Gene Structure And Functional Analysis Of Two Hereditary Hemolytic Anemias

Anemia is the most common blood disorder, which refers to a reduced concentration of hemoglobin, red blood cell count or hematocrit per unit volume of peripheral blood according to the normal standard of the same age, gender and region. Generally speaking, a diagnosis of anemia is grounded on hemoglobin concentration lower than120g/L, erythrocyte counts below4.5x1012/L and/or hematocrit below42%in adult males, and in adult females based on hemoglobin concentration lower than120g/L, erythrocyte counts below4.52×1012/L and/or hematocrit below42%. Hereditary hemolytic anemia is a group of hematological diseases consist of red blood cell membranes, metabolic enzymes as well as abnormal hemoglobin biosynthesis, leading to a shortened life expectancy of red blood cells. In this group of diseases, the red blood cells become very fragile, reduced deformability and are more susceptible to oxidative damage and accompanied with morphological changes. When hemolysis occurs, hematopoiesis are enhances in the bone marrow. If compensatory hematopoiesis in the bone marrow is inadequate to meet the body’s need, the body will eventually suffer from anemia. In this disease, a large number of red blood cells are destroyed prematurely, and inevitably lead to compensatory proliferation of red blood cells in the bone marrow. This marrow expansion may result in extra-medullary hematopoiesis in extra-medullary hematopoietic organs. In addition, metabolites of red blood cell accumulated in the body after erythrocyte disintegration which have potential to bring great harm to the body. Those hemolytic anemia have similar hematologic features. Among those disease, thalassemia and congenital dyserythropoietic anemia (CDA) are typically heritable hemolytic disease. Thalassemia is a world-wide inheritance disorder which could cause serious symptoms, and the CDA are a heterogeneous group of refractory anemia. This article will explain the clinical phenotype of the two diseases, the molecular basis and pathogenesis by several families. This study will focus on systematically examination of the clinical features and reveal the molecular basis and pathogenesis of the diseases.Section one:A novel fusion gene and a common α0-thalassemia deletion cause hemoglobin H diseaseBackgroundThalassemias are the most common recessive genetic disease in the world’s tropical and subtropical areas of high malaria with a major threat to human health. Among them, a-thalassemia is the most common. These inherited anemia syndrome mainly results from genetic defects of the human a-globin gene cluster mapped to chromosome16p13.3, and thus is characterized by a reduction or complete suppression of a-globin chain synthesis, which leads to a relative excess of β-globin chains in children and adults. While the dissociative beta globin chains are capable of forming insoluble tetramers (β4, or HbH), which are unstable and leading to the increase of erythrocyte damage in blood and various clinical manifestations. Hemoglobin H disease is a moderate type of a-thalassaemia caused by various defects in a globin genes and it is particularly prevalent in Southeast Asia including southern China. Interaction of--SEA allele with the silent a+-thalassemia deletions (such as-α37or-α4.2allele) or non-deletional a-thalassemia mutations in the a2-globin gene (such as aConstant Springa allele) have been extensively demonstrated to be causes of resulting in a Hb H disease phenotype. The extensive homologous sequences of the a-globin gene cluster comprise three segments of homology (X, Y and Z boxes), which are punctuated with non-homologous regions (I, II and III) at the fetal/adult a-globin gene (a2and al). A comparison of the nucleotide sequences of the ψal and a2-globin genes also show that they are approximately73%homologous. It has long been known that recombination between two misaligned wild-type a-globin gene clusters during meiosis may give rise to single a-globin gene deletions, such as-α42and-α3’7. Recently, another two a-globin hybrid genes α121and a212were identified. The origin of the two hybrid genes was most likely a single crossover between a normal allele and an existing recombination allele such as the-α3.7and aaaanti3.7alleles. Here we identified a novel a-globin fusion gene. We discussed its effect on gene transcription and possible origin.Materials and Methods1. Patient samples:The proband was a2-year-old girl from Guangxi Province of southern China. She showed typical hypochromic microcytic anemia. Hemoglobin electrophoresis showed Hb A22.01%, Hb F0.3%, Hb H11%. The girl was diagnosed with Hb H disease, moderate degree of anemia. During the