Carrier Detection Of Hemophilia Research Articles

Screening of intron 1 inversion and three intragenic factor VIII gene polymorphisms in Pakistani hemophilia A families

Indirect linkage analysis using highly informative polymorphic markers is the method of choice for carrier detection of hemophilia A in developing countries because direct DNA or mRNA sequence analysis is manifold costly and difficult than indirect gene tracking. Worldwide populations have revealed marked variation in the informativeness of polymorphic markers because of which each country has to select its own panel of markers for linkage analysis in hemophilia A families. The present study aimed at determining the informativeness of three factor VIII gene polymorphisms [intron 13(CA)n repeats, HindIII and AlwNI] in the Pakistani population. One hundred and forty-three individuals from 32 hemophilia A families and 68 unrelated anonymous females from the general population were screened for these polymorphisms using PCR and RFLP techniques. An inversion in intron 1 of the factor VIII gene causing 2-5% of severe hemophilia A cases was also screened in 128 Pakistani hemophilia A patients. None of the affected individuals carried the intron 1 inversion at least in peripheral blood leucocytes. The informativeness of intron 13 repeats, HindIII and AlwNI was 59.1% (13/22 hemophilia A families revealing five different alleles), 40.6% (13/32 hemophilia A families) and 6.25% (2/32 hemophilia A families), respectively. The cumulative informativeness of intron 13 repeats and HindIII was 63.6% (14/22 hemophilia A families), revealing strong linkage disequilibrium between these two polymorphic markers. These results suggest that there is a need to determine the informativeness of other polymorphic markers of the factor VIII gene to achieve 100% success rate for carrier detection of hemophilia A in Pakistan.

1. Intragenic polymorphism using PCR-RFLP in carrier detection of hemophilia a in Indian population

Background Hemophilia A is a common X linked recessive disorder. Carrier detection has been done using RFLP markers but heterozygosity rates vary in different ethnic groups. We examined usefulness of Intragenic markers including BclI, Hind III , Xbal for carrier detection in North Indian families with Hemophilia A. Design Prospective case series including 143 families, 150 Cases with Hemophilia and 238 relatives. On the basis of the FVIII bioassay study groups was divided into 80, 50 and 13 cases each of severe, moderate and mild hemophilia respectively. DNA was extracted using Qiagen DNA extraction Kit. Gene polymorphism was identified by the specific PCR/RFLP followed by gel electrophoresis. Expected frequency of Heterozygosity was calculated by using (1–a 2 +b 2) where a = positive allele frequency and b = negative allele frequency. Results Using PCR/RFLP polymorphism detection at Intron 18( Bcl1 ), Intron 19( HindIII ) and Intron 22 (Xba1 ) 101 of 132 families were informative. Conclusion Intragenic polymorphisms at B clI, Hind III, XbaI locus are informative in 78.5% Indian families with Hemophilia A. Although direct mutation detection is idea, linkage analysis is of value when mutation in families has not been identified. It is also useful for tracking of the origin of de novo mutations. The procedure is simple, cost effective, with low infrastructure and expertise requirement and can be implemented in developing countries for carrier screening and prevention of Hemophilia A.

Informativeness of St14 VNTR Polymorphic Marker in the Carrier Detection of Hemophilia A

AbstractHemophilia A is the most common hereditary severe disorder of blood clotting. In families affected with hemophilia, genetic analysis provides opportunities to prevent recurrence of the disease. This study establishes a diagnostic strategy for carrier-ship determination in Pakistani population using an extragenic polymorphic marker for the first time. The analysis of St14 VNTR (DXS52) was carried out by polymerase chain reaction (PCR), in order to determine its informativeness in terms of heterozygosity in Pakistani population. This may be a milestone for further analysis of other polymorphic markers for carrier detection and prenatal diagnosis of hemophilia. Seventy eight blood samples (Hemophiliac = 23, Normal = 55) from 15 families were analyzed for determining informativeness of St14 VNTR in carrier detection of hemophilia A. A total of nine alleles (2400, 2100, 1750, 1690, 1630, 1570, 1390, 1300, 1220 bp) was detected in the pool of subjects. 19 out of 40 females were found to be carriers with respect to the St14 VNTR polymorphic marker. The marker was informative in 73.33% of families. The expected heterozygosity rate of the St14 VNTR was 0.86 while the observed heterozygosity was 0.7. This shows that St14 VNTR is 70% informative in our population, allowing it to be a useful marker in carrier detection, as informativeness is the direct reflection of heterozygosity of a polymorphic marker.

