|Year : 2016 | Volume
| Issue : 1 | Page : 79-88
Genetic evaluation of children with ambiguous genitalia
Soheir S Abou El-Ella, Maha A Tawfik, Wafaa M Abo El-Fotoh, Ahmed Sh Abo Howla
Department of Pediatric, Faculty of Medicine, Menoufia University, Menoufia, Egypt
|Date of Submission||24-Nov-2014|
|Date of Acceptance||31-Dec-2014|
|Date of Web Publication||18-Mar-2016|
Ahmed Sh Abo Howla
MBBCh, Pediatric Department, Faculty of Medicine, Menoufia University, 32511 Menoufia
Source of Support: None, Conflict of Interest: None
The aim of the study was to conduct a clinical assessment of pediatric patients with ambiguous genitalia, perform molecular genetic studies for SRY and SOX9 genes, and provide genetic counseling for patients and their families.
Ambiguous genitalia, currently known as disorders of sex development (DSDs), are associated with atypical development of chromosomal, gonadal, or anatomical sexual characteristics, with an incidence of one in 4500 live births. Their early management is crucial for preventing complications, especially psychiatric impacts on the patient and the family, and should be decided after careful consideration of the child's welfare.
Materials and methods
Sixteen patients aged 1 day to 6 years were selected from the Genetic and Endocrinology Unit, Pediatric Department, Faculty of Medicine, Menoufia University, Egypt. All patients were subjected to detailed history taking, a thorough clinical examination, routine and hormonal investigations, imaging studies, and cytogenetic and molecular studies for SRY and SOX9 genes.
History revealed seven patients (43.75%) with positive consanguinity and five patients with similar conditions in their families. Hormonal study revealed five patients (31.25%) above normal ranges for serum 17-OH progesterone levels and two patients (12.5%) below normal ranges. Karyotyping revealed six patients with 46, XX DSD, eight with 46, XY DSD, one with 45, X, and another with 45, X/46, XY. On the basis of molecular studies, the SRY gene was positive for six patients with a normal male 46, XY karyotype and for one patient with 45, X karyotype (translocated SRY). SRY was negative for five patients with a normal female 46, XX karyotype and for one patient with 45, X/46, XY karyotype (deleted SRY). All patients were positive for the SOX9 gene and no deletions were detected.
Early identification of the genetic cause of DSD will in many cases streamline and direct clinical management of the patient with more focused endocrine and imaging studies and better surgical decision.
Keywords: Ambiguous genitalia, disorder of sex development, genetic counseling, SOX9, SRY
|How to cite this article:|
Abou El-Ella SS, Tawfik MA, Abo El-Fotoh WM, Abo Howla AS. Genetic evaluation of children with ambiguous genitalia. Menoufia Med J 2016;29:79-88
|How to cite this URL:|
Abou El-Ella SS, Tawfik MA, Abo El-Fotoh WM, Abo Howla AS. Genetic evaluation of children with ambiguous genitalia. Menoufia Med J [serial online] 2016 [cited 2019 Jun 27];29:79-88. Available from: http://www.mmj.eg.net/text.asp?2016/29/1/79/178991
| Introduction|| |
Sexual differentiation means the development of sexual characteristics that distinguish males from females. Sex and gender are important for health. The term gender defines those characteristics of females and males that are created by oneself (gender identity) and society (gender role), whereas sex involves those characteristics that are biologically determined since fertilization .
Disorders of sex development (DSDs) are complex, with a wide spectrum of phenotypes including 46, XY DSD (gonadal dysgenesis or undermasculinization due to defects in androgen synthesis or action), 46, XX DSD (gonadal dysgenesis or more commonly virilization due to prenatal or postnatal excess androgen exposure), and sex chromosome DSD. Congenital adrenal hyperplasia (CAH) and mixed gonadal dysgenesis are the most common causes, constituting approximately over 50% of all cases of genital ambiguity in the newborn period .
