Reliability of DHPLC in mutational screening of ?-globin (HBB) alleles

Colosimo, Alessia; Guida, Valentina; Luca, Alessandro De; Cappabianca, Maria Pia; Bianco, I; Palka, Giandomenico; Dallapiccola, Bruno

doi:10.1002/humu.10046

Cited by 48 publications

(31 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The present study adds to the growing number of reports showing the usefulness of DHPLC for mutation screening in disorders where disease-causing mutations are heterogeneous (Colosimo et al, 2002;Flex et al, 2002;De Luca et al, 2003). Nevertheless, DHPLC analysis of the entire NF1 gene in the present group of Italian patients disclosed a large number (32/75, 43%) of recurrent mutations suggesting the possibility of developing rapid tests focused on searching for these recurrent mutations as a first step in the routine diagnostic procedure.…”

Section: Discussionmentioning

confidence: 54%

Novel and recurrent mutations in theNF1 gene in Italian patients with neurofibromatosis type 1

et al. 2004

Self Cite

View full text Add to dashboard Cite

Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant disorders in humans, affecting 1 in 3500 individuals. NF1 is a fully penetrant exhibiting a mutation rate some 10-fold higher compared to most other disease genes. As a consequence, a high number of cases (up to 50%) are sporadic. Mutation detection is complex due to the large size of NF1 gene, the presence of pseudogenes and the great variety of lesions. In the present study we attempted to delineate the NF1 mutational spectrum in the Italian population reporting four-year experience with the direct analysis of the whole NF1 coding region in 110 unrelated subjects affected by NF1. For each patient, the whole coding sequence and all splice sites were studied for mutations, either by the protein truncation test (PTT), or, most often, by denaturing high performance liquid chromatography (DHPLC). Mutations were identified in 75 (68%) patients. Twenty-two mutations were found to be novel. The detection rate for the different methods was 7/18 (39%) for PTT, and 68/103 (66%) for DHPLC. The mutations were evenly distributed along the NF1 coding sequence. Thirty-two of the 75 unrelated NF1 patients in which germline mutations were identified (32/75, 43%) harbour 23 different recurrent mutations. Fifteen sequence variants likely to represent nonpathogenic polymorphisms were observed at the NF1 locus. Genotype-phenotype analysis was unable to detect any obvious correlation.

show abstract

Section: Discussionmentioning

confidence: 54%

Novel and recurrent mutations in theNF1 gene in Italian patients with neurofibromatosis type 1

et al. 2004

Self Cite

View full text Add to dashboard Cite

show abstract

“…However, because the pattern of bands obtained depends on the precise combination of alleles, the usefulness of this method for determining the genotype of b-thalassemic individuals is obviously limited by the great number of controls required. Moreover, the ability to detect mutations by SSCP is in general lower compared to other methods (e.g., DGGE or DHPLC, which are reported to detect 495% of the mutations present) (13). The SSCP method is nevertheless much easier to employ than other methods and is especially successful for b-thalassemic carriers.…”

Section: Discussionmentioning

confidence: 94%

“…The advantages and disadvantages of each of these methods are discussed on the European Molecular Genetics Quality Network website (11). Recently, systems based on a microelectronic chip (12) or denaturing high-performance liquid chromatography (dHPLC) (13) have been applied for large-scale, fast, and reliable mutational screening of known b-globin alleles.…”

mentioning

confidence: 99%

Identification of the four most common β‐globin gene mutations in Greek β‐thalassemic patients and carriers by PCR‐SSCP: advantages and limitations of the method

Kakavas

Noulas

Chalkias

et al. 2006

Clinical Laboratory Analysis

View full text Add to dashboard Cite

In the present study we investigated whether the single-strand conformational polymorphism (SSCP) method could be employed to identify (rather than simply detect) the four most common b-globin gene mutations in the Greek population: IVS-I-110, Cd39, IVS-I-1, and IVS-I-6. Using DNA from 50 b-thalassemic patients and carriers, we amplified by PCR the appropriate 238-bp region of the human bglobin gene, analyzed the reaction products by nondenaturing polyacrylamide gel electrophoresis, and visualized the bands by silver staining. Single-stranded DNA (ssDNA) fragments showed a reproducible pattern of bands that was characteristic of the mutations present. With the use of control samples containing six of the 10 possible combinations of the four most common b-globin gene mutations, we were able to predict the mutations present in a quarter of the patients studied. Our predictions were confirmed independently by the amplification refractory mutation system (ARMS) method. We conclude that this non-radioactive PCR-SSCP method can be used to reliably identify mutations in patients, provided that suitable controls are available. Moreover, the method is easy to apply to the identification of mutations in carriers, which makes it particularly useful for population screening.

show abstract

“…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

View full text Add to dashboard Cite

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.

show abstract

Reliability of DHPLC in mutational screening of ?-globin (HBB) alleles

Cited by 48 publications

References 18 publications

Novel and recurrent mutations in theNF1 gene in Italian patients with neurofibromatosis type 1

Novel and recurrent mutations in theNF1 gene in Italian patients with neurofibromatosis type 1

Identification of the four most common β‐globin gene mutations in Greek β‐thalassemic patients and carriers by PCR‐SSCP: advantages and limitations of the method

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

Contact Info

Product

Resources

About