Pavitt GD (2018) Protein Synthesis Initiation in Eukaryotic Cells. Cold Spring Harb Perspect Biol 10. doi: 10.1101/cshperspect.a033092 Mongan NP, Tadokoro-Cuccaro R, Bunch T, Hughes IA (2015) Androgen insensitivity syndrome. Best Pract Res Clin Endocrinol Metab 29: 569-80. doi: 10.1016/j.beem.2015.04.005 Nair RV, Bhavana S (2012) XY Female with Complete Androgen Insensitivity Syndrome with Bilateral Inguinal Hernia. J Obstet Gynaecol India 62: 65-7. doi: 10.1007/s13224-013-0379-1 Oakes MB, Eyvazzadeh AD, Quint E, Smith YR (2008) Complete androgen insensitivity syndrome--a review. J Pediatr Adolesc Gynecol 21: 305-10. doi: 10.1016/j.jpag.2007.09.006 Ono H, Saitsu H, Horikawa R, Nakashima S, Ohkubo Y, Yanagi K, Nakabayashi K, Fukami M, Fujisawa Y, Ogata T (2018) Partial androgen insensitivity syndrome caused by a deep intronic mutation creating an alternative splice acceptor site of the AR gene. Sci Rep 8: 2287. doi: 10.1038/s41598-018-20691-9 Philibert P, Audran F, Pienkowski C, Morange I, Kohler B, Flori E, Heinrich C, Dacou-Voutetakis C, Joseph MG, Guedj AM, Journel H, Hecart-Bruna AC, Khotchali I, Ten S, Bouchard P, Paris F, Sultan C (2010) Complete androgen insensitivity syndrome is frequently due to premature stop codons in exon 1 of the androgen receptor gene: an international collaborative report of 13 new mutations. Fertil Steril 94: 472-6. Pontén J (1967) Spontaneous lymphoblastoid transformation of long-term cell cultures from human malignant lymphoma. Int J Cancer 2: 311-25. doi: 10.1002/ijc.2910020406 Poujol N, Lumbroso S, Makni S, Terouanne B, Lobaccaro J, Bourguet W, Sultan C (2002) Pathophysiology of androgen insensitivity syndromes: molecular and structural approaches of natural and engineered androgen receptor mutations at amino acid 743. J Clin Endocrinol Metab 87(12): 5793-800. Quigley CA, De Bellis A, Marschke KB, el-Awady MK, Wilson EM, French FS (1995) Androgen receptor defects: historical, clinical, and molecular perspectives. Endocr Rev 16: 271-321. doi: 10.1210/edrv-16-3-271 Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, Grody WW, Hegde M, Lyon E, Spector E, Voelkerding K, Rehm HL (2015) Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 17: 405-24. doi: 10.1038/gim.2015.30 Sarpel U, Palmer SK, Dolgin SE (2005) The incidence of complete androgen insensitivity in girls with inguinal hernias and assessment of screening by vaginal length measurement. J Pediatr Surg 40: 133-6; discussion 136-7. doi: 10.1016/j.jpedsurg.2004.09.012 Sharma V, Singh R, Thangaraj K, Jyothy A (2011) A novel Arg615Ser mutation of androgen receptor DNA-binding domain in three 46,XY sisters with complete androgen insensitivity syndrome and bilateral inguinal hernia. Fertil Steril 95: 804 e19-21. doi: 10.1016/j.fertnstert.2010.08.015 Stepanenko AA, Dmitrenko VV (2015) HEK293 in cell biology and cancer research: phenotype, karyotype, tumorigenicity, and stress-induced genome-phenotype evolution. Gene 569: 182-90. doi: 10.1016/j.gene.2015.05.065 Sultan C, Philibert P, Gaspari L, Audran F, Maimoun L, Kalfa N, Paris F (2014) Androgen Insensivity Syndrome. Genetic Steroid Disorders Chapter 5: 225-237. Tadokoro-Cuccaro R, Hughes IA (2014) Androgen insensitivity syndrome. Curr Opin Endocrinol Diabetes Obes 21: 499-503. doi: 10.1097/med.0000000000000107 Yauy K, Baux D, Pegeot H, Van Goethem C, Mathieu C, Guignard T, Juntas Morales R, Lacourt D, Krahn M, Lehtokari VL, Bonne G, Tuffery-Giraud S, Koenig M, Cossée M (2018) MoBiDiC Prioritization Algorithm, a Free, Accessible, and Efficient Pipeline for Single-Nucleotide Variant Annotation and Prioritization for Next-Generation Sequencing Routine Molecular Diagnosis. J Mol Diagn 20: 465-473. doi: 10.1016/j.jmoldx.2018.03.009 Table Table 1 is available in the Supplementary Files section Additional Declarations No competing interests reported. Supplementary Files Table1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8151404","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":551611760,"identity":"6b4bd0fa-ceb8-430c-b434-ff5a10992372","order_by":0,"name":"Anne Bergougnoux","email":"","orcid":"","institution":"University of Montpellier","correspondingAuthor":false,"prefix":"","firstName":"Anne","middleName":"","lastName":"Bergougnoux","suffix":""},{"id":551611761,"identity":"5a0ba29b-4820-4f07-ae2b-5cfb7b9ff3a7","order_by":1,"name":"Guillaume Perez","email":"","orcid":"","institution":"University of Montpellier","correspondingAuthor":false,"prefix":"","firstName":"Guillaume","middleName":"","lastName":"Perez","suffix":""},{"id":551611762,"identity":"8cb29e3c-778f-436b-b0d6-d6768d023aa1","order_by":2,"name":"Abdelhay Boulahtouf","email":"","orcid":"","institution":"University of Montpellier","correspondingAuthor":false,"prefix":"","firstName":"Abdelhay","middleName":"","lastName":"Boulahtouf","suffix":""},{"id":551611763,"identity":"954780a5-294c-4147-9658-9c5e4391932a","order_by":3,"name":"Delphine Mallet","email":"","orcid":"","institution":"Hospices Civils de Lyon","correspondingAuthor":false,"prefix":"","firstName":"Delphine","middleName":"","lastName":"Mallet","suffix":""},{"id":551611764,"identity":"3a5629e0-8515-43c6-9b37-0b2bf301631e","order_by":4,"name":"Aurelie Gennetier","email":"","orcid":"","institution":"University of Montpellier","correspondingAuthor":false,"prefix":"","firstName":"Aurelie","middleName":"","lastName":"Gennetier","suffix":""},{"id":551611767,"identity":"7e0248db-e555-4037-9cda-5ff531ab788e","order_by":5,"name":"Patrick Balaguer","email":"","orcid":"","institution":"University of Montpellier","correspondingAuthor":false,"prefix":"","firstName":"Patrick","middleName":"","lastName":"Balaguer","suffix":""},{"id":551611768,"identity":"86cc57ba-4120-4a11-a8bc-fc59d07184a3","order_by":6,"name":"Frédérique Albarel","email":"","orcid":"","institution":"Aix-Marseille University","correspondingAuthor":false,"prefix":"","firstName":"Frédérique","middleName":"","lastName":"Albarel","suffix":""},{"id":551611769,"identity":"e6b64f0c-4323-46df-bcb5-8c88598777e5","order_by":7,"name":"Florence Compain","email":"","orcid":"","institution":"Centre Hospitalier Universitaire de Poitiers","correspondingAuthor":false,"prefix":"","firstName":"Florence","middleName":"","lastName":"Compain","suffix":""},{"id":551611770,"identity":"bc5abb17-7f06-4c71-b0b3-1e672e5cce26","order_by":8,"name":"Nadège Servant","email":"","orcid":"","institution":"University of Montpellier","correspondingAuthor":false,"prefix":"","firstName":"Nadège","middleName":"","lastName":"Servant","suffix":""},{"id":551611771,"identity":"c3833744-6304-4785-ab3e-7e851373bbe9","order_by":9,"name":"Nicolas Kalfa","email":"","orcid":"","institution":"University of Montpellier","correspondingAuthor":false,"prefix":"","firstName":"Nicolas","middleName":"","lastName":"Kalfa","suffix":""},{"id":551611774,"identity":"bcdcd5d0-fa12-4f55-a946-042fc92f95e8","order_by":10,"name":"Eric Renard","email":"","orcid":"","institution":"University of Montpellier","correspondingAuthor":false,"prefix":"","firstName":"Eric","middleName":"","lastName":"Renard","suffix":""},{"id":551611782,"identity":"49309263-c18e-4b8f-808f-db950298f7da","order_by":11,"name":"Laura Gaspari","email":"","orcid":"","institution":"University of Montpellier","correspondingAuthor":false,"prefix":"","firstName":"Laura","middleName":"","lastName":"Gaspari","suffix":""},{"id":551611786,"identity":"1734677b-dc09-4325-84c0-e029291ad227","order_by":12,"name":"Françoise