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.     

Diagnosis of two related carriers of severe haemophilia B with no family history

Haemophilia B is an X-linked disease affecting 1 in 30 000 males. Carrier diagnosis is usually carried out only in female relatives of haemophilic males, and the likelihood of discovering a carrier without a haemophilic male is very low. In this report we present the cases of two related women without a family history of haemophilia who were diagnosed as haemophilia B carriers. Following a minor haemorrhage in the proband, she and her mother were thought to be haemophilia B carriers because of a low factor IX level (16 and 23 IU dL-1, respectively; normal values >50 IU dL-1). The non-sense mutation C31118T, which is associated with severe haemophilia B, was detected in both women. This allowed us to diagnose them as being definite carriers of severe haemophilia B and give appropriate genetic counselling.     

Direct mutation analysis as the preferred method for carrier diagnosis in families with isolated cases of haemophilia B

Summary. Approximately one-third of haemophilia B cases are described as isolated due to their occurrence in families with no prior history of the disorder. In this report, two families with isolated haemophilia B were studied by the standard method of restriction fragment length polymorphism (RFLP) analysis coupled with factor IX activity and antigen levels with the aim of achieving carrier diagnoses. The limitations of using this approach in the determination of carrier status were highlighted by diagnostic problems arising in both families. The problems included difficulty in interpreting bioassay results, homozygosity for the RFLP marker in a key family member and the possibility of germline mosaicism. Unequivocal carrier diagnosis in the two families was ultimately achieved by direct mutation analysis.     

A haemophilia A and B molecular genetic diagnostic programme in Hungary: a highly informative and cost-effective strategy

Our aim was to set up a protocol in order to provide carrier and prenatal diagnosis to Hungarian haemophilia A (HA) and B (HB) patients and their relatives. For HA, a combination of direct mutation detection and some indirect marker analyses were used: the detection of the inversion mutation and analysis of three polymorphisms, BclI, IVS13 (CA)n and P39(CA)n. In severe cases, direct mutation detection was performed first. In inversion-negative severe cases and in moderate and mild cases, indirect methods were used. For carrier and prenatal diagnosis in HB, four polymorphisms, DdeI, TaqI, XmnI, and HhaI were examined. Our DNA bank contains samples from 50 HA families (34 severe, 15 moderate and one mild) and seven HB families from different parts of the country. In 100% of the HA cases either the gene inversion and/or at least one of the polymorphisms was found to be informative for carrier or prenatal diagnosis. In the HB cases, an informative marker was found in 95% of the cases (19 of 20). We conclude that these strategies are sufficient to make genetic diagnosis available to almost all HA and HB families in the region. This approach is highly informative and cost-effective, so it can be very useful in countries where direct sequencing of genes for factor VIII and IX is not available for routine diagnosis.     

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.     

Heteroduplex screening for molecular defects in factor IX genes from haemophilia B families

Summary. Heteroduplex screening of amplified fragments containing sequences of all known small haemophilic mutations in the factor IX gene localized mutations in 18 new families: 12 were at common recurrent sites; three were novel. Carriers and/or patients from each of 41 families with mutation in 7 exons and 5' and 3' non-coding regions were positive.     

Determination of haemophilia A carrier status by mutation analysis

A reliable method for determination of carrier status and genetic counselling is required for effective control of haemophilia. Linkage analysis is currently the most widely used method for this purpose; however, in cases where there is no prior family history and/or unavailability of informative polymorphic markers it is less applicable. Detection of a mutation characterized in each family may be an alternative method for determination of the carrier status. In this study, linkage analysis using four polymorphic DNA markers, and direct mutation analysis were compared to determine the carrier status in six unrelated Thai haemophilia A families, two with a family history and four without. In the two families with a family history of haemophilia A, the carrier and noncarrier statuses could readily be determined in eight females by either linkage or direct mutation analysis. In the four families without a family history, the polymorphic DNA markers for linkage analysis were informative in two families and uninformative in the other two. The carrier status could be excluded in all four female siblings of the patients in the former. However, the specific FVIII gene mutation was not observed in the mother of one patient, who should have carried the mutation. In the remaining two families with uninformative polymorphic DNA markers, the carrier and noncarrier statuses of four female members could only be determined by direct mutation analysis. Therefore, direct mutation analysis could circumvent the limitations of linkage analysis in the determination of haemophilia A carrier status in families without a previous history or informative polymorphic markers.     

