Prenatal diagnosis of a 16p11.2p11.1 mosaic small supernumerary marker chromosome (sSMC)

Background. Small supernumerary marker chromosome (sSMC) is a challenge in prenatal diagnosis. Its’ presence is associated with advanced maternal age and distinct ultrasound findings, prediction of postnatal clinical consequences and prenatal counselling is difficult. Exact characterization of an sSMC is augmented by the application of novel molecular methods such as fluorescent in situ hybridization (FISH) and array comparative genome hybridization (aCGH). Case presentation. Chorion villous sampling of a fetus of a 42-year old secundipara was carried out due to advanced maternal age and the conventional banding cytogenetic examination revealed a mosaic sSMC. Detailed prenatal ultrasound scan showed no fetal malformations. The karyotype from confirmatory amniocentesis was 47,XY,+mar[45]/46,XY. The level of mosaicism was 70% from chorionic villus and amnion cells as well. By using FISH and SNP based aCGH the exact origin of the marker was described and the final prenatal karyotype was determined as The increase in DNA dosage was 4,29 Mb affecting 20 genes with two OMIM ones ( ZNF267 and TP53TG3 ), the SNP analysis excluded the possibility of uniparental disomy (UPD) of the chromosome. The application of the molecular cytogenetic methods allowed us the differentiate the mosaic marker chromosome from the known 16p11.2 duplication syndrome in close neighboring that is connected to developmental delay and autism spectrum disorder. The present case was identified as a harmless de novo euchromatic variant (EV) of the short arm of chromosome 16. Postnatal karyotyping from neonatal peripheral blood confirmed the presence of prenatally of

4 the marker. FISH analysis with probe D16Z2 on cultured lymphocyte and buccal smear samples revealed a 64% and 45% mosaic state of the sSMC, respectively. The precise karyotype was finally defined as 47,XY,+min (16) Conclusions. Novel microarray methods combined with molecular cytogenetic analysis is particularly effective in the rapid and accurate diagnosis of sSMCs, especially in a prenatal situation when the exact characterization of a genomic imbalance is of utmost importance. Precise diagnosis facilitates proper genetic counseling, allows informed decision making and helps avoiding unnecessary pregnancy termination. background Small supernumerary marker chromosome (sSMC) is a structurally abnormal chromosome that cannot be clearly characterized by conventional banding cytogenetics and is equal or smaller in size than a chromosome 20 of the same metaphase spread [1]. Supernumerary marker chromosomes occur at a rate of 0.44 per 1000 newborns and at 0.72-0.75 per 1000 fetuses in prenatal settings possibly caused by higher rate of spontaneous loss and termination of pregnancy (TOP) [2,3].
The incidence of sSMCs has been estimated higher in prenatal cases with ultrasound anomalies [2], but the most frequent reason leading to the prenatal identification of an sSMC is advanced maternal age (AMA) [4]. Clinical phenotypes associated with a marker chromosome are highly variable from normal to severely affected patients.
It is generally accepted that about 70% of de novo sSMC carriers are phenotypically normal and less than one third of the patients show clinical symptoms [2]. The risk 5 of phenotypic abnormalities associated with an sSMC depends on many factors, including the chromosomal imbalance, the mode of inheritance, the chromosomal origin, the morphology, content and structure of the marker and the imprinting effects of uniparental disomy (UPD) [5]. The vast majority of marker chromosomes originate from acrocentric chromosomes, almost 30% from chromosome 15 [1].
Approximately two thirds of sSMCs are de novo and a significant proportion of them are mosaics [6].
Proper genetic counselling of a de novo sSMC remains a challenge for clinicians and it is especially true in a prenatal situation when the clinical outcome is uncertain and the time that is necessary for the genetic diagnosis is of utmost importance.
Except for certain sSMCs when the outcome is well established (such as isochromosome 12p-Pallister-Killian syndrome, tetrasomy 18p, i(5p), i(9p)) [7], accurate characterization of the marker chromosome is mandatory. The presence of a sSMC is usually identified by G-banding, however in most cases application of molecular cytogenetic techniques is necessary for proper diagnosis. Fluorescence in situ hybridization (FISH) based methods have been considered as the gold standard for decades in the evaluation of the origin of the marker [8,9]. To overcome the limitations of FISH (such as accuracy or resolution) array comparative genome hybridization (aCGH) has been applied in the diagnostic of sSMCs that can also determine the origin of the marker chromosome, moreover can also detect the exact size, the breakpoints, the genomic copy number changes and the genes involved [10,11].
Here we report a case of a prenatally diagnosed case of mosaic sSMC derived from chromosome 16 and characterized by molecular cytogenetic methods.

