Identification of a Rare Variant in the SRD5A2 Gene in Siblings With 46,XY Disorders of Sexual Development

Disorders of Sex Development.

Disorder of Sex Development (DSD) is a congenital condition in which development of chromosomal, gonadal or anatomical sex is atypical. They are complex condition related to social stigma, cultural and beliefs in Indonesian population. In Indonesia, there is only a few study regarding characteristic of DSD patients. The aims of our study was to evaluate the characteristic of patients with DSD in Cipto Mangunkusumo Hospital, Jakarta, Indonesia. We retrospectively reviewed the medical records of patients followed up over the period from 2010 to 2012 in Endocrinology Pediatric Clinic, Cipto Mangunkusumo Hospital, Jakarta, Indonesia. We found 74 patients with DSD. In this paper we excluded 29 patients confirmed as Congenital Adrenal Hyperplasia and 3 patients with Turner syndrome. Of 42 patients, 23 (54.8%) patients had 46,XY DSD; 11 (26.2%) patients had sex chromosome DSD; 3 (7.1%) patients had 46,XY DSD, and 5 patients have not done chromosomal analysis. The age when first diagnosed was ranging from newborn to 14 years old. Most cases referred by pediatrician (33.3%) and urologist (23.8%) with frequent complaint were ambiguous genitalia (52.4%) and severe hypospadia (14.3%). The most frequent of phenotype was hypospadia (66.7%). Twenty five (59.5%) of patients have been raised as boy prior to diagnosis. Twelve patients were raised as girls have 46, XY karyotype and 2 patients raised as boys have 46, XX karyotype. Definite diagnosis of 23 patients with 46, XY DSD was found only in 4 (17.4%) patients. The problem regarding lack of definitife diagnosis was related with the high cost of laboratory examination and socioeconomic problem in Indonesia. Our study suggest that the most common cause of DSD in our clinic was 46, XY DSD. Karyotype and clinical phenotype may help in establishing the diagnosis.

Disclosure: J. Kim: None. S. Park: None. S. Hwang: None. J. Yoon: None. G. Kim: None. H. Yoo: None. J. Choi: None. Background: Disorders of sexual development (DSDs) is a group of congenital conditions associated with discordant development of chromosomal, gonadal, and anatomical sex. Sex development is a complex process with a precise synergistic temporal-spatial transcriptional regulation of multiple genes. Advances in next-generation sequencing (NGS) techniques have achieved genetic diagnosis for up to 40 % of 46,XY DSDs. This study investigated the etiological distribution of DSDs identified by NGS. Methods: This study included 70 patients with DSDs (56 patients with 46,XY DSD and 14 patients with 46,XX DSD). Molecular genetic analysis was performed using targeted gene panel sequencing (n = 43) and whole-exome sequencing (WES, n = 26), including 25 patients for the first-line test and one without sequence variants by targeted gene panel sequencing. Whole-genome sequencing (WGS) was performed on 7 patients, including two patients for the first-line test and five patients without sequence variants by targeted gene panel sequencing or WES. Results: Patients with DSDs presented in infancy with ambiguous genitalia and at older children with aberrant pubertal development or discordance between karyotype and external genitalia. Twenty-four of 56 patients (42.9%) with 46,XY DSD were reared female, and 5 of 14 patients (35.7%) with 46,XX DSD were raised as a male. Pathogenic or likely pathogenic variants were identified in 21 of 56 patients with 46,XY DSD (37.5%); however, no rare sequence variants were found in patients with 46,XX DSD. The AR variants were the most common (8/70, 11.4%), followed by CYP17A1 (4/70, 5.7%), SRD5A2 (4/70, 5.7%), NR5A1 (2/70, 2.8%), GATA4 (1/70, 1.4%), MYRF (1/70, 1.4%), and deletion of DMRT1/2 (1/70, 1.4%). Mutation-positive patients with 46, XY DSD were assigned as female more frequently than mutation-negative patients (19/21 vs. 6/35, P-value <0.001). The lower diagnostic yield was observed in patients with a less severe phenotype, such as hypospadias or micropenis. Conclusions: This study demonstrated that genetic etiologies could be identified in 37.5% of patients with 46,XY DSD, whereas there were no genetic variants in patients with 46,XX DSD. Patients with a more severe phenotype were more likely to have a conclusive genetic diagnosis. NGS is a helpful technique to improve diagnostic efficiency in patients with DSD because of phenotypic and genetic heterogeneity. Presentation: Thursday, June 15, 2023

