ESHG Posters 12

A. Colosimo¹^,2, V. Guida¹, A. De Luca¹, G. Palka², M. Cappabianca³, I. Bianco³, B. Dallapiccola¹^,4;
¹CSS-Mendel Institute, Rome, ITALY, ²University of Chieti, Department of Biomedical Sciences, Chieti, ITALY, ³Associazione Nazionale Microcitemie in Italia, Rome, ITALY, ⁴University of Rome, Department of Experimental Medicine and Pathology, Rome, ITALY.

b-Thalassemia, one of the most common hereditary disease worldwide, results from a reduced or absent b-globin chain synthesis, due to over 200 identified mutations in the b-globin locus. The high prevalence of b-thalassemic carriers in Mediterranean countries presses for the development of genetic counseling and postnatal/prenatal molecular diagnosis programs. The major goal of this study was to develop a feasible protocol for the postnatal and prenatal diagnosis of b-thalassemia in the Italian population, based on a Denaturing High Performance Liquid Chromatography (DHPLC) assay. First of all, empirical optimization of DHPLC parameters was set up in a total of 40 Italian heterozygous carriers, whose 26 different b-globin mutations had been previously identified by ARMS-PCR. A group of 30 normal individuals was used as control. Secondly, the DHPLC optimized parameters were successfully applied to the mutational analysis of b-thalassemic patients, both compound heterozygotes (12 subjects) and homozygous patients (4 subjects). In addition,12 chorionic villi samples were subjected to DHPLC analysis upon molecular characterization of each parental b-globin alleles. Two other mutation detection methods (i.e. ARMS-PCR and allele-specific reverse dot-blot) plus direct sequence analysis were used in parallel to the DHPLC method, showing an accuracy rate of 100%. No misdiagnosis occurred. In summary, DHPLC has been shown to be a reliable, sensitive and rapid screening method to perform postnatal and prenatal diagnosis of b-thalassemia in the Italian population, within 2-3 days of sampling.

F. Sahebjam¹, C. Oberkenins², A. Tabarroki³, M. Neishabouri¹, A. Moritz⁴, S. Taimourian³, M. K. Javan¹, H. Najmabadi¹^,3;
¹University of Welfare and Rehabilitation Sciences, Tehran Iran, Tehran, IRAN (ISLAMIC REPUBLIC OF), ²Vienna Lab, Vienna Austria, Viena, AUSTRIA, ³Karimi-nejad Pathology and Genetic Center, Tehran Iran, Tehran, IRAN (ISLAMIC REPUBLIC OF), ⁴Vienna Lab, Vienna Austria, Vienna, AUSTRIA.

Beta-thalassemia is a major health problem in Iran and it is estimated that more than two million carriers of beta thalassemia live in Iran. Although currently the battery of 22 mutations in form of Beta strip assay (Vienna Lab) are used for mutation detection and prenatal diagnosis, the mutations of more than 20 percent of DNA samples still remain unknown by these probes. We have selected 72 samples of unidentified beta thalassemia cases, representing different ethnical and geographical areas of Iran. Samples were sequenced for beta globin gene. Results revealed one new mutation of Codon 95(A-T). We found three east-Asian mutations of IVS II-654 (C-T), Codon 24/25 (-GGT), and IVS II-850 (G-T) and also one individual with an unusual mutation of IVS I-2 (T-C), which have not been reported in non-black population. We also found other rare mutations of IVS I-130 (G-C), cd 82/83 (-G), cd 16 (-C), -88 (C-A), cd 15 (G-A), 5’UTR+22(G-A), Cap+1 (A-C), IVS II-2, 3 (+11, -2), cd 67 (T-G), cd 42/43 (+T), which are reported previously in European and Mediterranean populations. In nine carrier of beta thalassemia no mutations where found in beta globin gene , this may be result of very large deletions in beta or deletion in LCR region. These findings indicate that Iranian population shows a wide variety of thalassemia allelic distribution and also helps prenatal diagnosis program in Iran.

Differential Effects of the XMNI Site in cis to the Gg Globin Genes between Newborn Hb F Malta I Heterozygotes and Anaemic Adult Thalassaemia Homozygotes.

