XY Female Patient Research Articles

Identification of a novel mutation (Ala66Thr) of SRY gene causes XY pure gonadal dysgenesis by affecting DNA binding activity and nuclear import

Sex-determining region of the Y chromosome (SRY) gene plays a crucial role in male sexual differentiation and development. Several mutations in the SRY gene have been reported in the high mobility group (HMG) box domain and can cause gonadal dysgenesis symptoms. In this study, we report that a novel missense mutation in the SRY gene, a G to A transition within the HMG box, causes the Ala66Thr amino acid substitution in a female patient presenting 46,XY karyotype with pure gonadal dysgenesis. The G to A base transition was not found in the SRY sequence after the screening of 100 normal males. Furthermore, Ala66Thr mutation drastically reduced the binding capacity of SRY to DNA sequences, whereas wild-type SRY protein showed the normal binding capacity to DNA sequences in vitro. We also found that the mutant SRY protein was partly localized in cytoplasm, whereas wild-type SRY protein was strictly localized in cell nucleus. In addition, we analyzed the three-dimensional structure of SRY protein by homology modeling methods. In conclusion, we identified a novel SRY mutation in a 46,XY female patient with pure gonadal dysgenesis, demonstrating the importance of the Ala66Thr mutation in DNA binding activity and nuclear transport.

Functional Analysis of Mutations at Codon 127 of the SRY Gene Associated with 46,XY Complete Gonadal Dysgenesis

Complete gonadal dysgenesis (CGD) is characterized by an incomplete differentiation of the genital organs in a patient with a 46,XY karyotype. It is induced by mutations in the sex-determining region Y (SRY) gene which plays a key role in testis-determining pathways. The aim of this study was to investigate the possible pathogenic nature of a novel SRY mutation (p.Y127H) identified in a 46,XY female patient. To determine the effect of this mutation on SRY function, we studied its impact on DNA interaction by electrophoretic mobility shift assays. Since tyrosine 127 is close to the C-terminal nuclear localization signal of SRY, we conducted HA-SRY protein expression to observe the impact of the mutation on the nuclear import function in transfected cells. Our results showed that the Y127H mutation nearly abolishes the DNA-binding capacity of SRY and strongly impairs the nuclear localization of the mutated protein. Together with a previously described mutation analyzed in parallel in this paper (p.Y127C), our results highlight this tyrosine residue as a crucial structural determinant of the high mobility group box domain. This is the first report to explain the molecular mechanism of the Y127H mutation causing sex reversal and gives new insights for clinical practice to benefit patients with disorders of sex development.

Three novel and two known androgen receptor gene mutations associated with androgen insensitivity syndrome in sex-reversed XY female patients.

Molecular characterization of 23 cytogenetically confirmed XY females was attempted by screening coding regions of SRY and androgen receptor (AR) genes. Five of the index cases showed sequence variations in various exons of the AR gene: a deletion (n.1911delG) and substitutions n.1761G>A and n.1317C>T in exon 1; n.3510C>T transition in exon 6 and deletion mutation (n.3672delT) in exon 7. Four mutations identified here lead to the formation of truncated receptor protein, involving a substantial loss of AR functional domains which explains the phenotype in the subjects. The n.1761G>A substitution has been previously reported in cases with mild androgen insensitivity. Although the ligand-binding domain was considered as the mutational hot spot in AR gene, we report here 3/5 variations in the N-terminal domain emphasizing the significance of considering the N-terminal domain of AR as well for mutation screening. Our present observation also strengthens the role of AR gene and its direct association with AIS.

A novel mutation in the SRY gene of a Chinese 46, XY female patient with unilateral mixed germ cell tumor

Objective To determine the nosogenetic factors of a 46,XY female with primary amenorrhea and unilateral mixed germ cell tumor. Methods Eight genes associated with 46,XY gonadal dysgenesis were detected in the patient and her parents by target region captured-next generation sequencing. Results An insertion of a single nucleotide (adenine) at the coding site 230 (c.230_231insA) located in the high mobility group (HMG) domain of SRY was revealed, which led to a truncated protein (p.Lys77fsX27). This mutation was at position 2655414 of the Y chromosome, supported with 127 unique mapped reads, however, this mutation was not found in the in-house dataset of 1 092 controls. Additionally, none of the candidate gene was detected in the patient’s parents, which indicated that it is a de novo mutation. Conclusion A novel SRY sporadic mutation due to a single nucleotide insertion at position 230 (c.230_231insA) was identified as the cause of the disease in this patient. Target region captured-next generation sequencing was found to be an effective method for the molecular genetic testing of 46,XY complete gonadal dysgenesis (46,XY CGD).

