Klinefelter Syndrome Mosaicism 46,XX/47,XXY: A New Case and Literature Review

Abstract

Most cases of Klinefelter syndrome (KS) have 47,XXY karyotype. We reported the first case of 46,XX/47,XXY KS whose genital ambiguity was detected prenatally with postnatal confirmation of the mosaicism and ovotesticular disorder of sex development (OT-DSD). The paternal origin of the extra X chromosome was identified using trio cytogenomic single-nucleotide polymorphism array. Additional 18 cases were also reviewed. The clinical presentation of 46,XX/47,XXY is age-dependent with two age peaks, including ambiguous genitalia during infancy and gynecomastia with or without cyclical hematuria and left scrotal pain and mass in adolescence. The 46,XX is the predominant karyotype both in peripheral blood and gonadal tissue. The risk of germ cell tumor is very high throughout life in these individuals. Individuals with 46,XX/47,XXY mosaicism should be treated more as OT-DSD other than a simple mosaic KS. A multidisciplinary approach and long-term monitoring are necessary.

Introduction

Klinefelter syndrome (KS), 47,XXY, is a common sex chromosome aneuploidy with an approximate prevalence of 1 in 660 live born males. ¹ The phenotypes of the syndrome can vary according to the age of diagnosis, and 10% of cases have been accidentally identified at a prenatal stage without abnormal ultrasound findings. ¹ Almost 30% of cases are late diagnoses in adults who have varying symptoms of androgen deficiency or fertility problems. ¹ Around 80% of the karyotypes identified in KS individuals are 47,XXY with the remainder being 48,XXXY, 48,XXYY, 49,XXXXY, and 46,XY/47,XXY mosaicism. ¹

Disorders of sex development (DSD) are congenital conditions involving those with atypical chromosomal, gonadal, or anatomical sex development. ² The estimated incidence of DSD is 1 in 4,500 to 5,500. ² Ovotesticular DSD (OT-DSD) is a rare disorder of sexual differentiation, accounting for 3 to 10% of all DSD cases. OT-DSD is characterized by the presence of both ovarian tissue (as determined by the presence of primordial follicles) and testicular tissue (containing seminiferous tubules) in the same individual. These tissues may be present together unilaterally or bilaterally in the same gonad(s) or separately with ovarian tissue on one side and testicular tissue on the other, also known as lateral OT-DSD. The most common karyotype found in OT-DSD is 46,XX, followed by 46,XX/46,XY mosaicism and 46,XY, in that order. ³

KS mosaicism 46,XX/47,XXY is a very rare condition. One case was diagnosed prenatally due to cystic hygroma. ⁴ Herein, we reported the first case of KS mosaicism 46,XX/47,XXY with ambiguous genitalia detected during prenatal stage. In addition, the origin of the extra X chromosome was identified. Clinical features and gonadal karyotypes and histology of the published cases were also reviewed.

Subjects and Methods

Retrospective Study and Literature Review

Medical records including clinical data, prenatal and postnatal ultrasound reports, karyotype, and pathological findings were retrospectively analyzed. Papers of English language describing patients with 46,XX/47,XXY were reviewed.

Karyotyping and Chromosomal Microarray Analysis

Standard G-banding metaphase chromosome analysis was conducted on the excised and biopsy specimens obtained during reconstructive surgery, following established protocol. To determine the origin of the extra X chromosome, a trio chromosomal microarray analysis (CMA) analysis of the specimens from peripheral blood of the patient and the parents was performed. The CMA was performed using the Affymetrix CytoScan 750K microarray platform consisting of 550,000 unique nonpolymorphic probes and 200,436 single-nucleotide polymorphism (SNP) probes (Affymetrix; Santa Clara, California, United States). This platform was designed based on UCSC hg19 (University of California Santa Cruz, Human Genome 19) (NCBI Build 37, February 2009). The results were analyzed using Chromosome Analysis Suite software, version 3.2 (Affymetrix).

This study was approved by the Ramathibodi Hospital Institutional Review Board (protocol ID1085 and 076169) and complied with the Declaration of Helsinki. Written informed consent was obtained from the parents as for themselves and on behalf of the child for publication of the individual data and genetic analyses.

Results

A New Patient and the Parental Origin of the Extra X Chromosome

The patient was the first child of a 27-year-old Thai woman who had no history of medical or hormonal treatments. Family history was unremarkable. Fetal ultrasound scan performed at 20 weeks revealed normal male genitalia but a follow-up scan at 27 weeks showed bifid scrotum or labia separated by a prominent and elongated phallic structure, prompting the diagnosis of DSD. Due to the advanced gestational age, the parents opted for continuation of pregnancy and declined fetal chromosome study.