The differential effects of the “regulatory” XMNI site 5’ to the Gg globin gene were studied among newborn Hb-F-Malta-I heterozygotes and adult thalassaemia homozygotes.
Hb F Malta I was quantified among 135 heterozygote newborn (= 26% +/- 3.0). Genotyping of the “regulatory” XMNI site in trans excluded marked effects on the neonatal globin phenotype irrespective of gender: -- = 26.0%(n=116); -+ = 24.0% (n=19).
Quantification of Hb F parameters among 7 adult anaemic thalassaemia homozygotes / double heterozygotes showed tight dependence on the XMNI genotype. Patients with b+IVS-I,6C in association with the b+ IVS-II,1A mutations and -+ at the XMNI site had high values; Hb F = 54%, Gg = 0.71, F-cells = 3.0 x 1012/l, Hb F / F-cell = 17.7pg while one patient with the same mutations and a hybrid haplotype resulting in cis – at the XMNI site (XMN I genotype = --) had lower values; Hb F = 13%, Gg = 0.37, F-cells = 0.12 x 1012/l, Hb F / F-cell = 11.4pg. The main effect appears to be on the decreased level of F-cell numbers.
Hydroxyurea increased F cell number with constant HbF/Fcell resulting in elevated HbF (% and g/dl) and total Hb (g/dl) among b+ IVS-I,6C homozygotes with XMNI --
The data suggested independent control of cellular commitment and expression of g and b globin genes subject to the XMNI genotype in adults but not in neonates.

T. Parsa¹, S. Teimourian², M. Neishaboury¹, M. Neishaboury¹, A. Moritz³, C. Oberkanins³, W. klukelberg³, W. Krugluger³, H. Najmabadi¹^,2;
¹Genetics Research Center the Social Welfare and Rehabilitation University, Tehran, IRAN (ISLAMIC REPUBLIC OF), ²Karimi-Nejad Pathology and Genetics Center, Tehran, IRAN (ISLAMIC REPUBLIC OF), ³Institute of Clinical Chemistry, Rudolfstiftung Hospital, Vienna, AUSTRIA.

A study of molecular lesions of beta-thalassemia in Iran showed a highly heterogenous spectrum of mutations.
More than 100 beta-globin alleles from 50 unrelated thalassemia patients were analyzed for mutations by amplification refractory mutation system (ARMS) or a reverse-hybridization Strip Assay (Vienna lab).
Haplotype analysis using 5 restriction sites was performed on the following 5 mutations: cd 44 (-C), cd 22 (7bp del), IVSI-1 (G-A), cd 36-37 (-T), IVS II-1 (G-A).
Using DNA sequencing 6 different polymorphisms in 9 frameworks were detected in the beta-globin gene of these samples.
Comparison of these frameworks and haplotypes with Mediterranean types reveals several findings: (1) the same polymorphisms are associated with more frameworks in the Iranian thalassemic population than in the Mediterranean, suggesting that the Iranian thalassemic population is more ancient than the Mediterranean; (2) the haplotype associated with the IVS II-1 mutation is in a different framework than the Mediterranean IVS II-1 mutation, indicating the probability of different origins for the IVS II-I mutation.

A. Tordai, H. Andrikovics, V. Homolya, N. Erdei, L. Kalmár, A. Bors;
National Medical Center, Institute of Hematology and Immunology, Budapest, HUNGARY.

Hereditary haemochromatosis (HH) characterised by iron overload is thought to be caused by mutations (C282Y or H63D) of the HFE gene. HH was shown to be of Celtic origin and HFE mutations spread remarkably in the Caucasian population. Beta thalassemia minor (BTM), a mild form of anaemia, also shows a characteristic geographical distribution with high prevalence in the Mediterranian region. Due to ineffective erythropoesis, BTM is associated with iron overload. The goal of the current study was to examine the potential interaction between these two genetic disorders, both with significant prevalence in Hungary. 97 unrelated, consecutive patients (males/females:47/50, average age:33.5 years) with altered laboratory tests characteristic to BTM were examined for the presence of C282Y and H63D mutations of the HFE gene by PCR-RFLP. For the laboratory detection of the BTM phenotype, we used haemoglobin F (HbF), haemoglobin A2 (HbA2) and permeability tests. Each patient showed elevated HbA2 and either elevated HbF or altered permeability. The HFE genotyping showed 5 (5.2%) C282Y-heterozygous and 30 (30.9%) H63D heterozygous patients, two of them compound heterozygous. The C282Y allele frequency (2.6%) was lower than the reference value for Hungary (3.8%), the difference was not significant. The H63D allele frequency (15%) was similar to the reference value (14%). We are currently collecting additional clinical data to assess the iron homeostasis of BTM-patients with different HFE-gentoypes. Our data indicate a tendency towards decreased allele frequency of the C282Y variant among patients with BTM, suggesting a possible negative interaction between the two genetic disorders.

