74 | Slovenska pediatrija 2023; 30(2) Case report / Prikaz primera Abstract Introduction: Differences of Sex Development (DSD) occur in approximately 1/5000 live births. One of the recently found genetic causes for 46, x Y DSD is NR5A1 gene variants, respon- sible for a broad phenotypic spectrum. Case Report: We present a case of a full-term newborn with ambiguous genitalia: one gonad located in the urogenital fold, the other inguinal, absence of wrinkling or hyperpig - mentation of the urogenital folds’ skin, the short genital tubercle, and labio-scrotal urethral meatus (External Geni- tal Score of 4). Male-type urethra and the absence of a uterus or ovaries were determined by ultrasound. The karyotype was 46, x Y, and a pathogenic heterozygous single nucleotide duplication 614dupC in the NR5A1 gene was found by NGS. The decided gender of rearing was male. Orchidopexy was performed at age 14 months. The histology of the gonad was indicative of a prepubertal testis. Discussion: NR5A1 variants have variable expressivity and incomplete penetrance. 46, x Y patients with a pathogenic variant in the NR5A1 gene range from ambiguous genitalia to normal female external genitalia with virilization at puber- ty. Pubertal development does not strongly correlate to the degree of virilization at birth, with the majority showing signs of virilization at pubertal age. Keywords: Differences of Sex Development, Next-Genera- tion Sequencing, NR5A1 Izvleček Uvod: Motnje v razvoju spola (DSD) se pojavijo pri približno 1/5000 živorojenih otrok. Med nedavno odkritimi genetskimi vzroki za 46,x Y DSD so različice v genu NR5A1, ki so odgovor- ne za širok fenotipski razpon. Prikaz primera: Predstavljamo primer donošenega novoro- jenčka z dvoumnimi genitalijami: levostransko tipno gona- do v levi urogenitalni gubi volumna 1 ml, ter desnostransko tipno gonado v dimljah, odsotnost gubanosti in hiperpi- gmentacije kože urogenitalnih gub, kratek genitalni tuberkel dolžine 1,5 cm, labioskrotalni meatus sečnice (ocena zuna- njih genitalij 4). Ultrazvok je pokazal prisotnost spolnih žlez, ki so kompatibilne s testisi, moški tip sečnice in odsotnost maternice ali jajčnikov. Kariotip je bil 46,x Y in NGS je odkril patogeno heterozigotno podvajanje enega nukleotida 614dupC v genu NR5A1. Glede na to je bil določen moški spol in pri 8 mesecih je imel majhno strukturo penisa z dolžino <2 cm, brez razvitega skrotuma in labioskrotalno hipospadijo. Orhidopeksija je bila opravljena v starosti 14 mesecev. His- tologija gonad je kazala na prepubertetni testis. Razprava: Različice NR5A1 imajo spremenljivo ekspresivnost in nepopolno penetranco. Klinična slika pri bolnikih 46,x Y s pato- geno različico v genu NR5A1 sega od dvoumnih genitalij do nor - malnih ženskih zunanjih genitalij z virilizacijo v puberteti. Zdi se, da razvoj v puberteti ni močno povezan s stopnjo virilizacije ob rojstvu, saj velika večina kaže znake virilizacije v puberteti. Ključne besede: motnja v razvoju spola, Sekvencioniranje naslednje generacije, NR5A1 Management of a child with a difference in sex development caused by a NR5A1 pathogenic variant Obravnava otroka z motnjo v razvoju spola, kot posledica patološke variacije v genu NR5A1 Maria João Gaia, Jasna Šuput Omladič, Mojca Kavčič, Maruša Debeljak, Robert Kordič, Primož Kotnik Slovenska pediatrija 2/2023.indd 74 22/05/2023 07:07 Slovenska pediatrija 2023 | 75 Introduction The term Differences of Sex Develop - ment (DSD) encompasses conditions with atypical genital appearance or discordance between phenotypic and chromosomal sex (1). These are uncommon and occur in approximate- ly 1/5000 live births (2, 3) One of the genetic causes of DSD dis - covered relatively recently in individu- als with 46, x Y karyotype is mutations in the NR5A1 gene (2, 4). It encodes the Steroidogenic Factor-1 (SF-1), a tran- scription factor vital to steroidogenesis and gonadal and adrenal development (3, 5, 6). During fetal sexual