Marina Kleanthous

A MULTI-CENTER STUDY IN ORDER TO FURTHER DEFINE THE MOLECULAR BASIS OF β-THALASSEMIA IN THAILAND, PAKISTAN, SRI LANKA, MAURITIUS, SYRIA, AND INDIA, AND TO DEVELOP A SIMPLE MOLECULAR DIAGNOSTIC STRATEGY BY AMPLIFICATION REFRACTORY MUTATION SYSTEM-POLYMERASE CHAIN REACTION

The spectrum of the β-thalassemia mutations of Thailand, Pakistan, India, Sri Lanka, Mauritius and Syria has been further characterized by a multi-center study of 1,235 transfusion-dependent patients, and the mutations discovered used to assess the fidelity of a simple diagnostic strategy. A total of 44 β-thalassemia mutations were identified either by allele-specific oligonucleotide hybridization, amplification with allele-specific primers, or DNA sequencing of amplified product. The results confirm and extend earlier findings for Thailand, Pakistan, India, Mauritius and Syria. This is the first detailed report of the spectrum of mutations for Sri Lanka. Two novel mutations were identified, codon 55 (−A) and IVS-I-129 (A → C), both found in Sri Lankan patients. Two β-thalassemia mutations were found to coexist in one β-globin gene: Sri Lankan patients homozygous for the β0 codon 16 (−C) frameshift were also homozygous for the β+ codon 10 (C → A) mutation. Studies of Sri Lankan, Pakistani, and Indian carriers suggest the codon 10 (C → A) mutation is just a rare polymorphism on an ancestral allele, on which the β0 codon 16 (−C) mutation has arisen. Each country was found to have only a few common mutations accounting for 70% or more of the β-thalassemia alleles. A panel of primers to diagnose the majority of the mutations by the amplification refractory mutation system was developed, enabling a simple molecular diagnostic strategy to be introduced for each country participating in the multi-center study.

The impact of genotype on endocrine complications in thalassaemia major

Abstract:  Background: The clinical severity in thalassaemia major (TM) depends on the underlying mutations of the β‐globin gene and the degree of iron overload. Objective: The aim of the study was to investigate the impact of genotype on the development of endocrine complications in TM in our center. Subjects and methods: 126 (62 males, 64 females) thalassaemic patients of Greek Cypriot origin with a mean age of 31.2 (17–68) yr were included in the study. All patients, who were on the standard treatment protocol, were subsequently divided into two groups according to their genotype, group A (92): TM with no mitigating factor and group B (34): TM carrying one or more mitigating factors in the β‐ and/or α‐globin genes. Iron overload calculation was based on the amount of red cell consumption and the mean ferritin level over a 12‐year period. Statistical analysis was performed with the SPSS program. Results: Patients in group A, who were consuming larger amounts of blood on transfusions, were more likely to develop hypogonadism (P = 0.001) compared with patients in group B, despite their similar mean ferritin levels. The incidence of other endocrinopathies (short stature, hypothyroidism, and diabetes mellitus) was similar in the two groups. The prevalence of hypothyroidism in splenectomized patients was significantly higher (P = 0.005), whereas the presence of hypogonadism, impaired glucose homeostasis and insulin resistance, although more frequent, was not statistically significant. The clinical severity of TM had no impact on bone mineral density (BMD) in both men and women. BMD was only influenced by gonadal function. Conclusions: This study demonstrates that the underlying genetic defect in TM is a contributing factor for gonadal dysfunction, because the patients with the more severe defects have a greater rate of iron loading through higher red cell consumption.

Arrayed Primer Extension for the Noninvasive Prenatal Diagnosis of β-Thalassemia Based on Detection of Single Nucleotide Polymorphisms

β‐Thalassemia is one of the most common autosomal recessive single‐gene disorders in Cyprus. Development of a noninvasive prenatal diagnostic (NIPD) assay for β‐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.

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.

AN OVERVIEW OF CURRENT MICROARRAY-BASED HUMAN GLOBIN GENE MUTATION DETECTION METHODS

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.

Next generation sequencing of SNPs for non-invasive prenatal diagnosis: challenges and feasibility as illustrated by an application to β-thalassaemia.

β-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 β-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 β-thalassaemia using targeted NGS and haplotype analysis. Preliminary results show that NGS is effective in detecting paternally inherited alleles in the maternal plasma.

IthaGenes: An Interactive Database for Haemoglobin Variations and Epidemiology

Inherited haemoglobinopathies are the most common monogenic diseases, with millions of carriers and patients worldwide. At present, we know several hundred disease-causing mutations on the globin gene clusters, in addition to numerous clinically important trans-acting disease modifiers encoded elsewhere and a multitude of polymorphisms with relevance for advanced diagnostic approaches. Moreover, new disease-linked variations are discovered every year that are not included in traditional and often functionally limited locus-specific databases. This paper presents IthaGenes, a new interactive database of haemoglobin variations, which stores information about genes and variations affecting haemoglobin disorders. In addition, IthaGenes organises phenotype, relevant publications and external links, while embedding the NCBI Sequence Viewer for graphical representation of each variation. Finally, IthaGenes is integrated with the companion tool IthaMaps for the display of corresponding epidemiological data on distribution maps. IthaGenes is incorporated in the ITHANET community portal and is free and publicly available at http://www.ithanet.eu/db/ithagenes.

Oxidative stress in β-thalassaemia and sickle cell disease

Sickle cell disease and β-thalassaemia are inherited haemoglobinopathies resulting in structural and quantitative changes in the β-globin chain. These changes lead to instability of the generated haemoglobin or to globin chain imbalance, which in turn impact the oxidative environment both intracellularly and extracellularly. The ensuing oxidative stress and the inability of the body to adequately overcome it are, to a large extent, responsible for the pathophysiology of these diseases. This article provides an overview of the main players and control mechanisms involved in the establishment of oxidative stress in these haemoglobinopathies.

Hb Bart's Levels in Cord Blood and α-Thalassemia Mutations in Cyprus

The purpose of this study was to examine the frequency of α-thalassemia in the population of Cyprus using cord blood samples. The levels of Hb Barts were compared with the hematological indices and the results correlated with the presence of α-thalassemia mutations. The protocols for the polymerase chain reaction detection of the six most common α-globin mutations encountered in Cyprus were optimized, and the frequency of each mutation was determined through the screening of 495 random cord blood samples. The total allele frequency for the mutations examined was 10.6%, of which 1% is due to the triplication of the α-globin genes. The -α(3.7 kb) deletion accounts for 72.8% of all detectable mutations, while the –MED-I and -(α)-20.5 kb mutations account for 7.8%. The level of Hb Barts and the MCV and MCH values incord blood samples were found to correlate closely with the severity of α-thalassemia, although the -α(3.7 kb) deletion and perhaps other mild α-thalassemia mutations may not give detectable Hb Barts levels. A reasonably accurate estimate of the α-thalassemia carrier frequency may be obtained from cord blood studies if Hb Barts estimates are combined with hematological indices. When molecular methods are added, these give the best way to use cord bloods to survey populations for α-thalassemia.

Noninvasive Prenatal Diagnostic Assay for the Detection of β‐Thalassemia

Abstract:  The development of a noninvasive method for detection of β‐thalassemia in the population of Cyprus is based on the detection of paternally inherited single nucleotide polymorphisms (SNPs) as well as β‐thalassemia (β‐thal) mutations. We selected 11 informative SNPs for the Cypriot population linked to the β‐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 β‐thal mutation.