Prenatal diagnosis and molecular cytogenetic identification of small supernumerary marker chromosomes: analysis of three prenatal cases using chromosome microarray analysis

Karyotype, CMA, and FISH results

In fetus 1, the karyotype of amniocytes revealed to be 47,XY,+mar (Figure 1A). C-banding identified the marker as pseudoisodicentric (psu idic) chromosome, and N-banding demonstrated a bisatellited chromosome (Figure 1B, 1C). Thus, the sSMC was characterized as a pseudoisodicentric and bisatellited chromosome fragment. The pregnant couple had normal karyotypes, indicating that the marker was newly mutated.

To further characterize the origin and gene content of the sSMC, SNP array was performed, the results of which showed a gain of 1.5 Mb in chromosome 22q11.1q11.21 (with a copy number of 4), which contains SLC25A18, IL17RA, BID, XKR3, PEX26, CECR1, MICAL3, CECR2, TUBA8, USP18, and ATP6V1E1 11 Online Mendelian Inheritance in Man (OMIM) genes, of which two (CECR1 and CECR2) are the primary genes associated with cat eye syndrome (CES) (Figure 1D). Subsequently, confirmatory FISH using the BAC probe RP11-958H20 identified four signals on the marker chromosome in all metaphase and interphase cells, which agreed with the CMA findings (Figure 1E, 1F). Based on the SNP array, FISH, and cytogenetic analyses, the final karyotype of case 1 was defined as 47,XY,+idic (22q11.1q11.21) (Table 1).

In case 2, standard chromosomal karyotyping analysis of cultured amniocytes showed a 45,X mosaic karyotype (46,X,+mar[32]/45,X[68] (Figure 2A, 2B). After genetic counseling, the parents of fetus 2 consented to undergo percutaneous umbilical blood sampling. Cytogenetic analysis of umbilical cord blood showed mosaicism 46,X,+mar [22]/45,X[20]. The parental karyotypes were normal. An SNP array analysis revealed a 50 Mb genomic loss at Xp22.33q11.1 (spanning 290 OMIM genes), and a 58 Mb genomic loss at Xq21.31q28 spanning 296 OMIM genes (Figure 2C), including X-inactive specific transcript (XIST) gene. Metaphase FISH using the centromeric probes D18Z1, DXZ1, and DYZ3, and a sequence-specific DNA probe for RB1 and 21S259/D21S341/D21S342, which are located on 21q22.13, confirmed the SNP array findings (Figure 2D, 2E). Furthermore, FISH analysis identified the karyotype as 46,X,+mar.ish r(X)(DXZ1+)[8]/45,X[6]. By combining the CMA and FISH results, the marker chromosome was determined to be derived from the ring X chromosome, encompassing XIST gene, thus, the karyotype of fetus 2 was defined as 45,X/46,X,r(X) (p11.1q21.31) (Table 1).

In case 3, cytogenetic analysis using cultured amniocytes revealed that the karyotype of fetus 3 was 45,X[62]/46,X,+mar[9]. Moreover, 12.7% cells were found to harbor an sSMC and 87.3% of cells had 45,X (Figure 3A, 3B). Subsequent SNP array analysis of amniocytes revealed a 2.3 Mb genomic gain in Yq11.221q11.222 (encompassing one OMIM gene) and a 6.1 Mb genomic loss in Yq11.222q11.23, spanning 18 OMIM genes and encompassing HSFY1, PRY,DAZ1, AZFb, and AZFc, which are associated with azoospermia, oligospermia, and infertility (Figure 3C). Further FISH analysis using X, Y, and 18 chromosomal centromeric probes defined the karyotype of fetus 3 as 45,X(DXZ1×1, DYZ3×0)[22]/46,X,idic(Y)(q11.2?) (DXZ1×1, DYZ3×2)[2]/47,X,idic(Y)(q11.2?)×2(DXZ 1×1, DYZ3×4)[1]), which generally agreed with the SNP array results (Table 1).

