Laboratory Protocols
- Best practice guidelines
- Checking for maternal DNA contamination by STR analysis
- Checking for maternal DNS contamination by Variable number tandem repeat (VNTR) analysis
- Real time PCR
- Single cell PCR
Usefulness of collected protocols
The key to identifying the globin gene mutations in carriers and affected patients is an understanding of the genotype/phenotype relationships of the various globin gene mutations and the effects of interaction when several mutations are co-inherited. Often the quickest way to identify the mutations in an affected patient is to study the haematology of the patient’s parents and other family members and to screen them for single mutations. Carrier screening and mutation identification also forms one of the cornerstones of any prevention programme for the haemoglobin disorders. The strategy for carrier screening and mutation analysis is based on that fact that although heterozygotes are symptom free, they present specific haematological characteristics that are useful for their identification.
The accurate determination of the carrier phenotype is essential for the selection of the appropriate molecular tests to determine the carrier genotype for patient diagnosis or research studies involving new haemoglobinopathies. The basic haematological tests required are the measurement of the mean corpuscular volume (MCV), the mean corpuscular haemoglobin (MCH) value and the quantity of Hb A2 and Hb F. In addition, the haemoglobin pattern needs to be examined, by electrophoresis methods and HPLC chromatography. Other parameters also need to be examined in many cases, such as iron status and the stability of suspected Hb variants. All these procedures are to be described and posted onto the portal. In addition best practice guidelines will be posted which present the consensus of the expert laboratories involved in the Ithanet project.
The second phase of any investigation is to look at the haemoglobinopathy disorder at the molecular level. This can be either at the level of mRNA expression in the red cell or at the level of the DNA sequence change which is responsible for the disorder. There are three methods used in some laboratories to investigate globin chain synthesis by the measurement of mRNA levels and these protocols will be posted on the portal.
A variety of techniques based on the amplification of DNA by the polymerase chain reaction (PCR) have been developed to identify the globin gene mutations. The techniques commonly used are dot blot analysis, reverse dot blot analysis, the amplification refractory mutation system (ARMS), denaturing gradient gel electrophoresis, gap PCR, MLPA and restriction endonuclease analysis. Each method has its own advantages and disadvantages and all are recommended for use in best practice guidelines. The particular ones chosen by a laboratory depends not only on the technical expertise available in the diagnostic laboratory but also on the type and variety of the mutations likely to be encountered in the individuals being screened. Hence it is necessary to have procedures for all these techniques, both for known mutations and unknown mutations.
The haemoglobinopathies are a diverse group of inherited recessive disorders consisting of the structural hemoglobin variants and the thalassaemias. Together they form the commonest single-gene disorder in the world population and they are a serious public health problem in many countries. Although there is no definitive cure for the haemoglobinopathies, the methods of clinical management have improved considerably over the last few years and the life expectancy of affected individuals has been significantly increased. However, the treatment required is very expensive and is not a realistic means of controlling the disorders for many countries. Therefore most countries, especially those with a high incidence of β-thalassaemia, are applying prevention programme which involves screening the population for carriers, identifying the couples at risk, and providing a prenatal diagnosis service. Co-ordination of best practice is essential for prenatal diagnosis laboratories to provide a quality service and thus the Ithanet portal will contain essential up to date protocols for prenatal diagnosis of the haemoglobinopathies. This comprises of best practice guidelines and methods to determine possible error caused by DNA contamination of fetal samples, and the latest new diagnostic techniques of single cell PCR and real time PCR, which are essential for laboratories wishing to research into or to establish pre-implantation genetic diagnosis for haemoglobinopathies.