Molecular diagnosis in haemophilia A.GS Pandey, B Mittal
Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India., India
Correspondence Address: Source of Support: None, Conflict of Interest: None PMID: 11832649
Source of Support: None, Conflict of Interest: None
Haemophilia A is the commonest cause of X-linked inherited bleeding disorder. Due to inadequate medical facility for management of the disease, the DNA based genetic diagnosis has assumed great importance. Ideally, the direct detection of mutations is the most accurate and reliable approach for carrier detection and prenatal diagnosis. However, mutation detection is possible only in limited number of cases. In majority of haemophiliacs, no common mutation is easily identifiable. The limitation has been over come by the use of linkage-based analysis using polymorphic DNA markers in the factor VIII gene. Some of these markers can be identified by restriction enzymes and are called RFLP markers. Other markers are a class of short tandem repeats sequences which result in differences in the number of CA repeats in different individuals. The combined use of these markers has made it possible to identify carriers and provide prenatal diagnosis in upto 95% of families having affected individuals. Therefore, the recurrence of the disease can be prevented to a great extent in the haemophilia A affected families.
Keywords: Female, Genetic Markers, genetics,Hemophilia A, diagnosis,genetics,Human, Male, Molecular Diagnostic Techniques,
Haemophilia A is the commonest cause of inherited bleeding disorder resulting from defect in factor VIII gene located on long arm of X-chromosome at Xq28 locus. The disorder affects about one in 5,000 male births. Haemophilia A exhibits wide range of clinical severity and the diagnosis is based on quantitation of factor VIII: C antigen assay in the blood plasma. Symptoms of bleeding into joints and muscles correlate closely and inversely with residual procoagulant activity. Clinical severity depends on the activity of factor VIII in the blood plasma. Severe haemophilia A is presented with frequent spontaneous bleeding into joints, muscles, internal organs and associated with less than 1% normal factor VIII activity. Moderately severe patients with 2-5% of residual factor VIII activity show some spontaneous bleeds or bleeding after minor trauma. Mildly affected individuals show bleeding episodes only after significant trauma and have 6-50% of residual factor VIII activity in their blood plasma. Haemophilia A is generally represented with true breeds within a family. The clinical presentation of the disease is greatly influenced by the nature of the mutations in the factor VIII gene. Therefore, genetic information regarding mutation type may be very useful in genetic counselling.
Due to the lack of social support system and inadequate diagnostic facilities for haemophilia A, genetic counselling based on DNA diagnostic approaches have assumed great importance. The genetic testing involves carrier analysis and prenatal diagnosis. Mother of an affected boy can be obligate carrier if she has a haemophiliac father or more than one haemophiliac son or well documented maternal history. However, many families seeking genetic counselling have only one affected child. Female relatives of affected males are therefore, potential candidates for carrier assessment. The carrier diagnosis in most of the X-linked recessive genetic disorders is relatively difficult because females are asymptomatic and the information provided by coagulation assay is not equivocal due to the random X-chromosome inactivation or lyonisation.
In recent years, advances in DNA based diagnostics have made it possible to identify the carriers and provide prenatal diagnosis with 95% confidence. Therefore, the recurrence of the disease can be prevented to a great extent in the affected families. In this review, we have discussed various approaches to provide genetic counselling in haemophilia A affected patients and families by using polymorphic DNA markers as well as direct mutation detection in the factor VIII gene. We have also highlighted the current status of haemophilia A diagnosis in India.
The Factor VIII gene is one of the largest gene with a complex genomic organization comprising 186 kb with 26 exons ranging in size from 50bp to 3.2kb. It represents 0.1% of X-chromosome and codes for a 9 kb mRNA. The intron 22 of factor VIII contains another gene transcribed in the opposite direction which is expressed in a variety of cells. A large number of heterogeneous point mutations (>150) in the factor VIII gene have been identified in patients all over the world. Majority of mutations are single nucleotide substitutions or small deletions or insertions in the coding region of the gene. Approximately 50% of the severe haemophilia A patients show intron 22-inversion mutation.
