MicroRNAs (miRNAs) are small non-coding RNAs crucial for post-transcriptional and translational regulation of cellular and developmental pathways. The study of miRNAs in erythropoiesis elucidates underlying regulatory mechanisms and facilitates related diagnostic and therapy development. Here, we used DNA Nanoball (DNB) small RNA sequencing to comprehensively characterize miRNAs in human erythroid cell cultures. Based on primary human peripheral-blood-derived CD34+ (hCD34+) cells and two influential erythroid cell lines with adult and fetal hemoglobin expression patterns, HUDEP-2 and HUDEP-1, respectively, our study links differential miRNA expression to erythroid differentiation, cell type, and hemoglobin expression profile. Sequencing results validated by reverse-transcription quantitative PCR (RT-qPCR) of selected miRNAs indicate shared differentiation signatures in primary and immortalized cells, characterized by reduced overall miRNA expression and reciprocal expression increases for individual lineage-specific miRNAs in late-stage erythropoiesis. Despite the high similarity of same-stage hCD34+ and HUDEP-2 cells, differential expression of several miRNAs highlighted informative discrepancies between both cell types. Moreover, a comparison between HUDEP-2 and HUDEP-1 cells displayed changes in miRNAs, transcription factors (TFs), target genes, and pathways associated with globin switching. In resulting TF-miRNA co-regulatory networks, major therapeutically relevant regulators of globin expression were targeted by many co-expressed miRNAs, outlining intricate combinatorial miRNA regulation of globin expression in erythroid cells.

Haemoglobinopathies, including thalassaemias and sickle-cell syndromes, are demanding, lifelong conditions that pose a significant burden to patients, families, and healthcare systems. Despite the therapeutic advances and the resulting improvements in prognosis accomplished in past decades, these patients still face important challenges, including suboptimal access to quality care in areas with developing economies, changing epidemiology due to massive migration flows, an evolving clinical spectrum due to ageing in well-treated patients, and limited access to novel high-cost therapies. We herein describe the organization of healthcare services for haemoglobinopathies in Cyprus—with particular focus on beta-thalassaemia, the most prevalent condition in this region—along with selected patient outcomes. This report aims at underscoring the fact that nationally funded and well-coordinated prevention and care programmes for chronic and complex conditions, such as haemoglobinopathies, with active involvement from patient organizations lead to effective disease control and excellent outcomes in survival, quality of life, social adaptation, and public health savings, and allow timely and effective responses to emerging crises, such as the COVID-19 pandemic. The Cyprus paradigm could therefore serve as a blueprint for the organization or adaptation of haemoglobinopathy programs in other countries since these disorders are still widely occurring.

Non-invasive prenatal testing (NIPT) has been widely adopted for the screening of chromosomal abnormalities; however, its adoption for monogenic disorders, such as β-thalassaemia, has proven challenging. Haemoglobinopathies are the most common monogenic disorders globally, with β-thalassaemia being particularly prevalent in Cyprus. This study introduces a non-invasive prenatal haplotyping (NIPH) assay for β-thalassaemia, utilizing cell-free DNA (cfDNA) from maternal plasma. The assay determines paternal inheritance by analyzing highly heterozygous single-nucleotide variants (SNVs) in the β-globin gene cluster. To identify highly heterozygous SNVs in the population, 96 randomly selected samples were processed using Illumina DNA-prep NGS chemistry. A custom, high-density NGS genotyping panel, named HAPLONID, was designed with 169 SNVs, including 15 common pathogenic ones. The AmpliSeq for Illumina assay was then applied to cfDNA to evaluate the panel’s efficiency in performing NIPT for β-thalassaemia. Analysis revealed 219 highly polymorphic SNVs, and the sequencing of 17 families confirmed successful paternal allele determination. The NIPH assay demonstrated 100% success in diagnostic interpretation. This study achieved the advancement of an integrated NGS-NIPT assay for β-thalassaemia, bringing it one step closer to being a diagnostic assay and thereby enabling a reduction in the number of risky invasive prenatal sampling procedures in Cyprus and elsewhere.

Iron overload-driven liver fibrosis is a major concern in β-thalassaemia patients, but non-invasive or minimally invasive biomarkers for fibrosis staging remain limited. This study evaluated five plasma microRNAs (let-7a, miR-21, miR-29a, miR-34a, and miR-122) as potential markers for distinguishing liver fibrosis stages in β-thalassaemia. Plasma samples from 40 patients with fibrosis stages F0–F1 to F4 were analysed using RT-qPCR, normalised against the arithmetic mean of reference miRNAs miR-16 and miR-221. Expression levels of candidate miRNAs showed no statistically significant variation across stages, and logistic regression and ROC analyses revealed fair discriminatory performance for individual miRNAs and their combinations in selected stage comparisons. Notably, while for the discrimination of different fibrosis stages all five candidate miRNAs tested showed fair area-under-the-curve values between 0.7 and 0.8 individually and up to 0.917 in combination, none of these findings reached statistical significance. These results suggest that while the selected set of miRNAs reflects liver injury, its performance for precise fibrosis staging in β-thalassaemia is limited. A key cause for the low discriminatory power of these miRNAs may be the overall change of the blood miRNA transcriptome in haemoglobinopathies. The results indicate the need for validation in larger cohorts based on larger miRNA panels or the use of alternative source materials to improve diagnostic performance.