Denaturing HPLC Procedure for Factor IX Gene Scanning

Castaldo, Giuseppe; Nardiello, Paola; Bellitti, Fabiana; Rocino, Angiola; Coppola, Antonio; Minno, G. Di; Salvatore, Francesco

doi:10.1373/49.5.815

Cited by 14 publications

(11 citation statements)

References 12 publications

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“…Glu280, together with the other three acidic moderately hydrophilic amino acid (Glu270, Asp272, and Glu275) to form the Ca2 þ binding domain 1 domain, provide a calcium ion-binding site for the FVII heavy chain, which is essential for the activation of the FVIIa's limited proteolysis function, when the Glu280 change to an Ala, from an acidic moderately hydrophilic amino acid to a nonpolar hydrophobic amino acid, it will damage the CAI domain. There are four different missense mutations present in the same residue of FIX (F9 p.Glu291Lys, p.Glu291Val, p.Glu291Asp, and p.Glu291Gly) reported to cause hemophilia B [14,19], and two of them -Gly and Val -were nonpolar hydrophobic amino acids too, similar to Ala. So, the Glu220Ala substitution may play a big part in the patient ' s FVII deficiency.…”

Section: Discussionmentioning

confidence: 99%

Phenotypic and genotypic characterization of four factor VII deficiency patients from central China

Liu

Wang

Cheng

et al. 2015

Blood Coagulation &Amp; Fibrinolysis

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Hereditary coagulation factor VII deficiency (FVIID) is a rare autosomal, recessive inherited hemorrhagic disorder related to a variety of mutations or polymorphisms throughout the factor VII (FVII) gene (F7). The aims of this study were to characterize the molecular defect of the F7 gene in four unrelated patients with FVIID and to find the genotype-phenotype correlation. All nine exons, exon-intron boundaries, and 5' and 3'-untranslated regions of the F7 gene were amplified by PCR and the purified PCR products were sequenced directly. Suspected mutations were confirmed by another PCR and sequencing of the opposite strand. Family studies were also performed. A total of five unique lesions were identified, including three missense mutations (c.384A>G, c.839A>C, c.1163T>G, predicting p.Tyr128Cys, p.Glu280Ala and p.Phe388Cys substitution, respectively) and two splice junction mutations (c.572-1G>A, c.681+1G>T), among which two (p.Glu280Ala, p.Phe388Cys) were novel. A previously reported mutation p.Tyr128Cys was seen in the homozygous state in two unrelated patients. The other two cases were both compound heterozygotes of a missense mutation and a splicing site mutation. Multiple sequence alignment using DNAMAN analysis showed that all the missense mutations were found in residues that highly conserved across species and vitamin K-dependent serine proteases. Online software Polyphen and SIFT were used to confirm the pathogenic of the missense mutation. p.Tyr128Cys seems to be a hotspot of the F7 gene in ethnic Han Chinese population.

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Section: Discussionmentioning

confidence: 99%

Phenotypic and genotypic characterization of four factor VII deficiency patients from central China

Liu

Wang

Cheng

et al. 2015

Blood Coagulation &Amp; Fibrinolysis

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“…Mutation detection by DHPLC is over 95%, and DHPLC is very sensitive since it may detect a 5% diluted mutated DNA as in a mosaic status [6,7]. During preparation of this article another protocol has been published [8]. The DHPLC conditions presented by Castaldo et al .…”

Section: Nucleotide Changes and Mutations Identified By Dhplc In A Comentioning

confidence: 99%

Evaluation of denaturing high‐performance liquid chromatography (DHPLC) in the screening of mutations in hemophilia B patients

Herbert

Trossaërt

Boisseau

et al. 2004

Journal of Thrombosis and Haemostasis

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D in other cell types, e.g. keratinocytes [6] and osteoblasts [7]. Elevated PAI-1 levels have been implicated as a risk factor for cardiovascular events including myocardial infarction and stroke [8]. Patients with ESRD are known to have impaired fibrinolysis [9], in part, due to increased circulating levels of active PAI-1 [10].In summary, the expression of PAI-1, but not other gene products, was markedly reduced in paricalcitol vs. calcitrioltreated cells at concentrations of paricalcitol (4 nM) that are observed in the sera of HD patients. Because elevated PAI-1 levels are associated with increased cardiovascular mortality in patients undergoing HD, reduced expression of PAI-1 could contribute to the survival advantage observed in HD patients treated with paricalcitol. Determining PAI-1 levels in ESRD patients treated with paricalcitol and calcitriol is necessary to confirm these findings.

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“…Therefore, gene scanning procedures like DHPLC [5] or gene sequencing must be carried out so that the family mutation (or the haplotype) is known at the time of PD. In this context, also the following factors are essential for PD: 1) carrier diagnosis (and cascade screening) of all consanguineous women once a new severe haemophilic patient is identified, and again the multidisciplinary counselling plays a relevant role in promoting this activity; and 2) construction of a database of mutations of all known severe haemophilic patients in each region, which will include novel pathogenic mutations [7,10,22].…”

Section: Discussionmentioning

confidence: 99%

“…In particular, we analysed two short tandem repeats (STR) in introns 13 and 22 by fluorescent PCR (a single run under the same conditions) followed by capillary electrophoresis. For HB we analysed all gene exons using DHPLC [5], or gene sequencing. In families in which the mutation was unknown we used linkage analysis to identify intron 1 and exon 8 microsatellites by fluorescent PCR followed by capillary electrophoresis [2].…”

Section: F8/f9 Mutation Analysismentioning

confidence: 99%