The patient was born vaginally at 37 weeks of gestation without obstetric complications.

Physical examination revealed a short penis with length of 1.7 cm (normal range 2.3–3.8), labioscrotal fusion, and hypospadias, a 0.5-mL (normal range 1–2) right gonad located in the lower inguinal canal, and an unidentified left gonad. Ultrasonographic studies indicated normal kidneys, a normal sized and shaped uterus with a gonad measuring 0.8 × 0.6 cm at the left adnexa, the upper part of the vagina, and a right inguinal testis measuring 1.4 × 0.7 cm.

On day 2 of life, serum levels of total testosterone were measured at 257 ng/dL (normal range for male newborns 75–400), follicle stimulating hormone (FSH) at 0.09 mIU/mL (normal range 0.16–4.1 at 4 weeks–11 months), and luteinizing hormone (LH) at < 0.09 mIU/mL (normal range 0.02–7.0). Peripheral blood karyotype showed mosaicism 46,XX [36]/47,XXY [15]. Cystoscope and cystogram performed at 1 year showed a normal bladder, complete separation of the vaginal and urethral openings, and no vesicoureteral reflux.

He was followed by a multidisciplinary team approach involving pediatric endocrinology, pediatric genetics, urology, gynecology, plastic surgery, adolescent medicine, and child psychiatry. Sex assignment, gender choices, hormonal treatment, reconstructive surgery, urogenital, and reproductive functionalities were extensively discussed with the parents. The team suggested raising the child in a neutral environment (such as toys, clothes, and play), with close parental observation of the child's behavioral and gender tendency before making a decision on sex assignment, which can wait until the age of 4 years. The parents felt confident that their child had shown male predilection and, therefore, decided to have early reconstructive surgery performed. At 1 year and 10 months, surgical intervention including laparoscopic total hysterectomy, left salpingo-oophorectomy, biopsy of the right inguinal gonad, first-stage repair of hypospadias, right orchidopexy, total vaginal obliteration, and urethral lengthening was done. Testosterone was also administered before and after the surgery.

Gross and histopathological analyses of the excised specimens revealed underdeveloped uterus and adnexal organs ( Fig. 1A ). The left gonad consisted of ovarian and immature testicular tissue, whereas the right gonad contained testicular tissue with few seminiferous tubules and few spermatogonia ( Fig. 1B–D ). Karyotype result of gonadal tissue is described in Table 1 . The uterus tissue contained only 46,XX karyotype.

Fig. 1.

Internal genital organs and gonads. ( A ) Uterus and left adnexal, noted the left adnexa containing ovarian tissue, fallopian tube, and testicular excretory part. ( B ) Histology of the left gonad showing ovarian tissue (black arrow), ciliated tubal epithelium (yellow arrow), tubo-ovarian vessel (blue arrow), the testicular excretory part, and epididymis (orange arrow) (hematoxylin and eosin [H&E], 100×). ( C ) The ovary noted primordial and primary follicle at the subcortical area (black arrow), very few seminiferous tubules with a minimal amount of intratubular germ cells at the center (asterisk) (H&E, 400×). ( D ) Right gonad, noted few seminiferous tubules containing mostly Sertoli cells with few spermatogonia, and no spermatogenesis identified (H&E, 40×).

Table 1. Clinical presentations, external and internal genital organs, gonadal histology, and karyotypes of patients with 46,XX/47,XXY mosaicism.

