Candidate colorectal cancer predisposing gene variants in Chinese early-onset and familial cases

Table 1 Clinical characteristics and family histories of 23 early-onset and familial colorectal cancer patients

Patient ID	Gender	Patient's history	Family history
43-1A	Female	RC at 37 yr	Brother RC at 53 yr
43-2A	Male	RC at 53 yr	Sister RC at 37 yr
49-4A	Male	CC at 30 yr	Brother CC at 43 yr; sister CC at 23 yr
49-5A	Female	CC at 23 yr	Brother CC at 43 yr; brother CC at 30 yr
50-11A	Male	CC at 34 yr and relapse at 36	Father CRC at 35 yr and death at 52 yr; brother CC at 34 yr and death at 36 yr
54-2A	Female	RC at 44 yr	Sister CRC; Brother CC at 76 yr and death
66-1-1A	Female	CC at 47 yr	Sister CC at 51 yr
71A	Female	RC at 57 yr	Sister RC at 53 yr
77-1A	Female	CRC at 38 yr	Father EC at 64 yr and death; uncle CRC at 68 yr and death
102-1A	Male	RC at 25 yr
103-1A	Male	CC at 53 yr	Brother CC at 36 yr and death at 48 yr; mother IO at 63 yr and death
106-2A	Male	JC at 34 yr, CC at 39 yr, KC at 44 yr and PC at 45 yr	Father EC and death; mother RC at 42 yr and death; Sister CP
108-1A	Male	RC at 33 yr
110-1A	Male	CC at 36 yr
116-1A	Female	CC at 31 yr and HC at 57 yr	Brother intussusception and death at 40 yr; Brother CC at 50 yr, RC and SMT at 58 yr; brother IC at 50 yr, CC at 53 yr and RC at 61 yr; sister GC at 56 yr
120-1A	Female	RC at 36 yr
142-1A	Male	RC at 34 yr
149-1A	Male	CRC at 31 yr	Father EC and death, mother GC at 56 yr
154-1A	Female	CRC at 40 yr	Father HC, RC and death at 57 yr
156-1A	Female	CRC at 54 yr	Sister CP at 54 yr; sister CP; mother CC at 48 yr; grandfather EC and death.
164-1A	Male	CC at 30 yr	Uncle colonitls at 42 yr
165-1A	Male	CRC at 43 yr	Sister RC at 31 yr and death; grandmother RC at 65 yr and death.
180-1	Male	CRC at 40 yr	Sister CP at 46 yr

CRC: Colorectal cancer; CC: Colon cancer; RC: Rectal cancer; IC: Ileocecus carcinoma; IO: Intestinal obstruction; JC: Jejunum cancer; KC: Kidney cancer; PC: Pulmonary carcinoma; CP: Colonic polyps; HC: Hepatic carcinoma; GC: Gastric cancer; EC: Esophageal cancer; SMT: Splenic metastatic tumors.

Table 2 Alignment and coverage statistics for 23 early-onset and familial colorectal cancer patients

