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Haemophilia is a medical condition in which a person's blood does not thicken or clot properly when they are injured resulting in continuous bleeding (haemophilia.org). In severe cases the disease can cause internal bleeding of muscle and in joints which gives symptoms of stiffness and swelling leading to joint damage over time. In addition, haemophilia is a genetic hereditary disease which is sex-linked as the genes encoding for the clotting factor proteins are found on the x-chromosome. There is deficiency of or heterogenous mutations in the Factor VIII gene which causes haemophilia A (HA) or in the Factor IX (Christmas factor) gene causing haemophilia B (HB). Haemophilia A is the most frequently occurring of the two types. Mutations can occur during meiosis and DNA replication which may lead to deletions, insertions, amplifications, amorphic mutations and point mutations in the protein sequences that code for the clotting factors. A person who suffers from haemophilia is described by the term haemophiliac.
Figure 1: The FVIII protein (www.nwabr.org)
The clotting disorder mainly affects more males than females, 1 in 5000 males will be born with HA (Beard, 2010) due to the disease being an x-linked recessive trait. Recessive mutations inactivate the affected gene and lead to loss of function. Females are mostly carriers as they have two copies of the x-chromosome so the presence of a recessive copy is masked. This is because a female can only get the disease when both parents have the recessive allele of the clotting factor protein. However a male will get the disease if only the mother has a copy of the recessive allele. Fathers who are haemophiliacs cannot pass on the disease to their sons because the sons get the x-chromosome from their mothers, but all the daughters will be carriers if the mother is not a haemophiliac. Haemophilia has been inherited and passed down for generations in the royal families of Europe (Lodish, 2005).
Figure 2: The pedigree for an x-linked recessive disorder. The circles represent female and the squares are male. Affected individuals with the recessive trait are shaded. (www.migeneticsconnection.org)
Figure 3: An affected father cannot pass on the disorder to his sons if the mother is not a haemophiliac but all the daughters will be carriers. http://ocw.tufts.edu)
Identification of mutations in the Factor VIII gene
There are several mutations that take place in the FVIII gene causing haemophilia A and there are several methods which can be used in their identification. The FVIII gene is first amplified using PCR to enable the detection of unknown mutations (Bagnall, 2002). Simple detection methods include PCR, Southern blotting and dHPLC. After PCR direct DNA sequencing is performed to detect mutations (www.mun.ca/biology). Denaturing high performance liquid chromatography (dHPLC) separates hetero and homoduplexes on the basis of their melting behaviour. The mutant DNA causing haemophilia will have a different retention time to the normal DNA. Chemical cleavage detection of mismatched base pairs is based on the ability of hydroxylamine and osmium tetroxide to modify single base mismatches which is then used in the identification of mutations in the amplified gene. This can be further confirmed by sequencing. DNA heteroduplexes are run on a ureapolyacrylamide gel and mutations identified by the presence of cleaved DNA fragments. The position of the mutation is determined by the size of the cleaved fragments. SSCP - single strand conformational polymorphisms uses the principles of mobility in ssDNA which is determined by fragment length and secondary structure which is in turn sequence dependent. The fragment undergo electrophoresis and conformations are indicated by bands in the gel which show shifts where a mutated sequence is present because single base changes alter secondary structure which affects mobility.
Figure 4: The PCR amplification products of 5 patients with severe haemophilia A.
Journal of the American Society of Hematology (bloodjournal.hematologylibrary.org)
Mutations in the Factor VIII
There are now several known mutations that have been discovered in the FVIII gene. These include deletions, point mutations and gene rearrangements. Absent or altered intron splicing can also cause mutation in FVIII gene due to the transition of cytosine to thymine at CpG sites. These sites have been recognised as hot spot for mutations in the human genome (Peake, 1998).
Deletions and insertions cause frame-shift patterns which are changes to the reading frame that may introduce unrelated amino acids into the FVIII protein and can also result in the formation of stop codons (Bao-lai, 2010). Deletions are characterized into large deletions where more than 100bp are missing and less than 100bp are classed as small deletions. The deletions can range from less than 1kb to the deletion of a whole gene (Peake, 1998). There are many reported base pair deletions of nucleotide repeat sequences in exon 14 which encodes for the B domain of the FVIII gene. Although insertions takes place in the gene sequences of HA they are rare compared to deletions. The most common is the insertion of A to other A's already in the sequence. This is observed in exon 14 where an A is added to a string of 9A's resulting in moderate to severe HA (hadb.org.uk).
