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Table of Contents   
CASE REPORT
Year : 2023  |  Volume : 28  |  Issue : 2  |  Page : 154-159
 

A rare differences of sex development: male sex reversal syndrome (NonSyndromic 46, XX with Negative Sex-Determining Region of Y Chromosome Gene)


1 Department of Endocrinology and Metabolism, Institute of Post Graduate Medical Education and Research, SSKM Hospital, Kolkata, West Bengal, India
2 Institute of Child Health, Kolkata, West Bengal; Organization of Rare Disease, Bengaluru, Karnataka, India

Date of Submission12-Aug-2022
Date of Decision10-Sep-2022
Date of Acceptance30-Sep-2022
Date of Web Publication30-Nov-2022

Correspondence Address:
Pankaj Singhania
Department of Endocrinology and Metabolism, Institute of Post Graduate Medical Education and Research, SSKM Hospital, 244, AJC Bose Road, Kolkata - 700 020, West Bengal
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jiaps.jiaps_109_22

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   Abstract 


46, XX testicular differences of sex development (DSD) is a rare cause of DSD presenting as a phenotypical male with chromosomal sex of 46, XX. Sex-determining region of the Y chromosome (SRY)-positive 46, XX DSDs have a well-characterized pathogenetic mechanism, whereas in SRY-negative 46, XX DSDs, the pathogenesis is not clearly delineated. Herein, we present a case of a 3½-year-old child who presented with ambiguous genitalia and bilateral palpable gonads. On the basis of a karyotype and fluorescent in situ hybridization, we arrived at a diagnosis of SRY-negative 46, XX testicular DSD. Basal serum estradiol and human menopausal gonadotrophin stimulated estradiol levels and inhibin A blood levels were against the presence of any ovarian tissue. Imaging of the gonads showed bilateral normal-looking testis. A clinical exome sequencing revealed a heterozygous missense variant NR5A1:c275G>A (p. Arg92gln) located at exon 4 in the affected child. Protein structure analysis was further performed, and the variant was found to be highly conserved. Sanger's sequencing showed that the mother was heterozygous for the variant detected in the child. This case highlights the rarity of SRY-negative 46, XX testicular DSD with a unique variant. Largely under characterized, this group of DSDs needs to be reported and analyzed to add to the spectrum of presentation and genetic characteristics. Our case is expected to add to the database, knowledge, and approach to cases of 46, XX testicular DSD.


Keywords: 46, XX, differences of sex development, sex-determining region of Y chromosome negative, testicular


How to cite this article:
Singhania P, Ghosh A, Das D, Neogi S, Bhattacharjee R, Datta D. A rare differences of sex development: male sex reversal syndrome (NonSyndromic 46, XX with Negative Sex-Determining Region of Y Chromosome Gene). J Indian Assoc Pediatr Surg 2023;28:154-9

How to cite this URL:
Singhania P, Ghosh A, Das D, Neogi S, Bhattacharjee R, Datta D. A rare differences of sex development: male sex reversal syndrome (NonSyndromic 46, XX with Negative Sex-Determining Region of Y Chromosome Gene). J Indian Assoc Pediatr Surg [serial online] 2023 [cited 2023 Mar 29];28:154-9. Available from: https://www.jiaps.com/text.asp?2023/28/2/154/362390





   Introduction Top


Differences of sex development (DSDs) are rare disorders caused by abnormal chromosomal, anatomical, or gonadal development. DSDs are divided into 46, XY DSD caused by androgen biosynthetic defects, a defect in androgen action, or gonadal dysgenesis; 46, XX DSD resulting from androgen excess or disorders of gonadal development; and chromosomal DSD such as Turner syndrome, Klinefelter syndrome, mixed gonadal dysgenesis, and ovotesticular DSD.

46, XX testicular DSD is a rare cause of DSD presenting as a phenotypical male with chromosomal sex of 46, XX. This rare cause of DSD with varying incidence worldwide presents differently in different stages of life. In infancy, it can present with ambiguous genitalia with palpable gonads and in adults with primary infertility.[1] Also known as de La Chapelle syndrome, named after the first case reporting in 1964, few cases have been reported since then.[2] Ninety percent of the XX males are sex-determining region of the Y chromosome (SRY) positive and the rest are SRY negative. In the SRY-negative group, because of a lack of distinction of the Y chromosome or the SRY gene in these subjects, the characterization of SRY-negative XX males has been largely sketchy.[3]

Unlike SRY-positive 46, XX DSDs where the pathogenesis involves translocation of the SRY gene from Y to X chromosome, the cause in SRY-negative cases is not clearly defined. An imbalance between protestes and anti-testes genes has been implicated in some series. Overexpression of protestes genes such as SOX-3, SOX-9, NR5A1, and underexpression of anti-testes genes such as DAX-1 and WNT-4 may explain the etiopathogenesis.[4]

In view of the above information and the rarity of this entity, we present a case of 46, XX testicular DSD with genetically proven sex reversal to add to the few cases reported in the literature.