Growth morbidity in patients with cloacal exstrophy: a 42-year experience

PurposeCloacal exstrophy is associated with multiple comorbidities that affect growth. This report describes long-term growth outcomes in a large cohort of patients with cloacal exstrophy and explores associated comorbidities. MethodsRecords of 71 patients with cloacal exstrophy who were treated between 1974 and 2015 were reviewed, and 62 patients with growth data from 2 to 20years of age were included. Genetic sex, gender of rearing, and all heights, weights, and comorbidities were noted for each patient. Height-for-age, weight-for-age, and body mass index z-scores (HAZ, WAZ, and BMIZ) were determined using US Centers for Disease Control 2000 growth data, and average patient z-scores were calculated. ResultsThere were 904 height and 1301 weight measurements available for 62 patients. 31 were genetically 46,XY, 21 of whom underwent gonadectomy in infancy and were raised female. 46,XX patients, 46,XY male patients, and 46,XY female patients all had median HAZ and WAZ substantially lower than the general population, with median HAZ less than −2, while maintaining normal BMIZ. Short bowel syndrome and enterocystoplasty with intestine were associated with lower z-scores for all parameters. ConclusionsPatients with cloacal exstrophy have significant multifactorial long-term growth failure. These benchmark data can be used to further optimize management.Level of evidence: 2b

Mutational screening of the NR5A1 in azoospermia.

Nuclear receptor subfamily 5 group A member 1 (NR5A1) encodes a nuclear receptor that regulates transcription of multiple genes involved in adrenal and gonadal development, steroidogenesis and the reproductive axis. Human mutations in NR5A1were initially found in two 46, XY female patients suffering from severe gonadal dysgenesis and primary adrenal failure. However, more recent case reports have suggested that heterozygous mutations in NR5A1 may also contribute to the male infertility aetiology. We have analysed the coding sequence of NR5A1 in a cohort of 90 well-characterised idiopathic Iranian azoospermic infertile men versus 112 fertile men. Heterozygous NR5A1 mutations were found in 2 of 90 (2.2%) of cases. These two patients harboured missense mutations within the hinge region (p.P97T) and ligand-binding domain (p.E237K) of the NR5A1 protein. In silico analysis of the mutations showed that founded mutations could be detrimental. In conclusion, findings of the current and previous studies suggest that mutations in the NR5A1 gene are not common in azoospermia, and male infertility and inclusion of NR5A1 mutation screening in the diagnostic workup of male infertility may seem unnecessary.

Gonadal tumor incidence in 45,X/46,XY and 46,XY female patients: experience from one clinical center

Gonadal tumor incidence in 45,X/46,XY and 46,XY female patients: experience from one clinical center

A novel mutation (c.T3816 > C) in the androgen receptor gene in a 46,XY female patient with androgen insensitivity syndrome

Androgen receptor (AR) gene mutations are the most frequent cause of 46,XY disorders of sex development (DSD), and are associated with a variety of phenotypes ranging from phenotypic women (Complete Androgen Insensitivity Syndrome or CAIS) to milder degrees of undervirilisation (Partial Androgen Insensitivity Syndrome or PAIS) or men with infertility only (Mild Androgen Insensitivity Syndrome or MAIS). In this paper, we present the results of clinical, endocrine and molecular trials in a patient hospitalised because of primary amenorrhoea with typical phenotype of CAIS. The main objective of this study was to determine the molecular cause of androgen insensitivity syndrome in a 46,XY female patient. Molecular analysis of the AR gene was conducted using MSSCP (Multitemperature Single Strand Conformation Polymorphism) and sequencing methods. MSSCP analysis showed changes in electrophoretic mobility in exon 8 of the AR gene. Sequencing analysis revealed a missense mutation which has not been previously described. This is a c.T3816 > C transition mutation which causes a S901P substitution in the amino acid chain (based on the latest NCBI reference sequence NM 000044.2). The identified c.T3816 > C mutation in AR gene provides further evidence for the correlation between specific AR mutations and phenotype corresponding to androgen insensitivity.

