Analysis of polymorphisms in genes associated with the FA/BRCA pathway in three patients with multiple primary malignant neoplasms

Figures & data

Figure 1. Analytical process for genetic susceptibility of three patients.

Figure 2. Clinical information and pedigree of three cases of MPMNs. There are three parts, the left side is the site of the disease of three multiple primary cancer patients, which are represented by the 3 D body graph, and the arrow points to the lower right, which is the histogram of the survival time of each patient. The survival time is the period from the diagnosis of the tumour to death or end of follow-up. The end of follow-up of all three patients in this study was 6 November 2017. Corresponding to the upper right is the pedigree for each of the patient.

Table 1. Clinical information of three cases of MPMNs.

Sample	Tumor types	Order	Treatments	Status	Onset time	Survival time(month)
Patient 1	Endometrial cancer	1^st cancer	Surgery + chemotherapy	alive	Aug-1994	279
	Ureter cancer	2^nd cancer	Surgery + chemotherapy	alive	Apr-2005	151
	Bladder cancer	3^rd cancer	Surgery + chemotherapy	alive	Jan-2006	142
	Colon cancer	4^th cancer	Surgery + chemotherapy (including targeted therapy) + endocrine therapy	alive	Apr-2008	115
	Breast cancer	5^th cancer	Surgery + chemotherapy	alive	Oct-2013	49
Patient 2	Endometrial cancer	1^st cancer	Surgery alone	alive	Aug-2009	99
	Ovarian cancer	2^nd cancer	Surgery + chemotherapy + perfusion therapy	alive	Aug-2009	99
	Kidney cancer	3^rd cancer	Surgery + chemotherapy	alive	Aug-2009	99
	Breast cancer	4^th cancer	Surgery + chemotherapy (including targeted therapy) + endocrine therapy	alive	Nov-2013	48
Patient 3	Breast cancer	1^st cancer	Surgery + chemotherapy + radiotherapy + endocrine therapy	alive	Jun-1995	269
	Colon cancer	2^nd cancer	Surgery + chemotherapy	alive	Aug-2008	111
	Ovarian cancer	3^rd cancer	Surgery + chemotherapy + perfusion therapy	alive	Nov-2010	84

Table 2. The quality of sequencing data.

Sample	Raw data (G)	Effective (%)	Error (%)	Q20 (%)	Q30 (%)	GC (%)
Patient1	90.13	99.71	0.02;0.06	96.35;91.22	91.67;82.04	42.61;42.46
		99.71	0.02;0.06	96.45;92.42	91.59;83.67	42.61;42.46
		99.68	0.02;0.05	96.76;92.66	92.34;84.26	42.65;42.50
		99.77	0.02;0.05	96.33;92.53	91.38;83.97	42.66;42.50
Patient2	94.16	99.09	0.02;0.06	96.45;91.13	91.89;81.96	42.08;41.99
		99.17	0.02;0.06	96.55;92.33	91.84;83.58	42.07;41.99
		99.05	0.02;0.05	96.85;92.57	92.57;84.17	42.13;42.04
		99.27	0.02;0.05	96.43;92.41	91.64;83.82	42.12;42.03
Patient3	107.31	98.8	0.02;0.06	96.40;91.34	91.75;82.22	42.46;42.35
		98.93	0.02;0.05	96.51;92.55	91.71;83.85	42.45;42.34
		98.78	0.02;0.05	96.83;92.80	92.47;84.48	42.52;42.42
		99.03	0.02;0.05	96.38;92.63	91.49;84.12	42.49;42.36

Figure 3. Gel electropherogram and the quality of sample DNA for Sequencing.

Figure 4. Quality evaluation of sequencing data. The upper graph indicates the coverage depth (left coordinate) and coverage (right coordinate) of each chromosome. The abscissa indicates the chromosome number, the left ordinate indicates the average coverage depth, and the right ordinate indicates the coverage. When calculating the depth of coverage for each chromosome, the formula is: Sequencing data volume/The total length of per chromosome. When calculating coverage, the formula is: the total length covered/the total length of each chromosome. The two images below show the depth of sequencing. The left panel shows the base ratios for different sequencing depths, the abscissa indicates the depth of sequencing, and the ordinate indicates the proportion of bases with a depth of x in all bases; the image is generally distributed around the average depth. The graph on the right shows the cumulative base ratio at different depths. The abscissa indicates the depth of sequencing. The ordinate indicates the proportion of bases with a depth greater than x in all bases. For example, the ratio of bases with a depth of 0X corresponds to 100%. Indicates that there are 100% bases with a sequencing depth greater than 0X.The results show that the coverage of the sequencing data is over 99.8% and the depth is about 30×.