Informativeness of St14 VNTR Polymorphic Marker in the Carrier Detection of Hemophilia A

Hemophilia A is the most common hereditary severe disorder of blood clotting. In families affected with hemophilia, genetic analysis provides opportunities to prevent recurrence of the disease. This study establishes a diagnostic strategy for carrier-ship determination in Pakistani population using an extragenic polymorphic marker for the first time. The analysis of St14 VNTR (DXS52) was carried out by polymerase chain reaction (PCR), in order to determine its informativeness in terms of heterozygosity in Pakistani population. This may be a milestone for further analysis of other polymorphic markers for carrier detection and prenatal diagnosis of hemophilia. Seventy eight blood samples (Hemophiliac = 23, Normal = 55) from 15 families were analyzed for determining informativeness of St14 VNTR in carrier detection of hemophilia A. A total of nine alleles (2400, 2100, 1750, 1690, 1630, 1570, 1390, 1300, 1220 bp) was detected in the pool of subjects. 19 out of 40 females were found to be carriers with respect to the St14 VNTR polymorphic marker. The marker was informative in 73.33% of families. The expected heterozygosity rate of the St14 VNTR was 0.86 while the observed heterozygosity was 0.7. This shows that St14 VNTR is 70% informative in our population, allowing it to be a useful marker in carrier detection, as informativeness is the direct reflection of heterozygosity of a polymorphic marker.

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.

Hemophilia A: role of FVIIIC/vWF Ag in assisting linkage analysis for carrier detection.

Carrier detection for hemophilia A was carried out in 52 females from 30 families presenting to the Haematology Department AIIMS, using linkage analysis and factor VIIIC (FVIIIC)-von Willebrand factor (vWF) antigen assay. The allelic frequency for the marker Bcl 1 and Xba 1 was 0.58 and 0.54, respectively, for the positive alleles and 0.42 and 0.46, respectively, for the negative alleles. The heterozygosity frequency of Bcl I and Xba 1 was 0.65 and 0.55, respectively. Of the 52 females, 30 were mothers of hemophilic patients and 22 were sisters of hemophilic patients. Of the 30 mothers, positive family history was present in 14. In these patients, the defective X chromosome was tracked in 10, and in four the defective X chromosome could not be tracked because the mothers were homozygous for the marker used. Of the 16 mothers without a family history of disease, three were observed to be carriers based on linkage analysis and reduced factor VIII levels in mother/sister. Possible defective X chromosome was tracked in 11 mothers and five were noninformative because they did not show heterozygosity for the markers used. Using linkage analysis, nine of the 22 sisters were found to be definite carriers, 10 noncarriers, and three were noninformative. It is thus concluded that using Bcl 1 and Xba 1 linkage analysis, carrier status can be definitely ascertained in 50% females and this level of information can be increased to 61.5% by measuring FVIIIC/vWF antigen levels in them.

Advances in carrier detection in haemophilia.

Increasing worldwide use of molecular genetic analysis is enabling accurate carrier detection for the haemophilias to be made more widely available. Use of DNA polymorphisms in linkage analysis is an accurate method for carrier detection applicable to the majority of families. For those families with severe haemophilia A, the inversion mutation can be sought by most molecular genetics laboratories. For families remaining uninformative by these procedures, a range of point mutation screening techniques is available. Dedicated electrophoresis equipment is enabling use of these techniques to become more widespread.