Disorders of sex development (DSD) are defined as congenital conditions in which development of chromosomal, gonadal or anatomical sex is atypical. In 46, XY DSD, the genotype is XY, but the external genitalia is incompletely virilised, ambiguous, or completely female. The objectives of this prospective study are to evaluate the testicular Sertoli and Leydig cell functions, to establish the genetic basis and to determine the relative prevalence of etiologies in Chinese patients with 46,XY DSD in Hong Kong. All patients with 46,XY DSD (either new or known) presented to five paediatric departments in Hong Kong from July 2009 till June 2011 were recruited. They were assessed by paediatric endocrinologists. Comprehensive evaluation of testicular and adrenal functions was performed using serum hormonal assays and urine steroid profiling. Based on the hormonal results, mutational analyses of the candidate genes by polymerase chain reaction and direct DNA sequencing were conducted to delineate the genetic basis of the etiologies. Sixty-five patients (54 male and 11 female) with 46,XY DSD were recruited. Their age ranged from birth to 27 years. Sixty-one (94%) patients presented with ambiguous external genitalia, two presented with delayed puberty and one each with primary amenorrhoea and inguinal hernia. Definitive diagnoses were made in 25 (38%) patients. Eleven (17%) patients had 5-alpha reductase 2 deficiency. Androgen insensitivity was confirmed by genetic analysis in eight (12%) patients. There was one patient with each of the following etiologies: Swyer syndrome, SF-1 mutation, Frasier syndrome, cholesterol side-chain cleavage deficiency, persistent Mullerian duct syndrome and mixed gonadal dysgenesis. Genetic basis of the etiologies was delineated in 23 (35%) patients. A total of 10 novel mutations were identified. The longest follow up period was 27 years, none of the patients requested change of gender sex so far. In conclusion, 46,XY DSD is a heterogeneous group with diverse etiologies. Although 5-alpha reductase 2 deficiency is believed to be rare, it is not uncommon in Hong Kong.

Disorders of sex development (DSD) is a group of pathological conditions where there is a discrepancy between the chromosomal, gonadal and phenotypic sex. Objective — to investigate the clinical and molecular genetic characteristics of DSD in children in Ukraine. Materials and methods. We carried out a retrospecti­­ve analysis of 106 medical records of patients with DSD during period from 2000 to 2019 and analyzed the results of clinical data, medical history, hormonal, genetic, functional and instrumental investigations. All patients (from birth to 18 years old) underwent cytogenetic (karyotyping according to the standard methods) and, if necessary, molecular genetic studies (FISH method). Molecular genetic testing was performed in a selected group of patients with 46,XY and 46,XX DSD (n = 49) using whole exome sequencing (WES). Results and discussion. Among the examined 106 patients with DSD, chromosomal DSD was diagnosed in 17.0 % (n = 18) of patients, 46,XY DSD — in 68.9 % (n = 73) and 46,XX DSD — in 14.1 % (n = 15) cases. The majority of children (60 %) out of 46,XY DSD cohort are brought up in a female civilian sex, and 57.1 % of patients out of 46,XX DSD cohort — as males. Among children with 46,XY, and 46,XX DSD genetic testing (WES) found pathogenic mu­­tations in 46.9 % of cases, variants of unknown significan­­ce — in 30.6 % of cases, in 6.1 % of patients the identified mutations were not the cause of DSD, and in 16.3 % of patients WES didn’t find the genetic cause of their disease. According to the data of the Registry, the largest group of DSD in Ukrainian children (68.9 %) is 46,XY DSD, and the most common genetic cause of DSD in them (26 %) are mutations in the androgen receptor gene. Conclusions. All patients with 46,XY and 46,ХХ DSD require genetic testing (in particular WES) to verify the clinical diagnosis, justify treatment tactics and further observation.

