Down syndrome is the most common chromosomal disorder associated with intellectual disability, with an estimated prevalence of approximately 1 in 643 live births in the United States [1]. Worldwide prevalence varies considerably, reflecting differences in genetic background, maternal age distribution, access to prenatal screening, and diagnostic reporting systems [2]. In Mali, the true prevalence of Down syndrome remains unknown due to the absence of systematic surveillance and limited access to cytogenetic diagnostic services, although the condition is frequently encountered in clinical practice.

Cytogenetically, Down syndrome results from the presence of an additional copy of chromosome 21 and occurs in three major cytogenetic forms. Free trisomy 21, caused by meiotic nondisjunction, accounts for the majority of cases. Translocation trisomy 21, most commonly involving Robertsonian translocations, represents a smaller but clinically important proportion, while mosaic trisomy 21 is characterized by the presence of both normal and trisomic cell lines. These cytogenetic subtypes have distinct implications for recurrence risk, clinical presentation, and genetic counseling, underscoring the importance of precise chromosomal characterization [2]-[5].

Clinically, individuals with trisomy 21 typically present with intellectual disability, developmental delay, and characteristic craniofacial features. The condition is frequently associated with a wide spectrum of congenital and acquired medical complications. Congenital heart defects affect approximately 40% - 50% of individuals, gastrointestinal anomalies such as duodenal atresia occur in 5% - 10% of cases, and endocrine disorders, particularly congenital hypothyroidism, are also common [2][6][7]. Although the underlying chromosomal abnormality is irreversible, early diagnosis enables timely multidisciplinary interventions, including physical, speech, and cognitive therapies, which can significantly improve developmental and functional outcomes [7].

Definitive diagnosis of trisomy 21 relies on cytogenetic or molecular techniques, most commonly conventional karyotyping and fluorescence in situ hybridization (FISH). These methods not only confirm the presence of an extra chromosome 21 but also allow precise identification of the underlying chromosomal mechanism, which is essential for clinical management and genetic counseling [2][4]. However, in many low-resource settings, including Mali, access to such diagnostic technologies remains limited, resulting in delayed or incomplete diagnosis and suboptimal patient care [8][9].

The present study aimed to establish and evaluate the use of FISH for the diagnosis of trisomy 21 in Mali. Implementing this cytogenetic approach represents a critical step toward strengthening national diagnostic capacity, enabling earlier detection, improving genetic counseling, and supporting more targeted clinical management.

The study included twelve participants (seven males and five females) with a median age of 9.5 years (range: 0.3 - 14 years) (Table 1). All individuals were referred by the Malian Association of Trisomy 21 based on clinical suspicion of Down syndrome, characterized by key phenotypic features including intellectual disability, global developmental delay, and characteristic craniofacial dysmorphisms such as upslanting palpebral fissures, a flat nasal bridge, and epicanthic folds.

Table 1. The median age of the individuals was 9.5 years, ranging from 0.3 to 14 years. Patients P11 and P12 were those carrying the Robertsonian translocation rob (21; 21) (q10; q10) associated with trisomy 21.

Trisomy 21 was confirmed in all twelve cases either by chromosomal structural analysis using DAPI banding or by RUNX1 copy number assessment with FISH. Metaphase chromosome preparations were successfully obtained in eleven of the twelve cases (91.7%). In one case (Patient P2), microcoagulation during blood collection precluded metaphase harvest. In all eleven analyzable samples, karyotypic analysis identified nine cases of free trisomy 21 (47, XX, +21 or 47, XY, +21) and two cases of Robertsonian translocations: 46, XX, rob (21; 21) (q10; q10), +21 and 46, XY, rob (21; 21) (q10; q10), +21 (Table 1). In the two cases of Robertsonian translocations, parental karyotyping revealed normal chromosomal complements in both families, suggesting that the rearrangements occurred de novo. Given the relative rarity of Robertsonian translocations, the two cases were investigated and described in greater detail in [10].

