Genes to Cells (2006) 11, 95-97. doi:10.1111/j.1365-2443.2005.00920.x
© 2006 Blackwell Publishing or its licensors
Evaluation of somatic alterations of Pcdh-
transcripts in the brain by cDNA analysis without PCR
Teruyoshi Hirayama1,2,
Ayako Sasaki1,2 and
Takeshi Yagi1,2,*
1 KOKORO-Biology Group, Laboratories for Integrated Biology, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka, 565-0871, Japan
2 CREST, Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
Here we evaluate our previous results, ‘Somatic mutations of synaptic cadherin (CNR family) transcripts in the nervous system,’ published in Genes to Cells (Hirayama et al. 2001). The Protocadherin (Pcdh)-
, -ß, -
family exists as gene clusters. Using RT-PCR, we identified possible somatic mutations in Pcdh- 10. Unfortunately, however, PCR products include PCR errors. To evaluate the previous alterations of Pcdh- transcripts we had found by RT-PCR, we investigated cDNA libraries from mouse cerebral cortexes at E18.5, P1, P7, P10, and P60, without using PCR methods. Our method is shown in Supplementary materials. We constructed Pcdh--enriched cDNA libraries using a Pcdh--specific reverse primer, which was designed to anneal to a sequence in the constant region of exon 1 from which the extended sequence would cross the intron-exon boundary. We also added specific reverse primers for the control transcripts, N-cadherin and ß-actin, when we constructed the Pcdh--enriched cDNA libraries. To isolate the Pcdh--positive clones, we used a probe that was designed to anneal to the Pcdh-10 EC4-5 region, which is highly conserved among the Pcdh- transcripts from 1 to 12. All the types of Pcdh- isoforms were obtained and sequenced, with a total of 620 independent clones (1 303 062 bp) from all the developmental stages combined. From the same cDNA libraries, we also obtained 430 N-cadherin (914 795 bp) and 302 ß-actin (325 624 bp) independent clones as controls. Except at P1, the frequency of base substitution for Pcdh- was not significantly different from that of the control N-cadherin(Pcdh- vs. N-cadherin): E18.5 (0.6 x 10–5 vs. 1.6 x 10–5), P7 (1.5 x 10–5 vs. 1.4 x 10–5), P10 (2.2 x 10–5 vs. 2.4 x 10–5), P60 (1.2 x 10–5 vs. 2.3 x 10–5) (Table 1). In contrast, at P1 we found 16 single-base substitutions in 16 of 161 Pcdh--positive clones. The frequency of base substitution was 4.8 x 10–5. In the same libraries, N-cadherin and ß-actin transcripts had substitution rates of 1.4 x 10–5 and 1.8 x 10–5, respectively (Table 1). There were significant differences (P < 0.01) between the substitution rates of Pcdh- and those of the control molecules. The G to A transition bias at P1, a rate of 25%, was higher than the 6.3% found for N-cadherin at all stages or the 6.7% found for the Pcdh- transcripts except at P1 (Table 2). In our previous study, we reported a significantly increased frequency of base substitution, not only at P1 but also at P60, using RT-PCR methods. Here, analyzing the same developmental stages using cDNA libraries, we failed to detect a high frequency of substitution in the P60 Pcdh- transcripts; the 1.2 x 10–5 frequency was similar to the level of technical error. Therefore, our non-PCR results did not confirm the previous alterations in Pcdh- transcripts that we found using the PCR method. Therefore, we conclude that a large number of alterations identified in the previous report were introduced by artificial PCR errors, and this note corrects our earlier results. However, neither the significantly high sequence alteration rate nor the unusual ratio of G to A base substitutions in the Pcdh- transcripts at P1 ruled out the possibility of actual mutations in this gene in the brain.
Footnotes
Communicated by: Fumio Hanaoka
* Correspondence: E-mail: yagi{at}fbs.osaka-u.ac.jp
References
Hirayama, T., Sugino, H. & Yagi, T. (2001) Somatic mutations of synaptic cadherin (CNR family) transcripts in the nervous system. Genes Cells
6, 151–164.[Abstract]
Received: 12 August 2005
Accepted: 12 October 2005
