b-Thalassaemia is one of the most common autosomal recessive single-gene disorder worldwide, with a carrier frequency of 12% in Cyprus. Prenatal tests for at risk pregnancies use invasive methods and development of a non-invasive prenatal diagnostic (NIPD) method is of paramount importance to prevent unnecessary risks inherent to invasive methods. Here, we describe such a method by assessing a modified version of next generation sequencing (NGS) using the Illumina platform, called 'targeted sequencing', based on the detection of paternally inherited fetal alleles in maternal plasma. We selected four single-nucleotide polymorphisms (SNPs) located in the b-globin locus with a high degree of heterozygosity in the Cypriot population. Spiked genomic samples were used to determine the specificity of the platform. We could detect the minor alleles in the expected ratio, showing the specificity of the platform. We then developed a multiplexed format for the selected SNPs and analysed ten maternal plasma samples from pregnancies at risk. The presence or absence of the paternal mutant allele was correctly determined in 27 out of 34 samples analysed. With haplotype analysis, NIPD was possible on eight out of ten families. This is the first study carried out for the NIPD of b-thalassaemia using targeted NGS and haplotype analysis. Preliminary results show that NGS is effective in detecting paternally inherited alleles in the maternal plasma.

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.

beta-Thalassemia is one of the most common autosomal recessive single-gene disorders in Cyprus. Development of a noninvasive prenatal diagnostic (NIPD) assay for beta-thalassemia is based mostly on the detection of paternally inherited single nucleotide polymorphisms (SNPs) using the arrayed primer extension (APEX) method. Eleven SNPs with high degree of heterozygosity in the Cypriot population were selected and analyzed on 34 families and the informative SNPs were determined. The APEX assay was used on maternal plasma of seven families using the informative SNPs; paternal allele of the fetus was noninvasively detected in five families.

The panoply of human globin gene mutation detection methods could become significantly enriched with the advent of microarray-based genotyping platforms. The aim of this article is to provide an overview of the current medium and high-throughput microarray-based globin gene mutation detection platforms, namely the microelectronic array, the "thalassochip" arrayed primer extension (APEX) technology and the single base extension methods. This article also outlines an emerging method based on multiple ligation probe amplification (MLPA) and discusses the implications of customized solutions for resequencing of genomic loci in relation to molecular genetic testing of hemoglobinopathies.

The development of a noninvasive method for detection of beta-thalassemia in the population of Cyprus is based on the detection of paternally inherited single nucleotide polymorphisms (SNPs) as well as beta-thalassemia (beta-thal) mutations. We selected 11 informative SNPs for the Cypriot population linked to the beta-globin locus. Two different approaches were used: allele-specific polymerase chain reaction (AS-PCR) and the arrayed primer extension (APEX) method. The AS-PCR approach is being standardized, and the method was applied in two families. The paternally inherited allele was noninvasively detected with the AS-PCR approach on maternal plasma. Some preliminary tests were performed with the APEX method on genomic DNA of parents carrying the beta-thal mutation.