following year, she received periodic blood transfusion once every two months. She was referred for molecular testing to confirm the diagnosis at the age of2years. She and her family members’blood samples were collected using EDTA as anticoagulant after informed consent was obtained.2. Hematological analysis:Hematological parameters were determined by an automated cell counter and hemoglobin analysis was conducted with high performance liquid chromatography.3. Molecular diagnosis:Genomic DNA was isolated from blood leukocytes by a standard phenol/chloroform method. The common defects of both a-globin genes in the Chinese population were determined using gap-PCR. The17known β-thalassemia mutations in the Chinese population were detected by the RDB assay. Sanger sequencing of the entire a-globin genes was performed to identify an unknown mutation. Multiplex ligation-dependent probe amplification (MLPA) was performed to determine the gene dosage of the rare or unknown a-gene cluster variation, according to the manufacturer’s instructions. According to the above molecular detection techniques, A genetic variation located in the3’-untranslation region of a2-globin was identified. A pair of primers was designed to amplify the variation and its flanking sequence and the fragment was determined by Sanger sequencing. Specific primers for the variation were designed to screen the general population.Results and discussionHematology analysis showed that the girl was moderate degree of anemia with microcytosis and hypochromia (Hb91g/L, MCV76fL and MCH25.3pg). Her Hb was86g/L, Hb H was11%, and HbA2was1.8%. The proband’s erythrocyte characteristics and Hb electrophoresis were consistent with Hb H disease. There was no history of jaundice and hepatosplenomegaly at birth. The girl was noticed with an abnormally pale face at11months of age. Both of the proband’s mother and father had a-thalassemic trait, with2.5%HbA2and microcytosis. Results of molecular studies showed that the proband’s mother carried the Southeast Asian a-thalassemia deletion, which the proband inherited, whereas her father had the common-α4.2deletion, which the proband did not inherit.The MLPA peak pattern the proband was compound heterozygosity for (-SEA/) and a variation on exon3of a2gene and her father was a heterozygote for (-α4.2/) and a variation on exon3of a2gene. DNA sequence analysis revealed there were seven conserved single bases in the1.7-kb fragment, which were obviously diverse from the corresponding nucleotide positions on the normal a2gene. This mutation was not present in the National Center for Biotechnology Information database (NCBI) and Globin Gene Server database (http://globin.cse.psu.edu/). A sequence alignment of exon3of a2gene from various mammalian species showed the conservation of the bases. After carrying out a search in the database BLAST search for homology detection showed that the seven base substitutions were almost identical with the ψal gene. Sequence analysis showed that αl and β-globin genes of the family members were negative for any mutations, and there were no copy number variations According to MLPA pattern. An extensive transcript of the fusion gene read through the inter-genic sequence was determined and confirmed by DNA sequence analysis. Further, a new case with the fusion gene and β41-42mutations was identified. He had no anemia, with Hb14.7g/dL, MCV69.4fl, and MCH21pg. The fusion gene identified here enriched thalassemia gene type. Our study performs a new insight to the thalassemia in the world. This variation identified here provides a necessary foundation for molecular diagnostics.Section two:Mutations in human KLF1cause autosomal recessive thalassemia-like congenital dyserythropoietic anemiaBackgroundCongenital derythropoietic aemias (CDAs) are a group of blood disorder characterized by genetic heterogeneity and phenotypic diversity. Usually, CDAs can be clinically divided into several types, including CDA I, CDAII, CDA III and other variants based on morphological abnormalities characteristic of erythroblasts. It has been long recognized that understanding of complex disease associates with the advancing insights of its clinically different phenotypes. The decades of researches have gradually uncovered the molecular mechanisms of those mystery congenital blood diseases. Today, researches are focus on identifying more variants and their molecular mechanisms. Currently, the causative genes for CDA I, CDA II and CDA III have been identified, and their pathogenesis is still a matter of debate. In addition, several subtypes have been described, researches are also needed to systematically elaborate clinically phenotypes and reveal the molecular mechanisms.A recent