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

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.

Download Full-text

Detection of Intron22 Mutations in Iraqi Female Carriers in Wasit City with Hemophilia A

Global Journal of Medical Research ◽

10.34257/gjmrfvol17is1pg13 ◽

2017 ◽

pp. 13-24

Author(s):

Maysoon Mohammed Hassan

Keyword(s):

Factor Viii ◽

Hemophilia A ◽

Coagulation Factor ◽

Gene Identification ◽

Severe Hemophilia ◽

Factor Viii Gene ◽

Intron 1 ◽

Detection Mechanisms ◽

Female Carriers ◽

Chromosomal Recombination

The background:One of the prevalent main concerns in the medical world is the identification of Intron22 mutations in the Factor VIII gene carried by Iraqi patient in Wasit town, in Iraq suffering Hemophilia A (classical hemophilia) which is related to a X-chromosome recessive haemorrhage afflictions as the result of a flaw in the coagulation factor VIII (FVIII). It is essentially related with F8 mutations of Intron22 in version which forms the most typical kind of mutations of blood afflictions worldwide involving half the patients suffering from severe Hemophilia A that possesses mutations, in addition to Intron 1 inversion suffered by 5% of severe Hemophilia A patients.All of the inversion mutations are suffered mainly by males,and uncommonly by females due to the intra chromosomal recombination among the homologous areas, in inversion 1 or 22, with extragenic copy posited the telomeric to the Factor VIII gene. Unfortunately, there is an absence in Iraq on researches pertaining blood affliction gene identification in persons who carries the Intron22 mutations exception in the current research.Aims of study:The objectives of the research is to to analyze through the detection mechanisms, the existence of Intron 22 mutations in the Factor VIII gene of 10 Hemophilia A Iraqi carriers cohort families. The hypothesis and anticipated result is that there will be a minimal margin of hazardous possibility for the recurrence. The hereditary F8 mutation is unknown to be present on the maternal side of the patient sufferer due to the possibilty of germline mosaics that exists within the community.

Download Full-text

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

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615637 ◽

2001 ◽

Vol 85 (04) ◽

pp. 580-583 ◽

Cited By ~ 37

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.

Download Full-text

Faculty Opinions recommendation of AAV5-Factor VIII Gene Transfer in Severe Hemophilia A.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.732279394.793544825 ◽

2018 ◽

Author(s):

Valder Arruda ◽

Ben Samelson-Jones

Keyword(s):

Gene Transfer ◽

Factor Viii ◽

Hemophilia A ◽

Severe Hemophilia ◽

Factor Viii Gene

Download Full-text

Molecular characterization of severe hemophilia A suggests that about half the mutations are not within the coding regions and splice junctions of the factor VIII gene.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.88.16.7405 ◽

1991 ◽

Vol 88 (16) ◽

pp. 7405-7409 ◽

Cited By ~ 87

Author(s):

M. Higuchi ◽

H. H. Kazazian ◽

L. Kasch ◽

T. C. Warren ◽

M. J. McGinniss ◽

...

Keyword(s):

Factor Viii ◽

Molecular Characterization ◽

Hemophilia A ◽

Severe Hemophilia ◽

Factor Viii Gene ◽

Coding Regions ◽

Splice Junctions

Download Full-text

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

10.1055/s-0038-1644010 ◽

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.

Download Full-text