Background

Small supernumerary marker chromosomes (sSMC) are structurally aberrant chromosomes that are equal in size or smaller than chromosome 20 of the same metaphase spread and cannot be detected or characterized clearly by traditional cytogenetic banding alone. Molecular cytogenetic techniques (including array-based comparative genomic hybridization) are required for their characterization because they are too small to be examined for their chromosomal origin using classic banding techniques [1]. In prenatally determined de novo cases with sSMC, the probability of an aberrant phenotype is estimated to be 13%. This has been refined to 7% for sSMC from chromosome 13, 14, 15, 21, or 22and 28% for all non-acrocentric autosomes [2], with a new suggestion of 30% for all sSMC carriers [3]. Recently, familial sSMC has been discovered to be passed primarily through the maternal line [4]. sSMC is scientifically fascinating since their manner of formation, karyotypic evolution, and the fact that their existence can cause chromosomal imbalances (partial tri-, tetra-, or hexasomies) with no observable clinical effects are still incompletely understood [1, 3, 5]. Investigations in the recent decade have demonstrated the genesis of numerous sSMCs using polymorphic short tandem repeat (STR) markers or SNPs in patients. They've shown that sSMCs can be either maternal or paternal in origin [6,7,8]. If the origin is maternal, however, it is the product of meiosis I and II errors [9]. Usually, derivative 22 resulting from non-Robertsonian translocation—t(11;22)(q23;q11.2) is observed running in families and gives rise to an extra chromosome in the probands resulting in Emanuel syndrome [10,11,12]. Palindromic AT-rich regions (PATRR) are found at the breakpoint chromosome 22q11.2, implying that the center of the PATRRs is linked to double-strand breaks (DSBs) that lead to translocation. Various partner chromosomes have also been reported in the previous studies that include chromosomes 3, 4, 6, 8, 9,12, 14,15, 16,17, 19, and X [13,14,15,16,17,18,19]. Here we have reported a novel partner chromosome 20, which resulted in der(22) as sSMC, and present as 47,XY, + der(22)t(20;22)(q13;q11.2)dmat.

Case presentation

A 4 years old boy was referred to the Cytogenetics Laboratory at JSS Hospital, Mysore with a history of developmental delay, low set ears, and facial dysmorphism (Fig. 1). He began sitting at the age of two and stood at the age of three years and six months, but he is still unable to stand independently and completely. The child showed a periauricularpit (Fig. 2) and was unable to freely move his right hand. Downward slanting eyes, medially flared eyebrows, downturned mouth corners, and micrognathia were observed. He was the first alive child of the non-consanguineous healthy couple with 30 years old mother, 40 years old father, earlier they had three spontaneous abortions during the first trimester; in the fifth pregnancy, fetal anomalies were discovered at 14.1 weeks, including hypoplastic nasal bone and nuchal thickness of 2.8 and hence the fetus was terminated. The height, weight, and head circumference of the boy were 90 cm (cms), 11.85 kg(kgs), and 47 cm respectively. He had congenital heart defect with atrial septal defect (ASD), ventricular septal defect (VSD), and central nervous system (CNS) anomalies with the gross cranium.MRI findings show supratentorial chronic infarcts in bilateral parieto occipital and superior frontal lobes, as well as volume loss and lateral ventricle dilation in the watershed territory of the brain.

Fig. 1
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Low set ears

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Fig. 2
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Showing periauricular pit

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Methodology

Cytogenetic analysis

Lymphocytes were cultured from whole blood and stimulated with Phytohemagglutin—M (PHA-M) for 72 h at 37 °C. Colcemid was used to arrest the metaphases in the cultures. The metaphases were isolated using a hypotonic solution(0.075MKCl) and Carnoy's fixative. After GTG banding, chromosomal analysis was performed using Metasystems' Ikaros software.

Fluorescence in situ hybridization (FISH)

Fluorescence In Situ Hybridization was performed on cultured pellet from proband to confirm the derivative chromosome 22 using the BCR/ABL1 FISH probes (Metasystems).

High-resolution chromosomal microarray (CMA)

To ascertain the chromosomal origin and segmental composition of the derivative 22 chromosome, Chromosomal microarray analysis (CMA) was performed using an AffymetrixCytoScan™ 750 K array. The microarray consists of 750 K oligonucleotide probes across the genome, including 550 K unique non-polymorphic probes, and 200 K bi-allelic SNP (single nucleotide polymorphism) probes. Genomic DNA (250 ng) was digested with Nsp1 and ligated using an Nsp1 adapter. Titanium Taq amplified PCR products of size 120 to 2000 bp were purified using AMP pure beads and fragmented to the product size of 25 bp to 125 bp, biotin-labeled, hybridized on CytoScan 750 K gene chip. Data were analyzed using Chromosome Analysis Suite (ChAS) based on the human reference genome (GRCh37/hg 19).

