Carrier detection and prenatal diagnosis of hemophilia B with more advanced techniques

Summary We used the PCR to amplify three polymorphic regions of Factor IX gene on 35 Italian families: DdeI intron 1, MnlI exon f, and the polymorphism HhaI located 8 kb at the 3 end of FIX gene. We analyzed the MnlI and HhaI markers on DGGE and DdeI polymorphism on agarose gel. We reached an informativity of 78% and we found one mutation at codon 145 (exon f) during the screening for MnlI polymorphism. Furthermore, we performed 16 prenatal diagnoses on chorionic villus samples; five were female and 11 male. Four were uninformative three healthy and one affected male fetus were recognized by PCR techniques, two healthy and one affected fetus by Southern analysis. In three pregnant women examined for the first time during pregnancy, the PCR technique allowed us to perform a rapid diagnosis of noncarrier status, avoiding the fetal sampling procedures.     

Recombinant DNA methods in hemophilia A: carrier detection and prenatal diagnosis

Hemophilia A is a serious inherited bleeding disorder of man that is caused by deficiency of blood coagulation Factor VIII. Major clinical problems in the treatment of hemophilia A include the transmission of disease by therapeutic blood products and the development of alloantibody inhibitors to transfused Factor VIII. The genetic counseling of affected families is made more difficult by the inherent inaccuracy of carrier detection based on plasma Factor VIII levels. The cloning of genomic DNA and cDNA for human Factor VIII has been a starting point for at least a partial solution to each of these problems. Determination of the Factor VIII gene structure has elucidated the cause of hemophilia A in several patients. RFLPs within or near the Factor VIII gene have provided genetic markers that allow unambiguous assignment of carrier status and accurate prenatal diagnosis. This is generally accomplished by restriction enzyme digestion and Southern blotting of genomic DNA with Factor VIII probes. At present, a high degree of skill is required to perform and interpret these tests. The use of the so-called PCR method for the amplification of specific genomic DNA fragments promises to make these analyses faster and less technically demanding.     

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.     

Hemophilia A: carrier detection and prenatal diagnosis by DNA analysis

In this study, we used DNA polymorphisms for carrier detection and prenatal diagnosis of hemophilia A in a large group of Italian families. The restriction fragment length polymorphisms (RFLPs) investigated were the intragenic polymorphic Bc/I site within the factor VIII gene; the extragenic multiallelic Taq I system at the St14 locus; and the extragenic Bg/II site at the DX13 locus. The factor VIII probe was informative in 30%, St14 in 82%, and DX13 in 60% of obligate carriers. The combination of factor VIII-Bc/I and St14-Taq I showed that 91% of obligate carriers were heterozygotes for one or both; with all three probes, only 4% of obligate carriers were noninformative. In families clearly segregating for hemophilia A, RFLP analysis allowed us to define the carrier status for the hemophilia A gene in all 27 women tested. RFLP analysis allowed us to exclude the carrier status in 39 of 45 female relatives of sporadic patients. The combination of RFLP analysis and biological assay of factor VIII allowed us to identify a de novo mutation in the maternal grandfather in 7 of 12 of the families with sporadic cases, for which members of three generations were available for study. Nine of 10 couples requesting prenatal diagnosis provided informative RFLP DNA pattern. Carrier status was excluded in two women, two fetuses were shown to be female, and prenatal diagnosis was carried out in five pregnancies by DNA analysis. Prenatal testing was successful in three instances and failed in two because a sufficient amount of chorionic villous DNA was not obtained for the analysis.     

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.     

Carrier Screening and Prenatal Diagnosis of Hemoglobinopathies. A Study of Indigenous and Immigrant Couples in Northern Greece,Over the Last 5 Years

Hemoglobinopathies constitute the most frequent monogenic disorders worldwide and thalassemias are the most frequent genetic disorders in Greece. Over a 5-year period (2002–2006), 1,375 couples were screened for hemoglobinopathies and counseled at our Thalassaemia Prevention Unit, Hippokration Hospital, Thessaloniki, Greece. In 148 cases (10.7%), both partners carried an abnormal hemoglobin (Hb) gene and genetic counseling was offered. One hundred out of 116 pregnancies were at-risk of giving birth to an offspring carrying either the homozygous or double heterozygous forms of the mutations under discussion. The remaining 16 pregnancies involved couples who were heterozygous for mutations that did not cause severe clinical disease, and were exempted from prenatal diagnosis. Twenty-six fetuses were found to be homozygotes or double heterozygotes for clinically significant mutations. These couples were informed of the danger of having an affected child but the termination or continuation of the pregnancy was left to the couples to decide. Nevertheless, all the couples preferred to terminate the pregnancies. The National Thalassaemia Prevention Programme has effectively decreased the incidence of thalassemia major and sickle cell syndromes in Greece.     