The heterogeneous nature of mutations and complex genomic organization of factor VIII gene has made the direct detection of mutation difficult for genetic diagnosis. In view of the problems, majority of laboratories involved in genetic testing for potential carrier detection and prenatal diagnosis have relied upon linkage analysis employing DNA polymorphic markers. Polymorphic markers are slight sequence variations usually present in the non-coding regions of a gene in the population. The sequences are quite stable and inherited in a Mendelian fashion. Some of these markers can be identified by restriction enzymes and are called RFLP markers such as Bcl 1, Hind III and Xba1. Other markers are a class of short tandem repeat sequences which result in differences in the number of CA repeats in different individuals. These markers are randomly distributed in the whole genome and being routinely used in genetic diagnosis of various genetic diseases such as Duchenne and Becker muscular dystrophy., Selection of markers for a disease depends on the close association of the marker with the gene of interest so that there is a minimum possibility of genetic recombination between the marker and the gene carrying a mutation. In linkage analysis, blood samples of mother and affected sons are required but samples from father and unaffected sons can further aid in the analysis. In order to differentiate one of the two X-chromosome of the mother carrying the mutation gene, it is essential to have one or more heterozygous polymorphic marker in the mother. To carry out efficient tracking, the informativity of individual polymorphic markers i.e. the heterozygosity rate of each marker should be studied in the same population so that suitable markers for genetic diagnosis can then be identified for the population. The informativity of various polymorphic markers linked to factor VIII gene have been studied in different ethnic groups and found to differ significantly., Multi-allelic CA repeat markers occur within intron 13 and 22 of the gene with heterozygosity of 80% and 45% respectively., The Bcl1 RFLP marker is found to be more informative in Mediterreans and Japanese than Caucasians, while the Hind III marker show higher heterozygosity in Asian Indians. The Bgl1 RFLP heterozygosity varies from 30 % (Japanese) to 50 % (Caucasians). The multiallelic polymorphic locus DXS52 (st14), extragenic to factor VIII gene has been found to highly polymorphic (88%) in Indians than in other populations. The probability of meiotic recombination should be taken into consideration while using linked markers for DNA diagnosis. Extragenic polymorphic markers like Bgl 1 and st 14 show approximately 4.5% chance of recombination that increases the possibilities of diagnostic errors.
Recent studies indicate that dinucleotide CA repeat markers are highly informative and should be the starting point for carrier analysis. However, the analysis of the dinucleotide repeat markers is relatively difficult and generally requires radioactive PCR followed by separation on sequencing gels. Therefore, majority of the laboratories give priority for intragenic RFLP markers. If all RFLP markers on both X-chromosome of mother are similar, then the RFLP markers are noninformative for the family. In such cases CA repeat markers should be analyzed. Using RFLP and CA repeat markers; carrier analysis and prenatal diagnosis can be provided in upto 95% of families. There are some reports available regarding informativity of Factor VIII intragenic polymorphic markers in India. The heterozygosity of various markers in Indian population is represented in [Table - 1].
Using four common markers (Hind III, Bcl 1, Intron 13 and 22), we have been able to determine carrier status in 37 out of 40 families registered with us. The combination of Hind III and Bcl 1 alone showed 70 % (28/40) informativity in affected families. Therefore, RFLP markers in combination with CA repeats markers are needed to provide genetic diagnosis in more than 90% of the affected families in India.
[Figure - 1] depicts the application of linkage analysis for carrier identification. In this family, the mother was an obligate carrier due to one affected son and maternal history of the disease. Genetic analysis was carried out to determine the carrier status of the three daughters. The DNA samples from all family members were amplified by PCR for Hind III, Bcl 1, Intron 13 and 22 markers. The mother was found to be heterozygous for all four markers. The haplotype b of the mother inherited by the proband was found to be linked with the disease gene, while haplotype c was normal. By following the segregation of haplotype b in the offspring it was found that two female sibs inherited the maternal haplotype b, thereby suggesting that two of three daughters were carriers.
The linkage methodology is straightforward, rapid and inexpensive to perform and many families requesting genetic diagnosis for haemophilia A can benefit. All linkage-based methods assume that mother of the patients is carrier of the mutations but it may not be true particularly in sporadic cases without any family history because of new mutations. Also some families may not be informative for any of the available markers. The chance of recombination between the markers and mutation may also lead to small diagnostic error. Therefore the direct mutation detection methods are always superior to linkage-based methods for more accurate genetic diagnosis.
The haemophilia A affected patients are presented with heterogeneous nature of mutations in the factor VIII gene. These mutations are deletions, inversion, insertion and point mutations. The direct mutation analysis greatly increases the reliability of carrier detection in families with isolated cases of haemophilia A. It has been reported that around 50% severely affected haemophiliacs have inversion mutations in the intron 22 of the factor VIII gene. The mutation results from homologous recombination between the gene A located in intron 22 of factor VIII gene and one of the two homologous distal A gene copies, thus disrupting the coding sequence of the gene., Due to the large size of inversion, PCR based diagnosis is not easily feasible. The inversion mutation detection is carried out by conventional Southern hybridisation. In this method, the genomic DNA is digested with Bcl 1 restriction endonuclease electrophoresed and blotted on a nylon membrane. The blot is then hybridised with radio-labelled probe. There are two types of inversion, distal and proximal and both can be detected by the same procedure. The mothers of patients are generally carriers for inversion mutation. In India, about 40-45% of severely affected haemophiliacs have been found to carry intron 22 inversion mutations. Therefore, the direct detection of common inversion mutation should be the first steps for genetic diagnosis in the severely affected haemophilia A patients. Other mutations responsible for haemophilia A are mostly point mutation in the factor VIII gene and their spectrum is quite complex. Some 5% of patients have a mutation involving deamination of GC dinucleotide, which changes the Taq 1 restriction site and can be identified by Southern blot analysis., The mutations not involving changes in restriction sites cannot be recognized by routine procedures. In these cases mutation detection will require special techniques such as single strand conformational polymorphism (SSCP), conformation sensitive gel electrophoresis (CSGE), amplification and mismatch detection (AMD), denaturing gradient gel electrophoresis (DGGE) followed by DNA sequencing. However, none of the above mentioned techniques are suitable for routine diagnosis even in sophisticated laboratories because of technical complications.
