Dr. Ayman Mohsen Mashi, MBBS Consultant Hematology & Blood Transfusion Department Head, Laboratory & Blood Bank King Fahad Central Hospital, Gazan,

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1 Dr. Ayman Mohsen Mashi, MBBS Consultant Hematology & Blood Transfusion Department Head, Laboratory & Blood Bank King Fahad Central Hospital, Gazan, KSA 24/02/2018

2 β-thalassemia syndromes are a group of hereditary disorders characterized by a genetic mutation that leads to a complete absence, severe or mild deficiency of β-globin chains synthesis. The complete absence or severe deficiency results in thalassemia major or thalassemia intermedia.

3 Homozygous and compound heterozygous state (thalassemia major/intermedia) causes severe, transfusion-dependent anemia, whereas heterozygous state (β-thalassemia trait) causes mild-to-moderate microcytic anemia. The molecular bases of β thalassemia are very heterogeneous. The great majorities of β-thalassemia cases are caused by point mutations, affecting the coding region of critical areas of the β-globin gene and are only rarely produced by gross-gene rearrangements.

4 Mutations causing complete inactivation of β genes (such as deletion, initiation codon, nonsense, frameshift, or splicing mutations) will make the gene unable to produce any β-globin chain resulting in β0 thalassemia. Although some other mutations cause partial inactivation of the β genes causing reduction in β-globin chain synthesis resulting in β+ or β++ (silent), thalassemia depends on the degree of reduction of the β chains production.

5 Absence or reduction of β-globin chain will increase the accumulation of free α chain within the erythroblasts and the red blood cells. The major consequences of this pathophysiology are ineffective erythropoiesis, splenomegaly, and tissue hypoxia due to increased hemoglobin F and deformities of the skull and facial bone marrow. The severity of the disease differs according to the ratio between α-globin/non-α-globin chain synthesis and excessing in free α-chain.

6 The β-globin (HBB) genes are located in the short arm of chromosome 11 and controlled by single locus control region. The HBB gene contains three exons, two introns, and both 5 and 3 untranslated regions as well as it contains 146 amino acids with a molecular weight of around 1.6 Kb.

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8 Worldwide, 3% of the populations (~150 million people) are carriers of the β-thalassemia gene. Over 300 mutations in β-globin gene have been characterized globally with a subset of ~40 mutations responsible for the majority of cases, according to population studies.

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10 The mutations are population specific and each country has its own unique and frequency of β-globin mutations. The prevalence of β thalassemia is high in Mediterranean countries, the Middle East, Central Asia, India, Southern China, and the Far East as well as countries along the north coast of Africa, and in South America.

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12 In addition, it is encountered in diverse frequencies in all Arab countries with carrier rate of 1 to 11%. The prevalence of β thalassemia in Saudi Arabia varied significantly in different parts of the country, with the highest prevalence being in the Eastern province of the country (around Jubail, Qateef, Dammam, and Hofuf) and along the coastal strip of the Red Sea.

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25 Samples submitted for molecular screening for β thalassemia during the past 6-year period, 2008 to 2013 from the Medical Genetics and Hematology clinics in King Faisal Specialist Hospital and Research Center (KFSH & RC) (General Organization), Riyadh. Patients who were diagnosed with β thalassemia through clinical suspicion, family history, and have hypochromic microcytic anemia and/or high HbA2 or HbF level in hemoglobin electrophoresis were included for analysis. Family studies were excluded from this cohort. This retrospective review study was approved by the Research Advisory Council and the hospital ethical committee under the RAC# The samples represent cases from all over the kingdom that is referred to KFSH & RC for treatment options. Selection criteria were applied.

26 This study was carried out on a total number of 131 patients who were recruited for clinical investigation from the Hematology clinic at KFSH & RC for a 6-year period. Out of the total population, 28 (21%) were undetectable cases and 103 (79%) were detectable cases for β globin chain mutations.

27 The male gender represented 57/131 patients (43.5%), whereas female patients were 74/131 (56.5%). The pediatric patients (15 years or less) are account for 52% and adult patients (more than 15 years) account for 48%).

