Genetic screening of Fabry patients with EcoTILLING and HRM technology

<p>Abstract</p> <p>Background</p> <p>Anderson-Fabry disease (FD) is caused by a deficit of the α-galactosidase A enzyme which leads to the accumulation of complex sphingolipids, especially globotriaosylceramide (Gb3), in all the cells of the body, causing the onset of a multi-systemic disease with poor prognosis in adulthood. In this article, we describe two alternative methods for screening the <it>GLA </it>gene which codes for the α-galactosidase A enzyme in subjects with probable FD in order to test analysis strategies which include or rely on initial pre-screening.</p> <p>Findings</p> <p>We analyzed 740 samples using EcoTILLING, comparing two mismatch-specific<ul/>endonucleases, CEL I and ENDO-1, while conducting a parallel screening of the same samples using HRM (High Resolution Melting). Afterwards, all samples were subjected to direct sequencing. Overall, we identified 12 different genetic variations: -10C>T, -12G>A, -30G>A, IVS2-76_80del5, D165H, C172Y, IVS4+16A>G, IVS4 +68 A>G, c.718_719delAA, D313Y, IVS6-22C>T, G395A. This was consistent with the high genetic heterogeneity found in FD patients and carriers. All of the mutations were detected by HRM, whereas 17% of the mutations were not found by EcoTILLING. The results obtained by EcoTILLING comparing the CEL I and ENDO-1 endonucleases were perfectly overlapping.</p> <p>Conclusion</p> <p>On the basis of its simplicity, flexibility, repeatability, and sensitivity, we believe that<ul/>HRM analysis of the <it>GLA </it>gene is a reliable presequencing screening tool. This method can be applied to any genomic feature to identify known and unknown genetic alterations, and it is ideal for conducting screening and population studies.</p

Hepatitis C Genotyping by Denaturing High-Performance Liquid Chromatography

Determination of the hepatitis C virus (HCV) genotype for infected patients increasingly has become accepted as the standard of care. Genotype assignment helps in assessing disease prognosis and assists in establishing the appropriate duration of treatment. The great genetic diversity of HCV, with 11 major genotypes and >70 subtypes, contributes to the technical difficulty of genotype testing. While the “gold standard” for testing is nucleic acid sequencing, a variety of hybridization assays, including the line probe assay, have been developed to provide more rapid and accessible forms of testing. The aim of this study was to determine whether denaturing high-performance liquid chromatography (dHPLC) could be used as a clinical method for distinguishing HCV genotypes 1, 2, 3, and 4. A portion of the 5′ untranslated region of the HCV genome was amplified by heminested multiplex reverse transcription PCR. The two amplicons then were analyzed by dHPLC analysis and compared to the genotypes determined by sequence analysis. After 115 specimens were analyzed as standards, 200 masked specimens (specimens whose identity was not known before testing) were analyzed to determine the concordance of the assay. The assay had a concordance of 96% at the genotype level and a concordance of 87% at the subtype level. However, the dHPLC method was not as accurate as other reported methods of HCV genotyping. This is the first time that HCV genotyping has been performed by dHPLC

Evaluation of the COBAS Amplicor HBV Monitor Assay and Comparison with the Ultrasensitive HBV Hybrid Capture 2 Assay for Quantification of Hepatitis B Virus DNA

Performance characteristics of the COBAS Amplicor HBV Monitor test (Roche Diagnostics), which measures hepatitis B virus (HBV) DNA quantitatively, were evaluated and compared with the Ultrasensitive HBV Hybrid Capture 2 (HC2; Digene Corporation) assay. Linearity and within-run precision were assessed for both methods by using eight HBV DNA-positive samples serially diluted to obtain a range of <100 to 500,000 HBV DNA copies/ml and run in triplicate. Agreement between the methods was studied with 100 clinical samples. HC2 assay performance near the limit of detection was investigated through repeat testing of 149 samples with HC2 and testing of 37 samples with HC2 results of <4,700 HBV DNA copies/ml by Amplicor assay and a qualitative PCR assay. The linearity experiment for Amplicor had regression of observed values compared to expected values (y = 1.073x − 0.247; R(2) = 0.993, n = 32; for HC2, y = 0.855x + 0.759, R(2) = 0.729, n = 18). Within-run standard deviation of log HBV DNA copies/ml ranged from 0.003 to 0.348 (Amplicor) and 0.027 to 0.253 (HC2). Agreement assessed by Deming regression was poor [Amplicor = 1.197(HC2) − 0.961; R(2) = 0.799, standard error of the estimate (SEE) = 0.710, n = 94]. Near the lower limit of detection, 32 of 149 repeat HC2 results were <4,700 HBV DNA copies/ml. Of the 37 samples with HC2 results of <4,700 HBV DNA copies/ml, HBV DNA was not detected in 15 samples, while HBV DNA was detected by at least one PCR method in 12 samples. Amplicor is linear from 200 to 200,000 HBV DNA copies/ml with undiluted samples, and this range can be expanded through dilution. Inconsistent HC2 results near the limit of detection justify use of a grey zone