Paris","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDklEQVRIiWNgGAWjYBAC9gYQacAgxyABF2NuPJAAl8IEPAcgWoxhWoAUYwNYC0QKlxYGhsQGFC3IUhhaGHgPfq4oqEvvn91jwHSj4k6dOXtjw4EHFfcYeKSx6+Fh4EuWPGNwOHfGnTMGzDlnnklY9hwEOuxMMQMPXwJWLfbybwwkGwwO5G6QyDH/ndt2WMLgRmLDgcS2BAZ7HlwO4zH+2WBQl24gkWPADNZy/yFQy78EBh7cWsyAtjAnILTcAHo/sQGfFr40ywaDw4YzbqQVAP1yWHJnD9BhCccSeHBr4T18s+FPnTz/jOQNzDkVh/nN2Q8ffPijJkEOlxaQJlRggEOcCC2jYBSMglEwCmAAAIC3WmICePeiAAAAAElFTkSuQmCC","orcid":"","institution":"University of Montpellier","correspondingAuthor":true,"prefix":"","firstName":"Françoise","middleName":"","lastName":"Paris","suffix":""}],"badges":[],"createdAt":"2025-11-19 06:08:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8151404/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8151404/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":97120421,"identity":"79b203fe-1e21-44ab-bbcf-d7358380d2ae","added_by":"auto","created_at":"2025-12-01 07:49:48","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":566234,"visible":true,"origin":"","legend":"","description":"","filename":"ARpromoternov2025FPallDefform.docx","url":"https://assets-eu.researchsquare.com/files/rs-8151404/v1/c6aa07c3ac3bbb540ad01fcd.docx"},{"id":97140692,"identity":"ccad7c9e-5ec0-49d8-a18f-5e1ec2571e11","added_by":"auto","created_at":"2025-12-01 10:05:36","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":12922,"visible":true,"origin":"","legend":"","description":"","filename":"2513e61d854d4d449e13c4a02c222240.json","url":"https://assets-eu.researchsquare.com/files/rs-8151404/v1/b0006023804df0ae4011e3dd.json"},{"id":97120422,"identity":"8929c13f-45a6-4e00-8b2c-e1cba27a66df","added_by":"auto","created_at":"2025-12-01 07:49:48","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":105724,"visible":true,"origin":"","legend":"","description":"","filename":"2513e61d854d4d449e13c4a02c2222401enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8151404/v1/94648cdeb5753a99dcdbbbfb.xml"},{"id":97142014,"identity":"5439a79b-2370-479c-8894-8bfcc735475e","added_by":"auto","created_at":"2025-12-01 10:07:16","extension":"jpeg","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":452944,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8151404/v1/f10bf00c8435aaa3a87153f7.jpeg"},{"id":97142658,"identity":"a339871a-92f9-480e-9c36-b881f752d072","added_by":"auto","created_at":"2025-12-01 10:07:48","extension":"jpeg","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":597245,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8151404/v1/aca05f88318c57a93a34bedf.jpeg"},{"id":97141204,"identity":"d08f0b2f-3c0f-44c8-89b4-94e56173fd6e","added_by":"auto","created_at":"2025-12-01 10:06:24","extension":"jpeg","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":621717,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8151404/v1/67c1ef0854a18c0c051a24ef.jpeg"},{"id":97120428,"identity":"9203f0a2-9bd5-4667-aa1e-2d63eb54d869","added_by":"auto","created_at":"2025-12-01 07:49:49","extension":"jpeg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":650725,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8151404/v1/3e4bb9acaa7f55525b33d06e.jpeg"},{"id":97120424,"identity":"9828dbfe-c3c8-42a6-9d76-4e5fae70165e","added_by":"auto","created_at":"2025-12-01 07:49:49","extension":"jpeg","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":257485,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8151404/v1/f0a8c4296e7e65db6c325de9.jpeg"},{"id":97120429,"identity":"c90ea891-8438-4711-9c95-b2a566f24a1d","added_by":"auto","created_at":"2025-12-01 07:49:49","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":146459,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8151404/v1/f92b9d84864540b2509d31a4.png"},{"id":97120433,"identity":"bbf24e75-bdb7-4a86-88e7-2d6f9d956f46","added_by":"auto","created_at":"2025-12-01 07:49:49","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":110382,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8151404/v1/0f9a5d65d236235afb41b039.png"},{"id":97120432,"identity":"907e2454-5f60-4588-a17c-354159eb385f","added_by":"auto","created_at":"2025-12-01 07:49:49","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":155851,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8151404/v1/1d94bc5e0a6d30ffe1b4e44e.png"},{"id":97120430,"identity":"ca2432e9-35d4-4d2a-bfea-67648df888f8","added_by":"auto","created_at":"2025-12-01 07:49:49","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":163260,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8151404/v1/5390192514c8b7cc61908814.png"},{"id":97120425,"identity":"7083b87d-b168-4543-b4c7-6371856db93f","added_by":"auto","created_at":"2025-12-01 07:49:49","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":87436,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8151404/v1/20aeb6afaf5cf4ac6dd7a8a1.png"},{"id":97120426,"identity":"9fe53543-b184-4ffd-86f4-b3f49d2200d1","added_by":"auto","created_at":"2025-12-01 07:49:49","extension":"xml","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":102362,"visible":true,"origin":"","legend":"","description":"","filename":"2513e61d854d4d449e13c4a02c2222401structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8151404/v1/0b1c788a3bd0fd9e730b016e.xml"},{"id":97140753,"identity":"d2bdfeb2-1d1c-479e-bed7-82f4de6480fc","added_by":"auto","created_at":"2025-12-01 10:05:42","extension":"html","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":114881,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8151404/v1/62f8e1ad4a14e06bb6892c25.html"},{"id":97120416,"identity":"51cf6099-ec1c-4ce7-918d-ce2cdb4f9d55","added_by":"auto","created_at":"2025-12-01 07:49:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":140838,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8151404/v1/211db33c6298e67a405a8a02.png"},{"id":97120417,"identity":"5c7a5e08-4a58-4cab-9755-9f145b38a9b6","added_by":"auto","created_at":"2025-12-01 07:49:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":126976,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8151404/v1/0e4e4231910ce47b1f94ea69.png"},{"id":97140567,"identity":"20befb8f-9481-4b49-acea-7ad468a985ab","added_by":"auto","created_at":"2025-12-01 10:05:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":113069,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8151404/v1/f9fe089334a83524ef587b75.png"},{"id":97120420,"identity":"a813044f-3038-4497-8eea-9db23a123e5b","added_by":"auto","created_at":"2025-12-01 07:49:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":49248,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8151404/v1/53d9b3e7047afb877efa6225.png"},{"id":98626477,"identity":"6840ad47-c517-407c-ac5c-d6394722d79f","added_by":"auto","created_at":"2025-12-19 17:09:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":997100,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8151404/v1/8f1279dc-37d0-4b29-a14e-a38eee174cd7.pdf"},{"id":97120415,"identity":"2ccd7566-c905-42ff-87c0-2df2c596ac95","added_by":"auto","created_at":"2025-12-01 07:49:48","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":62208,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8151404/v1/6ea422530c61039eec2a0b72.