Optimal care of patients with DSD requires a multidisciplinary team and begins in the newborn period. Assessment of family history and prenatal history, a general physical examination with attention to any associated dysmorphic features, and an assessment of the genital anatomy are the first steps toward a correct diagnosis. The diagnostic evaluation of DSD includes hormone measurements, imaging, and cytogenetic and molecular studies for mutations (SRY, SF1, SOX9 and others). However, current molecular diagnosis is limited by cost, accessibility, and quality control ,.
The aim of this study was to conduct a clinical assessment of pediatric patients with ambiguous genitalia, perform genetic studies including karyotyping, molecular studies for SRY and SOX9 genes, and finally conduct genetic counseling for the patients and their families.
| Materials and methods|| |
The study was conducted on 16 children with ambiguous genitalia, who were selected from the Genetics and Endocrinology Unit, Pediatric Department, Menoufia University Hospitals, Egypt. They comprised seven apparently female patients and nine apparently male patients. Their ages ranged from 1 day to 6 years. The study was approved by the ethical committee of the hospital and the patient gave an informed consent.
After obtaining their consent, all patients and controls were subjected to the following:
- A detailed history including maternal diseases resulting in virilization, and exposure to androgens and endocrine disrupters (phenytoin); family history including consanguinity, similar abnormalities, recurrent miscarriages, and unexplained infant death; and neonatal history of failure to thrive, vomiting, pigmentation, and progressive virilization .
- A thorough physical examination (vital signs, anthropometric measurements, other congenital abnormalities, dysmorphic features, hirsutism, or abnormal body pigmentations) .
- A careful examination of the external genitalia, including:
- Size of the phallus,
- Examination of gonads (palpable or not, their size, location, and texture),
- Prader score for degree of virilization in female patients ,
- Calculation of the external masculinization score in male patients ,
- Tanner staging system for pubertal changes .
- Routine investigations including complete blood picture, serum electrolytes especially Na and K, and glucose levels for cases with salt-losing CAH .
- Hormonal studies involving serum levels of 17-OH progesterone (17-OHP), cortisol, and adrenocorticotropic hormone (ACTH) and others according to suspected diagnosis and interpreted in relation to specific reference ranges .
- Imaging studies including abdominopelvic ultrasonography, computed tomography, and/or MRI for visualization of the internal genitalia, gonadal site, and structure if present .
- Genetic studies including chromosomal karyotyping using G banding , and molecular studies for SRY (sex determining region Y) and SOX9 (SRY-box 9) genes for both patients and controls. The SRY gene is the main regulator of sexual differentiation, which subsequently activates the SOX9 gene to complete differentiation into the male line . The following steps were applied in the genetic lab of our unit:
- DNA extraction from blood samples .
- PCR for SRY and SOX9 genes .
- Gel electrophoresis .
- Genetic counseling was conducted for all families, including clinical aspects and diagnostic approach for the child's condition, available treatment options, and methods for communication and follow-up.
| Results|| |
The results of this study are illustrated in [Table 1],[Table 2] and [Table 3] and [Figure 1],[Figure 2],[Figure 3],[Figure 4],[Figure 5] and [Figure 6].
We must note that molecular studies were performed for all patients except 4, 11, and 13, who refused sample withdrawal.
We studied 16 patients aged 1 day to 6 years. Their presenting manifestations varied from ambiguous genitalia to vomiting and dehydration attacks, abnormal body hair growth, abnormal body pigmentations, and abnormal urine stream.
Demographic data of the studied patients showed that seven patients (43.75%) had a positive consanguinity and two patients (patients 14 and 15) were cousins. Five patients (31.25%) had possible similar conditions in their families [Table 1].
Anthropometric measurements showed that four patients (25%) were below the third percentile for weight and below −2 SD for BMI and three of them were below the third percentile for length.
Clinical examination of external genitalia showed that the external genital manifestations of the female patients were mainly clitoromegaly and fused labia, with no palpable gonads, and two of them had abnormal pubic hair growth, with scores of 1, 2, and 4 on the Prader system. The external genital manifestations of the male patients were mainly bifid scrotum and urethral opening at the base of the phallus. Three had microphallus. Two had only one palpable gonad and one patient (patient 12) looked apparently female [Table 2],[Table 2] and [Table 3] and [Figure 1],[Figure 2].
The external masculinization score was applied to all nine male patients, and their total scores were below normal (≥11).