Results

Cytogenetic analysis

The GTG banded metaphases were examined. The patient's karyotype was initially reported as abnormal—47,XY, + mar in all analyzed metaphases (Fig. 3). We advocated for parental karyotype to determine the carrier status of the supernumerary marker chromosome. The mother showed an abnormal female karyotype with a balanced translocation between the long arm of chromosome 20 at q13 and the long arm of chromosome 22 at q11.2—46,XX,t(20;22)(q13;q11.2) (Fig. 4), whereas, father’s karyotype was normal confirming that the mother was a carrier and transmitted the supernumerary derivative 22 chromosome to the child. Thereafter, the karyotype of the child was considered to be 47,XY, + der(22)t(20;22)(q13;q11.2)dmat.

Fig. 3
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Karyotype of the patient—47,XY, + der(22)t(20;22)(q13;q11.2)dmat

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Fig. 4
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Karyotype of the mother—46,XX,t(20;22)(q13;q11.2)

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Fluorescence in situ hybridization (FISH)

We used a BCR/ABL1 dual fusion probe with an orange probe hybridizing to the ABL1 gene region at 9q34.1 and a green probe hybridizing to the BCR gene region at 22q11.2. Metaphase revealed three green signals indicating that sSMC is corresponding to chromosome 22q11.2 (Fig. 5).

Fig. 5
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FISH showing 3 green signals (BCR gene on 22q11.2)

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Chromosomal microarray (CMA)

The cytogenomic microarray analysis revealed a 10.5 Mb gain involving chromosome 22 within cytoregion 22q11.2q12.1, indicating trisomy of this region (Fig. 6 and 7). There are 110 OMIM genes in this region. Developmental delay, intellectual disability/learning disability, and congenital heart anomaly are all symptoms of chromosome 22q11.2q12.1duplication syndrome. The majority of the genes are linked to the DiGeorge Syndrome/Velocardiofacial Syndrome genes.

Fig. 6
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Karyoview

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Fig. 7
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Detailed view_22q11.2q12.1gain

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KaryoviewCNVs6

See Figs. 6 and 7.

Discussion

Approximately 9% of all sSMCs arise from chromosome 22 [17].The supernumerary-der(22) syndrome has been thought to be due to the error in the meiosis. The widely known recurrent non-Robertsonian translocation in humans is the result of 3:1 meiotic malsegregation in carriers of the constitutional t(11;22)(q23;q11.2) translocation [20].In around 99% of cases, the extra derivative 22 chromosome rises during 3:1 meiotic malsegregation in one of the parents [11]. Usually, supernumerary derivative 22 chromosome is resulting in parents who had t(11;22)(q23;q11.2). This chromosomal rearrangement is a very common non-Robertsonian translocation and the carrier of the proband's derivative 22 chromosome.

Female carriers of the t(11;22) have a nearly 4% chance of having children with the supernumerary derivative chromosome 22 syndrome, while male carriers have a 0.7 percent chance [21]. The palindromic AT-rich regions (PATRR) have been located at both breakpoints of chromosome 11q23 and chromosome 22q11.2 [14, 16, 18]. Chromosome 22 at q11.2 breakpoint is involved in translocations with other partner chromosomes 4, 6, 8, 9, 12, 14, 15, 16, 17,19 and X with translocations at t(4;22)(p15.2;q11.2),t(6;22)(p22.1;q11.2), t(8;22)(q24.13;q11.2),t(8;22)(p22;q11.21), t(9;22)(p13;q11.2), t(12;22)(p12-p13.3;q11.2), t(14;22)(q31;q11.2), t(15;22)(q26.1;q11.21), t(16;22)(p13.3;q11.21), t(17;22)(q11;q11.2), t(19;22)(q13.42;q11.2) and t(X;22)(p22.3;q11.2) respectively [13, 14, 16, 17, 23,24,25,26,27,28,29,30,31]. In addition, another chromosomal rearrangement t(3;22)(q28;q13.3) is resulting into sSMC other than break point 22q11.2 [15]. Previously reported case shows sSMC resulting due to paternal uniparental disomy of chromosome 22 [32]. Present finding t(20;22)(q13;q11.2) is the new partner chromosome mediating to form supernumerary derivative chromosome 22 in offspring. Several clinical features of the patient overlap with those of the Emanuel syndrome. Overdosage of genes on the derivative 22 chromosome affects clinical features such as facial dysmorphism, developmental delay, micrognathia, congenital heart defect, and CNS malformation.

Conclusion

The clinical aspects of derivative 22 chromosome syndrome overlap with those of Emanuel syndrome, regardless of carrier chromosomal rearrangement. Most of the supernumerary der(22) chromosome is forming due to any one of the parents being the carrier. A non-Robertsonian translocation involving 22q11.2.A translocation had occurred in the mother between chromosomes 20 and 22 in our case. The parental karyotype analysis was effective in identifying the carrier of the supernumerary der(22).To minimize future pregnancies with defective progeny, prenatal karyotyping is essential.