Rarity of Somatic Mutation and Frequency of Normal Sequence Variation Detected in Sporadic Colon Adenocarcinoma Using High-Throughput cDNA Sequencing.

We performed high-throughput cDNA sequencing in colorectal adenocarcinoma and matching normal colorectal epithelium. All six hundred three genes in the UCSC database that were expressed in colon cancers and contained open reading frames of 1000 nucleotides or less were selected for study (total basepairs/bp, 366,686). 304,350 of these 366,686 bp (83.0%) were amplified and sequenced successfully. Seventy-eight sequence variants present in germline (i.e. normal) as well as matching somatic (i.e. tumor) DNA were discovered, yielding a frequency of 1 variant per 3,902 bp. Fifty-one of these sequence variants were homozygous (26 synonymous, 25 non-synonymous), while 27 were heterozygous (11 synonymous, 16 non-synonymous). Cancer tissue contained only one sequence-altered allele of the gene ATP50, which was present heterozygously alongside the wild-type allele in matching normal epithelium. Despite this relatively large number of bp and genes sequenced, no somatic mutations unique to tumor were found. High-throughput cDNA sequencing is a practical approach for detecting novel sequence variations and alterations in human tumors, such as those of the colon.

Introduction

It is widely believed that somatic as well as germline mutations play important roles in the origin and progression of colorectal cancers (Calvert and Frucht, 2002). Many genes have been investigated for mutation to elucidate mechanisms of colorectal cancer development, with these investigations demonstrating the involvement of mutations in colorectal carcinogenesis and progression. Samuels et al. reported that PIK3CA, a catalytic subunit of the class IA phosphatidylinositol 3-kinases, was somatically mutated in 32% of colorectal cancers, resulting in the attenuation of apoptosis and facilitated tumor invasion(Samuels et al. 2004). A comprehensive study entitled, “The Tyrosine Phosphatome” was accomplished by sequencing all genes involved in tyrosine phosphorylation in a large cancer cohort consisting of 175 colorectal cancer patients(Wang et al. 2004). Most mutational studies, however, have been preoccupied with the prevalence of somatic mutations in a specific single candidate gene in relatively small colorectal cancer patient cohorts. Recently, Sjoblom et al. reported the genome-wide frequencies of somatically mutated genes in human breast and colorectal cancers(Sjoblom et al. 2006). However, the methods these used were extremely expensive, time-consuming, and labor-intensive for a typical laboratory to perform. More practical strategies, amenable to smaller laboratories with more conservative budgets, would be of great value in the continuing quest to answer questions in the fields of tumor genomics and mutatomics. To this end, we present herein a circumscribed, practical mutational study employing high-throughput cDNA sequencing in colon adenocarcinoma, in which we demonstrate the eminent feasibility and results of determining sequence variation efficiently and at low cost.

Materials and Methods

Tissue samples

Colorectal cancer and its matching normal colonic mucosa from a patient undergoing surgical resection at the Baltimore VA Hospital after signing informed research consent was used for this study. Clinicopathological data were as follows: 75 year-old male; moderately-differentiated colorectal adenocarcinoma of the ascending colon; tumor size, 2.5 × 1.1 × 0.5 cm; TNM stage (Fifth Edition of the TNM classification of the UICC, 1997), T2N0MX, without any other malignancies. Both colorectal adenocarcinoma and normal colonic epithelium (obtained at the location within the surgically resected specimen furthest from the tumor) were cut into smaller pieces and frozen in liquid nitrogen immediately after removal. A frozen aliquot of each specimen was crushed into pieces and lysed immediately in either TRIZOL reagent (Invitrogen Corp., Carlsbad, CA,) to extract total RNA, or lysis buffer of a DNeasy Tissue kit (QIAGEN Inc., Valencia, CA) to extract DNA, according to these manufacturers’ instructions.

Cell lines

HeLa S3, HT29, HCT15, HCT116, LoVo, CaCo2, LS174T, LS411N, and DLD1, purchased from the American Type Culture Collection (ATCC), and KYSE30, 70, 110, 150, 220, 410, 770, 850 and OE33, obtained from Dr. Yutaka Shimada at Kyoto University in Japan (Shimada et al. 1992), were enrolled in the current study in order to validate our findings in the ATP50 gene. Culture conditions for each cell line were according to ATCC and the establisher’s recommendations. All cell lines were supplemented with 10% fetal bovine serum plus an appropriate concentration of penicillin and streptomycin.

Gene selection

To increase our chances of successfully amplifying and sequencing cDNAs, we restricted our study to genes that are known to be expressed in colorectal cancer cells, based on a gene expression database at the University of California, Santa Cruz (UCSC) [http://genome.ucsc.edu/index.html]. From among this gene set, we selected a subset of genes (approximately 600) containing open reading frames (ORFs) 1000 nucleotides or shorter in length. To automate design of the large number of primer sets required, we developed an in-house primer design algorithm based on the publicly available primer design software program, Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_slow.cgi). PCR products were designed to range from 300 to 500 bp in length. ORFs of cDNAs longer than 500 bp were divided into 2 or 3 fragments; primers were then designed with adjacent fragments overlapped, in order to completely cover these longer ORFs. Finally, for each heterozygous sequence alteration, genomic DNA primers (available on request) were designed to confirm cDNA sequencing results.