Sample ID	Total reads	Total mapped	Reads mapped to genome	Covered≥4×	Covered≥10×	Covered≥20×	Average target coverage
43-1A	62997602	52130593	45528212	93.30%	85.80%	74.30%	55.88×
43-2A	57099664	50367772	43924906	93.60%	86.40%	75.00%	54.94×
49-4A	67025248	51978393	45418701	93.30%	85.70%	74.10%	55.30×
49-5A	60632336	51598017	45450431	92.60%	85.00%	73.40%	55.49×
50-11A	68991044	58507454	51033221	94.00%	87.20%	76.80%	60.71×
54-2A	68459832	57860336	50820626	93.40%	86.50%	76.10%	61.71×
66-1-1A	69759994	58838035	51472112	94.10%	87.60%	77.50%	61.82×
71A	68055130	58181783	51012277	94.00%	87.60%	77.70%	61.50×
77-1A	65956248	56894265	49817369	93.80%	87.30%	77.10%	61.06×
102-1A	64702600	57086284	49672873	94.40%	87.90%	77.70%	59.92×
103-1A	66004146	55109962	48218769	93.80%	86.90%	76.40%	59.28×
106-2A	61956558	54367359	47567033	93.80%	86.80%	76.00%	57.97×
108-1A	64764180	56469665	49473520	94.00%	87.20%	76.50%	57.52×
110-1A	68883264	56962439	49975545	94.10%	87.30%	76.90%	59.31×
116-1A	68975484	60681318	53305706	93.70%	86.70%	75.60%	55.86×
120-1A	64307066	56593051	49900259	94.20%	87.30%	75.90%	53.02×
142-1A	72999930	65321752	57822754	94.70%	87.60%	76.20%	53.19×
149-1A	69636008	59789305	52641740	93.90%	87.20%	76.90%	61.19×
154-1A	80632788	63934448	56196297	94.40%	88.30%	78.90%	64.69×
156-1A	94340904	82125696	73199086	94.20%	87.10%	76.10%	56.48×
164-1A	67813680	58837471	51779826	93.60%	86.50%	75.80%	58.83×
165-1A	68657326	59845292	52561646	94.30%	87.80%	77.70%	60.99×
180-1	65727112	57908057	50742938	94.40%	87.90%	77.50%	59.01×
Average	68190354	58321250	51197211	93.90%	87.03%	76.35%	58.51×

Table 3 Prioritization scheme for exome data analysis of all 23 patients

Type of prioritization filter	Remaining variants (n)
All variants	1106642
Coding region and canonical splice site variants after quality filtering (total ≥ 10 reads, ≥ 5 variant reads and ≥ 20% variant reads)	13819
Non-synonymous variants, canonical splice site variants	9833
Variants that result in alterations in protein function (protein truncation, splice site defects and missense mutations at highly conserved (phyloP ≥ 3.0) nucleotide positions.Not in in-house database and MAF ≤ 0.001 in dbSNPv138	44321
	2883
Variants in known CRC predisposing genes and genes likely to play a role in CRC development (MAF ≤ 0.001 in ESP and 700 control Chinese exome data sets)	61
Variants/genes validated by Sanger sequencing	39 (32 different variants in 23 genes)

¹Including 172 nonsense variants, 188 frame shift variants, 943 canonical splice site variants, 237 in-frame deletions, 191 in-frame insertions and 2701 missense variants with highly conserved (phyloP ≥ 3.0); In-house database: 1302 in-house analyzed exomes, mostly from European ancestry. MAF: Minor allele frequency; ESP: Exome Sequencing Project database (6503 exomes, http://evs.gs.washington.edu/EVS/); 700 control Chinese exome data sets: Chinese subjects with Han ancestry (Juan Tian and Zhi-Min Feng, BGI, personal communication).

Table 4 Identification of germline mutations in known colorectal cancer predisposing genes

Sample ID	Gene name	Gene ID	Genomic change	cDNA change	Protein change	Pathogenicity
43-1A	MLH1	NM_000249	g.chr3:37053590G>A	c.677G>A1	p.Arg226Glna	Yes[38,44]
106-2A	MLH1	NM_000249	chr3:g.37048555G>T	c.453+1G>T	SSM	Yes[42]
116-1A	MLH1	NM_000249	g.chr3:37042445G>A	c.208-1G>A	SSM	Yes[43]
180-1	MUTYH	NM_001128425	g.chr1:45797972G>A	c.799C>T	p.Gln267*	Yes[39]
180-1	MUTYH	NM_001128425	g.chr1:45797914C>T	c.857G>A	p.Gly286Glu	Yes[40]
49-4A	MLH1	NM_000249	g.chr3:37067252_37067253insT	c.1163_1164insT	p.Arg389Profs*6	NR
49-5A	MLH1	NM_000249	g.chr3:37067252_37067253insT	c.1163_1164insT	p.Arg389Profs*6	NR
49-4A	MSH6	NM_000179	g.chr2:48027422C>G	c.2300C>G	p.Thr767Ser	NR
50-11A	MSH2	NM_000251	g.chr2:47641406A>T	c.793-2A>T	SSM	NR

¹This substitution results in a complete loss of exon 8 of MLH1 by RNA analysis[38]. NR: Not reported; SSM: Splice site mutation.

