In the current survey, we collected 16 sSMC prenatal samples, with an overall frequency of 0.0787%, which is close to that of other research [1, 3]. The origins of the sSMCs among our cases included acrocentric chromosomes (1/16), nonacrocentric chromosomes (4/16) and no Y chromosomes, significantly different from other research by Leda Dalpra et al. and Bing Huang et al. [13, 14]. This may be due to the small sample size of our study.
Reportedly, sSMCs deriving from acrocentric chromosomes with chromosome 15 are the most common type ; however, there was only one acrocentric chromosome with chromosome 22 in our study. This may be due to the limited number of sSMC cases we examined, and six of them lacked SNP or CNV-seq tests.
For case 7 to 11, the sSMCs contained negative genomic contents. The reason for the negative results was uncertain, but it may be due to sSMCs being derived from an acrocentric chromosome and containing only centromere heterochromatin without euchromatic regions.
For Case 12, the gain in the 9p12p21 region was evaluated as a variant of unknown significance. Supersonic ultrasound examination showed that the fetus was normal throughout the whole pregnancy. The baby grew normally and is at present a 2-year-old girl without any abnormalities. Therefore, we declare that the sSMC with a gain of approximately 10 Mb of the 9p12p21 region is benign. For Case 13, the CNV-seq result showed a triple-dosage increase of 18p11.32p11.21. This repetitive fragment encompasses 64 coding genes, of which 56 are OMIM genes, and there is insufficient evidence for triplosensitivity. Case ID  is included in the DECIPHER database with a repetitive fragment slightly smaller than 18p11.32p11.21, and the main clinical symptom was central hypotonia. In Case ID , the main clinical symptom was pathogenic. The DECIPHER database contains many cases that involve slightly larger repeats than the repeated fragment in case 14. The clinical manifestations included abnormal auricle, abnormal EEG, arched eyebrows, intellectual disability and multiple other malformations    (likely pathogenic). The DGV database does not include reports of 18p11.32p11.21 duplication. Comprehensive analysis suggests that 18p11.32p11.21 is likely pathogenic.
For Case 14, there was a duplication of 9.26 Mb at the 12p13.33p13.31 region (spanning 100 OMIM genes). Case ID , with similar repeated fragments, is included in the DECIPHER database and likely pathogenic (de novo constitutive). For case ID , clinical manifestations included EEG abnormalities, intellectual disability, prominent fingertip pads, ptosis, short nose, and type I diabetes mellitus. The DGV database does not include reports of 12p13.33p13.31 duplication. According to comprehensive analysis, the clinical significance of 12p13.33p13.31 gain was evaluated as a variant of unknown significance. However, the woman chose termination of pregnancyif the baby had any abnormality. We could not obtain definite clinical significance.
For Case 15, the karyotype was mosaicism 47,XN,+mar/46,XN. SNP array revealed a duplication of 8.9 Mb in the 9p21.1p13.1 region in 40% of the fetal cells, with the duplicated chromosomal section containing 68 OMIM genes, including ACO1, ALDH1B1 and APTX. In addition, there was insufficient evidence for triplosensitivity. The gain was evaluated as a variant of unknown significance. There were no abnormal ultrasonic manifestations throughout the whole pregnancy. The family continued the pregnancy, and the baby is now approximately 3.5 years old, with normal intellectual and physical development. Therefore, in our study, we could state that this gain is likely benign.
For Case 16, the SNP array detected a duplication of 1.8 Mb at the 22q11.1q11.21 region in fetal cells. The duplicated chromosomal section, that is, the sSMC, contained the cat eye critical region (CECR) (Type I) on chromosome 22q11.21, which is definitely pathogenic.
The effect of sSMCs commonly depends on their origin, size, content, structure and proportion of mosaicism, as well as the amounts of euchromatin . Others have reported that the risk of an abnormal phenotype may be associated with the size of the euchromatin region (even less than 1 Mb) or the number of genes (even less than 10). Nevertheless, when sSMCs contain some euchromatin fragments that are inherited from a healthy parent, we usually predict that they are likely benign. As the morphology of sSMCs is highly heterogeneous, the genotype-phenotype correlation is exceedingly problematic in diagnostics.
Up to day ten sSMC-specific syndromes were described in literature [18-21]. In our study we could identify two of them: tetrasomy 18p syndrome (Case 13) and Cat eye syndrome(Case 16). Tetrasomy 18p is a rare disorder. The frequency of tetrasomy 18p is 1/140,000 in live newborns , which is the most common type among all these isochromosomes . Their clinical characteristics include moderate to severe intellectual disability, developmental delay, microcephaly, typical dysmorphic features, and other anomalies, such as muscle tone abnormalities [24, 25]. Cat eye syndrome (CES) is a rare developmental disorder with an incidence of 1/150,000 to 1/50,000 in liveborn infants. CES occurs with a bisatellite-dicentric sSMC(22), resulting in trisomy or partial tetrasomy of chromosome 22. The clinical presentations of CES include high forehead, downslanting palpebral fissures, epicanthus, microphthalmia, cataract, and strabismus. Intellectual deficits, congenital heart defects and renal malformations may be involved in some severe cases [28-30].
It has been assumed that de novo sSMCs, particularly those with UPD, result from incomplete trisomy rescue. However, to determine whether UPD coincides or is a consequence, further study should be performed. As chromosomes 6, 7, 14, 15, 16, and 20 contain imprinting genes, UPDs of these chromosomes are most commonly reported in sSMC cases . When sSMCs contain only heterochromatic regions, it is necessary to exclude whether an imprinted chromosome is involved in UPD . This would be necessary due to the prognostic outcome, even though we have not found UPD in cases analyzed by SNP-array (cases 15 and 16). Other cases (7-14) in our study were analyzed by CNV-seq which is not capable to detect UPD and this represent shortcoming in our research.Mosaic conditions were present in 5 cases (29.4%), which was slightly lower than that reported in 2005 . At the same time, the sSMC cell line level varied from 4% to 74%, similar to previous literature .
Other researchers found that mosaicism is the reason for the normal or mild clinical manifestations in carriers of sSMCs with well-defined syndromes. No clinical manifestation is too expected in low-mosaic samples . In our study, five of 16 cases involved mosaic sSMCs(Case 1, 3, 10,12 and 15). The children of Case 10, 12 and 15 are with normal phenotypes up to now. The mosaicism level of Case 12 was 70%, and the sSMC results in a normal phenotype. Similarly, in Case 16, duplication of the DNA sequence was approximately 8.9 Mb. Most likely involving mosaicism, the child is approximately 3.5 years old without any adverse clinical signs. Our results seemingly confirm the previous conclusion that mosaicism results in normal or minor clinical signs in carriers of sSMCs. Of course, long term development should be supervised, maybe the CNVs effects of these duplication regions has not been shown.
SNP array or CNV-seq enables describing the genetic content of sSMCs, making it easier for genetic counselors to establish genotype-phenotype correlations. Therefore, it is an outstanding technique and will be implemented in prenatal genetic diagnostics.
Notably, detailed molecular characterization of sSMCs might contribute to precise prenatal counseling. There are some limitations in the current study. First, consequent CNV-seq analyses of fetal tissues after termination of pregnancy were not conducted. Second, in Cases 8-11, CNV-seq analyses could not exclude the probability of UPD on the initial chromosome. Third, we could not obtain karyotyping using peripheral blood or other body tissues in Cases 12 and 15.
In conclusion, our research emphasizes the combination of traditional cytogenetic and further molecular cytogenetic methods in the characterization of small marker chromosomes, which might facilitate a better understanding of the relationships between sSMCs and resulting phenotypes.