A Newborn with Genital Ambiguity, 45,X/46,XY Mosaicism, a Jumping Chromosome Y, and Congenital Adrenal Hyperplasia.

Disorders of sex development (DSD), formerly termed "intersex" conditions, arise from numerous causes. CAH secondary to 21-hydroxylase deficiency is the most common cause of DSD. Sex chromosome disorders, including sex chromosome mosaicism, are the second most common cause of DSD. We discuss a medically complex neonate with DSD presenting with ambiguous genitalia. Hormone levels suggested 21-hydroxylase deficiency. Molecular analysis revealed compound heterozygous mutations in the 21-hydroxylase gene (CYP21A2), confirming the diagnosis of CAH. Chromosome analysis revealed sex chromosome mosaicism with three cell lines: 45,X[8]/45,X,tas(Y;16)(p11.32;p13.3)[8]/45,X,t(Y;8)(p11.32;p23.3)[4] with the Y chromosome in telomere association with chromosomes 8p and 16p in different cell lines, a "jumping translocation." Histologically, the right gonad had irregular, distended seminiferous tubules with hyperplastic germ cells contiguous with ovarian stroma and primordial follicles. The left gonad had scant ovarian stroma and embryonic remnants. Chromosome analyses showed mosaicism in both gonads: 45,X[17]/45,X,tas(Y;8)(p11.32;p23.3)[3]. This is the first case of coexisting CAH and 45,X/46,XY mosaicism reported in the English literature and the third case of a constitutional chromosome Y "jumping translocation." Our report documents the medical and genetic complexity of children such as this one with ambiguous genitalia and discusses the need for a multidisciplinary team approach.

1. Introduction

Ambiguous genitalia in a newborn represent a medical and family psychological emergency. Ambiguous genitalia, a common presenting feature in most disorders of sex development (DSD), suggest a complex differential diagnosis that must be promptly and thoroughly investigated to determine the etiology. Guidelines for a quick, accurate diagnosis are critical for optimal patient management and gender assignment. The recognition of the complexity in diagnosis, terminology, and management of these conditions prompted the Lawson Wilkins Pediatric Endocrine Society (LWPES) and the European Society for Pediatric Endocrinology (ESPE) to issue a consensus statement on the classification of these disorders. The 2006 document proposed a new terminology nomenclature and classified DSD in three broad categories: (1) sex chromosome DSD (45,X Turner and variants, 47,XXY Klinefelter and variants, 45,X/46,XY mixed gonadal dysgenesis (MGD) and ovotesticular DSD (OT-DSD), 46,XX/46,XY chimeric type, or mosaic type OT-DSD), (2) 46,XY DSD (disorders of testicular development or disorders in androgen synthesis/action), and (3) 46,XX DSD (disorders of ovarian development or fetal androgen excess) [1, 2].

Congenital adrenal hyperplasia (CAH) falls into the 46,XX DSD consensus statement DSD category. CAH comprises a group of autosomal recessive disorders that result from either partial or complete deficiency of specific enzymes in the steroidogenesis pathway. The most common form, 21-hydroxylase deficiency, accounts for 90% to 95% of cases and is caused by mutations within the CYP21A2 gene [3]. CAH leads to virilization and genital ambiguity in females, whereas males exhibit no genital ambiguity.

45,X/46,XY mosaicism is classified within the sex chromosome DSD category. The clinical phenotype of patients with 45,X/46,XY mosaicism is broad, ranging from women, with or without Turner syndrome stigmata, to apparently normal males, with intervening variable ambiguous phenotypes [4]. Gonad histology associated with 45,X/46,XY mosaicism is also variable with partial, complete, mixed, or asymmetric gonadal dysgenesis [5].

“Jumping translocations” (JTs) are rare chromosomal phenomena where the same portion of one donor chromosome translocates to two or more different recipient chromosomes. JTs are mainly described in hematological malignancies and rarely observed in the constitutional karyotype. So far approximately 50 cases of constitutional JTs have been reported with the majority involving at least one acrocentric chromosome [6, 7].

Here we describe a full term neonate with genital ambiguity, CAH resulting from compound heterozygous mutations in the CYP21A2 gene, a mosaic 45,X/46,XY karyotype, a jumping chromosome Y translocation to nonacrocentric chromosomes, and a bilateral mosaic 45,X/46,XY gonadal karyotype exhibiting the same chromosome Y translocation.

2. Case Report

The patient was born at 39-week gestation to a 28-year-old G1P1 mother. Father was 27 years old. Pregnancy was complicated by chorioamnionitis. Mother had a history of polycystic ovarian syndrome with no other androgen exposure. The family history was negative for consanguinity or infantile deaths.

