Rapid Hemophilia A Molecular Diagnosis by a Simple DNA Sequencing Procedure: Identification of 14 Novel Mutations

2001 ◽  
Vol 85 (04) ◽  
pp. 580-583 ◽  
Author(s):  
Francisco Vidal ◽  
Elisenda Farssac ◽  
Carme Altisent ◽  
Lluís Puig ◽  
Dominique Gallardo
Keyword(s):  
Dna Sequencing ◽  
Factor Viii ◽  
Molecular Diagnosis ◽  
Hemophilia A ◽  
Genetic Defect ◽  
Cost Effective ◽  
Factor Viii Gene ◽  
Intron 22 Inversion ◽  
Pcr Products ◽  
Sequencing Procedure

SummaryWe here describe a simple, efficient DNA sequencing procedure for hemophilia A molecular diagnosis. In severe patients we first test for the presence of factor VIII gene intron 22 inversion using a recently described single-tube PCR method. In moderate, mild, or inversion-negative severe patients we systematically sequence the promoter, all exons and splice junctions of factor VIII gene. Specially designed primers allow amplification of 23 PCR products under the same salt conditions and thermocycling parameters. The whole sequencing procedure, from blood extraction to mutation identification, can be readily done within 42 h when using regular instruments or in just 14 h when using a high-throughput sequencer. Thus, this is a versatile and cost-effective strategy with little hands-on time requirements. Since its implementation we have identified mutations in 45/46 hemophilia A patients, 14 of which are novel. Once the genetic defect has been identified, accurate genetic counseling is then easily performed.

scholarly journals Analysis of intron 22 inversions of the factor VIII gene in severe hemophilia A: implications for genetic counseling

Blood ◽  
1994 ◽  
Vol 84 (7) ◽  
pp. 2197-2201 ◽  
Author(s):  
PV Jenkins ◽  
PW Collins ◽  
E Goldman ◽  
A McCraw ◽  
A Riddell ◽  
...  
Keyword(s):  
Factor Viii ◽  
Genetic Counselling ◽  
Hemophilia A ◽  
Family Members ◽  
Rflp Analysis ◽  
Homologous Region ◽  
Severe Hemophilia ◽  
Factor Viii Gene ◽  
Intron 22 Inversion ◽  
Blotting Technique

Abstract Intrachromosomal recombinations involving F8A, in intron 22 of the factor VIII gene, and one of two homologous regions 500 kb 5′ of the factor VIII gene result in large inversions of DNA at the tip of the X chromosome. The gene is disrupted, causing severe hemophilia A. Two inversions are possible, distal and proximal, depending on which homologous region is involved in the recombination event. A simple Southern blotting technique was used to identify patients and carriers of these inversions. In a group of 85 severe hemophilia A patients, 47% had an inversion, of which 80% were of the distal type. There was no association with restriction fragment length polymorphism (RFLP) haplotypes. The technique has identified a definitive genetic marker in families previously uninformative on RFLP analysis and provided valuable information for genetic counselling information may now be provided for carriers without the need to study intervening family members and the diagnosis of severe hemophilia A made in families with only a nonspecific history of bleeding. Analysis of intron 22 inversion should now be the first-line test for carrier diagnosis and genetic counselling for severe hemophilia A and may be particularly useful when there is no affected male family member or when intervening family members are unavailable for testing.

scholarly journals A Closed-Tube Nested Quantitative PCR Assay for Rapid Detection of Intron 22 Inversions in the Factor VIII Gene

2020 ◽  
Author(s):  
Shengnan Jin ◽  
Qingjuan Shang ◽  
Weijiang Jin ◽  
Liuqing Yang ◽  
Qian Ye ◽  
...  
Keyword(s):  
Factor Viii ◽  
Quantitative Pcr ◽  
Molecular Diagnosis ◽  
Rapid Detection ◽  
Hemophilia A ◽  
Clinical Use ◽  
Factor Viii Gene ◽  
Pcr Assay ◽  
Closed Tube ◽  
Female Carriers

Abstract Background An inversion of intron 22 in the Factor VIII gene (Inv22) is the causative mutation for 45% of severe hemophilia A cases. Available methods for molecular diagnosis of Inv22 are generally tedious and not ideal for routine clinical use. Methods We report here a new method using a single closed-tube nested quantitative PCR (CN–qPCR) for rapid detection of Inv22. This method combines a 12-cycle long-distance PCR (LD–PCR) amplifying the int22h regions, followed by a duplex qPCR targeting two specific regions close to the int22h regions. All reagents were added to a single PCR mixture for the closed-tube assay. Sequential LD–PCR and qPCR was achieved by designing primers at substantially different melting temperatures and optimizing PCR conditions. Results Seventy-nine male hemophilia A patients of different disease severity were tested by both the CN–qPCR assay and the standard LD–PCR assay. CN–qPCR successfully made calls for all samples, whereas LD–PCR failed in eight samples. For the 71 samples where both methods made calls, the concordance was 100%. Inv22 was detected in 17 out of the 79 samples. Additionally, CN–qPCR achieved clear separation for 10 female carriers and 10 non-Inv22 females, suggesting the assay may also be useful for molecular diagnosis of female carriers. Conclusions This new CN–qPCR method may provide a convenient and accurate F8 Inv22 test suitable for clinical use.

scholarly journals Analysis of intron 22 inversions of the factor VIII gene in severe hemophilia A: implications for genetic counseling

Blood ◽  
1994 ◽  
Vol 84 (7) ◽  
pp. 2197-2201
Author(s):  
PV Jenkins ◽  
PW Collins ◽  
E Goldman ◽  
A McCraw ◽  
A Riddell ◽  
...  
Keyword(s):  
Factor Viii ◽  
Genetic Counselling ◽  
Hemophilia A ◽  
Family Members ◽  
Rflp Analysis ◽  
Homologous Region ◽  
Severe Hemophilia ◽  
Factor Viii Gene ◽  
Intron 22 Inversion ◽  
Blotting Technique

