Using SNP microarray to distinguish mosaicism from chimerism in a phenotypically normal male newborn with prenatal 46,XX/46,XY karyotype

Abstract

Background

The coexistence of 46,XX/46,XY cells in cultured amniotic fluid sample is not uncommon. The vast majority of these cases are the result of contamination by maternal cells in an otherwise normal male fetus. However, this finding might be true chimera or mosaicism, which can be associated with sexual ambiguity. Conventional cytogenetic study could not exclude these abnormalities. Here, we present a case of 46,XX/46,XY at amniocentesis and ascertained a mosaicism by postnatal single nucleotide polymorphism (SNP) array in a phenotypic male newborn.

Case presentation

A fetus of a 37-year-old nulliparous woman underwent amniocentesis at 19 weeks of gestation because of advanced maternal age. Chromosome analysis of the first and second samples revealed a mixture of 46,XX and 46,XY cell lines. Prenatal ultrasound examination showed normal male genitalia. After birth, peripheral blood samples were analyzed by karyotyping, fluorescence in situ hybridization (FISH), SNP array, and short tandem repeat (STR) analysis. These studies confirmed 46,XX/46,XY mosaicism in a phenotypically normal male newborn.

Conclusion

Our case is the second report of 46,XX/46,XY mosaicism noted by prenatal cytogenetic study and confirmed by postnatal SNP array with normal male genitalia. When encountering XX/XY in prenatal diagnosis, SNP array is a useful tool to distinguish maternal cell contamination from chimera or mosaicism. Detailed prenatal ultrasound for the genitalia is required for the prediction of prognosis. The fetus may have a favorable outcome without ultrasound abnormalities.

Background

The coexistence of 46,XX/46,XY cells in cultured amniotic fluid sample is not uncommon. It has been reported to occur at a frequency of about 1.5 per 1,000 [1–4]. This finding may be attributed to several factors, including maternal cell contamination (MCC), a vanished twin, true fetal chimerism, or mosaicism. These conditions may result in a spectrum of genital phenotypes ranging from normal to ambiguous, depending on the underlying etiology. Among these possibilities, maternal cell contamination accounts for most cases. However, the possibility of chimera or mosaicism could not be excluded by conventional cytogenetic study. Here, we present a case of prenatally diagnosed 46,XX/46,XY and ascertained a mosaicism by postnatal single nucleotide polymorphism (SNP) array in a phenotypic normal male newborn.

Case presentation

A fetus of a 37-year-old nulliparous woman underwent amniocentesis at 19 weeks of gestation because of advanced maternal age. Cytogenetic analysis of amniotic fluid revealed two cell lines, 46,XX[18]/46,XY[11]. The parents were healthy and nonconsanguineous, and there was no contributory family history of hereditary diseases or congenital anomalies. The family was referred to our center for genetic counseling. A repeat amniocentesis performed at 21 weeks of gestation again demonstrated 46,XX[13]/46,XY[10], suggesting possible chimerism or mosaicism. Aside from the above, the pregnancy was uneventful except that the mother presented with gestational diabetes mellitus and was under diet control during pregnancy with satisfactory blood sugar levels. Prenatal ultrasound examination revealed normal male genitalia and no other abnormalities. The baby was delivered vaginally at 38 + 5/7 weeks of gestation with a birth weight of 3,170 g and Apgar scores of 9 and 10 respectively at the 1st and 5th minute. Upon physical examinations, the baby had the phenotypic appearance of normal male external genitalia with normal-sized testes palpable bilaterally inside the scrotum. The measured anogenital distance was 2 cm. Abdominal ultrasonography and magnetic resonance imaging (MRI) demonstrated normal internal genitalia for a male infant. Postnatally, peripheral blood lymphocytes obtained from the patient showed a karyotype of 46,XX[28]/46,XY[22]. The child is now 4 years and 7 months old and is developing well without the need for specific medical intervention at present. Ongoing follow-up is planned.

Methods

DNA extraction

DNA was extracted from peripheral blood using the QIAamp^® DNA Blood Mini kits (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The spectrophotometer (BioTek uQuant, Winooski, VT, USA) was used to assess the genomic DNA concentration and purity according to the manufacturer’s protocol.