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A novel variant in the 5’ UTR of the androgen receptor gene without coding region alterations in three patients with complete androgen insensitivity syndrome","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAndrogen insensitivity syndrome is the most frequent cause of differences of sex development (DSD) in individuals with a 46,XY karyotype and normal/high testosterone concentration (Sultan et al. 2014). This syndrome is due to disease-causing variants in the androgen receptor (\u003cem\u003eAR\u003c/em\u003e) gene, the cellular mediator of androgen activity that is crucial for masculinization of the external genitalia during fetal life and puberty. The phenotype ranges from complete androgen insensitivity syndrome (CAIS), where androgen-dependent tissues do not respond at all to the androgenic effects of testosterone and dihydrotestosterone, to partial and mild androgen insensitivity syndrome, where response is partial. \u003c/p\u003e\n\u003cp\u003eThe estimated prevalence of CAIS ranges from 1/20,000 to 1/64,000 male births (Mongan et al. 2015; Quigley et al. 1995). The timing of referral to clinicians extends from birth, or even before birth, to adulthood. During fetal life, referral is usually based on a discrepancy between the 46,XY karyotype and the presence of female external genitalia on prenatal ultrasonography (US). As individuals with CAIS are born with unambiguous external female genitals, a DSD may not be suspected until puberty onset. Primary amenorrhea is the most typical presentation in a phenotypic female adolescent, with normal breast development, but very sparse axillary and pubic hair. Height is typically normal or slightly increased. The genitourinary exam reveals normal external genitalia and a rudimentary, blind-ending vagina without uterus at US. In the neonatal period and infancy, CAIS should be suspected in patients with inguinal hernia and female phenotype. Various data indicate that CAIS incidence in children with premenarchal inguinal hernia is 1.1% (Hurme et al. 2009; Sarpel et al. 2005), and that 90% of all girls with CAIS will develop inguinal hernia (Oakes et al. 2008; Sarpel et al. 2005; Sultan et al. 2014).\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eAR\u003c/em\u003e gene is located on the X chromosome (Xq11-12). This gene (90 kb in length) contains eight exons and encodes a 919-amino acid protein (Eisermann et al. 2013). The AR protein has four major functional domains: N-terminal domain (residues 1-556) encoded by exon 1, central DNA-binding domain (residues 557-627) encoded by exons 2 and 3, C-terminal ligand-binding domain (residues 670-919) encoded by exons 4 to 8, and hinge region (residues 628-669) that connects the DNA-binding and ligand-binding domains (Sultan et al. 2014). \u003c/p\u003e\n\u003cp\u003eTo date, more than 1,000 syndrome-causing \u003cem\u003eAR\u003c/em\u003e variants have been reported (Gottlieb et al. 2012), and more than 480 likely pathogenic and pathogenic variants were listed in the ClinVar database on July 2025 (www.ncbi.nlm.nih.gov\u0026lt;clinvar). Most variants are located in exonic regions, mostly exons 4-8 (Georget et al. 2006; Poujol et al. 2002), followed by exons 2-3, and then exon 1 (Philibert et al. 2010). In more than 95% of CAIS cases, a syndrome-causing \u003cem\u003eAR\u003c/em\u003e variant is identified in the coding sequence; however, in the other 5%, the coding sequence is normal, despite typical clinical and hormonal features of CAIS (Tadokoro-Cuccaro and Hughes 2014). Various hypotheses have been formulated to explain this observation, such as defects in AR protein expression (Mongan et al. 2015) or \u003cem\u003eAR\u003c/em\u003e transcriptional activity, linked to a defect in a co-regulator protein (Adachi et al. 2000). Few studies have described deep intronic \u003cem\u003eAR\u003c/em\u003e variants located relatively far from the exonic boundaries (Känsäkoski et al. 2016; Ono et al. 2018) and the only 5’UTR sequence variant in CAIS (c-547C\u0026gt;T) was reported by Holterhus’ team in 2016 (Hornig et al. 2016). Here, we describe three unrelated patients with CAIS harboring a new likely pathogenic Single Nucleotide Variant (SNV) in the 5’UTR of the \u003cem\u003eAR\u003c/em\u003e gene. \u003c/p\u003e"},{"header":"Patients and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eClinical presentation\u003c/h2\u003e\u003cp\u003ePatient #1 was Caucasian and was referred to the pediatric unit of Marseille University Hospital, France, for neonatal unilateral inguinal hernia. Clinical examination revealed a palpable gonad in the left inguinal position and moderate clitoromegaly. US confirmed the presence of a gonad in the left hernial sac, and the uterus absence. The basal plasma testosterone concentration was 0.3 ng/ml, and the luteinizing hormone (LH) and follicle-stimulating hormone (FSH) concentrations were 0.1 and 0.2 IU/L, respectively, AMH 106 ng/mL. The patient\u0026rsquo;s karyotype was 46,XY. The diagnosis of CAIS was considered on the basis of the clinical presentation, but could not be confirmed by Sanger sequencing of the \u003cem\u003eAR\u003c/em\u003e gene. In line with the guideline practices at that time, gonadectomy was performed at the age of 10 years. The normal structure of the two testes was confirmed by a pathologist. Female puberty induction, based on 17β estradiol supplementation, was started at the age of 11 years. After 2 years of supplementation, breast development was B5 and estrogen replacement therapy was continued. The patient was lost to follow-up until the age of 37 years, when she was referred to the adult endocrine unit for medical follow-up. The patient\u0026rsquo;s height was 168 cm, weight was 58 kg, and pubertal status was B5P2. The patient reported low adherence to the prescribed hormone replacement therapy. The patient was married and reported normal intercourse.\u003c/p\u003e\u003cp\u003ePatient #2 (North African origin) was referred to the endocrine unit of Montpellier University Hospital, France, at the age of 32 years because of primary amenorrhea. The patient was married, but had never been able to have normal intercourse and was initiating divorce proceedings. At the age of 32 years, her doctor encouraged the patient to seek medical advice for her gynecological problems. The patient\u0026rsquo;s height was 170 cm, and weight was 58 kg. The patient refused the gynecological examination, but breast development was B5, which had occurred spontaneously at puberty, and pubic hair was absent. The presence of bilateral inguinal scars was observed, and the patient reported surgery for inguinal hernia at the age of ~\u0026thinsp;10 years. The basal plasma testosterone concentration was 17 ng/ml, LH and FSH concentrations were 36 and 5.4 IU/L, respectively, and basal estradiol and anti-M\u0026uuml;llerian hormone (AMH) concentrations were 44 pg/mL and 114 ng/mL, respectively. The karyotype was 46,XY. Magnetic resonance imaging revealed a very short vagina with a vaginal wall and two intraabdominal gonads without cysts or adenoma, whereas M\u0026uuml;llerian structures were not identified. Sanger sequencing of the \u003cem\u003eAR\u003c/em\u003e, \u003cem\u003eSRD5A2\u003c/em\u003e, and \u003cem\u003eNR5A1\u003c/em\u003e gene coding regions did not lead to the detection of any pathogenic variant.\u003c/p\u003e\u003cp\u003ePatient #3 (Caucasian origin) was referred to the pediatric endocrine unit of Civil Hospices of Lyon during the neonatal period for bilateral inguinal hernia. US confirmed the presence of inguinal gonads and no uterus. The karyotype was 46,XY. Testosterone concentration was 0.4 ng/mL, LH and FSH concentrations were 0.6 and 1.1 IU/L, respectively, basal estradiol concentration was 4.6 pg/mL and AMH concentration was 118 ng/mL. Sanger sequencing of the \u003cem\u003eAR\u003c/em\u003e, \u003cem\u003eSRD5A2\u003c/em\u003e and \u003cem\u003eNR5A1\u003c/em\u003e gene coding regions did not detect any pathogenic variant. The patient presented spontaneous puberty at the age of 14 years when pubertal status was B4P2 according to the Tanner stages.