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Haemophilia is a bleeding disorder caused by defects in blood coagulation protein factor VIII (FVIII) or factor IX (FIX). As there is no permanent cure for this disease, prenatal diagnosis has become an important way to prevent the birth of children with haemophilia phenotype. Genetic method is an important part of prenatal diagnosis. PCR has play a fundamental role in this method and become the most widely used technique in haemophiliac diagnosis.
Haemophilia is a bleeding disorder caused by defects in blood coagulation protein factor VIII (FVIII) or factor IX (FIX)(Peyvandi et al., 2006). In genetic level, haemophilia is a result of mutations in FVIII gene and FIX gene. Due to the different locations of gene mutations, there are two types of haemophilia: type A and type B, both of which are X-link recessive inherited. Haemophilia A is caused by mutations in FVIII gene (186kb, 26 exons, located in Xq27), while haemophilia B is caused by mutations in FIX gene (33.5kb, 8 exons, located in Xq28)(Peyvandi et al., 2006). The two genes are separated by 35cM. The incidence of Haemophilia A and B in male birth is approximately 0.2° and 0.04° respectively.
Patients with haemophilia can not control blood clotting normally and thus suffer from different levels of internal or external bleeding due to severity. About 50% of haemophiliacs suffer from a severe form of haemophilia. Typical symptoms in these patients include spontaneous and frequent bleeding into joints and muscles and severe complications such as arthropathy, muscular pains, brain damage and death. Before 1960s, patients usually can not live after the age of 11. Although newly developed treatment methods largely increase the averge lifespan of haemophilias patients nowadays, there is no permanent cure for this disease. Severe haemophilia is still one of the leading causes of death all around the world. As a result, prenatal diagnosis has become an important way to prevent the birth of children with haemophilia phenotype.
Phenotypic diagnosis and genetic diagnosis are two main types of prenatal diagnosis.
In phenotypic diagnosis, the plasma level of FVIIIc in baby's cord blood is tested to determine if the FVIII gene is appropriate expressed. However, as the level of FVIIIc in cord blood varies in the entire embryo stage, it is difficult to set the normal reference values. Another problem is that there is a risk of abortion after sampling cord blood by amniocentesis. Phenotupic method is more commonly used in developing districts lacking necessary molecular equipments. On the other hand, genetic diagnosis is main method to diagnosis haemophilia in developed countries.
Genetic diagnosis is the main and pivot part of haemophilia diagnosis. There are two main approaches to genetic diagnosis of haemophilia: linkage analysis and direct mutation detection. Molecular biological techniques are applied in genetic diagnosis, including Southern Blot, PCR and Biochip. Southern blot is usually not applied in clinical practice for its complexity and the technique of biochip is still in progress. With the advantages of rapid speed, simplicity and high accuracy, polymerase chain reaction plays an important role in both approaches. In this essay, I will discuss the role of PCR in genetic prenatal diagnosis of haemophilia. As the techniques and principles with which the diagnostic methods are applied to haemophilia A and haemophilia B are similar, I will emphasize on the diagnosis of haemophilia A in the following discussions.
2 Determination of Diagnostic Strategy
The selection of diagnostic method of haemophilia is due to the severity of the disease. Haemophilia is cause by mutations in FVIII and FIX gene, including inversion, missen, insertions and splice site mutations. Researchers have reported that about 45%-50% of severe A-type haemophiliacs had either an inversion in FVIII gene intron 22 or FVIII gene intron 1. The other half of severe cases may have mutations althoughout FVIII gene. Moderate and mild cases are usually not related to specific types of mutation. The haemophilia A diagnosis verification according to the severity is demonstrate in Figure 1.
3 Genetic Diagnosis of Haemophilia by PCR
3.1 Linkage Analysis
Linkage analysis is a conventional method to detect the carrier status of possible female carriers in families with inherited disorders. The key point of linkage analysis is to select appropriate polymorphic marker. Single nucleotide polymorphisms (SNPs) or variable number tandem repeat (VNTR) markers are two of the commonly used polymorphic markers used in the detection of haemophilia. The defective
Fig.1 Direct Mutation Detection Strategies in FVIII gene.
X-chromosome within the family with haemophilia can be tracked through analyzing markers that closely linked to the mutation, i.e. SNPs and VNTR markers in the FVIII or FIX gene. In this way an accuracy up to 99% can be gained.
SNPs are single nucleotide changes that have no influence on the sequence of amino acid. They occur everywhere in human genome. In conventional protocols of linkage analysis, agarose gel electrophoresis and southern blotting were commonly used to identify the intragenic polymorphism in FVIII and FIX gene. Nowadays, southern blotting is rarely used for its complexity and PCR has taken its place. SNPs are digested with a restriction enzyme of endonuclease and then detect by PCR-amplification. The low cost and simplicity of PCR have make linkage analysis become available even in developing districts.
However, linkage analysis has its limitations. It can not be applied in families lacking
prior family history or key pedigree members. And the selection of markers depend on its heterozygosity in the female of prior case also restrict its applications.
In these cases, direct mutation detection should be used.
3.2 Direct Mutations Detection
Levinson et al. (1987) demonstrated that using mismatch analysis of PCR-amplification, it is possible to directly detect two previously known TaqI site (TCGA) mutations in the factor VIII gene. In the next year, Reitsma et al. (1988) performed FIX gene analysis by PCR amplification. Although southern blotting had been used to detect mutation site at that time, PCR-amplified product has the advantage of permitting both carrier detection and prenatal diagnosis without the identification of southern blotting. Nowadays, direct mutation detection, with an accuracy of approximately 100%, is becoming more and more commonly used in the genetic diagnosis of haemophilia.
The protocols of direct detection involves with PCR-amplification of FVIII or FIX gen, and then DNA screening or sequencing methods to detect existing mutations.
To diagnose haemophilia with direct genetic detection, it is important to understand the mutation strategies of FVIII and FIX gene. It is reported that about half of severe cases of haemophilia A is caused by a large scale of gene inversion in FVIII, i.e. a reverse in the long arm of the X-chromsome with a break point in intron 22. Although the inversion can be detected with southern blotting, the introduce of PCR improved the yield of mutations.
Long PCR and inverse PCR are two commonly used protocols to detect the intron 22 inversion. Long PCR method is dependent upon multiplex PCR and the results can be obtained in twenty-four hours. PCR bands that are generated in all templates act as a control to show if the reaction is performed efficiently. The size of bands amplified can be up to 12kb. Inverse PCR, on the other hand, is more complex and time-consuming, but more reliable than long PCR. In inverse PCR method, DNA is digested with restriction enzyme and then ring ligated to produce the material for inverse PCR. PCR products are run on standard agarose gel electrophoresis.
PCR has play an important role on diagnosis of haemophilia. The major diagnostic methods involving PCR technique includes linkage analysis and direct mutation detection. Alternative methods other than PCR do exist, such as southern blotting. However, with the advantages of low cost, rapid speed and high accuracy, PCR a fundamental role in genetic diagnosis of haemophilia and has become a most commonly used diagnostic protocol in this area.