develop- ment, SF-1 regulates the bipotential gonad differentiation toward the tes- tes or ovaries (2). In addition, it stimu- lates the expression of multiple genes responsible for the male differentiation cascade, promoting the differentiation of the precursor cells toward Sertoli cells, virilization through steroidogen- esis in Leydig cells, and the regression of the paramesonephric ducts (2, 4, 5). In patients with 46, x Y DSD, mutations in the NR5A1 gene are responsible for a broad phenotypic spectrum, from iso- lated hypospadias to ambiguous exter- nal genitalia or entirely female external genitalia, with or without adrenal insuf- ficiency (2, 3, 4). Case report Neonatal period W e p r e s e n t a c a s e o f a f u l l - t e r m newborn from a third spontaneous pregnancy (one abortion) without com- plications. There was no family history of consanguinity or relevant diseas- es. The delivery was through elective cesarean section due to a previous cesarean section. Birth weight was on the 10-50th percentile, and birth length was on the 1st-3rd percentile. On phys - ical examination at birth, the following abnormalities of the external genitalia were observed: palpable gonad in the left urogenital fold with a volume of 1 mL, no palpable gonad in the right uro- genital fold with the gonad palpable inguinal, absence of wrinkling or hyper - pigmentation of the urogenital folds’ skin, presence of a penile structure/ genital tubercle with a length of 1.5 cm and diameter of 5-6 mm, labio-scro- tal urethral meatus and an anogenital distance of 2 cm. The External Genital Score (EGS) was 4 (1). There were no other relevant physical examination findings. An abdominal and genital ultrasound using the transperineal approach was performed on the fourth day of life. It revealed the presence of gonads in the urogenital folds compatible with testi- cles, the urethra in the genital tubercle morphologically according to the male type, and the absence of the uterus or ovaries. In addition, hydronephrosis of the left kidney (UTD-P3) with a wide ureter was diagnosed. The total testos- terone value was 1.0 nmol/L (reference for male infants under ten days old 1.0- 11.5 nmol/L) with free testosterone of 2.3 pmol/L, and AMH was on the low- er normal level (8.25 µg/L; reference FIGURE 1. C ELLULAR FUNCTIONS OF THE STEROIDOGENIC FACTOR-1 (SF-1). SLIKA 1. FUNKCIJE STEROIDOGENEGA FAKTORJA-1 (SF-1) V CELICI. NR5A1 SF-1 Differentiation and development of ovaries in prenatal Basal expression of steroidogenic Gonadotropin Induced expression of steroidogenic Regulators of ovaries Slovenska pediatrija 2/2023.indd 75 22/05/2023 07:08 76 | Slovenska pediatrija 2023; 30(2) range for male infants 10.64 to 161.84 µg /L). Normal basal and stimulated values of cortisol were determined by the ACTH test. The karyotype was avail - able on the 12th day of life and was 46, xY on all examined cells. The intact SRY gene on the Y chromosome was determined. A Voiding Cystourethrography (VCUG) showed VUR grade IV on the left kidney with the suitably wide urethra, with- out communication with surrounding structures. A heart ultrasound showed an open oval window and accessory tissue in the outflow tract of the right ventricle, without signs of obstruction, that normalized by the age of 4 months. In addition, the transfontanellar ultra- sound was normal. The multidisciplinary DSD team, together with both of the parents, suggested the gender of rearing to be male. Follow-up The genetic diagnosis was available at the age of 2 months. A hetero zy - gous single nucleotide duplication 614dupC in the NR5A1 gene was found by Next-Generation Sequencing (NGS), representing a premature transcription termination previously described as pathogenic (4). The same variant was found in the healthy mother. At three months, a GnRH test showed a basal LH of <0.1U/L and basal FSH of 4.0 U/L, increasing to 0.8 and 23.5 U/L, respectively, by 60 minutes. Inhibin B was