Pregnancy outcomes and follow-up study

Three de novo sSMCs were identified in three pregnancies. One pregnancy was continued, whereas two pregnancies, one with Turner syndrome (TS) and one with CES, were terminated by delivery through induced labor (Table 1, 2).

Case 1

After adequate genetic counseling, despite the ultrasound abnormalities and the unfavorable prognosis for fetus 1 of sSMCs associated with CES, the parents accepted the risk of an unpredictable degree of CES in the child and the pregnancy continued to term. At 38 weeks of gestation, a phenotypically abnormal boy was born, weighing 2900 g at birth, and Apgar scores of 8-9-10. The woman denied taking any teratogenic medicines or having any illness during the pregnancy. In addition to the prenatal findings, postnatal examination revealed bilateral preauricular skin tags, hypertelorism, left ear canal atresia, anal atresia, flattened nasal bridge, and bilateral low-set ear. Ophthalmological examination showed no anomaly; however, due to severe lung hypoplasia, the boy died at home on day 10 of life. Permission for an autopsy was not granted (Table 2).

Case 2

Normal female genitalia were observed sonographically. Given the chromosomal abnormality and the adverse prognosis, labor was induced at another hospital at 24⁺² weeks of gestation after adequate genetic counseling. Thus, clinical data were not available (Table 2).

Case 3

Normal male genitalia were observed sonographically. In the context of cardiovascular and chromosomal abnormalities, the pregnancy was electively terminated after the karyotype, SNP array, and FISH results were disclosed. A phenotypically normal male, weighing 250 g and showing no external malformations, was delivered through induced labor. Further post-mortem studies were declined. Thus, autopsy data were not available (Table 2).

sSMC in Turner syndrome case

TS occurs in 1.76 per 1,000 female fetuses upon mid-trimester amniocentesis [25], and the incidence of sSMCs in TS (sSMC^T) is 3.08% [26]. sSMC^T carriers primarily have a 45,X/46,X,+mar karyotype. Meanwhile, sSMC^T has been associated with three morphologies: diccentromeric (dic), ring chromosome (r), and small chromosomal segments with centromeres (min). Nearly all sSMC^Ts are confirmed to be a derivative of chromosome X or Y, and approximately 72.6% originate from Y, while most sSMC^T(Y) are from isodicentric chromosomes. A further 27% originate from the X chromosome with a majority of sSMC^Ts(X) determined to be ring chromosomes [27]. The remaining 0.4% are derived from autosomal chromosomes [28] and may be responsible for the TS phenotypes or gonadal dysgenesis.

The correlation analysis between sSMCs and clinical phenotypes not only contributes to the elucidation of the mechanism and genetic effect of sSMCs, but also has important clinical implications for the genetic consultation and treatment selection of TS fetuses. Phenotypes of individuals carrying mos45,X/46,X,+mar differ depending on the gene content, mosaicism proportion, size, X chromosome breakpoint location, and XIST-mediated silencing. Patients carrying sSMC^T(X) may present a mild variant TS phenotypes if sSMC^T(X) is inactivated [29]. Meanwhile, patients with a 45,X/46,XY karyotype are at significantly increased risk for gonadoblastoma. Both gonadoblastoma and in situ carcinoma can progress to non-invasive or invasive malignant germ cell tumors [30]. Some researchers consider that the GBY on Yp11-q11 may lead to the occurrence of malignant gonadal tumors [31, 32]. In our present study, cases 2 and 3 with sSMC^T were detected prenatally (Table 1). Since the prognosis of patients carrying different chromosome sources of sSMC^T vary greatly, once TS with sSMC^T is diagnosed prenatally it is necessary to further characterize the origin and morphology of sSMC^T using molecular genetic technologies.