Patient	Age	Clinical presentation	External genitalia			Location of Rt/Lt gonad	Internal organ		Histology of Rt/Lt gonad	Percent of 46,XX			Hormonal level	Reference
			Phallus	Hypospadia	Vagina		Ut	Duct Rt/Lt		PB	Rt gonad	Lt gonad	TT/FSH/LH
1	27 wk GA	Genital ambiguity	Ambiguous	+	+	I/A	+	NA/V,F	T/OT	71	62	82	NL/low/low	Present case
2	12 wk GA	Abnormal ultrasound and PND a	Male	–	NA	S/S	–	NA	NA	53	NA	NA	Low/NL/NL	Low et al 4
3	21 wk GA	Abnormal PND	NA	+ b	NA	A/A b	NA	NA	T/T	56	61	57	NA	Cheng et al 5
4	4 m	Genital ambiguity	Ambiguous	+	–	S/A	+	V/F	T/O	62	75	74	NA	Butler et al 6
5	5 m	Genital ambiguity	Ambiguous	+	+	S/I	+	NA/F	T/O	NA	66 c	84 c	NA	Ozsu et al 7
6	1.7 y	Genital ambiguity	Ambiguous	NA	NA	A/I	+	F/V	OT/T	NA	NA	NA	NA	Mao et al 8
7 d	5 y	Behavioral, cognitive, speech dis.	Male	–	–	I/I	–	NA	NA	81	NA	NA	Low/low/low	Tachon et al 9
8 d	5 y	Behavioral, cognitive, speech dis.	Female	–	+	A/A	+	NA	NA	81	NA	NA	NL/low/low	Tachon et al 9
9	13 y	Lt. scrotal pain, gynecomastia	Male	–	–	S/I	+	V/F	T/O	70	60	NA	NA	Kanaka-Gantenbein et al 10
10	14 y	Lt. scrotal pain, gynecomastia	Ambiguous	+	NA	I/I	NA	V,F/ V,F	OT/OT	50	NA	NA	Low/NL/high	Bergmann et al 11
11	14.5 y	Lt. scrotal pain, cyclic hematuria, gynecomastia	Male	+	NA	S/S	+	NA/F	T/OT	85	81	42	Low/high/high	Isguven et al 12
12	15 y	Lt. scrotal mass, gynecomastia	Male	NA	NA	S/S	+	NA/F	OT/O	87	NA	NA	Low/NL/NL	Chouhan et al 13
13	16 y	Gynecomastia	Male	NA	–	S/A	+	NA/F	OT/O	72	NA	NA	Low/NL/NL	Pérez-Palacios et al 14
14	16 y	Cyclic hematuria, gynecomastia	Male	–	NA	S/A	+	NA/F	T/O	80	NA	NA	Low/NL/NL	Talreja et al 15
15	16 y	NA	Ambiguous	+	NA	NA	NA	F/F	OT/O	72	NA	NA	Low/high/high	Torres et al 16
16	18 y	Germ cell tumor (mediastinum), gynecomastia	Ambiguous	NA	NA	S/S	–	NA	NA	50	NA	NA	NL/high/high	Song et al 17
17	25 y	Gender discrepancy, gynecomastia	Female	NA	+	A/A	+	F/F	O/O	81	NA	NA	NA	Hamlett et al 18
18	29 y	Infertility	Male	NA	NA	S/S	NA	NA	NA/T	80	NA	0	Low/high/high	Velissariou et al 19
19	62 y	Germ cell tumor (Rt testis)	Male	NA	NA	S/S	NA	NA	T/NA	NA	NA	NA	Low/high/high	Matsuki et al 20

Abbreviations: A, abdominal cavity; F, fallopian tube; FSH, follicular stimulating hormone; GA, gestational age; I, inguinal canal; Lt, left; LH, luteinizing hormone; m, months; NL, normal; NA, not available/not applicable; O, ovary; OT, ovotestis; PB, peripheral blood; PND, prenatal diagnosis; Rt, right; S, scrotum; T, testes; TT, testosterone; Ut, uterus; V, vas deferens; wk, weeks; y, years; +, yes; –, no.

^aLarge cystic hygroma and generalized edema which regressed completely at GA 19 weeks.

^bFrom autopsy after medical abortion.

^cBoth gonads consisted of 45,X/46,XX/47,XXY mosaicism.

^dCases 7 and 8 were monozygotic twins.

Results of the trio CMA demonstrated (1–22,X) × 2 and (1–22) × 2,(X,Y) × 1 in the mother and the father, respectively, and showed that the patient shared SNPs of the paternal X chromosome and half of the maternal X chromosomes, indicating paternal origin of the extra X chromosome.

Clinical Presentations, Genital Phenotypes, Gonadal Histology, and Hormonal Data of 19 Patients with 46,XX/47,XXY

Clinical phenotypes and related findings of the present case and 18 additional patients identified from the literature during 1969 to 2019 were analyzed, as shown in Table 1 . ^{4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20} The age of presentation ranged from prenatal stage to 62 years. All but two patients were raised as male. ^{9 18} Locations of gonads, histology, and body orientation are listed in Tables 2 and 3 .

Table 2. Location of gonad and its histology in 19 cases with 46,XX/47,XXY mosaicism.

Location	Gonadal tissue				Total
	Testis	Ovotestis	Ovary	NA
Scrotum	7	3	1	6	17
Abdomen	2	2	5	2	11
Inguinal	2	2	2	2	8
NA	0	1	1	0	2
Total	11	8	9	10	38

Abbreviation: NA, not available.

Table 3. Histology of the gonad in body orientation in 19 cases with 46,XX/47,XXY mosaicism.

