WWOX expression in relation to the rs11644322 SNP site

The SNP WWOX rs11644322 with a minor allele frequency (MAF) of 26.1 % is located in the immense intron 8 (776656 bp long) separating exon 8 and 9 (see Figure 16 for full length transcript). GeneBank entries (http://www.ncbi.nlm.nih.gov/gene/) suggest several alternative transcripts of WWOX terminating within intron 8.

100 kbp GRCh38.p2). The coding region contains 9 exons, the first and the last one flanked by the 5' and the 3'-untranslated region (UTR), respectively. The vertical lines represent the exons. The location of the index SNP rs11644322 in intron 8 is marked. Proportionality of sizes and distances are retained. The vertical lines denoted with E1 - E9 represent the protein coding exons.

4.1.2.2

As illustrated in Figure 16, rs11644322 is flanked by the exons 8 and 9 of WWOX.

Transcription of this region was compared with that of exon 4-6, considered as core WWOX region. For absolute quantification of the expression ratios between these two WWOX coding regions a cDNA comprising entire WWOX, was cloned as reference (see

Results

92 3.5.1). Expression analysis (see 3.6.4) in 88 LCLs (for one cell line reverse transcription failed) identified a mean transcription rate of 67 % for exon 8-9 compared to the core coding region (see Figure 17, bar plot), indicating the presence of the last exon in the majority of WWOX transcripts. In addition, a substantial intra-cell line correlation between the expression of these two WWOX regions was verified, which even increased upon gemcitabine exposure (Figure 17).

Figure 17: Expression of the last exon in relation to that of the core WWOX coding region. The mRNA expression of the terminal exon 9 (captured by an exon 8/exon 9-spanning primer pair) was compared with the major part of the coding region (represented by an exon 4/exon 5/exon 6-spanning primer pair). The graphs summarize the data obtained with 88 lymphoblastoid cell lines (for one cell line reverse transcription failed) treated either with PBS only (baseline) or with 30 nM of gemcitabine at 37 °C for 24 h. The scatter plot illustrates expression correlation between regions 4-6 and 8-9. Both axes are displayed in log10-scale, by which normal distributions of the data could be assumed. The respective regression lines with the Pearson correlation coefficient r are indicated. All expression data were referred to the cell line with the lowest transcript numbers for exon 4-6 under basal conditions (set to “1”). To account for inter-sample heterogeneity, expression data were normalized to a weighted mean of five reference genes (B2MG, GAPDH, HPRT1, UBC, 36b4). The lower right insert illustrates the quantitative transcript numbers of the last WWOX exon in relation to the core coding region of which the mean over the entire LCL cohort was set to “1” (error symbols denote one SD). This figure was generated with Sigma Plot version 12.

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4.1.2.3 Impact of rs11644322 SNP on WWOX regional transcription

It should be delineated whether the WWOX SNP rs11644322 affects transcription of exon 4-6 and 8-9. Presence of the AA genotype at the index SNP site was accompanied by lower transcription of both WWOX regions, with and w/o gemcitabine (Figure 18).

However, no significant change in WWOX gene expression could be detected between GG and GA genotypes in the present experimental setting with short-term incubation time of 24 h. upon 30 nM gemcitabine incubation for 24 h at 37 °C. The median value for each group is highlighted by a horizontal grey-shaded line. Statistical differences between two groups were assessed by the non-parametric Mann-Whitney U test. The lower line of p-values refers to testing between GG and GA genotype, the upper one between combined GG and GA versus AA configuration. This figure was generated with Sigma Plot version 12.

4.1.2.4 Whole transcriptome analysis around the WWOX index SNP

As located far distant from exon 8 and 9, it was suggested that the rs11644322 site might be involved in regulation of non-coding RNA expression. To discover non-coding RNAs vicinal to the index SNP, whole transcriptome analysis (RNAseq, see 3.6.5) was undertaken. For two pooled RNA probes from LCLs, carrying the GG vs. AA genotype at rs11644322, there was no coverage around the index SNP site (see Figure 19).

Results

94 This observation demonstrates that there are no transcripts encoded in the genetic vicinity of rs11644322.

WWOXSNP rs11644322 LCL Gallele sample

LCL Aallele sample

Figure 19: Whole transcriptome analysis around rs11644322. Data were analyzed by RNA sequencing of two pooled LCL samples containing the homozygous G (upper panel) or the A (lower panel) allele at rs11644322 with each pool consisting of RNA of five LCLs (cell identifiers at the Coriell institute for G allele: HG00096, HG00109, HG00120, HG00244, and HG00258; for A allele: HG00100, HG00108, HG00122, HG00245, and HG00265). Genomic sequences ± 5000 bp around the index SNP (marked with a dashed line) are shown.

Likewise, in the pancreatic cancer cell lines PaTu8988t, MiaPaca-II, and AsPC1 no reads or only reads at very low amounts, not distinguishable from technical noise (< 0.5 reads/kilobase of transcript/per million mapped reads), could be observed within a range of ±1 Mbp referred to the index SNP rs11644322.

4.1.2.5 Global transcriptome stratified for rs11644322

Five pooled LCLs each with GG or AA genotype at rs11644322, not exposed to gemcitabine, were subjected to global transcriptome analysis. Out of all identified and annotated transcripts only six showed differential expression of more than 2-fold (see Table 64). Compared with GG, transcription in cells with AA genotype was lower for TIMP2 and SEMA3C and higher for RNA5-8SP2, IGHA1, AL161626.1, and RNA5-8SP6. The most pronounced ratio was observed for RNA5-8SP2 (ribosomal pseudogene). For this transcript, which is located on chromosome 16 like WWOX, correlation with EC50 values for gemcitabine and with WWOX expression was evaluated in the entire set of 89 LCLs.

However, expression of RNA5-8SP6 was neither related to EC50 for gemcitabine nor to WWOX expression (core region and last exon).

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Table 64: Expression profile in LCL samples in dependence of WWOX rs11644322. Five non-treated LCLs each with homozygous wild-type (Coriell ID HG00096, HG00109, HG00120, HG00244, and HG00258) and homozygous variant allele (HG00100, HG00108, HG00122, HG00245, and HG00265) configuration at rs11644322 were pooled.

This table lists all transcripts differing by a log2-fold change of at least 2.0 between these two groups. Data were obtained by sequencing of total RNA. Transcript data refer to RPKM values. RPKM-normalized transcripts for AA at rs11644322 were divided by those for GG genotype.

Transcript notation Rs11644322_GG [RPKM] Rs11644322_AA [RPKM] Ratio AA/GG

TIMP2 1.50 0.18 0.12

4.1.3 Consequences of SP1 overexpression for cytostatic drug sensitivity