A rare case of male sex reversal syndrome (46, XX) with negative SRY gene: a disorder of sexual differentiation (DSD)

Background Male sex reversal syndrome is a rare genetic cause of male infertility with an overall incidence of 1/20,000–1/100,000 males. There is mismatching between the genetic make-up and the apparent clinical features. The clinical presentation of such cases is variable ranging from ambiguous genitalia at birth, failed puberty, up to normal male phenotype with infertility and hypogonadism. The exact molecular and genetic bases of this syndrome are still unclear. Most of the recorded cases were SRY positive (i.e. representing 80–90% of all cases), and they showed translocated SRY gene on the Y chromosome. Moreover, fewer cases of male sex reversal (46, XX) were SRY negative. Case presentation Herby, we report a rare case of a 35-year-old infertile male patient who presented with azoospermia, hypergonadotropic hypogonadism, and abnormal classical (46, XX) karyotype, as well as negative FISH for SRY gene. He had a previous negative biopsy and was asking for redoing micro-TESE, whoever he was discouraged as chances to find sperm is eventually nil, and instead, he was prescribed testosterone replacement therapy to correct hypogonadism. Conclusion Therefore, any case of non-obstructive azoospermia should be offered genetic testing trying to exclude non-treatable cases and for genetic counseling.

A Novel SRY Pathogenic Variant from a 46,XY Female Harboring a Nonsense Point Mutation (G to A) in Position 293

SRY gene mutation is a common cause of 46,XY female. We report a 46,XY female with a novel mutation of SRY c.293G>A (p.Trp98ter). Our report provides evidence for a pathogenic role of the SRY gene c.293G>A mutation in an individual and enlarges the spectrum of molecular diagnosis for these patients.

22q13 Duplication in Newborn With Dysmorphic Features: The Role of SOX10 in Disorders of Sex Development

Background: The 46,XX testicular disorder of sex development (DSD), also known as 46,XX male reversal, is a rare form of DSD and clinical phenotype shows complete sex reversal from female to male. The sex-determining region Y (SRY) gene can be identified in most 46,XX testicular DSD patients; however, approximately 20% are SRY-negative. Here we present a 2 week old with discrepant prenatal karyotype and infant phenotype. Case: This 2-week-old had dysmorphic features (bitemporal narrowing, broad and flat nasal bridge, bilateral epicanthal folds) and multiple congenital anomalies; IUGR, hypertelorism, cleft lip and palate, ASD, small kidneys, sacral dimple. Physical exam revealed palpable inguinal masses and microphallus without hypospadias. Postnatal karyotype showed a 46,XX chromosome complement with duplication of 22q13-qter. He was negative for SRY gene by FISH. Microarray analysis confirmed the duplication encompassing the SOX10 gene. Lab evaluation showed LH 10.81 mIU/ml, FSH 3.21 mIU/ml and total testosterone 241 ng/dl. These values are consistent with activation of the hypothalamic–pituitary–gonadal axis during the neonatal period in males. Conclusion: Our case along with previous cases supports the existence of a gene on chromosome 22q that can trigger testis determination in the absence of SRY. Potential mechanisms responsible for ovotesticular disorder in the XX (SRY−) individual could involve activation of testis specifying genes in the absence of SRY and/or inadequate expression of pro-ovary/anti-testis genes such as SOX 8 and SOX9. SOX10, a gene closely related to SOX9 and SOX8, and its overexpression has been suggested as a candidate for 46,XX DSD. Over-expression of SOX10 in mice resulted in the XX DSD. The spectrum of sex reversal phenotypes and gonadal asymmetry seen in the Sox10 transgenic mice closely mirrors the range of gonadal and reproductive tract anomalies seen in cases of partial duplication of human chromosome 22q13. Although SRY-negative 46,XX testicular DSD is a rare condition, an effort to make an accurate diagnosis is important for the provision of proper genetic counseling and for guiding patients in their long-term management.

Knockout of the HMG domain of the porcine SRY gene causes sex reversal in gene-edited pigs

The sex-determining region on the Y chromosome (SRY) is thought to be the central genetic element of male sex development in mammals. Pathogenic modifications within the SRY gene are associated with a male-to-female sex reversal syndrome in humans and other mammalian species, including rabbits and mice. However, the underlying mechanisms are largely unknown. To understand the biological function of the SRY gene, a site-directed mutational analysis is required to investigate associated phenotypic changes at the molecular, cellular, and morphological level. Here, we successfully generated a knockout of the porcine SRY gene by microinjection of two CRISPR-Cas ribonucleoproteins, targeting the centrally located “high mobility group” (HMG), followed by a frameshift mutation of the downstream SRY sequence. This resulted in the development of genetically male (XY) pigs with complete external and internal female genitalia, which, however, were significantly smaller than in 9-mo-old age-matched control females. Quantitative digital PCR analysis revealed a duplication of the SRY locus in Landrace pigs similar to the known palindromic duplication in Duroc breeds. Our study demonstrates the central role of the HMG domain in the SRY gene in male porcine sex determination. This proof-of-principle study could assist in solving the problem of sex preference in agriculture to improve animal welfare. Moreover, it establishes a large animal model that is more comparable to humans with regard to genetics, physiology, and anatomy, which is pivotal for longitudinal studies to unravel mammalian sex determination and relevant for the development of new interventions for human sex development disorders.