P. C. Giordano, C. L. Harteveld;
Leiden University Medical Center, Leiden, NETHERLANDS.

Hemoglobinopathy (HbP) prevention, achieved at high level in several Mediterranean countries, remains a neglected issue in many immigration areas of Northern Europe. The causes of such a shortcoming are related to the following factors. Lack of awareness by the majority of the population at risk and by the first (G.P.'s and midwives) and second line of healthcare (obstetricians, neonatologists paediatricians). Moreover, know-how and technology for carrier diagnostics is not always available in the laboratory. Carriers, whenever diagnosed, are not sufficiently informed about genetic risk and prevention and not referred for counselling. Finally, requests for financial support to the Dutch prevention foundation, for the implementation of a national prevention strategy, are systematically rejected. In order to offer better healthcare to the population at risk, we have created Hemoglobinopathies Work Groups intended to deal with these problems from different angles. We are trying to improve specific genetic education in the public by spreading leaflets and providing websites. The problem of professional information is tackled by symposia, seminars, publications in professional editiorials, frequent training sessions and patient discussion. We are organizing a nework of labs capable of producing a diagnosis for the 6 basic traits (HbS, E, C, D, b- and a-thalassemia) and to ad trait related genetic information to positive carrier diagnoses. Our aim is to implement carrier diagnostics at the pre-marital or pre-parental stage by intervention of GP or specialist; in early pregnancy, by intervention of midwives and obstetricians and at the neonatal level (newborn screening). Preliminary results are presented.

A. Saxena¹, S. R. Phadke²;
¹Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, INDIA, ²Sanjay Gandhi PGIMS, Lucknow, INDIA.

Cyprus and Greece have shown that by mass screening of general population birth of thalassemic children can be prevented. In India thalassemia is a serious public health problem. Since India is a large country, therefore screening of general population for carrier status is not feasible. Hence, screening has to be restricted to high risk groups. Extended family screening (EFS) means screening relatives of the affected child for carrier status. Objective: To explore if EFS for carrier detection was feasible in India, if not, then what are the barriers to its acceptance. Methods: Hundred couples with a thalassaemic child were interviewed using a predesigned questionnaires. Parents of affected child were given information on disease and its transmission and asked if they had conveyed to their relatives the possibility of their giving birth to a similarly affected child. Results: 96 couples had no reservation in sharing information about their thalassaemic child with relatives. Relatives of 62 couples accepted risk of being carriers but only 14 families got themselves tested. Another 34 families were willing to get tested but because of non - availability of screening facility in near by town, cost of test and lack of sufficient motivation did not get themselves tested. Conclusion: Majority of parents have no reservations in sharing information about the affected child. Communication needs to be improved for all families at risk to accept the risk of having a thalassaemic child. Screening should be more readily available and high risk groups should be motivated through awareness programmes.

A. D. Toraman¹, O. G. Kayisli¹, I. Keser¹, R. Cosan², D. Canatan², G. Luleci¹;
¹Akdeniz University, Faculty of Medicine, Department of Medical Biology and Genetics, Antalya, TURKEY, ²Antalya State Hospital, Thalassemia Center, Antalya, TURKEY.

Beta-Thalassemia, an autosomal recessive disease, is characterized by defects in beta-globin chain production.
Nearly 200 different mutations affecting various different processes in globin gene expression, have been reported as the cause of Beta-Thalassemia, which is a serious health problem in Turkey. Patients with Beta-Thalassemia major have severe clinical symptoms including hepatosplenomegaly, anemia and deformability predominantly in cranial and facial bones. Osteoporosis is emerging as a major cause of morbidity in patients with Beta-Thalassemia major. Polymorphism at the sp1-binding site of the COLIA1 gene is thought to be an important factor in the development of osteoporosis.
We amplified the region including the intronic polymorphism of COLIA1 gene to investigate sp1 gene polymorphism in Beta-Thalassemia major patients with osteoporosis. Amplified gene products were then digested by Bal1 restriction enzyme to detect the base substitution (G-A) in the sp1 binding site. 12 patients out of 15 studied were found to have an SS genotype, while an Ss genotype was observed in the other 3. The distribution of the genotypes was proportionately similar to those reported by others, although no ss genotype was observed in our study, which is the unfavorable genotype, possibly due to low number of subjects studied. Although there are findings to suggest a possible link between the COLIA1 polymorphism with increased rates of osteoporotic fracture, our results, to be extended, suggest careful interpretation of the effect of the ss genotype on bone fractures.

A. Morgado, I. Picanço, A. Miranda, T. Seixas, J. Lavinha, L. Romão, P. Faustino;
Instituto Nacional de Saúde Dr Ricardo Jorge, Lisboa, PORTUGAL.