The birth weight, length, and head circumference were 2830 g (50% ile), 49.5 cm (8% ile), and 33.5 cm (14.5% ile), respectively. BP was normal. Physical examination was normal with the exception of the genitourinary system. The external genitalia were ambiguous. The phallus was 2 cm in length with a blind dimple on the glans and a urethra that opened ventrally at the base, consistent with Prader 4 hypospadias. The clitoral index was 0.8. The anogenital ratio was 0.83 (Figure 1). No gonads were palpable in the labial/scrotal tissue or in either inguinal canal.

Figure 1

Photo of ambiguous genitalia in this patient.

Initial blood work showed normal electrolytes and thyroid function and undetectable levels of LH and FSH. Peripheral blood (PB) fluorescence in situ hybridization (FISH) analysis using probes for chromosomes X and Y centromeres and the SRY gene found a single X chromosome in 64–74% of nuclei and an XY SRY+ genotype in 26–36% of nuclei. FISH results became available on day of life 3 (DOL 3) (Figure 2(a)). Total testosterone on DOL 5 was 808 ng/dL (normal newborn female 20–64 ng/dL). A care conference with the family to discuss the hormonal and cytogenetic results, which appeared to fully explain the genital ambiguity, was scheduled. However, the state newborn screen returned suspicious for congenital adrenal hyperplasia (CAH). An unstimulated 17-hydroxyprogesterone was 2,800 ng/dL (normal < 90 ng/dL), plasma renin activity was 29.8 ng/mL/hr (normal 2–35 ng/mL/hr), and 11-deoxycorticosterone was slightly elevated at 72 ng/dL (7–49 ng/dL) (as may paradoxically be seen in 21-hydroxylase deficiency). The infant was provisionally diagnosed with coexisting CAH and mixed gonadal dysgenesis (MGD). Hydrocortisone and fludrocortisone were initiated. The total testosterone fell to 89 ng/dL on DOL 10, 4 days after the initiation of glucocorticoids.

Figure 2

Illustration of mosaicism for different cell lines. (a) FISH analysis of interphase nuclei with probes for chromosome X centromere (green) and the SRY gene (red). Partial karyotypes (b, c, d) show (b) chromosomes 8, t(Y;8), 16, and X in 46,X,tas(Y;8) cell line. (c) Partial karyotype shows chromosomes 8, 16, t(Y;16) and X in 46, X,tas(Y;16) cell line. (d) Partial karyotype shows chromosomes 8, 16, and X in 45,X cell line.

Conventional G-banded chromosome analysis showed a mosaic karyotype with three cell lines: 8 cells with a 45,X karyotype, 8 cells with a 46,X,tas(Y;16)(pter;pter) karyotype, and 4 cells with a 46,X,tas(Y;8)(pter;pter) karyotype: mos 45,X[8]/45,X,tas(Y;16)(p11.32;p13.3)[8]/45,X,tas(Y;8)(p11.32;p23.3)[4] (Figures 2(b), 2(c) and 2(d)). The Y chromosome was in telomeric association (tas) with chromosome 16 in 8 cells and with chromosome 8 in 4 cells. This unusual chromosome anomaly is the result of a chromosome Y “jumping translocation.” A 46,XY cell line was not found with analysis of additional 44 metaphase cells. Metaphase FISH analysis using a Yqh probe, Xp/Yp subtelomeric probes, and 8p and 16p subtelomeric probes showed all subtelomeric regions present with no loss of chromosome material secondary to the telomeric associations, tas(Y;16) and tas(Y;8) (Figures 3(a), 3(b), 3(c), and 3(d)). Paternal chromosome analysis was normal. Microarray CGH analysis using Baylor 4 × 180 Exon Array v8.1 chip showed sex chromosome mosaicism (arr Xpterqter(pter-qter)x1,Ypterqter(pter-qter)x0-1) with no other clinically relevant copy number variation.

Figure 3

FISH analysis of tas(Y;8) and tas(Y;16) with subtelomeric probes. (a) FISH with 8pter (green) and 8qter (red) probes shows signal on the junction of tas(Y;8) (arrow) and normal chromosome 8. (b) FISH with XYpter (green) and XYqter (red) probes shows signal on the junction of tas(Y;8) (arrow) and normal chromosome X. (c) FISH with 16pter (green) and 16qter (red) probes shows signal on the junction of tas(Y;16) (arrow) and normal chromosome 16. (d) FISH with XYpter (green) and XYqter (red) probes shows signal on the junction of tas(Y; 16) (arrow) and normal chromosome X.

CAH gene-targeted mutation analysis for 21-hydroxylase-related CAH revealed the patient to be a compound heterozygote for the following mutations in the CYP21A2 gene: IVS2-13C/A>G and p.R356W, a gene pattern expected to result in CAH with classical symptoms.

