In the current survey, we collected 17 sSMC prenatal samples, and the overall frequency was 0.0836%, which was close to that of other’s research1; 3 Herein, the origins of the sSMC included acrocentric chromosome (1/17), non-acrocentric chromosome (5/17) and no Y chromosome, which was significantly different from others’ research observed by Leda Dalpra et al and Bing Huang et al7; 8. This may due to the small sample size.
Reportedly, sSMC derived from acrocentric chromosome with Chromosome 15 was the most common type9, however, there was only one acrocentric chromosome with Chromosome 22 in our data. This may due to the limited amounts of sSMC cases in our report, and six of them lack of SNP or CNV-seq test.
For case 12, the gain in 9p12p21 region was evaluated as a variant of unknown significance. Whilst supersonic ultrasound examination displayed the fetus was normal throughout the whole pregnancy. The baby grew up and was a 2 years old girl without any abnormalities. So we could declare that the sSMC with a gain of about 10Mb of the 9p12p21 region was benign.
For case 13, the CNV-seq revealed a partial loss of X chromosome, (seq[hg19] 46,XX;
Xp22.33p11.1 (2700000–58560000)x1, Xq25q28 (125100000–154940000)x1). The first part Xp22.33p11.1 had a heterozygous loss of about 55.86Mb. The deleted fragment contains 316 protein-coding genes, including 263 OMIM genes. This deletion covers steroid sulfatase deficiency (STS), which manifests with ichthyose changes and was X-linked dominant inherited. It also contains multiple different dominant inherited genes, among which BCOR gene is related to microphthalmia syndrome OMIM, CDKL5 gene is related to epileptic encephalopathy OMIM. DECIPHER database collected the female cases with similar Xp22.33p11.1 deletion, with DECIPGE ID  (pathogenic), and it showed abnormal heart morphology, mitral valve malformation, heart disease, unilateral ventricle, etc.
In the meantime, the second part Xq25q28 had a heterozygous loss of about 29.84Mb. The deleted fragment encompasses 207 protein-coding genes and 169 OMIM genes. Likewisely, it contains multiple dominant inherited genes. Among them, FMR1 gene is related to Fragile X syndrome, with the OMIM ID. FHL1 is related to reducing body myopathy, with the OMIM ID . To sum up, the comprehensive analysis revealed that Xp22.33p11.1 deletion and Xq25q28 deletion were evaluated as pathogenic. The pregnancy was terminated.
For case 14, the CNV-seq result showed a triple-dosage increase on 18p11.32p11.21. (seq[hg19] 18p11.32p11.21(120000-14980000) x4). This repetitive fragment encompasses 64 coding genes, of which 56 are OMIM genes, and there is insufficient evidence for Triplosensitivity. The case ID  included in the DECIPHER database with a repetitive fragment slightly smaller than 18p11.32p11.21, the main clinical symptom is central hypotonia. And in case ID , the main clinical symptom is Pathogenic. The DECIPHER database contains many cases that are slightly larger than the repeated fragment in case 14. The clinical manifestations includes abnormal auricle, abnormal EEG, arched eyebrows, mental retardation and other multiple malformations (likely pathogenic). The DGV database does not include 18p11.32p11.21 duplicate reports. Comprehensive analysis suggested that 18p11.32p11.21 was likely pathogenic.
The G-banding analysis described the fetus’ karyotype as 47,XN,+mar without mosaicism in case 15. There was a duplication of 9.26Mb at seq[hg19] 12p13.33p13.31(160000–9420000)x3 (spanning 100 OMIM genes). Thus the sSMC originated from chromosome 12. Case ID  of similar repeated fragments is included in DECIPHER database, the phenotype is likely pathogenic (De novo constitutive). For case ID , their clinic manifestation includes EEG abnormality, intellectual disability, prominent fingertip pads, ptosis, short nose, type I diabetes mellitus. The DGV database does not include 12p13.33p13.31 duplicate reports. According to the comprehensive analysis, the clinical significance of 12p13.33p13.31 gain was evaluated as a variant of unknown significance. However, the woman choosed an abortion in case the baby has any abnormality. We could not get the definite clinical significiance.
For case 16, the traditional karyotype analysis showed 47,XN,+mar accounted for 32% of all cultured amniocytes and 68% of cultured cells was 46,XN, that is the karyotype was mosaicism 47,XN,+mar/46,XN. SNP array revealed that there was a duplication of 8.9 Mb at arr[hg19]9p21.1p13.1(30498773–39411673) in 40% of the fetal cells and the duplicated chromosomal section contained 68 OMIM genes including ACO1, ALDH1B1 and APTX. And 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 their pregnancy, and the baby was about 3.5 years old with normal intellectual and physical development. Therefore, in our study we could state that this gain was likely benign.
For case 17, the standard karyotype analysis displayed the fetus with abnormal karyotype of 47,XN,+mar. The SNP array reported that there was a duplication of 1.8Mb at arr[hg19]22q11.1q11.21(16878002–18656495) in the fetus cells. The duplicated chromosomal section, that is the sSMC, contained the cat eye critical region (CECR) (Type I) on chromosome 22q11.21, which was definitely pathogenic.
