Jill Finlayson

Thalassemia in Western Australia: 11 novel deletions characterized by Multiplex Ligation-dependent Probe Amplification

The number of immigrants in Western Australia from many different areas where hemoglobinopathies are endemic has increased dramatically since the 1970s. Therefore, many different thalassemia mutations have been introduced in the country, which add a technological diagnostic problem to the serious burden of hemoglobinopathy management and to public health care. Recently, we have developed a rapid and simple technique based on Multiplex Ligation-dependent Probe Amplification to detect deletions causing alpha-and beta-thalassemia, deltabeta-thalassemia and Hereditary Persistence of Fetal Hemoglobin. A screening for (unknown) deletions was performed in a cohort of patients of different ethnic backgrounds preselected for their thalassemia phenotype, in which common deletions and point mutations were excluded. Out of 37 cases suspected to carry a deletion, 27 were found to carry 17 different deletion types of which 6 causing alpha-thalassemia and 5 causing beta-thalassemia were novel. For 3 of the deletions, we have been able to characterize the exact breakpoint sequences by long-range PCR and direct sequencing. These results show that MLPA is a suitable technology to detect unknown and uncommon deletions. These could represent a diagnostic problem when offering prevention to couples at risk presenting with unclear phenotypes and might result in a serious fetal problem when the deletion involves embryonic genes.

Transfusion-related acute lung injury in a neutropenic patient

Transfusion‐related acute lung injury (TRALI) is a leading cause of transfusion related morbidity and mortality. Current concepts regarding the pathogenesis of this disorder imply a “two‐hit” model in which neutrophils are sequestered in the pulmonary capillary bed, and subsequently activated by substances in the transfused blood product. We report a case of TRALI in a patient with neutropenia and discuss the possible factors contributing to the respiratory symptoms in this patient. We also emphasise the importance of recognising mild cases of TRALI in order to investigate the implicated donor/s appropriately, and to minimise the risk for more severe episodes in other patients.

Two New α1-Globin Gene Point Mutations: Hb Nedlands (HBA1:c.86C>T) [α28(B9)Ala→Val] and Hb Queens Park (HBA1:c.98T>A) [α32(B13)Met→Lys]

We report two new point mutations of the α1-globin gene found in a Greek and a Burmese patient, both living in Western Australia. The patients were initially selected for their microcytic hypochromic parameters as belonging to a group suspected for uncommon (deletion) defects. Gap-polymerase chain reaction (gap-PCR) and multiplex ligation-dependent probe amplification (MLPA) technologies were applied, and in those cases not showing deletions, direct sequencing was performed. We have found 1) HBA1:c.86C>T, Hb Nedlands [α28(B9)Ala→Val] which, based on the red cell indices and phenotype prediction scores, is presumed to be clinically silent, and 2) HBA1:c.98T>A, Hb Queens Park [α32(B13)Met→Lys] which seems to be associated with a mild α-thalassemia (α-thal) phenotype. The phenotype/genotype correlation is briefly described.

A Moderately Severe α-Thalassemia Condition Resulting From a Combination of the α2 Polyadenylation Signal (AATAAA→AATA– –) Mutation and a 3.7 Kb α Gene Deletion in an Australian Family

We have recently studied a family with a rare combination of two abnormal α-globin genes. The combination of a two-base (AA) deletion in the α2 polyadenylation signal (poly A) (AATAAA→AATA– –) and a 3.7 kb α gene deletion, found in two children, resulted in a moderately severe thalassemic condition. Both parents and three siblings were tested and the hematological condition and molecular findings are presented. The father was born in India with Portuguese and British ancestry; the mother is of Dutch ancestry. All three siblings were born in Australia.

