INTRODUCTION First described in 1964, 46, XX male syndrome is a rare disorder of sex development (DSD) characterized by a discordance between a female karyotype and male phenotype. 1, 2 Its prevalence is estimated to be 3 to 4 cases per 100, 000 live male births. 3, 4 Clinically, the affected individuals can be grouped into three categories: phenotypically normal males with infertility, males with genital ambiguity, and true hermaphrodites. Previous reports have linked this condition to autoimmune thyroiditis and myelodysplastic syndromes. 5, 6 Here, we describe the case of a child with sex-determining region Y (SRY) -negative 46, XX male syndrome who presented with bone marrow failure despite a normal male phenotype. Unlike most SRY-negative patients, who often exhibit genital anomalies detectable at birth, 7, 8 this patient had normal height, bilateral testes of typical volume, and no hypospadias or micropenis. To our knowledge, no previous cases of SRY-negative 46, XX male syndrome associated with congenital bone marrow failure have been reported. Whole-exome sequencing (WES) excluded SRY mutations but uncovered variants within multiple genes governing ovarian repression, testis determination, and steroid receptor function. These alterations may collectively disrupt gonadal development, thereby producing a male phenotype despite the absence of Y chromosomal material. Moreover, additional variants of uncertain significance suggested a possible genetic contribution to bone marrow failure. 2. CASE PRESENTATION In May 2017, an 8-year-old boy was referred to a local hospital for evaluation of pancytopenia accompanied by recurrent epistaxis. Initial hematological analysis revealed leukopenia (white blood cell count WBC 3. 4 × 109/L, neutrophils 1. 22 × 109/L), anemia (hemoglobin Hb 96 g/L), and severe thrombocytopenia (platelets 44 × 109/L). Bone marrow smear examination demonstrated reduced myeloid proliferation, and biopsy confirmed hypoplastic hematopoiesis with absent megakaryocytes. Screening for paroxysmal nocturnal hemoglobinuria clones yielded negative results. Based on these findings, the patient was diagnosed with aplastic anemia. The patient received dexamethasone (0. 75 mg daily, discontinued after two months), stanozolol (2 mg twice daily), and cyclosporine (50 mg every 12 h). The patient had a satisfactory clinical response: white blood cell count and hemoglobin normalized, though platelet levels fluctuated between 23 and 59 × 109/L. One year later, in June 2018, the child was admitted to our department due to a platelet count drop to 10 × 109/L with recurrent nasal bleeding. Physical examination revealed mild anemia, scattered ecchymoses, and normal-sized testes without hypospadias or micropenis. No hepatosplenomegaly was detected. Anthropometric measurements indicated a weight of 31 kg and a height of 135 cm. Additional features included a small head, narrow palpebral fissures, and right thumb malformation, whereas café-au-lait spots were absent. His gross motor development was normal despite an in-toeing gait. Ultrasonography of the urinary system revealed no structural anomalies, and serum testosterone level was within the reference range. Viral serology and autoimmune panel test results were negative. The patient was born to a gravida 3, para 2, abortus 1 (G3P2A1) mother. The mother’s obstetric history included a first trimester induced abortion (G1), the birth of the current index case (G2P1), and a subsequent healthy female sibling (G3P2). Delivery was performed by cesarean section due to preterm labor associated with abdominal trauma. The patient’s birth weight was 2. 4 kg. The family history was unremarkable and his younger sister was healthy. 3. MATERIALS AND METHODS 3. 1. Diagnosis of bone marrow failure Colony-forming assays demonstrated markedly reduced levels of hematopoietic progenitors, colony-forming unit-erythroid (CFU-E), burst-forming unit-erythroid (BFU-E), and colony-forming unit-granulocyte-macrophage (CFU-GM). Repeat marrow aspirations confirmed hypocellularity with dyserythropoiesis, granulocytic hypoplasia, and rare megakaryocytes. A trephine biopsy revealed marked hypocellularity without fibrosis. Comprehensive testing, including immunophenotyping, fusion gene analysis, fluorescence in situ hybridization, and comet assays, yielded negative results. 3. 