Robert J. Pryor

High-Resolution Genotyping by Amplicon Melting Analysis Using LCGreen

BACKGROUND High-resolution amplicon melting analysis was recently introduced as a closed-tube method for genotyping and mutation scanning (Gundry et al. Clin Chem 2003;49:396-406). The technique required a fluorescently labeled primer and was limited to the detection of mutations residing in the melting domain of the labeled primer. Our aim was to develop a closed-tube system for genotyping and mutation scanning that did not require labeled oligonucleotides. METHODS We studied polymorphisms in the hydroxytryptamine receptor 2A (HTR2A) gene (T102C), beta-globin (hemoglobins S and C) gene, and cystic fibrosis (F508del, F508C, I507del) gene. PCR was performed in the presence of the double-stranded DNA dye LCGreen, and high-resolution amplicon melting curves were obtained. After fluorescence normalization, temperature adjustment, and/or difference analysis, sequence alterations were distinguished by curve shape and/or position. Heterozygous DNA was identified by the low-temperature melting of heteroduplexes not observed with other dyes commonly used in real-time PCR. RESULTS The six common beta-globin genotypes (AA, AS, AC, SS, CC, and SC) were all distinguished in a 110-bp amplicon. The HTR2A single-nucleotide polymorphism was genotyped in a 544-bp fragment that split into two melting domains. Because melting curve acquisition required only 1-2 min, amplification and analysis were achieved in 10-20 min with rapid cycling conditions. CONCLUSIONS High-resolution melting analysis of PCR products amplified in the presence of LCGreen can identify both heterozygous and homozygous sequence variants. The technique requires only the usual unlabeled primers and a generic double-stranded DNA dye added before PCR for amplicon genotyping, and is a promising method for mutation screening.

Rapid Genetic Analysis of X-Linked Chronic Granulomatous Disease by High-Resolution Melting

High-resolution melting analysis was applied to X-linked chronic granulomatous disease, a rare disorder resulting from mutations in CYBB. Melting curves of the 13 PCR products bracketing CYBB exons were predicted by Polands algorithm and compared with observed curves from 96 normal individuals. Primer plates were prepared robotically in batches and dried, greatly simplifying the 3- to 6-hour workflow that included DNA isolation, PCR, melting, and cycle sequencing of any positive products. Small point mutations or insertions/deletions were detected by mixing the hemizygous male DNA with normal male DNA to produce artificial heterozygotes, whereas detection of gross deletions was performed on unmixed samples. Eighteen validation samples and 22 clinical kindreds were analyzed for CYBB mutations. All blinded validation samples were correctly identified. The clinical probands were identified after screening for neutrophil oxidase activity. Nineteen different mutations were found, including seven near intron-exon boundaries predicting splicing defects, five substitutions within exons, three small deletions predicting premature termination, and four gross deletions of multiple exons. Ten novel mutations were found, including (c.) two missense (730T>A, 134T>G), one nonsense (90C>A), four splice site defects (45 + 1G>T, 674 + 4A>G, 1461 + 2delT, and 1462-2A>C), two small deletions (636delT, 1661_1662delCT), and one gross deletion of exons 6 to 8. High-resolution melting can provide timely diagnosis at low cost for effective clinical management of rare, genetic primary immunodeficiency disorders.

Microfluidic Genotyping by Rapid Serial PCR and High-Speed Melting Analysis

BACKGROUND Clinical molecular testing typically batches samples to minimize costs or uses multiplex lab-on-a-chip disposables to analyze a few targets. In genetics, multiple variants need to be analyzed, and different work flows that rapidly analyze multiple loci in a few targets are attractive. METHODS We used a microfluidic platform tailored to rapid serial PCR and high-speed melting (HSM) to genotype 4 single nucleotide variants. A contiguous stream of master mix with sample DNA was pulsed with each primer pair for serial PCR and melting. Two study sites each analyzed 100 samples for F2 (c.*97G>A), F5 (c.1601G>A), and MTHFR (c.665C>T and c.1286A>C) after blinding for genotype and genotype proportions. Internal temperature controls improved melting curve precision. The platforms liquid-handling system automated PCR and HSM. RESULTS PCR and HSM were completed in a total of 12.5 min. Melting was performed at 0.5 °C/s. As expected, homozygous variants were separated by melting temperature, and heterozygotes were identified by curve shape. All samples were correctly genotyped by the instrument. Follow-up testing was required on 1.38% of the assays for a definitive genotype. CONCLUSIONS We demonstrate genotyping accuracy on a novel microfluidic platform with rapid serial PCR and HSM. The platform targets short turnaround times for multiple genetic variants in up to 8 samples. It is also designed to allow automatic and immediate reflexive or repeat testing depending on results from the streaming DNA. Rapid serial PCR provides a flexible genetic work flow and is nicely matched to HSM analysis.

Rapid Molecular Analysis of the STAT3 Gene in Job Syndrome of Hyper-IgE and Recurrent Infectious Diseases

With the recent discovery of mutations in the STAT3 gene in the majority of patients with classic Hyper-IgE syndrome, it is now possible to make a molecular diagnosis in most of these cases. We have developed a PCR-based high-resolution DNA-melting assay to scan selected exons of the STAT3 gene for mutations responsible for Hyper-IgE syndrome, which is then followed by targeted sequencing. We scanned for mutations in 10 unrelated pedigrees, which include 16 patients with classic Hyper-IgE syndrome. These pedigrees include both sporadic and familial cases and their relatives, and we have found STAT3 mutations in all affected individuals. High-resolution melting analysis allows a single day turn-around time for mutation scanning and targeted sequencing of the STAT3 gene, which will greatly facilitate the rapid diagnosis of the Hyper-IgE syndrome, allowing prompt and appropriate therapy, prophylaxis, improved clinical outcome, and accurate genetic counseling.

Family clusters of variant X-linked chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a rare inherited immunodeficiency disorder. The clinical presentation is varied depending on the degree of involvement of the NADPH oxidase system responsible for the oxidative burst of neutrophils. We present 3 cases of variant X-linked CGD in an effort to introduce the disease and highlight the importance and limitations of CGD screening. The variant X-linked form of CGD results in a less severe phenotype and frequently presents later in life. Variant X-linked CGD is difficult to diagnose, but is becoming more readily recognized based on improved testing methods. A high index of suspicion in the setting of unusual infections such as Burkholderia cepacia pneumonia is essential to make the diagnosis. Family screening can lead to early intervention, prophylaxis, and appropriate genetic counseling.