There are 615 single base substitutions reported in the HA database (www.hadb.org.uk). Amino acid substitutions lead to low levels or undetectable FVIII activity in plasma. Single base substitutions usually result in nonsense mutations or termination codons which replace an amino acid leading to premature termination of translation and an incomplete non functional protein. A single base substitution can also result in a missense mutation where the FVIII protein is present but with an amino acid substituted for another. It is very rare for this type of mutation to cause severe HA as a whole amino acid is substituted and is very unlikely to be of major function in the protein.
Gene rearrangements are also responsible for HA. There have been many studies on the inversions of intron 1 and intron 22 which account for 50% of severe HA cases. The structure of intron 22 makes it a hot spot for large gene rearrangements. Intron 22 of FVIII has a 9.5 kb region present outside of the gene near the telomere of the X-chromosome (Andrikoviks, 2003). The high degree of identity and opposite direction of the extragenic copies compare to the intragenic homologous region promotes intrachromosomal recombination during gametogenesis. This leads to increased frequency in chromosomal arrangements in the intron 22 region. The mutation results in less than 1% clotting factor activity leading to severe HA. Inversions can either be distal or proximal, distal being the most common.
Mutations in the Factor IX gene
The molecular basis for HB shows considerable mutational heterogeneity. The mutations in the FIX gene vary between different populations. The FIX gene was cloned and sequenced between 1982 to 1983 allowing studies into the causes of HB. The gene is located on the long arm of the x-chromosome at Xq27. The gene is 34 kb long and is composed of 8 exons. The gene contains many transcription sites due to the absence of a TATA box or initiator element in the 5' end region of the gene. Most mutations are found in exon 8, the largest region of the gene. About 90% of HB is caused by point mutations and deletions observed in 5-10% of HB patients (Nawaz, 2008). Founder mutations are responsible for 20-30% of HB. The most common being missense mutations of Gly60Ser, Arg248Gln, Thr296Met and Ile397Thr which are estimated to be responsible for a third of all mild HB in American Caucasians. Missense mutations at codon 10 where Ala is replaced by Thr or Val results in increased warfarin sensitivity with normal FIX levels in the anticoagulant state and reduced when taking vitamin K anticoagulants leading to episodes of spontaneous bleeding (Lillicrap, 1998).
There are several screening methods used for the detection of mutations in the HB gene. The easiest method for finding mutations in the gene is an immunological assay because a third to half of HB patients have higher FIX antigen level than activity levels. After this analysis further more detailed screening can be performed. Just like in FVIII, PCR technology is used to screen for FIX mutations. There are 597 recorded mutations in the haemophilia b database (kcl.ac.uk). Many repeated nucleotides involve the conversion of CG to TG or CA. This is not at all surprising as CG sites are known to be hot spots for mutations (Lillicrap, 1998). Missense mutations in exon 1 at codon 17 where GTT is changed to ATT - Val to Ile. The change of CGG to TGG at codon 4 of exon 2 in FIX gene prevents the cleavage of 12 glutamic acid residues of the propeptide and results in circulation of pro-FIX in blood. The replacement of Arg by the stop codon TGA in exon 2, the GLA domain is an example of a nonsense mutation which results in truncated protein which is non-functional. Another example is observed in exon 8 at codon 333 in the catalytic domain of FIX which also results in a truncated protein (Nawaz, 2008).
Deletions and rearrangement only account for 3% of all HB mutations. Lys at codon 316 of exon 8 determines reactivity of FIXa to natural FIX. Deletion of the residue results in severe HB. There are 18 mutations which occur in the regulatory regions of HB protein. These mutations lead to HB Leiden which has the effect of increasing FIX levels by 25% after puberty and into adulthood. These mutations appear with 40 nucleotides close to a start site for transcription. A mutation in the 5' non-coding region of FIX gene results in the disruption of the CCAAT enhance the binding of protein. There are assumptions that steroid hormones may be the influencing factor for the increase observed in FIX levels post puberty (Lillicrap, 1998).