RT-PCR

Total RNA extracted from colorectal adenocarcinoma and normal colonic epithelium was reverse-transcribed using a SuperScript III First-Strand kit (Invitrogen, Carlsbad, CA), and respective cDNA pools were made. RT-PCR was performed using an AccuPrime Supermix I Kit (Invitrogen). The PCR protocol was as follows: 1 min at 96 °C followed by 35 cycles of 30 sec at 94 °C, 45 sec at 58 °C, and 1 min at 72 °C. Secondary PCR was performed on purified template from the first RT-PCR product, using the same protocol.

Sequencing

A BigDye Terminator v3.1 Kit (Applied Biosystems, Foster City, CA) was used for the sequencing reaction, and sequence products were read on an SCE 9610 automated 96-capillary sequencer (Spectruby BaseSpectrum v2.10 (SpectruMedix) and analyzed with Mutation Surveyor v2.2 (SoftGenetics LLC, State College, PA). Each time candidate sequence alterations were discovered in cDNA from colorectal cancer tissue, identical procedures were followed in matched normal epitheliam to confirm whether or not they represented somatic alterations. After candidate alterations were confirmed, the entire procedure was repeated separately on a fresh aliquot of cDNA from both the cancer and normal specimens in order to exclude amplification or technical errors due to two-stage PCR. Genomic DNA sequencing was also performed on heterozygous sequence variants to confirm that identical sequence alterations were present in genomic DNA.

Methylation-specific PCR (MSP)

Because the gene ATP50 was apparently mutated, raising the possibility that it was a tumor suppressor gene, we evaluated this gene for alternative inactivation via promoter hypermethylation. MSP primer sequences of ATP50 for the methylated reaction were: forward (5′-CGAGTGGGAGC-GATTTAGGAC-3′) and reverse (5′-AACGC-CAAAATTACGACACG-3′), which amplify a 94-bp product. β-actin was selected as an internal control gene, using previously published MSP primers (Eads et al. 2001). CpGenome Universal Methylated DNA (Chemicon International, Inc., Temecula, CA) was used as a positive control. The detailed MSP procedure has been previously published (Sato et al. 2002).

Microsatellite instability (MSI) assay

MSI at each locus was determined by analyses of the length of each PCR-amplified microsatellite. MSI status was confirmed by MSI assays at five consensus loci (BAT25, BAT26, D2S123, D5S346, and D17S250) according to criteria from a National Cancer Institute workshop (Boland et al. 1998). Detailed procedures were as previously described (Mori et al. 2001).

Results

Project overview

A total of 603 genes (S-Table 1) were selected based on their length (under 1,000 bp) and their predicted expression in colorectal cancers according to the UCSC database. One thousand thirty-eight primer pairs (available on request) were designed to cover the entire ORFs (total bp, 366,687) of these 603 genes. Sequence data from 862 (83.0%) of these 1,038 primer sets were successfully analyzed, meaning that approximately 304,350 total bp were successfully sequenced (all primer sets for RT-PCR and cDNA sequencing are available on request).

S-Table-1.

