Letter to the Editor Reply to the Letter to the Editor from Wang In Response: To date, all studies of the 15q25 locus have reported statistical associations of several single nucleotide polymorphisms (SNP) mapping in the CHRNA5 locus region with either lung cancer risk, smoking habit, or CHRNA5 mRNA levels, although the functional SNP(s) and the pathologic mechanism(s) have not been clarified. In our work, we reported the novel observation of an associ- ation between CHRNA5 mRNA levels in normal lung and the D398N variant (rs16969968), a coding change in the nicotinic receptor that we confirmed to be associated with lung cancer risk (1) and that was linked to ∼2.5-fold lower CHRNA5 mRNA levels in individuals homozygous for the risk allele (N398; n = 69; P = 8.04e-06; Fig. 4 in ref. 1). Because the 15q25 region con- tains several polymorphisms showing high linkage disequilibri- um with each other, we did not make conclusions about the existence of any biological association between D398N and CHRNA5 mRNA levels, as Wang et al. (2) have interpreted. Wang et al. (2, 3) recently showed that several variants either 5′ upstream of or within the CHRNA5 gene are more strongly associated with the variability in CHRNA5 mRNA expression than is the rs16969968 SNP (D398N) in the human frontal cortex, with the insertion/deletion variant rs38411324, located in the 5′ region near the gene, showing the strongest association with CHRNA5 mRNA expression. The authors selected the tagged SNP rs588765, mapping in the intron 1 of the gene, for genotyping in all of the case-control series because of a high- er genotyping success rate. Using the same 69 samples, we previously analyzed and the PCR primers described by Bierut et al. (4), we carried out an association analysis of rs3841324 with CHRNA5 mRNA levels in normal lung; indeed, we found a stronger association of the rs3841324 than the rs16969968 (D398N; P = 1.25e-08; Fig. 1), consistent with the findings of Wang et al. (3) in brain tissue. However, given the small (only 25 kb) distance between rs3841324andrs16969968,aswellastheexpectedhighlysignif- icant linkage disequilibrium of these polymorphisms (D' = 1.0; r^2 = 0.45; n = 69; analysis by JLIN; ref. 5), the higher association of the rs3841324 than of the D398N with CHRNA5 mRNA levels would be better interpreted as an indication of a closer distance of the first polymorphism with the functional polymorphism affecting CHRNA5 mRNA levels, and not as a proof of biological association. Moreover, the diplotype analysis proposed by Wang et al. (2) cannot be considered sufficient to provide biological mechanism, because it also consists in an asso- ciationanalysisanditsinterpretationiscomplicatedbythesmall number of samples and the variability of the mRNA expression. Inconclusion,wethinkthatfurtherstudiesareneededtoiden- tify the functional variant(s) modulating CHRNA5 mRNA levels. Felicia S. Falvella Antonella Galvan Elisa Frullanti Tommaso A. Dragani Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy Disclosure of Potential Conflicts of Interest Nopotentialconflictsofinterestweredisclosed. References 1. FalvellaFS,GalvanA,FrullantiE,etal.Transcriptionderegulationatthe 15q25 locus in association with lung adenocarcinoma risk. Clin Cancer Res 2009;15:1837–42. 2. WangJC,GruczaR,CruchagaC,etal.GeneticvariationintheCHRNA5 gene affects mRNA levels and is associated with risk for alcohol depen- dence. Mol Psychiatry 2009;14:501–10. 3. WangJC,CruchagaC,SacconeNL,etal.Riskfornicotinedependence andlungcancerisconferredbymRNAexpressionlevelsandaminoacid changeinCHRNA5.HumMolGenet2009;18:3125–35. 4. BierutLJ,StitzelJA,WangJC,etal.Variantsinnicotinicreceptorsand riskfornicotinedependence.AmJPsychiatry2008;165:1163–71. 5. CarterKW,McCaskiePA,PalmerLJ.JLIN:ajavabasedlinkagedisequi- librium plotter. BMC Bioinformatics 2006;7:60. Published OnlineFirst 8/25/09. F 2009AmericanAssociationforCancerResearch. doi:10.1158/1078-0432.CCR-09-1441 Fig. 1. Quantitative analysis of CHRNA5 mRNAlevelsasafunctionofthe rs3841324 22-bp insertion/deletion (I/D)polymorphismin69normallung tissuesamples(P =1.25e-08).Thelinewithineachboxrepresentsthe medianRQvalue;upperandloweredgesofeachboxrepresentthe75th and25thpercentile,respectively;upperandlowerbarsindicatethehighest andlowestvaluesdetermined,respectively.Pairwisecomparison(Tukey's test)indicatedthateachgenotypedifferedstatisticallyfromtheother two(P <0.05). 5599 Clin Cancer Res 2009;15(17) September 1, 2009 www.aacrjournals.org Downloaded from http://aacrjournals.org/clincancerres/article-pdf/15/17/5599/1986065/5599a.pdf by guest on 10 March 2023