Mutation detection in the alpha-1 antitrypsin gene (PI) using denaturing gradient gel electrophoresis

Lodewyckx, L; Vandevyver, Caroline; Vandervorst, C.; Steenbergen, Werner Van; Raus, Jef; Michiels, Luc

doi:10.1002/humu.1180

Cited by 12 publications

(15 citation statements)

References 18 publications

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“…Although standard diagnostic serology procedures, relying on IEF of serum AAT, have proven to be efficient to date, a previous study comparing DGGE to IEF showed this method of genotyping to be highly conclusive compared to only 85% efficiency of IEF [Lodewyckx et al, 2001]. The major disadvantage of using IEF is that AAT variants are not always distinguishable due to insufficient differences in gel migration patterns as a result of minor charge changes [Nukiwa et al, 1987].…”

Section: Discussionmentioning

confidence: 97%

“…Of the PCR gelbased pre-screening techniques of mutation detection currently available, DGGE is believed to be the most powerful provided the assay is designed using optimal conditions of primer design [Wu et al, 1998] and gel conditions [Hayes et al, 1999]. The advantage of using the described assay, as opposed to that reported by Lodewyckx et al [2001], is the use of broad-range DGGE, thus making it more cost-effective and highthroughput.…”

Section: Discussionmentioning

confidence: 97%

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Genetic diversity of the alpha-1-antitrypsin gene in Africans identified using a novel genotyping assay

Hayes

2003

Hum. Mutat.

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The highly polymorphic human alpha-1-antitrypsin (AAT) gene, more recently named SERPINA1, codes for the most abundant circulating plasma serine protease inhibitor, protease inhibitor 1 (PI). Most studies determining AAT haplotype frequencies have been restricted first by the limited accuracy of the phenotypic method used and secondly by the analysis of predominantly Caucasian populations. Limited studies have been performed on African-based populations. Here a new comprehensive assay for genotyping the entire coding region, including splice junctions, of the AAT gene was designed. This assay, based on denaturing gradient gel electrophoresis (DGGE), allows for the complete analysis of a single individual in two lanes on a gel. Application of the assay resulted in the identification of nine known AAT variants as well as 13 novel sequence variants, five of which are single nucleotide polymorphisms (SNPs), occurring exclusively in the African-based populations. This is the first comprehensive analysis of the genetic diversity of the AAT gene in a cohort from sub-Saharan Africa.

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Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 97%

Genetic diversity of the alpha-1-antitrypsin gene in Africans identified using a novel genotyping assay

Hayes

2003

Hum. Mutat.

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“…Genotyping is also currently in use, particularly for the detection of deficiency variants. This standard method is not always accurate, because the phenotypes determined by IEF and by a 1 -AT serum concentration vary due to external factors such as inflammation and injury (Lodewyckx et al 2001). …”

Section: Discussionmentioning

confidence: 99%

Analysis of the Alpha-1-Antitrypsin Deficient Alleles M3S, MZ, and ZZ by Biochemical and Molecular Methods: A Family Study

et al. 2008

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Deficiency of alpha-1-antitrypsin (alpha(1)-AT, a major protease inhibitor controlling tissue degradation) is a genetic disorder transmitted in a codominant autosomal form. It has more than 100 genetically determined variants. This study attempted to determine the degree of association between serum alpha(1)-AT levels and phenotypes and to provide a strategy for reliable laboratory evaluation of deficiencies. The study group consisted of a 38-year-old male proband with clinical features of emphysema, his first-degree relatives, and healthy controls. Family history revealed a four-generation pedigree. Genomic DNA was isolated from peripheral blood leukocytes. Alpha-1-AT levels were determined from human serum by immunonephelometry. Phenotypes were determined by isoelectric focusing of blood samples. DNA sequences of coding exons were analyzed by the amplification DNA technique and direct sequencing. Inheritance and plasma levels of the ZZ, MM, M3S, and MZ phenotypes were confirmed by the family study. In the family members with deficiencies, plasma concentrations were 22.55% +/- 5.15 (ZZ), 84.18% +/- 5.18 (M3S), and 61.06% +/- 7.15 (MZ) of the normal MM level. We found a close association between alpha(1)-AT level and genotype. A combination of genotyping, quantification, and phenotyping is the optimal strategy for the laboratory evaluation of alpha(1)-AT deficiency.

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“…These techniques include RFLP (Restriction of Fragment Length Polymorphism) [21][22][23], SSCP (Single Strand Conformation Polymorphism) [24] and DGGE (Denaturing Gradient Gel Electrophoresis) [25], based on the comparative analysis of electrophoresis profiles of DNA fragments, which have different sizes depending on the alleles present. The allelespecific PCR is also commonly used, possibly in multiplex [26]; amplification and hybridization kits with PI S and PI Z allele-specific labelled probes are also marketed [27].…”

Section: Methodsmentioning

confidence: 99%

Diagnosis of alpha-1 antitrypsin deficiency: Modalities, indications and diagnosis strategy

Balduyck

Odou

Zerimech

et al. 2014

Revue des Maladies Respiratoires

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Mutation detection in the alpha-1 antitrypsin gene (PI) using denaturing gradient gel electrophoresis

Cited by 12 publications

References 18 publications

Genetic diversity of the alpha-1-antitrypsin gene in Africans identified using a novel genotyping assay

Genetic diversity of the alpha-1-antitrypsin gene in Africans identified using a novel genotyping assay

Analysis of the Alpha-1-Antitrypsin Deficient Alleles M3S, MZ, and ZZ by Biochemical and Molecular Methods: A Family Study

Diagnosis of alpha-1 antitrypsin deficiency: Modalities, indications and diagnosis strategy

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