Sequence variations of the ?-globin genes: Scanning of high CG content genes with DHPLC and DG-DGGE

Lacerra, Giuseppina; Fiorito, Mirella; Musollino, Gennaro; Noce, Francesca Di; Esposito, Maria Valeria; Nigro, Vincenzo; Gaudiano, Carlo; Carestia, Clementina

doi:10.1002/humu.20088

Cited by 30 publications

(31 citation statements)

References 27 publications

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“…Previous reports have shown that DGGE run at optimised conditions can be a highly sensitive tool for such fragments [Hayes et al, 1999;Lacerra et al, 2004;Wu et al, 1999], and is therefore an appropriate platform for assessing new methods for this application. One report, which discusses HRM curve analysis of GC-rich fragments [Reed and Wittwer, 2004], suggests that GC content has no effect on HRM curve analysis, and also that biphasic melting curves may be easier to analyze than those with a single melt domain.…”

Section: Discussionmentioning

confidence: 99%

Assessing high-resolution melt curve analysis for accurate detection of gene variants in complex DNA fragments

et al. 2009

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Mutation detection has, until recently, relied heavily on the use of gel-based methods that can be both time consuming and difficult to design. Nongel-based systems are therefore important to increase simplicity and improve turn around time without compromising assay sensitivity and accuracy, especially in the diagnostic/ clinical setting. In this study, we assessed the latest of the nongel-based methods, namely high-resolution melt (HRM) curve analysis. HRM is a closed-tube method that incorporates a saturating dye during DNA amplification followed by a monitoring of the change in fluorescence as the DNA duplex is denatured by an increasing temperature. We assessed 10 amplicons derived from eight genes, namely SERPINA1, CXCR7, MBL, VDR, NKX3A, NPY, TP53, and HRAS using two platforms, the LightScanner s System using LC Green s PLUS DNA binding dye (Idaho Technology, Salt Lake City, UT, USA) and the LightCycler s 480 using the HRM Master dye (Roche Diagnostics, Indianapolis, IN, USA). DNA variants (mutations or polymorphims) were previously identified using denaturing gradient gel electrophoresis (DGGE) a method, similarly to HRM, based upon the different melting properties of doublestranded DNA. Fragments were selected based on variant and fragment complexity. This included the presence of multiple sequence variants, variants in alternate orientations, and single or multiple variants (constitutional or somatic) in GC-rich fragments. We demonstrate current limitations of the HRM method for the analysis of complex DNA regions and call for caution when using HRM as the sole method to make a clinical diagnosis based on genetic analysis.

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Section: Discussionmentioning

confidence: 99%

Assessing high-resolution melt curve analysis for accurate detection of gene variants in complex DNA fragments

et al. 2009

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“…On this basis, no functional consequences of the p.Ala27Thr substitution can be predicted according to known site mutation determinants (Storz and Moriyama 2008;Weber and Campbell 2011). The same amino acid replacement at the same position has already been detected in α-chain globin variants from goat breeds present in southern Italy (Puglia and Sardegna) (Pieragostini et al 2005;Pirastru et al 2009) and from human families in Caserta (Campania) (Lacerra et al 2004). Either in Agerolese bovine, Apulian/Sardinian goat, and human Casertian α-globin variants, this substitution involves a CpG dinucleotide that is considered as a mutation hotspot (Perutz 1990).…”

Section: Resultsmentioning

confidence: 86%

Bovine hemoglobin polymorphism: a novel alpha-globin variant identified in the Agerolese breed from southern Italy

et al. 2015

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This study describes the results of molecular screening in the Agerolese cattle breed from southern Italy, which constitutes an economical source and contributes to the regional biodiversity. Integrated electrophoretic, chromatographic, mass spectrometric, and DNA sequencing procedures were used to qualitatively and quantitatively evaluate globin chain polymorphism in a population of animals from different farms. In addition to α-, β A -, and β B -globin-bearing animal phenotypes, our data demonstrate the occurrence of individuals characterized by the presence of a novel α-globin variant, whose primary structure differs from the normal counterpart for the p.Ala27Thr substitution. The SNP c.79G>A located at the exon 1 of the HBA2 gene is responsible for this amino acid change and it represents a third example of α-globin gene polymorphism observed in Bos taurus. This phenomenon was ascertained only in two Mediterranean breeds from southern Italy and highlights the uncommon occurrence of α-globin variants in this geographical region, similarly to what has already been described in goat and human.

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“…In general, DHPLC is a rapid and cost-effective method because no particular manipulations or reagents are required pre-or post-PCR amplification. However, existing DHPLC mutation screening protocols available for b-globin gene are based on the Transgenomic Wave TM analyzer [8,9], requiring a significant capital injection, which can be particularly problematic for small laboratories. The DHPLC methodology described herein allows its implementation in every laboratory in possession of a HPLC instrumentation, since only the purchase of the separation column is required and the resulting chromatograms are generated equally fast as with the Transgenomic setup.…”

Section: Resultsmentioning

confidence: 99%

“…Denaturing high-performance liquid chromatography (DHPLC) is a cost-effective methodology that has been successfully applied for mutational detection of several genes [7], including the a-and b-globin genes [6,8,9], approaching 90-100% in sensitivity and specificity. However, the earlier-mentioned protocols are based on the Transgenomic Wave TM DHPLC system, which is expensive and therefore poses a hurdle for its implementation in low-budget laboratories, particularly those in developing countries.…”

Section: Introductionmentioning

confidence: 99%

A versatile denaturing HPLC approach for human β‐globin gene mutation screening

Bournazos¹,

Tserga²,

Patrinos

et al. 2006

American J Hematol

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Hemoglobinopathies represent the most common genetic disorder worldwide, with a higher prevalence among populations with a history of malaria endemicity. More than 690 mutations in the human b-globin gene are usually the cause of b-type hemoglobinopathies. Here, we report a rapid and highly sensitive b-globin gene mutation screening approach based on denaturing high-performance liquid chromatography (DHPLC), which contrary to the previously described ones can be used in every HPLC apparatus. The sensitivity and specificity of the method were tested in 120 healthy Greek subjects and 25 b-thalassemia heterozygotes and homozygotes, in which 11 different b-globin sequence variations had been previously characterized by denaturing gradient gel electrophoresis. Using this method, we were able to rapidly identify the commonest b-globin gene mutations, accounting for more than 90% of the mutant b-globin alleles reported for the Hellenic population. Compared to classical mutation screening approaches, our DHPLC approach provides the means for rapid, highly sensitive, cost-effective, and semi-automated simultaneous mutational scanning of a large number of samples. Am. J. Hematol. 82:168-170, 2007. V V C 2006 Wiley-Liss, Inc.

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Sequence variations of the ?-globin genes: Scanning of high CG content genes with DHPLC and DG-DGGE

Cited by 30 publications

References 27 publications

Assessing high-resolution melt curve analysis for accurate detection of gene variants in complex DNA fragments

Assessing high-resolution melt curve analysis for accurate detection of gene variants in complex DNA fragments

Bovine hemoglobin polymorphism: a novel alpha-globin variant identified in the Agerolese breed from southern Italy

A versatile denaturing HPLC approach for human β‐globin gene mutation screening

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