Table 5 Characteristics of 24 variants identified in 19 novel genes likely to play a role in colorectal cancer development

Sample ID	Gene name	Gene/pathway involved	cDNA change	Protein change	rs ID in dbSNP138	MAF (700 Chinese exomes)	MAF (NHLBI ESP)	MAF (1000 genome)
102-1A	ATM	Cancer gene, DNArep	c.1402_1403del	p.Lys468Glufs*18	NR	NR	NR	NR
66-1-1A	PARP1	DNArep	c.758dup	p.Lys254Glufs*6	NR	NR	0.000077	NR
66-1-1A	MAX	Cancer gene	c.181del	p.Leu61Serfs*15	NR	NR	NR	NR
106-2A	BUB1	Cancer gene	c.46C>T	p.Gln16*	NR	NR	NR	NR
149-1A	BUB1	Cancer gene	c.2844del	p.Gln949Argfs*3	NR	NR	NR	NR
165-1A	LIG3	DNArep	c.218del	p.Phe73Serfs*41	NR	NR	NR	NR
54-2A	MCC	Transposon studies	c.1355+1_1355+2ins14	SMM	NR	NR	NR	NR
49-4A	EIF2AK4	GWAS related	c.2214_2215insCGACGA	p.Glu738_Asp739insArgArg	NR	NR	NR	NR
71A	EIF2AK4	GWAS related	c.2214_2215insCGACGA	p.Glu738_Asp739insArgArg	NR	NR	NR	NR
103-1A	EIF2AK4	GWAS related	c.2214_2215insCGACGA	p.Glu738_Asp739insArgArg	NR	NR	NR	NR
108-1A	EIF2AK4	GWAS related	c.2214_2215insCGACGA	p.Glu738_Asp739insArgArg	NR	NR	NR	NR
120-1A	EIF2AK4	GWAS related	c.2214_2215insCGACGA	p.Glu738_Asp739insArgArg	NR	NR	NR	NR
154-1A	EIF2AK4	GWAS related	c.2214_2215insCGACGA	p.Glu738_Asp739insArgArg	NR	NR	NR	NR
164-1A	EIF2AK4	GWAS related	c.2214_2215insCGACGA	p.Glu738_Asp739insArgArg	NR	NR	NR	NR
77-1A	LRP5	WNT	c.2156A>G	p.Tyr719Cys	NR	NR	NR	NR
43-1A	LRP5	WNT	c.3536G>A	p.Arg1179His	NR	NR	0.000077	NR
54-2A	LRP5	WNT	c.3919C>T	p.Arg1307Trp	NR	NR	0.000077	NR
110-1A	RPS6KB2	PI3K/AKT	c.331A>G	p.Lys111Glu	NR	0.00075	NR	NR
43-1A	RPS6KB2	PI3K/AKT	c.683C>A	p.Thr228Asn	rs183360785	NR	NR	0.001
43-1A	RYR2	Somatic mutation gene	c.2701G>A	p.Gly901Ser	NR	NR	NR	NR
103-1A	RYR2	Somatic mutation gene	c.6457A>G	p.Lys2153Glu	NR	NR	NR	NR
102-1A	RYR3	Somatic mutation gene	c.13507G>A	p.Val4503Met	NR	NR	NR	NR
71A	ETV4	Cancer gene	c.991G>A	p.Glu331Lys	NR	NR	NR	NR
103-1A	PRDM1	Cancer gene	c.1499A>G	p.Gln500Arg	rs201512476	NR	NR	0.001
164-1A	TSC2	Cancer gene, PI3K/AKT	c.5200G>A	p.Asp1734Asn	NR	NR	NR	NR
71A	MTOR	PI3K/AKT	c.5857G>T	p.Val1953Leu	NR	0.000714	NR	NR
154-1A	DAAM1	WNT	c.667G>A	p.Val223Met	NR	NR	NR	NR
71A	FZD10	WNT	c.1341C>G	p.Phe447Leu	NR	NR	NR	NR
164-1A	TCF7	WNT	c.572G>T	p.Arg191Met	NR	NR	NR	NR
71A	MAST2	Transposon studies	c.3482A>G	p.Asn1161Ser	NR	NR	0.000077	NR