A novel mutation (c. 341A>G) in the SRY gene in a 46,XY female patient with gonadal dysgenesis

SRY (sex-determining region Y) gene, MIM 480000, NM_005634) is crucial for sex differentiation which encodes the protein responsible for initiating testis differentiation. SRY mutations are associated with the presence of XY gonadal dysgenesis symptoms.We studied a 46,XY female patient with primary amenorrhoea and negative family history. The clinical, endocrine, histopathologic and cytogenetic data are consistent with gonadal dysgenesis.Using a molecular analysis, a novel (c.341A>G, p. N65D) missense mutation within the HMGbox of SRY gene was detected.Escherichia coli expression of SRY study showed reduced expression of the mutated protein and gel retardation assay method revealed lowered DNA-binding ability in N65D variant of SRY.The novel mutation detected in the SRY gene may be an aetiopathogenic factor in clinically defined 46,XY complete gonadal dysgenesis (CGD).Because of an increased risk of gonadoblastoma, proper early diagnosis and treatment prevent development of malignancies.

Novel mutation of the sex‐determining region on the Y chromosome in a 46,XY female patient with monolateral dysgerminoma: A case report

Patients with 46,XY complete gonadal dysgenesis (Swyer syndrome) are characterized by the presence of female internal genital tract and bilateral streak gonads in a phenotypic female. These women have a high risk of developing rare type II malignant germ cell tumors. We report a rare case of a 33-year-old 46,XY female patient, who presented with an adnexal mass suspected for dysgerminoma, with a novel mutation of the sex-determining region on the Y chromosome consisting in the variant c.301C> G (p.L101V). Considering that effective screening is not available and the high risk of developing malignant neoplasm, prophylactic gonadectomy is mandatory.

A novel mutation of SRY gene identified in a 46,XY complete gonadal dysgenesis patient

The clinical and genetic characteristics in a patient with 46,XY complete gonadal dysgenesis was investigated. Clinical features and laboratory data were collected from the patient and the family. The exon of SRY gene was amplified by PCR and sequenced. The patient presented with primary amenorrhea, nonambiguous female external genitalia, slight breast development, and no axillary hair or pubic hair. The female internal reproductive organs consisted of uterus and streaks of ovarian tissue. Howerver, the chromosome karyotype was 46,XY. A missense mutation of A66T in SRY gene was identified, which was not previously reported. The novel SRY mutation caused the sex reversal in this 46,XY female patient. Key words: Sex reversal; Complete gonadal dysgenesis; Swyer syndrome; SRY gene; Mutation

Three novel CYP17A1 gene mutations (A82D, R125X, and C442R) found in combined 17α-hydroxylase/17,20-lyase deficiency

The aim of this study was to analyze the structure and functional consequences of 3 novel mutations (A82D, R125X, and C442R) of the CYP17A1 gene found in 2 patients with combined 17α-hydroxylase/17,20-lyase deficiency (17OHD). Two Chinese 46, XY female patients were diagnosed with 17OHD based on clinical findings and biochemical results. The CYP17A1 gene was analyzed by polymerase chain reaction direct sequencing. An in vitro expression system was performed in HEK293 cells to analyze the mutant P450c17 activity compared with the wild type. Analysis of the CYP17A1 gene sequences in patient 1 showed compound heterozygous mutations A82D (g.417 C > A) in exon 1 and Y329fs (g.4869 T > A, 4871del) in exon 6; DNA sequencing analysis in patient 2 revealed compound heterozygous mutations R125X (g.2045 C > T) in exon 2 and C442R (g.6457 T > C) in exon 8. The mutations A82D, R125X, and C442R have not been reported previously. The functional study demonstrated that the A82D, R125X, and C442R mutations almost completely eliminate enzymatic activity. These results, which indicate that Ala 82 and Cys 442 are crucial for both 17-hydroxylase and 17,20-lyase activities, help define the structure-function relationship of the CYP17A1 gene. The novel mutations A82D, R125X, and C442R further clarify the patients' clinical manifestations of combined 17OHD.

A novel SRY missense mutation affecting nuclear import in a 46,XY female patient with bilateral gonadoblastoma

