Single Alpha-globin Gene Deletion Research Articles

KLF1 Mutations Are Not Common in Israel but Can Explain Occasional Cases of Elevated HbA2 or Very Elevated Fetal Hemoglobin

Introduction: Mutations in KLF1 (Kruppel Like Factor1) have been noted to cause a number of different phenotypes of erythrocyte abnormalities, as KLF1 is known as a master regulator of many genes expressed in red blood cells. One important manifestation caused by KLF1 mutations is the upregulation of γ- or δ-globin genes, with associated microcytosis, thus mimicking beta thalassemia (β-thal) trait. Such findings cause difficulty in counseling couples referred for prenatal diagnosis of β-thal. KLF1 mutations have been reported to be more frequent in geographic regions where β-thal is common. Therefore we undertook to analyze for the presence of KLF1 mutations in Israeli individuals.Materials and Methods: We selected 100 individuals for analysis belonging to one of 4 different groups: 1. Individuals with isolated elevated HbA2 (n=14), or isolated elevation of HbF (n=13) or elevation of both HbA2 and HbF (n=6), who are not carriers of a β-thal mutation by sequence analysis. 2. Individuals with β-thal trait (n=19) who have a higher HbF (and/or HbA2) than is expected for β-thal trait, who do not have β-thal intermedia on clinical criteria (blood count, peripheral smear, spleen size). These individuals carried one of 8 mutations known to cause β-thal in our region 3. Two patients (pts) with a history of transfusions (one with massive splenomegaly and sickle trait, suspected to have coinherited CDA, and one with β-thal intermedia with HbF elevation unexpectedly high for his mutation (TATA box -28 A to C; HBB:c.-78A>C). This patient had HbF levels ranging from 23-33% and HbA2 ranging from 7.8-8.9% over years of followup from age 39-50 years. 4. Anonymous controls (n=46) who are pts with a hematological malignancy not suspected of carrying a hemoglobinopathy. KLF1 (exons 1, 2 and 3) was amplified using PCR (exon 1: 564 base pair product and exons 2 and 3: 1703 base pair product). PCR products were subjected to DNA sequence analysis using an Applied Biosystems ABI apparatus. The sequence obtained was analyzed using BLAST alignment and deviations from the published sequence were analyzed using the Mutation Taster program.Results: Two pts were found to carry substitutions with possible or proven clinical significance. One pt is heterozygous for a substitution at c.972C>A (codon 324 exon 3, E324D, aspartic acid to glutamine) which has not been previously reported. According to Mutation Taster this substitution may have clinical significance. This pt, who has normal hematological parameters, had isolated HbA2 elevation (4.1%) with no β-thal mutation identified. The second pt was found to be heterozygous for a substitution at c.901C>T (codon 301 exon 2, R301C, arginine to cysteine). This substitution has been reported by Gallienne et al, 2012, to be associated with elevated Hb F. This young pt was found to have 28% Hb F with a low HbA2 level of 1.4% and MCV of 63.8. His father also had 28% HbF. No β-thal mutation was identified in the pt or his father but the patient was found to be homozygous for a single alpha globin gene deletion. In addition to these 2 substitutions, many pts and controls carried known polymorphisms in KLF1. These polymorphisms are: c.304T>C, exon 2 residue 102 (S102P) (found in 24 pts and 19 controls); -148(G>A), in the upstream noncoding region (found in 11 pts and 8 controls); c.544T>C codon 182 exon 2 (F182L) (found in 3 pts and 4 controls); and c.115A>C, codon 39 in exon 2, M39L (found in 1 pt and 3 controls). One rare, previously unreported substitution was found in heterozygous form in the thal intermedia pt, located at c.259C>G codon 87 exon 2, P87A (proline to alanine). This was not found in the controls, however according to the location in the gene, and to Mutation Taster, this is not suspected to be a functional variant.Conclusions: Functional variants in KLF1 were rarely found in this group of patients. One out of 14 (7%) of individuals with isolated elevated HbA2 and 1/13 patients with isolated HbF elevation (7.7%) were identified as carrying heterozygous KLF1 functional variants. Two novel substitutions were found: E324D which is suspected to have clinical significance and P87A which is not presently suspected to be a functional variant. We conclude that KLF1 analysis may explain occasional individuals with high HbA2, or marked HbF elevation, in the absence of β-thal trait in our patient population. Larger studies are needed to confirm these findings. DisclosuresNo relevant conflicts of interest to declare.

