SRY-negative disorder of sex development in two biological brothers with SOX3 mutations

INTRODUCTION

Disorders of sex development (DSD) occur in approximately 1 in 1000–4500 live births, with a very large range of clinical presentations and underlying pathology.^[1]

Normal sexual differentiation is a very complex and genetically diverse process. One important genetic component of sexual differentiation is the SRY (sex-determining region of Y) gene, which is normally found on the Y chromosome. When present, the SRY gene acts to drive the development of testes, while the absence of the gene leads to the development of ovaries.^[1]

A well-described DSD presentation of phenotypic males is a scenario in which the SRY gene is translocated onto an X chromosome, leading to a phenotypic male with a 46,XX karyotype.^[2]

Less commonly, cases occur in which 46,XX phenotypic males do not have the presence of an SRY gene. In these cases, it has been hypothesized that the overexpression of the SOX3 (SRY-box transcription factor 3) gene leads to the male phenotype.^[3-5]

At present, there are 6 total case reports describing this phenomenon.^[3,4,6-9]

Here, we present the first case of biological siblings with male phenotype who were found to have SRY-negative, XX male phenotypes with SOX3 duplications. This further supports the theory of SOX3 as a functional SRY gene and provides a basis for a possible hereditary component of the mutation.

CASE REPORT

A 17-year-old boy presented to the pediatric endocrinology clinic with concerns for lack of phallus virilization. He had a history of hypospadias with webbing requiring 3 surgical repairs with pediatric urology. Upon presentation, he had been cleared by pediatric urology for several years. He had no concerns with behavior, growth, or development. He developed pubic and axillary hair in the 4^th/5^th grade and noted a growth spurt in the 7^th/8^th grade. He reported voice deepening and needing to shave a mustache recently. There were no headaches, vision changes, fatigue, polyuria, polydipsia, abdominal pain, nausea, vomiting, constipation, diarrhea, skin changes, fevers, or joint pain. He endorsed a normal sense of smell. The patient reported his gender identity as male. He denied sexual activity but was able to ejaculate with masturbation. One of his brothers had hypospadias; his family history was non-contributory. Physical examination was notable for Tanner 5 pubic hair. Testes measured 6 cc bilaterally. Stretched penile length was 5.2 cm with chordee present. Otherwise, his physical examination was within normal limits.

To further investigate this undervirilized, phenotypic male, laboratory tests and imaging were ordered. His bone age was congruent with his chronological age of 17. Thyroid function studies and insulin-like growth factor-1 level were normal. Gonadotropins revealed a follicle-stimulating hormone (FSH) level that was within normal limits at 8.35 mIU/mL (reference range for late pubertal males approximately 1.5–12.4 mIU/mL)^[10] and a luteinizing hormone (LH) level that was mildly elevated at 5.73 mIU/mL (reference range approximately 1.7–8.6 mIU/mL).^[10] Anti-Müllerian hormone was normal at 34.00 ng/mL (<128.29 for males).^[10] A morning testosterone level was 353 ng/dL, correlating with Tanner Stage 5 (reference range for Tanner V males approximately 300–1000 ng/dL).^[10] Dihydrotestosterone was within normal limits at 18 ng/dL (12–65 ng/dL). Inhibin B was 104 pg/mL (reference range for adolescent males approximately 80–300 pg/mL).^[10]

These laboratory evaluations were not clearly suggestive of either hypogonadotropic hypogonadism or hypergonadotropic hypogonadism. A karyotype analysis was sent as part of a complete workup in view of his age, lack of virilization, and history of hypospadias which was found to be 46,XX. A comprehensive DSD gene panel was performed through prevention genetics using next-generation sequencing with copy number variant (CNV) analysis. Chromosomal analysis confirmed a 46,XX karyotype without detectable Y chromosomal material. CNV analysis identified a duplication involving the SOX3 gene located on Xq27.1. No SRY gene sequences were detected, confirming an SRY-negative 46,XX testicular DSD. He was referred to Pediatric Urology to identify possible ovarian tissue or ectopic testicular tissue, given the unknown risk for gonadoblastoma with this genetic abnormality. Abdominal ultrasonography demonstrated normal renal anatomy with no evidence of congenital renal anomalies, and no ovarian or ectopic gonadal tissue was identified.

The patient was offered a semen analysis to estimate his fertility potential which was declined as per the wishes of the subject.

The patient has two younger brothers, one of whom also had hypospadias requiring surgical correction by pediatric urology. Given the diagnosis in the older sibling, this brother was referred to the clinic at 12 years of age for further evaluation. Following the index patient’s diagnosis, this patient underwent a targeted endocrine and genetic evaluation. His parents reported normal growth (height at 70^th percentile and weight at the 12^th percentile), normal development, pubertal onset (pubic hair stage 2 with 5 mL testes and penile length of 6.5 cm), and no pertinent history. Evaluation revealed karyotype analysis 46,XX, LH level of 0.2 mIU/mL (reference range for early pubertal males approximately 0.1–6.0 mIU/mL),^[10] FSH of 0.81 mIU/mL (reference range for early pubertal males approximately 0.3–4.6 mIU/mL),^[10] and total testosterone of 60 ng/dL (reference range for Tanner II males approximately 20–150 ng/dL).^[10]

The prevention genetics DSD panel performed in both siblings revealed an SRY-negative pattern with SOX3 duplication.

