Fine mapping of chromosome 6q23-25 region in familial lung cancer families reveals RGS17 as a likely candidate gene

Abstract

Purpose: We have previously mapped a major susceptibility locus influencing familial lung cancer risk to chromosome 6q23-25. However, the causal gene at this locus remains undetermined. In this study, we further refined this locus to identify a single candidate gene, by fine mapping using microsatellite markers and association studies using high-density single nucleotide polymorphisms (SNP).

Experimental design: Six multigenerational families with five or more affected members were chosen for fine-mapping the 6q linkage region using microsatellite markers. For association mapping, we genotyped 24 6q-linked cases and 72 unrelated noncancer controls from the Genetic Epidemiology of Lung Cancer Consortium resources using the Affymetrix 500K chipset. Significant associations were validated in two independent familial lung cancer populations: 226 familial lung cases and 313 controls from the Genetic Epidemiology of Lung Cancer Consortium, and 154 familial cases and 325 controls from Mayo Clinic. Each familial case was chosen from one high-risk lung cancer family that has three or more affected members.

Results: A region-wide scan across 6q23-25 found significant association between lung cancer susceptibility and three single nucleotide polymorphisms in the first intron of the RGS17 gene. This association was further confirmed in two independent familial lung cancer populations. By quantitative real-time PCR analysis of matched tumor and normal human tissues, we found that RGS17 transcript accumulation is highly and consistently increased in sporadic lung cancers. Human lung tumor cell proliferation and tumorigenesis in nude mice are inhibited upon knockdown of RGS17 levels.

Conclusion: RGS17 is a major candidate for the familial lung cancer susceptibility locus on chromosome 6q23-25.

Figure 1

Familial lung cancer pedigrees and linkage fine mapping. (a) Six familial lung cancer pedigrees used for fine mapping analysis. Filled circles (females) or squares (males) represent affected members within lung cancer families. Numbers below each individual correspond to the sample identifiers in the pedigree used for fine mapping studies. (b) Fine linkage mapping with six multigenerational pedigrees at an increased average density of 2.4 cM per marker. LOD scores for individual families were estimated with Simwalk2 under the autosomal dominant model as used previously. The region of haplotype sharing by affected members within each of the families covers a region of ~3 cM centering on the marker D6S2442 (154.10 cM), spanning 152.0 to 154.2 Mb on chromosome 6q.

Figure 2

Schematic view of linkage and association mapping of familial lung cancer on chromosome 6q23-25. (a) Linkage scan results for 6q23-25 from the GELCC lung cancer families (12). (b) Association mapping in 24 independent 6q-linked cases and 72 controls using 3,957 SNPs from Affymetrix 500K chipsets. (c) Enhanced view of association mapping in 153–154 Mb region. All three significant SNPs were located in the first intron of RGS17. (d) Physical map and LD blocks. Pairwise LD, measured as D′, was calculated from the HapMap CEU population using the methods of Gabriel (34) as implemented in Haploview (35). Shading represents the magnitude and significance of pairwise LD, with a white-to-red gradient reflecting lower to higher LD values. All three SNPs rs4083914, rs9479510 and rs6901126 were located within block 12 with a size of 43 kb.

Figure 3

RGS17 mRNA expression is increased in lung tumor tissue, and RNAi knock-down of RGS17 transcript inhibits tumor cell proliferation and tumor growth. (a) Expression of RGS17 in human lung adenocarcinomas and adenosquamous carcinomas relative to patient matched normal lung tissue controls. Green: normal tissues; red: tumor tissues. (b) RGS17 knock-down inhibits proliferation. Cell growth is measured by MTT viable cell staining over 10 days. shRNA knock-down of RGS17 transcript in H1299 human lung tumor cells was measured by quantitative real-time PCR (inset). (c) RGS17 knock-down inhibits colony formation. Colonies were stained after 10 days in culture. (d) Nude mouse tumorigenesis assays. 3 × 10⁶ cells were injected s.c. on the left (H1299-shRGS) and right (H1299-vector) flanks. (e) Tumor volume was monitored until mice were sacrificed 28 days post injection.