2. Karyotyping Metaphase chromosomes from cultured bone marrow and peripheral blood lymphocytes were examined by Wright G-banding. 3. 3. Genetic analysis Genomic DNA was extracted using the Universal Genomic DNA Kit (CWBIO, Beijing, China). DNA was fragmented into 180–280 bp fragments using Covaris, captured with the Agilent SureSelect Human All Exon V6 kit, and sequenced on the Illumina HiSeq PE150 platform. The reads were aligned to the GRCh37/hg19 reference genome. Bioinformatics processing was conducted using Novogene and MyGenostics. Variant calling included the detection of structural variants (SVs), single-nucleotide variants (SNVs), and insertions/deletions (INDELs). 4. RESULTS 4. 1. Karyotype analysis Chromosomal analysis of the bone marrow confirmed a 46, XX karyotype without structural abnormalities, excluding mosaicism and Y-chromosome material. The peripheral blood analysis yielded concordant results (Fig. 1). Figure 1.: G-banding karyotype of the patient. The analysis reveals a 46, XX karyotype, confirming the absence of a Y chromosome. 4. 2. Whole-exome sequencing WES excluded SRY and SOX (SRY-box) family gene variants, but revealed multiple alterations in genes involved in gonadal differentiation and steroidogenesis (Table 1; Figure 2; Table S1, https: //links. lww. com/BS/A142). Table 1. - Filtered SNVs, INDELs, and SVs identified in the SRY-negative 46, XX male syndrome with bone marrow failure. Chr Gene Name HGNC (gene) OMIM (gene) Start End SV Reference sequence dbsnp150 SNV/INDEL Locus (human) zygosity 1000g2015augₐll SIFT Mutation Taster Genes associated with loss-of-function in female pathway 1 WNT4 12783 603490 8446628 60870960 INS — — — — — — — — 1 RSPO1 21679 609595 8446628 60870960 INS — — — — — — — — 3 CTNNB1 2514 116806 28882969 49052285 DEL — — — — — — — — Genes associated with 46, XX disorders of testis development 9 DMRT1 2934 602424 841971 841971 — NM₀21951 rs3739583 exon1: c. 133T>A: p. S45T 9p24. 3 heterozygosis 0. 224641 0. 474, T 1, P 9 DMRT1 2934 602424 967981 967981 — NM₀21951 rs279895 exon5: c. 968-4G>C: splicing 9p24. 3 homozygous 0. 9361 — — 8 FOG2 16700 603693 68297270 129244929 DEL — — — — — — — — Sex differentiation (eg, steroid synthesis/receptors) 10 CYP17A1 2593 609300 62512366 123228974 DEL — — — — — — — — 8 STAR 11359 600617 25828254 39487128 INS — — — — — — — — 8 STAR 11359 600617 38002807 38002807 — NM₀00349 rs537903614 exon6: c. 677T>C: p. V226A 8p11. 23 heterozygosis 0. 0002 0. 001, D 1, D 2 SRD5A2 11285 607306 31805880 31805880 — NM₀00348 rs142200057 exon1: c. 89₈9 + 1insC 2p23. 1 homozygous 1. 0000 — — X AR 644 313700 66766356 66766356 — NM₀00044 rs760580125 exon1: c. 1368₁369insGGC: p. G456delinsGG Xq12 heterozygosis 0. 1976 — — X AR 644 313700 66765158 66765158 NM₀00044 unknown exon1: c. 170₁71insGCAGCAGCAGCAGCAGCAGCAGCA: p. L57delinsLQQQQQQQQ Xq12 heterozygosis unknown — — Chr = chromosome, DEL = deletions, INDEL = insertion and deletions, INS = insertion, SNV = single-nucleotide variation, SV = structural variation. Figure 2.: Proposed molecular mechanisms underlying SRY-negative 46, XX male syndrome with bone marrow failure. Pathogenic variants in ovarian-specifying genes (WNT4, RSPO1, and CTNNB1) perturb canonical Wnt/β-catenin signaling, thus compromising ovarian differentiation and failing to suppress the testicular program. Simultaneously, alterations in testis-favoring regulators (GATA4, FOG2, and DMRT1) activate testis-specific transcriptional networks while further repressing ovarian gene expression. Moreover, abnormalities in steroidogenic and androgen-associated genes (STAR, CYP17A1, SRD5A2, and AR) impair androgen biosynthesis and signaling, thereby influencing downstream gonadal maturation. Collectively, these multilayered genetic perturbations—despite an XX karyotype and absence of SRY—synergistically produce a fully masculinized phenotype with bone marrow failure. Variants in female pathway genes included WNT4 (wingless-type MMTV integration site family member 4), RSPO1 (R-spondin family member 1), and CTNNB1 (catenin beta 1), key regulators of ovarian differentiation through the Wnt/β-catenin pathway. 9 Disruption of this signaling axis may attenuate ovarian development and permit the ectopic activation of testicular pathways. 