SNP array

Single nucleotide polymorphism (SNP) array analysis was performed using the CytoScan 750 K array (Thermo Fisher Scientific, Chelmsford, MA, USA). Array results were analyzed using Chromosome Analysis Suite (ChAS; version 4.1). All genomic coordinates were taken from the December 2013 (GRCh38) human reference sequence (NCBI Build 38). The Affymetrix CytoScan 750 K Array contains approximately 750,000 markers, including 550,000 nonpolymorphic probes used for copy number analysis and 200,000 SNP probes for copy number as well as heterozygosity analysis. SNP probes can be used to identify absence of heterozygosity (AOH) regions, a type of genomic alteration characterized by a long contiguous region of homozygous alleles, within a chromosome. The underlying etiology of AOH, which includes identity by descent and uniparental disomy, could be further distinguished by comparison with parental SNP array data. Parent–child trio analysis can also be used to determine the parental origin of chromosomal inheritance.

Microsatellite genotyping

Short tandem repeat (STR) analysis was performed using GenePhile G-Plex PCR Amplification Kit (GenePhile Bioscience Co., Ltd., Taipei, Taiwan). The multiplex polymerase chain reaction (PCR) assay for STR loci was applied with 15 autosomal markers (loci D3S1744, D4S2366, D8S1110, D10S2325, D12S1090, D13S765, D14S608, Penta E, D17S1294, D18S536, D18S1270, D20S470, D21S1437, Penta D and D22S683). Genotype was analyzed using GeneMapper ID software (Applied Biosystems) through comparison with the allelic ladder and reference DNA control samples.

FISH (fluorescence in situ hybridization)

Fluorescence in situ hybridization (FISH) analysis was performed using the Vysis AneuVysion Multicolor DNA Probe Kit (Abbott, Inc.). The AneuVysion kit contains centromeric probes for chromosomes X (CEPX, DXZ1), Y (CEPY, DYZ3), 18 (CEP18, D18Z1), and an LSI 13/21 probe. In the present study, only the CEPX (Xp11.1–q11.1, SpectrumGreen) and CEPY (Yp11.1–q11.1, SpectrumOrange) probes were analyzed to evaluate sex chromosome constitution.

Cytogenetic analysis

Conventional cytogenetic analysis was performed on heparinized peripheral venous blood samples obtained from the patient. Lymphocyte cultures were established in RPMI medium supplemented with phytohemagglutinin (PHA) according to standard cytogenetic procedures and incubated at 37 °C for 72 h. Metaphase cells were harvested following colcemid treatment, exposed to hypotonic solution, and fixed with methanol: acetic acid (3:1). Chromosome preparations were subjected to G-banding using Wright stain. G-banded metaphase chromosomes were analyzed at a resolution of greater than 400 bands per haploid karyotype. A total of 20 metaphase cells were routinely evaluated, and karyotypes were interpreted according to ISCN 2020 nomenclature.

Results

Postnatal peripheral blood karyotyping revealed a karyotype of 46,XX[28]/46,XY[22] (Fig. 1A). Confirmation of SRY (sex-determine region of Y-chromosome) gene by PCR amplification showed a positive result in this patient. SNP array analysis revealed a consistent finding of sex chromosome mosaicism based on the copy number (CN) profile of the sex chromosomes (CN of X = 1.44; CN of Y = 0.56) (Fig. 1B). In addition, the B-allele frequency (BAF) plot demonstrated an AOH of the X chromosome, which is suggestive of a single origin for the X chromosomes rather than from two genetically distinct cell lines. Analysis of the X chromosome revealed 623 informative markers from a total of 5,717 SNP probes. The exclusive maternal inheritance of these markers indicated that the X chromosomes were entirely originated from the mother. The allele difference plots of the autosomes did not demonstrate additional AOH regions or allelic patterns suggestive of two genetically distinct cell populations (Fig. 1A and C).

Fig. 1.