BackgroundOver 100 mutations in the SRD5A2 gene have been identified in subjects with 46,XY disorder of sex development (DSD). Exploration of SRD5A2 mutations and elucidation of the molecular mechanisms behind their effects should reveal the functions of the domains of the 5α-reductase 2 enzyme and identify the cause of 46,XY DSD. Previously, we reported a novel compound heterozygous p.Q6X/p.H232R mutation of the SRD5A2 gene in a case with 46,XY DSD. Whether the compound heterozygous p.Q6X/p.H232R mutation in this gene causes 46,XY DSD requires further exploration.MethodsThe two 46,XY DSD cases were identified and sequenced. In order to identify the source of the compound heterozygous p.Q6X/p.H232R mutation, the parents, maternal grandparents, and maternal uncle were sequenced. Since p.Q6X mutation is a nonsense mutation, p.H232R mutation was transfected into HEK293 cells and dihydrotestosterone (DHT) production were analyzed by liquid chromatography–mass spectrometry (LC–MS) for 5α-reductase 2 enzyme activities test. Apparent michaelis constant (Km) were measured of p.H232R mutation to analyze the binding ability change of 5α-reductase 2 enzyme with testosterone (T) or NADPH.ResultsThe sequence results showed that the two 46,XY DSD cases were the compound heterozygous p.Q6X/p.H232R mutation, of which the heterozygous p.Q6X mutation originating from maternal family and heterozygous p.H232R mutation originating from the paternal family. The function analysis confirmed that p.H232R variant decreased the DHT production by LC–MS test. The Km analysis demonstrated that p.H232R mutation affected the binding of SRD5A2 with T or NADPH.ConclusionsOur findings confirmed that the compound heterozygous p.Q6X/p.H232R mutation in the SRD5A2 gene is the cause of 46,XY DSD. p.H232R mutation reduced DHT production while attenuating the catalytic efficiency of the 5α-reductase 2 enzyme.

Background: Disorders of sex development (DSD) encompass a wide range of conditions associated with numerous causative genes. In about 50-60% of 46,XY DSD individuals, the underlying molecular cause remains uncertain. GATA4 haploinsufficiency has been described in patients with congenital heart defects (CHD), while only a few studies reported mutations related to 46,XY DSD phenotype. This study investigated clinical phenotypes and molecular characteristics of two 46,XY DSD patients with GATA4 mutations. Methods: Mutation analysis was performed in patients with 46,XY DSD by whole exome sequencing (WES) using Illumina NextSeq platform. Clinical and endocrine characteristics were reviewed retrospectively. GATA4 variants identified by WES were verified by Sanger sequencing. Functional activity of GATA4 variants was tested by luciferase reporter assay on the SRY and AMH promoter using two different cell systems including HEK293 and NCI-H295R. Results: Subject 1 presented with micropenis and hypospadias at the age of 5 months. Karyotype was 46,XY. Mullerian duct remnants were not found in pelvic ultrasound. The patient underwent urethroplasty at the age of 10 months and was reared as a male. Subject 2 with complete female external genitalia was referred to our hospital because of 46,XY karyotype on G-scanning. The patient underwent laparoscopic orchiectomy at the age of 1.8 years and was assigned as a female. Both patients were responsive to hCG stimulation tests and did not have CHD. Subject 1 harbored a novel heterozygous variant of c.643A>G (p.R215G)] in GATA4, whereas a previously reported variant of c.1220C>A (p.P407Q) was identified in Subject 2. In vitro luciferase reporter assays using SRY and AMH promoter revealed decreased transcriptional activity of both p.P407Q and p.R215G. Conclusions: This study expanded phenotypic spectrum of mutations in GATA4 in patients with 46,XY DSD without CHD. GATA4 mutations in patients with 46,XY DSD may not be associated with CHD. Possible explanations for phenotypical variability comprise incomplete penetrance, variable expressivity, and oligogenic mechanisms.