10 In testis development genes, a missense mutation in DMRT1 (doublesex- and mab-3-related transcription factor 1) and a large fragment deletion of FOG2/ZFPM2 (friend of GATA2/zinc finger protein, FOG family member 2) were identified. Both genes act within the GATA4 (GATA Binding Protein 4) –FOG2–DMRT1 network, which is essential for testicular differentiation, suggesting aberrant activation of pro-testicular transcriptional programs in the absence of SRY. 11, 12 Deletions or mutations in STAR (steroidogenic acute regulatory protein), CYP17A1 (cytochrome P450 family 17 subfamily A member 1), SRD5A2 (steroid 5 alpha-reductase 2), and AR (rogen receptor) were detected in steroidogenesis and androgen receptor genes. These variants likely compromised androgen biosynthesis or signaling, contributing to the patient’s incomplete virilization despite testicular gene activation. Collectively, these findings indicate that dysregulation of the Wnt/β-catenin and GATA4–FOG2–DMRT1 pathways, compounded by impaired steroidogenesis, underlies the patient’s SRY-negative 46, XX male phenotype. 4. 3. Diagnosis and treatment Based on the combined genetic and clinical evidence, the patient was diagnosed with SRY-negative 46, XX male syndrome complicated by bone marrow failure. Treatment with cyclosporine (50 mg q12h), stanozolol (2 mg daily), and levamisole (50 mg daily) stabilized peripheral counts: WBC 4. 25 × 109/L, Hb 106 g/L, and platelets 60 × 109/L. The hematological response suggests androgen-mediated stimulation of erythropoiesis along with immunomodulatory effects. 5. DISCUSSION The 46, XX male syndrome represents an uncommon DSD, with an estimated frequency of 1 in 20, 000 to 25, 000 live male births. 13 It is defined as a discordance between the chromosomal and phenotypic sex. Clinically, three major categories have been described: infertile but otherwise phenotypically normal males, individuals with genital ambiguity (eg, hypospadias, micropenis, and hyperclitoridy), and true hermaphrodites characterized by the coexistence of ovarian and testicular tissues. 4, 14 Our patient had the classical form, presented as a phenotypically normal male despite an XX karyotype, and uniquely exhibited co-existing bone marrow failure. In most cases, SRY translocation accounts for testicular development in individuals with 46, XX karyotype. However, approximately 10% of cases are SRY-negative, suggesting alternative molecular mechanisms. 15 Gain-of-function variants in pro-testicular genes (eg, SOX9, SOX3, SOX8, and SOX10) or loss-of-function variants in ovarian-promoting genes (eg, WNT4, RSPO1, FOXL2 Forkhead Box L2, and CTNNB1) can initiate testicular differentiation in the absence of SRY. 16–20 In this patient, WES excluded pathogenic variants in SRY and SOX family genes, but revealed multiple variants within female pathway genes, testis-determining genes, and steroidogenic genes. The Wnt/β-catenin signaling pathway is essential for ovarian fate maintenance, as genetic ablation of its components (WNT4, RSPO1, CTNNB1, or RSPO1 putative receptor LGR4) results in partial sex reversal toward a testicular phenotype. 10, 21 Variants in WNT4 have been associated with Müllerian anomalies, hyperandrogenism, and Sex Reversion, Kidneys, Adrenal and Lung (SERKAL) dysgenesis syndrome. 22, 23RSPO1 mutations are linked to SRY-negative 46, XX testicular or ovotesticular DSD. 24 Loss of RSPO1 function alone was sufficient to induce complete female-to-male sex reversal in the absence of the testis-determining gene SRY. 25 In this case, SVs affecting WNT4, RSPO1, and CTNNB1 likely impaired canonical Wnt/β-catenin signaling, leading to female-to-male sex reversal. During early gonadal differentiation, GATA4 functions as a central transcriptional regulator by binding to DNA through its zinc finger domain and recruiting FOG2 via its N-terminal finger domain to form a core transcriptional complex. 26 The GATA4–FOG2 interaction is crucial for initiating testis formation, 11 and disruption of this complex can alter the balance of sex determination. Notably, large fragment deletions of FOG2 have not been previously reported in individuals with 46, XX DSD, suggesting that the novel deletion identified in our patient may have a functional impact on testicular differentiation. Downstream of this regulatory axis, GATA4 directly modulates the transcription of DMRT1, maintaining a high expression level required for testicular development. 12DMRT1 reinforces testicular fate by activating SOX9 and repressing ovarian-promoting genes, such as FOXL2 and WNT4. 