Initial cytogenetic and molecular studies showed XX/XY status. A The karyotype from amniotic fluid analysis showed 46,XX[18]/46,XY[11] (upper panel), and that from peripheral blood analysis showed 46,XX[28]/46,XY[22] (lower panel). B Results of the copy number status from a whole-genome view of the patient’s peripheral blood DNA. Data suggests normal copy number for the autosomes and copy number mosaicism for the sex chromosomes (copy number of X chromosome = 1.44; copy number of Y chromosome = 0.56). C Allele difference plot of the whole genome and B-allele frequency (BAF) plot of the X chromosome demonstrated an absence of heterozygosity (AOH) of the entire chromosome. The red, green and yellow data points depict the allele peaks. Allele peaks for the autosomes correspond to a normal diploid pattern (3 lines), whereas that for the X chromosome indicate an absence of heterozygosity (2 lines)

Further investigation through STR analysis (Fig. 2A and B) identified only one maternal and one paternal allele pattern. No additional alleles indicative of chimerism from two distinct zygotes were detected. Taken together, the SNP array and STR findings support a diagnosis of chromosomal mosaicism rather than chimerism.

Fig. 2.

Molecular studies confirming mosaic mechanism of XX/XY. A Genotype analysis of microsatellite markers within the autosomes in the father, the mother, and the patient indicates biallelic inheritance. B Representative example of the allele patterns of marker D6S474 from the father (lower panel, two alleles: 15, 16), the mother (middle panel, two alleles: 13, 14), and the patient (upper panel, two alleles: allele 14 from maternal heritage, allele 15 from paternal heritage). C Interphase fluorescence in situ hybridization (FISH) analysis was performed using CEPX (DXZ1) probes corresponding to Xp11.1-q11.1 labeled with SpectrumGreen and CEPY (DYZ3) probes corresponding to Yp11.1-q11.1 labeled with SpectrumOrange. Two distinct cell populations were identified: cells showing two green signals consistent with XX cell lines and cells showing one green and one orange signal consistent with XY cell lines. This supported the presence of XX/XY mosaicism in both buccal and urine-derived cells

FISH analysis using probes for the centromeres of X and Y chromosomes was performed on the urine and buccal mucosa cells (Fig. 2C). Probes were hybridized to the human Y chromosome at locus DYZ3 (CEP Y) over the region of Yp11.1-q11.1, and to the human X chromosome at locus DXZ1 (CEP X) over the region of Xp11.1-q11.1. Fifty-seven out of 100 (57%) urinary epithelial cells showed both X and Y signals and 43 out of 100 (43%) revealed two X signals. In addition, 82 out of 113 (73%) buccal epithelial cells contained both X and Y signals and the remaining cells had two X signals. FISH analysis on these tissues showed an XY predominance, in contrast to what were observed from amniocytes and leukocytes.

Discussion

The finding of a mixture of 46,XX/46,XY cells in amniotic fluid samples has been reported to occur at a frequency of about 1.5 per 1,000 [1–4]. The vast majority of these cases resulted from maternal cell contamination (MCC) in an otherwise normal male fetus. The incidence of MCC was more common in cultures established from bloody fluids. Approximately 35% of all cases were associated with bloody samples. The frequency of MCC can be reduced by using a smaller-gauge needle for amniocentesis (preferably 21 gauge or greater), initially having the stylet in place, and discarding or separating the first 1 or 2 milliliters of amniotic fluid. A comparison of chromosome polymorphisms in the satellite regions of the amniotic fluid cells with the mother’s cells can be informative. If uninformative, molecular MCC analysis should be considered. Although nearly all cases with an admixture of XX and XY cells turn out to be MCC of amniotic fluid from normal male fetuses, the possibility of XX/XY chimerism or mosaicism still exists.

