George Christopoulos

Molecular Characterization of β-Thalassemia in Syria

This study concerns the determination of β-thalassemia alleles and other hemoglobin variants in 82 patients from Syria. We have characterized 146 chromosomes and found 17 different β-thalassemia mutations, and one β-globin chain variant that gives rise to the abnormal Hb S. the eight most common β-thalassemia mutations were the IVS-I-110 (G→A), IVS-I-1 (G→A), codon 5 (-CT), -30 (T→A), codon 39 (C→T), IVS-I-6 (T→C), IVS-II-1 (G→A), and codon 15 (TGGT´G). These mutations accounted for almost 75% of the total β-thalassemia chromosomes. We identified 34 different genotypes with a high level of homozygosity. the various β-thalassemia mutations were characterized using gene amplification with specific oligonucleotide primers, restriction enzyme analysis, denaturing gradient gel electrophoresis and direct sequencing. by combining these three approaches we were able to detect mutations in almost 90% of the chromosomes studied. Our findings provide a sound foundation on which to base a preventive program for thalassemia and we believe that the data that we present will facilitate the improvement of medical services such as carrier screening, genetic counseling, and prenatal diagnosis. Furthermore a detailed knowledge of the molecular pathology of β-thalassemia will strongly improve the prenatal diagnosis services in Syria.

Hb Agrinio [α29(B10)Leu→Pro (α2)] in Combination with – –MED I Results in a Severe Form of Hb H Disease

We report two cases of compound heterozygote patients for the – –MED I and Hb Agrinio [α29(B10)Leu→Pro (α2)] anomalies in two unrelated Greek Cypriot families. The first patient had a serious form of Hb H disease and died at the age of 21 due to complications arising during an operation. The second patient showed a severe hematological picture and has been regularly transfused since an early age. This patient exhibits bone abnormalities as well as hepatosplenomegaly. The severity of these two incidences emphasizes the need for the inclusion of a screening test for the – –MED I/αAgrinioα genotype among those already offered during prenatal diagnosis. Two homozygotes, as well as a number of simple, compound, and double heterozygotes for Hb Agrinio have been identified in Cyprus and their hematological indices are presented.

Identification of α‐Thalassemia Mutations in Iranian Individuals with Abnormal Hematological Indices and Normal Hb A2

To define the spectrum of a-thalassemia (thal) mutations in Iran, we have studied 69 subjects with abnormal hematological indices (low MCV, low MCH) but with normal Hb A2 levels, for seven common a-thal mutations ( a , a , a , a 5 a, aa, , and MED ). Four different mutations, a , a , a 5 a, and aa, were found in 17 chromosomes, of which 13 were of the a 3.7 type. Iran, with a population of 65 million, consists of heterogeneous population groups and an estimated three million carriers of b-thal and 20,000 thalassemia patients (1). Premarital carrier screening has now been routinely performed since 1997, although it was originally initiated within the thalassemia prevention program in 1992. During this, we encountered a substantial number of subjects with abnormal hematological indices (low MCV and MCH) but with a normal range of Hb A2, which suggested the presence of iron deficiency or a-thal, or both. Since the determination of iron deficiency is not that straightforward, we offered to explore the spectrum of a-thal mutations in this targeted population, as reliable data for Iran is not available. Using DNA analysis, we screened for the presence of seven common a-thal mutations, namely a , a , a , a 5 a, aa, , and MED .

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 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.

Microsatellite Markers Within the α-Globin Gene Cluster for Robust Preimplantation Genetic Diagnosis of Severe α-Thalassemia Syndromes in Mediterranean Populations

In this study we report the development of a generic protocol for preimplantation genetic diagnosis (PGD) of severe α-thalassemia (α-thal) syndromes in α-thal carrier couples of Mediterranean origin. The in silico identification and design of primers for multiplex analysis of short tandem repeats (STRs), was followed by the optimization of polymerase chain reaction (PCR) conditions for multiplexed STR analysis within the α-globin gene cluster (16p3.3) and subsequent optimization and validation of a single-cell multiplex reaction including the selected STRs. Three simple dinucleotide repeats were selected based on their rate of heterozygosity, multiplex PCR efficiency and product size, and location within the α-globin gene cluster. The multiplex PCR was optimized in single lymphocytes with PCR efficiency ranging from 92.5 to 98% and an allele drop-out (ADO) rate of 0 to 9.0% for the three loci. The optimized method was applied in two clinical PGD cycles and genotypes were achieved in 17 out of 18 blastomeres (94%). Transfer of unaffected embryos led to a singleton pregnancy in one of the two couples. The triplex PCR validated for Greek and Cypriot populations is a robust generic method for α-thal PGD.