27 A novel de novo DMRT1 duplication was identified in an SRY-negative 46, XX male, potentially causing male sex determination by escaping transcriptional repression. 28 The variant identified in our patient (p. S45T) could potentially shift this regulatory balance toward testis formation. Epigenetic and coregulatory mechanisms further fine-tune this pathway. GATA4 expression can be downregulated by promoter hypermethylation and histone hypoacetylation (H3/H4), 26 whereas p300 overexpression has been shown to relieve FOG2-mediated repression of GATA4 transactivation. 29 Collectively, these findings underscore the intricate, multilayered regulation of GATA4–FOG2–DMRT1 signaling in human testis determination. Despite the established importance of the GATA-FOG complex in hematopoiesis, 30 a causal link to bone marrow failure has not been established. Epigenetic mechanisms such as dimorphic miRNAs and DNA methylation regulate gonadal development and may contribute to sex determination. However, their direct role in 46, XX DSD has not been confirmed. 31 The patient harbored steroidogenic gene variants, including a large fragment deletion in CYP17A1 and alterations in STAR, SRD5A2, and AR. The identified pathogenic STAR variant (p. A218V) has been shown to disrupt cholesterol transport, leading to steroidogenic failure and causing 46, XY DSD combined with congenital lipoid adrenal hyperplasia. 32 Although CYP17A1 mutations typically disrupt androgen biosynthesis, our patient retained normal external genitalia, suggesting either residual enzyme activity or compensatory steroidogenic pathways maintaining androgen levels. 33 Notably, previous reports have described hypospadias in individuals harboring dual variants of AR and SRD5A234; however, our case presented with a completely masculinized phenotype. This highlights the variability in genotype–phenotype correlations among 46, XX DSD patients. Long-term follow-up is essential to monitor for potential manifestations including gynecomastia or adrenal insufficiency during adolescence. To date, few studies have documented hematological abnormalities in patients with 46, XX male syndrome. Our case was notable for the congenital bone marrow failure accompanied by dysmorphic features (small head, thumb anomaly, and in-toeing gait). Although the pathogenic link between sex reversal syndromes and bone marrow dysfunction remains unclear, cryptic genetic variants affecting hematopoietic regulation cannot be excluded. Chromosomal instability, as observed in hematological malignancies, may contribute to overlapping mechanisms. Further genomic and functional studies are required to confirm these associations. This case supports the diagnostic utility of WES in uncovering complex genetic underpinnings of DSD. 35, 36 Comprehensive genomic analyses can delineate rare variants, inform prognosis, and guide multidisciplinary management encompassing hematology, endocrinology, and genetics. Long-term surveillance is warranted to address the evolving endocrine, hematological, and psychosocial outcomes in these patients. 6. CONCLUSION Here, we report a rare case of SRY-negative 46, XX male syndrome associated with congenital bone marrow failure. WES identified multiple variants disrupting ovarian pathways, testis differentiation, and steroid receptor function, which collectively explain the male phenotype, despite the absence of Y chromosomal material. In addition, undefined variants may contribute to bone marrow failure, suggesting a novel link between sexual development and hematopoietic regulation. Our findings emphasize the value of WES as an effective diagnostic approach in complex DSD cases and highlight the need for continued investigation of the molecular and clinical interplay between gonadal and hematopoietic development. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foun-dation of China (82200193, 82572140). We express thanks to MyGenostics for their technical assistance. We want to acknowledge the patient and his family for participating in the follow-up. AUTHOR CONTRIBUTIONS A. Z. and L. L. conceived the study, analyzed data, and drafted the manuscript. Y. Z. , W. Y. , and X. Z. reviewed it. M. R. , L. Z. , Y. Z. , and Y. C. provided clinical data. L. C. and X. Z. supervised the work and critically revised the manuscript. All authors approved the final version.
Zhang et al. (Wed,) studied this question.