Hb A2 Episkopi - a novel δ-globin chain variant [HBD:c.428C>T] in a family of mixed Cypriot-Lebanese descent.

OBJECTIVES: Thalassaemia is a potentially lethal inherited anaemia, caused by reduced or absent synthesis of globin chains. Measurement of the minor adult haemoglobin Hb A2, combining α- with δ-globin, is critical for the routine diagnosis of carrier status for α- or ß-thalassaemia. Here, we aim to characterize a novel δ-globin variant, Hb A2 Episkopi, in a single family of mixed Lebanese and Cypriot ancestry with mild hypochromic anaemia and otherwise normal globin genotype, which also presents with a coincidental 0.78-Mb sequence duplication on chromosome 1 (1q44) and developmental abnormalities. METHODS: Analyses included comprehensive haematological analyses, cation-exchange high-performance liquid chromatography (CE-HPLC), cellulose acetate electrophoresis (CAE), Sanger sequencing and structure-based stability predictions for Hb A2 Episkopi. RESULTS: The GCT > GTT missense mutation, underlying Hb A2 Episkopi, HBD:c.428C > T, introduces a cd142 codon change in the mature protein, resulting in reduced normal Hb A2 amounts and a novel, less abundant Hb A2 variant (HGVS: HBD:p.A143V), detectable as a delayed peak by CE-HPLC. The latter was in line with structure-based stability predictions, which indicated that the substitution of a marginal, non-helical and non-interface residue, five amino acids from the δ-globin chain carboxy-terminus, was moderately destabilizing. DISCUSSION: Detection of the new variant depends on the diagnostic set-up and had failed by CAE and on an independent CE-HPLC system, which, in unfavourable circumstances, may lead to misdiagnoses of ß-thalassaemia as α-thalassaemia. Given the mixed background of the affected family, the ethnic origin of the mutation is unclear, and this study thus suggests awareness for possible detection of Hb A2 Episkopi in both the Cypriot and the Lebanese populations.

The molecular spectrum and distribution of haemoglobinopathies in Cyprus: a 20-year retrospective study.

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 ß-thalassaemia patients and 595 Hb H disease patients in Cyprus. Moreover, we report the molecular spectrum of α-, ß- 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 ß- 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.

Prevalence of RhD status and clinical application of non-invasive prenatal determination of fetal RHD in maternal plasma: a 5 year experience in Cyprus.

BACKGROUND: After the discovery that cell-free fetal DNA (cffDNA) is circulating in the maternal plasma of pregnant women, non-invasive prenatal diagnosis for fetal RhD in maternal plasma in RhD negative women at risk for haemolytic disease of the newborn (HDN) was clinically established and used by many laboratories. The objectives of this study are: (a) to assess the feasibility and report our experiences of the routine implementation of fetal RHD genotyping by analysis of cffDNA extracted from maternal plasma of RhD negative women at risk of HDN, and (b) to estimate the RhD phenotype frequencies, the RHD genotype frequencies and the RhD zygosity in the Cypriot population. METHODS: cffDNA was extracted from maternal plasma of 73 RhD negative pregnant women. Real-Time Multiplex-PCR was used to amplify regions of RHD gene in exons 4, 5 and 10. RhD phenotypes were determined on 445 random samples using conventional agglutination slide test. RESULTS: The fetus was predicted to be positive in 53 cases and negative in 18 cases. Two of cases were identified as D-variants, weak D type-1 and 11. The frequency of RhD negative homozygosity in the Cypriot population was estimated to be 7.2%, while the frequencies of RHD hemizygosity and RhD positive homozygosity was calculated to be 39.2 and 53.6%, respectively. CONCLUSION: Fetal RHD genotyping can be accurately determined using cffDNA from maternal plasma. The implementation of the test has eliminated all use of unnecessary anti-D and reduced the total use of anti-D by 25.3% while achieving appropriate management of the RhD negative pregnancies.

Delta-thalassemia in Cyprus.

To help clarify the hematological picture of patients who may be positive for beta- and delta-globin gene mutations, the following study was carried out. Our aim was to identify the delta-globin gene mutations found in the Greek Cypriot population, their frequencies and the Hb A2 values associated with them. Seventy-four samples were selected from a random sample of 5,030 individuals, and the database of the Molecular Genetics Thalassaemia Department containing diagnostic analyses data was also mined for relevant information. Four novel for Cyprus delta-globin gene mutations: -30 (T-->C), Hb A2-Wrens [delta98(FG5)Val-->Met, GTG-->ATG], IVS-I-2 (T-->C) and Hb A2-Yokoshima [delta25(B7)Gly-->Asp (GGT-->GAT)] were identified. Hb A2-Yialousa [delta27(B9)Ala-->Ser, GCC-->TCC], Hb A2-Yokoshima, Hb A2-Troodos [delta116(G18)Arg-->Cys, CGC-->TGC], Hb A2-Pelendri [delta141(H19)Leu-->Pro, CTG-->CCG], codon 4 [delta4(A1)Thr-->Ile], codon 59 (-A), Hb A2-Wrens, IVS-II-897 (A-->G), IVS-I-2, -55 (T-->C) and -30 bring the total to 11 delta-globin alleles found in the Greek Cypriot population. Hb A2-Yialousa is the most common mutation followed by codon 4, with frequencies of 60.7 and 17.8%, respectively.Hb A2 levels above 1.9% have been found to indicate a significantly reduced possibility for the presence of a delta-globin gene mutation in this population. For Hb A2 levels of 1.7 and 1.8% the possibility of a delta-globin gene mutation rises to 90.9% and reaches 100% for lower Hb A2 levels. The frequency of all the mutant delta-globin chromosomes in the sample is 0.0067 and the carrier frequency is 1.26%.