Carrier detection for hemophilia B: evaluation of multiple polymorphic sites

DNA analysis was performed in families with hemophilia B. Restriction fragment length polymorphisms (RFLPs) produced by endonucleases Taql, Xmnl, and Ddel were studied by two factor IX genomic probes, F9(VIII) and F9(XIII). Fifty-seven subjects from ten families were investigated; of them, 31 were carriers (11 obligate and 20 potential). Of the potential carriers, ten displayed laboratory features allowing for a phenotypic diagnosis of heterozygosity. Segregation analysis of the markers was informative in 19/20 potential carriers, which belong to nine of the ten studied families. Among the potential carriers, Taql allowed the carriership assessment in 15 (78.9%), Xmnl in 15 (94.7%), and Ddel in two (10.4%). Diagnosis was not possible in one family since a homozygosity in the key individuals with all the employed enzymes (Taql, Xmnl, Ddel, + BamHI) was found. Hemophilia B syndrome in two families likely results from a new mutation. In one family, a first-trimester prenatal diagnosis was performed. The use of RFLP analysis allowed us to improve genetic counseling as compared with the phenotypic evaluation by clotting factor assays. Indeed, evaluation of RFLP increased by 26% the carriership assessment of the potential carriers of the hemophilia B trait.     

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.     

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.     

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.     

Allele frequencies of three factor VIII gene polymorphisms in Iranian populations and their application in hemophilia A carrier detection

Hemophilia A is an X-linked recessive bleeding disorder caused by a quantitative or qualitative deficiency of blood coagulation factor VIII (FVIII). ARMS (amplification refractory mutation system) primers were designed to determine allele frequencies of three FVIII gene linked markers, IVS7 nt 27 G/A SNP, BclI/intron 18, and HindIII/intron 19 among 85 normal Iranian women from unrelated families. Then same method was applied to perform carrier detection for hemophilia A families. The allele frequencies of IVS7 nt 27 "G"/"A" allele, BclI "T"/"A" allele, and HindIII "C"/"T" allele among normal women were 0.88/0.12, 0.52/0.48, and 0.48/0.52, respectively. The three polymorphisms were found to be in strong linkage disequilibrium, which decreased the overall heterozygosity to 51%. Twenty-one women from 15 unrelated hemophilia A families were referred to us for hemophilia A carrier detection. Taking advantage of these three biallelic polymorphisms in conjunction with multiallelic St14 VNTR (locus DXS52), IVS13 (CA)n STR, and IVS22 (CA)n STR, carrier status was determined in 16 women (16/21 or 76% of the at-risk women) from 11 families (11/15 or 73% of the families). The used ARMS methods are rapid and can easily be applied in conjunction with other FVIII gene linked polymorphisms for indirect mutation detection of hemophilia A where they are informative.     

Non-catheter associated venous thrombosis in hemophilia A and B. A critical review of all reported cases

All reported cases of non-catheter induced venous thrombosis in patients with hemophilia A or B have been carefully evaluated. A total of 27 cases were reported,12 patients with hemophilia A and 15 patients with hemophilia B. The age of patients varied between 9 and 67 years. There were 10 cases of deep vein thrombosis, 8 patients with pulmonary embolism accompanied or not by deep vein thrombosis, 5 cases of superficial vein thrombosis. In addition, there were 3 cases of thrombosis in unusual sites (1 retinal central vein thrombosis and 2 portal vein thrombosis). Finally, in one case, venous thrombosis was multiple. There was a fatality in a hemophilia B patient with pulmonary embolism. The most frequent risk or triggering factor in hemophilia A was the administration of Feiba or rFVIIa concentrates in patients with inhibitors. Surgery together with Prothrombin Complex concentrates was the most frequent cause in hemophilia B patients. Congenital associated prothrombotic risk factors were present in two patients. No or very few therapeutic procedures were initiated in these patients but for a suspension or reduction of concentrates infusion. In a few instances low molecular weight heparin was given for a few days. The frequent association of venous thrombosis with infusion of concentrates indicates the need for a careful evaluation of patients about to receive such therapy.     

Prenatal and molecular diagnosis of hemophilia B

Prenatal diagnosis was carried out on a woman who had previously given birth to a son with a spontaneous mutation of C → T transition at nt 31133 of the factor IX (F.IX) gene. The diagnosis was performed on chorionic villi sampling by the method of amplification-created restriction site (ACRS). It revealed a female fetus with a normal F.IX gene, as confirmed by DNA sequencing after delivery. Meanwhile, a survey using the ACRS method to evaluate the inheritance of 63 individuals from 8 hemophilia B families was done. A different single-point mutation in each family was proved by DNA sequencing. One individual had a mutation with a naturally-created restriction site. In each of the remaining patients, we were able to show an enzyme-cutting site in their DNA amplification product for ACRS with the designed mutagenesis primers. All patients and carriers could be diagnosed accurately by comparing ACRS results with clinical and laboratory findings. There were new novel mutations among the patients. © 1996 Wiley-Liss, Inc.     

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.     