In case 2, karyotyping analysis of amniocytes revealed that the fetus was 32% mosaic (46,X,+mar[32]/ 45,X[68] (Figure 2). SNP array revealed that the derivate corresponded to the Xp11.1-q21.31 region, which contains the XIST gene (Xq13.2), and its preferential inactivation is possible. Subsequently, FISH (VYSIS, Inc) analysis using a DXZ1 probe indicated that the marker chromosome originates from ring X chromosome [r(X)]. As a result, the karyotype was defined as mos 45,X[68]/46,X,r(X)(p11.1q21.31)[32], which is diagnosed as TS. The r(X) with XIST expression can cause random X inactivation. However, the abnormal severe TS phenotypes may be caused by the inactivation bias of r(X). The X inactivation test was not carried out for fetus 2 as per the mother’s instructions. Fetus 2 had no major structural anomalies apart from hydrops fetalis based on the SNP array result. We, therefore, postulate that XIST may mediate sSMC^T(X) inactivation in this specific case.

Supernumerary ring chromosomes constitute approximately 10% of SMCs [33]. Abnormally sized r(X) accounts for 5% of TS patients [34]. In fact, most individuals with larger r(X) tend to have TS phenotypes, without mental retardation. However, a severe TS phenotypes (mental retardation/developmental delay) may also be due to the inactivation bias of X chromosome [35]. Most of these patients require therapeutic intervention for infertility. However, Yuge et al.[36, 37] reported a normal pregnancy, that did not require therapeutic intervention for infertility, in a patient diagnosed with TS that had mos 45,X/46,X,r(X) karyotype.

It is impossible to determine the mosaicism proportion in different endoderm/ /mesoderm/ectoderm tissues, however, the relatively high proportion of amniocytes with an abnormal karyotype in case 2 (32%) could be a poor prognostic sign. Moreover, the haploinsufficiency effect of SHOX gene at Xp22.3 in fetus 2 may lead to early fusion of distal limb growth as well as short stature. The female ovarian function maintenance region is primarily located at Xp11 and Xq13-q26, while deletion in Xq28 can cause premature ovarian failure and infertility, of which, the involved genes POF1 and POF2 are located in Xq26-q28 and Xq13.3, respectively [37]. Therefore, we propose that fetus 2 might present with short stature, primary amenorrhea, premature ovarian failure, uterine dysplasia, breast dysplasia, and infertility since puberty. Finally, the pregnancy was terminated electively after genetic counseling.

In case 3, the pregnant woman performed amniocentesis due to high risk of serological Down syndrome screening in the second trimester, meanwhile, prenatal ultrasound showed fetal tricuspid regurgitation with broad inner diameter of the right pulmonary artery. Karyotype analysis of amniocytes showed 45,X[62]/46,X,+mar[9], of which, 45,X cell lines accounted for 87.3%. The family history was unremarkable. Subsequent SNP array analysis of amniocytes revealed a 2.3 Mb genomic gain in Yq11.221q11.222 (encompassing one OMIM gene) and a 6.1 Mb genomic loss in q11.222q11.23 (containing AZFb and AZFc) encompassing 20 OMIM genes, including HSFY1, PRY, and DAZ1, which can cause oligospermia and infertility in men (Figure 3C). In addition, to clarify the origin and morphology of the sSMC, FISH assay was carried out using DYZ3 (red) centromeric probe located at Yp11.1-q11.1. In the metaphase cells, four red signals were detected on the sSMC, indicating that the sSMC was isodicentromeric of Yp chromosome. This provides a strong basis for elucidating the recombination mechanism of sSMC. Ultimately, the sSMC was determined as idic (Y)(q11.2).

Although the mechanism of idic(Y) formation remains unknown, Beaulieu et al. [38–40] studied patients with idic(Y) karyotype, and proposed that Y chromosome may not be separated during phase I and II of meiosis as well as sister chromosome monomer during mitosis, which is an important mechanism for idic(Y) formation. Moreover, Mekkawy et al. [41] suggested that breakpoints of idic(Y) often occur in regions of the repeat sequence, and considered that the special structure in these regions could easily cause non-allelic exchange recombination within chromosomes, resulting in the deletion, inversion, repetition, and idic(Y), and subsequent deletion of sperm-related genes.

