Haemoglobinopathies are the most common monogenic diseases, posing a major public health challenge worldwide. Cyprus has one the highest prevalences of thalassaemia in the world and has been the first country to introduce a successful population-wide prevention programme, based on premarital screening. In this study, we report the most significant and comprehensive update on the status of haemoglobinopathies in Cyprus for at least two decades. First, we identified and analysed all known 592 β-thalassaemia patients and 595 Hb H disease patients in Cyprus. Moreover, we report the molecular spectrum of α-, β- and δ-globin gene mutations in the population and their geographic distribution, using a set of 13824 carriers genotyped from 1995 to 2015, and estimate relative allele frequencies in carriers of β- and δ-globin gene mutations. Notably, several mutations are reported for the first time in the Cypriot population, whereas important differences are observed in the distribution of mutations across different districts of the island.

Summaryβ-thalassaemia is one of the commonest autosomal recessive single-gene disorders worldwide. Prenatal tests use invasive methods, posing a risk for the pregnancy itself. Development of a noninvasive prenatal diagnostic method is, therefore, of paramount importance. The aim of the present study is to identify high-heterozygote informative single-nucleotide polymorphisms (SNPs), suitable for the development of noninvasive prenatal diagnosis (NIPD) of β-thalassaemia. SNP genotyping analysis was performed on 75 random samples from the Cypriot population for 140 SNPs across the β-globin cluster. Shortlisted, highly heterozygous SNPs were then examined in 101 carrier families for their applicability in the noninvasive detection of paternally inherited alleles. Forty-nine SNPs displayed more than 6% heterozygosity and were selected for NIPD analysis, revealing 72.28% of the carrier families eligible for qualitative SNP-based NIPD, and 92% for quantitative detection. Moreover, inference of haplotypes showed predominant haplotypes and many subhaplotypes with sufficient prevalence for diagnostic exploitation. SNP-based analyses are sensitive and specific for the detection of the paternally inherited allele in maternal plasma. This study provides proof of concept for this approach, highlighting its superiority to NIPD based on single markers and thus providing a blueprint for the general development of noninvasive prenatal diagnostic assays for β-thalassaemia.

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Sirs,The idiopathic nephrotic syndrome is a common clinicopathological entity characterized by massive proteinuria, hypoalbuminaemia, hyperlipidaemia, oedema, and various glomerular changes, occurring mainly in children in 15-20% of whom the condition is steroid-resistant. About 85% of patients with steroid-resistant nephrotic syndrome (SRNS) exhibit renal histology of focal segmental glomerulosclerosis (FSGS), and the rest exhibit mesangial proliferative glomerulonephritis (MsPGN) or other rarer histological phenotypes [1]. Mutations in the NPHS2 gene, encoding podocin, which is one of the important proteins of the slit diaphragm, are a frequent cause of sporadic SRNS in children, occurring in 2.8-28% of the cases [2-4]. Mutations in exons 8 and 9 of the WT1 gene have also been reported (more frequently in girls) with isolated SRNS [5]. Other genes that are responsible have been recently reported, accounting for rare cases of SRNS.Idiopathic nephrotic syndrome is a frequent glomerular disease in Cyprus and Greece, where 15-20% of cases are steroid resistant, in accordance with the literature. In this work we studied for the first time in Greece and Cyprus a cohort of 24 children (ten boys, 14 girls) with SRNS. Renal histology, based on 1-3 biopsies, showed changes in FSGS in 21 children and MsPGN in three children. We investigated these children at the molecular level by searching for mutations in the NPHS2 and WT1 (exons 8 and 9) genes. In doing so, we used, for the first time to our knowledge, SURVEYOR endonuclease (Transgenomic, UK), an enzyme that cleaves double-stranded DNA at positions of heteroduplex mismatches, as a method for identifying mutations and/or polymorphic variants. For this, genomic sequences, amplified by polymerase chain reaction (PCR) and encompassing the exons, the consensus exon−intron splice junctions and flanking intronic sequences (primers available on request), were denatured by being heated to Pediatr Nephrol (

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.