Haemoglobin variants, iron status and anaemia in Sri Lankan adolescents with low red cell indices: A cross sectional survey

Iron deficiency complicates the use of red cell indices to screen for carriers of haemoglobin variants in many populations.In a cross sectional survey of 7526 secondary school students from 25 districts of Sri Lanka, 1963 (26.0%) students had low red cell indices. Iron deficiency, identified by low serum ferritin, was the major identifiable cause occurring in 550/1806 (30.5%) students. Low red cell indices occurred in iron-replete students with alpha-thalassaemia including those with single alpha-globin gene deletions. Anaemia and low red cell indices were also common in beta-thalassaemia trait. An unexpected finding was that low red cell indices occurred in 713 iron-replete students with a normal haemoglobin genotype.It is common practice to prescribe iron supplements to individuals with low red cell indices. Since low red cell indices were a feature of all forms of α thalassaemia and also of iron deficiency, in areas where both conditions are common, such as Sri Lanka, it is imperative to differentiate between the two, to allow targeted administration of iron supplements and avoid the possible deleterious effects of increased iron availability in iron replete individuals with low red cell indices due to other causes such as α thalassaemia.

Effectiveness of Imatinib Therapy for a Patient with Sickle Cell Anemia and Chronic Myelocytic Leukemia.

Abstract 2559Poster Board II-536 Background:To date, hydroxyurea (HU) is the only FDA-approved agent to treat sickle cell anemia (SCA). There is a need to identify alternative agents to prevent the complications of sickle cell disease. Clinical observations of patients receiving medications for other indications may provide insights into new opportunities. We present the clinical course of a patient with SCA and chronic myelocytic leukemia (CML) treated with imatinib. Case Report:A 24 year old woman with hemoglobin (Hb) SS had a variably elevated white blood cell (WBC) count for 3 years. WBC counts ranged from 11–39 × 103/cu mm; differential typically included 65–75% neutrophils, 15–20% lymphocytes, 1–13% metamyelocytes, and 1–3% myelocytes. Consistent with previous values, Hb ranged from 9–11 g/dL, and platelet counts from 250–400 × 103/cu mm. Real-time PCR for BCR-ABL obtained from the peripheral blood was positive. Bone marrow biopsy showed a markedly hypercellular marrow with granulocytic hyperplasia. Blasts were not increased in number. Cytogenetic studies revealed 46 XX, t(9;22)(q34;q11.2) in 20/20 analyzed cells. Further characterization by fluorescence in situ hybridization (FISH) technique demonstrated the loss of chromosome 9 long arm material. BCR-ABL interphase FISH confirmed the presence of 9/22 chromosomal translocation, found in 279/300 interphase nuclei. The patient was diagnosed with SCA in early childhood. Molecular analysis of genomic DNA revealed homozygosity for Hb S and single alpha-globin gene deletion of the rightward type. There was no evidence for typical deletions associated with HPFH or delta-beta zero thalassemia. The C>T polymorphism at nt −158 in the G-gamma-globin gene promoter region and other known HPFH point mutations in the G- or A-gamma globin genes were not identified. Her clinical course has not been complicated by stroke, pulmonary hypertension or acute chest syndrome. Radiographic studies revealed evidence of multiple bony infarcts. She was hospitalized with painful episodes 1-2 times per year, until the age of 16. At that time, her frequency of hospitalizations for painful episodes increased to 3-5 times per year, and showed a temporal relationship to her menses. She was started on oral contraceptives and hydroxyurea (HU) at the age of 18. Subsequently, hospitalizations for painful episodes decreased to 0-2 per year. At the age of 22, hydroxyurea was held during pregnancy. After delivery, HU was reinitiated and continued until the diagnosis of CML. She was hospitalized for painful episodes two times in the 12 months preceding the diagnosis of CML. Imatinib mesylate monotherapy at 400 mg/day was initiated. Two weeks later, a hematologic response was documented. At one year, bone marrow cytogenetic and molecular complete responses were documented. In the 17 months since initiation of imatinib, the patient has had a significant reduction in the frequency of painful episodes. No hospitalizations have been required and the patient has had a reduction in daily pain allowing discontinuation of a transcutaneous electrical nerve stimulation unit. During imatinib therapy Hb levels ranged from 8.0 to 9.2 g/dL, WBC counts from 3.3–6.1 × 103/cu mm, and platelet counts from 114–205 × 103/cu mm. Hemoglobin electrophoresis results are shown in the table.Hb FHb SPre-HU*26%71%Post-HU (n=7, mean)*35%63%Post-imatinib (n=2, mean)*34%64%Post-imatinib (by HPLC)20%74%*by gel electrophoresis Clinical Implications:To our knowledge, the clinical course of patients with SCA treated with imatinib has not previously been reported. Imatinib targets the BCR-ABL fusion tyrosine kinase, c-kit, and platelet-derived growth factor receptor. Myelosuppression is frequently noted as a side effect of this therapy. The improvement in this patient's sickle cell symptoms may be attributable to a reduction in the neutrophil count, as observed with HU therapy, but it is possible imatinib therapy had a positive effect on release of cytokines or other factors involved in vaso-occlusion. The Hb F level was not significantly increased with imatinib therapy. Evaluation of small molecule tyrosine kinase inhibitors in experimental models of SCA should be considered. Disclosures:No relevant conflicts of interest to declare.