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.     

A rapid method for haemophilia B mutation detection using conformation sensitive gel electrophoresis

Conformation sensitive gel electrophoresis (CSGE) was confirmed as an effective procedure for screening the factor IX (FIX) gene by detecting 10/10 previously known FIX gene mutations. The FIX genes of a further 11 haemophilia B patients with unknown mutations were then screened and an abnormal CSGE profile was identified in all cases. Subsequent DNA sequencing demonstrated one of these to be a novel mutation (31133insT, Arg338Fs), the remaining 10 having been previously reported on the haemophilia B database. Mutation screening of the FIX gene using CSGE was demonstrated to be a rapid and efficient means of carrier analysis in families with haemophilia B.     

Moderate haemophilia B in a female carrier caused by preferential inactivation of the paternal X chromosome.

The case of a female with moderate haemophilia B is reported. She is the only affected member of her family, and factor IX RFLP analysis shows her to have inherited no maternal markers for polymorphisms located in the first intron and 8 Kb 3' of the polyadenylation signal (DdeI and HhaI, respectively). This clearly indicates a deletion involving at least the last 7 exons of the factor IX gene. Her other factor IX gene inherited from her healthy father is normal as her son is also healthy. This suggests the patient's haemophilia to be due to gross bias in the proportion of factor IX-producing cells with an inactive paternal X chromosome. Methylation studies on the 5' region of the PGK gene show that virtually all the patient's lymphocytes carry a hypermethylated and presumably an inactive paternal X chromosome. The reason for this bias in the activity of her two X chromosomes is not clear, as no chromosomal alterations were found.     

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.     

Factor IX gene mutations in haemophilia B: a New Zealand population-based study

Summary. Haemophilia B (Christmas disease) is an X-linked bleeding disorder resulting from an inherited deficiency of coagulation factor IX activity. Due to the heterogeneity of mutations within the factor IX gene there is a marked clinical variability in disease severity. By applying techniques of mutational analysis and direct sequencing of PCR products it is now potentially possible to determine the pathogenic gene defect in entire haemophilia B populations. We report here characterization of the factor IX gene defect in all the haemophilia B patients in New Zealand as part of a nationwide approach towards providing efficient and cost-effective haemophilia B genetic counselling services for these families. Twenty-six different mutations were identified in 32 unrelated haemophilia B families. Three defects at nucleotide positions +8,6659 and 17696 are novel mutations which have not been reported by other laboratories. A PCR-based diagnostic screening test for direct mutational analysis could be performed in most cases; 17 of the 26 mutations altered a restriction enzyme recognition sequence and, with the exception of the total gene deletion, base changes not affecting a restriction enzyme site could be detected by allele-specific PCR.     

Variability in bleeding phenotype in Amish carriers of haemophilia B with the 31008 C→T mutation

