Rapid genotyping of F8 intron 22 inversion by nested PCR based on long-distance PCR

F8 intron 22 inversion (Inv22) accounts for about 40% of severe hemophilia A (HA) cases and is mainly genotyped by long-distance PCR (LD-PCR) or inverse-PCR (I-PCR). These methods require long separation times or enzymatic digestion. We aimed to shorten the separation time of LD-PCR. Long-read seque...

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Veröffentlicht in:Journal of thrombosis and thrombolysis Jg. 49; H. 4; S. 591 - 601
Hauptverfasser: Wang, Xiong, Hu, Weihong, Gao, Yong, Li, Dengju, Lu, Yanjun
Format: Journal Article
Sprache:Englisch
Veröffentlicht: New York Springer US 01.05.2020
Springer Nature B.V
Schlagworte:
Adolescent
Adult
Aged
Cardiology
Child
Child, Preschool
Factor VIII - genetics
Female
Genotyping
Genotyping Techniques
Hematology
Hemophilia
Humans
Infant
Male
Medicine
Medicine & Public Health
Middle Aged
Polymerase chain reaction
Polymerase Chain Reaction - methods
Sensitivity and Specificity
Sequence Inversion
Young Adult
Long-read sequencing
Nested PCR
intron 22 inversion
Long-distance PCR
Hemophilia A
F8 intron 22 inversion
ISSN:0929-5305, 1573-742X, 1573-742X
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Zusammenfassung:F8 intron 22 inversion (Inv22) accounts for about 40% of severe hemophilia A (HA) cases and is mainly genotyped by long-distance PCR (LD-PCR) or inverse-PCR (I-PCR). These methods require long separation times or enzymatic digestion. We aimed to shorten the separation time of LD-PCR. Long-read sequencing was applied for LD-PCR products from 20 Inv22 patients and 4 controls to validate the differences between products generated using P-Q and P-B primer pairs in LD-PCR. We then confirmed two unique regions (chrX: 154879481–154880814, chrX: 155376388–155376505, GRCh38) in the PCR products from P-Q and P-B primer pairs, respectively. The nested PCR P1, Q1, and B1 primers were located near the homologous sequence and two unique regions, respectively. The P1-Q1 and P1-B1 primer pairs generated 1621 bp and 540 bp fragments, respectively, and the Inv22 carriers produced both fragments. In total, 228 previously diagnosed subjects including 39 Inv22 carriers, 52 Inv22 patients, 82 Inv22 negative males, and 55 Inv22 negative females were genotyped using nested PCR, and the results revealed excellent sensitivity and specificity (100 and 100%, respectively). The separation time was shortened from 5 to 0.5 h. Therefore, we present a rapid genotyping method for F8 Inv22 by nested PCR based on LD-PCR.
Bibliographie:ObjectType-Article-1
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ISSN:0929-5305
1573-742X
1573-742X
DOI:10.1007/s11239-020-02043-5