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Journal of Postgraduate Medicine, Vol. 47, Issue 4, 2001 pp.274-280 Molecular Diagnosis in Haemophilia A Pandey GS, Mittal B Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India. Code Number: jp01081 Abstract
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.
Key Words: Factor VIII, inversion
mutation, polymorphic markers, carrier analysis, genetic counselling, linkage
analysis. 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.(3) 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. Factor VIII gene and Mutations 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(4) and codes for a 9 kb mRNA. The intron 22 of factor VIII contains another gene transcribed in the opposite direction(5) 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.(6) Approximately 50% of the severe haemophilia A patients show intron 22-inversion mutation.(7) Genetic prediction by Linkage analysis 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.(8) 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.(9,10) 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.(11,12) Multi-allelic CA repeat markers occur within intron 13 and 22 of the gene with heterozygosity of 80% and 45% respectively.(13,14) 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.(15) 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.(16) 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.(17) 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.
Limitations of linkage analysis
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.(20)
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.
Direct mutation Detection for
genetic testing
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.(21,22)
Due to the large size of inversion, PCR based diagnosis is not easily feasible.
The inversion mutation detection is carried out by conventional Southern hypidisation.
In this method, the genomic DNA is digested with Bcl 1 restriction endonuclease
electrophoresed and blotted on a nylon mempane. The blot is then hypidised with
radio-labelled probe.(23) 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.(15) 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.(24,25) 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),(26) conformation sensitive gel electrophoresis (CSGE),(27) amplification
and mismatch detection (AMD),(28) denaturing gradient gel electrophoresis (DGGE)(29)
followed by DNA sequencing. However, none of the above mentioned techniques are
suitable for routine diagnosis even in sophisticated laboratories because of technical
complications.
Prenatal diagnosis
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.(30)
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.
Genetic counselling
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.
Gene therapy in Haemophilia A
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.(31) This strategy was scaled up to a canine model for haemophilia
A(32) but expression was short lived, probably due to cellular and humoral immune
responses against adenoviral vectors and transgene products.(33) The factor
VIII gene has also been transduced in fipoblast; endothelial and myoblast cells
by retroviral vectors and significant levels of expression have been reported.(34)
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. Haemophilia Federation of India
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 ping 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.
Conclusions
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.
References This article is also
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