The Tanner staging system showed that all patients are in the first pubertal stage, except for two female patients (4 and 16) with pubic hair stage 2.
As regards serum electrolytes and blood glucose levels, all patients were within normal levels, except three patients (2, 12, and 14) who had hyperkalemia and hyponatremia, of whom only two (12 and 14) suffered from hypoglycemia.
Hormonal evaluation revealed seven patients (43.75%) with abnormal serum 17-OHP levels: five patients (31.25%) had above normal ranges and two (12.5%) had below normal ranges. In addition, among these seven patients, six had elevated serum ACTH levels and low serum cortisol levels. The mean serum 17-OHP level of all patients was 321.25 ± 603.57 ng/dl SD. The mean serum ACTH levels, a.m.: 101.56 ± 88.2 pg/ml, p.m.: 59.3 ± 51.98 pg/ml. Mean for serum cortisol levels, a.m.: 11.1 ± 8.31 mg/dl, p.m.: 10.87 ± 7.5 mg/dl [Table 2] and [Table 3].
With regard to imaging studies, all genetically female patients had internal female genitalia, except patient number 6 with the 45, X karyotype. Clinically, this patient had a bifid scrotum with a palpable right testis and phallus measuring 2.5 cm in length; imaging studies showed no internal female genitalia, but the presence of right scrotal and left inguinal testes. All genetic male patients showed no internal female genitalia, except patient number 7 with the 45, X/46, XY karyotype, who showed a uterus and bilateral pelvic streak gonads. Clinically, this patient was an apparent female on external genitalia with clitoromegaly, fused labia, identified vaginal and urethral orifices, and no palpable gonads.
Cytogenetic studies revealed six female patients with 46, XX DSD, eight male patients with 46, XY DSD, one with 45, X, and another with 45, X/46, XY.
Regarding molecular studies, SRY products were detected at the 418-bp position and SOX9 products at 270 bp compared with 100-bp DNA ladder on gel electrophoresis. Results for the SRY gene revealed that patient number 6 with the 45, X karyotype was positive for the SRY gene and patient number 7 with the 45, X/46, XY karyotype had a deleted SRY. The SOX9 gene was present in all studied patients and no deletions were detected [Figure 3],[Figure 4],[Figure 5] and [Figure 6].
|Figure 3: Gel electrophoresis for the SRY gene in the apparently male cases. First lane on the left shows 100-bp DNA ladder with two reference bands (1000 and 500 bp) for easy orientation as marked in the figure. Lanes 2– 7: the studied apparently male patients (P1– P12). SRY bands were detected at position 418 bp in the studied apparent male patients, including patient 6 who had a karyotype of 45, X (a translocated SRY for this pati ent).|
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|Figure 4: Gel electrophoresis for the SRY gene in the apparently female cases, F = female, M = male. First lane on the left shows 100-bp DNA ladder. Lane 2: female control. Lane 3: male control. Lanes 4– 9: the studied apparently female patients (P2– P16). SRY was negative for female controls and positive at position 418 bp for male controls; the studied apparently female cases showed no bands for the SRY gene, including patient 7 who had a karyotype of 45, X/ 46, XY (deleted SRY gene for this patient).|
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|Figure 5: Gel electrophoresis for the SOX9 gene in the apparently male cases. First lane on the left shows 100-bp DNA ladder. Lanes 2– 7: the studied apparently male patients (P1– P12). SOX9 bands were detected at position 270 bp in the studied apparently male cases and no deletions were dete cted.|
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|Figure 6: Gel electrophoresis for the SOX9 gene in the apparently female cases, F = female, M = male. First lane on the left shows 100-bp DNA ladder. Lane 2: female control. Lane 3: male control. Lanes 4– 9: the studied apparently female patients (P2– P16). SOX9 was positive for male controls, for female controls, and for all apparently female cases at position 270 bp, and no deletions were detected in the studied c ases.|
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Genetic counseling was conducted through frequent sessions and included the following:
- Discussion of clinical aspects and the diagnostic approach for the child's condition with the parents before rushing to sex selection.
- Documentation of family history and pedigree information.