   Case Report Top


This 3½-year-old child, the only issue of a nonconsanguineous marriage presented with ambiguous genitalia noted since birth (small phallus, bifid scrotum, and bilateral scrotal gonads). There was no history of the crisis, increasing pigmentation, or neonatal hypoglycemia. Maternal virilization or androgen exposure in pregnancy was also absent. The child was reared as a male. There was no history of similar complaints in any family member.

The child was born at term by cesarean section, had a birth weight of 2.2 Kg and the perinatal period was uncomplicated. Till this age of presentation, the developmental milestones were largely as per schedule.

Examination revealed a healthy-looking playful child weighing 14kgs and 91 cm tall. Blood pressure was normal for age. Genital examination revealed an External Masculinization Score of 3/12 (bilateral testis in labioscrotal folds; absent scrotal fusion; micropenis; and a proximal urethral meatus). The anogenital ratio was 0.6 [Figure 1].
Figure 1: Genitalia of proband showing micropenis, bifid scrotum, proximal hypospadias, and bilateral labioscrotal gonads

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Ultrasonography did not show any Mullerian structures and confirmed the presence of testes in the labioscrotal folds bilaterally [Figure 2]. The prostate was not clearly delineated. An MRI pelvis also corroborated these findings. A karyotype ordered next revealed 46, XX [Figure 3]. Routine investigations including electrolytes were essentially normal. Serum AMH was >23 ng/ml, serum LH 0.101 U/L (n < 0.3), serum FSH 0.951U/L, serum testosterone <20 mg/ml, serum 17-hydroxyprogesterone 0.92 ng/ml (n < 10) and serum estradiol <20 pg/ml. All these values were suggestive of functioning Sertoli cells and the prepubertal status of androgen synthesis. In view of the above, a working diagnosis of 46, XX testicular DSD was made, and fluorescent in situ hybridization (FISH) was ordered to look for SRY translocation. The FISH report for SRY translocation was negative.
Figure 2: USG showing bilateral hypoechoic oval teste without any Mullerian structures

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Figure 3: Karyotype of Index child (proband)

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Clinical exome sequencing

Focused exome sequencing of protein-coding regions in genes associated with the disease was performed using NGS. Heterozygous missense variant NM_004959.5 (NR5A1): c.275G>A (p. Arg92gln) located at exon 4 identified in the individual. This variant is classified as a likely pathogenic mutation. The variant has been reported to ClinVar as pathogenic with a status of (0 stars) no assertion criteria provided (Variation ID 12796 as of 2021-10-07). The p. Arg92gln variant though is novel (not in any individuals) in gnomAD, indigen, and in 1kG. There is also only one classified pathogenic variant (c.271G>A; p. G91S) within 6 amino acid positions of the variant p. Arg92Gln, providing insufficient evidence of being in a mutation hot spot. The protein structure analysis indicated, the variant is highly conserved and located in the Ftz-F1 region which is probably involved in DNA-binding specificity and stability. This residue is predicted to be functional and is exposed and hence may be tightly related to protein functions as understood by the ConSurf images [Figure 4]. Glutamine side chains do not penetrate as deeply into the minor groove as do the wild-type arginine side chains. We discovered that the mutation did not affect the stability of the isolated NR5A1 DNA-binding domain but may affect the interaction of the NR5A1 DNA-binding domain with its response elements as indicated by 9% lower (enthalpic) in silico binding energy. These effects may be variable and specific with different response elements. The above indicates that this variant can be significant in causing disease.
Figure 4: ConSurf image of the protein sequence indicating the conserved residues across species

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To detect the presence of functioning ovarian tissue in the gonads including the possibility of ovotesticular DSD, human menopausal gonadotrophin (hMG) stimulation test was conducted in the child with 150 IU hMG intramuscularly administered for 3 consecutive days. Twenty-four hours following the last injection, serum estradiol, and inhibin A were measured. Serum estradiol was <20 pg/ml and serum indibin A was <1 pg/ml. USG inguinoscrotal region was also done which revealed no follicular development. This further proved that the gonads were testicular and not ovotesticular.

A final diagnosis of SRY-negative 46, XX testicular DSD due to a heterozygous missense mutation in p. arg92gln region of NR5A1 was made.

