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.     

The status of carrier and prenatal diagnosis of haemophilia in China

Haemophilia A (HA) and haemophilia B (HB) are the most common X-linked inherited bleeding disorders. It is important to detect the carrier women in families with HA/HB and subsequent antenatal diagnosis of confirmed carriers. This study consists of 102 HA families which include 68 mothers for prenatal diagnosis and 107 female relatives for carrier diagnosis, and 29 HB families which include 16 mothers and 31 female relatives respectively. The rapid fluorescent PCR with two groups of different combined polymorphism markers was applied for linkage analysis in HA and HB families respectively. The Amelogenin gene was added to help the detection of gender diagnosis. Gene sequencing was also used to detect the mutations directly. There were 37 causative F8C mutations (23 novel) and 24 causative F9C mutations (eight novel) found in this cohort of patients. Few of the women could not be diagnosed due to homologous recombination and/or inability to locate the mutation. Complicated cases have been found in some families. With regard to carrier and prenatal diagnosis, it was considered that genetic diagnosis by linkage analysis and direct sequencing was successful. Some special families might require combination of the linkage analysis and gene sequence for a successful diagnosis. New intragenic SNP and STR sites special to Chinese population need to be discovered.     

Feasibility of prenatal diagnosis and carrier detection in South African haemophilia A patients

The feasibility of DNA diagnosis for haemophilia A was tested in South African patients and families by screening for the common inversion mutation in the factor VIII gene and for the intragenic microsatellite markers in introns 13 and 22. The allele frequencies at the two microsatellite loci were significantly different, with informativity being higher in the Negroid (100%) than the Caucasoid group (67%). In severely affected haemophiliacs the inversion was found in 43% (6/14) of Negroids but in only 32% (13/41) of Caucasoids. Presence of a second common unidentified mutation may account for the low frequency in the latter. Haplotype analysis shows a disproportionately high frequency of an (AC)₂₀ intron 13–(AC)₂₆ intron 22 inversion negative Caucasoid haemophilia chromosome, supporting a founder effect.     

A new strategy for prenatal diagnosis in a sporadic haemophilia B family

Although the quality of life for haemophiliacs has clearly improved in the last few years, haemophilia still remains a serious disorder justifying prenatal diagnosis (PD) and, if necessary, termination. Because chorionic villus sampling (CVS) is performed in the first trimester of pregnancy, an increasing number of carriers are interested in this test. It has been shown that waiting for the results is particularly distressing for pregnant women, therefore decreasing the diagnostic procedure time can be psychologically helpful. Here we report on PD in a sporadic haemophilia B family based on the direct identification of the pathogenic mutation in a CVS taken at the 12th gestational week. In order to hasten the results, we recovered DNA from a single villus fragment boiled in water and used it directly for PCR reaction. Conformation-sensitive gel electrophoresis (CSGE) was used to detect the mutation in the haemophilia carrier and in the foetus. This approach allowed us to obtain a diagnosis within 24 h of CVS, thus avoiding the long-term psychological effects on the pregnant woman.     

Identifying carriers at high risk for negative reactions when performing prenatal diagnosis of haemophilia

The aim of the current study was to determine which pregnant carriers of haemophilia are at particularly high risk for having notably negative psychological reactions in association with prenatal diagnosis (PND) by fetal blood sampling of offspring haemophilia. Among 29 carriers of haemophilia A or B, notable psychiatric or psychosomatic symptoms in association with PND were significantly related to the woman's having a negative view of herself in general and of being a gene carrier, a planned pregnancy, high education, a good general knowledge of haemophilia and a guiding philosophy of life. Women with strong negative reactions significantly more often reported signs of depressive mood at follow-up. The results suggest that giving more information and promoting increased knowledge about the disease and the PND procedure will, by itself, not help women to cope better emotionally. Provision of prospective psychosocial support is recommended.     

