Laboratory Issues

Optimal detection of mutations that predict a truncated neurofibromin polypeptide occurs when the nonsensemediated decay pathway is partially inhibited, thereby increasing the ratio of mutant transcripts with a premature termination codon to normal transcripts. A protein synthesis inhibitor, such as puromycin, in the culture medium is effective for Epstein-Barr virus (EBV)-transformed lym-phoblasts or phytohemaglutinin-stimulated primary lymphocytes.21,28 Furthermore, blood handling and shipping protocols must be used to reduce false positives in PTT resulting from environmental effects such as cold shock29 or delay in messenger RNA isolation.21,28

There is no standardized proficiency program of interlaboratory comparison for NF1 testing. Performance assessment must be conducted by participation in ungraded proficiency survey programs, split sample analysis with other laboratories, or other suitable and documented proficiency-testing methods. No NF1 testing kits, probes, or controls are approved or cleared by the Food and Drug Administration. Intronic primers for amplification of NF1 exons and associated splice junctions that apparently do not coamplify the NF1 pseudogene fragments have been reported.23,30 Two other factors require consideration during test development and interpretation. Reports in support of,31 and in opposition to,32 an apparent tandem duplication of the NF1 gene region have been published. In addition, transcriptional activity from NF1 pseudo-genes or pseudogene fragments has been reported.33 Some issues related to NF1 testing have been reviewed recently.34

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