Heterogeneity in α‐thalassemia interactions in Malays, Chinese and Indians in Malaysia

Interactions between different determinants of alpha-thalassemia raises considerable problems, particularly during pregnancies where antenatal diagnosis is necessary. This study aims to determine the different types of deletional alpha-thalassemia and Hemoglobin Constant Spring (HbCS), and their frequency in Malays, Chinese and Indians in Malaysia. DNA from 650 pregnant women from the Antenatal Clinic of the University of Malaya Medical Center in Kuala Lumpur, Malaysia who showed mean cell volume < or =89 fL and/or mean cell hemoglobin < or =28 pg were analyzed for the double alpha-globin gene South-East Asian deletion (--SEA), the -alpha3.7 and -alpha4.2 single alpha-globin gene deletions and HbCS. One hundred and three (15.8%) of the pregnant women were confirmed as alpha-thalassemia carriers: 25 (3.8%) were alpha-thalassemia-1 carriers with the --SEA/alphaalpha genotype, 64 (9.8%) were heterozygous for the -alpha3.7 rightward deletion (-alpha3.7/alphaalpha), four (0.6%) were heterozygous for the -alpha4.2 leftward deletion (-alpha4.2/alphaalpha), nine (1.4%) were heterozygous for HbCS (alphaCSalpha/alphaalpha) and one (0.2%) was compound heterozygous with the -alpha3.7/alphaCSalpha genotype. The double alpha-globin gene --SEA deletion was significantly higher in the Chinese (15%) compared to the Malays (2.5%) and not detected in the Indians studied. The -alpha3.7 deletion was distributed equally in the three races. HbCS and -alpha4.2 was observed only in the Malays. The data obtained gives a better understanding of the interactions of the different alpha-thalassemia determinants in the different ethnic groups, thus enabling more rapid and specific confirmation of alpha-thalassemia in affected pregnancies where antenatal diagnosis is necessary.