Comparison of attitudes towards prenatal diagnosis and termination of pregnancy for haemophilia in Iran and Italy

Prenatal diagnosis (PND) is an important issue in the comprehensive care of haemophiliacs. As a consequence of technological progress made in the field of PND, the early detection of an affected fetus provides the expectant couple with a chance to terminate pregnancy. This study was undertaken to assess the attitudes of two different haemophilic populations in Iran and Italy towards PND and termination of pregnancy. This study series included 59 Iranians (38 haemophilia A patients and 21 mothers) and 50 Italians (27 haemophilia A patients, 16 mothers and seven fathers). All the 109 participants received a questionnaire including demographic characteristics and evaluating the psychological effects stemming from PND and termination of pregnancy. Approximately 84.7% of the Iranians and 35.4% of the Italians were not familiar with the possibilities afforded by PND for haemophilia (P < 0.001). Termination of pregnancy appeared to be accepted by 58.2% of the Iranian and 16.7% of the Italian participants (P < 0.001). The greater rate of acceptability of abortion in Iranians may be due to differences in the quality of patient care in the two countries.     

Direct carrier testing of haemophilia B by SSCP

Summary. Haemophilia B is due to multiple molecular defects in the factor IX gene. Most of them are single base substitutions, and can now be identified by direct sequencing of the coding sequence of the factor IX gene, preceded or not by a screening strategy. In some instances the mutation alters an enzyme recognition site and this allows rapid and accurate carrier testing and prenatal diagnosis in the affected pedigree. This was not the case for the previously described nt 31119 (G– > A) mutation that we found in an extended haemophilia B pedigree, during the search for mutations in the factor IX gene in patients from Southern France. We first detected this mutation by single stranded conformation polymorphism (SSCP) and then identified it by DNA sequencing. Carriership could be easily determined in the females of the pedigree by analysis of the SSCP patterns. Our results indicate that the SSCP analysis of amplified genomic DNA fragments can be successfully used as a diagnosis approach for direct carrier testing and prenatal diagnosis.     

Prenatal diagnosis of haemophilia in Sweden now more commonly used for psychological preparation than termination of pregnancy

The aims of the study were to define the frequency, outcome and reasons for prenatal diagnosis (PND) in Sweden during a 30‐year period in order to study trends and changes. The study population, from the Swedish nationwide registry of PND of haemophilia, consisted of 54 women, compromising >95% of all, who underwent PND (n = 90) of haemophilia during 1977–2013. PND was performed by amniocentesis (n = 10), chorionic villus sampling (n = 64) or by analysis of foetal blood (n = 16). A total of 27/90 foetuses were found to have haemophilia. Sixteen went to termination and the remaining 11 were born during the end of the study period (2000–2013). Three of 90 pregnancies were terminated due to findings other than haemophilia and 3/90 PNDs led to miscarriage. In the 30 families with known haemophilia, PNDs (n = 55) were used in 27/55 cases for ‘psychological preparation’ and in 23/55 cases with the aim to terminate the pregnancy. A subgroup of women (n = 17) who consecutively underwent PND in the years 1997–2010 were further interviewed. For 11/17, being a carrier had a negative effect on the decision to become pregnant, and in 11 cases PND had influenced their decision to conceive. Our study show that PND of haemophilia is stable over time but increasingly used during the last decade as a psychological preparation for having a child with haemophilia as compared to earlier where more terminations of pregnancies were conducted.     

The use of the Port-A-Cath in children with haemophilia — a review

Summary. Port-A-Caths have been used increasingly in children with severe haemophilia. In non-inhibitor patients where Port-a-Caths were used to facilitate long-term prophylaxis, the infection rate is rather low and ranges in the various studies from 0 to 29%, with a median follow-up time of about 27 months. Patients that received the Port-A-Cath for the induction of immune tolerance (inhibitor patients) have a high infection rate of 50% to 83%. Although this percentage is high, good venous access is extremely important, especially in this group. The number of both inhibitor and non-inhibitor patients in the studies are very small, and a prospective survey is important to obtain more adequate data.     

