Prenatal Diagnosis Of Hemophilia Research Articles

Analysing two dinucleotide repeats of FVIII gene in Iranian population

Using dinucleotide repeats for carrier detection and prenatal diagnosis of haemophilia A patients, led us to find different alleles and their frequencies in Iranian population. Polymerase chain reaction (PCR) amplification of two short tandem repeat (STR) loci of factor VIII (FVIII) gene was performed, and the PCR products were resolved on 10% native polyacrylamide gel, and samples were analysed with sequenced DNA markers made of PCR cloning of the dinucleotide FVIII gene fragments. Seven different alleles were observed for intron 13 STR, having 18-24 (CA) repeating units and five alleles for intron 22 STR having 24-28 repeating units of (CACT). Bands produced during dinucleotide study were defined in detail so this could improve the genotyping of heterozygotes and homozygotes. Conformational band produced were characterized to specify the dinucleotide pattern. Our results confirm the Hardy-Weinberg proportions of the heterozygosity rate of the 85 analysed individuals. The observed heterozygosity rate for intron 13 and 22 was 52% and 59% respectively. Our data also indicate that our population is closer to caucasians than to any other populations. Finding different dinucleotide repeat alleles and their frequencies has made it possible to identify carriers and provide prenatal diagnosis with more confidence. This allows antenatal diagnosis to be performed in the vast majority of carriers.

Prenatal diagnosis of haemophilia A by using intron 1 inversion detection

Prenatal diagnosis of haemophilia A by using intron 1 inversion detection

New FACTOR IX linked marker alleles in African Haemophilia B patients*

Three markers, one restriction length polymorphism (RFLP) (MseI) and two microsatellite markers (Intron 1 and 3'UTR), linked to the FACTOR IX gene, were assessed for the purpose of genetic testing for Haemophilia B families in South Africa. This was carried out using seven Haemophilia B families and fifty random control samples. We observed five new alleles for the Intron 1 marker within the black control and patient sample groups, and informativity in 89% (8/9) of all carrier females for at least one of the three markers observed. These markers are useful for carrier detection and prenatal diagnosis of Haemophilia B in the great majority of South African black and white families.

Robustness of factor assays following cordocentesis in the prenatal diagnosis of haemophilia and other bleeding disorders

Prenatal diagnosis is the generally accepted option for genetic disorders including haemophilias and other bleeding disorders. Cord blood analysis between 17.4 and 20.6 weeks of gestation was performed in 172 confirmed carriers belonging to families of haemophilia A, haemophilia B, von Willebrand disease (VWD), factor VII and X deficiency; 133 were carriers for haemophilia A, 30 for haemophilia B, six for type 3 VWD, two for FX deficiency and one for FVII deficiency. The approach to the cord was either transabdominal or transamniotic. The volume of blood collected varied between 1 and 2 mL. In case of haemophilias, the diagnosis was offered by factor VIII/IX:C activity and antigen assays wherever required. In case of VWD, the diagnosis was based on von Willebrand factor antigen assays as detected by ELISA along with FVIII:C assay while in cases of FVII and FX deficiency, the diagnosis was based on FVII:C and FX:C respectively. The factor levels were compared with the normal range established in the laboratory for different coagulation factors between 18 and 21 weeks of gestation in women tested for other haematological disorders. Only in two cases, the procedure had to be repeated for reasons of extensive maternal contamination. All the deliveries have been followed up and the diagnoses reconfirmed by repeat clotting factor assays and DNA analysis whenever informative. Simple precautions like collection of fetal blood samples in smaller volumes in separate tubes, assaying multiple coagulation factors in the fetal blood samples helped us to offer diagnoses in all the women analysed. No fetal death or abortion was reported following the procedure. We suggest that accurate fetal blood sampling is a safe technique for the diagnosis of many of the bleeding disorders in places where genetic diagnostic services are not available.