\u003c/p\u003e\u003cp\u003eThe patients\u0026rsquo; clinical features are summarized in Table\u0026nbsp;1.\u003c/p\u003e\u003cp\u003e All three patients (or the parents) gave their informed consent for genetic testing and publication.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eGenetic analyses\u003c/h3\u003e\n\u003cp\u003eThe massively parallel sequencing (MPS) design included a panel of genes (coding sequences and known regulatory regions, such as enhancers) (list of genes available on request) implicated in DSD, hypogonadotropic hypogonadism, steroidogenesis, or infertility (either isolated or as part of a syndromic disorder) based on studies in humans or knockout and/or knockdown studies in animal models showing consequences on sex determination or differentiation.\u003c/p\u003e\u003cp\u003eMPS libraries from blood samples of patients #1 and #2 were prepared following the manufacturer\u0026rsquo;s instructions, as briefly described below. The DNA libraries were prepared using SeqCap EZ probes and the KAPA HyperPrep Kits and HyperCap Target Enrichment Kits (Roche Diagnostics\u0026reg;, Basel, Switzerland). The libraries were sequenced in independent runs using a MiniSeq High Output Reagent Cartridge and Flowcell on a MiniSeq (Illumina, San Diego, CA, USA).\u003c/p\u003e\u003cp\u003eRead alignment was performed using Local Run Manager (Illumina, San Diego, CA, USA) or the MobiDL workflow developed by the MoBiDiC team (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/mobidic/MobiDL\u003c/span\u003e\u003cspan address=\"https://github.com/mobidic/MobiDL\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). This workflow is dedicated to next generation sequencing data based on capture libraries and focuses on gene panels/exomes. It uses the Genome Analysis ToolKit (GATK) 4 HaplotypeCaller and Google DeepVariant for variant calling. Copy number variants (CNV) were detected using the MobiCNV tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/mobidic/MobiCNV\u003c/span\u003e\u003cspan address=\"https://github.com/mobidic/MobiCNV\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe Captain Achab workflow and the MoBiDiC Prioritization Algorithm (Yauy et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) were used for SNV filtering and prioritization. The pathogenicity of rare variants was then predicted using the online MobiDetails DNA Variant Interpretation tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://mobidetails.iurc.montp.inserm.fr/MD/\u003c/span\u003e\u003cspan address=\"https://mobidetails.iurc.montp.inserm.fr/MD/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eFor patient #3, MPS was performed as previously described (Mazel et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) using a custom design based on a SeqCap EZ Solution-Based Enrichment strategy (Roche NimbleGen) and paired-end sequencing 2 \u0026times; 150 bp on a Nextseq 500 (Illumina). Targeted regions included coding exons\u0026thinsp;\u0026plusmn;\u0026thinsp;50 bp and the 5\u0026prime; and 3\u0026prime; UTRs (list of genes available on request). Genomic alignment against the hg19/GRCh37 assembly and variant calling were done using BWA-MEM v.0.7.12 and GATK HaplotypeCaller v.3.4 (Broad Institute), respectively.\u003c/p\u003e\u003cp\u003eVariants were classified according to the American College of Medical Genetics (ACMG) guidelines (Richards et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The suspected syndrome-causing variant was confirmed by Sanger sequencing with the following primers: forward 5\u0026rsquo;-GTGCTGTACAGGAGCCGAAG-3\u0026rsquo;; reverse 5\u0026rsquo;-GGTAACTCCCTTTGGCTGC-3\u0026rsquo;.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn silico\u003c/b\u003e \u003cb\u003epredictions of upstream Open Reading Frames (uORF)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe Open Reading Frame Finder from NCBI (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/orffinder/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/orffinder/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) predicts ORFs from a pasted sequence and provides the predicted protein sequences from newly detected ORFs.\u003c/p\u003e\n\u003ch3\u003ePlasmids and transient transactivation experiments\u003c/h3\u003e\n\u003cp\u003eThe AR 5\u0026rsquo;UTRwt-HIS-GFP plasmid that contains the 5\u0026rsquo;UTR of AR was a gift from PL Holtertus (Hornig et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The AR 5\u0026rsquo;UTRwt-luciferase plasmid was created by inserting by PCR the 5\u0026rsquo;UTR of AR (-1117; +3) from the AR 5\u0026rsquo;UTRwt-HIS-GFP plasmid in the Kpn1 and Nco1 sites of the pGL4 luciferase reporter vector (Promega, Charbonni\u0026egrave;res-les Bains, France). The AR 5\u0026rsquo;UTRc-829C\u0026thinsp;\u0026gt;\u0026thinsp;T-luciferase and AR 5\u0026rsquo;UTRc-547C\u0026thinsp;\u0026gt;\u0026thinsp;T-luciferase plasmids were constructed by overlap extension PCR. The used primers are listed in supplementary Table\u0026nbsp;1.\u003c/p\u003e\u003cp\u003eU2OS cells (Merck, France) were grown in DMEM-F12 medium (Merck, France) supplemented with 10% fetal bovine serum (Sigma-Aldrich/Merck, France). Typically, 100,000 cells/well were plated in 12-well plates and transfected the day after using JetPEI (Dutscher, Bernolsheim, France) and 1.9 \u0026micro;g of AR 5\u0026rsquo;UTR-AR-luciferase plasmid with 0.1\u0026micro;g Renilla-luciferase reporter vector (Thermo Fisher Scientific, USA). After 48h, Firefly/Renilla luciferase activity was measured following the Dual-Luciferase Reporter Assay protocol (Promega) and a MicroBeta Wallac luminometer (PerkinElmer).\u003c/p\u003e\n\u003ch3\u003eQuantitative RT-PCR analysis\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from foreskin fibroblasts of a man who underwent posthectomy (control #1), from skin fibroblasts of another man (control #2) and from genital skin (labia majora) fibroblasts of patient #3 using a standard TRIzol/propanol protocol.\u003c/p\u003e\u003cp\u003eThen, RNA was reverse transcribed with MMLV reverse transcriptase (Invitrogen, 28025013). Quantitative PCR was carried out with a LightCycler\u0026reg; 480 System from Roche, using the LightCycler 480 SYBR Green I Master (Roche, 04887352001). The primers used were in exon 5 of \u003cem\u003eAR\u003c/em\u003e: F/5\u0026rsquo;-CATCCCTAAGGATACCCAGGGACCATC-3\u0026rsquo;, R/ 5\u0026rsquo;-GGATGGTCCCTGGGTATCCTTAGGGGATG-3\u0026rsquo;.\u003c/p\u003e\u003cp\u003e A written consent for medical research was obtained for the two healthy donors and patient #3.