RESUMO
ß-Thalassemia is brought about by defective ß-globin (HBB [hemoglobin subunit ß]) formation and, in severe cases, requires regular blood transfusion and iron chelation for survival. Genome editing of hematopoietic stem cells allows correction of underlying mutations as curative therapy. As potentially safer alternatives to double-strand-break-based editors, base editors (BEs) catalyze base transitions for precision editing of DNA target sites, prompting us to reclone and evaluate two recently published adenine BEs (ABEs; SpRY and SpG) with relaxed protospacer adjacent motif requirements for their ability to correct the common HBBIVSI-110(G>A) splice mutation. Nucleofection of ABE components as RNA into patient-derived CD34+ cells achieved up to 90% editing of upstream sequence elements critical for aberrant splicing, allowing full characterization of the on-target base-editing profile of each ABE and the detection of differences in on-target insertions and deletions. In addition, this study identifies opposing effects on splice correction for two neighboring context bases, establishes the frequency distribution of multiple BE editing events in the editing window, and shows high-efficiency functional correction of HBBIVSI-110(G>A) for our ABEs, including at the levels of RNA, protein, and erythroid differentiation.
RESUMO
BACKGROUND: Thalassemias are inherited blood disorders and by far one of the most common monogenic diseases globally. Beta-thalassemia has a particularly high prevalence in Cyprus, with the IVSI-110 G>A (HBB:c.93-21G>A) pathogenic variation representing almost 79% of the total carriers. The discovery that 3% to 20% of cell-free fetal DNA (cffDNA) is present in the maternal plasma allowed the development of non-invasive prenatal diagnosis (NIPD) of monogenic diseases, like beta-thalassemia, avoiding the risks of invasive procedures. However, the development of NIPD holds major technical challenges and has not yet reached the clinical setting. METHODS: In this study, we apply droplet digital PCR (ddPCR) coupled with the relative variant dosage approach to develop a NIPD assay for IVSI-110 G>A beta-thalassemia. We have implemented an optimization process for ddPCR to address the challenges of ddPCR assays such as inconclusive rain droplets and thus increase the sensitivity and specificity of the assay. The established protocol was evaluated on 40 maternal plasma samples with a median gestational age of 10 weeks where both parents carried the same pathogenic variation. RESULTS: Thirty-three samples were correctly classified, 6 remained inconclusive, and 1 was misclassified. Our assay exhibited 97.06% accuracy (95% CI, 82.46-99.68), 100% sensitivity (95% CI, 76.84-100), and 95% specificity (95% CI, 75.13-99.87), demonstrating its efficiency for the non-invasive detection of both maternal and paternal alleles. CONCLUSIONS: We have developed an efficient, simple, and cost-effective ddPCR assay for the non-invasive determination of fetal genotype in couples at risk of IVSI-110 G>A beta-thalassemia, bringing NIPD of monogenic diseases closer to the diagnostic setting.