DISCUSSION

46,XX male DSD can be broadly classified based on the presence or absence of the SRY gene. Approximately 90% of cases are SRY-positive, most commonly due to translocation of Y chromosomal material containing the SRY locus onto the X chromosome during paternal meiosis, while the remaining cases are SRY-negative. Individuals with SRY-positive 46,XX DSD typically demonstrate normal external male genitalia and masculinization and are often diagnosed in late adolescence or adulthood during evaluation for infertility or small testicular size. In contrast, SRY-negative 46,XX DSD is rare and is thought to result from dysregulation of downstream testis-determining pathways.^[5]

The presence of increased SOX3 expression has been theorized to play a role as a functional SRY gene in cases of phenotypically male patients with 46,XX karyotype.^[1] While rare, 6 case reports to date have supported this possibility.^[3,4,6-9] Comparison with previously reported SRY-negative 46,XX individuals with SOX3 rearrangements demonstrates a wide phenotypic spectrum, ranging from ovotesticular DSD and ambiguous genitalia to phenotypic males with hypospadias and cryptorchidism. Most prior cases involved isolated patients with de novo SOX3 duplications.

One previous report by de Oliveira et al. described biological siblings with SOX3 duplication; however, both were 46,XX with differing phenotypes, including ovotesticular DSD in one sibling and atypical genitalia in the other.^[3,4,6-9] Our case of two biologic phenotypic males with these genetic findings further supports this hypothesis and raises the possibility of a hereditary component to the mutation rather than a spontaneous mutation. We report the first known case of biological siblings with male phenotypes. Summarizing the findings from the aforementioned sources plus our novel findings of two affected brothers, we postulate germline mosaicism as the likely pattern of inheritance. Germline mosaicism would result in cells of the gonads having differing genetics in various proportions. The other cells of the body, otherwise known as somatic cells, remain unaffected. This could probably explain the differing phenotypes of our two patients and others with the same genetic mutation. The parents of these two patients were not tested, but the parents studied by de Oliveira et al. demonstrated normal parental testing.^[4]

Notably, both brothers had normal testosterone, FSH, and LH levels for their respective ages. In SRY-positive 46,XX men, typically levels of testosterone are normal at the time of spermarche; however, they decrease in adulthood. This is followed by increasing gonadotropin levels resulting in hypergonadotropic hypogonadism in adulthood.^[9] This is different from the levels seen in Patient 1. At 17 years of age, puberty should be in a more advanced stage in this patient with a congruent bone age. The patient’s testosterone and FSH levels were normal for age. LH was elevated for age which is consistent with previous findings of patients with evolving hypergonadotropic hypogonadism.

Another point to be borne in mind is the possibility of gonadoblastoma or other tumors. Patients with DSD are at risk for gonadoblastoma and germ cell carcinoma in situ.^[11] Gonadoblastoma subtypes include seminoma, dysgerminoma, and different subtypes such as yolk sac carcinoma, embryonal carcinoma, choriocarcinoma, and teratomas. It is not known whether SOX3 upregulation would predispose to gonadoblastoma and it is an important point for potential future research.

Finally, limited data is known about the fertility of these patients. Semen analysis was offered to patient 1, but at this time, it has not been feasible. To date, fertility has not been studied in the aforementioned patients with SOX3 mutations.

It has been hypothesized that SOX3 is the evolutionary ancestor of the SRY gene. This case report would help support this hypothesis. It has been shown to affect sexual differentiation in mice.^[2] In the aforementioned case of biological siblings with SOX3 duplication, the patients’ parents underwent a complete microarray which showed no genetic anomalies.^[4] SOX3 is located on the X chromosome (Xq27.1) and follows an X-linked pattern of inheritance. Reported cases of SOX3 duplication may arise either de novo or through maternal transmission, particularly in families with multiple affected male offsprings. Paternal transmission is biologically unlikely, as fathers transmit a Y chromosome to male offspring and therefore would not be expected to pass an X-linked SOX3 duplication to sons. All current evidence supports the upregulation of SOX3 to be a germline mutation.^[3,4,8] The parents of these two patients were not tested, but the parents studied by de Oliveira et al. ^[4] demonstrated normal parental testing. These findings also highlight the value of combining sequencing-based approaches such as whole-exome sequencing with CNV analysis, as structural rearrangements like SOX3 duplications may be missed by sequencing alone.

CONCLUSION

In this case report, we describe two biological siblings with SRY-negative 46,XX testicular disorders of sex development associated with SOX3 duplication who both presented with a male phenotype and hypospadias. This case further supports the concept that SOX3 upregulation may function as an SRY-like mechanism in phenotypically male individuals with a 46,XX karyotype. The presence of the same SOX3 duplication in two biological siblings also raises the possibility of a hereditary mechanism, such as germline mosaicism, contributing to recurrence within a family.