Performance Characteristics of the COBAS Amplicor Hepatitis C Virus (HCV) Monitor, Version 2.0, International Unit Assay and the National Genetics Institute HCV Superquant Assay

The COBAS Amplicor Hepatitis C Virus (HCV) Monitor assay, version 2.0, which reports in international units per milliliter, was compared to the assay reported in copies per milliliter by analyzing dilution series and clinical plasma samples by both methods. In addition, the Amplicor international unit assay was compared to the National Genetics Institute HCV Superquant assay. The dilution series ranged from <100 to 5,000,000 HCV RNA copies/ml and consisted of 32 points, assayed in triplicate in each assay. Thirty clinical samples ranging from 1,000 to 1,000,000 HCV RNA copies/ml were assayed in duplicate. Deming regression analysis comparing the Amplicor HCV RNA international units-per-milliliter and copies-per-milliliter assays was calculated as follows: (Amplicor international units per milliliter) = 1.030(Amplicor copies per milliliter) − 0.392; R(2) = 0.981; n = 28; S(y/x) (standard error of the estimate) = 0.129. The linearity of the Amplicor international units-per-milliliter assay was as follows: observed = 0.886(expected) + 0.437; R(2) = 0.983; n = 30. The linearity of the Superquant assay was as follows: observed= 0.918 (expected) + 0.436; R(2) = 0.986; n = 32. Deming regression analysis comparing the Amplicor and Superquant assays was calculated as follows: Superquant = 1.066(Amplicor) − 0.0197; R(2) = 0.908; S(y/x) = 0.308; n = 28. The Amplicor and Superquant assays were linear through the range of 600 to 600,000 IU of HCV RNA/ml and ∼300 to 5,000,000 HCV RNA copies/ml, respectively. The narrow range of the Amplicor assay means that some samples will require dilution and retesting for accurate quantification above 600,000 IU of HCV RNA/ml. The Amplicor and Superquant assays agreed well within the range of 600 to 600,000 IU of HCV RNA/ml (∼1,000 to ∼1,000,000 HCV RNA copies/ml). Overall, the Amplicor and Superquant assays agree well, and results obtained in one assay could be expected to compare well with results from the other when reported in copies per milliliter

A Single-Tube Nucleic Acid Extraction, Amplification, and Detection Method Using Aluminum Oxide

A disposable 0.2-ml polymerase chain reaction (PCR) tube modified with an aluminum oxide membrane (AOM) has been developed for the extraction, amplification, and detection of nucleic acids. To assess the dynamic range of AOM tubes for real-time PCR, quantified herpes simplex virus (HSV) DNA was used to compare AOM tubes to standard PCR tubes. AOM PCR tubes used for amplification and detection of quantified HSV-1 displayed a crossing threshold (C(T)) shift 0.1 cycles greater than PCR tube controls. Experiments with HSV-1-positive cerebrospinal fluid (CSF) examined the extraction, amplification, and detection properties of the AOM tubes compared to the Qiagen DNA blood mini kit. The AOM extraction, amplification, and detection of HSV-1 in CSF displayed differences of less than one C(T) when compared to Qiagen-extracted samples. Experiments testing the AOM method using clinical CSF samples displayed 100% concordance with reported results. AOM tubes have no adverse effects on amplification or fluorescence acquisition by real-time PCR and can be effectively used for the extraction, amplification, and detection of HSV from CSF. The AOM single tube method is a fast, reliable, and reproducible technique for the extraction, amplification, and detection of HSV in CSF