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEx vivo AR\u003c/b\u003e \u003cb\u003emRNA quantification\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTotal RNA from foreskin fibroblasts of control #1, from skin fibroblasts of control #2 and from genital skin fibroblasts of patient #3 was isolated using the RNA Now kit (Biogentex Laboratories, Inc., League City, TX, USA). RNA was reverse transcribed using random hexamers with the GeneAmp\u0026reg; RNA PCR kit (Thermo Fisher Scientific) following the manufacturer\u0026rsquo;s instructions. Then, \u003cem\u003eAR\u003c/em\u003e cDNA was PCR amplified using primers in exon 1 (A9 Fw 5\u0026lsquo;-GACTTCACCGCACCTGATGTGTGG-3\u0026rsquo;) and exon 8 (H2 Rv 5\u0026rsquo;- TTCCCCAAGGCACTGCAGAGGA-3\u0026rsquo;), with the Taq CORE Kit (MP Biomedicals) and the following touch-down program: initial denaturation at 94\u0026deg;C for 3 minutes; 14 cycles with denaturation at 94\u0026deg;C for 20 seconds, hybridization at 63\u0026deg;C for 40 seconds (-0.5\u0026deg;C at each cycle), extension at 72\u0026deg;C for 2 minutes; 26 cycles with denaturation at 94\u0026deg;C for 20 seconds, hybridization at 56\u0026deg;C for 40 seconds, extension at 72\u0026deg;C for 2 minutes; final extension at 72\u0026deg;C for 7 minutes. PCR products (patient #3 and two controls) were analyzed and compared with the LabChip\u0026reg; 90 system (Caliper Life Science, Hopkinton, MA, USA). Direct Sanger sequencing was performed using the same primers and the BigDye 1.1 Terminator sequencing kit on a ABI 3130 automated sequencer (Applied Biosystems, Foster City, CA, USA). Sequences were aligned using SeqScape V2.5.\u003c/p\u003e\n\u003ch3\u003eWestern blot analysis\u003c/h3\u003e\n\u003cp\u003eProtein extracts (30 \u0026micro;g) from genital skin (labia majora) fibroblasts from patient #3 and of preputial skin (posthectomy) from control #1 were separated by SDS-PAGE on NuPAGE 7% gels (Thermo Fisher Scientific France, Illkirch-Graffenstaden). To identify the predicted shorter AR protein, NuPAGE 15% gels (Thermo Fisher Scientific France, Illkirch-Graffenstaden) were used. After transfer, PVDF membranes (Thermo Fisher Scientific France, Illkirch-Graffenstaden) were incubated with antibodies against native AR (D6F11, Cell Signaling Technology, USA; 1/2000) and the short AR form pAR-62AA (custom synthesis by Eurogentec, Belgium, H-CQSATLSQPPSPPFS-NH2; 1/1000, 1/10 and 1/2).\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cb\u003eIdentification of the same 5\u0026rsquo;UTR mutation in the\u003c/b\u003e \u003cb\u003eAR\u003c/b\u003e \u003cb\u003egene in all three patients\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGenetic analysis of the three 46,XY patients with DSD revealed a novel c.-829C\u0026thinsp;\u0026gt;\u0026thinsp;T point mutation in the 5\u0026rsquo;UTR sequence of the \u003cem\u003eAR\u003c/em\u003e gene (RefSeq NM_000044.6) (Fig.\u0026nbsp;1A). According to gnomAD V3, this variant has not been reported in the general population, whatever the ethnicity.\u003c/p\u003e\u003cp\u003eNo other variant detected in the patients\u0026rsquo; DNA was considered pathogenic (data available on request).\u003c/p\u003e\u003cp\u003eAccording to the ACMG classification criteria, the new c.-829C\u0026thinsp;\u0026gt;\u0026thinsp;T variant could be considered as likely pathogenic (class 4, PS4, PM2, PS3 and PP4).\u003c/p\u003e\u003cp\u003eThe ORF Finder tool detected a new uORF due to the creation of an initiation codon in the presence of the c.-829C\u0026thinsp;\u0026gt;\u0026thinsp;T variant (Fig.\u0026nbsp;1B). This new AUG was located 296 bp downstream of the transcription start site (TSS) of the \u003cem\u003eAR\u003c/em\u003e gene. The new AUG is embedded in a vertebrate Kozak sequence (i.e., cXXATGG), with an expected A/GXXATGG consensus sequence. This could allow the recognition of the newly identified AUG by the translation machinery, theoretically leading to the production of a short protein of 62 amino acids, called AR-p62AA.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFunctional analysis of the\u003c/b\u003e \u003cb\u003eAR\u003c/b\u003e\u003cb\u003e-5\u0026rsquo;UTR mutation\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo functionally characterize the impact of the c-829C\u0026thinsp;\u0026gt;\u0026thinsp;T point mutation, the wild type (wt) or mutated (c-547C\u0026thinsp;\u0026gt;\u0026thinsp;T and c-829C\u0026thinsp;\u0026gt;\u0026thinsp;T) AR 5\u0026rsquo;UTR was cloned directly upstream of the luciferase reporter gene and the obtained plasmids were transfected in U2OS cells (Fig.\u0026nbsp;2A-B). Luciferase activity was decreased in cells that express the AR 5\u0026rsquo;UTRc-829C\u0026thinsp;\u0026gt;\u0026thinsp;T compared with the AR 5\u0026lsquo;UTRwt (Fig.\u0026nbsp;2C) and also compared with the previously described disease-causing AR 5\u0026rsquo;UTR variant (AR 5\u0026rsquo;UTRc-547C\u0026thinsp;\u0026gt;\u0026thinsp;T), used as positive control.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAR\u003c/b\u003e \u003cb\u003emRNA quantity and quality\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThen, RT-qPCR was used to quantify \u003cem\u003eAR\u003c/em\u003e mRNA quantity in foreskin and skin fibroblasts from controls #1 and #2, respectively, and genital skin fibroblasts of patient #3. This analysis did not show any significant difference in \u003cem\u003eAR\u003c/em\u003e mRNA quantity (Fig.\u0026nbsp;3A). To determine whether any additional syndrome-causing variant located in intronic regions in linkage disequilibrium with the c.-829C\u0026thinsp;\u0026gt;\u0026thinsp;T mutation was present, \u003cem\u003eAR\u003c/em\u003e transcripts in genital fibroblasts from patient #3 and in foreskin and skin fibroblasts from controls #1 and #2, respectively, were quantitatively analyzed by touch-down PCR to cover all exons (Fig.\u0026nbsp;3B). No additional peak was detected compared to the \u003cem\u003eAR\u003c/em\u003e transcript pattern in the two controls, confirming that only the full \u003cem\u003eAR\u003c/em\u003e transcript was transcribed, without any detectable aberrant spliced form (Fig.\u0026nbsp;3C).\u003c/p\u003e\n\u003ch3\u003eAR protein analysis\u003c/h3\u003e\n\u003cp\u003eGiven the discrepancy between the severe phenotype with complete feminization and the low effect of the mutation on AR transcriptional activity and mRNA quality and quantity, AR protein expression was assessed by western blotting. AR protein level (native form; 140 kDa) was decreased in genital skin fibroblasts from patient #3 compared with control #1 (Fig.\u0026nbsp;4A). In addition, the intensity of a high molecular weight (~\u0026thinsp;170 kDa) band was significantly increased in patient #3. However, as the average weight of an amino acid is \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\sim110\\:\\text{D}\\text{a},\\)\u003c/span\u003e\u003c/span\u003e the putative AR-p62AA protein should approximatively weight \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:62\\cdot\\:110\\:\\text{D}\\text{a}\\:=\\:6.82\\:\\text{k}\\text{D}\\text{a}\\)\u003c/span\u003e\u003c/span\u003e. Western blot analysis using an antibody against the AR-p62AA protein did not detect any band between 5 kDa and 10 kDa (Fig.\u0026nbsp;4B).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study describes a new 5\u0026rsquo;UTR variant in the \u003cem\u003eAR\u003c/em\u003e (c.