Haemophilia A diagnosis by automated fluorescent DNA detection of ten factor VIII intron 13 dinucleotide repeat alleles.
L. KochhanM. R. A. LALLOZJohannes OldenburgJohn H. McVeyK. OlekHans‐Hermann BrackmannEdward G. D. TuddenhamR. Schwaab
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Abstract:
Haemophilia A is a recessive X linked bleeding disorder caused by deficiency or functional abnormality of coagulation factor VIII. This disease usually has no visible phenotype in female carriers; hence, great efforts are made to offer all haemophilia A families accurate carrier diagnosis. Significant progress in this direction was made with the identification of the intron 13 variable number tandem repeat (VNTR), which is hitherto the most informative single marker within the factor VIII gene. The authors have established intron 13 VNTR detection in their laboratory by adapting its analysis to an automated sequencer using different primers of which one is fluorescent dye labelled. With this method, which is more rapid and convenient than that originally described, 67 haemophilia A families of German origin were screened and two new alleles (alleles 17 and 25) were identified. The informativeness of the VNTR in these families based on the patients maternal X chromosomes (134) is about 67%.Keywords:
Minisatellite
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Abstract A novel mutation was detected in the Factor VIII gene of a sporadic case of severe haemophilia A. The lesion, a CGA → TGA transition, converts Arg 795 to Term and adequately accounts for the severe phenotype observed. PCR/direct sequencing was used to confirm the carrier status in the mother. Exclusion of haemophilia A in an at‐risk pregnancy was then achieved by demonstration of the absence of this lesion in fetal DNA from a chorionic villus sample. The mutation was also detectable by chemical cleavage of mismatch (CCM), which both confirmed the prenatal diagnosis and established the carrier status of the proband's sister. This example therefore serves to illustrate the potential of direct gene analysis in sporadic cases of haemophilia A and/or in families uninformative for known RFLPs.
Proband
Chorionic villus sampling
Chorionic villi
Haemophilia B
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The aim of this study was to establish a simple, rapid carrier detection and prenatal diagnosis system for hemophilia A. Intron 22 inversion in FVIII gene was directly examined by long-distance polymerase chain reaction. Polymorphisms of factor VIII intragenic restriction fragment length polymorphism of Bcl I, short tandem repeat (STR) within intron 13 and 22, and extragenic DXS 52 (St 14) variable number tandem repeats (VNTR) loci were assessed by hereditary linkage analysis. The diagnostic rates for these loci were 47.6% (intron 22 inversion), 27.8% (Bcl I, 28.6% and 29.4% (STR within intron 13 and 22), and 81.3% (DXS52), respectively. The overall diagnostic rate in 21 families was 94.7%. The diagnosis in hemophilia A patients or carriers can be made if intron 22 inversion is present. The intragenic and extragenic loci hereditary linkage analysis could be used to establish the diagnosis in intron 22 inversion-negative patients.
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Summary. Genetic diagnosis of haemophilia A has been studied in two aspects. One is to directly identify the mutations in the factor VIII genes of the affected probands, and the other is to examine the usefulness of several intragenic factor VIII markers for gene tracking. Direct mutational analysis by PCR‐SSCP (polymerase chain reaction‐single‐strand conformation polymorphism) has been accomplished previously in 87 haemophilia A patients, accounting for nearly 10% of cases in Taiwan. Of the 87 cases, 46% were with point mutations, short deletions or insertions, and most of the remaining were with gene inversion readily identified by Southern blotting. Further examination of 112 patients has estimated a 33% incidence for gene inversion in all the patients with haemophilia A, or 37% in severe cases. Since the direct mutational detection described above cannot be used in all Chinese families with haemophilia A, genetic markers were also investigated. The two CA repeat markers located at intron 13 (CA‐13) and intron 22 (CA‐22), respectively, were amplified and analysed simultaneously. Seven different alleles with 18‐24 CAs have been identified for CA‐13. Alleles of 20 and 21 CAs are the most common and their population frequency was 0.68 and 0.24, respectively. The CA‐22 marker contained a repetition of (GT) n (AG) n as was identified in the white European but not in the Canadian population. Alleles with 25 and 26 GT/ AGs account for 18% and 75% of this group of samples, respectively. The expected rate of heterozygosity for either CA markers was 68%, although a value of 57% was observed by haplotype analysis, indicating an association of the two repeat markers. Nevertheless, the study of 62 females showed that with the combined use of CA‐13 and CA‐22 with Bell, approximately 71% would be informative for these markers. This number may increase to 81% if X bal polymorphism is added. We propose that a better genetic diagnosis procedure for Chinese individuals would be first to look for the inversion mutation, secondly for one of the intragenic markers, and then at the PCR‐SSCP analysis.
Single-strand conformation polymorphism
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Haemophilia B
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Hypervariable region
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Southern blot
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Summary. Haemophilia A is a bleeding disorder caused by defects in the gene coding for the co‐factor, factor VIII (FVIII). The few available intragenic restriction fragment length polymorphisms (RFLPs) currently used in carrier detection and prenatal diagnosis of haemophilia A are informative in only about 65% of cases. We earlier reported a multi‐allelic dinucleotide tandem repeat, (CA) n , specific to intron 13, which remains the single most informative marker within the FVIII gene. We here report a second informative dinucleotide repeat of the form (GT) n (AG) n , located to intron 22 of the FVIII gene. The polymerase chain reaction (PCR) method was used to examine the variability of the repeat in 60 individuals (75 X‐chromosomes) and revealed four alleles. The calculated heterozygosity rate is 45%, and family studies showed X‐linked mendelian inheritance. The intron 22 dinucleotide repeat is tightly linked with established RFLPs and tracks with haemophilia A in family studies. We now show that by simultaneous amplification of the intron 13 and 22 repeats using PCR all alleles for both markers are detectable on a single polyacrylamide gel. The information thus obtained from a single multiplexed analysis is greater than from multiple RFLP analyses. Hence, rapid haplotype determination by simultaneous amplification and detection of two intragenic dinucleotide repeats should supersede less informative RFLP analysis.
Dinucleotide Repeat
Mendelian inheritance
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Human genetics
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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.
Factor IX
genomic DNA
Southern blot
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Haemophilia B
Factor IX
Restriction site
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Abstract Background Hemophilia A is a common X‐linked recessive bleeding disorder caused by deleterious mutations in the gene encoding factor VIII. Though direct mutation analysis is the common practice in most of the developed countries, restriction fragment length polymorphism (RFLP) analysis using common polymorphic markers of factor VIII gene is still the most practical and feasible method in developing countries like India. Method We investigated the utility of a Bsl I polymorphism (A3864C) in exon 14 of factor VIII gene for the genetic diagnosis of hemophilia A families by polymerase chain reaction (PCR) followed by digestion with enzyme Bsl I. Results Out of 213 unrelated women examined for heterozygosity of this marker, 69 were found to be informative (32.4%), with frequencies of 0.32 and 0.68 for the ‘+ ’ and ‘− ’ alleles respectively. Subsequently 13 hemophilia A families, which were not informative with any of the common markers routinely used in genetic diagnosis of hemophilia and which were also negative for intron 1 and 22 inversions were analyzed. Three were found to be informative exclusively with this marker (23%). Conclusion Hence it is a highly useful marker in the genetic diagnosis of hemophilia A families in India. Copyright © 2008 John Wiley & Sons, Ltd.
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