Echocardiogram and renal ultrasound studies to assess cardiac, aortic, and kidney structure were negative for anomalies associated with the 45,X karyotype. Cystoscopy, vaginoscopy, and laparoscopy on DOL 9 revealed bilateral fallopian tubes, streak gonads, a normal-appearing uterus, vagina, and cervix. The gonads were removed and sent for histological analysis. The right gonad measured 1.5 × 0.3 × 0.2 cm, and the left gonad measured 1.4 × 0.7 × 0.3 cm. The histological examination of the right gonad revealed slightly irregular and distended seminiferous tubules with hyperplastic germ cells contiguous with ovarian stroma exhibiting several primordial follicles. The pathologist interpreted these findings as consistent with ovotestis (Figure 4). The left gonad showed scant ovarian stroma and embryonic remnants focally present with no seminiferous tubules or peritesticular adnexal structures consistent with gonadal dysgenesis. Chromosome analysis of tissues from both gonads showed a mosaic karyotype: mos 45,X[17]/45,X,tas(Y; 8)(p11.32; p23.3)[3]. The tas(Y;16) was not found in gonadal tissues. FISH analysis of the gonads using FFPE tissue showed a single X chromosome in 80% of nuclei and an XY genotype in 20% of nuclei. This mosaic genotype was seen in both the areas with the seminiferous tubules and the areas with the ovarian stroma.

Figure 4

Gonadal histology. (a) Ovarian tissue identified in the right gonad revealed characteristic ovarian stroma and the presence of several scattered primordial follicles (H&E ×100). (b) In addition to the ovary, the right gonad also revealed testicular tissue with numerous seminiferous tubules that appeared dysmorphic (H&E ×200). (c) Immunohistochemistry with inhibin stain highlights the seminiferous tubules that display irregular branching and anastomosis (Inhibin ×200).

A gender assignment care conference was held with the family and our multidisciplinary team. Parents had bonded with the infant as a female, as the second trimester ultrasound had identified the fetus as a girl. The team concurred with the family's wish for female gender assignment. The possibility of gender dysphoria was discussed, and the team recommended strongly against early feminizing or tissue reduction surgery. The possibility of future gender reassignment was also discussed.

The patient is followed in both the pediatric endocrinology clinic and the multidisciplinary clinic for disorders of sexual development. She continues on hydrocortisone and fludrocortisone. Her height is at the 3rd percentile at 6 months, which may reflect the 45,X cell line. Growth hormone therapy is anticipated in the near future [8]. The patient and family are reassessed with the help of a psychologist and a social worker at each multidisciplinary clinic visit.

3. Discussion

No reports of a patient with coexisting 45,X/46,XY mosaicism and CAH were found in the English language literature. One report in the French literature [9] described a newborn with 45,X/46,XY mosaicism and ambiguous genitalia with a moderate elevation of blood 17-OH progesterone, consistent with CAH secondary to 21-hydroxylase deficiency. One gonad was palpable, and histological examination revealed the presence of a testis and a streak gonad. The authors considered mixed gonadal dysgenesis as the cause of sexual ambiguity in their patient.

Both the congenital adrenal hyperplasia and the sex chromosome mosaicism could have contributed to the genital ambiguity in our patient. The initial total testosterone was in the range for a newborn male at 808 ng/dL at 5 days of age. It fell to 89 ng/dL by DOL 10, the day of surgery (which was also 4 days after the initiation of glucocorticoids). A repeat testosterone level two days after surgery was 110 ng/dL. Normally the nadir of testicular testosterone production occurs between 4 and 7 days of life. Thus, it is difficult to determine whether the fall in testosterone was a result of suppression of adrenal androgen in response to hydrocortisone treatment or the normal physiological nadir.

The existence of two separate diagnoses, either of which could have contributed to genital ambiguity, complicated gender assignment. Psychosexual follow-up studies of individuals with disorders of sexual development have yielded varied results [10-13]. CAH secondary to 21-hydroxylase deficiency is the best-studied form of DSD with respect to long-term psychological follow-up. Studies have shown a small, but clinically substantial risk for gender dysphoria in patients with CAH. Dessens et al. performed a meta-analysis of the literature on gender identity, gender identity problems, gender dysphoria, and gender change in chromosomal females with congenital adrenal hyperplasia from years 1950 to 2005. The authors showed that gender dysphoria varied depending on the sex of rearing. Of the 250 CAH 46,XX individuals raised as female, 13 (5.2%) reported gender dysphoria, whereas, of 33 raised as male, 4 (12.1%) exhibited gender dysphoria. The authors recommended female gender assignment in 46,XX newborns with CAH even in the presence of significant virilization [14].