-829C\u0026thinsp;\u0026gt;\u0026thinsp;T) detected in three patients with CAIS without mutations in \u003cem\u003eAR\u003c/em\u003e coding region. The three patients had inguinal hernia: in patients #1 and #3 it appeared in the neonatal period and in patient #2 at the age of 10 years. In the presence of inguinal hernia, CAIS was only considered in patients #1 and #3. Patient #2 did not seek medical attention until much later, although she had primary amenorrhea. The molecular diagnosis of CAIS was only confirmed in adulthood in patients #1 and #2 (37 and 32 years, respectively), because patient #1 was lost to follow-up for several years and patient #2 was too embarrassed to consult earlier about her sexual difficulties. The association between inguinal hernia and CAIS in girls is well known (Sultan et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and was a major reason for early referral in several case reports (Konar et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Listyasari et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Nair and Bhavana \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Sharma et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and in large series (Hurme et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Sarpel et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). For instance, Deeb found that 57% of 120 patients with CAIS presented with inguinal hernia, thus the most frequent mode of clinical presentation of CAIS in childhood (Deeb and Hughes \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Inguinal hernia in CAIS is explained by the role of testosterone and its receptor AR in the second phase of testicular descent (Hutson et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Therefore, irrespective of the age at which the inguinal hernia appears, the diagnosis of CAIS should be considered.\u003c/p\u003e\u003cp\u003eFor the three patients reported here, \u003cem\u003eAR\u003c/em\u003e coding sequence was first analyzed using the Sanger method, but no syndrome-causing variant was identified. However, the clinical, radiological and hormonal phenotype clearly indicated resistance to androgens. Thus, MPS was used. This approach allowed identifying the c.-829C\u0026thinsp;\u0026gt;\u0026thinsp;T variant in the 5\u0026rsquo;UTR of \u003cem\u003eAR\u003c/em\u003e. The absence of other potentially causative variants and of any aberrant splicing pattern was confirmed by MPS and touch-down PCR.\u003c/p\u003e\u003cp\u003eVariants in the non-coding sequences of the \u003cem\u003eAR\u003c/em\u003e gene have been rarely detected in patients with androgen insensitivity syndrome. The McGill database (Gottlieb et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) contains only few DNA variants in the 5\u0026rsquo;UTR of \u003cem\u003eAR\u003c/em\u003e involved in prostate cancer. A pathogenic insertion of a LINE-1 retrotransposon in the 5\u0026rsquo;UTR of \u003cem\u003eAR\u003c/em\u003e has been associated with partial androgen insensitivity syndrome in nine XY individuals from the same family (Batista et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). To our knowledge, a syndrome-causing SNV in this region has been reported only once in two unrelated patients with CAIS (Hornig et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The authors showed that this upstream variant (c.-547C\u0026thinsp;\u0026gt;\u0026thinsp;T) creates a novel translation start site (AUG) located downstream the TSS, thus limiting the translation of the native AR form. Here, \u003cem\u003ein silico\u003c/em\u003e prediction tools suggested that the novel c.-829C\u0026thinsp;\u0026gt;\u0026thinsp;T variant induces the recognition of another alternative AUG (located upstream the native AUG) and the production of a short protein of 62 amino acids, called AR-p62AA. However, this short protein could not be detected by western blotting. Yet, the three patients had a markedly severe CAIS phenotype. Therefore, it seems evident that AR biology was strongly affected. Western blot analysis of genital skin fibroblasts from patient #3 showed the presence of a band of the expected molecular weight for AR, the intensity of which was strongly decreased compared with the control. This suggests a decrease of the AR protein amount in patient #3, which could partly explain the observed phenotype. In addition, a high molecular weight band (~\u0026thinsp;170 kDa) was over-represented compared with the control. This high-molecular-weight band may indicate the presence of polyubiquitinated AR. Ubiquitination is a post-translational modification that involves the conjugation of ubiquitin, a highly conserved peptide of 76 residues, alone or in a polymerized chain (Komander and Rape \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Ubiquitination has many functions, ranging from labeling proteins to be degraded to modifying the interaction pattern of the ubiquitinylated protein. Considering the observed phenotype, this finding could suggest AR polyubiquitination, resulting in increased AR degradation. Furthermore, polyubiquitination has other effects that could influence AR functionality. These include confinement of proteins to the cytosol and impairment of the interaction with some partners. This could affect AR binding to HSP90, which is required to regulate AR binding affinity. Additionally, if due to confinement in the cytosol, AR could not enter the nucleus and dimerize, it would lose its transcription-activating function. These effects would reinforce the particularly low bioavailability of AR, which could be consistent with the patients' severe phenotype. The presence of this high-molecular weight band suggests that other post-translational modifications (e.g. acetylation or phosphorylation) are not implicated, but does not exclude sumoylation. This modification is nuclear and is mostly initiated by DNA damage and stress (Hendriks and Vertegaal \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). As sumoylation modifies the interacting characteristics of the protein, AR could no longer undergo dimerization, resulting in impaired transcriptional activity, which may partly explain the observed phenotype. However, the decrease in AR protein amount clearly observed in patient #3 tends to exclude sumoylation.\u003c/p\u003e\u003cp\u003eThe western blot analysis and the luciferase activity assays gave contradictory results. However, the luciferase assays were obtained in U2OS cells that like other cell lines (e.g., HEK 293 cells) generally used for this kind of assay, are immortalized or cancer cells with karyotypic and/or genetic alterations and are considered \u0026ldquo;protein-producing machines\u0026rdquo; (Pont\u0026eacute;n \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1967\u003c/span\u003e; Stepanenko and Dmitrenko \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The quality-controls for RNAs and proteins, as well as the regulatory mechanisms for transcription and translation, may be modified in U2OS cells, which makes it challenging to extrapolate the results to \u003cem\u003ein vivo\u003c/em\u003e conditions.\u003c/p\u003e\u003cp\u003eAlthough ubiquitination is the most attractive hypothesis, the precise relationship between ubiquitination and the mutation remains unclear because the \u003cem\u003ein silico\u003c/em\u003e methods predicted the emergence of a short protein variant that was not identified by western blotting. The \u003cem\u003ein silico\u003c/em\u003e analysis, which was not exhaustive, may have overlooked alternative translation initiation sequences, apart from the Kozak sequences, which may be dependent on the cell type or cellular conditions. Moreover, putative new motifs that can act as recognition motifs for degradation-associated enzymes and accessory proteins were not actively searched.