Multiple Xbal polymorphisms for carrier detection and prenatal diagnosis of haemophilia A

Three XbaI restriction fragment length polymorphisms (RFLPs) can be detected using the factor VIII-intron 22 probe (p482.6) in a XbaI-KpnI double digest of genomic DNA. The XbaI (A) site had been reported by Wion et al (1986) to be in intron 22, while the two additional sites. XbaI (B) and XbaI (C), are shown here to be X-linked and close to the XbaI (A) site. The frequencies of heterozygosity for these three sites are 0.49, 0.18 and 0.30 respectively. In 75 females the observed heterozygosity rate for the XbaI (A) site is 0.41 and this increased to 0.57 with the two additional sites. Care should be exercised when interpreting the XbaI RFLPs, since the 1.4 kb XbaI/KpnI fragment and the 4.8 kb XbaI fragment are associated with both positive XbaI (A) and XbaI (B) sites. By the combined use of the multiple XbaI polymorphisms with the BclI site in intron 18, the carrier detection rate would increase to 67%. Four prenatal diagnoses had been performed using the multiple XbaI polymorphisms.     

Hemophilia B with associated factor VII deficiency: A distinct variant of hemophilia B with low factor VII activity and normal factor VII antigen

Summary Factor VII activity and cross-reacting material was assayed in fresh and deep frozen non-contacted plasma in 43 patients with Hemophilia B belonging to different kindreds.Factor VII activity was found to be slightly decreased (about of 50% normal) in 12 patients, regardless of the thromboplastin used. In an additional patient (hemophilia B_m) factor VII was slightly decreased in 1 10 diluted plasma but was normal in further diluted plasma. In the remaining 30 patients factor VII activity was normal. No significant variation was found between fresh and deep frozen plasmas. Factor VII antigen or cross-reacting material was normal.These patients with associated factor VII defect represent a distinct variant of hemophilia B. The defect seems to be due to a faulty activation of factor VII but the underlying mechanism remains unclear.This study was supported in part by a grant from the M.P.I., Rome (grant 1592-77) and by a grant from the Venetian Region, Venice.     

Gene analysis and prenatal diagnosis for two families of congenital factor V deficiency

Summary. We studied two families in which the probands had severe bleeding tendency and showed low plasma levels of coagulation factor V (FV) antigen and activity. Sequence analysis of the FV gene on proband 1 demonstrated a novel G16088C homozygous missense mutation in exon 3 resulting in an Asp 68 to His substitution and on proband 2, a C69969T homozygous missense mutation in exon 23 leading to Gly2079Val. The parents of both families were each heterozygous for the corresponding FV gene defect. During their second pregnancy, the two families requested prenatal diagnosis. Chorionic villi were analysed at 12 weeks of gestation and cord blood samples were tested at 22 weeks. Microsatellite analysis performed in family 1 showed that the foetus sample was not contaminated by maternal tissue. The foetus 1 was found to be heterozygous for the familiar G16088C mutation with lower FV activity in the cord blood; the foetus 2 was a normal one. The diagnosis was confirmed after the birth. This is the first report of prenatal diagnosis for FV deficiency.     

Initial results of genetic carrier diagnosis in risk pedigrees with hemophilia A and B in East Germany

In 7 families at risk for hemophilia A 42 individuals were evaluated by the Taq I polymorphism of the extragenic probe St 14.1 and the Bcl I as well the Hind III polymorphism of the intragenic probe F8e16-19. 15 out of 20 females of the core families were identified as carriers, under them all of the obligate heterozygotes. 5 individuals were excluded as carriers. The heterozygosity for each of the RFLPs was found to be between 30% and 79%. Combining the single data in 96% heterozygosity was found under the individuals tested. A linkage disequilibrium was found between the Bcl I and Hind III polymorphismus of the probe F8e16-19. A family at risk for hemophilia B including 5 individuals was studied using the Taq I and Xmn I polymorphisms of the probe P1. In one of two females the carrier state could be excluded in the other one confirmed.     

Application of molecular genetics to prenatal diagnosis and carrier detection in the hemophilias: some limitations

Prenatal diagnosis and carrier detection in the hemophilias have received much attention in recent years. The error rate in prenatal diagnosis by fetoscopy is less than 1%; fetoscopy is not possible, however, until the second trimester of pregnancy. Carrier detection based on bioassays of plasma has an irreducible error rate (approximately 5%?), because of the "lyonization" phenomenon in heterozygous women, and the final results are always probabilistic. New DNA methods promise to alleviate these difficulties. Prenatal diagnosis can be accomplished in the first trimester. "Lyonization" is bypassed in carrier detection, and the results may sometimes be essentially nonprobabilistic. But the DNA methods have certain limitations of their own which are not widely appreciated. Aside from cost and the necessity to adopt a new technology, there are inherent genetic problems: mothers must be heterozygous for both a disease gene and a marker gene, final results are probabilistic if the marker gene lies outside the disease gene, and multiple marker genes are often in linkage disequilibrium. We have concluded that a clinical unit planning to use the DNA methods must also maintain the conventional methods at a high level of performance.