Carrier status detection
The detection of carrier status in at risk females is important in haemophilia care. In a family were there are affected males, they are the first to be screened for mutations which will then be compared to female relatives and confirm carrier or non-carrier status. Carriers can also be identified if they symptoms of mild haemophilia such as excessive bleeding after an injury, childbirth or a surgical procedure (Goodeve, 1998). Measuring the content of FVIII in plasma can confirm suspected carrier status. A family pedigree can be drawn to aid the identification of possible carriers or non-carriers (haemalliance.org.uk). The severity of the disease in a carrier can be established from the status of affected males and females. Since most cases of HA arise from intron 1 and intron 22 inversions these are the first mutations to be screened for in carrier status detection.
Linkage analysis, direct analysis or intragenic polymorphism can also be used to detect carrier status. PCR is carried out to amplify coding genomic DNA or mRNA of the male haemophiliac. The PCR sequences will then be screened fro mutations which are then characterized as a polymorphism or point mutation in both FIX and FVIII. The female family members can then be screened for the mutation and carrier status determined.
There are 10 known DNA sequence polymorphisms which can be used in linkage analysis of the FVIII gene in order to define carrier status. Different techniques for carrier detection may need to be applied for different ethnic groups. This is because different polymorphisms appear at varying frequencies in ethnic groups. Polymorphisms in the intron 13 and 22 where there repeated dinucleotides are also used as carrier status detection. However the intron 13 polymorphism which has 20 repeats of the dinucleotide CA was found to be a more frequent allele in haemophiliacs of Caucasian origin (Goodeve, 1998).
Conformation sensitive gel electrophoresis (CSGE) methods can be used in the detection and analysis of carrier status of HA. In CSGE, DNA heteroduplexes are detected by their migration on a denaturing polyacrylamide gel. The DNA of an affected individual and that who needs their carrier status to be determined are amplified by PCR, heteroduplexed, undergo electrophoresis and staining by ethidium bromide. Mutations in the DNA cause changes in gel migration. These changes are seen as shifted bands on a membrane. The method can detect alterations in sequence lengths of between 200 to 800bp. Denaturing gradient gel electrophoresis (DGGE) is similar to CSGE but does not need radioactive labelling and detects carrier status in both HA and HB. It separates DNA on the basis of melting point and has a mutation sensitivity of 85%.
Prenatal diagnosis of Haemophilia
The prenatal diagnosis of haemophilia is an important decision which can be exercised by prospective parents with haemophilia or if the mother is a carrier (Shenfield, 2002). This allows haemophilia to be screened and detected in early stages of pregnancy. If the mother is a carrier the parents are given the choice to terminate pregnancy. When a foetus is found to be affected by CVS diagnosis pregnancy can be terminated in the first trimester. Chorionic villi sampling (CVS) is a technique were foetal extracts are obtained through transabdominal or transcervial CVS in weeks 10-12 of gestation. This also enables the determination of foetal sex and if the foetus is female further analysis need not be done. Male foetuses need detailed analysis to determine their haemophilia status. If there is a known mutation in the family, it is sequenced and then diagnosed using PCR and RFLPs. This process may take approximately 7-9 days. Blastocytes of an embryo can be isolated and the DNA amplified then analysed allowing diagnosis in early pregnancy and for IVF treatment. It may not be a very common technique now but could be used more in the near future.
Where genetic analysis has proved impossible or inconclusive, cellular material obtained at amniocentesis can be used for genetic diagnosis in the second trimester usually after gestation week 16. Foetal blood can also be obtained by ultrasound guided cordocentesis and analysed for presence of haemophilia causative mutations. These two techniques carry a 1-2% risk to the foetus hence they are only sought for after failure of other diagnosis.
The direct amplification of pathogenic mutation only requires detection of mutation from one member of the family. Thereafter no other family member's DNA needs be sequenced unless if suspected to be a carrier. In HA analysis the first analysis is that of the most common mutation of the intron inversions in the FVIII gene which is detected by southern blotting methods. However since homogeneity of mutations in FIX gene is low, a mutation has to be sought for in an affected family member. This provides genetic markers for the localisation of the mutation and the type of mutation present. Other techniques include AMD and SSCP.