Small supernumerary marker chromosomes (sSMCs) are structurally abnormal chromosomes that cannot be identified unambiguously by conventional cytogenetic analysis alone, usually due to their small size and complex structure [1]. They are characterized as additional centric chromosome fragments, equal to or smaller than chromosome 20 in size on the same metaphase spread [2]. Around 3.3 million humans in the current global population are sSMC carriers, and sSMCs can originate from any human chromosome [3]. sSMCs are found in 0.072-0.075% of prenatal cases and 0.044% of live births, although the rate is increased to 0.288% in intellectually disabled patients and to 0.125% among people with infertility issues [4,5,6]. Defining the phenotype-genotype correlation of sSMCs remains a challenge due to their complex origins and genetic materials. The phenotype of sSMCs is variable (mental and growth retardation, craniofacial and urogenital abnormalities, and cardiac anomalies). It depends on their size, genetic content, and degree of mosaicism, while the presence of uniparental disomy can also influence the associated phenotypes [2,6,7,8]. sSMCs composed predominantly of heterochromatic material are often associated with normal development, while those containing euchromatic, gene-rich regions may lead to congenital anomalies or developmental delay. Remarkably, only 30% of sSMC carriers manifest clinical symptoms [4,9]. The application of modern prenatal diagnostic techniques such as chorionic villus sampling (CVS), amniocentesis (AFT), and molecular cytogenetic methods, including fluorescence in situ hybridization (FISH), chromosomal microarray analysis (CMA), and next-generation sequencing (NGS), has improved the characterization of sSMCs. Markedly, a combination of CMA and FISH or NGS and FISH is often required to determine the chromosomal origin of an sSMC, identify the genes involved, and predict the potential phenotype [10,11]. Mosaicism adds a layer of uncertainty: low-level mosaic sSMCs detected in prenatal CVS samples may reflect true fetal mosaicism, confined placental mosaicism, or technical/biological sampling variability, and the fraction of abnormal cells in amniotic fluid or CVS often does not predict tissue distribution after birth. Additionally, the tissue distribution of the abnormal cell line (fetal vs. placental, organ-specific), and the genetic content of the sSMC (heterochromatic vs. euchromatic, gene-containing segments) are very important for predicting the phenotypic outcome [12,13]. Low-level mosaicism (commonly described in the literature as <10-20% of cells in the tested sample) is often associated with favorable outcomes, particularly when the marker is predominantly heterochromatin; however, exceptions exist, especially when gene-rich regions are involved. Prior studies of feto-placental mosaicism further highlight the complexity of genotype-phenotype correlations in prenatal sSMCs and the limitations of predicting clinical outcome solely from mosaicism level [12,13,14]. This diagnostic uncertainty complicates both prognostic assessment and genetic counseling, as the phenotypic outcome cannot be accurately predicted based solely on the cytogenetic finding [15]. Given this heterogeneity, comprehensive cytogenetic and molecular characterization together with systematic postnatal clinical follow-up are essential to refine prognosis and provide informed genetic counseling [16,17].

Here, we present a fetus with a low-level mosaic sSMC detected and investigated during prenatal testing. Postnatal clinical follow-up was performed in the newborn boy to assess the persistence and phenotypic consequences of the abnormality. This case highlights the variability in clinical outcomes associated with low-level mosaic sSMCs and underscores the importance of integrated prenatal and postnatal follow-up.

The mechanisms leading to the formation of small supernumerary marker chromosomes (sSMCs) are heterogeneous and remain incompletely understood. Several mechanisms have been proposed, including meiotic nondisjunction, trisomy rescue, postzygotic chromosome mis-segregation, and, in selected complex cases, chromothripsis [19]. However, the underlying mechanism cannot be determined in the present case.

Until today, >7,500 cases of sSMCs have been reported in the literature [18]. In general, sSMCs represent a very heterogeneous group of structural chromosomal abnormalities with highly variable clinical consequences. Common clinical features that are recorded in sSMC(8) cases include developmental delay, mental retardation, intellectual disability, severe hypotonia, attention deficit hyperactivity disorder (ADHD), skeletal abnormalities and atypical facial appearance, in different severities. Multiple factors such as chromosomal origin, gene content, level of mosaicism, and tissue distribution of the abnormal cell line can affect the subsequent phenotype [4,12,20]. According to [18], the triplo-sensitive region in the short arm of chromosome 8 starts between 40.23 and 40.75 Mb. Thus, the sSMC(8) observed here does not appear to include dosage-dependent genes, which could lead to phenotypic abnormalities.

Only a few cases of low-level mosaic sSMCs have been reported prenatally, and their postnatal outcomes range from normal development to mild or more severe manifestations. The overall risk of abnormal phenotypic outcomes in prenatal cases with sSMC presence is estimated at approximately 13% [3].