Disorders of sex development (DSD) are rare disorders defined as discrepancy of chromosomal, gonadal, or anatomic sex. Prenatal lack of androgens or partial androgenization results in atypical development of the genitalia in individuals with XY DSD. In the past, most XY DSD individuals with ambiguous genitalia were assigned to the female sex and often underwent early feminizing treatment, including constructive genital surgery to allow heterosexual intercourse, gonadectomy, and hormone therapy. Few outcome studies of the effect of early feminizing genital surgery on sexual satisfaction and performance among adults with XY DSD have been performed. There is increasing evidence, primarily from DSD patients, that questions the benefit of early feminization treatment. The aim of this study was to evaluate satisfaction with reconstructive genital surgery and sexual life among adults with XY DSD. A total of 57 individuals with XY DSD enrolled in the German multicenter clinical evaluation study of DSD were interviewed using a condition-specific questionnaire assessing standardized measures of satisfaction with treatment. The sexual life and sexual problems evaluated included the following: satisfaction with overall sex life, present partnership, previous sexual partners, decreased sexual desire, problems with sexual arousal, and pain during intercourse (dyspareunia). The individuals were divided into 3 subgroups: (1) XY DSD complete females (XY DSD-CF) or individuals with complete androgen insensitivity syndrome, raised as females; (2) XY DSD partially androgenized females (XY DSD-PF), raised as females; and (3) XY DSD partially androgenized males, assigned to the male gender. Some comparisons were done with a control group of normal individuals surveyed about sexual satisfaction. There was substantial dissatisfaction with overall treatment among individuals with XY DSD-PF (43.3%) and XY DSD-CF (56.2%). XY DSD-PF individuals who had feminization surgery expressed high dissatisfaction, with the functional result (47.1%) and clitoral arousal (47.4%) compared with controls. Individuals with XY DSD-PF reported a substantially higher proportion of having female sexual partners (9.1%) or partners of both sexes (18.2%) and dyspareunia (56.5%) compared with female controls. XY DSD-CF individuals had decreased sexual desire (70.6%), arousal (52.9%), and dyspareunia (56.3%) compared with controls. Most individuals with complete androgen insensitivity syndrome reported significant sexual problems compared with controls; there was decreased desire (81.8%), arousal (63.6%), and dyspareunia (70%) (all P < 0.05). These findings indicate that use of early feminizing surgery in XY DSD individuals should be avoided or minimized, and that surgical correction should be performed primarily in puberty or adulthood with the parents and patients consent. The decision of sex of rearing might not have been appropriate in some cases, and the choice of gender of rearing can be postponed in some families. Information about the disorder should be provided to DSD-affected individuals and their families in an age-appropriate and sensible manner. Patients and their families should be followed from childhood to adolescence and adulthood, and should receive multidisciplinary care with psychological support and input from other patients and families with DSD.

Inhibin B is a hormone produced by the Sertoli cells that can provide important information for the investigation of disorders of sex development (DSD) with 46,XY karyotype. The aim of this study is to compare two enzyme-linked immunosorbent assay (ELISA) assays for dosage of serum inhibin B in patients with 46,XY DSD with normal testosterone secretion. Twenty-nine patients with 46,XY DSD and normal testosterone secretion (partial androgen insensitivity syndrome [PAIS] [n=8]; 5α-reductase deficiency [n=7] and idiopathic 46,XY DSD [n=14]) were included. Molecular analysis of the AR and SRD5A2 genes were performed in all patients and the NR5A1 gene analysis in the idiopathic group. Measurements of inhibin B were performed by two second-generation ELISA assays (Beckman-Coulter and AnshLabs). Assays were compared using the interclass correlation coefficient (ICC) and the Bland-Altman method. ICC was 0.915 [95% confidence interval (CI): 0.828-0.959], however, a discrepancy was observed between trials, which is more evident among higher values when analyzed by the Bland-Altman method. It is recommended to perform the inhibin B measurement always using the same ELISA kit when several evaluations are required for a specific patient.