In vitro characterization of the α-thalassemia point mutation HBA2:c.95+1G>A [IVS-I-1(G>A)(α2)]

The α-thalassemias are a group of disorders occurring as a result of decreased synthesis of α-globin chains, most commonly due to deletions of α-globin genes. Detection of α-thalassemia (α-thal) caused by point mutations has increased during the past few years and more than 70 different point mutations have been reported for the α1- and α2-globin genes. The mutation at the splice donor site of the first intervening sequence [IVS-I-1 (G>A)] of the α2-globin gene, HBA2:c.95+1G>A, is thought to cause a thalassemic phenotype by interfering with and preventing the normal splicing of pre-mRNA. We developed an in vitro expression system to study α-globin gene point mutations at the molecular and cellular levels. The expression vector carrying the HBA2:c.95+1G>A mutation (α2GIVS-I-1G>A) was created using site-directed mutagenesis of a wild type (WT) construct of the α2-globin gene (α2G2034WT). Gene expression experiments in human bladder carcinoma 5637 cells were carried out using sequence verified WT and mutated clones. Complementary DNA synthesis and polymerase chain reaction (PCR) analysis showed normal α2-globin transcripts from cells transfected with the WT vector, but aberrant transcripts from cells transfected with the mutated vector carrying the splice donor site mutation. In the presence of the G>A mutation, normal splicing does not occur, and a cryptic splice site 49 bp upstream of the normal site is used. The translation of this product produces a premature termination codon, thus resulting in a thalassemic phenotype.

Hematologic Features of Hepatosplenic T-Cell Lymphoma

To the Editor We read with interest the article by Chen and colleagues1 in which they describe the clinical and pathologic characteristics of 7 cases of the rare CD4–/CD8– γδ subtype of T-cell large granular lymphocytic (T-LGL) leukemia. The authors state that 16 cases of CD4–/CD8– γδ T-cell neoplasm had initially been identified, but 9 had been excluded because they represented “various other types of γδ T-cell lymphoma/leukemia involving the blood and bone marrow.”1 It is unfortunate that the data on which these exclusions were made was not provided. This information would have been valuable, especially in determining whether any of the 9 excluded cases were, in fact, hepatosplenic T-cell lymphoma (HSTL), another rare disease with phenotypic similarities with CD4–/CD8– γδ T-LGL leukemia. The data from the resulting 7 cases described in the published series infer that the lack of isochromosome (7q), a feature of HSTL, was a major discriminator, as 6 of 6 tested cases were negative. However, the authors downplay the significance of cytogenetics by only stating that it “may be helpful in difficult cases” in distinguishing between CD4–/CD8– γδ T-LGL leukemia and HSTL. Instead they claim that, although the distinction between these 2 entities can be difficult, it may be readily achieved on morphologic studies and the pattern of splenic and marrow infiltration.1 We want to make particular comment on the …

Differential gene expression analysis in early and late erythroid progenitor cells in β-thalassaemia

β‐ thalassaemia is a disorder of globin gene synthesis resulting in reduced or absent production of the β‐globin chain in red blood cells. In this study, haematopoietic stem cells were isolated from the peripheral blood of six transfusion dependent β‐thalassaemia patients and six healthy controls. Following 7 and 14 d in culture, early‐ and late‐ erythroblasts were isolated and purified. No morphological difference in maturation was observed following 7 d in culture, while a delayed maturation was observed in the patient group after 14 d. Following RNA isolation and linear amplification, gene expression analyses were performed using microarray technology. The generated data were analysed by two methods: the BRB‐ArrayTools platform and the Bioconductor platform using bead level data. Following 7 d culture, there was no difference in gene expression between the control and patient groups. Following 14 d culture, 384 differentially expressed genes were identified by either analysis. A subset of 90 genes was selected and the results were confirmed by Quantitative‐Real‐Time‐polymerase chain reaction. Pathways shown to be significantly altered in the patient group include apoptosis, MAPKinase and the nuclear factor‐κB pathway.