We, therefore, speculated that fetus 3 carrying SMC(Y), encompassing GBY, had an increased risk of gonadal tumor occurrence. Hence, should the couple have opted to continue the pregnancy, the fetus would have to be followed-up closely. Further karyotype analysis performed on skin and gonad tissue would also be required and dysplastic gonad would have to be removed laparoscopically as soon as possible to avoid tumorigenesis in the case of the presence of Y chromosome.

Isodicentric Y chromosomes are unstable and are generally lost during mitosis, producing a 45, X cell line accounting for majority proportion, thus forming a mosaic 45, X/46, X, Idic(Y) [42]. The karyotype 45,X/46,X,idic(Y) is rarely detected prenatally and the phenotypes of these patients, ranging from TS to sexual developmental disorder, sex ambiguity to azoospermia, and mental retardation, may be influenced by the location of Y chromosome breakpoints, the proportion of abnormal cell lines, and whether the SRY gene is lost [43–45]. In addition, clinical phenotypes are not necessarily related to the proportion of the two cell lines, since the karyotype in the fetal sex gland system may differ entirely from that in amniotic fluid cells. Thus, the clinical manifestations of patients carrying mos45,X/idic(Y) are congenital gonadal dysplasia and genital malformation [46]. Moreover, AZFb, AZFd, and AZFc regions are absent in patients with idic(Y), resulting in spermatogenesis disorder, and potentially azoospermia. Willis et al. [47] demonstrated that most individuals diagnosed as idic(Y) prenatally, will be male with a normal phenotype. However, affected individuals may be at risk for growth deficiency, developmental delay, and infertility [43–44]. We detected a breakpoint on Y(q11.2) and a large deletion spanning many genes in case 3. Some of these genes, including PRY, HSFY1, and DAZ, are strongly linked to male infertility [48, 49]. Specifically, the HSF family members, HSF1 and HSF2, are likely to play important roles in human spermatogenesis as some azoospermia patients harbor deletion of HSFY on AZFb [49, 50]. In fetuses with 45,X/46,X idic(Y) karyotype, the 45,X cell line may play a more significant role in sexual differentiation, independent of the percentage of abnormal Y cell lines in the prenatal sample [47]. However, to accurately predict its future phenotypes, long-term longitudinal follow-up studies on the fetus carrying identical karyotypes are necessary. Given that fetus 3 had a high risk for gonadoblastoma and infertility, after adequate genetic counseling and informed consent, the pregnancy was terminated.

In conclusion, phenotype–karyotype correlations of sSMCs pose a major challenge in prenatal diagnosis. Combined use of cytogenetics, FISH, and CMA can clarify the origin and pathogenicity of sSMCs, which is not only helpful to characterize sSMCs formation mechanisms and genetic effects, but also has important clinical significance for fetal genetic consultation, treatment selection, and ultimately, parental decision making.

Case presentation

Case 1

A 37-year-old, gravida 4, para 1, pregnant woman underwent amniocentesis at 19⁺¹ weeks of gestation due to advanced maternal age. She and her husband were healthy and nonconsanguineous, and they had a 9-year-old healthy son. There was no family history of congenital malformations. Prenatal ultrasound examination at 20 weeks of gestation revealed fetal right dominant heart, ventricular septal defect, aortic dysplasia, left ventricular punctate echogenicity, and single umbilical artery (Table 2). No other abnormalities were observed.

Case 2

A 27-year-old, gravida 2, para 0, pregnant woman was referred for amniocentesis at 18⁺⁴ weeks of gestation for a positive first-trimester maternal serum screening for Down syndrome (pregnancy-associated plasma protein A: 524 mU/L[0.365 MOM (0.45–2.0)], and free β-human chorionic gonadotrophin (hCG): 147 ng/mL[1.42 MOM (0.25–2.0)), screening was suggestive of increased risk for trisomy 21 (1/198). Cytogenetic analysis showed a 45,X mosaicism karyotype (46,X,+mar[32]/45,X[68]). After adequate genetic counseling, the parents of fetus 2 consented to undergo percutaneous umbilical blood sampling. Cytogenetic analysis using umbilical cord blood showed mosaicism 46,X,+mar[22]/45,X[20]. The pregnancy was uncomplicated. There was no family history of congenital anomalies. A second ultrasound examination at 22 weeks of gestation revealed patent foramen ovale and aortic stenosis (Table 2). Normal female genitalia were observed sonographically.

