Interphase or nuclear in situ hybridization (nuc ish) on uncultured amniocytes revealed nuc ish(D21S342/D21S341/D21S259 × 1). Hybridization with a DNA probe directed at chr 21q22.13-q22.2 produced one signal (Fig. 1). The absence of one signal for chr 21 may indicate either a complete loss of chr 21 (monosomy 21), or a deletion/unbalanced translocation involving chr 21q22.13-q22.2.
Figure 1 FISH analysis using probes designed to detect the region 21q22.13-q22.2 on cultured amniocytes showed loss of one signal for chr 21 or on 21q22.13-q22.2.
Chromosome analysis on cultured amniocytes shows the presence of two abnormal cell lines: mos 45,XX,-21/46,XX,del(21)(q22)dn; see karyotype (Fig. 2). Of the 30 cells examined from four independent cultures, 10 cells showed one copy of chr 21 (monosomy 21), and 20 cells had 46 chromosomes with a loss of the distal part of the long (q) arm of chr 21 at band q22. Parental chromosome analyses showed normal karyotypes. The parents did not have a rearrangement involving 21q22 and thus the deletion in this prenatal specimen was apparently de novo (dn) in origin. The abnormal cell lines were observed in four independent cultures and fit the definition of true mosaicism.
Figure 2G-banded karyotype of the fetus showing the full and partial monosomy of chr 21 in two different metaphases.
DNA oligo-probes and SNP microarray analysis on uncultured amniocytes from amniotic fluid showed a pathogenic partial terminal deletion of 10 Mb on the long arm (q) of chr 21 (Fig. 3). This result confirmed the FISH findings of non-mosaic loss of 21q22 with no evidence of complete loss of chr 21.
Figure 3 Chromosomal SNP microarray using DNA from uncultured amniocytes showing a terminal deletion of chr 21q22.12q22.3.
The identified deletion is described as arr[GRCh37] 21q22.12q22.3(36285036_48090317)x1. Current evidence obtained from reputable databases and peer-reviewed literature indicated that this deletion is causative of partial chr 21q monosomy.
Follow-up microarray analysis on cultured amniocytes showed mosaicism for a full chr 21 deletion and the partial chr 21q deletion (Fig. 4). The ISCN of the microarray is arr[GRCh37] 21q11.2q22.12(15412676_36272993)x1 ~ 2,21q22.12q22.3(36431283_47612400)x1. This result confirmed the karyotype finding of cultured amniocytes.
Figure 4 Chromosomal SNP microarray using DNA from cultured amniocytes showing the full and terminal deletion of chr 21q22.12q22.3. The B-allele frequency (BAF) representing the mosaic nature of this finding.
In this case report the fetus was first screened with NIPT which returned no indication of aneuploidy for the target chromosomes of that analysis. Ultrasonography at 18-weeks of gestation revealed a duplication of SVC. Following a subsequent amniocentesis, the fetus was determined to have mosaicism for both partial and full chr 21. The parents’ karyotypes were normal, suggesting this chromosomal 21 anomaly was de novo and the mosaic pattern was apparently of post-zygotic origin. Although the mother was 37 years old and thus considered to be of advanced maternal age, increased maternal age is not in itself an effective screen for aneuploidy . In contrast, structural chromosomal abnormalities, including microdeletions and microduplications, do not increase in frequency with maternal age . During genetic counseling the parents were informed about the recurrent risk of future pregnancies along with laboratory test artifacts and nondisjunction risk. The patient terminated the pregnancy following the abnormal molecular and cytogenetic results due to the possible challenges the baby would face if carried to full term or live birth.
Full monosomy of chr 21 is rare and most often is lethal in intra-uterine life. However, partial monosomy 21 is even rarer and only a few patients have been reported in the literature . Correlations between clinical phenotype and genotype were hard to determine due to the variability of reported deletion breakpoints . Neonates with partial 21q deletions display multiple dysmorphic features at birth such as cardiac, pulmonary, renal, skeletal and genitourinary abnormalities. These infants often fail to thrive and if they did survive, they displayed intellectual disabilities, congenital malformations of the heart and several other physical disabilities and physiological disorders.