Less is known about gender dysphoria in patients with sex chromosome mosaicism, as few studies exist. In a psychosexual follow-up study with sex chromosome mosaicism, 19 young adults were studied. Nine were raised as females and 10 as males. All patients raised as male exhibited male gender identity. Two out of nine women raised as female (22%) did not identify with the female gender [11].

Sex chromosome mosaicism is among the more difficult of all DSDs with respect to gender assignment. By definition, there are variations in the numbers of X and Y chromosomes in different tissues leading to the entire spectrum of male and female phenotypes, hormonal levels, and gender identity in patients with this single diagnosis [4, 5].

Histopathological examination of the gonads from our patient showed ovarian stroma with primordial follicles and seminiferous tubules with germ cells on the right and ovarian stroma without testicular tubules or peritesticular adnexa on the left. The pathognomonic histologic feature of OT-DSD is the presence of seminiferous tubules and ovarian follicles or oocytes, representing testicular and ovarian tissue in the same individual. MGD may feature streak gonads, dysgenetic testes, or asymmetric gonads with streak on one side and dysgenetic testis on the other. The interpretation of a right ovotestis stimulated a query regarding the classification of ovotestis given the pathological findings in this very young child. A study that found a greater density of primordial follicles in the youngest of girls with TS and 45,X karyotypes [15] raises the question of whether the gonad would continue to show follicles and still be consistent with an ovotestis if examined at an older age.

In addition to having a mosaic sex chromosome complement, our patient's Y chromosome showed telomeric association with two different chromosomes, such that the child possessed three constitutional cell lines. The attachment of one chromosome to two or more other chromosomes in different cell populations is referred to as a jumping translocation (JT). This type of anomaly may be seen in various types of malignancies, but it is a very unusual constitutional chromosome anomaly. A literature search found two reports of a jumping translocation (JT) involving the Y chromosome. Sawyer et al. described the first chromosome Y JT in an Ullrich-Turner syndrome patient with a mosaic 45,X/46,X,tas(Y;21)(q12;p13) karyotype in tissue samples from skin, peritoneum, fascia, and left and right gonads. Two additional cell lines were seen in the left gonad: 46,X,tas(Y; 21)(q12; p13),−22 and 46,X,tas(Y; 21)(q12; p13),+tas(Y; 14)(q12; p13),−22. The Yqter was in telomere association with chromosome 21 short arm in the skin, peritoneum, fascia, and right gonad and in tas with chromosomes 14 and 21 short arms in the left gonad [16]. Huang et al. described a unique telomeric association involving the Y chromosome that “jumped” during meiosis from chromosome 19 in the father, tas(Y;19)(pter;pter), to chromosome 15 in his son, tas(Y;15)(pter;pter). Father and son were normal phenotypic males [17]. In our patient, the Ypter was in telomeric association with the nonacrocentric chromosomes 8 and 16 in different cell lines. Both gonads showed the tas(Y;8). Paternal chromosome analysis was normal indicating that the telomeric associations were de novo. The genetic content of the cells with 46,X,tas(Y;8) and 45,X,tas(Y;16) in our patient is equivalent to normal 46,XY cells as microarray CGH and subtelomeric FISH analyses showed no evidence for gain or loss of genetic material at the associated telomeres.

The exact mechanism that leads to chromosome telomeric association is unknown. It is known, however, that telomeres are very important for maintenance of chromosome stability. Telomeres cap the ends of chromosomes and prevent chromosome fusion [7, 18, 19]. We can only speculate on how the Yp telomere had become attached to the chromosome 8 or 16 telomere. Possibly the association was mediated through telomere recombination to gain stability during meiosis or when the zygote was formed. Chromosomes in telomeric association have two centromeres (dicentric). During mitosis, the configuration of the telomerically associated dicentric chromosome would pose problems to segregation. The chromosomes in association may be “pulled apart” with detachment of the Y chromosome. The detached Y chromosome may be lost with the formation of a 45,X cell line or may reattach to the telomere of a second chromosome, resulting in the formation of a 45,X cell line and either a 46,X,tas(Y;16) or 46,X,tas(Y;8) cell line.

In summary, disorders of gonadal development exhibit a wide clinical, cytogenetic, and histopathological spectrum making gender assignment difficult. An experienced multidisciplinary team approach is necessary early in the course of DSDs, as there are many factors that must be taken into account prior to assigning gender. These include gonadal function, phenotype, internal genitalia (i.e., presence of uterus), potential of fertility and sexuality, gonadal histopathology and risk of future gonadal malignancy, and prenatal brain virilization. Our patient with ambiguous genitalia exemplifies the complexity that may be found during the complete endocrine and genetic evaluation and highlights the necessity for a multidisciplinary team in making gender assignment and management decisions.