Alpha-thalassaemia, a common genetic disorder leading in its most moderate form to a hypochromic microcytic anaemia, is mainly the result of deletions on chromosome 16. Current testing for a-thalassaemia is based on an algorithm of exclusion-testing, which necessitates a wide range of dianostic procedures. Such procedures are labour-intensive, time-consuming and not 100% specific. As a result, there is a need for a general, rapid and efficient screening method. This article discusses the use of real-time quantitative PCR detection of deletions as one possible solution.
by Dr S. Brunner-Agten and Dr A. R. Huber
Alpha-thalassaemia
a-thalassaemia is a common genetic disorder, which leads in its most moderate form to an asymptotic anaemia with persistent hypochromia microcytosis. The clinical outcome of more severe cases leads to very severe transfusion-dependent anaemia or hydrops fetalis.
The condition is mainly the result of deletions on chromosome 16, which contains, at its telomeric region, two highly homologous and closely linked genes (al- and a2-gene) encoding the a globin chains. During meiosis, misalignment of chromosome homologues followed by reciprocal recombination at three highly homologous segments (named X, Y, and Z and separated by non-homologous segments [Figure 1]), results in various
deletion-duplication events.
The outcomes of the genetic disorder are diverse and the severity is correlated with the number of affected a globin loci and the exact nature of the gene deletion [Figure 2].
The phenotypes of a-thalassaemia represent two clinically significant forms, which are Hb Bart hydrops fetalis (Hb Bart) syndrome and haemoglobin H (HbH) disease. In HB Bart, all four a-globin alleles are deleted or inactivate (--/--) and death in the prenatal or neonatal period is inevitable. HbH, however, is the result of the deletion or dysfunction of three of the four a-globin alleles (--/-a). It is characterised by microcytic hypochromic haemolytic anaemia, hepatosplenomegaly, mild jaundice and sometimes thalassaemia-like bone changes.
The phenotypes of a+-thalassaemia and a0-thalassaemia, where just one or two a-globin-genes are affected, are more common. a+-thalassaemia results from deletion or dysfunction of one a-globin allele (a a/-a), e.g. by reciprocal recombination between the Z region, 3.7 kb apart, or between the X region, 4.2 kb apart, giving rise to the -a3.7kb and -a4.2kb deletion, respectively [Figures 1 and 2]. Carriers of a+-thalassaemia, also known as a-thalassaemia silent carrier, may have a silent haematological phenotype or present a moderate thalassaemia-like haematological picture. aº-thalassaemia however, may be caused by extended deletions varying from 100 kb to more than 250 kb resulting in deletion or dysfunction of two a-globin alleles (homozygotes (-a/-a) or heterozygotes (a a/--), e.g. -a SEA, -a TAI, -a FIL, -a MED, -(a)20.5kb. Carriers of aº-thalassaemia, also known as a-thalassaemia trait, show microcytosis (low MCV), hypochromia (low MCH) and normal percentages of HbΑ2 and HbF [1].
It is estimated that there are at least 200 million people worldwide affected by thalassaemia. In Switzerland, after iron deficiency, thalassaemia is the most prevalent cause of hypochromic anaemia [2, 3]. To offer genetic counselling for couples who wish to start a family, or to avoid unnecessary iron substitution, it is important to also identify the heterozygote carriers of a-thalassaemia who have mild or even no symptoms.
Available methods for alpha-thalassaemia screening
Current testing for a-thalassaemia is based on an algorithm of exclusion-testing (i.e. to exclude iron deficiency and β-thalassaemia and other haemoglobinopathies), which requires a wide range of procedures such as hematological testing of red blood cell indices, peripheral blood smears, supravital staining to detect RBC inclusion bodies, qualitative and quantitative haemoglobin analysis, bone marrow examination, and the in vitro synthesis of radioactively-labelled globin chains in affected individuals. However, the final proof of the presence of an a-thalassaemia is only obtained using biomolecular diagnostics [2]. This includes polymerase chain reaction (gap-PCR) amplification of the normal or aberrant a-globin gene [4,5], ELISA for the detection of a–globin chains in circulation [6] and hybridisation assays with a-strips.
Current technologies are, however, labour-intensive and time-consuming, and may still not provide an accurate analysis of all variants of the diseases. There is a great need for a general, rapid and efficient screening method, which is completely standardised and suitable for the routine laboratory.
Quantitative real-time PCR as an a-thalassaemia screening method
Real-time Quantitative PCR technique (RT-PCR) has been applied in different investigations including pathogen detection, allelic discrimination, gene expression and gene regulation [7-9], as well as for the detection of duplications and deletions, e.g. in Duchenne and Becker muscular dystrophies, cystic fibrosis and neuroblastomas [10-12]. However, while RT-PCR has also been applied for the detection of a-thalassaemia [13], current methods only allow for detection of several restricted mutations such as the southeast Asian type deletion, or a group of three different deletions (-a 3.7kb, -a SEA and -a MED).
We are now evaluating a new screening assay (patent pending) for the detection of a-thalassaemia-causing deletions using multiple primer sets, which enables classification of the genotype of an individual by performing only one (or maximally two) single RT-PCR run.