Prevalence of α-Globin Gene Deletions Among Patients with Unexplained Microcytosis in a North-American Population

Increasing multi-ethnicity is likely to make alpha-thalassemia (alpha-thal) more prevalent in Western metropolitan areas. Multiplex polymerase chain reaction (m-PCR) allows rapid and precise identification of most of alpha-thal carriers. With this method, we sought to determine the prevalence of alpha-thal and the corresponding genotype, among all non repetitive consecutive blood samples that had an unexplained microcytosis. These specimens had been sent to the hematology laboratory for a blood count analysis, found to be microcytic, and secondarily tested for ferritin level and hemoglobin (Hb) high performance liquid chromatography (HPLC) profile. Five hundred and sixteen microcytic blood samples were evaluated and 197 samples with normal ferritin and Hb HPLC were studied by m-PCR. Among 196 interpretable PCRs, 48 alpha-thal cases (24.5%) were identified: 28 with a single alpha-globin gene deletion and 20 with two alpha-globin gene deletions. Of these 20 cases, six showed two deletions in cis. None of the erythrocytic parameters studied predicted the presence of alpha-thal deletions. We conclude that a significant proportion (24.5%) of blood counts with microcytosis not explained by an iron deficiency, an inflammatory state or an abnormal Hb on HPLC, are caused by an alpha-globin gene deletion. The pertinence of genetic counseling for alpha-thal based on molecular diagnosis should be evaluated more formally in urban centers where this genetic condition is likely to have an increasing prevalence and clinical relevance.

Prevalence study and molecular characterization of alpha-thalassemia in Filipinos.

In order to determine the prevalence and molecular basis of alpha-thalassemia (thal) among Filipinos, a total of 2954 Filipinos in Taiwan were enrolled in this study. A complete blood count was done for every subject. Those with microcytosis (MCV less than 82.5 fl) were studied with hemoglobin (Hb) high-performance liquid chromatography to determine the levels of Hb A2 and Hb F, and with an enzyme immunoassay to determine plasma ferritin levels. Those who had microcytosis and normal or low levels of Hb A2 and Hb F were further studied with molecular methods for alpha-globin gene mutations. We used Southern blot hybridization and/or the polymerase chain reaction to detect Southeast Asian deletion, Filipino deletion, rightward and leftward single alpha-globin gene deletions, and Hb Constant Spring and Hb Quong Sze. Specific amplification and direct DNA sequencing of the alpha2- and alpha1-globin genes were carried out in apparent alpha-thal carriers without any of the above-mentioned mutations. Our results showed that in Filipinos the prevalence of alpha-thal 1 was 5% (147 carriers) and that of alpha-thal 2 was 1.7% (49 carriers); two had Hb H disease. Among the alpha-thal 1 carriers, 89 had the Southeast Asian deletion and 58 had the Filipino deletion. Among the alpha-thal 2 carriers, 48 had a rightward deletion and one had a leftward deletion. None had Hb Constant Spring or Hb Quong Sze. Specific amplification and DNA sequencing in five apparent alpha-thal carriers did not reveal mutations in the 2-kb region spanning the alpha2- and alpha1-globin genes. The molecular defects of alpha-thal in Filipinos were different from those in the neighboring ethnic groups. Elucidation of the alpha-thal mutations in Filipinos is useful in the genetic counseling and prenatal diagnosis of this common disease.

Genotype of subjects with borderline hemoglobin A2 levels: Implication for, β‐thalassemia carrier screening

In this study, we have defined by molecular analysis, the alpha, beta, and delta globin genotype in a group of individuals with normal or thal-like red cell indices but borderline hemoglobin (Hb)A2 levels, who were identified in a program for beta-thal carrier screening. In 37 of 125 individuals with borderline HbA2 levels, we detected a molecular defect in the beta, in both the delta and the beta, or in the alpha globin gene. Specifically seven of these subjects were carriers of the -101 C T mutation, ten of the IVSI nt6 T C mutation, 16 were double heterozygotes for delta and beta thal, and two had the triple alpha globin gene and two the single alpha globin gene deletion. From these results, we may conclude that subjects with borderline HbA2, particularly when they marry a typical beta-thal carrier, should be extensively investigated in order not to miss heterozygous beta-thalassemia.