normal, and AMH low (7.88 µg/L). T h e d e c i d e d g e n d e r o f r e a r i n g w a s male, and at eight months of age, the child presented with urogenital folds (without developed scrotum), a small penile structure with <2 cm length and 5-6 mm diameter, and underdeveloped root, labio-scrotal hypospadias and inguinal positioned gonads (Figure 2). The child had a bilateral orchidopexy. The goal of the procedure was to fix the gonads inside the urogenital folds out- side of the abdominal cavity. A biopsy of the right gonad was performed dur- ing the procedure. Histologically, sper- matogonia in the seminiferous tubules were described alongside clusters of Leydig cells in the interstitium. Discussion Variants in the NR5A1 gene are pre- dominantly inherited dominantly with variable expressivity and incomplete penetrance. 90% of patients have 46, xY karyotype (5). There is no concise phenotype–genotype correlation, with a spectrum varying from com- plete female to complete male appear - ance (5). Case series of 46, xY patients with NR5A1 variants report a wide vari- ety of presenting phenotypes, rang- ing from ambiguous genitalia and low EGS at birth to normal female exter - nal genitalia, but with signs of virili- zation at puberty or pubertal delay. Even if external genitalia is apparent- ly female, most patients have no resid- ual Müllerian structures (uterus and fallopian tube) (2, 4, 5). AMH is respon - sible for their regression during pre- natal development. In x Y fetuses, it is secreted from the Sertoli cells of the testis. Patients with 46, x Y DSD may present normal AMH levels at birth, without a uterus or fallopian tubes, or low concentrations of AMH and detectable Müllerian structures. The reported patient presented without Müllerian structures and a low normal AMH value at birth and low value at age two months, suggesting that AMH was secreted prenatally, with a decrease in secretion postnatally (4). Inhibin B lev- els in male subjects reflect the function of the Sertoli cell in the testis. The lev- els in patients with NR5A1 pathogenic variations are mostly very low (2). In the presented case, inhibin B was nor- mal. Inhibin B and AMH values below the male reference range despite the absence of Müllerian structures and decrease in testicular volume through- out puberty suggest progressive Serto- li cell failure over time and highlight the importance of keeping the possibility of early spermiogram and cryopreser- vation in patients with an NR5A1 muta- tion in mind (2, 4, 7). In most cases with pathogenic variants in the NR5A1 gene, testosterone level is low during the neonatal period, which was also determined in the present case (4). Therefore, hypogonadotropic hypogonadism with normal/low nor- mal testosterone levels is expected, but low and extremely low testoster- one concentrations may also be deter- mined (2, 4, 5). Monig et al. described that despite normal testosterone lev- e l s , L H l e v e l s w e r e e l e v a t e d i n t h e vast majority of patients and postu- lated that this could be due to Leydig cells having a relevant preserved func- tion, but higher LH levels are need- ed to maintain enough testosterone, possibly because of impaired SF-1 stimulation activity or partial gonad- al dysgenesis due to the NR5A1 muta- tions (2). Increased FSH concentrations and low LH/FSH ratios have also been reported (4), with our patient present- ing this hormonal pattern. Pubertal development does not strong - ly correlate to the degree of virilization of the external genitalia at birth in 46, xY DSD patients with NR5A1 muta- tions. The great majority of patients, even with very low EGS, show signs of virilization at pubertal age, though in many cases, the gonadal volume stays below average (2, 4). In a few cases, viri- lization might not occur, and spontane- ous breast development might even be present (2). Patients with ambiguous genitalia at birth and male sex assign- ment who showed