Carrier detection of hemophilia by immunological and coagulation assays in a selected sample of the Mexican population.

Hemophilia is a recessive, hereditary disease linked to the X chromosome affecting primarily males, with the most common form being type A. The purpose of this investigation was to detect carriers related to hemophiliac patients identified in the northeastern part of Mexico. Twenty-one families of hemophilic patients were studied, including a total of 60 women, 22 obligatory carriers (OC), and 38 possible carriers (PC). Results obtained in the quantification of factor VIII:C by a one-stage assay and of von Willebrand factor (FvW:Ag) by immunoelectrophoresis were compared with data obtained in a control group. The results were analyzed by discriminant analysis, linear regression, and the ratio, VIIIC/FvW:Ag, obtaining the following percentages of identification: 91% of OC and 66% of PC with discriminant analysis; 86% of OC and 27% of PC with linear regression, and 64% of OC and 18% of PC with the ratio. The control group was not positive for the test. The techniques used in this study were trustworthy, reproducible, and of low cost, so that procedures used in this investigation for the detection of hemophilia carriers may be implemented in a clinical laboratory as an alternative to the high cost of molecular studies. With a specificity of 91%, although these tests are inexpensive, they contain significant risk of misclassification from a genetic counseling standpoint. A negative result or a positive result with a positive family history would be useful. However, trying to identify a new mutation in a family with only a single affected son might be problematic. Am. J. Hum. Biol. 9:173–178, 1997. © 1997 Wiley-Liss, Inc.

Carrier detection of hemophilia by immunological and coagulation assays in a selected sample of the Mexican population

Carrier detection of hemophilia by immunological and coagulation assays in a selected sample of the Mexican population

Carrier detection of hemophilia by immunological and coagulation assays in a selected sample of the Mexican population

Carrier detection of hemophilia by immunological and coagulation assays in a selected sample of the Mexican population

Factor IX gene haplotypes in Brazilian Blacks and characterization of unusual Ddel alleles

Analysis of factor IX gene polymorphisms is considered the best approach for prenatal diagnosis and carrier detection of haemophilia B when the identification of the gene mutation is not possible. Studies involving factor IX gene polymorphisms in Black populations are scarce and essentially restricted to the North-American Black population whose composition is substantially different from that of the Brazilian and presumably other Black populations of South America. In this paper we report the analysis of eight factor IX gene polymorphisms in Brazilian Blacks: 5' BamHI, DdeI, intron 2 BamHI, XmnI, TaqI, TaqI, MspI, MnlI and HhaI. Characterization of the VNTR-like DdeI polymorphism revealed six different alleles: B, AB, A2B, A2B2, A3B and A5B, the last being described here for the first time. The 5' BamHI, DdeI, MspI and HhaI polymorphisms showed the highest heterozygosities (0.40-0.50) and are in linkage equilibrium with one another. 19 complete haplotypes could be identified in this population. Based on the results we propose a systematic strategy for carrier detection and prenatal diagnosis of haemophilia B in this population. The combined analysis of four polymorphisms (5' BamHI, HhaI, MspI and DdeI) provided an informative genetic marker in 85% of the females. The use of all eight polymorphisms allows information in 95% of females. Additionally, differences in gene frequencies and haplotype distribution suggest dissimilarities in factor IX gene polymorphisms between the Brazilian and the North-American Black populations.