Identification of fetal gender in maternal blood is a helpful tool in the prenatal diagnosis of haemophilia

Fetal DNA identification in maternal circulation has provided a new approach for non-invasive prenatal diagnosis. However, fetal DNA can persist in maternal blood long after the delivery, severely hampering this possibility. We addressed the issue of fetal DNA persistence in maternal blood. Thus, we investigated cell-free fetal DNA as a reliable approach in prenatal diagnosis of haemophilia. Forty non-pregnant women, who had had at least a male fetus, 29 control pregnant women, and 14 pregnant women, carriers of hemophilia A or B. The assessment of Y-chromosomal sequences was performed by analysing SRY and amelogenin genes using PCR-based techniques. A protocol consisting of double centrifugation at full speed followed by plasma filtration hampered the detection of Y chromosome-specific sequence in non-pregnant women. In 29 control pregnant women, blinded determination of fetal sex confirmed the specificity and sensitivity of the method applied. In 14 pregnant carriers of hemophilia, the investigation revealed a male fetus in nine pregnancies. Excluding the three cases in which a spontaneous miscarriage occurred, the sensitivity and specificity of fetal sex prediction by SRY and amelogenin gene analyses were both 100% as compared with the invasive approach and the fetal sex outcome at birth (six males and five females). Because of its high accuracy in prediction, fetal gender determination with cell-free fetal DNA in maternal plasma may be a useful tool in prenatal diagnosis of haemophilia allowing for the avoidance of invasive procedures for female fetuses.

A highly informative, multiplexed assay for the indirect detection of hemophilia A using five-linked microsatellites.

Hemophilia A is a severe bleeding disorder caused by almost 1000 different known mutations in the F8C gene. Direct mutation analysis is sometimes difficult for this disorder. When a mutation cannot be found, linkage analysis can be used for prenatal and carrier diagnosis. To develop a rapid and effective system for carrier detection and prenatal diagnosis of hemophilia A based on a single-multiplexed polymerase chain reaction (PCR) reaction utilizing five microsatellite markers. Two intronic microsatellites and three other markers flanking the factor VIII gene were ascertained, and primers were designed for multiplex PCR amplification. A kindred with Hemophilia A was tested for linkage using the panel of primers, and informativity in the general population was ascertained by testing 50 unrelated females. Co-amplification of all microsatellites was optimized using DNA extracted by standard methods. Rapid detection and sizing of products were carried out using an automated DNA sequencer. The combined microsatellite panel was informative in each of the kindreds tested, and in 100% of the 50 unrelated females (95% CI 94.2-100%). This method enables the indirect detection of hemophilia A for patients in whom mutations cannot be found, facilitating carrier testing and prenatal analysis. It is rapid and straightforward compared with many other published protocols, and offers a high degree of informativity.

Carrier Detection and Prenatal Diagnosis of Hemophilia in Developing Countries

Hemophilia A and B, inherited as X-linked recessive traits, are the most common hereditary hemorrhagic disorders caused by a deficiency or dysfunction of coagulation factor VIII (FVIII) or FIX, respectively. Hemophilia is prevalent worldwide, without ethnic or geographic limitations, and remains a life-threatening and often disabling condition. Advances in molecular medicine in this century have markedly improved hemophilia treatment. However, management is still largely inadequate in developing countries. Therefore, carrier detection and prenatal diagnosis remain the key steps for the prevention of the birth of children with hemophilia in developing countries where patients with this coagulation disorder rarely live beyond childhood. Carrier detection and prenatal diagnosis are possible by direct or indirect genetic analysis of the F8 or F9 genes. In countries with more advanced molecular facilities and higher budget resources, the most appropriate choice in general is a direct strategy for mutation detection by prescreening techniques or direct mutation detection. However, in countries with limited facilities and low budget resources, carrier detection and prenatal diagnosis are usually performed by linkage analysis with genetic markers. This article suggests the possibilities of genetic diagnosis and a feasible strategy for carrier detection and prenatal diagnosis in families with hemophilia A and B in developing countries.

Prenatal diagnosis of hemophilia.

There are widely differing views about the acceptability of prenatal diagnosis and selective abortion for hemophilia both among affected families and healthcare professionals. This debate is beyond the scope of this chapter, but it is essential that the mechanistic approach outlined here is not seen in isolation, but as part of a coordinated package of laboratory tests and “nondirective” counseling.

Prenatal diagnosis in hemophilia A using factor VIII gene polymorphism--Indian experience.