Validation of the immunochromatographic strip for α-thalassemia screening: a multicenter study

α(0)-Thalassemia occurs from a deletion of 2 linked α-globin genes and interaction of these defective genes leads to hemoglobin (Hb) Barts hydrops fetalis, the most severe and lethal thalassemia syndrome. Identification of α(0)-thalassemia carriers is thus essential for the prevention and control program. An immunochromatographic (IC) strip test was developed for rapid screening of α(0)-thalassemia by testing for Hb Barts in the blood samples using a specific monoclonal antibody against Hb Barts. To evaluate its sensitivity and specificity, the IC strip test was assessed in a cohort with various thalassemia genotypes from 4 different laboratories in Thailand and Australia. The result showed 97% sensitivity in α-thalassemia carriers with 2 α-globin genes deletion and Hb H disease. This is, in particular, the useful rapid screening test for regions where β-thalassemia and homozygous Hb E are also common. Similar hematologic and Hb data make it impossible to address the concomitant inheritance of α(0)-thalassemia in these samples without polymerase chain reaction (PCR)-based techniques, leading to misdiagnosis of the risk of having Hb Barts hydrops fetalis. However, α-globin genotyping should be carried out in samples with positive IC strip as positive reactivity was also observed in homozygous α(+)-thalassemia carriers who have 2 trans α-globin gene deletions. These results indicate that in combination with red blood cell indices, the IC strip test could rule out mass populations for further α(0)-thalassemia detection by PCR-based analysis. The Alpha Thal IC strip also has the potential to replace testing for Hb H inclusion bodies, as it appears to be more sensitive, specific, and less labor intensive.

Alpha thalassaemia due to non-deletional mutations on the -3.7 alpha globin fusion gene: laboratory diagnosis and clinical importance

Aims: Alpha (&agr;) thalassaemia may be caused by large deletions of the &agr; globin gene(s), or rarely, non-deletional mutations. Both types of mutations may co-exist, and if located on the same allele (&agr;0), produce a reproductive risk of hydrops fetalis. We illustrate how clinical-laboratory correlation and accurate &agr; gene sequencing are essential in identifying such patients. Method: Nine asymptomatic patients with -&agr;3.7 thalassaemia trait were noted to have significant microcytosis that was insufficiently explained by a single &agr; deletion. Hence &agr;1 and &agr;2 globin gene sequencing were performed, which detected a non-deletional mutation in all patients. A new set of &agr;1 specific primers were designed for separate sequencing of the &agr;1 gene and the -&agr;3.7 fusion gene, respectively, so that the non-deletional mutation could be localised to the correct allele. Results: In six of nine patients tested, the non-deletional mutation was on the &agr;1 globin gene. In three patients the mutation was located on the -&agr;3.7 fusion gene. The latter group functionally has an &agr;0 allele (&agr;&agr;/–) with a reproductive risk for Hb Barts hydrops fetalis. Conclusion: Non-deletional mutations can occur on the &agr; globin gene or a fusion gene such as the -&agr;3.7 allele. Identification and accurate localisation of these mutations is important as this can have significant reproductive implications.

A molecular tool to assess the pathological relevance of alpha-globin DNA variants.

Aim: While the phenotype for heterozygous beta-thalassaemia is straightforward, it is more difficult to confirm a causative relationship for mutations in the alpha-globin genes. The aim of this study was to generate an in vitro system to evaluate the pathological relevance of &agr;-globin mutations. Methods: The novel variant HBA1:c.301-3C>G was used as a model. In silico analysis predicted an aberrant acceptor splice site in the mutant sequence. Subsequent in vitro studies included generation of and transfection of an expression vector carrying the HBA1:c.301-3C>G mutation, RNA purification, reverse-transcription polymerase chain reaction (RT-PCR) and cDNA sequencing. Immunofluorochemistry (IFC) with antibodies specific to the N- and or C- terminal of the &agr;-globin protein was used in protein detection. Results: In vitro molecular characterisation of this point mutation confirmed the preferential utilisation of a cryptic splice site at intron 2 of the pre-mRNA, resulting in a shift in the reading frame causing a premature termination codon (PTC) at codons 101/102 and generation of a truncated protein. Conclusion: We have described here a molecular tool to study mutations that affect &agr;-globin pre-mRNA splicing and translation. We confirm in silico predictions of the consequences of the HBA1:c.301-3C>G mutation, proving aberrant RNA splicing and the production of a truncated &agr;-globin protein.