Gene list

GenBank Accession No.	Gene Symbol	GenBank Accession No.	Gene Symbol	GenBank Accession No.	Gene Symbol
NM_000100.2	CSTB	NM_198057.1	TSC22D3	NM_002811.3	PSMD7
NM_000999.2	RPL38	NM_022652.2	DUSP6	NM_002812.3	PSMD8
NM_001827.1	CKS2	NM_021121.2	EEF1B2	NM_002813.4	PSMD9
NM_001863.3	COX6B1	NM_001412.2	EIF1AX	NM_170750.1	PSMD10
NM_001866.2	COX7B	NM_004094.3	EIF2S1	NM_004577.3	PSPH
NM_001867.2	COX7C	NM_001423.1	EMP1	NM_175847.1	PTBP1
NM_002489.2	NDUFA4	NM_001424.3	EMP2	NM_198974.1	PTK9
NM_002491.1	NDUFB3	NM_001425.1	EMP3	NM_002823.2	PTMA
NM_002966.1	S100A10	NM_207168.1	ENSA	NM_002824.4	PTMS
NM_003009.2	SEPW1	NM_001005915.1	ERBB3	NM_133377.1	RAD1
NM_003063.1	SLN	NM_001983.2	ERCC1	NM_153824.1	PYCR1
NM_003095.1	SNRPF	NM_001984.1	ESD	NM_000320.1	QDPR
NM_003133.1	SRP9	NM_001439.1	EXTL2	NM_004161.3	RAB1A
NM_003498.3	SNN	NM_001997.2	FAU	NM_004162.3	RAB5A
NM_003746.1	DYNLL1	NM_005247.2	FGF3	NM_002868.2	RAB5B
NM_003860.2	BANF1	NM_002007.1	FGF4	NM_198896.1	RAB6A
NM_003945.3	ATP6V0E	NM_002010.1	FGF9	NM_002870.2	RAB13
NM_004045.2	ATOX1	NM_023108.1	FGFR1	NM_183235.1	RAB27A
NM_004485.2	GNG4	NM_001449.3	FHL1	NM_201434.1	RAB5C
NM_004541.2	NDUFA1	NM_054014.1	FKBP1A	NM_198829.1	RAC1
NM_004772.1	C5orf13	NM_002013.2	FKBP3	NM_002872.3	RAC2
NM_005274.1	GNG5	NM_016725.1	FOLR1	NM_133630.1	RAD51L3
NM_005517.2	HMGN2	NM_004477.1	FRG1	NM_002881.2	RALB
NM_005694.1	COX17	NM_000146.2	FTL	NM_006325.2	RAN
NM_005770.3	SERF2	NM_198903.1	GABRG2	NM_002884.1	RAP1A
NM_005887.1	DLEU1	NM_000166.2	GJB1	NM_015646.3	RAP1B
NM_005949.1	MT1F	NM_024009.2	GJB3	NM_032626.5	RBBP6
NM_005954.2	MT3	NM_002061.2	GCLM	NM_181558.1	RFC3
NM_005978.3	S100A2	NM_006708.1	GLO1	NM_181578.1	RFC5
NM_006156.1	NEDD8	NM_002066.1	GML	NM_134427.1	RGS3
NM_006274.2	CCL19	NM_016592.1	GNAS	NM_005614.2	RHEB
NM_006304.1	SHFM1	NM_005301.2	GPR35	NM_000326.3	RLBP1
NM_006353.2	HMGN4	NM_002083.2	GPX2	NM_002938.2	RNF4
NM_006698.2	BLCAP	NM_002084.2	GPX3	NM_183045.1	RNF6
NM_006829.2	C10orf116	NM_203506.1	GRB2	NM_002946.3	RPA2
NM_007233.1	TP53AP1	NM_001512.2	GSTA4	NM_002947.3	RPA3
NM_007281.1	SCRG1	NM_147149.1	GSTM4	NM_033301.1	RPL8
NM_012456.1	TIMM10	NM_145871.1	GSTZ1	NM_033251.1	RPL13
NM_012458.2	TIMM13	NM_004492.1	GTF2A2	NM_000985.2	RPL17
NM_012460.2	TIMM9	NM_002095.3	GTF2E2	NM_000984.3	RPL23A
NM_013332.1	HIG2	NM_000858.3	GUK1	NM_000992.2	RPL29
NM_013343.1	LOH3CR2A	NM_005318.2	H1F0	NM_001001.3	RPL36AL
NM_014041.1	SPCS1	NM_002106.3	H2AFZ	NM_021029.3	RPL36A
NM_014051.2	TMEM14A	NM_005324.3	H3F3B	NM_001002.3	RPLP0
NM_014221.1	MTCP1	NM_005326.3	HAGH	NM_002949.2	MRPL12
NM_014356.2	C6orf123	NM_005327.1	HADHSC	NM_001007.3	RPS4X
NM_014445.2	SERP1	NM_005330.3	HBE1	NM_001015.3	RPS11
NM_014624.3	S100A6	NM_004494.1	HDGF	NM_001019.3	RPS15A
NM_014792.2	KIAA0125	NM_139011.1	HFE	NM_001020.3	RPS16
NM_016096.2	ZNF706	NM_005340.3	HINT1	NM_001022.3	RPS19
NM_016305.1	SS18L2	NM_002118.3	HLA-DMB	NM_001023.2	RPS20
NM_016565.2	CHCHD8	NM_002128.3	HMGB1	NM_001025.3	RPS23
NM_020142.3	LOC56901	NM_002129.2	HMGB2	NM_002960.1	S100A3
NM_020179.1	FN5	NM_004965.6	HMGN1	NM_005620.1	S100A11
NM_020181.1	C14orf162	NM_002131.2	HMGA1	NM_000664.3	ACACA
NM_020248.1	CTNNBIP1	NM_173158.1	NR4A1	NM_198970.1	AES
NM_020408.3	C6orf149	NM_002136.1	HNRPA1	NM_001636.1	SLC25A6
NM_021104.1	RPL41	NM_031314.1	HNRPC	NM_001001787.1	ATP1B1
NM_021127.1	PMAIP1	NM_002138.3	HNRPD	NM_001687.4	ATP5D
NM_021177.3	LSM2	NM_021644.2	HNRPH3	NM_001002256.1	ATP5G3
NM_023937.2	MRPL34	NM_006896.2	HOXA7	NM_004047.2	ATP6V0B
NM_031286.2	SH3BGRL3	NM_153715.1	HOXA10	NM_198589.1	BSG
NM_031287.2	SF3B5	NM_156037.1	HOXB6	NM_004927.2	MRPL49
NM_032412.2	ORF1-FL49	NM_004502.2	HOXB7	NM_006136.2	CAPZA2
NM_032574.1	LOC84661	NM_024016.2	HOXB8	NM_022845.2	CBFB