Gonad	Side		Total
	Right	Left
Testis	8	3	11
Ovotestis	5	3	8
Ovary	1	8	9
NA	5	5	10
Total	19	19	38

Abbreviation: NA, not available.

Only the present case and one from the literature have described the origin of the extra X chromosome in XX/XXY individuals, and both cases confirmed paternal origin. ¹⁹

Discussion

Among 19 cases studied, there were two age peaks for diagnosis with different manifesting symptoms and signs. The first peak was during infancy because of ambiguous genitalia and the second peak at adolescence due to gynecomastia with or without history of cyclical hematuria and left scrotal pain. The other presenting features were germ cell tumor, disorders of behavioral and psychomotor development, and gender discrepancy.

The most common appearance of external genitalia was male (50%), followed by ambiguous (39%) and female type (11%). Ambiguous genitalia was detected postnatally in all but one patient. When present, ambiguous genitalia was accompanied by hypospadias and the upper part of vagina, whereas those with male phenotype were often found to have hypospadias. Although fetal anatomical DSD can be diagnosed at around 13 to 15 weeks' gestation, the detection rate improves with advanced gestational age, a more experienced sonographer, and using a higher resolution ultrasound, as also observed in the current patient. Unilateral and bilateral cryptorchidism were common at 65%. ^{5 6 7 8 9 10 11 14 15 18}

Uterus was found in 79% (11/14) of the cases with male or ambiguous external genitalia (5 and 4). These uteri were bi or unicornuated and always possessed a fallopian tube on at least one side. In cases where uterus was present, gonads were mostly (7/10) proven to be pure ovary on at least one side which complemented with pure testicular tissue (4), ovotestis (2), or unmixed ovary on the other, while the remainders (3/10) were found to have ovotestis on one side and pure testis on the other ( Table 1 ). Data about the duct was available in 12 patients, including six bilaterally and six left-only. The right ducts showed no predilection of being the vas deferens, fallopian tube, or mixed fallopian-vas deferens. In contrast, 75% of left ducts were fallopian tube only, with the remainder being mixed fallopian-vas deferens or vas deferens only. Vas deferens was found in 41% (5/12), with accompanying testis or ovotestis. These findings suggest that having male genital phenotype does not exclude the presence of uterus and related organs. Theoretically, testis and ovotestis produce no or insufficient Mullerian inhibitory factor to the level that can suppress the development of Mullerian structures; therefore, the uterus, upper part of vagina, and the uterine tube can develop in such cases.

Among patients with available data, OT-DSD was present in most cases (85% or 11/13), including unilateral (6), lateral (4), and bilateral (1). Among those with unilateral OT-DSD, the other gonad is equally found to be ovarian or testicular tissue. As for the location of the gonad, scrotal gonad is more likely to be, though not always, a pure testis than being ovotestis and pure ovary (S: T > OT > O). Contrarily, abdominal gonad is more likely to be a pure ovary than ovotestis/testis (A: O > OT/T), whereas inguinal gonad does not show any specific pattern of tissue type. Regarding body orientation, the right gonad is more likely to be a testis than ovotestis or ovary (Rt: T > OT > O), while the left gonad is more likely to be an ovary than ovotestis and pure testis (Lt: O > OT/T; Table 3 ). In other words, the pure ovary is often on the left, similar to those found in the common OT-DSD. ³

The 46,XX karyotype was found to be more predominant than the XXY in the peripheral blood chromosome complement (88%) and the gonads(83%), regardless of the type of external genitalia and the gonadal histology. One might predict that the masculine features may correlate with the percentage of XXY element, but it was not the case here.

Among nonmosaic KS, approximately 50% of cases are a result of nondisjunction during paternal miosis I. ²¹ The result of the present case and that described by Velissariou et al, suggest that the extra X chromosome in XX/XXY individuals is a result of paternal meiosis I nondisjunction followed by postzygotic elimination of the Y chromosome. ¹⁹ We cannot conclude whether or not the paternal origin of the extra X chromosome in 46,XX/47,XXY plays a role in leading to this rare mosaicism, due to very small number of cases. Additional cases and further investigation are required before a conclusion can be made.

In most cases where data was available, the levels of testosterone, FSH, and LH were low in prepubertal age while the testosterone level was low with elevated FSH and LH concentrations in adolescent/adult patients ( Table 1 ). This data indicates that the altered hormonal functions in XX/XXY are similar to those observed in XXY KS.