case presentation
The 42-year-old gravida (G:2, P:1) was referred to our prenatal center at 13 weeks 1 day of the gestation because of advanced maternal age (AMA). First trimester ultrasound scan showed normal fetal anatomy, nuchal translucency fell into normal range (NT:1.65 mm), nasal bone was visible, crown-rump length (CRL) was 70 mm,    [36]. In the evaluation of the final karyotype we took into consideration the notions of Liehr regarding the nomenclature problems of ISCN in the definition of sSMCs [12].

discussion and conclusion
Prenatal identification and characterization of an sSMC and proper genetic counselling is a challenging task for clinicians. Although it is regarded that about 30% of all de novo sSMC carriers have associated abnormalities, prenatal prediction of the phenotypical consequences is problematic. The first study evaluating the overall risk for an abnormal phenotype in case of a de novo sSMC was based on a large number of amniocentesis samples and found it to be 13% prenatally [13]. By utilizing molecular genetic methods such as FISH, Crolla et al. confirmed that, excluding chromosome 15 derived markers, the risk increased to 28% [14]. A study of 108 prenatally detected cases of marker chromosomes collected from 12 laboratories found that the risk for phenotypical abnormality was 26 % and it was reduced to 18% when the prenatal ultrasound was normal [15]. They reported the highest chance of abnormalities in case of a ring chromosome probably due to the largest amount of genetic material necessary to form the ring appearance. The risk was significantly reduced when the marker appeared in a monocentric and nonsatellited form, while it was higher with bisatellited dicentrics, acentrics or isodicentrics.
It seems that somatic mosaicism is often associated with sSMCs. Chromosomal mosaicism is one of the main difficulties in prenatal diagnosis. It represents the phenomenon of the presence of two or more chromosomally different cell lines in an individual arising from a single zygote. The main mechanism of mosaicism forming an sSMC involves the maternal meiosis I. or II. chromosomal non-disjunction error followed by incomplete trisomy rescue in the dividing pre-implantation embryo [16].
It is predicted that meiosis II. segregation errors occur more frequently then meiosis I. errors and it is strictly connected to AMA. It has been established that ovarian aging is the most important factor not only in aneuploidies but in the formation of de novo sSMCs as well [17]. Chromosomal mosaicism in CVS and in amniocytes is well-known and occurs in 1-2% of CVS and 0.1-0.3% of amniocentesis samples [18].
The differentiation of the cells and the tissues begins at the early post-fertilization stage. The distribution of normal and abnormal cell lines in the fetus and the placenta depends on the stage and the mechanism of the differentiation. When trisomy rescue occurs soon after fertilization, the mosaic formation regards both placental and fetal tissues, when it occurs at a later stage (following the separation of the fetal and the placental compartments), the aneuploid cell line can be confined to the placenta, to the fetus or both. The prenatal study by Graf et al.
reported a total of 61% rate of mosaicism in sSMC and found no difference between the groups with or without phenotypic abnormality [15]. According to a large review that studied 3124 sSMC cases previously reported in the literature, the authors found 52% overall rate of somatic mosaicism. Non-acrocentric derived sSMCs were more involved in mosaicism. The authors emphasized that in the vast majority of the cases there was no correlation between the grade of somatic mosaicism detected in the peripheral blood or in amnion cells and the severity of the clinical status [19]. In a recent survey of 143.000 consecutive prenatal diagnosis the frequency of overall mosaic sSMC was 69% and the risk of confirmation in amniotic fluid following mosaic CVS result was 33.3%, suggesting a high rate of confined placental mosaicism (CPM) [3]. The main indication for the invasive procedure was AMA and ultrasound anomaly. It seems that sSMCs derived from chromosome 16 are relatively rare and it was found that 91% were mosaics [19]. sSMCs can be associated with uniparental disomy, either in complete or segmental forms, as a result of trisomic zygote rescue [4,5]. Mosaic trisomy with UPD occur at a significantly high frequency from chromosome 16. However, chromosome 16 does not seem to be involved in imprinting mechanisms with clinical consequences.