NM_032747.1	USMG5	NM_014620.2	HOXC4	NM_001760.2	CCND3
NM_052871.2	MGC4677	NM_153693.1	HOXC6	NM_171827.1	p32/CD8A
NM_052971.1	LEAP-2	NM_006897.1	HOXC9	NM_001773.1	CD34
NM_080677.1	DYNLL2	NM_014212.2	HOXC11	NM_000611.4	CD59
NM_138448.2	ACYP2	NM_024501.1	HOXD1	NM_001780.3	CD63
NM_139286.3	CDC26	NM_134421.1	HPCAL1	NM_004359.1	CDC34
NM_194327.1	GALIG	NM_182638.1	HPS1	NM_058197.2	p16/CDKN2A
NM_198835.1	ACACA	NM_005524.2	HES1	NM_001280.1	CIRBP
NM_020115.3	ACRV1	NM_198431.1	HSPA4	NM_001833.1	CLTA
NM_001124.1	ADM	NM_001540.2	HSPB1	NM_022645.2	CSH2
NM_000674.1	ADORA1	NM_005528.1	DNAJC4	NM_012140.3	SLC25A10
NM_000676.2	ADORA2B	NM_181353.1	ID1	NM_148979.1	CTSH
NM_001630.1	ANXA8	NM_002166.4	ID2	NM_000396.2	CTSK
NM_001154.2	ANXA5	NM_174856.1	IDH3B	NM_001336.2	CTSZ
NM_080649.1	APEX1	NM_004508.2	IDI1	NM_001915.2	CYB561
NM_000041.2	APOE	NM_005533.2	IFI35	NM_004418.2	DUSP2
NM_152876.1	FAS	NM_021068.1	IFNA4	NM_004427.2	PHC2
NM_000486.3	AQP2	NM_000612.2	IGF2	NM_001970.3	EIF5A
NM_053286.1	AQP6	NM_001552.1	IGFBP4	NM_001419.2	ELAVL1
NM_001659.1	ARF3	NM_000576.2	IL1B	NM_198194.1	STOM
NM_001660.2	ARF4	NM_172200.1	IL15RA	NM_202001.1	ERCC1
NM_001663.2	ARF6	NM_005536.2	IMPA1	NM_023110.1	FGFR1
NM_001664.2	RHOA	NM_014214.1	IMPA2	NM_201557.1	FHL2
NM_004040.2	RHOB	NM_198219.1	ING1	NM_004468.3	FHL3
NM_175744.3	RHOC	NM_198337.1	INSIG1	NM_057092.1	FKBP2
NM_005168.2	RND3	NM_002198.1	IRF1	NM_016730.1	FOLR1
NM_001665.2	rho G	NM_004030.1	IRF7	NM_004477.1	FRG1
NM_004309.3	ARHGDIA	NM_181493.1	ITPA	NM_002032.1	FTH1
NM_001177.3	ARL1	NM_002228.3	JUN	NM_002035.1	FVT1
NM_004311.2	ARL3	NM_002231.2	CD82	NM_001487.1	BLOC1S1
NM_004314.1	ART1	NM_004137.2	KCNMB1	NM_004483.3	GCSH
NM_032468.2	ASPH	NM_033360.2	KRAS	NM_004124.2	GMFB
NM_005171.2	ATF1	NM_002295.2	RPSA	NM_000581.2	GPX1
NM_004024.2	ATF3	NM_005563.3	STMN1	NM_002085.1	GPX4
NM_001677.3	ATP1B1	NM_005564.2	LCN2	NM_147148.1	GSTM4
NM_001679.2	ATP1B3	NM_005566.1	LDHA	NM_002095.3	GTF2E2
NM_001001977.1	ATP5E	NM_201544.1	LGALS8	NM_002107.3	H3F3A
NM_001002015.1	ATP5F1	NM_004987.3	LIMS1	NM_005342.1	HMGB3
NM_005175.2	ATP5G1	NM_005574.2	LMO2	NM_002133.1	HMOX1
NM_001002258.1	ATP5G3	NM_002346.1	LY6E	NM_002134.2	HMOX2
NM_001003701.1	ATP5J	NM_002353.1	TACSTD2	NM_156036.1	HOXB6
NM_001694.2	ATP6V0C	NM_014220.1	TM4SF1	NM_024017.3	HOXB9
NM_001697.2	ATP5O	NM_002354.1	TACSTD1	NM_000194.1	HPRT1
NM_004322.2	BAD	NM_030885.2	MAP4	NM_005343.2	HRAS
NM_053056.1	CCND1	NM_203378.1	MB	NM_174856.1	IDH3B
NM_138578.1	BCL2L1	NM_002386.2	MC1R	NM_000628.3	IL10RB
NM_004050.2	BCL2L2	NM_182763.1	MCL1	NM_181431.1	FOXK2
NM_000713.1	BLVRB	NM_012328.1	DNAJB9	NM_181468.1	ITGB4BP
NM_005180.5	PCGF4	NM_005370.4	RAB8A	NM_201543.1	LGALS8
NM_004331.2	BNIP3L	NM_177524.1	MEST	NM_002359.2	MAFG
NM_032515.3	BOK	NM_005371.3	METTL1	NM_004528.2	MGST3
NM_004332.1	BPHL	NM_017459.1	MFAP2	NM_022792.2	MMP19
NM_007306.1	BRCA1	NM_145791.1	MGST1	NM_002448.1	MSX1
NM_198590.1	BSG	NM_002413.3	MGST2	NM_005962.3	MXI1
NM_001207.3	BTF3	NM_002414.3	CD99	NM_079424.1	MYL6
NM_001731.1	BTG1	NM_002415.1	MIF	NM_032104.1	PPP1R12B
NM_007311.2	BZRP	NM_022791.2	MMP19	NM_004547.4	NDUFB4
NM_172369.1	C1QG	NM_002434.1	MPG	NM_182739.1	NDUFB6
NM_001217.2	CA11	NM_021126.3	MPST	NM_005005.1	NDUFB9
NM_000387.3	SLC25A20	NM_012331.2	MSRA	NM_020529.1	NFKBIA
NM_006888.2	CALM1	NM_002451.3	MTAP	NM_002520.4	NPM1
NM_005184.1	CALM3	NM_015675.1	GADD45B	NM_002607.2	PDGFA
NM_005185.2	CALML3	NM_001002841.1	(MYL4	NM_005022.2	PFN1
NM_001745.2	CAMLG	NM_079423.1	MYL6	NM_000942.4	PPIB
NM_001003962.1	CAPNS1	NM_002478.3	MYOD1	NM_206873.1	PPP1CA
NM_004346.2	CASP3	NM_032103.1	PPP1R12B	NM_183079.1	PRNP
NM_001755.2	CBFB	NM_005594.2	NACA	NM_002765.2	PRPS2
NM_004059.3	CCBL1	NM_182744.1	NBL1	NM_145888.1	KLK10
NM_001759.2	CCND2	NM_014222.2	NDUFA8	NM_002790.2	PSMA5