The classical phenotype of heterozygous beta-thalassemia (beta-thal) can be modified by a number of environmental and genetic interacting factors eg, the cotransmission of a delta-thalassemia determinant reducing the typical increased hemoglobin (Hb) A2 to normal or borderline values.
In this study, we have defined by molecular analysis, the beta- and delta-globin genotypes in a group of 44 individuals with beta-thal-like red cell indices but normal or borderline Hb A2 levels (2.3-3.5%), who were detected in a beta-thal carrier screening program in the Portuguese population. They were tested by ARMS for common Portuguese beta-thal mutations. Delta-globin gene mutations were identified by a PCR-SSCP scanning method followed by sequencing.
We detected a beta-thal mutation in all subjects: 26 were carriers of the beta+IVS-I-6T>C mutation, 7 beta0Cd39C>T, 6 beta0IVS-I-1G>A, and 5 beta+IVS-I-110G>A. Two individuals were double heterozygotes for beta0Cd39C>T in trans with delta+27G>T, and other two individuals were double heterozygotes for beta0IVS-I-1G>A or beta+IVS-I-110G>A and delta+27G>T, respectively. Futhermore, one novel base substitution within the delta-gene promoter region, -80G>A, whose functional consequence is under investigation, was also detected in cis with the delta+27G>T, in one individual with the beta0Cd39C>T.
Another result of this study was the molecular characterization of three putative delta-chain hemoglobin variants detected by isoelectrofocusing or low-pressure ion exchange chromatography: i) delta-Cd16,GGC>CGC(Gly>Arg) - Hb B2 - was identified in heterozygosity in two microcytic and hypochromic individuals presenting also the -alfa3.7kb deletion; ii) delta-Cd136,GGT>GAT(Gly>Asp) - Hb A2-Babinga - was identified in heterozygosity in a hematologically normal individual.

Screening of beta-globin gene mutations causing beta-thalassemia in Romanian population

R. Talmaci, L. Gavrila, L. Dan;
University of Bucharest, Bucharest, ROMANIA.

Beta-thalassemia is a group of inherited recessive disorder in with a defect in synthesis of beta-globin polypeptide chain of hemoglobin is present. From people in all over the world, more than 200 different thalassemia mutations in beta-globin gene have been reported. The aim of our research is to search for distribution of beta-thalassemia mutations in Romanian population. Beta-thalassemia is a frequent genetic disorder in Romania. Therefore, the molecular diagnosis is at present a primary goal for heterozygotes screening and diagnostic confirmation. As being the only center in Romania, having such task, we started mutation screening and molecular diagnosis of beta-thalassemia in Romanian patients. Using direct detection by PCR based methods like ARMS-PCR and RFLP-PCR we have found out that the most frequent gene mutations in Romania are IVS1-6, IVS1-1, IVS2-745, cd 39, IVS1-110, cd 6 and IVS2-1(in order to decreased frequencies). Our data indicated that the mutations identified in Romanian population are of the Mediterranean type. Furthermore, these data will be used in future prenatal diagnosis of beta-thalassemia and for current screening of specific mutations in Romania.

Identification of two novel beta thalassemia mutations and a novel compound heterozygosity in Antalya population: Hb Antalya, Cod 3 (+T)/ IVS1.110, Hb Tyne/Hb S

O. G. Kayisli¹, A. D. Toraman¹, I. Keser¹, A. Yesilipek², D. Canatan³, G. Luleci¹;
¹Akdeniz University, Faculty of Medicine, Department of Medical Biology and Genetics, Antalya, TURKEY, ²Akdeniz University, Faculty of Medicine, Department of Pediatric Heamatology and Oncology, Antalya, TURKEY, ³Antalya State Hospital, Thalassemia Center, Antalya, TURKEY.

Beta-Thalassemia, an autosomal recessive disease, is characterized by reduced synthesis of beta globin gene. Until now nearly 200 different mutations, affecting many different processes in globin gene expression, have been reported. b-Thalassemia and Sickle cell anemia create a serious health problem in Antalya, southern part of Turkey. In this investigation, three patients who were clinically diagnosed as b-Thalassemia minor, intermedia, and Sickle cell anemia/b-Thalassemia, were firstly screened to detect the known common beta globin gene mutations in Mediterranean Region, using reverse dot blot hybridization (RDBH), and the amplification refractory system (ARMS). However, no common mutations were observed in the b globin gene of these patients by the above mentioned methods. For this reason, DNA sequence analyses were performed to detect the sequence changes in b globin gene. We found two novel mutations; Hb Antalya, a partial frameshift mutation in codon3-5 of b globin gene leading to unstable globin chains in a patient with b-Thalassemia minor; a frameshift mutation in Codon 3 (+T) in compound heterozygosity with IVSI.110 in a patient with b-Thalassemia intermedia, and also found a novel compound heterozygosity for Hb Tyne/Hb S in a patient with b-Thalassemia/Sickle cell anemia. We belive that such cases may be considered to be important examples for understanding both the molecular mechanisms of genetic heterogeneity and genotype-phenotype interaction in b-Thalassemia.