In most reported cases, individuals who carry the 46,XX/46,XY karyotype were the result of processes considered chimera rather than mosaic. Chimerism is defined as the presence of two or more cell lines derived from different zygotes within the same individual. Mosaicism, on the other hand, is defined as the presence of more than one cell line derived from a single zygote within an individual. The distinction between chimerism and mosaicism has not been made with certainty in the era when molecular techniques were unavailable as conventional cytogenetic karyotyping could only reveal the presence of multiple cell lines without identifying their parental origins [5, 6]. In recent decades, however, the implementation of advanced molecular diagnostic tools, such as STR typing and SNP microarrays, has allowed investigators to definitively distinguish between the two phenomena by allele counts. Consequently, an increasing number of chimeric cases have been successfully identified through routine prenatal diagnosis [7]. Chen et al. [8, 9] presented one case and reviewed four previous reports. On the other hand, Yaron et al. [10]. described the first application of molecular techniques to elucidate the origin of the two cell lines from a single zygote. They explained two theoretical mechanisms of 46,XX/46,XY mosaicism. In one of the two, a nondisjunction occurs at meiosis II during oogenesis, leading to a 24,XX oocyte, which develops into a 47,XXY zygote after fertilization with a normal 23,Y sperm cell. This zygote simultaneously loses one X chromosome in one cell line and one Y chromosome in the other cell line during the first postzygotic mitotic cell division (Fig. 3A). The other mechanism might involve postzygotic nondisjunction during early embryogenesis, in which a simultaneous mitotic nondisjunction, involving both the X and Y chromosomes, occurs in a 46,XY embryo. This would lead to the coexistence of 46,XX and 46,YY cell lines, with subsequent death of the nullisomic X cell line (Fig. 3B).

Fig. 3.

Mechanisms for 46,XX/46,XY mosaicism arising from meiotic and mitotic nondisjunction. A A nondisjunction event during oogenesis results in the formation of a 24,XX oocyte. Following fertilization by a normal 23,Y sperm, a 47,XXY zygote is formed. During the first postzygotic mitotic division, mitotic errors result in the loss of one X chromosome in one daughter cell line and the Y chromosome in the other. B Postzygotic nondisjunction during early embryogenesis in a 46,XY embryo may involve simultaneous mitotic errors affecting both the X and Y chromosomes, leading to the generation of 46,XX and 46,YY cell lines. The nullisomic X (46,YY) cell line is presumed to be nonviable and is therefore subsequently lost

In our case, mosaicism was detected in cells derived from multiple embryonic lineages. Buccal epithelial cells, which originate from the ectoderm [11], as well as peripheral blood cells and urine-derived cells, which primarily consist of urothelial cells and renal tubular epithelial cells originating from mesodermal derivatives [12, 13], all demonstrated the presence of sex chromosome mosaicism. The involvement of tissues derived from distinct germ layers suggests that the mosaic event occurred at a very early stage of embryonic development, prior to germ layer differentiation. In contrast, if mosaicism occurs after germ layer differentiation, the mosaicism would be expected to be confined to a particular germ layer and therefore to specific tissue types, rather than being detected across multiple tissues types.

SNP array analysis alone could not definitively distinguish between the possible mechanistic origins of mosaicism in this case. The widespread distribution of mosaicism across multiple germ layer–derived tissues suggests an early postzygotic event, likely occurring during the first or second mitotic division after fertilization. Based on the available data and the general incidence of chromosomal events, the mechanism illustrated in Fig. 3A, involving meiotic II nondisjunction followed by postzygotic mitotic rescue, is considered the more plausible explanation. In this model, the initial embryonic cell line would be 47,XXY, a relatively common aneuploidy with an estimated incidence of approximately 1 in 500–1,000 male births. Subsequent chromosome loss or anaphase lag in independent daughter cell populations could then give rise to 46,XY and 46,XX cell lines without requiring simultaneous chromosomal errors. In contrast, the alternative mechanism (Fig. 3B), in which a normal 46,XY zygote generates both 46,XX and 46,YY cell lines during a single mitotic event, would require concurrent nondisjunction of both X and Y chromosomes, with coordinated segregation of sister chromatids to opposite daughter cells. Such a double-segregation error is considered highly unlikely from a probabilistic standpoint.