NM_199246.1	CCNG1	NM_004548.1	NDUFB10	NM_152255.1	PSMA7
NM_001239.2	CCNH	NM_004549.3	NDUFC2	NM_176783.1	PSME1
NM_001763.1	CD1A	NM_002496.1	NDUFS8	NM_183236.1	RAB27A
NM_000733.2	CD3E	NM_181827.1	NF2	NM_004583.2	RAB5C
NM_001769.2	CD9	NM_001001716.1	NFKBIB	NM_133629.1	RAD51L3
NM_005191.2	CD80	NM_005008.2	NHP2L1	NM_021033.4	RAP2A
NM_152942.1	TNFRSF8	NM_198175.1	NM23A/NME1	NM_002899.2	RBP1
NM_001244.2	TNFSF8	NM_000904.1	NQO2	NM_000976.2	RPL12
NM_001001392.1	CD44	NM_000270.1	NP	NM_001016.2	RPS12
NM_198793.1	CD47	NM_199185.1	NPM1	NM_000331.2	SAA1
NM_000560.2	CD53	NM_006172.1	NPPA	NM_005981.3	TSPAN31
NM_203330.1	CD59	NM_002524.2	NRAS	NM_002970.1	SAT
NM_004357.3	CD151	NM_004559.2	YBX1	NM_006745.2	SC4MOL
NM_001786.2	CDC2	NM_007105.1	SLC22A18AS	NM_006746.3	SCML1
NM_033534.1	CDC2L2	NM_005602.4	CLDN11	NM_001037.3	SCN1B
NM_052827.1	CDK2	NM_175568.1	P2RX4	NM_003000.1	SDHB
NM_000075.2	CDK4	NM_175081.1	P2RX5	NM_183352.1	SEC13L1
NM_078467.1	CDKN1A	NM_002567.2	PBP	NM_014563.2	TRAPPC2
NM_004064.2	CDKN1B	NM_002573.2	PAFAH1B3	NM_003016.2	SFRS2
NM_000077.3	CDKN2A	NM_181696.1	PRDX1	NM_152235.1	SFRS8
NM_078626.1	CDKN2C	NM_000281.2	TCF1/PCBD1	NM_004593.1	SFRS10
NM_005195.2	CEBPD	NM_032403.1	PCDHGC3	NM_173217.1	ST6GAL1
NM_001806.2	CEBPG	NM_002592.2	PCNA	NM_170679.1	SKP1A
NM_001809.2	CENPA	NM_033023.1	PDGFA	NM_005984.1	SLC25A1
NM_004365.2	CETN3	NM_002608.1	PDGFB	NM_022875.1	SMN2
NM_005507.2	CFL1	NM_213612.1	SLC25A3	NM_004596.3	SNRPA
NM_001817.1	CEACAM4	NM_002642.3	PIGC	NM_198216.1	SNRPB
NM_152253.1	CHKB	NM_002648.2	PIM1	NM_198220.1	SNRPB2
NM_013324.4	CISH	NM_006224.2	PITPNA	NM_177542.1	SNRPD2
NM_001281.2	CKAP1	NM_002653.3	PITX1	NM_004175.3	SNRPD3
NM_001284.2	AP3S1	NM_000929.1	PLA2G5	NM_022807.2	SNRPN
NM_001288.4	CLIC1	NM_001005376.1	PLAUR	NM_000454.4	SOD1
NM_001291.2	CLK2	NM_021910.1	FXYD3	NM_006943.2	SOX12
NM_001293.1	CLNS1A	NM_021105.1	PLSCR1	NM_001047.1	SRD5A1
NM_007097.2	CLTB	NM_153321.1	PMP22	NM_003132.1	SRM
NM_004368.2	CNN2	NM_174930.2	PMS2L5	NM_003135.1	SRP19
NM_007310.1	COMT	NM_001003686.1	PMS2L3	NM_003144.2	SSR1
NM_001300.3	KLF6	NM_022716.1	PRRX1	NM_003155.1	STC1
NM_001861.2	OX4I1	NM_002696.1	POLR2G	NM_177528.1	SULT1A2
NM_001305.3	CLDN4	NM_006232.2	POLR2H	NM_004177.3	STX3A
NM_001306.2	CLDN3	NM_006233.4	POLR2I	NM_004604.3	STX4A
NM_001307.3	CLDN7	NM_021129.2	PPA1	NM_003164.2	STX5A
NM_001878.2	CRABP2	NM_203430.1	PPIA	NM_177534.1	SULT1A1
NM_004379.2	CREB1	NM_000943.4	PPIC	NM_003166.2	SULT1A3
NM_001310.2	CREBL2	NM_177951.1	PPM1A	NM_181491.1	SURF5
NM_181571.1	CREM	NM_177969.1	PPM1B	NM_014231.3	VAMP1
NM_005206.3	CRK	NM_206877.1	PPP1CB	NM_014232.1	VAMP2
NM_005207.2	CRKL	NM_002710.1	PPP1CC	NM_005638.3	SYBL1
NM_001889.2	CRYZ	NM_006241.3	PPP1R2	NM_006754.2	SYPL1
NM_139014.1	MAPK14	NM_002715.1	PPP2CA	NM_003187.3	TAF9
NM_177436.1	CSE1L	NM_178002.1	PR 53/PPP2R4	NM_005643.2	TAF11
NM_022644.2	CSH2	NM_000945.3	PPP3R1	NM_172208.1	TAPBP
NM_177560.2	CSNK2A1	NM_005399.3	PRKAB2	NM_134324.1	TARBP2
NM_001320.5	CSNK2B	NM_207578.1	PRKACB	NM_201437.1	TCEA1
NM_001321.1	CSRP2	NM_212461.1	PRKAG1	NM_213648.1	TCF7
NM_000396.2	CTSK	NM_138981.1	MAPK10	NM_181738.1	PRDX2
NM_004394.1	DAP	NM_002756.2	MAP2K3	NM_201443.1	TEAD4
NM_020548.4	DBI	NM_002764.2	PRPS1	NM_003201.1	TFAM
NM_001924.2	GADD45A	NM_139277.1	KLK7	NM_174886.1	TGIF
NM_004083.4	DDIT3	NM_002774.2	KLK6	NM_003255.3	TIMP2
NM_001355.2	DDT	NM_213633.1	PSG4	NM_003270.2	TSPAN6
NM_030655.2	DDX11	NM_203287.1	PSG11	NM_003271.3	TSPAN4
NM_213566.1	DFFA	NM_148976.1	PSMA1	NM_021137.3	TNFAIP1
NM_000791.2	DHFR	NM_002789.3	PSMA4	NM_000363.3	TNNI3
NM_007326.1	CYB5R3	NM_002791.1	PSMA6	NM_005079.1	TPD52
NM_138281.1	DLX4	NM_002794.3	PSMB2	NM_003287.2	TPD52L1
NM_203316.1	DPAGT1	NM_002801.2	PSMB10	NM_199362.1	TPD52L2