The heterogeneous nature of the mutations, the size, and the complexity of the factor VIII gene makes direct mutation analysis in hemophilia A families in India an option that is not very feasible and practical. Thus, carrier screening and prenatal diagnosis of hemophilia A often depends on haplotype analysis using restriction fragment length polymorphisms (RFLP) and short tandem repeat (STR) markers to track the defective factor VIII gene within a family. The main objective of this present study was to assess the utility of using polymerase chain reaction (PCR)-based five polymorphic markers: four intragenic Hind III, Bcl I, intron 13, and intron 22 STRs and one extragenic marker St14 in prenatal diagnosis. Forty-one chorionic villus samples (CVS) were studied from 41 families with a history of hemophilia A. PCR and RFLP were used for screening. Intron 22 STR showed the highest informativeness (60.9%), followed by Hind III (51.2%), Bcl I (46.3%), & intron 13 STR (51.2%); the extragenic marker St14 (VNTR) was informative in 46.3% of families. Linkage analysis, with the combined use of these five PCR-based polymorphic markers, gives good informativeness of 87.8% in the Indian population. Of the 41 CVS tested, 21 were found to be male fetuses and of these 13 were found likely to be affected with hemophilia A. Only in 12.2% of the families were none of the markers informative.

Carrier testing and prenatal diagnosis of haemophilia B by SSCP in an Iranian family

Haemophilia B is an X-linked recessive bleeding disorder caused by multiple molecular defects in factor IX gene. During our genotyping programme of haemophilia B patients, a family from the west of Iran was referred to us. We first detected the mutation in the index case of the family by single-strand conformation polymorphism (SSCP) technique. This technique was then applied in carrier detection and prenatal diagnosis for females of this family. Sequencing later revealed the mutation as being G to A at 20519.

Carrier detection and prenatal diagnosis of hemophilia A.

The aim of this study was to establish a simple, rapid carrier detection and prenatal diagnosis system for hemophilia A. Intron 22 inversion in FVIII gene was directly examined by long-distance polymerase chain reaction. Polymorphisms of factor VIII intragenic restriction fragment length polymorphism of Bcl I, short tandem repeat (STR) within intron 13 and 22, and extragenic DXS 52 (St 14) variable number tandem repeats (VNTR) loci were assessed by hereditary linkage analysis. The diagnostic rates for these loci were 47.6% (intron 22 inversion), 27.8% (Bcl I, 28.6% and 29.4% (STR within intron 13 and 22), and 81.3% (DXS52), respectively. The overall diagnostic rate in 21 families was 94.7%. The diagnosis in hemophilia A patients or carriers can be made if intron 22 inversion is present. The intragenic and extragenic loci hereditary linkage analysis could be used to establish the diagnosis in intron 22 inversion-negative patients.

St 14 (DX S52) VNTR polymorphism in the Indian population and its application in carrier detection and prenatal diagnosis of haemophilia A families.

The frequency of different polymorphic variants of the multi-allelic locus DXS52 (St14) of the human X chromosome, adjacent to the factor VIII gene, was evaluated by means of PCR for the heterogeneous population of India. It was shown that the heterozygosity index of this polymorphism in the studied population of 282 unrelated subjects was much higher (88%) than reported elsewhere. Two new alleles (1,750 bp and 1,420 bp) were detected during this study. Out of 65 families studied using this polymorphism for carrier detection and antenatal diagnosis, 58 were informative with this polymorphism. thus indicating that this polymorphism can serve as an important marker in the carrier detection and prenatal diagnosis of haemophilia A families.

Carrier detection and prenatal diagnosis of hemophilia A in a Korean population by PCR-based analysis of the BclI/intron 18 and St14 VNTR polymorphisms.

We have undertaken this study to identify the usefulness of polymerase chain reaction (PCR)-based analysis of DNA polymorphisms in the BclI/intron 18 and St14 variable number of tandem repeats (VNTR) loci for carrier detection and prenatal diagnosis of hemophilia A in the Korean population. We have analyzed these polymorphisms in members of 105 unrelated Korean families with severe hemophilia A. The observed heterozygosity rates for the BclI/intron 18 and St14 VNTR polymorphisms were 21.0% and 71.3%, respectively. The BclI/intron 18 polymorphism was less informative in Koreans when compared with Caucasians and Japanese. The allele frequencies for St14 VNTR in Koreans were different from those in Caucasians. Compared with Caucasians, there was a markedly higher occurrence of low molecular weight alleles in Koreans. The observed heterozygosity for the St14 VNTR polymorphism in combination with the BclI/intron 18 polymorphism was 81.9%. These two polymorphisms were applied to determine the carrier status of 107 women from 65 unrelated families, and to assess fetal status in 37 pregnancies. So far, we have experienced one case of misdiagnosis of carriership. Our study demonstrated that the PCR-based analysis of the BclI/intron 18 and St14 VNTR polymorphisms was useful in the carrier detection and prenatal diagnosis of hemophilia A in the Korean population.

