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Spectrum of Alpha Thalassemia Alleles in Kuwait

Background: The frequency of alpha thalassemia trait is about 40% in the Kuwaiti population, but there has been no comprehensive study of the prevalent alleles. This is a report of the patients who were referred for molecular diagnosis over a 20-year period. Methods: Blood samples from suspected cases were sent to the Hemoglobin Research Laboratory of the Department of Pediatrics, Kuwait University. A retrospective study of the molecular characterization of samples from 1994 to 2015 was carried out. The alpha globin genotypes were determined by a combination of PCR, allele-specific oligonucleotide hybridization and reverse dot-blot hybridization (Vienna Lab Strip Assay). Results: 400 samples were characterized and analyzed from individuals aged <1 month to 80 years, with a median of 6 years (~60% children and adolescents). Most (90.8%) were Kuwaiti nationals. The common genotype was homozygosity for the polyadenylation-1 mutation (αPA-1α/α PA-1α) in 33.3%, followed by heterozygosity (αα/α PA-1α) for the same mutation in 32.3%. The PA-1 was therefore the most frequent allele (0.733). The frequency of the α0, –MED was 0.19. Rare alleles that were found in very low frequencies included the α0 (--FIL) in a Filipino child, Hb Constant Spring, Hb Adana,and Hb Icaria. Conclusion: There is a wide variety of alpha thalassemia alleles among Kuwaitis, but the nondeletional PA-1 is by far the most common cause of moderate to severe HbH disease phenotype. The α0 (–MED) allele is also encountered, which has implications for pre-marital counselling especially the possibility of having babies with alpha thalassemia major (Barts hydrops fetalis).

in the desert areas to as high as 60% in the agricultural zones of Eastern Saudi Arabia [3,4].
The normal individual has a complement of four α-globin genes (αα/αα), any one of which can be absent, producing varying degrees of alpha thalassemia [1]. In α 0 alleles, both genes on a chromosome are deleted (--), while in α + , a variable portion of the α2 or α1 gene is deleted, resulting in reduction in the chain synthesized. The size of the deletion in α 0 alleles is variable and each is named for the part of the world where it is prevalent.
Heterozygotes for these alleles have a thalassemia minor phenotype, but homozygotes and compound heterozygotes often have HbH disease of varying severity and blood transfusion requirements.
Alpha thalassemia frequency is about 30-40% in Kuwait and HbH disease is mostly linked to the polyA (α PA− 1 ) mutation [15,16]. However, there has been no comprehensive study of the whole spectrum of alpha thalassemia alleles. In particular, it is important to establish the prevalence of the α 0 allele in the population to determine the chance of having the more severe alpha thalassemia major (Bart's hydrops fetalis). To this end, we have analyzed all cases of suspected α-thalassemia referred for molecular diagnosis over Blood was obtained by venipuncture into EDTA, complete blood count was done using ABX Pentra 120 cell counter (ABX France, Montpellier). The hemoglobin quantitation was determined using cation-exchange high performance liquid chromatography (HPLC) -Shimadzu LC-20AT, Shimadzu Corporation, Kyoto, Japan. DNA was isolated from peripheral leucocytes using phenol extraction. All the samples were screened for the α-thal-2 (-3.7 kb) deletion [17] and the α2-globin gene polyadenylation site (AATAAA→AATAAG) mutation using a PCR method and allele-specific oligonucleotide hybridization respectively as previously reported [17,18]. All other samples that were not solved with these methods, were subjected to reverse-dot blot hybridization using the Vienna Lab StripAssay technique (Vienna Lab Diagnostics, Vienna, Austria).
The ethnic origins of the patients were documented by their nationalities. The different αglobin genotypes were identified and the allele frequencies calculated. The mean Hb level and RBC indices compared among patients with different genotypes. Clinical data were collected from the HbH disease patients, especially whether they were being followed in a hematology clinic and if they were on any medications or how often they were transfused with blood.
Data are presented as means ± SD or as percentages as appropriate. Statistical differences between mean values among the major groups were tested using Student t test or ANOVA. Continuous variables were subjected to Chi-square test to determine statistically significant differences.

Results
Over the period of the study from 1994 to 2015, there were 400 samples received as suspected moderate to severe alpha thalassemia or HbH disease. Of these, 183 were females and 217 males, with ages ranging from 1 to 80 years and median of 6 years, although more than half (60%) of the patients were below 20 years of age. Most (90.8%) patients were Kuwaitis with a few other nationalities as shown in Table 1. Table 2 shows the distribution of α-globin genotypes in the study. The most common (33.3%) was homozygosity for the α PA− 1 (c.*94A > G), nondeletional α-thal allele, which gives a HbH disease phenotype, followed by heterozygosity for the same allele (32.3%).
The compound heterozygosity of -α − 3.7 /α PA− 1 was the next common at 20.5%. None of the other frequencies reached double digits. Table 3

Discussion
This report is limited to patients who were suspected to have moderate to severe alpha thalassemia based on microcytic, hypochromic anemia, no laboratory evidence of iron deficiency and normal hemoglobin electrophoresis or HPLC. In addition, they had H inclusion bodies and/or H band on HPLC.
The study has revealed the extent of the diversity among individuals with alpha thalassemia in Kuwait. As expected, the vast majority of the patients had the α PA− 1 nondeletional allele, which has been reported as the commonest cause of HbH disease in Kuwait. The frequency of the allele in this study was 0.733. It was found as homozygote in 33.3% of the patients, while heterozygotes accounted for 32.3%. It was present as a compound heterozygote with -α-3.7 kb in 20.5% and in combination with other alleles like -MED , -α − 5nt , α cd59 and α PΑ− 2 .
The third most frequent allele in this study turned out to be the α0, --MED, which was found in 13 patients, in whom it presented a HbH disease phenotype in 10. Of the latter, it was in compound heterozygosity with the α2 cd19 − G in 6 patients from 2 related families, with the -α − 3.7kb , in 2 patients, the α IVS1− 5nt and the α PA− 1 in 1 patient each.
The α PA− 1 allele was first described from Eastern Saudi Arabia[10] and previous studies from Kuwait [2,16] had highlighted its role in the etiology of HbH disease among our patients. It has also been reported as the commonest cause of HbH disease in Saudi Arabia [19], Jordan, [20], Bahrain [21] and UAE [22]. The allele is therefore widespread in the Arabian Gulf, especially in the countries adjoining Saudi Arabia. It is uniformly associated with a moderate thalassemia intermedia phenotype with only occasional blood transfusion requirement, usually with intercurrent infection [16]. HbH disease is mainly a disease of childhood in Kuwait. Most patients are referred to the clinic for investigation of persistent microcytic, hypochromic anemia not responding to iron therapy. Indeed, the anemia tends to improve as the child stops rapid grow the and enters puberty.
This study is the first to report the presence of the α 0 , -MED allele among Kuwaiti patients. In 3 cases, it was as simple heterozygotes while in 10 cases, it was as compound heterozygotes with the pentanucleotide (5nt) deletion in the α IVS1− 5nt in 1, the α PA− 1 mutation in 1, the -α − 3.7 in 2, and the α cd19 (G/A) in 6 (from 2 related families). In all these instances, the phenotype was mild with moderate anemia. However, in 2 patients with the -MED / α cd19 α genotype, the patients were stunted 8 in growth and were on regular transfusion. The other rare alleles found in the study included the α 0