Sequence variants

Seventy-eight sequence variants within 50 genes were found among the 603 genes studied (Table 1) (S-Table 2 for detailed information). Thus, the frequency of sequence variants was 1 per 3,902 bp (78 total variants/304,350 total bp). Of these 78 sequence alterations, 51 were homozygous (26 synonymous, 25 non-synonymous) and 27 were heterozygous (11 synonymous, 16 non-synonymous). All sequence alterations were detected in both colorectal cancer tissue and matched normal colonic epithelium, with the exception of an alteration in the gene ATP50 (NM_001697), which manifested a unique expression mechanism (Fig. 1). Forty-four sequence alterations had been previously reported, but 34 sequence alterations were completely novel, having never been reported in the SNP database at The National Center for Biotechnology Information (NCBI).

Table 1.

Sequence variants

	Homozygous		Heterozygous
	Synonymous	Non-synonymous	Synonymous	Non-synonymous
Number of alterations	26	25	11	16

S-Table 2.

Sequence variants

		Homozygous alteration				Heterozygous alteration
GenBank Accession No.	Gene Symbol	Synonymous alteration	NCBI SNP Database	Non-Synonymous alteration	NCBI SNP Database	Synonymous alteration	NCBI SNP Database	Non-Synonymous alteration	NCBI SNP Database
NM_000320	QDPR Leu132Leu	G396A,	rs2597775
NM_000331	SAA1			C209T, Ala70Val	rs1136743
				T224C, Val75Ala	rs1136747
NM_001007	RPS4X Leu164Leu	G492A,	rs7580
NM_001020	RPS16 Gly5Gly	C15T,	rs17626
	T27G, Ser9Ser		rs17628
NM_001047	SRD5A1					A309AG, Pro103Pro	rs3822430
						G348GA, Ala116Ala	rs8192186
NM_001320	CSNK2B Tyr46Tyr	T138C,	rs14365
NM_001636	SLC25A6 Phe136Phe	T408C,	rs7205
NM_001697	ATP50 Gly36Gly	T108C,	rs17728665	A218AG, Lys73Arg	*
NM_001760	CCND3							T775TG, Ser259Ala	rs1051130
NM_001817	CEACAM4			T668A, Val223Glu.	*
NM_001861	COX4I1							G7GA, Ala3Thr	rs17855751
NM_001889	CRYZ Gly18Gly	G54A,	rs4650284			T138C, Gly46Gly	*
NM_002131	HMGA1 Ser2Ser	T6C,	*	G49A, Glu17Lys	*
	G78T, Arg26Arg	*		G112A, Gly38Arg	*
	C255A, Gly85Gly	*	Pro48Leu	C143T,	*
			C217T, Arg73Gly	*
			C236T, A237G,	*
			Pro79Leu G286A,	*
			Glu96Lys
NM_002136	HNRPA1 Gly248Gly	C744T,		*
NM_002414	CD99					C369CT, Ala121Ala	rs4575010
NM_002642	PIGC Gly89Gly	T267C,	rs2230471
NM_002813	PSMD9			T50C, Val17Ala	rs2230681
NM_003144	SSR1			C388T, His130Tyr	*
NM_003255	TIMP2					G303GA, Ser101Ser	rs2277698
NM_004064	CDKN1B							T326TG, Val109Gly	rs2066827
NM_004137	KCNMB1							G193GA, Glu65Lys	rs11739136
NM_004175	SNRPD3 Ala101Ala	T303C,	rs3176991
NM_004365	CETN3							G28GC, Val10Leu	rs4873
NM_004468	FHL3 Pro180Pro	G540A,	rs7366048
NM_004549	NDUFC2							C136CG, Leu46Val	rs8875
NM_005171	ATF1					C327CT, Tyr109Tyr	rs1129406
NM_005191	CD80					G135GA,	rs2228017
					Val45Val
NM_005301	GPR35							G85GA, Ala29Thr. A880AC,	*rs3749172
NM_005342	HMGB3 Asn186Lys	C558G,	*					Ser294Arg
NM_005594	NACA					T543TA, Ile181Ile	rs4788
NM_005984	SLC25A1 Lys277Lys	A831G,	*
NM_006353	HMGN4 Gly66Gly	G198A	rs4871
NM_006896	HOXA7 Ala32Ala	T96G,	rs2301720	G52A, Ala18Thr	rs2301721
NM_007310	COMT His12His	C36T,	rs4633	G322A, Val108Met	rs4680
NM_007311	BZRP His53Arg			A158G, Ala68Ala	rs6971	G204GA,	rs6972
NM_012328	DNAJB9 Pro61Pro	G183A,	rs1043615
NM_013332	HIG2 Glu28Glu	A84G,	*
NM_014212	HOXC11 Ser12Ser	T36G,	rs4759315
NM_014232	VAMP2			T346A, Ser116Thr	*
NM_021068	IFNA4							A146AC,	*
							His49Pro G178GC,	*
							Gly60Arg T190TA,	*
							Phe64Ile G187GC,		rs3203576
NM_024009	GJB3						Glu63Gln C357CT,	*
						Asn119Asn
NM_033251	RPL13			G334A, Ala112Thr	rs9930567
NM_052871	MGC4677			G28T, Ala10Ser	rs28673896	C12CT, Thr4Thr	*	C109CT, Arg37Cys	*
NM_058197	CDKN2A	Arg54Gly		A160T,			*
NM_145888	KLK10	A318C, Gly106Gly	rs2075688	T347C, Leu116Pro			*
		C336G, Thr112Thr	rs1061368
		G423A, Leu141Leu	rs2075689		`
NM_172200	IL15RA			C248T, Pro83Leu	`		*
				A337C, Thr113Pro			*
NM_172369	C1QG	Gly215Glu		G644A,			*
NM_181571	CREM	Ile137Thr		T410C,			*
NM_198970	TEAD4			Pro194Leu				C580CT,	*
NM_201544	LGALS8			Met56Val.				A166AG,	rs1041937
				G542GC, Gly181Ala	*