In order to carry out the assay, genomic DNA from human blood is extracted using a manual or automated DNA purification method. Photometric quantification of genomic DNA is performed on the NanoDrop liquid handling device (Thermo Fisher Scientific, Inc.) and only samples within a defined range of DNA purity (260nm: 280nm ratio) are selected for the experiments. A Light Cycler System is used for RT-PCR. The specificity of the obtained amplicons is analysed through melting curves, gel electrophoresis and/or sequencing. Further quantification in reference to endogenous controls (reference genes) allows identification of the relative quantity of the amplified gene. Through analysis of the obtained amplification pattern we are able to define the genotype of the individual. In the case of an aberrant genotype (positive screening result), subsequent analysis, e.g. sequencing, allows further characterisation of the exact nature and location of the mutation (if this is relevant information for the clinic). In the case of negative screening results, no additional work-up in the alpha-gene-cluster is necessary.
Conclusion
With this quantitative RT-PCR assay we have developed a new, completely standardised method for routine laboratory alpha-thalassaemia-screening, which enables the genotype of each patient to be classified by performing one single RT-PCR run.
The implementation of this new method in a diagnostic laboratory requires the assessment of a new algorithm for testing. The two first steps normally carried out, namely the determination of the haemogram and the iron metabolism parameters, would be retained. However, after iron deficiency and b-thalassaemia and haemoglobinopathies are excluded, the new a-thalassaemias screening method can be used instead of the common molecular biological tests used to date.
The quantification of the a-globin gene will allow the determination of the real prevalence of a-thalassaemia, with the detection of all carriers who may otherwise be subject to mis- or nondiagnosis. This helps to provide genetic advice and (prenatal) diagnostics. Beyond this, the method can help to minimise unnecessary, potentially toxic and expensive iron substitution in patients who have a-thalassaemia, rather than iron deficiency anaemia.
References
1. Herklotz R, Risch L and Huber AR. Hemoglobinopathies--clinical symptoms and diagnosis of thalassemia and abnormal hemoglobins. Ther Umsch 2006; 63(1): 35-46.
2. Huber AR et al. Anomales Hämoglobin: Erscheinungsbilder und Abklärung. Swiss Medical Forum, 2004.
3. Huber AR et al. Thalassämie-Syndome: Klinik und Diagnose. Swiss Medical Forum, 2004.
4. Chang JG et al. Rapid diagnosis of alpha-thalassemia-1 of southeast Asia type and hydrops fetalis by polymerase chain reaction. Blood 1991; 78(3): 853-4.
5. Ko TM et al. Carrier detection and prenatal diagnosis of alpha-thalassemia of Southeast Asian deletion by polymerase chain reaction. Hum Genet 1992; 88(3): 245-8.
6. Ausavarungnirun R. et al. Detection of zeta-globin chains in the cord blood by ELISA (enzyme-linked immunosorbent assay): rapid screening for alpha-thalassemia 1 (Southeast Asian type). Am J Hematol 1998; 57(4): 283-6.
7. Bowie LJ et al. Detection of alpha-thalassemias by multiplex polymerase chain reaction. Clin Chem 1994; 40(12):2260-6.
8. Das H et al. Quantitation of Fas and Fas ligand gene expression in human ovarian, cervical and endometrial carcinomas using real-time RT_PCR. Br J Caner 2000; 82(10): 1682-8.
9. Fujii K et al. Mutation detection by TaqMan-allele specific amplification: application to molecular diagnosis of glycogen storage disease type Ia and medium-chain acyl-CoA dehydrogenase deficiency. Hum Mutat 2000; 15(2): 189-96.
10. Jancourt F et al. Rapid identification of female carriers of DMD/BMD by quantitative real-time PCR. Hum Mutat 2004;. 23: 385–391.
11. Schneider M et al. Detection of exon deletions within an entire gene (CFTR) by relative quantification on the LightCycler. Clin Chem 2006; 52(11): 2005-12.
12. DePeter K et al. Quantification of MYCN, DDX1, and NAG gene copy number in neuroblastoma using a real-time quantitative PCR assay. Mod Pathol 2002; 15(2): 159-66.
13. Armour JA et al. The detection of large deletions or duplications in genomic DNA. Hum Mutat 2002; 20(5): 325-37.
The authors
Dr Saskia Brunner-Agten and Prof. Andreas R. Huber
Cantonal Hospital Aarau (KSA)
Switzerland
Address for correspondence:
Prof. Dr. med. Andreas R. Huber
Zentrum für Labormedizin
Kantonsspital Aarau AG
Tellstrasse
CH-5001 Aarau
Switzerland
Tel :+41 62 838 53 01
E-mail: andreas.huber@ksa.ch
The coinheritance of beta- and alpha- thalassaemia: a review of one patient and her family
The diagnosis and management of alpha-thalassaemia may be complicated by the variability of the phenotype, which is due to the interaction of coinherited alpha-thalassaemia and the variable severity of beta-thalassaemia mutations. A well-documented case of complex beta- and alpha-thalassaemia coinheritance is described in this paper. Laboratory and clinical data for the patient and her family were reviewed. The patient was an asymptomatic girl, one of identical twins. She presented at one month of age for follow-up of an abnormal newborn-screening result (haemoglobin F only), which initially suggested homozygosity for beta-thalassaemia. Extensive studies on the patient and family revealed that she had coinherited alpha-thalassaemia traits and homozygous beta-thalassaemia. This case demonstrates the interaction of coinherited alpha- and beta-thalassaemia with the resultant amelioration of the clinical phenotype. It also highlights the importance of family studies and close follow-up in diagnosing complex haemoglobinopathies.
Mast KJ, Hammond S, Qualman SJ, Kahwash SB. Lab Hematol. 2009;15(3):30-3.