A rare form of the 46,XY disorders of sex development (DSD), 5α-reductase deficiency was first described in patients with pseudovaginal perineoscrotal hypospadias, microphallus, and cryptorchid testes in 1974 by Imperato. This undervirilization in the male is due to an alteration in the 5α-reductase type 2 gene (SRD5A2), which encodes for 5α- reductase activity. Our registry of 750 patients with DSD showed no definitive diagnosis in 80% of cases with 46,XY DSD. Our aim is to identify mutations in SRD5A2 gene and to describe phenotype of detected mutative cases. Mutation analysis was performed for genomic DNA extracted from WBC of 10 patients with 46,XY DSD using PCR and direct sequencing. We identified mutations of SRD5A2 gene in two cases. The first case presented with isolated micropenis at birth, two palpable testes in the normal scrotum. Pelvic ultrasound showed no ovaries and uterus, karyotype was 46,XY and SRY was positive. Serum FSH level was 2.4 UI/L; LH level was 0.9 UI/L and testosterone level was 0.4 nmol/l at 8 years of age. A homozygous missense mutation (p.R237G) was identified in the SRD5A2 gene. The second case presented with microphaslus, penoscrotal hypospadias, gonad bilateral in labioscrotal folds. No uterus and ovaries were found by pelvic ultrasound. Karyotype was 46,XY and SRY was positive. A novel homozygous missense mutation (c.659C>T; p.S220L) was identified in the SRD5A2 gene. Mutation analysis of SRD5A2 gene helps to make definitive diagnosis for patients with 46,XY DSD.

46,XY disorders of sex development (DSD) present with diverse phenotypes and complicated genetic causes. Precise genetic diagnosis contributesto accurate management, and targeted next-generation sequencing (NGS) and whole-exome sequencing are powerful tools for investigating DSD. However, the prevalent variants resulting in 46,XY DSD remain unclear, especially those associated with mild forms, such as isolated hypospadias, inguinal cryptorchidism, and micropenis. From 2019 to 2021, 74 patients with 46,XY DSD (48 typical and 26 mild) from the First Affiliated Hospital of Sun Yat-sen University were enrolled in our cohort study for targeted NGS or whole-exome sequencing. Our targeted 46,XY DSD panel included 108 genes involved in disorders of gonadal development and differentiation, steroid hormone synthesis and activation, persistent Müllerian duct syndrome, idiopathic hypogonadotropic hypogonadism, syndromic disorder, and others. Variants were classified as pathogenic, likely pathogenic, variant of uncertain significance, likely benign, or benign following the American College of Medical Genetics guidelines. As a result, 28 of 74 (37.8%) patients with pathogenic and/or likely pathogenic variants acquired genetic diagnoses. The Mild DSD patients acquired a diagnosis rate of 30.7%. We detected 44 variants in 28 DSD genes from 31 patients, including 33 novel and 11 reported variants. Heterozygous (65%) and missense (70.5%) variants were the most common. Variants associated with steroid hormone synthesis and activation were the main genetic causes of 46,XY DSD. In conclusion, 46,XY DSD manifests as a series of complicated polygenetic diseases. NGS reveals prevalent variants and improves the genetic diagnoses of 46,XY DSD, regardless of severity.