Long-term psychological effects of carrier testing and prenatal diagnosis of haemophilia: comparison with a control group

The long-term psychological effects resulting from carrier testing and prenatal diagnosis (PD) of haemophilia were evaluated by comparing mental symptomatology scores (Symptom Check List, SCL-90) for 50 carriers of haemophilia who had undergone PD about five years earlier, 55 carriers who had not undergone PD and 262 control women who were not carriers. All of the women had children. Carrier testing for haemophilia per se and in combination with PD does not appear to have negative long-term psychological effects. A low tendency for somatization seems to be a factor characteristic of carriers considering PD for haemophilia and it was particularly salient when carriers had to consider the more invasive late PD procedures (amniocentesis and fetal blood sampling). Women who performed late PD appeared to represent a positive selection of carriers for whom a good sense of coherence and social support protected them from negative long-term psychological effects. It is reassuring that early PD by CVS, which is the current method of choice for PD for haemophilia, did not appear to have negative long-term psychological effects, even for women who had had an early abortion following PD.

Long‐term psychological effects of carrier testing and prenatal diagnosis of haemophilia: comparison with a control group

Long‐term psychological effects of carrier testing and prenatal diagnosis of haemophilia: comparison with a control group

Carrier Detection and Prenatal Diagnosis of Hemophilia A. Status of The Art

Carrier Detection and Prenatal Diagnosis of Hemophilia A. Status of The Art

Prenatal diagnosis of haemophilia.

Genotype assessment based on direct identification of the pathogenic mutation in a chorionic villi sample obtained in the 11-12th gestational week is the most reliable method for prenatal diagnosis and should be used if available. Genetic linkage studies of polymorphisms should be the second choice in the assessment of carriers and in prenatal diagnosis. Carriers of haemophilia should be offered adequate psychosocial support before, during and after the prenatal diagnostic procedures.

How do carriers of hemophilia and their spouses experience prenatal diagnosis by chorionic villus sampling?

A semistructured personal interview with 29 female carriers of hemophilia and 23 of their spouses was performed at a median of 3 1/2 years after the first chorionic villus sampling (CVS) and gene analysis. Carriers with a hemophilic father or brother, had high sense of coherence (SOC) scores, and thus would be expected to have good ability to handle the stress of prenatal diagnosis (PD). Prenatal diagnosis of hemophilia by CVS was generally well accepted by the women and their spouses. However, the period while waiting for test results was experienced as emotionally troublesome by both the women and their spouses; women reporting significantly more psychiatric or psychosomatic symptoms than men. Positive effects resulting from participation by the spouse on how the women experienced PD could not be identified. Selective abortion after first trimester PD was clearly experienced as emotionally painful by both the women and their spouses, these reactions being similar to, but not as pronounced as those found after second trimester selective abortion. Notably, signs of depressive mood were observed several years after the abortion, and the provision of psychosocial support should be recommended for couples who decide upon termination of pregnancy following PD.

Carrier testing and prenatal diagnosis of haemophilia--utilisation and psychological consequences.

Attitudes towards prenatal diagnosis, and abortion vary widely between different countries, religions, cultures and over time. Carrier testing and prenatal diagnosis (PD) of haemophilia have become an integrated part of the comprehensive care for haemophilia in Sweden as well as in many other countries. Almost all carriers are interested in carrier testing if they are aware of the possibility. With the development of PD by chorionic villus sampling in the first trimester, the method became acceptable for many carriers, and it has in Sweden actually had an effect on the incidence of haemophilia in the 1990s. The use of PD is more common among women who perceive haemophilia as a very serious disease and who have a positive attitude towards legal abortion. The main reason for carriers not to use PD was that they do not find haemophilia to be a sufficiently serious disorder to justify an abortion. Women and their spouses are under a great deal of psychological pressure in association with the PD procedure, and the psychological consequences of having to terminate a pregnancy are long-lasting. At follow-up, about 6 years after PD and abortion, these women, however, do not have more signs of psychological distress than women without PD experience. Nevertheless, they must be offered qualified assistance both before and after PD as well as adequate follow-up after an abortion to help them cope with the emotional strain they are under.

Genetic services available for counselling and prenatal diagnosis of haemophilia.

Genetic services available for counselling and prenatal diagnosis of haemophilia.