Intrachromosomal recombinations involving F8A, in intron 22 of the factor VIII gene, and one of two homologous regions 500 kb 5′ of the factor VIII gene result in large inversions of DNA at the tip of the X chromosome. The gene is disrupted, causing severe hemophilia A. Two inversions are possible, distal and proximal, depending on which homologous region is involved in the recombination event. A simple Southern blotting technique was used to identify patients and carriers of these inversions. In a group of 85 severe hemophilia A patients, 47% had an inversion, of which 80% were of the distal type. There was no association with restriction fragment length polymorphism (RFLP) haplotypes. The technique has identified a definitive genetic marker in families previously uninformative on RFLP analysis and provided valuable information for genetic counselling information may now be provided for carriers without the need to study intervening family members and the diagnosis of severe hemophilia A made in families with only a nonspecific history of bleeding. Analysis of intron 22 inversion should now be the first-line test for carrier diagnosis and genetic counselling for severe hemophilia A and may be particularly useful when there is no affected male family member or when intervening family members are unavailable for testing.

Molecular analysis of FVIII gene in severe HA patients of Costa Rica

2010 ◽  
Vol 30 (S 01) ◽  
pp. S150-S152
Author(s):  
G. Jiménez-Cruz ◽  
M. Mendez ◽  
P. Chaverri ◽  
P. Alvarado ◽  
W. Schröder ◽  
...  
Keyword(s):  
Factor Viii ◽  
Coagulation Factor ◽  
Costa Rican ◽  
Factor Viii Gene ◽  
Intron 22 Inversion ◽  
Intron 1 ◽  
Polymerase Chain ◽  
Inversion Analysis

SummaryHaemophilia A (HA) is X-chromosome linked bleeding disorders caused by deficiency of the coagulation factor VIII (FVIII). It is caused by FVIII gene intron 22 inversion (Inv22) in approximately 45% and by intron 1 inversion (Inv1) in 5% of the patients. Both inversions occur as a result of intrachromosomal recombination between homologous regions, in intron 1 or 22 and their extragenic copy located telomeric to the FVIII gene. The aim of this study was to analyze the presence of these mutations in 25 HA Costa Rican families. Patients, methods: We studied 34 HA patients and 110 unrelated obligate members and possible carriers for the presence of Inv22or Inv1. Standard analyses of the factor VIII gene were used incl. Southern blot and long-range polymerase chain reaction for inversion analysis. Results: We found altered Inv22 restriction profiles in 21 patients and 37 carriers. It was found type 1 and type 2 of the inversion of Inv22. During the screening for Inv1 among the HA patient, who were Inv22 negative, we did not found this mutation. Discussion: Our data highlight the importance of the analysis of Inv22 for their association with development of inhibitors in the HA patients and we are continuous searching of Inv1 mutation. This knowledge represents a step for genetic counseling and prevention of the inhibitor development.

THE RELATIVE EFFICACY OF GENETIC ANALYSIS AND COAGULATION TESTING IN THE DIAGNOSIS OF CARRIERS OF HEMOPHILIA A

1987 ◽  
Author(s):  
D Lillicrap ◽  
A R Giles ◽  
J J A Holden ◽  
B N White
Keyword(s):  
Factor Viii ◽  
Gene Deletion ◽  
Hemophilia A ◽  
Fragment Length Polymorphism ◽  
Genetic Diagnosis ◽  
Prior History ◽  
Carrier Status ◽  
Factor Viii Gene ◽  
Coagulation Testing ◽  
History Of

This study has assessed the relative benefits of restriction fragment length polymorphism (RFLP) linkage and coagulation testing in the diagnosis of carriers of hemophilia A. 221 samples from 55 families have been studied for intragenic and flanking RFLPs. All samples were tested for the Factor VIII intragenic Bell RFLP and for the flanking marker St 14. 83% of obligate carrier females were heterozygous at oneor both of these two polymorphicsites. However, only38% of these women were heterozygous at the intragenic site and might safely be offered prenatal diagnosis using this marker for the hemophilia mutation. Carrier diagnosis was obtained in 52% of 81 potential carriers tested. Diagnosis wasbased on intragenic RFLP information in only 48% of these cases. Genetic diagnosis was possible in 27 atrisk women from families with no prior history of hemophilia. Four of these women were diagnosed as carriers on the basis of a gross Factor VIII gene deletion and the remaining 23 women were identified as non-carriers by the Bell (11) and Stl4 (12) RFLP data. 39 women remained undiagnosed after gene analysis studies. 23 of these women were female relatives of sporadic hemophiliacs and thus RFLP segregation analysis was inappropriate. A further 9 potential carriers were undiagnosed because of homozygosity in key individuals in their families. In 31 potential carriers we have quantitated Factor VIII:C (one stage assay) and vWf:Ag (Laurell and ELISA) and derived probabilities for carrier status. In 3 women there was conflicting genetic and coagulation data. Meanwhile, in 12 undiagnosed women from sporadic families, carrier diagnostic probabilities of > 0.9 were obtained. These studies indicate that optimal carrier detection for hemophilia A requires more intragenic and closely linked RFLPs and the continuance of coagulation testing to assist women from sporadic families.

Localization and Characterization of Mutations Within the Factor VIII Gene in a Cohort of 212 Patients with Hemophilia A

2001 ◽  
pp. 270-272
Author(s):  
J. Schröder ◽  
V. Ivaskevicius ◽  
S. Rost ◽  
A. Müller ◽  
H.-H. Brackmann ◽  
...  
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Factor Viii Gene
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