Factor VIII gene mutations found by a comparative study of SSCP, DGGE and CMC and their analysis on a molecular model of factor VIII protein

Schwaab, R.; Oldenburg, Johannes; Lalloz, M. R. A.; Schwaab, U.; Pemberton, S.; Hanfland, P.; Brackmann, H.‐H.; Tuddenham, Edward G. D.; Michaelides, K

doi:10.1007/s004390050636

Cited by 41 publications

(32 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…PCR amplification of all 26 F8 exons, including flanking intronic regions, was performed on 50-100 ng of extracted DNA using previously described amplification primers and cycling conditions [Bogdanova et al, 2002;Schwaab et al, 1997]. Amplicons were sequenced in both directions using the ABI PRISM Dye terminator cycle sequencing reaction kit (Applied Biosystems; www.appliedbiosystems.com), and electrophoresis was performed on an ABI-3700 genetic analyzer.…”

Section: Dna Extraction and Analysismentioning

confidence: 99%

Spectrum of molecular defects and mutation detection rate in patients with severe hemophilia A

Bogdanova

Markoff²,

Pollmann³

et al. 2005

Hum. Mutat.

View full text Add to dashboard Cite

Hemophilia A is the most frequently occurring X-linked bleeding disorder, affecting one to two out of 10,000 males worldwide. Various types of mutations in the F8 gene are causative for this condition. It is well known that the most common mutation in severely affected patients is the intron 22 inversion, which accounts for about 45% of cases with F8 residual activity of less than 1%. Therefore, the aim of the present study was to determine the spectrum and distribution of mutations in the F8 gene in a large group of patients with severe hemophilia A who previously tested negative for the common intron 22 inversion. Here we report on a mutation analysis of 86 patients collected under the above-mentioned criterion. The pathogenic molecular defect was identified in all patients, and thus our detection rate was virtually 100%. Thirty-four of the identified mutations are described for the first time. The newly detected amino acid substitutions were scored for potential gross or local conformational changes and influence on molecular stability for every single F8 domain with available structures, using homology modeling.

show abstract

Section: Dna Extraction and Analysismentioning

confidence: 99%

Spectrum of molecular defects and mutation detection rate in patients with severe hemophilia A

Bogdanova

Markoff²,

Pollmann³

et al. 2005

Hum. Mutat.

View full text Add to dashboard Cite

show abstract

“…PCR was performed on 50 -100 ng of extracted DNA essentially using the amplification primers and cycling conditions described by Schwaab et al (1997).…”

Section: Dna Extraction and Amplificationmentioning

confidence: 99%

Prevalence of small rearrangements in the factor VIII gene F8C among patients with severe hemophilia A

Bogdanova

Markoff²,

Pollmann

et al. 2002

Hum. Mutat.

View full text Add to dashboard Cite

Hemophilia A is a common X-linked bleeding disorder caused by various types of mutations in the factor VIII gene F8C. The most common intron 22-inversion is responsible for about 40% of the severe hemophilia A cases while large deletions, point mutations and small (less than 100 bp) deletions or insertions are responsible for the disease in the rest of patients. We report on nine novel (6 deletions, two indels and one partial duplication) and five recurrent small rearrangements identified in 15 German patients with severe hemophilia A, negative for the intron 22-inversion. c.2208-2214delTTATTAC/c.2207-2215insCTCTT and c.4665-4678del/c.4664-4678insAAGGAA identified in the present study are the first small indels described in the factor VIII gene. Our analyses suggest that the prevalence of this type of mutations (predominantly located in exon 14) among patients with severe phenotype and negative for the common intron 22-inversion, is about 30%. The correlation between these molecular defects and formation of factor VIII inhibitors as well as the parental origin of the de novo mutations are evaluated. Finally we show that denaturing HPLC (DHPLC) and classic heteroduplex analysis (HA) are able to detect these sequence alterations on 100% and could be preferred as a screening approach when analysing for mutations in factor VIII in severely affected patients.

show abstract

“…While conventional DNA sequencing typically detects a presence of minor allele at a concentration above 20%, techniques based on spatial separation of mutant and wild-type (such as denaturant gradient gel electrophoresis, DGGE, or single-strand conformation polymosphism, SSCP) are capable of detecting low-level mutations in fractions down to 10% or lower [18]. An example of detection sensitivity using CGCE is shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Application of cycling gradient capillary electrophoresis to detection of APC, K‐ras, and DCC point mutations in patients with sporadic colorectal tumors

et al. 2004

View full text Add to dashboard Cite

A previously introduced technique of cycling gradient capillary electrophoresis (CGCE) was applied to monitoring of molecular changes during adenoma-carcinoma transition in progression of sporadic colorectal cancer. The purpose of this work was optimization of separation parameters for selected mutation regions in tumor suppressor genes involved in the early stages of colorectal carcinogenesis, followed by scanning for these mutations in clinical tissue samples from patients with adenomatous polyps and early carcinomas. A total of 47 colorectal tumors in various stages of progression were examined. Main emphasis was given to evaluation of mutation detection sensitivity and specificity required for effective early disease detection. A total of 7 different somatic mutations was identified among 32 K-ras mutant samples, 1 inherited mutation and 5 somatic mutations were identified among 15 adenomatous polyposis coli (APC) mutated samples. None of the two previously reported "deleted in colorectal carcinomas" (DCC) mutations was found in any of the clinical samples. In addition to simple optimization of running conditions, CGCE has demonstrated sensitivity and selectivity allowing detecting small mutant fractions as well as combination of multiple mutants within a single target sequence.

show abstract

Factor VIII gene mutations found by a comparative study of SSCP, DGGE and CMC and their analysis on a molecular model of factor VIII protein

Cited by 41 publications

References 36 publications

Spectrum of molecular defects and mutation detection rate in patients with severe hemophilia A

Spectrum of molecular defects and mutation detection rate in patients with severe hemophilia A

Prevalence of small rearrangements in the factor VIII gene F8C among patients with severe hemophilia A

Application of cycling gradient capillary electrophoresis to detection of APC, K‐ras, and DCC point mutations in patients with sporadic colorectal tumors

Contact Info

Product

Resources

About