Patients with disorders of sex development (DSD), especially those with gonadal dysgenesis and hypovirilization, are at risk of developing the so-called type II germ cell tumors (GCTs). Both carcinoma in situ and gonadoblastoma (GB) can be the precursor lesion, resulting in a seminomatous or non-seminomatous invasive cancer. SRY mutations residing in the HMG domain are found in 10-15% of 46,XY gonadal dysgenesis cases. This domain contains two nuclear localization signals (NLSs). In this study, we report a unique case of a phenotypical normal woman, diagnosed as a patient with 46,XY gonadal dysgenesis, with an NLS missense mutation, on the basis of the histological diagnosis of a unilateral GB. The normal role of SRY in gonadal development is the upregulation of SOX9 expression. The premalignant lesion of the initially removed gonad was positive for OCT3/4, TSPY and stem cell factor in germ cells, and for FOXL2 in the stromal component (ie, granulosa cells), but not for SOX9. On the basis of these findings, prophylactical gonadectomy of the other gonad was performed, also showing a GB lesion positive for both FOXL2 (ovary) and SOX9 (testis). The identified W70L mutation in the SRY gene resulted in a 50% reduction in the nuclear accumulation of the mutant protein compared with wild type. This likely explains the diminished SOX9 expression, and therefore the lack of proper Sertoli cell differentiation during development. This case shows the value of the proper diagnosis of human GCTs in identification of patients with DSD, which allows subsequent early diagnosis and prevention of the development of an invasive cancer, likely to be treated by chemotherapy at young age.

A Novel Heterozygous Mutation of Steroidogenic Factor-1 (SF-1/Ad4BP) Gene (NR5A1) in a 46, XY Disorders of Sex Development (DSD) Patient without Adrenal Failure

Steroidogenic factor-1 [(SF-1/Ad4BP) (MIM184757)] is a nuclear receptor that regulates multiple genes involved in adrenal and gonadal development, steroidogenesis, reproduction, and other metabolic functions. Initially, mutations of SF-1/Ad4BP gene (NR5A1) in humans were identified in two 46, XY female patients with adrenal insufficiency and gonadal dysgenesis. However, recent studies have revealed that heterozygous mutations are more frequently found in 46, XY disorders of sex development (DSD) patients without adrenal failure than in 46, XY DSD patients with adrenal failure. We encountered a Japanese female patient of 46, XY DSD without adrenal failure and identified a novel mutation (V41G) of NR5A1. Functional analysis revealed that this mutant protein could not activate CYP19 promoter, indicating loss of function. In conclusion, we add a novel mutation of NR5A1 in 46, XY DSD patient without adrenal failure.

Novel mutation in the ligand-binding domain of the androgen receptor gene (1790p) associated with complete androgen insensitivity syndrome

Mutations in the X-linked androgen receptor (AR) gene cause androgen insensitivity syndrome (AIS), resulting in an impaired embryonic sex differentiation in 46,XY genetic men. Complete androgen insensitivity (CAIS) produces a female external phenotype, whereas cases with partial androgen insensitivity (PAIS) have various ambiguities of the genitalia. Mild androgen insensitivity (MAIS) is characterized by undermasculinization and gynecomastia. Here we describe a 2-month-old 46,XY female patient, with all of the characteristics of CAIS. Defects in testosterone (T) and dihydrotestosterone (DHT) synthesis were excluded. Sequencing of the AR gene showed the presence in exon 6 of a T to C transition in the second base of codon 790, nucleotide position 2369, causing a novel missense Leu790Pro mutation in the ligand-binding domain of the AR protein. The identification of a novel AR mutation in a girl with CAIS provides significant information due to the importance of missense mutations in the ligand-binding domain of the AR, which are able to induce functional abnormalities in the androgen binding capability, stabilization of active conformation, or interaction with coactivators.

Five novel mutations in steroidogenic factor 1 (SF1,NR5A1) in 46,XY patients with severe underandrogenization but without adrenal insufficiency

Steroidogenic factor 1 (SF1, NR5A1) is a nuclear receptor that regulates multiple genes involved in adrenal and gonadal development, steroidogenesis, and the reproductive axis. Human mutations in SF1 were initially found in two 46,XY female patients with severe gonadal dysgenesis and primary adrenal failure. However, more recent case reports have suggested that heterozygous mutations in SF1 may also be found in patients with 46,XY partial gonadal dysgenesis and underandrogenization but normal adrenal function. We have analyzed the gene encoding SF1 (NR5A1) in a cohort of 27 patients with 46,XY disorders of sex development (DSD) from the German network of DSD. Heterozygous SF1 mutations were found in 5 out of 27 (18.5%) of cases. Four patients with SF1 mutations presented with the similar phenotype of mild gonadal dysgenesis, severe underandrogenization, and absent Müllerian structures. Of these, two patients harbored missense mutations within the DNA-binding region of SF1 (p.C33S, p.R84H), one patient had a nonsense mutation (p.Y138X) and one patient had a frameshift mutation (c.1277dupT) predicted to disrupt RNA stability or protein function. One additional patient ([c.424_427dupCCCA]+[p.G146A]) displayed a more marked phenotype of severe gonadal dysgenesis, normal female external genitalia, and Müllerian structures. Functional studies of the missense mutants (p.C33S, p.R84H) and of one nonsense mutant (p.Y138X) revealed impaired transcriptional activation of SF1-responsive target genes. To date, adrenal insufficiency has not occurred in any of the patients. Thus, SF1 mutations are a relatively frequent cause of 46,XY DSD in humans.