- Taking consent for photography and blood sample withdrawal.
- Providing information on available options.
- Providing support in decision-making.
- Recognition of inheritance patterns and risk estimation for the disease if there is genetic basis.
- Determining methods for communication and follow-up.
| Discussion|| |
The present study included 16 patients: six patients (37.5%) had normal female 46, XX karyotype, eight patients (50%) had normal male 46, XY karyotype, and two (12.5%) had abnormal karyotype (one with 45, X and another with 45, X/46, XY). This was consistent with the results of Siklar et al. , who reported in a study on disorders of gonadal development that 51.4% of patients with DSD had the 46, XY karyotype, 34.9% had 46, XX, and 13.7% had other conditions; the limitations in our data collection may have led to estimation of less than the usual variation expressed by other authors.
The ages of our patients ranged from 1 day to 6 years, with a mean age at presentation of 2.2 ± 3.3 months. The most common age at presentation was in the neonatal period (11 cases, 68.75%), followed by infancy (five cases, 31.25%). Our results were in agreements with those of Hughes , who reported that DSD is typically diagnosed at birth in infants with ambiguous genitalia, whereas disorders associated with phenotypic males and females may be diagnosed much later.
With respect to family pedigree, five patients had possible similar conditions in their families and two of them were cousins. This was consistent with the approach taken by Clayton et al. , who recommended taking a full history from the parents and/or the patient, including the medical background of the family (paternal and maternal), and documenting it as an essential step in the management of DSD and associated genetic counseling.
Seven patients (43.75%) had a positive consanguinity and patients 14 and 15 were cousins. This was in line with a study conducted by Mazen et al.  on differential diagnosis of DSDs in Egypt, which showed a consanguinity rate of 61% in the affected families. As CAH (the most common cause of DSD and diagnosed in seven patients in this study) is autosomal recessive, the high prevalence of consanguinity in our cases is a significant risk factor in DSD cases, as also reported by Al Jurayyan  and Nimkarn et al. .
Presenting manifestations included ambiguous genitalia, recurrent vomiting, and dehydration (salt-losing crisis) in three patients (two females and one male), abnormal body hair growth in two female patients, abnormal body pigmentations in one female patient, and abnormal urine stream in two apparently male patients. These presentations were similar to those of Warne and Raza , who illustrated that the signs of DSD vary from being not recognized at all as in cases with salt-losing CAH to being completely ambiguous external genitalia.
As regards the anthropometric measurements, all patients were within normal ranges, except four, who were below the third percentile for weight and less than −2 SD for BMI, and three were below the third percentile for length. This is consistent with the significance of serial anthropometric measurements as an important tool in ensuring that DSD, especially salt-losing CAH, does not affect the child's normal growth pattern and in follow-up of treatment as recommended by Dauber et al. .
With regard to local examination, the external genital manifestations of the female patients were mainly clitoromegaly and fused labia, with no palpable gonads, and two of them had abnormal pubic hair growth, with scores of 1, 2, and 4 on the Prader system. In agreement with our results, Ogilvy-Stuart and Brain  demonstrated that any apparent female case with DSD may present with a wide range of presentations beginning from clitoromegaly, fused labia, a concealed vagina, or even a vestibule with single opening like a male with penioscrotal hypospadias.
In male patients, the external genital manifestations were mainly bifid scrotum and urethral opening at the phallus base. Three patients had microphallus. Two had only one palpable gonad and one patient looked apparently female (patient 12). In addition, the external masculinization score, which is used to describe the degree of ambiguity in an undervirilized boy, was applied and all male patients scored below 11, which indicates the need to be evaluated carefully for possibility of DSD as recommended by Ahmed and Rodie .
Regarding Tanner staging, most patients were in the first stage (prepubertal), except two, which was consistent with early age of presentation and evaluation in the majority of our cases depending on the etiological nature of the disease. This was in line with the observations of Traggiai and Stanhope , who illustrated the dependency of Tanner stages on hormonal effects as regards secondary sexual characteristics.