Molecular procedure for prenatal diagnosis is same as for carrier analysis. Prenatal diagnosis in carrier females can be performed in early pregnancy. Samples aspirated from the foetus by chorionic villi sampling (CVS) or amniocentesis is suitable for diagnosis. Before starting DNA analysis, sex of the foetus is determined by PCR using specific primers. If the foetus is female, no further investigations are required but in the case of male foetus linkage or inversion analysis is carried out. The usefulness of polymorphic markers in prenatal diagnosis is explained as a case report from our laboratory records. [Figure:2]
In this family, the mother was an obligate carrier due to two-affected sons and family history. The DNA samples from all family members and foetus were amplified by PCR for four markers. The mother was found to be heterozygous for Hind III, Bcl 1 and STR intron 22 markers. The haplotype b of the mother inherited by the proband (II-1) was found to be linked with the disease gene, while c haplotype was normal. By following the segregation of b in the offspring we could determine the status of foetus (II-3) which inherited the maternal haplotype b, thereby suggesting that the foetus would be affected with haemophilia A. The parents were counselled accordingly.
The basic aim of genetic counselling is to provide sufficient information regarding carrier testing and prenatal diagnosis. The counsellor should provide psychological/psychosocial support to patients and their families through the process of testing, so the unbiased choice should be made regarding their medical care. Genetic testing should test all carriers early in life. It is preferable that carrier testing should be completed before a potential carrier become pregnant. Since the carrier status of female foetus is not determined, all female foetuses are reported as unaffected for the disease. However, the parents should be asked to contact the centre for counselling before marriage. Technical aspects of both genotype and phenotype testing should be discussed with parents along with accuracy and limitations of the test. Women with positive carrier results should be advised of all reproductive choices including abortion, adoption and in vitro fertilization. Risk for each testing procedure should be discussed taking into consideration the testing facilities available to the patients.
Haemophilia A is supposed to be suitable genetic disorder for gene therapy because- i) The gene responsible for the disease has been cloned and well studied, ii) the factor VIII gene product does not require tight regulation in expression, iii) the clinical phenotypes can be improved by modest level increment in factor VIII protein level, iv) the haemophilia A condition is life long with severe effects, v) treatment is only possible by replacement of factor VIII which is not entirely satisfactory, expensive and are not free from all risks
The basic concept for gene therapy is to transfer normal functional gene in the somatic cells of the patients, so that it produces functionally active factor VIII protein. However, the problem in haemophilia A gene therapy is the size of cDNA which is 7 kb and most of the viral vectors have limitation on the size of the inserts.
During past few years, a major progress toward the goal of sustained high-level expression has been reported in animal models using both viral and non-viral gene delivery vehicles. The major problem in retroviral mediated gene transfer in liver was the low levels of expression and need for invasive procedures to induce cell division in hepatocytes. The long-lived therapeutic level (40ng/ml) of factor VIII expression was achieved in immuno competent mice by adenoviral-mediated transfer. This strategy was scaled up to a canine model for haemophilia A32 but expression was short lived, probably due to cellular and humoral immune responses against adenoviral vectors and transgene products. The factor VIII gene has also been transduced in fibroblast; endothelial and myoblast cells by retroviral vectors and significant levels of expression have been reported. The intraperitoneal injection of factor VIII gene in mice and human patients showed therapeutic circulating levels with transient expression. It is well established that infusion of human factor VIII or vectors expressing it result in the rapid generation of alloantibodies in the animals and it is a major limitation for gene therapy. In spite of availability of several gene delivery vehicles and injection routes in animal models, the persistent expression of therapeutic plasma levels of clotting factor without cellular immune response against the cells expressing factor VIII gene has to be optimised in order to provide the basis for potential clinical trials.
The burden of severe genetic diseases such as haemophilia A is heavy in our country because of poor awareness, inadequate diagnostic facilities and lack of social support system. There are several centres providing haemophilia A care in India but their number is not enough to bring about rapid development of a network of genetic services. There should be at least 100,000 people affected with haemophilia A, based on prevalence of about 1 in 10,000 in our population of 1 billion. The Haemophilia Federation of India (HFI) was established in 1983 and has chapters in different cities. Five thousand registered cases of haemophilia with the society represent only about 5% of the estimated number. This is because of lack of awareness among physicians and the public in general. The federation organises annual workshops and training centres for haemophilia affected patients all over the country and imports factor VIII concentrates for treatment. They also guide the affected families for genetic counselling. Further information regarding haemophilia management by HFI can be obtained from Internet web site http://www.chennaibest.com/cityresources/Health_and_Medicine/hemophilia.asp.
DNA based linkage analysis of factor VIII gene has enabled development of specific tests for carrier analysis and prenatal diagnosis in haemophilia A. Genetic counselling based on these protocols can help in significantly reducing recurrence of the disease in the affected families.
[Figure - 1][Table - 1], [Table - 2]