In view of the identified mutation in the child, Sanger sequencing for the parents was ordered which showed that the mother was heterozygous for the variant detected in the child. The father's report was normal [Figure 5]. The mother was then interrogated for any such symptoms or similar in her family members. Taking note of the fact that there is a phenotypical correlation of premature ovarian insufficiency with the variant in the discussion, the mother was questioned and investigated along these lines. The mother had no history of delayed puberty, amenorrhea, or irregular menstruation. She was a normal-looking female with tanner five breasts and normal genitalia. Her blood reports showed an early follicular phase serum LH of 6.7 mIU/ml and FSH of 5.6 mIU/ml. Her AMH level was 1.2 ng/ml, and she had a 46, XX karyotype.
Figure 5: Sanger sequencing data (electropherogram) for the mother's sample showing nucleotide change at chr9: c.275G>A, (p. Arg92Gln) in NR5A1 gene (a). Sanger sequencing data (electropherogram) for the father's sample showing no nucleotide change at chr9: c.275G>A, (p. Arg92Gln) in NR5A1 gene (b)

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A multidisciplinary medical board with members from endocrinology, urology, pediatric medicine, pediatric surgery, and radiology was constituted to discuss the treatment of this case. The parents were asked about their choice of sex of rearing. All pros and cons of either sex of rearing were clearly explained. The parents are yet to decide on the sex of rearing. Whenever agreeable, genital surgery will be undertaken with the help of pediatric surgery and urology experts.


   Discussion Top


This infant with bilateral palpable gonads was initially being considered a case of 46, XY DSD with gross undervirilization. Once the karyotype showed 46, XX chromosomal composition, a diagnosis of 46, XX testicular DSD was provisionally made.

46, XX testicular DSDs are rare and most cases are discovered while investigating infertility in azoospermic males with a 46, XX karyotype.

In our case along with the hormonal workup, a genetic analysis revelated a heterozygous mutation in NR5A1 (p. Arg92Gln) as a cause for this disorder. To our knowledge, this is the first case of this mutation in an Indian subject with SRY-negative testicular DSD. NR5A1 codes SF which regulates CYP11A1 a seminal step in testicular development.[3]

That the mother is also heterozygous and at present has no symptoms can be due to incomplete penetrance. She is young and it cannot be concluded if she has primary ovarian insufficiency as reported in a few cases. There have been numerous illustrations on intrafamilial phenotypical variability and incomplete penetrance, where a spectrum from unaffected mutation carriers to cases with 46, XX testicular DSD or ovotesticular DSD, have been described indicating that these conditions may represent a phenotypic spectrum of the same underlying defect. Incomplete penetrance has been shown to result from differences in spatiotemporal expression, in a 46, XY DSD mouse model (B6 XYpos). Further antagonism of the testis-and ovary-determining pathways during ovotestis development in mice have been delineated.[5]

NR5A1 has a role in both gonadal and adrenal development.[6] CYP11A1, CYP17A1, CYP21A2, CYP11B1, and CYP11B2.HSD3B1 STAR are the genes involved in the steroid synthetic pathway regulated by NR5A1.[4]

NR5A1 and SRY in synergy are responsible for testicular development through the upregulation of SOX9. Upregulation of SOX 9 is responsible for male reproductive tract development through AMH. On the other hand, NROB1 acts as a negative regulator of NR5A1 and SOX 9, thus facilitating female sexual differentiation. Homozygous NR5A1(-/-) knockout mice have revealed 46, XY sex reversal with adrenal insufficiency, whereas heterozygous mutations have shown milder phenotypes.[7]

More than 70 mutations in NR5A1 have been reported so far, the first being reported in a case of 46, XY sex reversal with adrenal failure.[8] It was a Gly35glu heterozygous mutation. The first report of SRY-negative NR5A1 in 46, XX individuals was done by Bashamboo et al. 2015.[4] This was a pArg92Trp heterozygous variant. Protein structure modeling of NR5A1 p.(Arg92Gln) was performed by Baetens et al., 2017 who showed that this mutated side chain binds in the DNA minor groove without hydrogen bond formation to the DNA.[9] Some studies have shown the role of the pArg92trp NR5A1 mutation in 46, XX sex reversal. NR5A1 is also less sensitive to NROB1 in this mutation, which increases the expression of SOX9 in 46, XX testicular DSD individuals. This suppression of NROB1 is through b-catenin suppression in the WNT signaling pathway. This suppression has also been shown to be present in heterozygous pArg92trp NR5A1 mutation. Hence, this variant suppresses ovarian pathways leading to testicular development in XX individuals who are SRY negative.