\u003c/p\u003e\u003cp\u003eOn the other hand, it could be hypothesized that the mutation does not affect the protein but the mRNA. For example, it would be conceivable that the emergence of novel secondary structures within the 5'UTR of mRNA may destabilize ribosomes and activate RNA quality control systems, leading to increased mRNA degradation. Although the eIF4A factor displays RNA helicase activity that can undo secondary structures on RNA (Merrick and Pavitt \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), a new structure may be interpreted as an anomaly and linked by factors to mRNA delivery and confinement in P-bodies. However, RT-qPCR did not find any difference in \u003cem\u003eAR\u003c/em\u003e mRNA levels between patient #3 and the healthy controls. Furthermore, cDNA sequencing did not reveal any mRNA sequence abnormality or aberrant splicing pattern. Collectively, these results tend to demonstrate that \u003cem\u003eAR\u003c/em\u003e transcription and mRNA are not affected by the c.-829C\u0026thinsp;\u0026gt;\u0026thinsp;T mutation. This reinforces the hypothesis that the problem concerns the AR protein metabolism/degradation. A significant decrease in AR protein bioavailability due to an increased degradation is biologically consistent with the CAIS phenotype.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study reports the discovery of a new point mutation, c.-829C\u0026thinsp;\u0026gt;\u0026thinsp;T, in the 5\u0026rsquo; UTR of the \u003cem\u003eAR\u003c/em\u003e gene in three unrelated patients with similar CAIS phenotype. Although the \u003cem\u003ein silico\u003c/em\u003e tools predicted that c.-829C\u0026thinsp;\u0026gt;\u0026thinsp;T induces the recognition of an alternative AUG and translation of a short protein of 62 amino acids called AR-p62AA, this new isoform could not be detected by western blotting. The presence of a high molecular AR form associated with a marked decrease in the native AR protein led to the hypothesis that AR ubiquitination is responsible for its increased degradation. However, the link between the c.-829C\u0026thinsp;\u0026gt;\u0026thinsp;T variant and ubiquitination was not demonstrated. As far as we know, this is the second report of patients with CAIS harboring a 5\u0026rsquo;UTR SNV of the \u003cem\u003eAR\u003c/em\u003e gene. This work contributes to increase awareness of CAIS with a normal AR coding sequence and underlines the need to analyze also non-coding regions of the \u003cem\u003eAR\u003c/em\u003e gene in typical CAIS. The presence of the c.-829C\u0026thinsp;\u0026gt;\u0026thinsp;T variant in the \u003cem\u003eAR\u003c/em\u003e gene should orient the diagnosis toward CAIS.\u003c/p\u003e\u003cp\u003eWe declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.\u003c/p\u003e\u003cp\u003eWe would like to warmly thank Professor Holterhus for help with the primer design as well as Professor Didier Bessis and Doctor Boris Delaunay for collecting the skin/foreskin samples from the healthy male controls.\u003c/p\u003e\u003cp\u003eThis research did not receive any specific grant from any funding agency in the public, commercial or non-profit sector.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eAnne Bergougnoux and Guillaume Perez are co-first authorsAll the authors contributed to writing the manuscript.Anne Bergougnoux, Guillaume Perez, Fran\u0026ccedil;oise Paris, Delphine Mallet, Aurelie Gennetier, Abdlhay Boulahtof, Patrick Balaguer and Nad\u0026egrave;ge Servant prepared the figures.Fran\u0026ccedil;oise Paris prepared the Table 1All authors reviewed the manuscript\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eWe would like to warmly thank Professor Holterhus for help with the primer design as well as Professor Didier Bessis and Doctor Boris Delaunay for collecting the skin/foreskin samples from the healthy male controls.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdachi M, Takayanagi R, Tomura A, Imasaki K, Kato S, Goto K, Yanase T, Ikuyama S, Nawata H (2000) Androgen-insensitivity syndrome as a possible coactivator disease. N Engl J Med 343: 856-62. doi: 10.1056/nejm200009213431205\u003c/li\u003e\n\u003cli\u003eBatista RL, Yamaguchi K, Rodrigues ADS, Nishi MY, Goodier JL, Carvalho LR, Domenice S, Costa EMF, Kazazian HH, Mendonca BB (2019) Mobile DNA in Endocrinology: LINE-1 Retrotransposon Causing Partial Androgen Insensitivity Syndrome. J Clin Endocrinol Metab 104: 6385-6390. doi: 10.1210/jc.2019-00144\u003c/li\u003e\n\u003cli\u003eDeeb A, Hughes IA (2005) Inguinal hernia in female infants: a cue to check the sex chromosomes? BJU Int 96: 401-3. doi: 10.1111/j.1464-410X.2005.05639.x\u003c/li\u003e\n\u003cli\u003eEisermann K, Wang D, Jing Y, Pascal LE, Wang Z (2013) Androgen receptor gene mutation, rearrangement, polymorphism. Transl Androl Urol 2: 137-147. doi: 10.3978/j.issn.2223-4683.2013.09.15\u003c/li\u003e\n\u003cli\u003eGeorget V, Bourguet W, Lumbroso S, Makni S, Sultan C, Nicolas JC (2006) Glutamic acid 709 substitutions highlight the importance of the interaction between androgen receptor helices H3 and H12 for androgen and antiandrogen actions. Mol Endocrinol 20(4): 724-34.\u003c/li\u003e\n\u003cli\u003eGottlieb B, Beitel L, Nadarajah A, Paliouras M, Trifiro M (2012) The androgen Receptotr gene mutations database: 2012 update. http://www.androgendb.mcgill.ca, last update 2013. Hum Mutat 33: 887-94. \u003c/li\u003e\n\u003cli\u003eHendriks IA, Vertegaal AC (2016) A comprehensive compilation of SUMO proteomics. Nat Rev Mol Cell Biol 17: 581-95. doi: 10.1038/nrm.2016.81\u003c/li\u003e\n\u003cli\u003eHornig NC, de Beaufort C, Denzer F, Cools M, Wabitsch M, Ukat M, Kulle AE, Schweikert HU, Werner R, Hiort O, Audi L, Siebert R, Ammerpohl O, Holterhus PM (2016) A Recurrent Germline Mutation in the 5\u0026apos;UTR of the Androgen Receptor Causes Complete Androgen Insensitivity by Activating Aberrant uORF Translation. PLoS One 11: e0154158. doi: 10.1371/journal.pone.0154158\u003c/li\u003e\n\u003cli\u003eHurme T, Lahdes-Vasama T, Makela E, Iber T, Toppari J (2009) Clinical findings in prepubertal girls with inguinal hernia with special reference to the diagnosis of androgen insensitivity syndrome. Scand J Urol Nephrol 43: 42-6. doi: 10.1080/00365590802299247\u003c/li\u003e\n\u003cli\u003eHutson JM, Li R, Southwell BR, Newgreen D, Cousinery M (2015) Regulation of testicular descent. Pediatr Surg Int 31: 317-25. doi: 10.1007/s00383-015-3673-4\u003c/li\u003e\n\u003cli\u003eK\u0026auml;ns\u0026auml;koski J, J\u0026auml;\u0026auml;skel\u0026auml;inen J, J\u0026auml;\u0026auml;skel\u0026auml;inen T, Tommiska J, Saarinen L, Lehtonen R, Hautaniemi S, Frilander MJ, Palvimo JJ, Toppari J, Raivio T (2016) Complete androgen insensitivity syndrome caused by a deep intronic pseudoexon-activating mutation in the androgen receptor gene. Sci Rep 6: 32819. doi: 10.1038/srep32819\u003c/li\u003e\n\u003cli\u003eKomander D, Rape M (2012) The ubiquitin code. Annu Rev Biochem 81: 203-29. doi: 10.1146/annurev-biochem-060310-170328\u003c/li\u003e\n\u003cli\u003eKonar S, Dasgupta D, Patra DK, De A, Mallick B (2015) Chromosomal Study is Must for Prepubertal Girl with Inguinal Hernia: Opportunity to Diagnose Complete Androgen Insensitivity Syndrome. J Clin Diagn Res 9: GD01-3. doi: 10.7860/jcdr/2015/11411.5750\u003c/li\u003e\n\u003cli\u003eListyasari NA, Robevska G, Santosa A, Bouty A, Juniarto AZ, van den Bergen J, Ayers KL, Sinclair AH, Faradz SM (2019) Genetic Analysis Reveals Complete Androgen Insensitivity Syndrome in Female Children Surgically Treated for Inguinal Hernia. J Invest Surg: 1-7. doi: 10.1080/08941939.2019.1602690\u003c/li\u003e\n\u003cli\u003eMazel B, Mallet D, Roucher-Boulez F, Signor CB, Bournez M, Darmency V, Bourgeois V, Poe C, El Khabbaz F, Vitobello A, Philippe