Prognostic evaluation and following genetic counseling are further complicated by low-level mosaicism, as the percentage of abnormal cells detected in amniocytes or CVS does not necessarily reflect the fetal tissue composition due to biological and sampling variability. Several studies have indicated that mosaic levels below 10-20% often have limited phenotypic expression, particularly when confined to extraembryonic tissues. The same studies show that low-level mosaicism is more often associated with favorable outcomes, particularly when the marker is largely heterochromatic [5,13]. Nevertheless, exceptions in the literature, especially when the sSMC contains euchromatic DNA with known dosage-sensitive genes, emphasize the need for individualized interpretation and thorough genetic counseling [15]. Therefore, both prenatal detection and postnatal confirmation are essential to accurately assess the clinical relevance. Additionally, although uniparental disomy (UPD) should be considered in selected cases with apparently de novo sSMCs, chromosome 8 is not associated with a well-established clinically relevant imprinting disorder. Therefore, additional UPD testing was not considered necessary in the present case.

Chromosome 8-derived sSMCs constitute a small subset of all reported cases (approximately 70 cases in the existing literature) and show diverse clinical presentations depending on the specific pericentromeric segment involved. For example, the involvement of 8q12.2 critical gene has been associated with severe phenotypic characteristics among the reported cases [21]. The pericentromeric regions of chromosome 8 consist mostly of heterochromatin, with limited euchromatin in 8p11-p12 and 8q11.1. Around 18 reported cases involving 8p11.2/q11.21 exist in the literature, with the severity of the clinical outcome relying solely on the precise size and gene content of the duplicated region [2,9,22,23,24].

In the present case, the sSMC was identified through conventional karyotype in 8% of analyzed metaphases (12 out of 150), consistent with a low-level mosaic abnormal cell line. FISH established its chromosomal origin and demonstrated that it arose from the pericentromeric region of chromosome 8, which was mostly heterochromatin and involved only minimal euchromatin. The BAC probe RP11-503E24 (8p11.21; hg19: 42,384,567-42,555,145) hybridized to the marker chromosome, whereas RP13-116A4 (8q11.21; hg19: 48,482,616-48,530,952) did not, indicating that the breakpoint lies between these two loci. Accordingly, based on the FISH mapping results, the retained euchromatic segment is not expected to encompass any currently established dosage-sensitive genes. As expected, CMA did not identify the marker chromosome, most likely because the abnormal cell line (8%) was below the detection threshold of the platform and because pericentromeric regions are suboptimally represented in array-based analyses. This case highlights the complementary role of conventional cytogenetics, FISH, and CMA in the investigation of prenatally detected sSMCs. While CMA is highly effective at detecting clinically relevant copy number imbalances, it may miss low-level mosaic marker chromosomes, making conventional karyotyping and FISH indispensable for their characterization. The subsequent postnatal normal phenotype is in agreement with this genomic low-mosaic result, in combination with the absence of ultrasound abnormalities during pregnancy. Additionally, the newborn did not show any congenital anomalies or dysmorphic features, and early developmental milestones remained age-appropriate during the first nine months of life, supporting the existing literature on the benign outcome of low-level mosaic pericentromeric sSMCs with limited euchromatic content [13]. Nevertheless, continued follow-up is required, as neurodevelopmental disorders associated with chromosome 8-derived sSMCs may become apparent later in childhood.

This case illustrates key challenges in genetic counseling for families when low-level mosaic sSMCs are identified prenatally. Although the combined use of conventional karyotyping, FISH and CMA significantly improves the characterization of prenatally detected sSMCs, each technique has specific limitations and they cannot fully predict the degree of postnatal tissue mosaicism or developmental outcome. Therefore, integrated prenatal and postnatal follow-up remains essential. Systematic neonatal examination and ongoing pediatric follow-up have so far shown no clinical consequences, providing reassurance to the family and contributing valuable evidence to the limited literature on chromosome 8-derived mosaic sSMCs. Continued long-term follow-up remains important to assess later neurodevelopmental outcomes.

This case report is subject to several limitations, including the inherent inability to determine tissue-specific mosaicism postnatally and the lack of whole-genome sequencing-based breakpoint resolution, which may have further clarified the exact euchromatic content of the marker. Nonetheless, the concordance of cytogenetic findings, normal CMA results, and postnatal development strongly supports the benign nature of this specific chromosomal variant.

In conclusion, the combination of conventional karyotyping, FISH, and molecular techniques such as CMA improves the characterization of sSMCs and guides genetic counseling, especially in prenatally detected sSMCs. In prenatal settings, uncertainty regarding prognosis can cause significant parental anxiety; thus, detailed postnatal follow-up and multidisciplinary communication are critical. This report adds evidence to the scientific community supporting that low-level mosaic sSMCs derived from pericentromeric regions-particularly of chromosome 8-are often associated with a normal clinical outcome. The favorable clinical outcome emphasizes the importance of cautious interpretation of such findings and highlights the need for continued data collection to refine genotype-phenotype correlations.