Genetic Basis and Carrier Detection of Hemophilia B of Chinese Origin

We have characterized the genetic defects of 17 hemophilia B patients of Chinese origin by means of the polymerase chain reaction (PCR) and direct sequencing. The single-strand conformation polymorphism (SSCP) was used as an initial screening method to analyze the entire coding region and the flanking introns of each individual's factor IX gene. The abnormal exons were subsequently amplified and the nucleotide sequence determined. Of the 17 patients studied, 16 had single point mutations and one had a gross gene deletion of exons VII and VIII of factor IX. Among these 16 factor IX variants with point mutations 13 were missense and two were nonsense mutations. The remaining one had a nucleotide deleted, resulting in frame shifting at amino acid residue 97. A total of ten novel mutations, including the one with gross gene deletion, are reported in this study which have not been described previously. Five of the remaining seven variants were missense mutations with novel amino acids substituted for residues 127, 132, 180, 207, and 215, respectively. Mutations containing different amino acid residues at those positions have been reported. The last two are variants that have already been described to contain mutations at amino acid residues 333 and 365, respectively. To evaluate the efficiency of SSCP analysis in assessing the mutated exons and to further confirm our results we sequenced the entire exons of all 17 factor IX genes. The mutations detected by SSCP method were indeed the only mutation identified in each factor IX variant.(ABSTRACT TRUNCATED AT 250 WORDS)

Further characterization of the new marker at DXS115 with regard to carrier detection in hemophilia A

Further characterization of the new marker at DXS115 with regard to carrier detection in hemophilia A

Usefulness of Determination of Factor VIII Intron 7 Polymorphism by a Non-Radioactive Technique for Carrier Detection of Hemophilia A

Usefulness of Determination of Factor VIII Intron 7 Polymorphism by a Non-Radioactive Technique for Carrier Detection of Hemophilia A

Direct carrier detection in hemophilia B kindreds: Use of modified primers (mutagenic primers) for enzymatic amplification of the factor IX gene

Rapid direct detection of point mutations in hemophilia B kindreds was performed by analyzing the restriction fragments of the factor IX gene amplified by polymerase chain reaction (PCR). The family members of the two patients, HB 5 and HB 6, whose mutant factor IX gene had previously been defined by sequence analysis, were examined in this study. Since there are no restriction endonucleases available for detecting each mutation directly, we designed modified primers which were substituted one nucleotide near the mutated positions. Following PCR with these primers, new Mbo I or Alu I cleavage sites for normal alleles were created. In each family, the restriction fragment revealed the carriership of each patient's mother. This simple methodology to detect mutations of interest is useful for genetic counseling in sporadic cases of hemophilia B.

PCR and specific oligonucleotide hybridization for the molecular characterization and carrier detection of hemophilia A

PCR and specific oligonucleotide hybridization for the molecular characterization and carrier detection of hemophilia A

Carrier detection in haemophilia A by direct analysis of factor VIII gene lesions

CGA----TGA (Arg----Term) transitions in the factor VIII gene causing severe haemophilia A were detected in two patients at codons 336 and 427 using a combination of oligonucleotide discrimination hybridization and DNA sequencing. Carrier detection analysis was then performed by polymerase chain reaction/direct sequencing of the appropriate region of the gene in female relatives of the probands.

A new marker at DXS 115 useful for carrier detection in hemophilia A

In this brief communication we report a new intergenic polymorphism at DXS115 as a marker for detection of heterozygotes in families at risk for hemophilia A. Total genomic DNA was isolated from white blood cells, double digested by KpnI and XbaI and hybridized with EcoRI/SstI fragment of the genomic probe p482.6. The incidence of the polymorphic 5.1-kb fragment was estimated as 0.069 in a German population. A technical advantage of using the XbaI/KpnI RFLP is that both the intragenic XbaI-RFLP in intron 22 of factor VIII gene and the new intergenic RFLP can be evaluated at the same time.

Improved carrier detection of haemophilia A using novel RFLPs at the DXS115 (767) locus

Two novel restriction fragment length polymorphisms (RFLPs) around the DXS115 (767) locus, detectable with the restriction enzymes MspI, are described. Since DXS115 is closely linked to the factor VIII gene (F8C), the MspI RFLP was employed in haemophilia A carrier detection. The utility of these RFLPs lies in the increased applicability and accuracy of diagnoses carried out in cases where available intragenic markers are uninformative.

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.