C, Duffourd Y, Thauvin-Robinet C, Faivre L, Nambot S (2022) Epileptic encephalopathy as a new feature of the sudden infant death with dysgenesis of the testes syndrome caused by TSPYL1 variants. Am J Med Genet A 188: 3540-3545. doi: 10.1002/ajmg.a.62966\u003c/li\u003e\n\u003cli\u003eMerrick WC, Pavitt GD (2018) Protein Synthesis Initiation in Eukaryotic Cells. Cold Spring Harb Perspect Biol 10. doi: 10.1101/cshperspect.a033092\u003c/li\u003e\n\u003cli\u003eMongan NP, Tadokoro-Cuccaro R, Bunch T, Hughes IA (2015) Androgen insensitivity syndrome. Best Pract Res Clin Endocrinol Metab 29: 569-80. doi: 10.1016/j.beem.2015.04.005\u003c/li\u003e\n\u003cli\u003eNair RV, Bhavana S (2012) XY Female with Complete Androgen Insensitivity Syndrome with Bilateral Inguinal Hernia. J Obstet Gynaecol India 62: 65-7. doi: 10.1007/s13224-013-0379-1\u003c/li\u003e\n\u003cli\u003eOakes MB, Eyvazzadeh AD, Quint E, Smith YR (2008) Complete androgen insensitivity syndrome--a review. J Pediatr Adolesc Gynecol 21: 305-10. doi: 10.1016/j.jpag.2007.09.006\u003c/li\u003e\n\u003cli\u003eOno H, Saitsu H, Horikawa R, Nakashima S, Ohkubo Y, Yanagi K, Nakabayashi K, Fukami M, Fujisawa Y, Ogata T (2018) Partial androgen insensitivity syndrome caused by a deep intronic mutation creating an alternative splice acceptor site of the AR gene. Sci Rep 8: 2287. doi: 10.1038/s41598-018-20691-9\u003c/li\u003e\n\u003cli\u003ePhilibert P, Audran F, Pienkowski C, Morange I, Kohler B, Flori E, Heinrich C, Dacou-Voutetakis C, Joseph MG, Guedj AM, Journel H, Hecart-Bruna AC, Khotchali I, Ten S, Bouchard P, Paris F, Sultan C (2010) Complete androgen insensitivity syndrome is frequently due to premature stop codons in exon 1 of the androgen receptor gene: an international collaborative report of 13 new mutations. Fertil Steril 94: 472-6.\u003c/li\u003e\n\u003cli\u003ePont\u0026eacute;n J (1967) Spontaneous lymphoblastoid transformation of long-term cell cultures from human malignant lymphoma. Int J Cancer 2: 311-25. doi: 10.1002/ijc.2910020406\u003c/li\u003e\n\u003cli\u003ePoujol N, Lumbroso S, Makni S, Terouanne B, Lobaccaro J, Bourguet W, Sultan C (2002) Pathophysiology of androgen insensitivity syndromes: molecular and structural approaches of natural and engineered androgen receptor mutations at amino acid 743. J Clin Endocrinol Metab 87(12): 5793-800.\u003c/li\u003e\n\u003cli\u003eQuigley CA, De Bellis A, Marschke KB, el-Awady MK, Wilson EM, French FS (1995) Androgen receptor defects: historical, clinical, and molecular perspectives. Endocr Rev 16: 271-321. doi: 10.1210/edrv-16-3-271\u003c/li\u003e\n\u003cli\u003eRichards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, Grody WW, Hegde M, Lyon E, Spector E, Voelkerding K, Rehm HL (2015) Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 17: 405-24. doi: 10.1038/gim.2015.30\u003c/li\u003e\n\u003cli\u003eSarpel U, Palmer SK, Dolgin SE (2005) The incidence of complete androgen insensitivity in girls with inguinal hernias and assessment of screening by vaginal length measurement. J Pediatr Surg 40: 133-6; discussion 136-7. doi: 10.1016/j.jpedsurg.2004.09.012\u003c/li\u003e\n\u003cli\u003eSharma V, Singh R, Thangaraj K, Jyothy A (2011) A novel Arg615Ser mutation of androgen receptor DNA-binding domain in three 46,XY sisters with complete androgen insensitivity syndrome and bilateral inguinal hernia. Fertil Steril 95: 804 e19-21. doi: 10.1016/j.fertnstert.2010.08.015\u003c/li\u003e\n\u003cli\u003eStepanenko AA, Dmitrenko VV (2015) HEK293 in cell biology and cancer research: phenotype, karyotype, tumorigenicity, and stress-induced genome-phenotype evolution. Gene 569: 182-90. doi: 10.1016/j.gene.2015.05.065\u003c/li\u003e\n\u003cli\u003eSultan C, Philibert P, Gaspari L, Audran F, Maimoun L, Kalfa N, Paris F (2014) Androgen Insensivity Syndrome. Genetic Steroid Disorders Chapter 5: 225-237.\u003c/li\u003e\n\u003cli\u003eTadokoro-Cuccaro R, Hughes IA (2014) Androgen insensitivity syndrome. Curr Opin Endocrinol Diabetes Obes 21: 499-503. doi: 10.1097/med.0000000000000107\u003c/li\u003e\n\u003cli\u003eYauy K, Baux D, Pegeot H, Van Goethem C, Mathieu C, Guignard T, Juntas Morales R, Lacourt D, Krahn M, Lehtokari VL, Bonne G, Tuffery-Giraud S, Koenig M, Coss\u0026eacute;e M (2018) MoBiDiC Prioritization Algorithm, a Free, Accessible, and Efficient Pipeline for Single-Nucleotide Variant Annotation and Prioritization for Next-Generation Sequencing Routine Molecular Diagnosis. J Mol Diagn 20: 465-473. doi: 10.1016/j.jmoldx.2018.03.009\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"androgen receptor, complete androgen insensitivity syndrome (CAIS), non-coding gene sequence, 5’UTR variant, differences of sexual development","lastPublishedDoi":"10.21203/rs.3.rs-8151404/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8151404/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA syndrome-causing androgen receptor (\u003cem\u003eAR)\u003c/em\u003e gene variant is identified in \u0026gt;\u0026thinsp;95% of 46,XY individuals with a female phenotype due to complete androgen insensitivity syndrome (CAIS). Here, we describe three patients (two adults, 37 and 32 years of age, and a 14-year-old teenager) with CAIS harboring a new 5\u0026rsquo;UTR variant of \u003cem\u003eAR\u003c/em\u003e. Sanger sequencing of the \u003cem\u003eAR\u003c/em\u003e coding region did not identify any known syndrome-causing variant. Massive parallel sequencing of genes, known to be involved in differences in sexual development, and their regulatory regions identified a novel c.-829C\u0026thinsp;\u0026gt;\u0026thinsp;T variant in the 5\u0026rsquo;UTR sequence of \u003cem\u003eAR\u003c/em\u003e in all three patients. The ORF Finder software predicted the use of a new AUG codon located 296 bp downstream of the transcription start site (not confirmed by western blotting). Luciferase activity was slightly decreased in U2SO cells after transfection of the AR 5\u0026rsquo;UTR-c.-829C\u0026thinsp;\u0026gt;\u0026thinsp;T construct, but this could not explain the CAIS phenotype. Western blotting with an anti-AR antibody showed increased expression of a high molecular weight band and a decrease of the native AR protein. Aberrant splicing and mRNA level alterations were not detected. This study identified the c.-829C\u0026thinsp;\u0026gt;\u0026thinsp;T AR variant in three unrelated patients with CAIS. The functional analysis suggests that a posttranslational modification in AR may increase its molecular weight. The reduced bioavailability of the native AR protein could explain CAIS in these three patients. This second 5\u0026rsquo;UTR-coding sequence variant highlights the need to analyze \u003cem\u003eAR\u003c/em\u003e exons and non-coding regions in all patients with CAIS.\u003c/p\u003e","manuscriptTitle":"A novel variant in the 5’ UTR of the androgen receptor gene without coding region alterations in three patients with complete androgen insensitivity syndrome","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-01 07:49:44","doi":"10.21203/rs.3.rs-8151404/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"74dff895-2ae0-42e8-9af3-45e33ef867eb","owner":[],"postedDate":"December 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-19T02:53:52+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-01 07:49:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8151404","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8151404","identity":"rs-8151404","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}