FISH analysis was first performed by testing the probe on a normal diploid control sample. Control experiments showed the expected two distinct RUNX1 signals in all 400 interphase nuclei analyzed, with no background fluorescence detected. In metaphase spreads from the control sample, two signals were consistently observed at the expected 21q22.12 loci, confirming both the specificity and sensitivity of the probe (Figure 1(A)). Subsequently, FISH interphase analysis was successfully performed on the twelve patient samples, achieving a 100% success rate, including in patient P2, whose karyotype analysis was inconclusive due to the absence of metaphase spreads. All 400 interphase nuclei analyzed per patient displayed three distinct green fluorescent signals, indicating the presence of three copies of chromosome 21. No nuclei showed fewer than three signals, excluding mosaicism above the detection threshold (≥1/400 cells). These findings are consistent with non-mosaic trisomy 21 within the limits of detection of the assay. In metaphase spreads, three signals were precisely localized to 21q22.12, confirming the presence of an extra copy of chromosome 21 (Figures 1(B)-(D)).

Results from both cytogenetic approaches were fully concordant in the eleven cases for which both FISH and karyotyping results were available, consistently showing three copies of chromosome 21. In the single case lacking metaphase data, FISH alone provided definitive diagnostic confirmation.

Figure 1.Representative images of fluorescence in situ hybridization analysis. (A) Metaphase spread from a healthy individual showing two distinct hybridization signals (indicated by red arrows) corresponding to chromosome 21. (B)-(D) Metaphase spreads from three different affected individuals showing three distinct signals on chromosome 21. In each case, the presence of three discrete hybridization signals (red arrows) confirms an additional copy of chromosome 21.

This study is the first to combine conventional karyotyping and FISH for the diagnosis of trisomy 21 in Mali, confirming the complementarity of these methods in a cohort of twelve clinically suspected cases. Karyotyping can reveal structural and numerical chromosomal abnormalities while FISH provided high-resolution quantitation of copy number and allowed detection or exclusion of low-level mosaicism [2].

Metaphase spreads were achieved in eleven of twelve samples (91.7% success). The lone failure was due to microcoagulation during blood collection, a known pre-analytical factor compromising lymphocyte culture, particularly in pediatric patients [11]. This underscores the necessity of stringent sample collection protocols, especially in low-resource settings where repeat sampling and experiment are resource demanding.

Among the eleven patients with analyzable metaphases, nine presented three distinct copies of chromosome 21, consistent with free trisomy 21 (47, XX, +21 or 47, XY, +21). The remaining two patients had trisomy 21 associated with a Robertsonian translocation, resulting in a total of 46 chromosomes due to fusion of two chromosome 21 homologs. These cases, including evidence supporting their de novo occurrence, are described in greater detail in [10]. Although rob (21; 21) accounts for only 1% - 1.5% of Down syndrome cases worldwide [12][13], its detection in 2 of 11 cytogenetically confirmed cases (18.18%) in this cohort likely reflects the small sample size, referral bias, and potential underdiagnosis of free trisomy 21. These findings underscore the importance of systematic cytogenetic screening in the Malian population.

FISH succeeded in all samples, reinforcing its utility when karyotyping is impeded by culture failure or poor sample quality. This reflects the growing technical expertise in our laboratory in applying FISH for both constitutional and acquired chromosomal disorders [14][15]. A major advantage of FISH is its ability to provide rapid, targeted analysis directly on interphase nuclei, eliminating the need for cell culture and making it valuable in urgent or technically challenging cases [16]. Using a chromosome 21-specific probe allowed precise enumeration of chromosomal copies across numerous nuclei, confirming trisomy 21 diagnoses and sensitively excluded mosaicism. This shows that FISH serves as both a practical alternative and a complementary tool to standard cytogenetics. Its ability to detect low-level mosaicism enhances diagnostic confidence, particularly in ambiguous or borderline cases [16]-[18].