The effect of sSMC commonly depends on its origin, size, content, structure and the proportion of mosaicism, as well as the amounts of euchromatin10. Others has reported that the risk of an abnormal phenotype could 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 the sSMC contain some euchromatin fragments which are inherited from the healthy parent, we usually predict it as likely benign11. As the morphology of sSMC is highly heterogeneous, therefore the correlation of genotype-phenotype is exceedingly problematic in diagnostics.
Ten sSMC-specific syndromes were introduced in detailed by Hamideh etal in 2015, including Turner syndrome, Marker chromosome 15 syndrome, Emanuel syndrome (derivative 11;22 syndrome), Cat Eye syndrome (CES), Der(22)t(8;22)(q24.1;q11.1) syndrome, Isochromosome i (5p), 9p isochromosome syndrome, Isochromosome 18p (tetrasomy 18p)syndrome Tetrasomy 15qter syndrome (neocentric sSMA), and Pallister-Killian syndrome4. Given the above theory, Turner syndrome (case 13), tetrasomy 18p syndrome(case 14) and Cat eye syndrome(case 17) were involved in our study. Patients with Turner syndrome have variable phenotypes, like short stature, infertility, cardiovascular health issues, neurocognitive and behavioral aspects12; 13. Tetrasomy 18p is a rare disorder. The frequency of tetrasomy 18p is 1/140000 in live newborns14, which is the most common type among all these isochromosomes15. Their clinical characteristics include moderate to severe mental retardation, development delay, microcephaly, typical dysmorphic features, and other anomalies like muscle tone abnormality16; 17. Cat eye syndrome (CES) is a rare developmental disorder with the incidence of 1/150000 to 1/50000 in liveborn infants18. CES is occured with a bisatellite-dicentric sSMC(22) resulting in a trisomy or partial tetrasomy of chromosome 2219. The clinical presentations of CES included high forehead, downslanting palpebral fissures, epicanthus, microphthalmia, cataract, and strabismus. Intellectual deficits, congenital heart defects and renal malformations may be involved in some severe cases20–22.
It has been assumed that de novo sSMC, particularly those with UPD, resulted from incomplete trisomy rescue23. However whether UPD is coincidence or consequence, further study should be performed to explore it. As chromosomes 6,7,14,15,16,20 contain imprinting genes, so UPD of these chromosomes are most reported in sSMC cases24. When the sSMC contained only heterochromatic regions, it is necessary to exclude whether an imprinted chromosome is involved for UPD25. This is necessary, even though, in our experience we have not found UPD cases for case 16 and 17 which were detected by SNP-array. We could not find UPD for cases 7–15 detected by CNV-seq, which is the big limitation in our research.
Mosaic condition was presented in 5 cases (29.4%), which was a little lower than that reported in 20057. At the same time the sSMC cell line level varied from 4–74%, which was similar to what displayed in previous literature7.
Earlier researchers found that mosaicism was the reason for the normal or mild clinical manifestations in carriers of sSMC with well-defined syndromes. In only 2% of cases, there were no clinical signs or much less severe outcomes than expected in low-mosaic samples26. Herein, five cases out of 17 were mosaic sSMC. The mosaic level of case 12 is 70%, and the sSMC contained a gain of about 10Mb with a variant of unknown significance which turned out to be normal phenotypes expressing since the child was about 2 years old with normal mental and physical development. Similarly, in case 16, the duplication of the DNA sequence is about 8.9Mb containing 68 OMIM genes. Most likely due to mosaicism, the child was about 3.5 years old without any adverse clinical signs. Our results seemingly confirmed the previous conclusion that mosaicism would result in normal or minor clinical signs in carriers of sSMC.
SNP array or CNV-seq enable us to detect the origin and actually describe the genetic content of sSMC, and so forth make it easier for genetic counselor to establish the genotype-phenotype correlations, therefore it is an outstanding technique and will be tendency in prenatal genetic diagnositics.
A total of 11 sSMC were successfully detected by SNP array or CNV-seq in our survey, 3 were likely pathogenic, 3 were a variant of unknow significance and 5 were likely benign. In clinical, the outcomes of many cases were difficult to predict, due to their unclear origins, or different proportions of mosaicism and the possibility of uniparental disomy (UPD).
What noteworthy is that detailed molecular characterization of sSMC could contribute to precise prenatal counseling and help precise decision makings of the fetuses with sSMC. Whiles, there are some limitations in the current study. Firstly, the consequent CNV-seq analyses of the fetal tissues after abortion were not conducted. Secondly, the CNV-seq analyses of case 8–11 could not exclude the probability of UPD in the initial chromosome. Thirdly, we could not get the children’s karyotyping of peripheral blood or other body tissues in case 12 and 16.
In conclusion, our research emphasize the combination of traditional cytogenetic and further molecular cytogenetic methods in characterization of small maker chromosome, which could make an easier understanding of the relationships between sSMC and the resulting phenotypes.