MAF: Minor allele frequency; NR: Not reported; DNArep: DNA repair pathway; WNT: WNT signaling pathway; SSM: Splice site mutation.

Table 6 In silico functional prediction of 16 missense variants

Sample ID	Gene name	Gene/pathway involved	cDNA change	Protein change	Domain	PhyloP score	Grantham score	Align GVGD	SIFT score	SIFT prediction	Polyphen2 score	Polyphen2 prediction
77-1A	LRP5	WNT	c.2156A>G	p.Tyr719Cys	LDLR class B repeat	4.751	194	C65	0.000	D	0.999	PrD
43-1A	LRP5	WNT	c.3536G>A	p.Arg1179His	LDLR class B repeat	3.712	29	C25	0.000	D	0.953	PrD
54-2A	LRP5	WNT	c.3919C>T	p.Arg1307Trp	LDLR class A repeat	3.172	101	C35	0.000	D	0.948	PrD
110-1A	RPS6KB2	PI3K/AKT	c.331A>G	p.Lys111Glu	Protein kinase, catalytic domain	4.639	56	C55	0.000	D	0.535	PoD
43-1A	RPS6KB2	PI3K/AKT	c.683C>A	p.Thr228Asn	Protein kinase, catalytic domain	5.062	65	C55	0.001	D	0.994	PrD
43-1A	RYR2	Somatic mutation gene	c.2701G>A	p.Gly901Ser	Ryanodine receptor	6.081	56	C55	0.010	D	1.000	PrD
103-1A	RYR2	Somatic mutation gene	c.6457A>G	p.Lys2153Glu	Intracellular calcium-release channel	5.067	56	C0	0.020	D	0.615	PoD
102-1A	RYR3	Somatic mutation gene	c.13507G>A	p.Val4503Met	Ryanodine Receptor TM 4-6	6.012	21	C15	0.000	D	1.000	PrD
71A	ETV4	Cancer gene	c.991G>A	p.Glu331Lys	PEA3-type ETS-domain transcription factor, N-terminal	6.424	56	C55	0.001	D	0.862	PoD
103-1A	PRDM1	Cancer gene	c.1499A>G	p.Gln500Arg	Zinc finger, C2H2	4.875	43	C0	0.050	D	0.570	PoD
164-1A	TSC2	Cancer gene, PI3K/AKT	c.5200G>A	p.Asp1734Asn	Rap/ran-GAP	5.538	23	C0	0.000	D	0.998	PrD
71A	MTOR	PI3K/AKT	c.5857G>T	p.Val1953Leu	PIK-related kinase	5.634	32	C0	0.001	D	0.827	PoD
154-1A	DAAM1	WNT	c.667G>A	p.Val223Met	Diaphanous GTPase-binding	6.347	21	C0	0.000	D	0.998	PrD
71A	FZD10	WNT	c.1341C>G	p.Phe447Leu	Frizzled protein	4.229	22	C15	0.000	D	0.984	PrD
164-1A	TCF7	WNT	c.572G>T	p.Arg191Met	High mobility group, HMG1/HMG2	4.202	91	C65	0.000	D	0.999	PrD
71A	MAST2	Transposon studies	c.3482A>G	p.Asn1161Ser	PDZ/DHR/GLGF	4.854	46	C0	0.000	D	0.999	PrD

D: Damaging; PoD: Possibly damaging; PrD: Probably damaging.