Optimization of Apolipoprotein E Genotyping.

Background: Three common alleles of apolipoprotein E (apoE) have been identified and are expressed codominantly to generate six genotypes. Different apoE genotypes are implicated in several cardiovascular and neurologic disorders. Testing for apoE genotypes has increasing diagnostic importance, particularly in the risk assessment of coronary artery disease. A reproducible and cost-effective assay was developed. Methods and Results: Polymerase chain reaction (PCR) amplification of the fourth exon of the apoE gene is performed in the presence of dimethyl sulfoxide using two-step thermal cycling. The PCR products are digested with HhaI restriction enzyme and analyzed by agarose gel electrophoresis to determine apoE genotypes. Effects of several factors, including dimethyl sulfoxide, DNA concentration, and PCR cycling conditions, on PCR specificity and efficiency have been determined and optimized. Conclusions: Apolipoprotein E genotyping by a PCR restriction fragment length polymorphism analysis has been optimized for use in a clinical diagnostic laboratory, allowing evaluation of up to 52 samples by one technician in one day.

Novel pncA Mutations in Pyrazinamide-Resistant Isolates of Mycobacterium tuberculosis.

The gene coding for pyrazinamidase (PZase) activity, pncA, in Mycobacterium tuberculosis was recently cloned, and several mutations which correlate with in vitro resistance to pyrazinamide (PZA) have been identified. During the development of a clinical molecular assay for the detection of PZA resistance, two previously unreported mutations in isolates of PZA-resistant M. tuberculosis were identified. The assay that uses automated DNA sequencing is relatively rapid and allows the detection of any mutations present in the coding region of the pncA gene. The new mutations are both point mutations resulting in amino acid substitutions; 241T --> G results in F80V, and 511G --> C results in A171P. The identification of these mutations accentuates the utility of automated DNA sequencing in the clinical laboratory.

A Novel Missense Mutation, R1283S, of the Cystic Fibrosis Transmembrane Conductance Regulator Gene in a 47-Year-Old African-American Patient.

Background: More than 600 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been described; however, at least 50% of the disease-associated mutations in the African-American population remain unknown. Reported here is a novel missense mutation, R1283S, in a 47-year-old African-American patient with mild cystic fibrosis. Methods and Results: The patient was screened for 27 common and less common CFTR mutations and 2 mutations were detected. Direct sequencing confirmed the presence of the DeltaF508 mutation and revealed the presence of a novel missense mutation, R1283S. Conclusions: R1283S appears to be a cystic fibrosis mutation associated with mild disease, and adds to the number of known mutations in African-Americans. R1283S can be confused with the more common mutation, W1282X, when polymerase chain reaction-restriction fragment length polymorphism analysis is used for detection.