spontaneous puber - ty and normal testosterone values have been reported, supporting the idea of preserved Leydig cell function later in life (4, 8, 9). Notably, the prevalence of adrenal insufficiency incidence is low (0.05%) in these patients (5). Most cohorts show no adrenal insufficiency (2, 4, Slovenska pediatrija 2/2023.indd 76 22/05/2023 07:08 Slovenska pediatrija 2023 | 77 5), although sporadic cases have been reported (6). In heterozygous Nr5a1+/− mice, adrenal insufficiency was deter- mined only during stress conditions. It was associated with significant adre- nal hyperplasia, demonstrating that a normal gene dosage of SF-1 is required for mounting an adequate stress response (10). One must be aware of possible acute adrenal insufficiency in NR5A1 subjects during stress condi- tions (4). Two additional features were deter- m i n e d i n t h e p r e s e n t e d c a s e : V U R grade IV on the left kidney and open oval window and accessory tissue in the outflow tract of the right ventricle. Both features do not seem to be associ - ated with NR5A1 mutation (2–5). The gender of rearing of DSD patients is a topic of debate. An experienced multi- disciplinary team should make optimal clinical management of individuals with DSD in close communication with the caregivers, and all patients should r e c e i v e a g e n d e r a s s i g n m e n t ( 1 1 ) . Factors influencing gender assign- ment include etiology, genital appear- ance, reproductive anatomy, surgical options, the need for lifelong replace- ment therapy, the potential for fertility, and parental/cultural factors (11, 12). Most 46, x Y subjects with NR5A1 muta- tions reared as girls will undergo progressive masculinization if the gonads are not removed, and boys with NR5A1 mutations can undergo spontaneous puberty as well as pre- served fertility, suggesting that a 46, x Y individual with an NR5A1 mutation reared as a boy has certain advantag- es (4, 8, 9, 13). In a literature review, 46% of patients with NR5A1 mutations were assigned female, and 54% were assigned male (5). Female-to-male sex reassignment was referred in 15% of patients, but only 1 case underwent male-to-female sex reassignment. In another cohort, 16 out of 19 patients assigned female gender at birth decid- ed to reassign to male gender (4). Giv- en all these factors, the reported child was assigned male gender at birth after extensive discussions and approval by the parents. The specific etiology of the DSD and reported evidence for future outcomes in these patients, the pres- ence of male gonads without Müllerian structures, and the hormonal profile lead us to believe this could be the best option for the patient. References 1. Chan YM, Levitsky LL. Evaluation of the infant with atypical genital appearance (difference of sex devel- opment). In: UpToDate, Shefner JM (Ed), UpToDate, Waltham, MA. 2. Mönig I, Schneidewind J, Johannsen TH, Juul A, Wer - ner R, Lünstedt R, Birnbaum W, Marshall L, Wünsch L, Hiort O. Pubertal development in 46, XY patients with NR5A1 mutations. Endocrine 2022;75(2):601-13. 3. Tantawy S, Mazen I, Soliman H, Anwar G, Atef A, El-Gammal M, El-Kotoury A, Mekkawy M, Torky A, Rudolf A, Schrumpf P, Grüters A, Krude H, Dumargne MC, Astudillo R, Bashamboo A, Liebermann H, Köhler B. Analysis of the gene coding for steroidogenic factor 1 (SF1, NR5A1) in a cohort of 50 Egyptian patients with 46, XY disorders of sex development. Eur J Endocrinol 2014; 10;170(5):759-67. 4. Song Y, Fan L, Gong C. Phenotype and Molecular Characterizations of 30 Children From China With NR5A1 Mutations. Front Pharmacol 2018; 30;9:1224. 5. Fabbri-Scallet H, de Sousa LM, Maciel-Guerra AT, Guerra-Júnior G, de Mello MP. Mutation update for the NR5A1 gene involved in DSD and infertility. Hum Mutat 2020;41(1):58-68. 