Lee et al.  recommended measurements of serum sodium, potassium, and blood glucose levels with a sense of urgency to exclude a salt-losing crisis in cases with CAH, the most common cause of DSDs worldwide. In this study, all patients were within normal levels except three (patients 2, 12, and 14), who had hyperkalemia and hyponatremia, of whom two (patients 12 and 14) suffered from hypoglycemia, which was consistent with laboratory parameters in patients suffering from salt-losing CAH as a result of associated adrenal insufficiency in these cases as demonstrated by Speiser and White .
With regard to imaging studies, seven patients with an apparently female phenotype had normal Mullerian structures and gonads, except patient 7, who had bilateral pelvic streak gonads. Nine patients with an apparently male phenotype showed normal inguinoscrotal gonads and no internal female genitalia. These findings were in line with those of Chavhan et al.  on imaging of ambiguous genitalia, who showed dependency of the results on the etiological nature of the disease.
With regard to hormonal studies, we found that seven patients (43.75%) had abnormal serum 17-OHP levels (five patients were above normal ranges and two were below normal ranges), which was associated with high serum ACTH levels and low serum cortisol levels in six of them. Five of them showed elevated serum adrenal androgen levels, and the other two showed decreased levels. These results were in agreement with laboratory findings of CAH as demonstrated by Wajnrajch and New , indicating the key role of these three hormones, especially 17-OHP, in establishing a diagnosis of CAH.
In patient 1, we found mildly elevated serum17-OHP levels, normal serum cortisol and ACTH levels, and low adrenal androgen levels (dehydroepiandrostenedione and androstenedione). These results agreed with a diagnosis of CAH due to isolated 17, 20-lyase deficiency, as illustrated by Geller et al. .
In patient 14, hormonal evaluation demonstrated low serum 17-OHP level and cortisol levels, elevated serum ACTH levels, elevated serum dehydroepiandrostenedione levels, and low serum androstenedione levels, which was consistent with laboratory findings in CAH due to 3-hydroxysteroid dehydrogenase deficiency, as illustrated by Speiser et al. .
Patient 12 presented with recurrent vomiting and dehydration attack at 3 weeks of age. On local examination, female-like external genitalia was seen but palpation identified bilateral labioscrotal gonads. This patient had hyperkalemia, hyponatremia, and hypoglycemia on routine laboratory investigations. Imaging studies confirmed bilateral labioscrotal gonads and absence of Mullerian structures. Hormonal evaluation showed decreased serum 17-OHP and serum cortisol levels with elevated serum ACTH levels and low adrenal androgen levels. These findings agreed with laboratory findings in cases with salt-losing CAH due to 20-22 desmolase deficiency, as demonstrated by Bhangoo et al. .
As regards other hormonal assays, we found that patient 3 had high serum testosterone levels, low serum dihydrotestosterone (DHT) levels, and a high T: DHT ratio (37.4) after hCG stimulation test. As this patient had a normal male karyotype, these findings were consistent with a diagnosis of 5-α reductase deficiency, as illustrated by Imperato-McGinley and Zhu .
The genetic study was divided into two main categories: karyotyping and molecular studies. Karyotyping of the patients revealed six patients with female 46, XX DSD, eight patients with male 46, XY DSD, and two patients with sex chromosome DSD (one had 45, X and the other had 45, X/46, XY). These results were in agreement with those of Lee et al. , who emphasized on the essentiality of karyotyping for provisional diagnosis and classification of DSDs.
With regard to molecular studies, we focused on two genes, the SRY gene and the SOX9 gene. Mutations of the SRY gene can result in 46, XY sex reversal or gonadal dysgenesis , whereas translocation of Y chromosomal material including the SRY gene can result in 46, XX male syndromes . Deletion or mutations of the SOX9 gene can result in severe skeletal malformation syndrome (campomelic dysplasia), which is frequently associated with male to female sex reversal .
Regarding the SRY gene, we detected the SRY gene band at the 418-bp position [Figure 3],[Figure 4] in the studied genetically male patients, except patient 7, who had a karyotype of 45, X/46, XY. No SRY gene bands were detected in the studied genetically female patients, except patient 6 who had a karyotype of 45, X. This is in line with the role of the SRY gene in sex differentiation toward the male line through the presence of the Y chromosome or its translocation to another chromosome, mostly the X chromosome, as illustrated by Huang and Yen  in the genetics of sex determination and differentiation.