All patients with genital ambiguity had been raised as male with subsequent surgical removal of Mullerian structures and unilateral gonadectomy ( Table 1 ). ^{6 7 8 10 11 12 13 14 15} The data suggested that urogenital reconstructive surgery is necessary, therefore a multidisciplinary team approach should be activated as soon as possible, once the diagnosis of 46,XX/47,XXY is made. This can lead to appropriate presurgical intervention for the patients and families. In general, sex assignment should be decided based on at least three sets of factors: (1) physical appearance and functionality including phallus, urethra, presence of vagina, and uterus; (2) gonadal and reproductive functionality in the future; and (3) gender tendencies and behavioral and psychological phenotypes of the child, rather than relying on the percentages of XX and XXY present in the peripheral and gonadal tissue.

Gender identity can be expressed before the age of 4 years, and may develop as early as 18 to 24 months of age. ²² Some studies suggest postponing the definitive urogenital reconstruction until 5 to 6 years or until patient are old enough to identify their own sex based on the external genital appearance. ²³

The reproductive functionality in 46,XX/47,XXY may have considerations that differ from those of 46,XY/47,XXY KS and the more common OT-DSD, namely 46,XX, 46,XX/46,XY, or 46,XY. There has been no report of successful reproduction in 46,XX/47,XXY whereas spontaneous fertility of KS has been described, though rarely. ¹ For the common OT-DSD, at least 21 pregnancies have been noted in OT-DSD individuals with 46,XX (8 individuals) and 46,XX/46,XY (1 individual), all of whom were raised as female. This is likely due to the fact that the ovarian tissue tends to be mature even when present in ovotestis, while the testicular tissue is usually immature when existing in persons with OT-DSD. ³

Assisted reproductive technology (ART), such as testicular sperm extraction, followed by intracytoplasmic sperm injection is shown to allow fatherhood in nonmosaic and mosaic 46,XY/47,XXY KS. ¹ In males, as the number of extra X chromosome increase, the severity of spermatogenic impairment and the difficulties in ART also increase. ¹ Given the absence of spermatogenesis commonly observed in the testicular histology of adult individuals with 46,XX/47,XXY, the chances of spontaneous fertility and effective ART seem to be modest. ^{10 12 19}

The most common late complications in 46,XX/47,XXY raised as male were gynecomastia (64%), followed by scrotal pain and mass and/or cyclical hematuria (50%); behavioral and speech problems; and germ cell tumor. The risk of gynecomastia in 46,XX/47,XXY is higher than that of 30% described in 47,XXY KS, reflecting a further decrease of androgen production in the XX/XXY. The left scrotal pain is explained by ruptured or unruptured hemorrhagic corpus luteal cysts during ovulation in the ovary or ovotestis. ^{10 13} The monthly cyclic hematuria is a result of menstrual blood from the uterus, passing through the cervix and vagina, urogenital sinus, and penile urethra. ¹⁵

In our review, we observed the occurrence of germ cell tumor at 18% (2/11) in adolescents and adult individuals, which is much higher than 1 in 4,000 reported among 47,XXY KS and 1 in 83,333 among the general population. ²⁴ Both 47,XXY KS and 46,XX/47,XXY KS are at higher risk of developing extragonadal germ cell tumors, especially at mediastinal sites with the majority (75%) of cases being mature teratomas. ¹ As for other types of malignancies, the risk of breast cancer in KS is 3% or 50 times higher than that of general male population. The increased risk of malignancies is attributed to an increased estradiol-to-testosterone ratio, the presence of two X chromosome (as 15% of the gene escaping X-inactivation on the additional chromosome), ^{1 24} and higher susceptibility to virus-induced cellular transformation of the XXY cells. ²⁵ This may explain the greater risk for tumor development in 46,XX/47,XXY, compared with those with 47,XXY. ^{17 20} To reduce the risk of tumor development, the following are recommended: removal of gonadal tissue and duct structure inappropriate to the sex of choice, orchidopexy, androgen replacement in hypogonadal males, and close follow-up until complete puberty development. ^{1 3 24}

Conclusion

Clinical presentations of 46,XX/47,XXY are age-dependent with two age peaks, infancy and adolescence. The 46,XX is the predominant karyotype both in peripheral blood and gonadal tissue. There is an extremely high risk of germ cell tumor throughout life in these individuals. It is unclear whether the paternal origin of the extra X chromosome is a predisposing factor leading to KS mosaicism, 46,XX/47,XXY. Further investigation is needed. The 46,XX/47,XXY mosaic KS should be treated more toward OT-DSD, rather than a simple mosaic KS. A multidisciplinary approach and long-term monitoring are necessary.

Funding Statement

Funding This work was supported by grants from the Department of Pathology, Faculty of Medicine Ramathibodi Hospital.