Batten disease is an autosomal recessive disorder also known as juvenile neuronal ceroid lipofuscinosis. The most common mutation for this disease is an approximately 1-kbp deletion in the CLN3 gene, which accounts for about 80 to 85% of the mutation load. We developed a rapid assay for this mutation using the PCR to produce amplicons that are detected by nucleobase quenching of the fluorescent signal from a probe labeled with a fluorescent dye. The probe overlaps the deletion breakpoint and is completely base paired to the mutant amplicon. However, three bases at the 5 end of the probe do not base pair with the wild-type amplicon. The alleles are distinguished by the different melting temperatures of the probe amplicon hybrids. Comparison of this new method with an allele-specific PCR and gel electrophoresis-based method showed 100% concordance in determination of the genotype for 30 specimens (11 homozygous mutant, 8 heterozygotes, and 11 homozygous normal). PCR followed by allele-specific melting curve analysis using nucleobase quenching has utility as a rapid method for detection of the most common mutation that causes Batten disease.
A biopsy of a nasal mass was received from another institution for a hematopathology consultation. The specimen had morphologic and immunostaining features consistent with a B-cell lymphoma, histologically low-grade, and suggestive for an extranodal marginal cell lymphoma of mucosa associated lymphoid tissue (MALT) type. We used PCR of the IgH gene to evaluate clonality on DNA derived from this specimen. The primers were from the BIOMED-2 report (van Dongen et al. 2003, Leukemia17:2257) and the amplicons were subjected to heteroduplex formation prior to PAGE. A homoduplex of approximately 140 bp was obtained reproducibly from the FR2 and JH primers, which is below the usually acceptable size limits of 250–295 bps. No homoduplex was obtained using FR3 and JH primers. We sequenced the FR2/JH amplicon using the PCR primers as sequencing primers. The amplicon was 137 bps, with 92 bps between the primers. After the upstream VH3 FR2 primer there were approximately 25 bps from the FR2 region of several members of the VH3 family, with VH3-49 (allele *03) being the best match. Adjacent to the downstream consensus JH primer there were approximately 30 bp from the J6 segment. Between the identifiable sequences there were 37 bp that we could not identify. Blast searches turned up several matches of 18 bp, but nothing that gave convincing evidence for its origin. We interpret these results as indicating a clonal IgH rearrangement followed by a deletion that removed most of the downstream portion of the V segment, including the FR3 region. It is likely that the 37 bp in between the identified IgH segments consists of randomly inserted nucleotides and IgH sequence that has been somatically mutated beyond recognition, although other interpretations are possible. However, the amplicon does appear to be derived from an IgH rearrangement, which is consistent with derivation from a monoclonal population of B-lymphocytes. This work illustrates that DNA fragments outside of the size range expected from PCR of the antigen receptor genes may still be consistent with a monoclonal result. Thus, this type of result should not be dismissed, but should be subjected to further analysis.
Chronic myelogenous leukemia (CML) is caused by a balanced translocation between chromosomes 9 and 22 that creates the BCR-ABL fusion gene (FG) and activates the ABL tyrosine kinase (TK). Imatinib inhibits this TK and causes long-term remission in most CML patients. Relapse during imatinib therapy is often associated with mutations in the BCR-ABL FG that cause resistance to inhibition by imatinib. The usual procedure for detecting imatinib resistance mutations involves reverse transcription, PCR of the BCR-ABL FG, then nested PCR of the ABL TK to generate a template for sequencing. This procedure is suboptimal in a clinical laboratory because of the risk of carryover contamination, and extra time and workload, all due to the use of nested PCR. We describe a procedure for preparing sequencing template from the BCR-ABL FG that does not require nested PCR. The initial PCR employs a mixture of thermostable DNA polymerases that increases the sensitivity of the reaction for limiting concentrations of the desired target. One round of PCR provides enough template for sequencing. The PCR products are sequenced directly without gel purification, using three internal primers plus the downstream PCR primer as sequencing primers. The internal primers provide additional specificity resulting in a high quality sequence even if the PCR produces undesired products. Using this methodology we analyzed a series of eleven CML patient specimens selected because of a four-fold or greater increase in expression of the BCR-ABL FG during treatment with imatinib. The expression of BCR-ABL RNA was measured using a reverse transcription Q-PCR assay. The leftover cDNA from the expression assay was used as template for the PCR to generate the sequencing template. Five patients lacked a detectable mutation, and six had a mutation in the BCR-ABL TK. The six mutations found, G279E, F317L, L248V, M351T, F359C, and H396R, had all been seen previously and associated with resistance to imatinib. We conclude that detection of imatinib resistance mutations in the BCR-ABL FG is feasible without using nested PCR, although with a reduced analytical sensitivity. Elimination of the nested PCR step makes the procedure easier, quicker and less likely to suffer from carryover contamination. This method is a viable alternative for the detection of imatinib resistance mutations in the clinical laboratory.
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