Previous studies have suggested KCNE1, RCAN1, CLC6, RUNX1 and DYRK1A as candidate genes for congenital cardiac anomalies residing on chr 21 [5–7]. It is highly anticipated that the loss of the chr 21 contributed to the fetal cardiac phenotype of SVC duplication. Lyle et al. suggested that terminal deletions on chr 21q in the range of 5.6–11 Mb show a relatively milder phenotype [12, 13]. The terminal deletion on chr 21q was comparable in size (10 Mb) to what was found in this case. The cardiac defects described for monosomy chr 21 include mostly pulmonary stenosis, patent ductus arteriosus and septal defects . Recently a case was reported of interrupted inferior vena cava with azygous continuation .
Bioinformatics analysis of this region through online databases were also performed. DECIPHER, ISCA and NCBI-dbVar databases were used to identify the loss of copy number variants and its phenotype . There were only a few pathogenic or likely pathogenic variants reported for the chromosomal region we have identified through aCGH (Fig. 5). Most of the patients reported have the terminal deletion of chr 21q. We reviewed only those cases that substantially overlapped with our aCGH findings i.e. 21q11.2q22.12 and 21q22.12q22.3.
To delineate the specific phenotype of SVC duplication relating to chr 21q11.2q22.12 and chr 21q22.12q22.3 deletions we also searched NCBI-PubMed database. There were only 8 patients described in the literature with the condition of “congenital heart defect” with variable 21q breakpoints [5, 6]. However, we could not identify any mosaic patients that resemble the genotype-phenotype findings we investigated here. The present case report is the first to describe superior vena cava duplication as a constituent in the spectrum of cardiac anomalies found in monosomy chr 21. Thus, this clinical feature with the cytogenetics results add to the body of knowledge of understanding the molecular pathogenies of monosomy 21/partial 21q monosomy/21q deletion syndrome.
Figure 5 Bioinformatics analysis of the deleted region at position: chr 21:15412676–48090317. A. DECIPHER CNVs filter as follows: Loss only – pathogenic, Likely pathogenic, all sizes, squish; ISCA filter: Loss only – pathogenic, squish. B. NCBI dbVar showing both gain and loss of pathogenic CNVs (Note: likely pathogenic is the same as pathogenic CNVs – see variant summary counts for nstd37 in dbVar)
The clinical use of NIPT to screen high-risk patients for the most common fetal chromosomal aneuploidies is becoming increasingly common. The American College of Medical Genetics and Genomics (ACMG) and the American College of Obstetricians and Gynecologists (ACOG) recommended NIPT as a screening test for common trisomies (i.e. 21, 18, and 13) and, if requested, sex chromosomal aneuploidies [3, 16]. NIPT screening test does not eliminate the possibility of other anomalies of the tested chromosomes like mosaicism, deletions or duplications . Therefore, USG at an appropriate gestational age should be performed and when fetal anomalies are detected, invasive diagnostic testing with chorionic villus sampling or amniocentesis, depending on gestational age, are recommended to detect those chromosomal abnormalities .
Despite negative NIPT results, the abnormal anatomy scan of the fetus in this report at 18-weeks of pregnancy prompted us to conduct further invasive diagnostic tests. The invasive diagnostic tests we performed were much more informative than the fetal cell-free DNA NIPT test. Results of FISH and aCGH from uncultured amniocytes were in agreement; subsequent karyotyping and aCGH on cultured amniocytes were concordant. These molecular and cytogenetic results reinforced that a single test does not always lead to a decisive diagnosis. The complete monosomy cell line in this case may have represented cell culture artifact; this cannot be ruled out. Accordingly, analysis of uncultured cells from amniocentesis should be the preferred cytogenetic technique. Moreover, during post-test follow-up, genetic counselors should make it clear that these cytogenetic testing artifacts could modify the recurrent risk of certain chromosomal abnormalities.
In conclusion, full and partial monosomy chr 21 is presumed to be lethal during the antenatal period. The fetus identified with this condition required a thorough investigation using a combination of conventional and molecular-cytogenetic techniques to exclude any ambiguity and determine the pathogenesis of the genomic imbalance.