study revealed a KLF1dominant mutation (E325K) account for a new CDA subtype. KLF1has long been known as a specific Erythroid-specific transcription factor. Studies have shown that natural KLF1mutations were existed in healthy peoeple. Further study revealed that KLF1involved in of various processes of gene expression during erythropoiesis. Although many natural mutations in KLF1were identified in human KLF1, only the dominant mutation E325K associated with human disease. However, KLF1is essential to erythropoiesis for klfl-null mouse died of anemia. Whether other KLF1mutations associated with human disease or not and their phenotypic features are not fully understood. This study derives from our research of thalassemia intermediate and major. We found two thalassemia-like patients with special phenotypes which are irrelevant to thalassemia. This paper aims to identify gene variation responsible for their phenotypes, analyze the impact of the mutations on gene expression, and systematically analyze the clinical features of this disease. We took a further step to propose the differential diagnosis with other similar genetic anemia.Materials and Methods1. Patient samples:Two patients (JK and GH) with congenital anemia of unknown origin, were collected to our lab for genetic diagnosis. Peripheral blood from the patients and other members of the families, and bone marrow samples from the patients and patient GH’s father were taken after parents’consent and approval from the Local Ethics Committee (303Hospital of People’s Liberation Army, China).2. Analysis of clinical manifestations and laboratory examinations:Peripheral blood was examined using automatic blood cell analyzer, ELISA technology and flow cytometry (FCM) to get the erythrocyte parameters, blood biochemical data and red blood cell surface protein expression levels. Erythroblast morphology was captured by a transmission electron microscope. Hemoglobin components (Hb A, Hb A2and Hb F) were determined by high performance liquid chromatography (HPLC), and globin chains were determined by Reverse-phase HPLC.3. Molecular diagnostic methods:Genomic DNA was extracted from peripheral blood leukocytes by standard phenol/chloroform method. Conventional PCR and Sanger sequencing were used to detect a-and β-thalassemia deletions and point mutations. Rare and novel mutations in α-and β-globin genes were screened by multiplex ligation-dependent probe amplification technology (MLPA). Candidate genes related to erythropoiesis were screened by deep sequencing, and the potentially pathogenic mutations by PCR as well as Sanger sequencing.3. Methods of functional analysis:Expression vectors which express a fusion protein tagged with GFP were constructed using the cDNA of potential disease-causing gene KLF1. Expression vectors were transfected into HEK-293cells and K562to analyze the cellular localization. Luciferase reporter vectors drived by β-globin gene, BCL11A and CD44promoters were constructed. Reporter vectors were regulated by KLF1in K-562when reporter vectors co-transfected with Expression vectors. The activity of KLF1and KLF1mutant was determined by co-transfected with Reporter vectors.Results and discussionClinical manifestations showed the probands had moderate hemolytic anemia with microcytosis and hypochromia. They presented with an increase of minor hemoglobin components, such as Hb A2and Hb F, which was very similar to thalassemia intermedia. We also found that the erythrocytes had CD44expression decreased as well as InLu rare blood type. Hemoglobinpathy was excluded by Sanger sequencing and MLPA technologies. Transmission electron microscopic analysis shows intermediate or late erythroblasts with cytoplasmic pseudopodia, distribution of chromatin, large cytoplasmic membrane bulges, and invagination of the nuclear membrane, which was in accordance with dysplastic erythropoiesis. We identified two compound heterozygotes for a previously described frameshift mutation (G176RfsX179) and one of two novel missense mutations (A298P and P338S) in KLF1. Further study showed that mutation in KLF1is probably responsible for this disease. KLF1-targeted promoter-reporter assay showed that the additive effects of the two KLF1variants could cause decreased expression of the HBB, BCL11A, and CD44genes, which are involved in erythropoiesis, with consequent dyserythropoiesis and an oc/non-a chain imbalance. This is the first report of an autosomal recessive thalassemia-like CDA in humans, manifesting as CDA, β-thalassemia, and other distinctive phenotypes. It provides further insight into the multiple roles of KLF1during erythropoiesis, and further improves clinical diagnosis, genetic counseling and prevention for CDA disease.