6. Lin L, Gu WX, Ozisik G, To WS, Owen CJ, Jameson JL, Achermann JC. Analysis of DAX1 (NR0B1) and steroi - FIGURE 2. T HE Ex TERNAL GENITALIA OF THE PATIENT AT EIGHT MONTHS OF AGE. SMALL GONADS WERE PALPA- BLE IN THE UROGENITAL FOLDS THAT DID NOT DEVELOP INTO THE SCROTAL FOLDS. THE PHALLIC STRUCTURE WAS POORLY DEVELOPED. THE ANOGENITAL DISTANCE WAS REDUCED. THESE FEATURES INDICATE SUBOPTIMAL DEVELOPMENT OF THE ExTERNAL MALE GENITALIA. SLIKA 2. ZUNANJE SPOLOVILO V STAROSTI 8 MESECEV. MAJHNE GONADE SO BILE TIPNE V UROGENITALNIH GUBAH, KI SE NISO RAZVILE V SKROTUM. FALIČNA STRUKTURA JE SLABO RAZVITA. ANOGENITALNA RAZDALJA JE BILA ZMANJŠANA. NAVEDENO KAžE NA SUBOPTIMALEN RAZVOJ ZUNANJEGA MOŠKEGA SPOLOVILA. Slovenska pediatrija 2/2023.indd 77 22/05/2023 07:08 78 | Slovenska pediatrija 2023; 30(2) dogenic factor-1 (NR5A1) in children and adults with primary adrenal failure: ten years experience. J Clin Endocrinol Metab 2006;91(8): 3048-54. 7. Philibert P, Polak M, Colmenares A, Lortat-Jacob S, Audran F, Poulat F, Sultan C. Predominant Sertoli cell deficiency in a 46, XY disorders of sex development patient with a new NR5A1/SF-1 mutation transmitted by his unaffected father. Fertil Steril 2011;95(5): 1788. e5-9. 8. Fabbri HC, de Andrade JG, Soardi FC, de Calais FL, Petroli RJ, Maciel-Guerra AT, Guerra-Júnior G, de Mello MP. The novel p.Cys65Tyr mutation in NR5A1 gene in three 46, XY siblings with normal testosterone levels and their mother with primary ovarian insufficiency. BMC Med Genet 2014; 10;15:7. 9. Gabriel Ribeiro de Andrade J, Marques-de-Faria AP, Fabbri HC, de Mello MP, Guerra-Júnior G, Maciel-Guerra AT. Long-Term Follow-Up of Patients with 46, XY Partial Gonadal Dysgenesis Reared as Males. Int J Endocrinol 2014;480724. 10. Bland ML, Jamieson CA, Akana SF, Bornstein SR, Eisenhofer G, Dallman MF, Ingraham HA. Haploin- sufficiency of steroidogenic factor-1 in mice dis- rupts adrenal development leading to an impaired stress response. Proc Natl Acad Sci U S A. 2000 Dec 19;97(26):14488-93. 11. Lee PA, Houk CP, Ahmed SF, Hughes IA. Internation - al Consensus Conference on Intersex organized by the Lawson Wilkins Pediatric Endocrine Society and the European Society for Paediatric Endocrinology. Con- sensus statement on management of intersex disor - ders. International Consensus Conference on Intersex. Pediatrics 2006;118(2): e488-500. 12. Mieszczak J, Houk CP, Lee PA. Assignment of the sex of rearing in the neonate with a disorder of sex develop- ment. Curr Opin Pediatr 2009;21(4): 541-7. 13. Yagi H, Takagi M, Kon M, Igarashi M, Fukami M, Hasegawa Y. Fertility preservation in a family with a novel NR5A1 mutation. Endocr J 2015; 62(3): 289-95. Maria João Gaia, dr. med. Pediatrics Department, Vila Nova de Gaia Hospital Center, Vila Nova de Gaia, Portugal Jasna Šuput Omladič Department of Pediatric Endocrinology, Diabetes, and Metabolism, Division of Pediatrics, University Medical Centre Ljubljana, Ljubljana, Slovenia Mojca Kavčič, dr. med. Department of Neonatology, Division of Pediatrics, University Medical Centre Ljubljana, Division of Pediatrics, University Medical Centre Ljubljana, Ljubljana, Slovenija doc. dr. Maruša Debeljak, spec. lab. med. gen. Unit for Special Laboratory Diagnostics, Division of Pediatrics, University Medical Centre Ljubljana, Ljubljana, Slovenija and Medical Faculty, University of Ljubljana, Ljubljana, Slovenia Robert Kordič, dr. med. Department of Urology, Division of Surgery, University Medical Centre Ljubljana, Ljubljana, Slovenia Primož Kotnik (kontaktna oseba / contact person) Department of Pediatric Endocrinology, Diabetes, and Metabolism, Division of Pediatrics, University Medical Centre Ljubljana, Ljubljana, Slovenia and Medical Faculty, University of Ljubljana, Ljubljana, Slovenia prispelo / received: 12. 1. 2023 sprejeto / accepted: 10. 2. 2023 João Gaia M, Kavčič M, Debeljak M, Kordič R, Kotnik P. Management of a child with a difference in sex devel - opment caused by a NR5A1 pathogenic variant. Slov Pediatr 2023; 30(3): 74−78. https://doi.org/10.38031/ slovpediatr-2023-2-04. Slovenska pediatrija 2/2023.indd 78 22/05/2023 07:08