We read with interest the paper of Pizzo et al. [1], confirming that adolescence is a key period for the diagnosis of 46,XY disorders of sex development (DSD) [2]. However, in our opinion, some points should be better addressed. Among these, we have the following. The authors stated that the reported girl showed “normal intellectual function,” but this information had little relevance because mental function is not impaired in females with 46,XY DSD, reaching adolescence without any clinical suspicions. Indeed, this statement might be stigmatizing for these persons. The statement that this adolescent was affected by hypergonadotropic hypogonadism (HH) is correct, according to the high LH and FSH values shown in Table 1 [1]. Very low levels of both 17β-estradiol and testosterone were also shown [1]. This endocrine pattern is not typical of late adolescent and young adult females with complete androgen insensitivity syndrome (CAIS) and intact testes. On the contrary, these persons show high/normal levels of both 17β-estradiol (for a person with a 46,XY karyotype) and testosterone [3–5]. In addition, adolescent/young adult women with CAIS did not show HH: LH is in high normal range or slightly elevated (due to the androgen resistance at central level), but FSH is in normal range (due to unaffected inhibin secretion from Sertoli cells) [3–5]. In addition, SHBG was reported within adult male range by Pizzo et al. [1], suggesting a normal sensitivity to the low levels of androgens in this girl [6]. Indeed, SHBG is within normal female range in women with CAIS, due to peripheral androgen resistance [5]. All these findings show poor agreement with the affirmed diagnosis of CAIS [1]. The clinical phenotype of the reported girl did not match with the phenotype of adolescents with CAIS; in fact, the latter shows normal breast development related to the relatively high normal estrogen levels and unopposed androgen action [3, 4, 7] and does not show hypotrophic breasts [1]. It is quite surprising that hormonal replacement therapy was started before any diagnosis was established. Histological findings do not completely agree with the diagnosis of CAIS, in particular the absence of Leydig cells, which are abundant in adolescent females with CAIS and sometimes formed aggregates up to 2 mm in diameter [8, 9]. 46,XY karyotype, female phenotype, and absence of mullerian derivatives may be present in several 46,XY DSD; they should be excluded before diagnosis of CAIS by optimal clinical, endocrine, and genetic investigations [10]. For example, the testosterone/Δ4-androstenedione ratio in the adolescent reported by Pizzo et al. [1] is very low (0.13; normal values >0.8 [11]). Thus, diagnoses of 17β-hydroxysteroid-dehydrogenase deficiency type 3 or 46,XY gonadal dysgenesis [11, 12] must be considered in the diagnostic process. Risk of gonadal cancer largely varies among 46,XY DSD. For example, in CAIS is very low at least during the first two decades of life [13–15]. Thus, delayed gonadal removal can be recommended to permit both full sexual development [15] and better bone health [16]. If diagnosis of CAIS is certain, surgery can be postponed—at least until the legal age at which the propositus can participate in decision making [1, 15, 17–19]. We are concerned and in complete disagreement with the decision to perform gonadectomy without full disclosure and assent of the adolescent [17–19]. In conclusion, some findings are poorly consistent with a diagnosis of CAIS, which should be confirmed by molecular analysis of androgen receptor gene [2, 7]. In our experience, more than 25% of the females referred to our departments with clinical/endocrine diagnosis of CAIS did not have this diagnosis confirmed by genetic analysis [20]. Clinical approach should be changed according to the new guidelines for management of persons with 46,XY DSD and directly involving the girl in the decision process [2, 18, 21]. We also stress that multidisciplinary team evaluation in tertiary centers with documented experience in this field must be guaranteed to each person with 46,XY DSD for optimal holistic management [21], especially before performing irreversible surgical procedures.