The pathogenesis of hypochromic anaemia in Saudi infants.

The pathogenesis of hypochromic anaemia was studied in 138 Saudi bedouin infants aged 9 months. Approximately 25 per cent had hypochromic anaemia, but less than 10 per cent had serum ferritin levels indicative of iron deficiency. A few infants had heterozygous beta-thalassaemia, but many infants with hypochromic anaemia had normal haemoglobin A2 levels together with serum ferritin levels above 20 micrograms/l. DNA analysis of cord blood taken from the hospital where the infants were born showed that the frequency of the single alpha-globin gene deletion type (-alpha 3.7) of alpha-thalassaemia is 0.13 in the bedouin population of Western Saudi Arabia. alpha-Thalassaemia probably accounts for much of the anaemia previously thought to be due to iron deficiency in Saudi infants. Studies of iron status and estimation of the frequency of genetic causes of hypochromic anaemia are important when assessing the need for widespread nutritional programmes to prevent iron deficiency and in the interpretation of reference ranges of red cell indices in populations from malarial areas.

Alpha-thalassemia in Papua New Guinea.

A study of the distribution of alpha-thalassemia in Papua New Guinea (PNG) was carried out by DNA analysis. A total of 664 DNA samples were screened for alpha-thalassemia 2 and alpha-thalassemia 1 caused respectively by either deletion of one or both of the duplicated alpha-globin genes. alpha-Thalassemia 2 was detected in high frequencies in coastal and lowland regions where malaria has been holo- to hyperendemic but in low frequencies in non-malarious highland regions. The highest frequency was observed in the north coast of PNG. The distribution of alpha-thalassemia 2 seems to be in accordance with other conditions such as ovalocytosis and G6PD deficiency which are also prevalent in this population, suggesting that they may interact in protection against malaria. However, it appears to be negatively correlated with beta-thalassemia and alpha-thalassemia 1, the latter being extremely rare in this population. Analysis of the types and subtypes of the single alpha-globin gene deletion revealed a predominance of the -alpha 4.2 type in general, except in some regions in the south where the -alpha 3.7 type is prevalent. The -alpha 3.7 I subtype is the common form of the -alpha 3.7 deletion in the PNG mainland. The -alpha 3.7 III subtype, previously reported to be unique in Melanesians and Polynesians, was detected in an offshore island of PNG. However, this subtype is very rare in Melanesians from the PNG mainland.

Alpha thalassaemia in an Italian population.

The incidence of alpha-thalassaemia in an Italian population has been determined by a survey of random cord bloods for the presence of Hb Bart's. 144 out of 4730 (3%) had detectable amounts of Hb Bart's. Furthermore, alpha-globin gene analysis of 100 random cord bloods showed that five out of 100 had the common type of alpha-thalassaemia caused by a single alpha-globin gene deletion (-alpha). The molecular basis of alpha-thalassaemia was also determined in a selected group of 34 newborns with detectable levels of Hb Bart's. 25 of these cases had the -alpha 3.7 deletion type of alpha-thalassaemia and nine had nondeletion types of alpha-thalassaemia in four of which the molecular defect was detectable directly by restriction enzyme analysis.

A genetic combination of silent beta-thalassaemia, high Hb A2 beta-thalassaemia, and single alpha globin gene deletion causing mild thalassaemia intermedia.

This paper reports a Sardinian patient, who was a compound heterozygote for silent beta-thalassaemia and high Hb A2 beta o-thalassaemia with the clinical phenotype of mild thalassaemia intermedia; alpha globin...