According to GRCh38/hg19 chromosome 16 has a size of 90.4 Mb. The proximal short arm of the chromosome contains a several copy number variation (CNV) hotspots that predispose to deletions and duplications. The chromosome 16p11.2 duplication syndrome (OMIM 614671) represent a continuous gene duplication syndrome with genomic coordinates (GRCh38:28,500,000-35,300,000). The typical region is an approx. 600 kb genomic duplicate/ deletion from 29,5-30,1 Mb associated with developmental delay and obesity [20]. More distant starting from the centromere is a large microscopically visible region of 8-9 Mb in 16p11.2-16p12.1 that was reported with developmental delay and autism spectrum disorder (ASD) [21,22]. Although most affected patients show different dysmorphic features, mental retardation and behavioral problems, the wide range of phenotypical spectrum refers to incomplete penetrance and variable expressivity of these genomic abnormalities [23]. Moreover, while patients with a deletion of that region has severe obesity besides developmental delay, affected individuals with a duplication are characterized by reduced postnatal weight and low BMI [24]. Thus, it seems that the phenotypes of the duplication carriers mirror those of the deletion carriers [25]. From centromere to telomere, the proximal part of the short arm close to the heterochromatic region is prone to CNV formation. The whole region can undergo duplication together with the heterochromatic blocks forming visible, unusual G-banding pattern [26]. The centric euchromatic region of chromosome 16 is in close proximity to the large block of heterochromatin and this centromere-near region colocalizes with an euchromatic variant (EV) [27]. The EV that is mapped to 14 However, one fetus (case 16-U-39) was diagnosed by amniocentesis with a mosaic marker forming a ring of the proximal short arm (r(16)(::p12.2→p11.2::)), that resembles the most in terms of the chromosomal region. The data of the clinical symptoms are not available. Albeit, the distal breakpoint of that sSMC was involved in 16p12.2 and the shape of the marker was a ring chromosome. It is also important to emphasize that supposedly the sSMC cases without clinical consequences are less likely reported in the literature.  [32]. It is demonstrated that ZNF267 mRNA is up-regulated in liver cirrhosis and may be a risk factor for hepatocellular carcinoma [33]. Tp53 target gene 3 (TP53TG3) was mapped to the proximal short arm of Chr16 and is located at GRCh38/chr16:32,673,518-32,676,128 [34]. It is one of the numerous TP53 genes, those transcription factors that are involved in cell cycle arrest, apoptosis, DNA repair, chromosomal stability, and inhibition of 15 angiogenesis. The TP53TG3 gene has no proven phenotypic gain of function effects described so far in the databases. The function of the other 18 genes in that region remains unknown.
Exact identification of an sSMC is especially important in a prenatal situation and the time necessary for the diagnosis is of great importance. Utilization of a clear algorithm and diagnostic protocol is a valuable tool in the management of a prenatally detected sSMC and can prevent the unnecessary delays during the diagnostic process [35]. Regarding our case, the characterization of the marker chromosome before the molecular genetic era would not have been carried out properly and in many instances that pregnancies would have been terminated. FISH with whole chromosome paints was very useful in the analytical processes to give information about the origin of sSCMs and might predict the euchromatic content of the markers. [36]. However, precise genotype-phenotype correlation can only be determined via chromosomal microarray technology. By applying SNP microarray analysis, we could exclude UPD, determine and specify the gene content and the region to be a harmless EV block. Our statement was also strengthened by the notion that the detailed fetal and fetal cardiac ultrasound examination did not confirm any malformation [16]. The child now is two months old and has not shown any sign of somatomental retardation or dysmorphic feature. However, we emphasize that those clinical symptoms such as developmental delay or ASD can manifest later in life. It is important to note that another prenatal case of a 16p copy number variation was diagnosed by our team recently. Genetic analysis of a fetus showing mild bilateral ventriculomegaly and partial dysgenesis of the corpus callosum (by ultrasound and MRI) at pregnancy week 20 revealed normal male karyotype and a microdeletion of 1.385 Mb of the short arm of Chr16 by aCGH 16 (Chr16:32,542,904-33,928,095). The genomic position of that microdeletion is inside the region of the child with the sSMC in the present case. The association between the CNV and the corpus callosum dysgenesis is uncertain, the pregnancy is ongoing now (unpublished own data, Figure 2.).
In summary, we present prenatal diagnosis and molecular cytogenetic characterization of an sSMC. Exact identification of the marker chromosome as an EV enabled us to provide proper genetic counseling, to allow informed decision making and to avoid the unnecessary pregnancy termination. ZT performing ultrasound, genetical counselling, invasive procedures and writing the manuscript. EPT genetical counselling, cytogenetic and data analysis, writing the manuscript. IB and ES literature research and data analysis. HP carrying aCGH and writing the manuscript. AB, JS, JK, VG and JD revision of the manuscript. All authors reviewed and approved the manuscript.

Funding
There were no sources of funding for this study.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
This study was performed with the approval of Medical Ethics Committee of the Institution.

Consent for publication
The patient in this report provided their consent for publication.

Competing interests
The authors have no conflict of interest to declare.