In the past, highly informative microsatellite markers provided a powerful tool for studying the transmission of chromosome homologues from parents to children. Niu et al. [14]. reported a cell lineage in a hermaphrodite infant showing 46,XX/46,XY karyotype. By using microsatellite markers, they identified one maternal and one paternal contribution to both the autosomes and the X chromosome, suggesting that both cell lines came from one single embryo rather than a chimeric event. Van Bever et al. [15]. presented a phenotypical male patient with 46,XX/46,XY tetragametic chimerism diagnosed by multiparameter investigation. A genome-wide SNP array analysis showed an aberrant B-allele pattern with broad band splitting, consistent with the presence of two genetically distinct cell populations, including a major and minor cell line. In contrast, our patient did not demonstrate additional allelic patterns on autosomal allele difference plots or extra parental alleles on STR analysis, thus arguing against tetragametic chimerism. Spier et al. [16]. described a male infant with paternal uniparental diploidy mosaicism and a 46,XX/46,XY karyotype, in whom SNP array analysis identified mosaic uniparental disomy and postzygotic chromosomal events. Similarly, the presence of X chromosome AOH with exclusively maternally derived SNP signals in our patient supports a postzygotic mosaic mechanism with a single parental origin of the X chromosome. Notably, if mosaicism or segmental AOH involves other autosomes, it may unmask or increase the risk of autosomal recessive disorders or imprinting-related diseases associated with uniparental disomy; however, no additional autosomal AOH regions were detected in our case. This is the first case of a phenotypic normal male newborn with prenatal diagnosis of 46,XX/46,XYand ascertained a mosaicism by SNP array postnatally.

The phenotypic spectrum of these patients is variable. It ranges from normal male or female genitalia to different degrees of ambiguous genitalia [17–20]. However, only one case of 46,XX/46,XY mosaicism ascertained prenatally with molecular analysis has been described in the literature [10]. The affected infant was found to have a normal male phenotype. The prenatal diagnosis of sex chromosome mosaicism involving both male and female cell lines presents a counseling dilemma. Up to this time, most information regarding outcome has been obtained from individuals who have come to professional attention after birth due to the presence of medical problems, which frequently involve abnormalities of sexual development. Consequently, there may be significant ascertainment bias. A review has yielded only one case report of a prenatally ascertained and postnatally confirmed 46,XX/46,XY in a normal male fetus, presumably a chimera [18].

Our case is the second reported prenatally diagnosed and postnatally confirmed case of 46,XX/46,XY in a male without genital abnormalities. The phenotypic findings associated with a 46,XX/46,XY karyotype, based on the current case and the other reported case, appear to be normal male. Both patients might manifest a subtle abnormality of secondary sexual development and fertility; therefore, close long-term follow-up may be indicated.

One limitation of this study is that CEP18 signals included in the AneuVysion probe set were not available as an internal ploidy control. Therefore, tetraploid cells with sex chromosome aneuploidy could not be excluded by FISH analysis alone. Nevertheless, the patient presented with a normal male phenotype and no major congenital anomalies at birth, these findings that are not typical of clinically significant tetraploidy. Moreover, the concordant results obtained from conventional karyotyping, SNP array analysis, STR analysis, and FISH performed on multiple tissue types collectively support the diagnosis of 46,XX/46,XY mosaicism in this patient.

In conclusion, we present the second case report of 46,XX/46,XY mosaicism noted by prenatal karyotyping and postnatal SNP array with normal male genitalia. When encountering XX/XY in prenatal diagnosis, SNP array is a useful tool to distinguish MCC from chimera or mosaicism. Detailed prenatal ultrasound for the genitalia is required for the prediction of prognosis. The fetus may have a favorable outcome without ultrasound abnormalities.

Abbreviations

MCC

Maternal cell contamination

SNP

Single nucleotide polymorphism

FISH

Fluorescent in situ hybridization

STR

Short tandem repeat

MRI

Magnetic resonance imaging

PCR

polymerase chain reaction

CN

Copy number

AOH

Absence of heterozygosity

UPD

Uniparental disomy

Author contributions

Study concept and design, collection and assembly of data: JC, PS, YJ. Data interpretation: PS, YJ, YL. Manuscript writing: YL, PS, YJ. Review and Editing of the manuscript: JC, PS, YJ, PC, SC.

Funding

Not applicable.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

This study was approved by the Ethics Committee of Chung Shan Medical university Hospital (CS1-23184).

Competing interests

The authors declare no competing interests.