281.A novel scavenger receptor domain containing genes differentially expressed in the embryonic mouse testis

Sex in mammals is determined by the constitution of the sex chromosomes: two X-chromosomes results in the development of ovaries and a female phenotype, while an X and a Y chromosome result in testis development and a male phenotype. The SRY gene on the Y chromosome has been identified as the genetic switch, which initiates testis formation. The importance of SRY and the related SOX9 gene in sex determination has been well characterised in humans and mouse. Mutations in SRY and SOX9 account for approximately 20% of sex reversed XY-female patients suggesting that other genes are required for normal testis development. The mouse model was used to identify and characterise differentially expressed genes in the embryonic testis/ovary that may represent new sex determining candidates. A suppression subtraction hybridisation screen was conducted on testes/ovaries mouse cDNA between 12.0-12.5 dpc. Differential clones were sequenced, bioinformatic analysis was performed and expression patterns verified by whole mount in situ hybridisation (WISH) on 12.5 dpc mouse partial embryos. We identified a novel gene (B7) containing a scavenger receptor domain of unknown function. B7 was expressed in the gonads of both sexes from 11.5-12.0 dpc. From 12.5 -13.5 dpc, B7 expression was present in the testis but not in the ovary. We termed B7 Testis Expressed Scavenger Receptor (Tesr). Expression of Tesr was also seen in the mouse brain, eye, head, heart, neural arch and cartilage primordium. Furthermore, Tesr expression was analysed in the chicken embryo. Low expression was seen in the male gonads and mesonephric tubules from day 3.5-7.5. Tesr was expressed in chicken embryo's heart, notochord, neural tube, dorsal aorta and the somites at day 3.5 and 4.5. Tesr may play a role in phagocytosis of apoptotic cells and may be involved in scavenger, adhesion, defence functions and immune responses. Another possible role for Tesr in the developing testis may be cholesterol delivery for steroidogenesis.

Two unbalanced translocations involving a common 6p25 region in two XY female patients

We report two 46,XY female patients with two different de novo unbalanced translocations, each involving the chromosomal region 6p25. The patient with a 46,XY,der(6)t(X;6)(p21.2;p25) karyotype had a sex reversal phenotype. The patient with a 46,XY,der(13)t(6;13)(p25;q33) karyotype had a male pseudohermaphrodite phenotype. Multi-paint fluorescent in situ hybridization was performed to determine the origin of the derivative material on 6p and 13q. The association of abnormalities of the 6p25 region with either an Xp duplication or a 13q deletion is reported here for the first time.

The Human Doublesex-Related Gene, DMRT2, Is Homologous to a Gene Involved in Somitogenesis and Encodes a Potential Bicistronic Transcript

Intense efforts are currently being pursued to identify autosomal genes associated with 46,XY male-to-female sex reversal. The genes DMRT1 and 2 are located on distal 9p, a region deleted in 46,XY sex-reversed patients. They are considered excellent candidates because of their homology to regulators of sex development in invertebrates. We present the genomic structure of DMRT2, showing that it generates several transcripts with distinct coding potential. In addition to the previously reported 226-amino-acid protein-encoding transcript, we describe other mRNA isoforms that are potentially bicistronic and are predicted to encode an additional 328-amino-acid polypeptide. Finally, a stop codon-containing exon (exon 4) can be skipped by alternative splicing and can generate a transcript that is predicted to encode a fusion protein. The latter shares 58% amino acid identity with a gene recently described in fish, termed terra. Differences in expression pattern exist for DMRT2 mRNA isoforms among the human adult tissues tested, between adult tissues and human embryos, and between DMRT2 and DMRT1 during embryonic development. We failed to detect mutations by sequencing of DMRT2 in a sample of 46,XY female patients. The interesting structure of DMRT2 coupled to preliminary functional studies in fish showing that terra is involved in somitogenesis suggests that validation or exclusion of this gene as a cause of sex reversal will require more in-depth investigations.

A case of premature menopause in an ovulating 46,XY female patient

We report the case of premature ovarian failure in a 37-year-old woman who was found to have an XY genotype and, apart from short stature, was a phenotypically normal female.