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Editor—The autosomal recessive disease β thalassaemia is a common single gene disorder that poses a serious health problem in many parts of the world. According to the Human Gene Mutation Database (http://www.uwcm.ac.uk/uwcm/mg/hgmd.html) and the β-Globin Gene Server (http://globin.cse.psu.edu) about 300 sequence variants in the β globin gene have been identified up to the present. Mutations in the β globin gene have been found at carrier frequency rates ranging from 1% in some areas of Saudi Arabia to 15% in others.1 Both β° and β+ thalassemia have been reported.2 Studies on the molecular pathogenesis of β thalassaemia have shown that the mutations encountered in Arab countries close to the Mediterranean basin are the same as those reported in other Mediterranean populations.3 In the Gulf region, in Saudi Arabia, UAE, and Iraq, the Asian pattern of mutations seems to be prevalent.4 5 The precise genetic changes prevalent in the different regions of the large country of Saudi Arabia and analysis of the genotype/phenotype relationship of the disease in Saudi patients still remain inadequately studied.
The present study aimed to investigate the mutational pattern of the β globin gene and to explore the relationship between these mutations and disease presentation in a group of patients with β thalassaemia major from the eastern region of Saudi Arabia. For this purpose, 31 children diagnosed with β thalassaemia major who over the past two years had regularly attended the paediatric clinics of Qatif Central Hospital or Dammam Maternity and Children Hospital were selected. Within this group of patients there were four pairs of sibs and one pair of first cousins. The whole β globin gene of all patients was amplified using standard PCR techniques and six specially designed different primers for amplification and sequencing. Nucleotide sequencing was performed by electroinjection of the PCR products into an automatic capillary ABI Prism Genetic Analyzer type 310 (Perkin-Elmer, USA).
Results of the nucleotide sequencing have enabled accurate identification of disease causing mutations both in the homozygous and heterozygous states in each of the 31 patients diagnosed with β thalassaemia major. In total, eight disease causing mutations were detected: CD39(C→T),6 IVS-1 3′end-25bp,7−2CD8(−AA),8 IVS-2+1(G→A),9−1CD44(−C),10 +1CD8/9(+G),11IVS-1+5(G→C),11 and IVS-1+5(G→T)12 that comprised allele frequencies of 32.1%, 22.6%, 15.1%, 15.1%, 7.5%, 3.8%, 1.9%, and 1.9%, respectively. An overall β thalassaemia detection rate of 100% was achieved (tables 1 and 2), thus reflecting the efficiency of the technique. The accuracy of the genetic analysis has a special diagnostic importance in view of the fact that certain haemoglobinopathies, for example, sickle cell anaemia, spherocytosis, and autoimmune haemolytic disease, are known to produce symptoms that mimic those of β thalassaemia major. The silent polymorphic mutation CD2(CAC→CAT) was very frequent among this group of children as it was encountered in 16 out of 31 patients with β thalassaemia major. On the other hand, the polymorphisms IVS-2+26 (T→G) and IVS-2+74(G→T) were less common and detected in only two patients each (Nos 29 and 37 and 17 and 45, respectively). A fourth polymorphism IVS-2+666(T→C) was also detected in two patients (Nos 17 and 45). These sequence variants are not shown in table 2.