No report found.

Figure 1.

cDNA sequencing of ATP50. Two different alteration sites were detected. At the 108th nucleotide, colorectal cancer tissue had only a mutant cytosine nucleotide, while normal colon contained both a thymine (wild) and a cytosine (mutant). Both codons GGT and GGC encoded glycine (synonymous alteration). At the 218th mucleotide, colorectal cancer tissue had only a mutant guanine nucleotide, while normal tissue contained both an adenine (wild) and a guanine (mutant). AAA encoded lysine and AGA encoded arginine (non-synonymous alteration). Gly, glycine; Arg, arginine.

Tumor-specific regulation of gene expression

Tumor-specific regulation of gene expression was found for NM_001697 (ATP50, Homo sapiens ATP synthase, H+ transporting, mitochondrial F1 complex, O subunit). The sequence alterations T108C (GGT to GGC, homozygous, Gly36Gly) and A218G (AAA to AAG, homozygous, Lys73Arg) were observed only in cancer-derived cDNA, while the alterations T108TC (CGT and GGC, heterozygous, 36Gly) and A218AG (AAA and AAG, heterozygous, 73Lys and 73 Arg) were observed in cDNA from normal epithelium. Surprisingly, both T108TC (CGT and GGC, heterozygous, 36Gly) and A218AG (AAA and AAG, heterozygous, 73Lys and 73 Arg), which were identical to the two alterations observed in normal cDNA, were observed in genomic DNA from both cancer and normal tissue (Figures 2, 3). This result implied that the cancer exhibited monoallelic expression from the variant allele of ATP50, while the normal epithelium manifested biallelic heterozygous expression, i.e. from both the reported normal allele and our discovered variant mutant allele simultaneously.

Figure 2.

Representative result of cDNA sequencing of ATP50 in normal and cancer tissues. The red box in the left panel shows the 36th codon, while the red box in the right panel shows the 73rd codon. All alterations were confirmed by both forward and reverse sequencing.

Figure 3.

Genomic DNA sequencing of ATP50 in normal and cancer tissues. Upper panels, forward sequencing; lower panels, reverse sequencing. The red boxes show the 36th and 73rd codons. Both normal and colorectal cancer tissues contain identical heterozygosities at the 108th and 218th nucleotides.

MSP

One possible mechanism for monoallelic expression observed for ATP50 was DNA methylation of its promoter region. MSP showed, however, that there was no methylation of the ATP50 promoter in colorectal cancer (S-Fig. 1).

Somatic mutations

There were no somatic mutations found among the 603 genes studied or within the p53 gene.

MSI status

MSI assays showed that there was no microsatellite instability in genomic DNA (S-Fig. 2).

Discussion

In the current study, we assumed that if a mutant protein was involved in carcinogenesis or tumor progression, this mutant would be expressed and therefore detectable in tumor mRNA. i.e. we assumed that somatic mutations involved in carcinogenesis or tumor progression would be detectable by direct cDNA sequencing. By using this strategy, we avoided the need for sequencing each exon of genomic DNA, reasoning that genes which are never expressed in normal or malignant colon probably do not participate in colorectal carcinogenesis. We discovered 78 sequence variants (44 of which had been previously reported as single-nucleotide polymorphisms, but 34 of which had never been reported) among the 603 genes (304,350 bp of ORFs) studied.