Towards achieving global haemophilia care — World Federation of Hemophilia programmes

Summary. The overall purpose of the “Decade Plan” published in 1992 was to do everything possible to make effective treatment available to people with haemophilia throughout the world. In 1990 it was estimated that there were 350,000 individuals with haemophilia worldwide, with 80% or 280,000 without treatment. By the year 2020 this may have reached the figure 440,000, or the population of Jerusalem. Earlier this century Carroll Birch reported a series of 113 patients where 82 died before their 15th year, and only six survived beyond 40. Thus as Jones has pointed out for the developing world this is a paediatric problem for the WFH to face now. The examples described in this paper illustrating the WFH programmes and the committment of many people from the 88 member organisations worldwide as well as the doctors and scientists shows what can be done through cooperation. There is no doubt that these achievements are capitalising from the unique quality of WFH from the beginning, that is a membership which includes both those with haemophilia and their families, and those with an interest in treating haemophilia.     

The impact of prenatal diagnosis on the incidence of haemophilia in Sweden

Summary A demographic survey was made of all children (n= 137) born with severe or moderate haemophilia in Sweden during the period 1970-92. Bn addition, all prenatal diagnoses (n= 86) performing during the period were evaluated. The annual incidence of severe and moderate haemophilia, having remained constant for decades, increased from 0.78/10,000 males in the 1970s to 1.34 in the 1980s, levelling off at 1.31 in the 1990s. Although prenatal diagnosis did not affect the incidence of haemophilia in the 1970s and 1980s, it did so in the 1990s, because the incidence would have been 40% higher (1.83) had not prental diagnosis been available and 16 affected fetuses been aborted. The average proportion of sporadic cases, 62%, remained almost unchanged during the study period, suggesting mutation rates to be constant. There were fewere children in families with known haemophilia than in sporadic families, but no evidence was found to suggest that the frequency of female offspring (i.e. potential carriers) born in haemophilia families had increased since the option of prenatal diagnosis was introduced.     

Genetic diagnosis of haemophilia and other inherited bleeding disorders

Summary.  Inherited deficiencies of plasma proteins involved in blood coagulation generally lead to lifelong bleeding disorders, whose severity is inversely proportional to the degree of factor deficiency. Haemophilia A and B, inherited as X-linked recessive traits, are the most common hereditary hemorrhagic disorders caused by a deficiency or dysfunction of blood coagulation factor VIII (FVIII) and factor IX (FIX). Together with von Willebrand's disease, a defect of primary haemostasis, these X-linked disorders include 95% to 97% of all the inherited deficiencies of coagulation factors. The remaining defects, generally transmitted as autosomal recessive traits, are rare with prevalence of the presumably homozygous forms in the general population of 1:500.000 for FVII deficiency and 1 in 2 million for prothrombin (FII) and factor XIII (FXIII) deficiency. Molecular characterization, carrier detection and prenatal diagnosis remain the key steps for the prevention of the birth of children affected by coagulation disorders in developing countries, where patients with these deficiencies rarely live beyond childhood and where management is still largely inadequate. These characterizations are possible by direct or indirect genetic analysis of genes involved in these diseases, and the choice of the strategy depends on the effective available budget and facilities to achieve a large benefit. In countries with more advanced molecular facilities and higher budget resources, the most appropriate choice in general is a direct strategy for 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 reviews the genetic diagnosis of haemophilia, genetics and inhibitor development, genetics of von Willebrand's disease and of rare bleeding disorders.     

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.     

Confronting the psychological burden of haemophilia

Summary. Providing comprehensive care, counselling and support to haemophilic patients, and their parents have always been quite complex for haemophilia treatment centres. Nowadays, starting with recent developments in genetic counselling, prenatal diagnosis and carrier testing, the psychological burden on patients and parents might possibly have increased, compared with even the recent past. The emotional strains and worries associated with a possibly affected newborn and his care through childhood and adolescence may also have a grievous impact on couple dynamics and marital relationship. The impact may be even higher in families in which haemophilia is newly diagnosed. The main psychological problems faced by parents and then by affected individuals are herein chronologically reviewed, starting from genetic counselling before conception through childhood, adolescence and adulthood. Aware of the psychosocial burden on patients and their families associated with haemophilia, from prenatal diagnosis and carrier testing until later stages of life of the affected individual, a board of Italian haemophilia specialists and psychologists is designing and organizing an innovative network of psychological support services in some Italian haemophilia centres and promoting specific educational programmes in this setting.     