28 Detectable cases (103 patients) were categorized into three groups according to allele zygosity: homozygous, heterozygous, and compound heterozygous; 47 (45.6%), 41 (39.8%), and 15 (14.6%), respectively. The majority of homozygous cases are discovered in pediatric pateints, whereas the majority of adult cases are heterozygous.

29 Nineteen mutations were identified in all detectable (103 patients) cases that are illustrated in Table 1.

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31 Three mutations (c.315+1g>a, c.118c>t, and c.92+5g>c) were detected in the majority of cases (66%). The c.315+1g>a, previously known as IVS-II-1G>A, is a splicing mutation that was most frequently encountered in our study with a frequency of 32%. Followed by truncating nonsense mutation, the c.118c>t (previously known as Q39X or p.gln 40X) 23% and the c.92+5g>c (previously known as IVS-I-5G>C) 11%. The c.315+1g>a and c.118c>t mutations were common in homozygous cases, whereas c.92+5g>c was more common in compound heterozygous cases.

32 Five novel mutations (c.410g>a, c.-31c>t, c.68_74delaagttgg, c.316-3c>a, and c.-151c>t) were identified for the first time in Saudi population. The former three mutations represent 1% of cases for each, whereas the later presented with a frequency of 2%.

33 More recent studies were conducted to provide a precise figures and frequencies of the β-thalassemia mutations in specific region of Saudi Arabia. All previous studies that reported different β-globin gene mutations in Saudi Arabia are summarized in Table 2.

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35 Abuzenadah et al. have recently identified 23 mutations responsible for β-thalassemia in western region of Saudi Arabia. Of these, there were seven common mutations with the most frequent one being IVS-I-5G>C and the other 16 mutations were less common, including one new mutation that has never been reported in the Saudi Arabia, the FS c20/21 mutation.

36 In other recent studies reported from the Eastern Province of Saudi Arabia by Al-Sultan et al., 14 mutations were identified. Among these, there were five frequent mutations with the more common one being IVS-II-1G>A [Table 2]. The author reported two novel mutations IVS-I-130 (G C) and IVS-I-110 (G A), which have not been previously reported in the population of the Eastern Province.

37 A total of 12 different mutations were reported in one more recent study from Riyadh by Warsy et al., in which the IVS-I-5G>C mutation was the most common.

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40 In this study, we present a comprehensive report on the spectrum of β-thalassemia mutations. Given that, KFSH and RC represents a tertiary specialized hospital in Saudi Arabia in which advance molecular technologies are available for detecting wide range of molecular abnormalities.

41 Our study more and less shows similar findings that had been observed by previous studies. A total of 19 mutations were identified in this retrospective report. Out of these, 14 mutations have been reported in the previous studies that have been conducted in Saudi Arabia and five novel mutations were reported for the first time in Saudi population.

42 It has been noticed that the frequencies and prevalence of the 14 mutations are different among the Saudi population who screened for these mutations in the previous studies. This diversity is mainly due to the unique geographical position of Saudi Arabia that lie between the Mediterranean and Southeast Asian region.

43 The most frequent mutations in our study are c.315+1g>a, c.118c>t, and c.92+5g>c. These findings were also reported by most of studies on Saudi population with differences in their frequencies. The clinical significance of c.315+1g>a and c.118c>t mutations is their ability to produce the β0 thalassemia phenotype. These mutations were reported to occur in most of the Mediterranean and Gulf countries (Warsy, Abuzenadah, AlSultan, AlAli), although the c.92+5g>c produces β+ thalassemia phenotype and frequently encountered in Asian Indian.

44 In comparing our data to previous studies, we found that five mutations appear to be fingerprints of Saudi population and were most frequently reported in all studies. These mutations are c.315+1g>a, c.118c>t, c.92+5g>c, c.93-22_del 25bp, and c.93-21g>a.

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46 We report five novel mutations (c.410g>a, c.-31c>t, c.68_74delaagttgg, c.316-3c>a, and c.-151c>t), which to the best of our knowledge, have not been described in Saudi Arabia.

47 In summary, this comprehensive analysis successfully identified 19 mutations, five of which are novel, and confirmed the previously published mutations among the Saudi population. The potential outcome of this study is to publish a list of more frequent β-globin gene mutations in Saudi β-thalassemia patients.

48 Thank you,