Disorders of sex development (DSD) can be classified as 46,XX DSD, 46,XY DSD, and sex chromosome DSD. Several underlying causes including associated genes have been reported. Steroidogenic factor-1 is encoded by the NR5A1 gene, a crucial regulator of steroidogenesis in the growth of the adrenal and gonadal tissues. It has been discovered to be responsible for 10 to 20% of 46, XY DSD cases. Here, we described a 2-month-old infant who had ambiguous genitalia and 46, XY. Using whole exome sequencing followed by polymerase chain reaction-Sanger sequencing, a novel heterozygous nonsense c.1249C > T (p.Gln417Ter) variant in the NR5A1 gene was identified. It is present in his mother but absent in his father and maternal aunt and uncle. At the age of 7 months, the patient received a monthly intramuscular injection of low-dose testosterone for 3 months in a row. His penile length and diameter increased from 1.8 to 3 cm and from 0.8 to 1.3 cm, respectively. The patient also had normal adrenal reserve function by adrenocorticotropic hormone stimulation test. This study identified a novel causative p.Q417X (c.1249C > T) variant in NR5A1 causing 46,XY DSD in a Thai boy which is inherited from his unaffected mother.

The medical, surgical and psychosocial management of individuals affected by disorders of sex development (DSD) is currently under scrutiny. Patients who possess a 46,XY chromosomal complement (i.e., those affected by 46,XY DSD) pose particular challenges to healthcare professionals because this group is most likely to report dissatisfaction with their sex of rearing as they progress from childhood through adulthood. The current review examines satisfaction with sex of rearing, degree of concordance between masculine/feminine behavior and sex of rearing, and sexual orientation in adults affected by 46,XY DSD, reared male or female. Such individuals may present with female external genitalia, ambiguous external genitalia or a micropenis. The impact of potentially confounding variables such as physical health, fertility and sexual function on psychosexual development in persons affected by DSD is considered when such data are available. Finally, observations from non-DSD clinical populations and animal models are discussed, including their appropriateness for informing healthcare professionals about behavioral outcomes in DSD patients. Rodent models that support the aromatization hypothesis or the independent influence of genes on the Y chromosome for brain/behavior masculinization do not generalize to human psychosexual development; however, the nonhuman primate literature is more instructive for understanding the direct impact of androgens on human gender development. Finally, discussion pertaining to the predominance of male gender development in cases of 5α-RD-2 and 17β-HSD-3 deficiencies is offered. Keywords: Intersex, DSD, gender, disorders of sex development, androgen insensitivity, gonadal dysgenesis, testosterone biosynthetic defect, ambiguous genitalia

Familial 46, XY Disorder of Sexual Development (DSD) was discovered in a Ph+, BCR::ABL1P210+ Acute Lymphoblastic Leukemia (ALL) female with RCBTB2::LPAR6 fusion gene. Siblings developing 46, XY DSD are extremely rare. Patients with 46, XY DSD have much higher rates of gonadal cancers. Nevertheless, the incidence of hematologic malignancies in patients with DSDs has received little attention. RCBTB2::LPAR6 is a rarely reported fusion gene in ALL. Herein, we report a rare case of a newly diagnosed Ph+, BCR::ABL1P210+ ALL patient who was 77 years old and female by social sex. Whole Exome Sequencing (WES) and RNA sequencing revealed TET2 and NF1 mutations in addition to a rarely reported RCBTB2::LPAR6 fusion gene and 17 other genes with uncertain clinical significance. The patient was surprisingly found to have a male karyotype. On ultrasound, neither the uterus nor the ovaries were discernible. A detailed family and marital history revealed that the patient had undergone surgery at an early age for an unexplained inguinal mass. She had slow pubertal development, scanty menstruation, and few overtly feminine characteristics. She had three marriages, but none succeeded in getting pregnant. The patient had never sought therapy for infertility due to the inaccessibility of medical treatment and a lack of medical knowledge. Her sister, 73 years old and female by social sex, who had amenorrhea in adolescence and was unable to conceive, had the same experience. To our surprise, she also had a male karyotype. Due to the absence of long-term social attention and follow-up, studies on the incidence of hematologic malignancies in patients with 46, XY DSD are incredibly uncommon. Siblings developing 46, XY DSD is extremely rare. We report the oldest patient diagnosed with 46, XY DSD. There have not yet been any reports of familial 46, XY DSD with a concurrent diagnosis of Ph+BCR::ABL1P210+ ALL with a rarely reported RCBTB2::LPAR6 fusion gene.