Mutation analysis and copy number alterations of KIF23 in non-small-cell lung cancer exhibiting KIF23 over-expression

Figures & data

Table 1 Characteristics of 15 NSCLC patients with elevated level of KIF23

Sample	Histology	Gender	Age (years)	Smoking (packs/year)	TNM	Stage
15	SCC	Male	69	50	pT2N0M1pul	IV
16	SCC	Male	64	50	pT2N0M0	Ib
52	SCC	Male	72	30	pT1N0M0	Ia
66	SCC	Male	69	30	pT3N0M0	IIb
67	SCC	Male	61	40	pT2N0M0	Ib
68	SCC	Male	80	62	pT2N0M0	Ib
73	SCC	Male	44	18	pT2N0M0	Ib
84	SCC	Male	72	55	pT2N2M0	IIIa
87	SCC	Male	77	40	pT2N0M0	Ib
103	SCC	Male	44	60	pT2N1M0	IIb
104	In situ adenocarcinoma	Female	62	30	pT1N0M0	Ib
105	SCC	Male	57	15	pT1N0M0	Ia
108	SCC	Male	75	50	pT3N2M0	IIIa
109	SCC	Male	68	50	pT2N0M0	Ib
114	In situ adenocarcinoma	Male	64	30	pT1N0M0	Ia

Abbreviations: SCC, squamous cell carcinoma; NSCLC, non-small-cell lung cancer.

Figure 1 Relative expression of KIF23 in NSCLC and noncancerous lung samples from the study.

Notes: NSCLC samples with the highest KIF23 expression analyzed in the present work are indicated with black circles. Expression level was detected using Illumina, Human-6 microarrays, quantile-normalized, and log2-transformed as described.³ Differential gene expression analysis comparing NSCLC samples to noncancerous lung tissue was performed using t-tests with an empirical Bayes correction from the Bioconductor Limma package. On average, the KIF23 expression in tumor samples was 2.79 times higher than in noncancerous samples. Matched samples (tumor and noncancerous tissue from the same individual) were available only for a minority of the cases. Data from Valk et al.³
Abbreviations: TU, tumor; NC, non cancerous; NSCLC, non-small-cell lung cancer.

Table 2 KIF23 mutations in cancer

Position GRCh37	DNA, amino acid change	rs ID	MAFa	Prediction	Genotype	Cancer type	References
69,709,610, intron 2	c.82–112C>G	rs150343049	G=0.0004 (1000G)	No predictable effect	C/G, case 68	NSCLC	This study
69,709,686, intron 2	c.82–36G>A	rs3759825	G=0.0749 (ExAC)	Splicing changeb	A/A, all cases	NSCLC	This study
69,714,651, intron 5	c.454–14delT	rs769142360	−=0.0186 (ExAC)	No predictable effect	−/T, all cases	NSCLC	This study
69,714,707, exon 6	c.496G>A, p.E166K	–	NA	Possibly damagingc Splicing changed		Melanoma	Cifola et al 2013^Citation24
69,715,488, intron 6	c.564–10C>T	rs7180624	C=0.2610 (ExAC)	No predictable effect	T/T, all cases	NSCLC	This study van de Wetering et al 2015^Citation25
69,715,488, intron 6	c.564 10delC	rs199725995	−=0.2552 (ExAC)	Splicing changee	−/−, all cases	NSCLC	This study
69,715,495, intron 6	c.564 3delT	–	NA	Splicing changef	−/T, all cases	NSCLC	This study
69,728,530, exon 13	c.1326A>C, p.(V442=)	rs11852675	C=0.0481 (ExAC)	Splicing changeg	A/C, cases 16, 108	NSCLC	This study
69,728,949, exon 14	c.1443A>G, p.(P481=)	rs3825858	A=0.1097 (ExAC)	No predictable effect	G/G, all cases	NSCLC	This study
69,729,001, exon 14	c.1495G>A, p.E499K	–	NA	Benignc Splicing changeh		Melanoma	Cifola et al 2013^Citation24
69,729,111, intron 14	c.1555+50delT	rs72161188	−=0.0044 (ExAC)	No predictable effect	−/T, all cases	NSCLC	This study
69,732,736, exon 17	c.1977C>T, p.(T659=)	rs937724	C=0.1095 (ExAC)	Splicing changei	T/T, all cases	NSCLC	This study
69,732,770, exon 17	c.2011C>T, p.R671W	rs377162847	T=7.7e-5 (EVS)	Probably damagingc Splicing changej		Colorectal cancer	DeRycke et al 2013^Citation23

Notes:

a Allele frequencies for all variants were retrieved from http://www.ncbi.nlm.nih.gov/variation/view using data from Exome Variant Server (http://evs.gs.washington.edu), Exome Aggregation Consortium (http://exac.broadinstitute.org) and a Deep Catalog of Human Genetic Variation (http://browser.1000genomes.org).

b Created splicing signal at branch point according to SpliceSiteFinder-like (from 0/100 to 72.3/100), and increased splicing signal according to Human Splicing Finder (from 71.2/100 to 79.5/100).

c According to PolyPhen2.

d Created splicing signal at intron 5 acceptor site according to NNSPLICE (from 0/1.0 to 1.0/1.0). Increased splicing signal in exon 6 according to MaxEntScan (from 2.9/12 to 5.2/12), and increased splicing signal nearby according to MaxEntScan (from 1.5/12 to 4.2/12). Abolished splicing binding site for splicing factor SF2/ASF in exon 6 according to ESE finder. Abolished splicing signal at intron 6 donor site according to NNSPLICE (from 1.0/1.0 to 0/1.0).