Although FISH has been used routinely for the detection of common chromosomal abnormalities such as trisomy for more than three decades in high-income countries, its clinical implementation in resource-limited settings, including Mali, remains limited. This study shows the feasibility of a robust FISH-based diagnostic workflow for trisomy 21 in resource-constrained environments. FISH is particularly well suited to such settings, as it enables reliable analysis even when sample quality or cell culture viability is suboptimal. Importantly, this work provides a practical proof of concept for integrating cytogenetic testing into both postnatal and prenatal diagnostic pathways in settings where advanced genomic technologies such as chromosomal microarray analysis or cell-free fetal DNA testing are not yet available, despite their routine use in developed countries [19][20]. In settings where chromosomal abnormalities remain prevalent, partly due to the absence of pregnancy termination for fetal aneuploidies, this successful implementation shows feasibility, reproducibility, and scalability, and establishes a foundation for expanding cytogenetic diagnostics and strengthening national clinical genetics services.

This study has several limitations. First, the referral-based recruitment may have introduced selection bias. Second, the absence of systematic early postnatal enrollment likely limited accurate estimation of the relative frequencies of cytogenetic subtypes of trisomy 21 and may have introduced survival bias due to early mortality. Third, the exclusively clinically selected, all-positive cohort limits generalizability and precludes assessment of diagnostic sensitivity and specificity.

To ensure long-term sustainability, continued efforts are required to address the challenges that limited earlier initiatives aimed at implementing cytogenetic diagnostic services for trisomy 21 in Mali more than two decades ago. Despite its pioneering contribution, that effort faced substantial barriers to continuity, including insufficient funding, inadequate infrastructure, and a shortage of qualified personnel, resulting in a prolonged absence of routine cytogenetic services in Mali [9]. Building on lessons learned from that experience, the present study serves as both a foundation and a catalyst for establishing durable, accessible, and sustainable cytogenetic diagnostics for trisomy 21 in the country. As genomic diagnostics continue to expand globally, particularly with the routine use of non-invasive prenatal testing in developed countries [19][21], the development of context-appropriate and affordable workflows remains essential to ensure equitable access to accurate diagnosis, recurrence risk assessment, and effective genetic counseling. Our findings show how conventional and molecular cytogenetic methods can be successfully integrated into resource-limited settings to deliver reliable, high-quality diagnostic care.

This study shows the practical feasibility of implementing cytogenetic diagnosis of trisomy 21 in Mali using a combined approach of karyotyping and FISH. The integration of these complementary techniques enhances diagnostic accuracy, informs patient management, and strengthens genetic counseling within clinical settings. Beyond the immediate diagnostic benefit, these results provide a critical foundation for the development of national cytogenetic diagnostic capacity and the broader integration of genetic medicine into Mali’s healthcare system.

We are grateful to the Association of Trisomy 21 of Mali for its support.

This work was supported by grant U01HG007044 from the National Institute of Neurological Disorders and Stroke (NINDS), USA, administered by the National Human Genome Research Institute (NHGRI) as part of the H3Africa initiative, and by grant CH2-116457 from the Canadian Institutes of Health Research (CIHR), administered through the Cancer Research Institute of the Canada Hope Program. We are deeply grateful to the patients and their families for their participation in this study.

OS: Conceptualization, formal analysis, funding acquisition, investigation, methodology, resources, validation, writing - original draft, writing - review & editing.

CO: Formal analysis, methodology, writing - original draft, writing - review & editing.

COS: Investigation, methodology.

OT: Investigation, writing - review & editing.

DS: Investigation, methodology.

MKG: Methodology, writing - review & editing.

AT: Investigation.

GL: Funding acquisition, resources, writing - review & editing.

MK: Methodology, writing - review & editing.

MT: Methodology, writing - review & editing.

All data generated or analyzed during this study are available upon reasonable requested from the corresponding author. included in this published article.

This study was conducted in full compliance of the declaration of Helsinki and the study protocol has been approved by the institutional Ethics Committee of Faculty of Medicine and Dentistry of the University of Sciences, Techniques and Technologies of Bamako, Mali, under approval number 2020/129/CE/FMOS/FAPH. Written consent and assent forms were obtained from all participants before enrollment and share of data.

Informed consent was obtained from all the legal guardian of participants involved in the study.

Samples used in this study are available upon reasonable request from the corresponding author.