br Introduction In myasthenia gravis MG autoantibodies
Introduction In myasthenia gravis (MG), autoantibodies against the native AChR conformation cause loss of AChR at the neuromuscular junction and fatigable muscle weakness (Newsom-Davis et al., 1993). The AChR is a pentameric transmembrane protein, with two splice forms of the α-subunit, P3A− and P3A+ (Beeson et al., 1990) in humans; during development, the fetal γ-subunit is replaced by the ‘adult’-specific ε-subunit to form the mature AChR (α2,β,δ,ε). Around 10% of MG patients have thymomas (Marx et al., 1997) which are epithelial neoplasms, with varying lymphocytic components, classified as WHO types A, AB, B1–3 (Strobel et al., 2004). It has been suggested that defective negative selection of the thymocytes leads to the export of potentially autoreactive T cells (Marx and Müller-Hermelink, 1999), but the strong association of MG with filgotinib to AChR and other muscle antigens suggests active sensitization of T cells to muscle antigens in the tumour (Nagvekar et al., 1998). Indeed, low levels of all five AChR subunit mRNAs have been detected using reverse transcription followed by PCR (RT-PCR), or nested PCR (Wilisch et al., 1999). RT-PCR, however, is extremely sensitive and the results may be misleading. We studied 22 MG, and 3 non-MG thymomas with the more accurate and quantitative method of RNAse protection.
Results Each of the five AChR subunit mRNAs could be detected by RT-PCR in normal thymus samples, as confirmed by Southern blotting and hybridization with an internal subunit-specific oligonucleotide (data not shown). These results confirm previous reports (Bruno et al., 2004) and also demonstrated the presence of AChR δ-subunit mRNA. We studied thymomas from 22 patients with MG and 3 without MG. Since AChR antibodies were present in all sixteen sera available for analysis, including two of those without MG (Table 1A), we pooled all the results for analysis. Expression of AChR α-, ε- and γ-subunits in thymomas by RT-PCR was positive in 21/25 (84%), 19/25 (76%) and 11/25 (44%) samples respectively (Fig. 1B). Both P3A+ and P3A− isoforms of the α-subunit were detected, although the P3A− signals were consistently stronger. AChR β- and δ-subunit mRNAs were also detected in many thymomas (data not shown). To obtain a more accurate measure of the mRNAs, we used RNase protection assays (RPA). None of the AChR subunits were detected by RPA in RNA derived from control thymus (n=3), or from human liver, lung, brain or from yeast. By contrast, RNA derived from partially denervated muscle gave strong RPA bands for all AChR subunits except ε (as predicted as the AChR would be predominantly of fetal type). Remarkably, in thymomas, RPA detected only the ε-subunit mRNA, with 13 (52%) of the 25 samples positive (Fig. 1A). Though variable, band intensities were often comparable with those from muscle. We did not detect signals for the α-, β-, γ- or δ-subunit mRNA by RPA, even in the thymomas positive by RT-PCR for AChR α- or γ-, β- or δ-subunit mRNAs. The results are detailed in Table 1A where they are listed according to the thymic pathology, and correlations with histology and RT-PCR results are summarized in Table 1B. All of the 13 thymomas that were positive for ε-subunit mRNA by RPA were also positive by RT-PCR, compared with only 6 of the 12 RPA-negatives (p=0.005, Fisher's exact test). Interestingly, ε-subunit mRNA expression by RPA was more frequent in tumours classified as WHO type A or AB than in those classified as predominantly B1–3 (Table 1B, Fisher's exact test p=0.020).
Discussion Despite many demonstrations of the AChR subunits in thymomas from MG patients, it has never been clear whether there is preferential expression of any one subunit, probably because the RT-PCR methods used were too sensitive. We used the RNase protection assay (RPA) which readily detects expression of all AChR subunit mRNAs in human muscle (MacClennan et al., 1997), and after denervation (Fig. 1A), but provides a more quantitative and accurate measure than RT-PCR. Surprisingly, we only detected the AChR ε-subunit mRNA; this was found in 12/22 (55%) of thymomas from MG patients and in one of three without MG. Moreover, ε-subunit mRNA expression was more common in thymomas of the A or AB classification than in those with the B classifications.