A modified I-PCR to detect the factor VIII Inv22 for genetic diagnosis and prenatal diagnosis in haemophilia A

To explore the effectiveness of modified inversion-polymerase chain reaction (I-PCR) to detect the factor VIII (FVIII) intron 22 inversion (Inv22) for genetic diagnosis and prenatal diagnosis in haemophilia A (HA). Both modified I-PCR and LD-PCR were applied to analyse the FVIII Inv22 for 24 patients with HA. Prenatal diagnosis was performed on six foetuses. Foetal blood samplings were carried out by cordocentesis from 22 to 26 weeks of gestation. Ten patients with FVIII Inv22 in 10 HA families were found, and the remaining 14 patients were found without the Inv22 in 19 HA families. Prenatal diagnosis confirmed that four foetuses were normal and all of them born normally. However, two foetuses had been identified as abnormal and undergone abortion. Compared with LD-PCR, modified I-PCR is more rapid and convenient for detecting the FVIII Inv22 in genetic diagnosis. It is recommended that a patient undergoes both modified I-PCR (to detect the FVIII Inv22) and biochemical assay (to measure the FVIII activity of umbilical cord blood) in prenatal diagnosis. When we have more experience, the DNA samples from chorionic villus or amniotic fluid can be analysed for prenatal diagnosis using the modified I-PCR alone.     

Blood smears and prenatal diagnosis

Fetal blood sampling is a widely used technique for both diagnostic and therapeutic purposes. However, despite the fact that examination of blood smears is a critical step in the study of a large variety of disorders, the place of fetal blood smears analysis has been overshadowed by more sophisticated diagnostic tools such as those offered by molecular biology. Nevertheless, as highlighted by four cases presented in this paper, the role of fetal blood smears examination must be re-emphasized. The recognition of abnormal cytological findings limits the number of possible aetiologies, thus elegantly contributing to the diagnosis without multiplying unnecessary test techniques.     

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.     

Origin of mutation in sporadic cases of haemophilia A

The aim of this study was to define the origin of mutation in sporadic cases of severe haemophilia A. The series was composed of 31 families with sporadic severe haemophilia A in the geographical catchment area of the Malmö haemophilia centre. The mutation was characterized in 29/31 families: inversion type 1 (n = 11), inversion type 2 (n = 3), other inversion (n = 1), small or partial deletion (n = 6), insertion (n = 2), non-sense mutation (n = 4) and mis-sense mutation (n = 2). Of 29 probands, eight carried a de novo mutation, whereas the proband's mother was found to carry the mutation in 21/29 families. Of the 21 carrier mothers, 16 had de novo mutations (i.e. the proband's maternal grandfather and grandmother were non-carriers). Owing to the lack of samples from the grandparents, origin could not be determined in the remaining five families. Polymorphisms of the FVIII gene were used to determine whether the de novo mutation of the carrier mother was of paternal or maternal origin. In 15/16 cases the mutation was of paternal origin and in 1/16 cases of maternal origin. In the series as a whole, mutation frequency was 6-fold higher in males than in females, but no differences in the ratio of sex-specific mutations rates was found among different types of mutation.     

Application of intron 9 and intron 25 dinucleotide repeats of the factor VIII gene for carrier diagnosis in haemophilia A

Summary.  We describe the usefulness of two dinucleotide repeats located in intron 9 and in intron 25 of the factor VIII gene for carrier diagnosis of haemophilia A. We analyzed 100 unrelated Spanish women and 34 women from haemophilia A (HA) families in whom known intragenic markers were unhelpful in determining their carrier status. The heterozygosity rate of intron 9 and intron 25 markers in the 100 control women was lower (0.28 and 0.38, respectively) than the values obtained with common markers routinely used in our laboratory. However, the application of intron 9 and intron 25 markers was effective in identifying the at-risk X chromosome in 11 of 34 (32%) of the uninformative women from HA families. The combined use of these repeats with current markers may facilitate the identification of the X chromosome in HA families for application in carrier, prenatal and pre-implantation diagnoses.