Hb Zürich – Altstetten (a2 142 TAA → CAT): A new hemoglobin variant with elongated a - chain analogous to Hb Constant Spring detected in a Thai woman

F. Dutly, H. Frischknecht;
IMD Institute for Medical and Molecular Diagnostics Ltd., Zürich, SWITZERLAND.

During routine hemoglobin screening by ion exchange HPLC we detected a fraction eluting shortly after Hb A2 . The relative concentration was 2 % of total hemoglobin. A 30 year old Thai female resident in Switzerland showed slight anemia, no microcytosis and only minimal hypochromia. The patient was clinically healthy and showed no additional hematological abnormalities.
To identify the mutant hemoglobin, we sequenced the alpha globin genes. The alpha 2 gene showed two transitions in codon 142: the first being the common T → C Hb Constant Spring mutation. Additionally we detected a A → T transition in the third base of codon 142, leading to a His instead of Gln and the additional 30 amino acids as in Hb Constant Spring. First attempts to analyze the mutant hemoglobin by mass spectrometry were not successful. This most likely reflects the instability of the mutant molecule.
The second transition most likely occurred on the background of the Hb Constant Spring mutation and thus should exhibit the same properties with regard to combinations with alpha thalassemia(s).

A Rare Mutation of Beta-Globin Gene (IVS 2-849 A->G) at Exon 2-Intron 2 Splice Site in a Turkish Patient with Beta-Thalassaemia Major

C. F. Sargin¹, A. E. Manguoglu Aydemir¹, N. Nal¹, I. Keser¹, A. Yesilipek², G. Luleci¹;
¹Akdeniz University, Faculty of Medicine, Department of Medical Biology an Genetics, Antalya, TURKEY, ²Akdeniz University,Faculty of Medicine,Department of Pediatric Haematology, Antalya, TURKEY.

Beta-Thalassaemia, among the most common hemoglobinopathies in Antalya, Turkey, is an autosomal recessive disease. Mostly, point mutations on beta-globin gene causes beta-thalassaemia, only in rare cases a deletion or an insertion is resposible for the disease. Reverse Dot Blot Hybridization (RDBH) method is used for screening of common mutations and sequence analysis and silver staining were performed to detect any uncommon mutation. Here, we report a rare variant -intervening sequence 2 (IVS2) 849 A->G- in a Turkish family. While, proband’s mother has IVS2.849 A->G, father has IVS1.1 genotype. The first child of the family has a IVS2.849 A->G/IVS1.1 genotype, with beta-Thalassaemia major phenotype. Prenatal diagnosis was performed for the second gestation and genotype of the fetus was found as IVS2.849 A->G/Normal and parents decided for the continiuation of the pregnancy. Clinical findings were compared with the previous reports . This first report of IVS2.849 A->G mutation in Turkish population, shows that there are many more mutations contributing the heterogeniety of mutation spectrum of beta-globin gene in Turkish population.

H. Najmabadi¹^,2, R. Karimi-Nejad², S. Teimourian², F. Sahebjam², M. H. Karimi-Nejad²;
¹Genetics Research Center, University of Social Welfare and Rehabilitation, Tehran, IRAN (ISLAMIC REPUBLIC OF), ²Karimi-Nejad Pathology and Genetic Center, Tehran, IRAN (ISLAMIC REPUBLIC OF).

Thalassemia is the most common genetic disorder in Iran, with over 2 million carriers of beta- thalassemia. As a first center in Iran, we established in 1990 a prenatal diagnosis for beta-thalassemia. During this period we have diagnosed total of 478 cases (225 amnion and 253 CVS samples). Two strategies direct and indirect (RFLPs) were used for diagnosis. In direct method Arms and beta globin strip assay (Vienna Lab) using 22 common beta globin gene mutation panels specific for Iranian population were performed. Using both techniques we were able to provide a reliable prenatal diagnosis for over 96% of the pregnancies. Out of these samples 21.9% normal for beta globin gene mutation, 46.8% trait, and 27.4 % were affected.
In 3.9 % of the cases we could not determine any mutation or establishing any informative RFLP system. Our data shows very close Mendelian distributions.