There have been theories linking the NR5A1 (also known as SF1) gene with various disorders such as infertility, adrenal disorders, ovarian development, and subsequently ovarian insufficiency.[10] A similar search was also carried out in our case. Although the mother was carrying the mutation, she has no phenotypical correlation including features of ovarian insufficiency.

This case report of a 46, XX testicular DSD with pArg92trp NR5A1 mutation highlights the importance of NR5A1 in gonadal development and will open up newer avenues for further characterization of 46, XX testicular DSDs.


   Conclusion Top


All DSDs with palpable gonads are not XY DSDs. In all cases of 46, XX testicular DSDs, SRY should be detected to classify as SRY-positive or SRY-negative 46, XX testicular DSD. The presentation of 46, XX testicular DSDs can be highly heterogenous from ambiguous genitalia to infertility in adulthood. NR5A1 which encodes SF1 is an important regulator of testicular development and mutations can cause 46, XX testicular DSDs. Apparently, normal females carrying the mutation should be followed up for ovarian insufficiency or premature menopause.

Informed consent

Taken from the child's parents.

Declaration of patient consent

The authors certify that they have obtained appropriate consent from parents. In the form, the legal guardian has given his consent for images and other clinical information to be reported in the journal. The guardian understands that names and initials will not be published and due efforts will be made to conceal patient identity, but anonymity cannot be guaranteed.

Learning points

  1. All differences of sex development (DSDs) with palpable gonads are not XY DSDs
  2. 46, XX testicular DSDs are mostly SRY positive and are well characterized
  3. SRY-negative 46, XX testicular DSDs are rare and less is known about them
  4. The heterozygous missense variant NR5A1: c275G>A (p. Arg92gln) is novel and not reported in gnomAD.


Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Akinsal EC, Baydilli N, Demirtas A, Saatci C, Ekmekcioglu O. Ten cases with 46, XX testicular disorder of sex development: Single center experience. Int Braz J Urol 2017;43:770-5.  Back to cited text no. 1
    
2.
Chen T, Tian L, Wu F, Xuan X, Ma G, Tang R, et al. Clinical and genetic analysis in males with 46, XX disorders of sex development: A reproductive centre experience of 144 cases. Andrologia 2019;51:e13232.  Back to cited text no. 2
    
3.
Vorona E, Zitzmann M, Gromoll J, Schüring AN, Nieschlag E. Clinical, endocrinological, and epigenetic features of the 46, XX male syndrome, compared with 47, XXY Klinefelter patients. J Clin Endocrinol Metab 2007;92:3458-65.  Back to cited text no. 3
    
4.
Bashamboo A, Donohoue PA, Vilain E, Rojo S, Calvel P, Seneviratne SN, et al. A recurrent p. Arg92Trp variant in steroidogenic factor-1 (NR5A1) can act as a molecular switch in human sex development. Hum Mol Genet 2016;25:3446-53.  Back to cited text no. 4
    
5.
Wilhelm D, Washburn LL, Truong V, Fellous M, Eicher EM, Koopman P. Antagonism of the testis- and ovary-determining pathways during ovotestis development in mice. Mech Dev 2009;126:324-36.  Back to cited text no. 5
    
6.
Jeyasuria P, Ikeda Y, Jamin SP, Zhao L, De Rooij DG, Themmen AP, et al. Cell-specific knockout of steroidogenic factor 1 reveals its essential roles in gonadal function. Mol Endocrinol 2004;18:1610-9.  Back to cited text no. 6
    
7.
Bland ML, Fowkes RC, Ingraham HA. Differential requirement for steroidogenic factor-1 gene dosage in adrenal development versus endocrine function. Mol Endocrinol 2004;18:941-52.  Back to cited text no. 7
    
8.
Ito M, Achermann JC, Jameson JL. A naturally occurring steroidogenic factor-1 mutation exhibits differential binding and activation of target genes. J Biol Chem 2000;275:31708-14.  Back to cited text no. 8
    
9.
Baetens D, Stoop H, Peelman F, Todeschini AL, Rosseel T, Coppieters F, et al. NR5A1 is a novel disease gene for 46, XX testicular and ovotesticular disorders of sex development. Genet Med 2017;19:367-76.  Back to cited text no. 9
    
10.
Lourenço D, Brauner R, Lin L, De Perdigo A, Weryha G, Muresan M, et al. Mutations in NR5A1 associated with ovarian insufficiency. N Engl J Med 2009;360:1200-10.  Back to cited text no. 10
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]



 

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