New Protocol for Detection of Intron 22 Inversion Mutation From Cases With Hemophilia A

Kumar, Praveen; Husain, Nuzhat; Soni, Priyanka; Faridi, Nuzhat Jahan; Goel, Sudhir K.

doi:10.1177/1076029613498817

Cited by 8 publications

(9 citation statements)

References 13 publications

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“…However, quantitative reverse transcription PCR without gel electrophoresis cannot be used to diagnose Inv22 carriers. 18 Gau et al reported a method that used a different set of primers to amplify an intact exon 22 to 23 splice site PCR product 23 ; a potential drawback to using this method is that lack of an exon 22 to 23 diagnostic band could also be caused by improper PCR conditions or other experimental artifacts.…”

Section: Discussionmentioning

confidence: 99%

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Accurate, simple, and inexpensive assays to diagnose F8 gene inversion mutations in hemophilia A patients and carriers

et al. 2016

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Key Points• Improved assays to detect intron 22 and intron 1 inversions in the F8 gene have been developed.• These assays can efficiently detect or rule out the most common genetic mutations resulting in hemophilia A.The most frequent mutations resulting in hemophilia A are an intron 22 or intron 1 gene inversion, which together cause ;50% of severe hemophilia A cases. We report a simple and accurate RNA-based assay to detect these mutations in patients and heterozygous carriers.The assays do not require specialized equipment or expensive reagents; therefore, they may provide useful and economic protocols that could be standardized for central laboratory testing. RNA is purified from a blood sample, and reverse transcription nested polymerase chain reaction (RT-NPCR) reactions amplify DNA fragments with the F8 sequence spanning the exon 22 to 23 splice site (intron 22 inversion test) or the exon 1 to 2 splice site (intron 1 inversion test). These sequences will be amplified only from F8 RNA without an intron 22 or intron 1 inversion mutation, respectively. Additional RT-NPCR reactions are then carried out to amplify the inverted sequences extending from F8 exon 19 to the first in-frame stop codon within intron 22 or a chimeric transcript containing F8 exon 1 and the VBP1 gene. These latter 2 products are produced only by individuals with an intron 22 or intron 1 inversion mutation, respectively. The intron 22 inversion mutations may be further classified (eg, as type 1 or type 2, reflecting the specific homologous recombination sites) by the standard DNA-based "inverse-shifting" PCR assay if desired. Efficient Bcl I and T4 DNA ligase enzymes that cleave and ligate DNA in minutes were used, which is a substantial improvement over previous protocols that required overnight incubations. These protocols can accurately detect F8 inversion mutations via same-day testing of patient samples.

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Section: Discussionmentioning

confidence: 99%

“…RNA-based methods to detect Inv22 and Inv1 mutations 10,17 have been used in research and have also been proposed for clinical genotyping of HA patients. 18 These methods have been based on the presence or absence of PCR-amplified complementary DNA products spanning the normal exon 22 to 23 or exon 1 to 2 splice site, respectively.…”

Section: Introductionmentioning

confidence: 99%

Accurate, simple, and inexpensive assays to diagnose F8 gene inversion mutations in hemophilia A patients and carriers

et al. 2016

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“…Since the publication of sequence FVIII gene by Gitschier et al (14) at Genetech in 1984, numerous mutations causing HA have been identified. The most common is the inversion of intron 22, which occurs in 40-50% of patients with severe HA (15), whereas the inversion of intron 1 is present in just 1-5% of patients (16).…”

Section: Introductionmentioning

confidence: 99%

Evaluation of factor VIII polymorphic short tandem repeat markers in linkage analysis for carrier diagnosis of hemophilia A

Sabina

Dong

et al. 2016

Biomedical Reports

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Abstract. Hemophilia A (HA) is the most common inherited X-linked recessive bleeding disorder caused by heterogeneous mutations in the factor VIII gene (FVIII). Diagnosis of the carrier is critical for preventing the birth of children affected by this coagulation disorder, which ultimately facilitates its management. Due to the heterogeneous nature of mutations, the large inversions and the complexity of the FVIII gene, direct recognition of the disease-associated mutation in HA is complex. Indirect linkage analysis using highly informative heterozygous polymorphic markers is an alternative method for determining the co-segregation of the mutant gene within a family for carrier detection of HA. The aim of the present study was to perform carrier diagnosis in a family with HA. Rapid multifluorescent polymerase chain reaction (PCR) was performed with six extragenic short tandem repeats (STRs), DXS1073, DXS15, DXS8091, DXS1227, DXS991, DXS993 and one intragenic marker, STR22 for linkage analysis in the HA family. All the STR markers employed in the present study were informative for linkages of pathogenic and healthy haplotypes among family members, particularly STR22, DXS1073 and DXS15. The STR marker, STR22, is within the FVIII gene while the DXS1073 and DXS15 markers are very close to the FVIII gene, where the chances of recombination are comparatively low, and provided the most accurate interpretation analysis, indicating that the proband's sister may have been the HA carrier. Rapid multifluorescent PCR using STR markers and linkage analysis was identified to be a simple method for performing HA carrier diagnosis.

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“…Furthermore, mutation detection allows for carrier and prenatal diagnosis to be conducted [5].The intron 22 inversion (inv22) was first detected by means of a Southern blot assay [2]. This technique proves to be accurate, but has the disadvantage of being time-consuming and labour intensive [6]. The availability of Southern blot reagents has also become a major obstacle.…”

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confidence: 99%

“…Newer methods such as long-distance polymerase chain reaction (LD-PCR) and inverse PCR (I-PCR) have been described to detect the inversion [7,8]. However, LD-PCR is reportedly difficult to standardize and is sensitive to reductions in DNA quality [6,9]. I-PCR involves three steps including restriction, self-ligation and standard PCR analysis.…”

mentioning

confidence: 99%

Rapid identification of the intron 22 inversion in haemophilia A

Kloppers

Rensburg

2016

Haemophilia

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New Protocol for Detection of Intron 22 Inversion Mutation From Cases With Hemophilia A

Abstract: Conventional long PCR and I-PCR methods are work intensive, prolonged, and sometimes difficult to be standardize. The cDNA method is short, involves 3 short-segment amplifications, and is easy to reproduce.

Cited by 8 publications

References 13 publications

Accurate, simple, and inexpensive assays to diagnose F8 gene inversion mutations in hemophilia A patients and carriers

Accurate, simple, and inexpensive assays to diagnose F8 gene inversion mutations in hemophilia A patients and carriers

Evaluation of factor VIII polymorphic short tandem repeat markers in linkage analysis for carrier diagnosis of hemophilia A

Rapid identification of the intron 22 inversion in haemophilia A

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