e Decreased splicing signal at acceptor site according to MaxEntScan (from 9.0/16 to 6.4/16).

f Decreased splicing signal at acceptor site according to MaxEntScan (from 9.0/16 to 7.6/16) and increased splicing signal according to GeneSplicer (from 7.8/15 to 8.5/15).

g Splicing binding sites for SC35 and SRp55 are changed to SF2/ASF and SF2/ASF (IgM-BrCa1) according to ESE finder.

h Created splicing binding sites for splicing binding factor SC35 and SRp40 in exon 14.

i Created splicing signal according to Human Splicing Finder (from 0/100 to 66.4/100). Binding sites for splicing factor SF2/ASF (IgM-BrCa1) were changed to a binding site for SRp40.

j Two splicing binding sites for each of splicing binding factor SF2/ASF, SF2/ASF (IgM-BrCa1), and SRp40 were abolished.

Abbreviation: NSCLC, non-small-cell lung cancer.

Figure 2 Sequences of case-specific heterozygous DNA variants present in 3 of 15 NSCLC samples.

Notes: (A) c.1326A>C in exon 13, case 16. The same change was seen in case 108 (C). (B) c.82–112C>G in intron 2, case 68. Upper panels show sequence of DNA from peripheral blood, and lower panels show sequence of tumor DNA.
Abbreviation: NSCLC, non-small-cell lung cancer.

Figure 3 DNA CNA of chromosome 15 profiles in NSCLC. Whole-genome genotyping was performed using Illumina, SNP-array. Analysis was done using GenomeStudio and cnvPartition v. 3.2.0 with manual revision of the SNP-array profiles. BAFs for SNPs along the chromosome 15 show additional copy of chromosome 15 in 9 NSCLC cases. Only case 87 demonstrates normal profile with BAF =0.5 indicating absence of CNA on chromosome 15.

Abbreviations: CNA, copy number alteration; NSCLC, non-small-cell lung cancer; SNP, single nucleotide polymorphism; BAF, B-allele frequency.

Figure S1 (A) Sequencing of cell cycle homology region (CHR) in the promoter of KIF23 in 9 NSCLC tumors. The consensus wild-type sequence TTTGAAA was present in all samples. (B) Correlation between TP53 and KIF23. Scatter plot of log2 mean-centered expression values of KIF23 and TP53, TP53: Pearson’s correlation 0.256, uncorrected p-value =0.008. Pearson product-moment correlation (r) and uncorrected p-value were calculated using R software package.

Abbreviation: NSCLC, non-small-cell lung cancer.

Figure S2 CNV region display from GenomeStudio performed by cnvPartition 3.2.

Table S1 Sanger sequencing and CNAs detection in NSCLC

Sample	Sex	Histology	Sanger sequencing	CNAs number detected by SNP-array
15	Male	SCC	+	153
16	Male	SCC	+	NAa
52	Male	SCC	+	148
66	Male	SCC	+	NA
67	Male	SCC	+	171
68	Male	SCC	+	172
73	Male	SCC	+	92
84	Male	SCC	+	97
87	Male	SCC	+	11
103	Male	SCC	+	NA
104	Female	In situ adenocarcinoma	+	68
105	Male	SCC	+	43
108	Male	SCC	+	NA
109	Male	SCC	+	NA
114	Male	In situ adenocarcinoma	+	164

Note:

a Data not available, SNP-array was not performed due to a lack of DNA.

Abbreviations: SSC, squamous cell carcinoma; CNA, copy number alterations; NSCLC, non-small-cell lung cancer.

Table S2 Chromosomal copy number alterations in NSCLC analyzed by genome wide genotyping using Illumina SNP-array

Chromosome case	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21	22
15	+1	+2	+3	+4	+5	+6	+7	+8	+9	+10	+11p	+12p, q	+13		+15	+16	+17	+18	+19	+20p	+21	+22
52	+1p	+2	+3	+4	+5q	+6			+9p				+13	+14	+15		+17p				+21
67	+1	+2	+3	+4	+5	+6	+7	+8	+9p	+10	+11	+12q	+13	+14	+15	+16	+17p, q	+18	+19	+20p	+21	+22q
68			+3p	+4				+8q	+9p, q		+11q	+12	+13		+15	+16	+17p	+18	+19	+20p		+22
73	+1	+2	+3p	+4q	+5p, q	+6	+7		+9p	+10	+11q	+12p	+13		+15	+16q	+17p	+18	+19		+21q	+22
84	+1q	+2	+3p, q	+4q	+5	+6	+7p	+8	+9	+10p	+11	+12	+13q	+14	+15	+16	+17	+18q	+19	+20	+21	+22
87	+1p		+3	+4q	+5q	+6p	+7p	+8p	+9	+10	+11p		+13	+14		+16p	+17p, q					+22
104	+1q	+2	+3	+4	+5	+6q	+7q	+8	+9		+11	+12p, q	+13	+14	+15		+17	+18	+19q	+20	+21	+22
105	+1q	+2q	+3q	+4	+5	+6p		+8	+9	+10		+12p	+13q	+14q	+15	+16p	+17p	+18	+19q	+20q	+21	+22q
114	+1	+2q	+3q	+4	+5	+6	+7q	+8	+9q	+10	+11	+12	+13	+14q	+15	+16	+17	+18q	+19		+21q	+22

Notes: Chromosome gains are marked with “+”. Partial gain of a short (p) or a long arm (q) of chromosome is indicated with +p or +q.

Abbreviations: NSCLC, non-small-cell lung cancer; SNP, single nucleotide polymorphism.

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