Recently, Sjoblom T. et al. performed genome-wide sequencing in breast and colorectal cancers, revealing that an average of 52 mutations occurred in each colorectal cancer(Sjoblom et al. 2006). According to the article by Sjoblom et al. the somatic mutation frequency in colon cancers was 3.2 somatic mutations/Mb, on average (Table 1 of their paper). Therefore, the probability of our finding zero somatic mutations among the 603 genes (304,350 bp) that we studied was 37.76% (please see formula below), suggesting that our findings were statistically quite consistent with Sjoblom’s results:

The Sjoblom team also defined “CAN-genes” (candidate cancer genes) as those that were frequently mutated in colorectal cancers, and found that 69 genes could be included in this category. Although the CAN-genes KRAS, GNAS and TP53 were studied by us, no somatic mutations were found in these genes. Furthermore, in addition to the genes mentioned above, NRAS, HRAS, p16, and p27 were included in the current study, but these genes also contained no somatic mutations. Finally, results of MSI assays revealed MS-stability (MSS), implying an absence of mutations in the major DNA mismatch repair genes (although these genes were not studied due to their long ORFs). It is possible that other molecular pathogenetic pathways were involved in this colorectal tumorigenesis, such as those containing APC, MCC, DCC, or the TGF-β cascade: these genes were also not examined in the current study due to ORF length.

Approximately 24,000,000 bp among the entire genomic DNA sequence are reported as ORFs in the UCSC database. The average density of each SNP is once per 1.9 kilobases (i.e. 1,419,190 SNPs/2.7 gigabases of human genome sequence)(Sachidanandam et al. 2001). We sequenced 304,350 bp of ORFs (viz., 1.26% of the total ORFs in the UCSC database: 304,350 bp/24,000,000 bp) and discovered 78 sequence variants, yielding a frequency of 1 alteration per 3,902 bp (78/304,350 bp). Our observed sequence variant distribution may provide a basis with which to estimate the number of SNPs in a single individual with colon cancer. That is, the SNPs reported above are one possible subset of the entire database; there is no guarantee that a given individual will always harbor all SNPs in the database.

The human ATP50 gene (X83218, NM_001697), encoding a 213-amino acid ATP synthase OSCP subunit, is a key structural component of the stalk of the mitochondrial respiratory chain F1F0-ATP synthase, which is a vital element in the cellular pathway of energy conversion (Senior, 1988). Although a mutant strain of yeast in which the delta subunit of F1F0-ATP synthase had been inactivated by insertional mutagenesis showed little or no ATPase activity(Giraud and Velours, 1994), and dysfunction of ATP synthase can cause a variety of degenerative diseases(Wallace, 1994), there have been no previous reports detailing a relationship between ATP synthase and tumorigenesis. We found restricted monoallelic (i.e. monoallelically silenced) expression of an altered allele from ATP50 in our colon cancer tissue, which would be expected to exert the same effect as would a somatic mutation of this gene. Genomic DNA sequencing of ATP50 revealed that this monoallelic expression was not due to LOH. We therefore studied the methylation status of the CpG island in the promoter region of ATP50 by MSP, but we found no methylation of this region. Other epigenetic mechanisms, such as histone deacetylation, might have contributed to monoallelic expression of ATP50. There was no monoallelic expression of ATP50 in 20 cancer cell lines that we examined. Although monoallelic expression of this altered ATP50 allele may be involved in a subset of colorectal cancers, further study is required to clarify the potential functional role of this gene in carcinogenesis.

This study poses several advantages as well as limitations. Firstly, it has been reported that some synonymous mutations may influence the stability of mRNA(Duan and Antezana, 2003; Chamary and Hurst, 2005) because they affect the thermodynamic stability of mRNA secondary structures (Fitch, 1974; Klambt, 1975). Nonsense-mediated mRNA decay (NMD) is also known as a surveillance pathway that rapidly degrades mRNAs containing premature termination codons(Culbertson and Leeds, 2003; Amrani et al. 2006). These mechanisms may cause instability of mRNA, accelerate the degradation of mRNA, and consequently result in difficulty in detecting sequence alterations by cDNA sequencing. Since we used cDNA as our starting material for sequencing, we may have ignored some key genes because of RNA degradation. Nevertheless, many sequence variants were detected reasonably well in the current study, suggesting that degradation of mRNA occurred rarely, if at all, as a consequence of sequence alterations. Instead, we considered it more important to increase our chances of finding sequence alterations by using cDNA rather than genomic DNA because of the lower cost, time, and labor involved in sequencing cDNA, as well as the increased relevance of only studying genes that are expressed in the colon.

Secondly, it is conceivable that we lost some gene sequence information due to extremely low expression levels. Therefore, we employed two-stage PCR to increase our chances of successful sequencing, thereby achieving a relatively high success rate of 862/1,038 reactions, or 83.0%. Possibly, this result still may have included genes that were not expressed in our particular colorectal cancer, even though we used the UCSC database to select genes that were purportedly expressed in colorectal cancers. Our sequencing success rate appears favorable when compared to genomic DNA sequencing, where 92% of genes were successfully analyzed (Wang et al. 2004). The total number of exons sequenced in our study was 2107, implying that at least 2107 primer pairs would have been necessary to conduct this study had it been attempted by genomic DNA sequencing; in contrast, we accomplished this task using only 1038 primer sets for cDNA sequencing. This contrast demonstrates that our method is useful to explore mutations because it is not only more cost-effective, but also less demanding in time and labor.