The spectrum of mutations identified here confirms the notion that, for historical reasons, there is an overlap between Mediterranean and Asian mutations in Saudi Arabia.1 Five of the eight mutations detected here have been reported to exist among patients from other parts of the country, albeit with different allele frequencies.1 We report here the novel identification in the Saudi population of three mutations: the Turkish frameshift mutation -2CD8(-AA),8 the Kurdish frameshift mutation -1CD44(-C),10 and the Mediterranean/Black consensus mutation IVS-1+5(G→T).12 It is highly unlikely that these mutations arose independently in the Saudi population. On the contrary, there is historical evidence that the Arabian, Turkish, and Kurdish populations have interacted in the past through trade and the introduction of Islam in Turkey. The introduction of the Mediterranean/Black mutation in Saudi Arabia can be explained by a similar historical reason, that is, gene flow facilitated by trade and expansion of Islam in the Mediterranean region and in Africa. Similarly, evidence of a common ethnic origin of the cystic fibrosis mutation 3120+1G→A in Arab, African, Greek, and African-American populations has recently been reported.13 However, we have detected the rare Asian Indian mutation +1CD8/9(+G) in the compound heterozygous state in two patients. It is likely that the Indian mutation may have been introduced to this region by gene flow since the mutation has recently been identified in patients from the geographically close island of Bahrain,14 which is a known historical centre for international trade between Arabia, India, and Asia. Similarly, we have found that the splice junction frameshift mutation IVS-1 3′end-25bp, first reported in Asia and India, was the second most prominent mutation with an allele frequency of 22.6% in this group of patients. This mutation has recently been reported as the most common mutation in Bahrain with the highest allele frequency of 40%.14 Therefore, all these findings provide genetic evidence that the eastern coast of Saudi Arabia, unlike the inner desert parts of the Arabian Peninsula, was particularly prone to gene flow from Turkey, Iran, and the Indian subcontinent. The most frequent mutation in the β globin gene detected in this study is the Mediterranean CD39(C→T) mutation with an allele frequency of 32.1%. This mutation has recently been reported to have an even higher allele frequency in Libya, where the mutation, together with IVS-1+6 (T→C) and IVS-1+110 (G→A), comprised an allele frequency of more than 90% in Libyan patients.15 The mutation IVS-1+5(G→C) is a known Asian mutation and has been detected in only one patient in this study. Interestingly, and by contrast, this mutation has recently been found to have the highest allele frequency of 61% in patients from the neighbouring Sultanate of Oman.16
The clinical presentation of β thalassaemia major in this group of children was mostly severe. Most patients suffered from failure to thrive leading to delayed puberty, increased plasma volume, pallor, lethargy, haemochromatosis, hepatosplenomegaly, and jaundice. Less common presentations of the disease noticed in this region of the country included cardiomegaly, repeated infections (such as pneumonia, peritonitis, and meningitis), deformity of the facial bones and teeth, osteoporosis, liver cirrhosis, ascites, and diabetes mellitus. In the clinical management of patients with β thalassaemia, blood transfusion comes second after bone marrow transplantation as the most delicate, laborious, and costly management of the disease. Therefore, for the sake of simplification, the presentation of the disease in this study is classified as severe, moderate, or mild if the number of annual blood transfusions needed is more than six, between six and three, or between two and none, respectively. Hence, 25 patients (81%) presented with a severe form, four patients (13%) with a moderate form, and only two patients (6%) presented with a mild form of the disease. The genotype/phenotype analysis indicated that the mutations CD39(C→T) and -1CD44(-C) in the homozygous state were consistently associated with severe presentation of β thalassaemia major. However we observed variability of disease presentation from severe to moderate and mild caused by the mutations IVS-2+1(G→A), IVS-1 3′end-25bp and -2CD8(-AA) in the homozygous state. It is possible that the phenotypic variation in patients with the same genotype may well find its basis in the number of active α globin genes. Genetic analysis of the α globin gene has not been performed in this study. In two patients who are carriers of the mutations IVS-1+5(G→C) and IVS-1+5(G→T), known to cause β+ thalassaemia, the mutations apparently did not confer protection against the adverse phenotypic expression of the disease. A possible explanation is that the two mutations are encountered here in the compound heterozygous state with CD39(C→T), which is known to cause a severe form of the disease.
In summary, we report the first identification of three mutations, the Turkish frameshift mutation -2CD8(-AA), the Kurdish frameshift mutation -1CD44(-C), and the Mediterranean/Black mutation IVS-1+5(G→T) in patients with β thalassaemia major from the eastern region of Saudi Arabia. Documentation of the spectrum of β thalassaemia mutations could facilitate national screening and educational programmes which would be important with respect to the problem of the haemoglobinopathies in this region.
We are grateful to Dr Samia Flimban of the Maternity and Children Hospital in Dammam for supporting this study. Our thanks are extended to Miss Michaela Finsel for her assistance in the preparation of the manuscript. We gratefully acknowledge the financial sponsorship of this work by the Alexander von Humboldt-Stiftung, Bonn (Bad Godesberg), Germany.