Case 3

A 30-year-old, gravida 2, para 1, pregnant woman was referred for amniocentesis due to abnormal triple marker screening in the second trimester, screening suggestive of a high risk of Down syndrome 1:204. Maternal serum uE3 was 3.97 nmol/L(0.56 MOM), α-fetoprotein was 43.3 U/mL (0.93 MOM), and free β-hCG was 32.2 ng/mL (2.62 MOM). Amniocentesis was performed at 18⁺¹ weeks of gestation. There was no other relevant family history and the couple was nonconsanguineous. A detailed fetal ultrasound at 22⁺⁵ weeks of gestation revealed fetal tricuspid regurgitation, as well as a broad inner diameter of the right pulmonary artery. No other malformations were detected (Table 2).

Chromosomal karyotyping

Amniotic fluid or fetal cord blood sample was obtained according to the invasive procedure protocol. Routine cytogenetic analysis by G-banding (C/NOR-banding when necessary) technique was performed according to standard laboratory protocols and ISCN 2016. Briefly, 20 mL of amniotic fluid was collected and subjected to in situ amniocyte culture. Parental blood samples were also collected for cytogenetic analyses. Amniocyte in situ culture and harvest, as well as G-banding were performed. Fifteen primary colonies were examined. If the available number of the primary colonies was fewer than 15, a total of 20 cells from both primary and trypsinized cultures were examined.

Single nucleotide polymorphism arrays

SNP arrays constitute one type of CMA technology capable of detecting genome wide CNVs. Here, a high-resolution SNP array was initially performed on three fetuses carrying sSMCs using an Affymetrix array (CytoScan® 750 K; Affymetrix/Thermo Fisher Scientific, Santa Clara, CA, USA) according to manufacturer’s instructions. The procedure included genomic DNA extraction, digestion and ligation, PCR amplification, PCR product purification, quantification and fragmentation, labeling, array hybridization, washing, and scanning, and the results were analyzed with CHAS software (Affymetrix/Thermo Fisher Scientific) using annotations of the genome version GRCh37. The reporting threshold was set at gains or losses ≥ 400 kb and loss of heterozygosity ≥ 10 Mb. For the interpretation of these results, our local database and the following public database were used: DGV (http://projects.tcag.ca/variation/), Cytogenomics Array Group CNV Database (http://www.cagdb.org/), Database of Chromosomal Imbalance and Phenotype in Humans using Ensembl Resources database (DECIPHER, http://decipher.sanger.ac.uk/), Online Mendelian Inheritance in Man (OMIM, http://www.omim.org). Parental analysis was performed to interpret VOUS when necessary. sSMCs were initially characterized using a SNP array and then were further confirmed by FISH.

Fluorescence in situ hybridization

FISH was carried out on the three cases. The probes were selected based on the CNVs detected by the SNP array. Commercial probes were used to target chromosomes 13/21, 14/22, 15/16, and 18, the chromosome X α-satellite centromere (DXZ1 at Xp11.1-q11.1), the Y α-satellite region (DYZ3 at Yp11.1-q11.1), and BAC clone probe RP11-958H20 at 22q11.1-22q11.2. FISH was performed on interphase/metaphase amniocytes according to the manufacturer’s instructions (Vysis, Downers Grove, IL, USA).

Data availability statement

The data that support the findings of this study are available from the corresponding author.

Statement of ethics

The study was approved by the ethics boards of Fujian Provincial Maternity and Children’s Hospital (No.12, No.11, and No.10), Each patient received written informed consent for participation. All procedures were performed in accordance with the Declaration of Helsinki.