Summary.  The aim of this study was to characterize the variability of bleeding phenotype and its association with plasma factor IX coagulant activity (FIX:C) in haemophilia B carriers in a large Amish pedigree with a unifying genetic mutation, C-to-T transition at base 31008 of the factor IX gene ( Xq27.1–27.2) . A cross-sectional survey of haemophilia B carriers included a multiple choice questionnaire evaluating symptoms of mucocutaneous bleeding, joint bleeding and bleeding after haemostatic stress [menstruation, postpartum haemorrhage (PPH), dental extractions and invasive surgeries]. Severity of bleeding was graded as 0 to 4, 0 being no bleeding whereas 4 being severe bleeding. Association between total bleeding scores and the FIX:C was evaluated. Sixty-four haemophilia B carriers participated in this study. Median age: 18 years (range 1–70 years); median bleeding score: 1 (range 0–8). Besides PPH, isolated symptoms of bruising, epistaxis, menorrhagia and postsurgical bleeding including dental extraction were not associated with lower FIX:C. Bleeding score ≥3 was associated with involvement of at least two bleeding sites and a lower mean FIX:C of 42 ± 10.3% (95% CI 36.4–47.7) while a score >3 had involvement of ≤2 sites and higher mean FIX:C of 54.9 ± 21.5% (95% CI 49–61), P  = 0.005. Subcutaneous haematoma formation and bleeding after haemostatic stress requiring treatment were associated with bleeding scores ≥3. Phenotypic variability existed among the carriers of haemophilia B who belonged to a single pedigree carrying a single unifying mutation. The utility of bleeding scores to define bleeding phenotype precisely in haemophilia B carriers needs further evaluation.     

The use of DNA markers for carrier detection and prenatal diagnosis of haemophilia A in Egyptian families

Summary. Haemophilia A is the most common inherited X‐linked recessive bleeding disorder. The aim was to investigate the usefulness of two DNA markers in linkage analysis, one intragenic BCL1 affecting restriction site in intron 18, and is detected as restriction fragment length polymorphism (RFLP), and one extragenic variable number of tandem repeat (VNTR) locus DXS52 (St14) to formulate an informative and accurate carrier detection and prenatal diagnosis. The study included 46 families with at least one child affected with haemophilia A, and 30 unrelated normal females as control group. Polymerase chain reaction (PCR) and restriction enzyme analysis were used to study the polymorphism in BCL1, and long‐distance PCR for detection of VNTR (ST14) alleles. The incidence of BCL1 (+) allele was 74%, 72% and 60% in patients, mothers and control group, respectively. Expected heterozygosity for BCL1 was 40% in mothers of affected cases compared with 48% in the female control group. However, observed heterozygosity was found to be 48% in the mothers of affected cases, compared with 60% in the control group. Thus, 48% of the studied families are informative for this marker alone. Nine different alleles of VNTR (St14) were observed in mothers and six alleles in affected cases and six in the control group. The most prevalent alleles were 1300 bp (45.5% and 34%) and 700 bp (13.6% and 20%) in patients and their mothers, respectively. Observed heterozygosity in mothers was 41% compared with 43.3% in controls. The combined use of both BCL1 and St14 markers raised the informative rate to 63.6%. Carrier detection and prenatal diagnosis is possible in haemophilia A families using both DNA markers. We suggest screening haemophilic families first for BCL1 polymorphism followed by analysis of St14 locus.     

Factor IX gene sequencing by a simple and sensitive 15-hour procedure for haemophilia B diagnosis: identification of two novel mutations

We have developed a simple, sensitive and cost-effective direct DNA sequencing procedure for the molecular diagnosis of haemophilia B. All factor IX gene essential regions were amplified under identical thermocycling parameters allowing mutation identification in less than 15 h from blood sample collection. Identical results in terms of accuracy and speed were obtained when using a single hair as the source of DNA. Using this approach, we have found mutations in 10 out of 10 haemophilia B patients, two of which are novel (P287T and S123C). Very suitable for individual studies, this procedure can be easily scaled up for higher throughput.     

Prenatal diagnosis of haemophilia B: the Italian experience

This article describes prenatal diagnosis (PND) of haemophilia B (HB) within the framework of Italian haemophilia centres and genetics laboratories. The study details the experience from six haemophilia genetic centres (three in the North, one in the Centre and two in the South of Italy) and summarizes the different techniques used to perform PND of HB during the last 15 years. To date, the Italian HB database includes 373 characterized unrelated patients and their genetic information has permitted the identification of 274 carriers of childbearing age. This database represents the main instrument for timely and precise PND. Sixty‐six prenatal diagnoses were performed on 52 HB carriers whose average age at the time was 34 (ranging from 24 to 44 years). In 44 cases, genetic counselling for carrier status determination was performed before pregnancy, while eight were not studied prior to pregnancy. Foetal samples were obtained by chorionic villus sampling in 52 cases, by amniocentesis in 12 while two were diagnosed by analysis of free foetal DNA obtained from maternal peripheral blood. In 35 (53%) pregnancies the foetus was female. For 31 men (47%), haemophilia status was determined by analysis of previously determined informative markers or familial mutations (12 affected and 19 unaffected). There may be more than one laboratory involved in the PND diagnostic pathway (providing DNA extraction, karyotype analysis, gender determination, maternal contamination detection, molecular diagnosis and sequencing). Good communication between all the parties, coordinated by the haemophilia centre, is essential for a successful and rapid process.     