Sex Determination, Gonadal Sex Differentiation and Pl­­asticity in Vertebrate Species

A diverse array of sex determination (SD) mechanisms, encompassing environmental to genetic, have been found to exist among vertebrates, covering a spectrum from fixed SD mechanisms (mammals) to functional sex change in fishes (sequential hermaphroditic fishes). A major landmark in vertebrate SD was the discovery of the SRY gene in 1990. Since that time, many attempts to clone an SRY ortholog from non-mammalian vertebrates remained unsuccessful, until 2002, when DMY/DMRT1BY was discovered as the SD gene of a small fish, medaka. Surprisingly, however, DMY/DMRT1BYwas found in only two species among more than 20 species of medaka, suggesting a large diversity of SD genes among vertebrates. Considerable progress has been made over the last 3 decades, such that it is now possible to formulate reasonable paradigms of how SD and gonadal sex differentiation may work in some model vertebrate species. This review outlines our current understanding of vertebrate SD and gonadal sex differentiation, with a focus on the molecular and cellular mechanisms involved. An impressive number of genes and factors have been discovered that play important roles in testicular and ovarian differentiation. An antagonism between the male and female pathway genes exists in gonads during both sex differentiation and, surprisingly, even as adults, suggesting that, in addition to sex-changing fishes, gonochoristic vertebrates including mice maintain some degree of gonadal sexual plasticity into adulthood. Importantly, reviewing various SD mechanisms among vertebrates suggest that this is the ideal biological event that can make us understand the evolutionary conundrums underlying speciation and species diversity.

PSX-32 Late-Breaking Abstract: Production of a Gene Knock-In Bull Calf by Embryo-Mediated Genome Editing

Genome editing offers an opportunity to introduce targeted gene insertions into livestock breeding programs. Molecular geneticists have typically employed a donor repair template and the homologous recombination (HR) pathway in somatic cells to introduce gene knock-ins into livestock genomes, followed by cloning. Editing embryos directly to achieve targeted gene knock-ins is inefficient, especially for introducing large DNA sequences. Here we report using a one-step method to produce a gene knock-in bull calf by cytoplasmic microinjection of CRISPR/Cas9 reagents into a bovine embryo. In vitro fertilized one-cell bovine zygotes were injected with a gRNA/Cas9 ribonucleoprotein complex and homology mediated end joining donor template containing the sex determining region Y (SRY) gene, the green fluorescent protein (gfp) reporter gene driven by the SV40 promoter, and one kilobase homology arms targeting the H11 safe harbor locus on bovine chromosome 17. Seven-day blastocysts were evaluated using fluorescent microscopy, and nine green fluorescent embryos were transferred to synchronized recipients. Ultrasound evaluation at 35 days revealed one pregnancy. In April 2020, a healthy 50 kg male calf was born. DNA was extracted from placenta, blood and a fibroblast line derived from the calf and analyzed for SRY-GFP knock-in, as well as genotypic sex. PCR and Sanger sequencing revealed the biallelic edit of the target location on chromosome 17, with the insertion of three or seven copies of the SRY-GFP construct in addition to donor plasmid backbone, or a 26 base pair insertion, and an XY genotype. Future analysis of the XX offspring inheriting the SRY gene on chromosome 17 from this knock-in bull will reveal whether inheritance of the bovine SRY gene is sufficient to trigger the male developmental pathway in cattle.

CLINICAL AND GENETIC CHARACTERISTICS OF 17α-HYDROXYLASE/17, 20-LYASE DEFICIENCY: c.985_987delTACinsAA MUTATION OF CYP17A1 PREVALENT IN CHINESE HAN POPULATION

Backgrounds: 17α-hydroxylase/17, 20-lyase deficiency (17-OHD) is a rare recessive hereditary disease, which could be attributed to cytochrome P450 17 α-hydroxylase (P450c17) deficiency caused by CYP17A1 gene mutations. Methods : A large cohort of 10 Chinese Han patients with 17-OHD from 2012 to 2020 were enrolled. The clinical and biochemical features were investigated and genetic mutations of CYP17A1 were analyzed by PCR-Sanger sequencing. Karyotype identification and SRY gene test were also carried out. In-silico analysis was used to predict the effects of genetic mutations on the protein function. Results: Gender was female in all the patients. The common complaints were hypertension, hypokalemia and primary amenorrhea. Karyotype was 46, XY and SRY gene was detected in 7 patients, and karyotype was XX in the remaining 3 patients. A total of 7 mutations including Y329N, Y329*, Y329Lfs*, R96W, A82D, S380N and A487_P489del have been identified in CYP17A1 gene. Y329Lfs* mutation was found in 9/10 (90%) patients with a high allele frequency of 70%. In-silico prediction showed that a novel variant of c.1139G>A (S380N) occurred at a conserved residue and should be disease-causing. Conclusion: We presented a detailed description of the clinical and genetic characteristics in Chinese patients with 17-OHD, and concluded that Y329Lfs* mutation of CYP17A1 prevalent in Chinese Han population. Therefore, hotspot screening by PCR-Sanger sequencing for exon 6 of CYP17A1 could contribute to the rapid diagnosis of 17-OHD in China. Genetic counseling based on the genetic diagnosis for at-risk relatives is advised. Abbreviations: 17-OHD = 17α-hydroxylase/17, 20-lyase deficiency; 17a-OHP= 17ahydroxyprogesterone; ACTH= adrenocorticotropic hormone; ACMG= American College of Medical Genetics and Genomics; CAH= congenital adrenal hyperplasia; CT= computed tomography; CYP17A1= cytochrome P450 family 17 subfamily A member 1 gene; DHEAS= dehydroepiandrosterone sulfate; EV= estradiol valerate tablets; HC= hydrocortisone; HGMD= Human Gene Mutation Database; P450c17= P450 17 α-hydroxylase; PCR= Polymerase chain reaction; PRED= prednisone; UFC= 24-h urinary free cortisone; SRY= sex-determining region Y gene.