Table 3. The mutation test of sequencing data.

		Samples
Mutation		Patient1	Patient2	Patient2
SNV	All	3091100	3093895	3097412
	Novel	40715	40878	40578
InDeL	All	395488	401784	406744
	Novel	278644	283835	288337
CNV	Loss	94	120	79
	Gain	122	89	140
SV	Deletion	1205	1183	1356
	Insertion	46	13	33
	Inversion	84	85	106
	Translocation	321	363	487

Table 4. List of polymorphism selected from three samples.

Sample	Chromosome	Genome location	Gene	Region	HGVS number	Amino acid change	Homozygous and Heterozygous	Variation type	1000 Genomes frequency	Rs number
Patient 1	17	59763347	BRIP1	Exonic	c.T898C	p.S300P	Homozygous	Missense	0.67	rs4986764
	9	35074965	FANCG	Exonic	c.A821G	p.K274R	Heterozygous	Missense	0	rs935074968/TFEC
	8	90965508	NBN	Exonic	c.C1809A	p.F603L	Heterozygous	Missense	0.0014	rs192236678
	17	63534302	AXIN2	Exonic	c.T1219C	p.S407P	Heterozygous	Missense	0.01	rs116931989
	2	31751329	SRD5A2	Exonic	c.C699G	p.F233L	Heterozygous	Missense	0.0014	rs9332966
	19	33792752	CEBPA	Exonic	c.568_569insCGCACC	p.S190delinsSHP	Heterozygous	insertion	0	.
Patient 2	10	88635779	BMPR1A	Exonic	c.C4A	p.P2T	Heterozygous	Missense	0.45	rs11528010
	3	10084828	FANCD2	Exonic	c.G983A	p.R328Q	Heterozygous	Missense	0.0032	rs35625434
	14	75513534	MLH3	Exonic	c.C2825T	p.T942I	Heterozygous	Missense	0.02	rs17102999
	13	32899249	BRCA2	Exonic	c.G353A	p.R118H	Heterozygous	Missense	0	rs80358603
	14	45628296	FANCM	Splicing	TTATATATATATATA/TTATATATATATA	–	Heterozygous	deletion	0	.
	14	45654415	FANCM	Splicing	TCTTACTTA/TCTTA	–	Heterozygous	deletion	0	.
Patient 3	11	64575505	MEN1	Exonic	c.G512A	p.R171Q	Heterozygous	Missense	0.01	rs607969
	11	108121673	ATM	Exonic	c.G1481A	p.G494D	Heterozygous	Missense	0	.
	5	80021361	MSH3	Exonic	c.C1430T	p.A477V	Heterozygous	Missense	0	rs580725542
	17	41244976	BRCA1	Exonic	c.C2572T	p.Q858X	Heterozygous	Nonsense	0	.
	2	58392880	FANCL	Exonic	c.A493G	p.T165A	Heterozygous	Missense	0.0041	rs149731356
	19	33792773	CEBPA	Exonic	c.546_548del	p.182_183del	Heterozygous	deletion	0	.
	16	89818581	FANCA	Exonic	c.C289T	p.R97C	Heterozygous	Missense	0	rs142377616

Figure 5. The patient-function network analysis. The circle represents the function of the variant gene. The more overlapping the function, the larger the circle.

Table 5. The functional analysis of crucial genes.