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 Italian haemophilia B mutation database: a tool for genetic counselling, carrier detection and prenatal diagnosis

Introduction

The Italian database of factor IX gene (F9) mutations has been built since 2001 and is, so far, the most practical instrument for comprehensive genetic counselling, carrier detection and prenatal diagnosis. Over time the haemophilia B database has been enriched by entries on a larger number of patients and molecular genetic data identifying heterogeneous mutations spanning the entire F9.

Methods

Conformation sensitive gel electrophoresis is a variant of heteroduplex analysis, which has been applied for screening F9 for mutations, which are further fully characterised by direct sequencing of the amplified mutated regions. This project has involved 29 Italian haemophilia centres and provides data concerning the analysis of a cohort of 306 unrelated patients with haemophilia B (191 with severe, 67 with moderate and 48 with mild disease, including 8 patients with severe haemophilia B with inhibitors). The recorded data include levels of factor IX clotting activity, inhibitor status and clinical severity.

Results

Detailed analysis of the mutations revealed 164 different mutations, that are considered as unique molecular events (8 large deletions, 11 small deletions, 1 combined deletion/ insertion, 2 insertions, 104 missense, 20 nonsense, 14 mutations in a splicing site, 3 in the promoter and 1 silent). The data recorded in the Italian F9 mutation database provided the basis to study 85 families with haemophilia B, involving 180 females (20 obligate carriers, 106 carriers and 54 non-carriers) and enabled 14 prenatal diagnoses to be made in 12 females.

Conclusions

Genetic analysis is required to determine female carrier status reliably. Female relatives may request carrier analysis, when a male relative is first diagnosed as having haemophilia or when they are pregnant. At present, the data collected in the Italian national register of mutations in haemophilia B provide the opportunity to perform prompt and precise determination of carrier status and prenatal diagnosis by specific mutation analysis.     

Multiplex analysis of two intragenic microsatellite repeat polymorphisms in the genetic diagnosis of haemophilia A

Summary. Use of molecular genetic studies for carrier detection and prenatal diagnosis of haemophilia A will be informative in approximately 75% of kindreds with a prior history of the disorder if previously reported bi-allelic intragenic polymorphisms are used. In this study we report the use of two multi-allelic, microsatellite repeat polymorphisms within the factor VIII gene in genetic testing for this disease. These two, dinucleotide repeat, polymorphisms have been analysed using a multiplex polymerase chain reaction (PCR) protocol, and results can be available within 3 d of receipt of samples. At the intron 13 polymorphic locus we have confirmed the original observation of eight alleles, whilst at the intron 22 locus, in contrast to a preliminary report of this polymorphism, we have seen five alleles. We have analysed 32 families (174 subjects) with the two microsatellite repeat markers and have found that using these two polymorphisms alone, 81% of families are informative for linkage analysis. The intron 13 repeat was informative in 22/31 families tested (71%) and the intron 22 polymorphism was informative in 12/17 families (71%). We have also found that in 11/32 families tested (34%) the microsatellite repeats were the only informative intragenic markers. In view of these observations, we believe that the most effective strategy for initiating haemophilia A genetic testing is to combine the multiplex PCR analysis of the intron 13 and 22 dinucleotide repeats with the PCR analysis of the intron 18 BclI marker. In our population this protocol provided informative results in approximately 88% of families and results can be available within 3 d of receiving the samples for testing.