As regards the SOX9 gene, the results demonstrated the presence of the SOX9 gene band at nearly the 270-bp position in all cases [Figure 5] and [Figure 6]. Therefore, no deletions for the SOX9 gene were detected in our studied cases, and this was in line with clinical manifestations of the studied cases in which no single case showed skeletal malformation. This agreed with the findings of Velagaleti et al. , who illustrated in a study on the SOX9 gene that heterozygous loss-of-function mutations in the human SOX9 cause campomelic dysplasia, which is frequently associated with male to female sex reversal in about 65% of cases.
The karyotype 45, X is usually associated with a female and only rarely with a male phenotype. Male individuals with a 45, X karyotype carry small fragments of Y-chromosomal material, including the testis-determining gene SRY, mostly due to translocations from the Y chromosome to an autosome ,,.
In this study, on local examination of patient 6 who presented to us with ambiguous genitalia on the first day of life we found a bifid scrotum with only palpable right testis and phallus measuring 2.5 cm in length with proximal hypospadias [Figure 1]. Imaging studies confirmed the presence of right scrotal testis, left inguinal gonad, and absence of Mullerian structures. Hormonal evaluation was normal for adrenal and testicular functions. These results were consistent with being an undermasculinized male, but cytogenetic studies including fluorescent in-situ hybridization revealed a case of 45, X. This directed evaluation toward the possibility of a translocated SRY gene, and the molecular study for the SRY gene revealed its presence in this case [Figure 3]. These findings agreed with those of Wimmer et al.  on the diagnosis of 45, X male syndrome.
Patient 7 was a clinically apparent female according to external genitalia who was stage 2 on the Prader scale [Figure 2]. Her laboratory evaluation was normal; imaging studies including MRI showed a uterus and bilateral pelvic streak gonads. These results were consistent with being a virilized female, but cytogenetic studies including fluorescent in-situ hybridization revealed a case of 45, X/46, XY, which was in line with a diagnosis of mixed gonadal dysgenesis as illustrated by Telvi et al. .
The predominance of the X or XY cell line determines the gonadal differentiation into a testis or a streak gonad , which was in line with the observation made in this patient, who had bilateral streak gonads and a 7% male cell line. In addition, the molecular study determined the absence of the SRY gene [Figure 4]. This suggests that the deleted SRY gene played a role in maintaining the female phenotype and Mullerian structures despite the presence of the Y chromosome. This was consistent with the report of Shahid et al.  on the occurrence of mutations in the SRY gene in patients with the 45, X/46XY karyotype. Similar observations were made by Dumic et al.  in a study on a family with multiple DSDs.
Selection of the best treatment options for the various DSDs in the studied patients was difficult, relating to sex of rearing, hormonal therapy and surgery. Treatment options as recommended by Lee et al.  included genetic counseling for the family, lifetime hormonal replacement therapy, salt-losing crisis management in cases with CAH, sex hormonal therapy, and surgical interventions.
During this study, the main challenges were posed by the parents of the studied cases, who showed marked irritability with their children's condition and the spectrum of DSDs, which posed a diagnostic dilemma and hampered the collection of study material. The parents were anxious about their children's condition and wanted a diagnosis as soon as possible, which was met from our side with assurance and the need for appropriate full anatomical, biochemical, and multidisciplinary evaluation before sex assignment. This considerable challenge was consistent with the culture of the Middle East, as illustrated by Al Jurayyan .
| Conclusion|| |
The birth of a child with ambiguous genitalia is a medical and social emergency that must be overcome in order to decide the appropriate sex of the child and to prevent complications. The matter must be handled with immediacy and sensitivity and should be based on sound knowledge of sex determination and differentiation, with efforts being made to evaluate the attitude of parents and provide adequate counseling. Examination of external genitalia with emphasis on gonadal detection and karyotyping is the first essential step in the evaluation of children with DSDs. Uniform classification of DSDs, good cytogenetic and molecular facilities, individualized approach with integrated team management, and supportive counseling form the mainstay of management of DSD.
| Acknowledgements|| |
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3]