Hb LIMASSOL [β8(A5)Lys → Asn]: A NEW HEMOGLOBIN VARIANT

Nearly 800 hemoglobin (Hb) variants have been reported worldwide (1). Although most cases were associated with hematological disorders, more recently, many non-pathological mutants were discovered by chance in the course of thalassemia carrier screening programs, or during diabetes control surveys. Four Hb variants have previously been reported at position 8(A5)Lys of the b-globin chain: Hb Rio Grande (!Thr) (2), Hb J-Luhe (!Gln) (3), Hb N-Timone (!Glu) (4), and Hb Nakano (!Met) (5). The present report describes a new nonpathological Hb variant, that we have named Hb Limassol [b8(A5) Lys !Asn]. It was found in the course of a thalassemia screening program in a young Greek Cypriot male who originated from Limassol, a town on the south coast of Cyprus. Hematological studies were done using routine methods and the hematological parameters of the proband were in the normal range: Hb 15.3 g=dL, RBC 5.47 l0=L, PCV 0.450 L=L, MCV 82.3 fL, MCH 28.0 pg, MCHC 34.0 g=dL. The morphology of the RBCs was also normal. Routine Hb analysis was performed by cation exchange high performance liquid chromatography (HPLC), using the routine VARIANT b-Thalassemia Program (Bio-Rad Laboratories, Hercules, CA, USA). The HPL chromatogram HEMOGLOBIN, 25(4), 421–424 (2001)

Novel splice site mutation at IVS8 nt 5 of HEXB responsible for a Greek-Cypriot case of Sandhoff disease

Sandhoff disease is caused by abnormalities in HEXB gene encoding the β‐subunit of β‐hexosaminidase. In this study, we analyzed the HEXB gene of a Sandhoff carrier in the Greek‐Cypriot community. A G to C transversion was identified in one allele of her HEXB gene at position 5 of the 5′‐splice site of intron 8 (IVS8 nt5). One of 13 cDNA clones derived from her lymphocyte HEXB mRNA lacked the last four nucleotides “GTTG” of exon 8, which created a premature termination codon at 11 codons downstream. In vivo transcription of the mutant HEXB gene fragment in CHO cells resulted in deletion of the “GTTG.” The mutation has not been found in 40 DNA samples from anonymous donors, indicating that this is not a polymorphism in the Cypriot population. These results clearly indicate that the splice site mutation at IVS8 nt5 is responsible for this case of Sandhoff disease. Hum Mutat 13:38–43, 1999.

The first family with Tay-Sachs disease in Cyprus: Genetic analysis reveals a nonsense (c.78G>A) and a silent (c.1305C>T) mutation and allows preimplantation genetic diagnosis.

Tay-Sachs disease (TSD) is a recessively inherited neurodegenerative disorder caused by mutations in the HEXA gene resulting in β-hexosaminidase A (HEX A) deficiency and neuronal accumulation of GM2 ganglioside. We describe the first patient with Tay-Sachs disease in the Cypriot population, a juvenile case which presented with developmental regression at the age of five. The diagnosis was confirmed by measurement of HEXA activity in plasma, peripheral leucocytes and fibroblasts. Sequencing the HEXA gene resulted in the identification of two previously described mutations: the nonsense mutation c.78G>A (p.Trp26X) and the silent mutation c.1305C>T (p.=). The silent mutation was reported once before in a juvenile TSD patient of West Indian origin with an unusually mild phenotype. The presence of this mutation in another juvenile TSD patient provides further evidence that it is a disease-causing mutation. Successful preimplantation genetic diagnosis (PGD) and prenatal follow-up were provided to the couple.