ID	Description	BgRatio	p value	p.adjust	q value	geneID	Count
GO:0042626	ATPase activity, coupled to transmembrane movement of substances	114/17354	6.51E-08	2.31E-06	1.03E-06	CEBPA/BMPR1A/MLH3/BRCA2/FANCD2	5
GO:0016820	Hydrolase activity, acting on acid anhydrides, catalyzing transmembrane movement of substances	116/17354	7.10E-08	2.31E-06	1.03E-06	CEBPA/BMPR1A/MLH3/BRCA2/FANCD2	5
GO:0015399	Primary active transmembrane transporter activity	123/17354	9.53E-08	2.31E-06	1.03E-06	CEBPA/BMPR1A/MLH3/BRCA2/FANCD2	5
GO:0015405	P-P-bond-hydrolysis-driven transmembrane transporter activity	123/17354	9.53E-08	2.31E-06	1.03E-06	CEBPA/BMPR1A/MLH3/BRCA2/FANCD2	5
GO:0043492	ATPase activity, coupled to movement of substances	129/17354	1.21E-07	2.35E-06	1.04E-06	CEBPA/BMPR1A/MLH3/BRCA2/FANCD2	5
GO:0016887	ATPase activity	436/17354	2.38E-06	3.84E-05	1.71E-05	CEBPA/BMPR1A/MLH3/BRCA2/FANCD2/FANCM	6
GO:0030145	Manganese ion binding	51/17354	1.58E-05	0.000218864	9.74E-05	MEN1/MSH3/BRCA1	3
GO:0042623	ATPase activity, coupled	357/17354	1.81E-05	0.000219261	9.76E-05	CEBPA/BMPR1A/MLH3/BRCA2/FANCD2	5
GO:0022804	Active transmembrane transporter activity	367/17354	2.07E-05	0.000222638	9.91E-05	CEBPA/BMPR1A/MLH3/BRCA2/FANCD2	5
GO:0004012	Phospholipid-translocating ATPase activity	20/17354	0.000169841	0.00164746	0.000732999	CEBPA/FANCD2	2
GO:0003785	Actin monomer binding	27/17354	0.000312496	0.002654882	0.001181228	MEN1/MSH3	2
GO:0005319	Lipid transporter activity	141/17354	0.000328439	0.002654882	0.001181228	CEBPA/MLH3/FANCD2	3
GO:0001784	Phosphotyrosine residue binding	35/17354	0.000527295	0.003934433	0.001750534	MEN1/MSH3	2
GO:0045309	Protein phosphorylated amino acid binding	44/17354	0.000834021	0.005778576	0.002571043	MEN1/MSH3	2
GO:0004715	Non-membrane spanning protein tyrosine kinase activity	51/17354	0.001119557	0.007239799	0.00322118	MEN1/MSH3	2
GO:0005548	Phospholipid transporter activity	56/17354	0.001348362	0.008174447	0.00363703	CEBPA/FANCD2	2
GO:0051219	Phosphoprotein binding	77/17354	0.00253112	0.014442271	0.006425753	CEBPA/MEN1	2
GO:0019905	Syntaxin binding	83/17354	0.002933605	0.01580887	0.007033789	CEBPA/MEN1	2
GO:0000149	SNARE binding	119/17354	0.005928601	0.03026707	0.013466629	CEBPA/MEN1	2
GO:0051015	Actin filament binding	156/17354	0.00999434	0.048146684	0.021421748	MEN1/MSH3	2
GO:0008201	Heparin binding	160/17354	0.010491032	0.048146684	0.021421748	BRIP1/ATM	2
GO:0002161	Aminoacyl-tRNA editing activity	12/17354	0.011695774	0.048146684	0.021421748	NBN	1
GO:0038191	Neuropilin binding	12/17354	0.011695774	0.048146684	0.021421748	MEN1	1
GO:0016874	Ligase activity	171/17354	0.011912582	0.048146684	0.021421748	NBN/FANCA	2

Figure 6. KEGG pathway of three samples. The top three graphs show the KEGG pathway for each patient. The graph at the bottom left shows network of all the post-screening variations involved. Overall, all variations were detected in genes involved in the FA/BRCA pathway to form a complex network system.

Table 6. KEGG pathway of crucial genes in three samples.

Sample name	Pathway ID	Pathway description	Observed gene count	Matching proteins in network	Matching proteins in network	The function of proteins
Patient 1	3460	Fanconi anemia pathway	2	ENSP00000259008	BRIP1	BRIP1 acts late in the Fanconi anemia pathway, after FANCD2 ubiquitination. Involved in the repair of DNA double-strand breaks by homologous recombination in a manner that depends on its association with BRCA1.
				ENSP00000367910	FANCG	FANCG is a DNA repair protein that may operate in a postreplication repair or a cell cycle checkpoint function. May be implicated in interstrand DNA cross-link repair and in the maintenance of normal chromosome stability.
Patient 2	3460	Fanconi anemia pathway	3	ENSP00000267430	BRCA2	BRCA2 involved in double-strand break repair and/or homologous recombination. recombinational DNA repair by promoting assembly of RAD51 onto single-stranded DNA (ssDNA).
				ENSP00000287647	FANCD2	FANCD2 involved in the repair of DNA double-strand breaks, both by homologous recombination and single-strand annealing. May participate in S phase and G2 phase checkpoint activation upon DNA damage. Plays a role in preventing breakage and loss of missegregating chromatin at the end of cell division, particularly after replication stress.
				ENSP00000369497	FANCM	ATPase required for FANCD2 ubiquitination, a key reaction in DNA repair.
Patient 3	3460	Fanconi anemia pathway	3	ENSP00000373952	BRCA1	BRCA1 plays a central role in DNA repair by facilitating cellular responses to DNA damage.The BRCA1-BARD1 heterodimer coordinates a diverse range of cellular pathways such as DNA damage repair, ubiquitination and transcriptional regulation to maintain genomic stability.
				ENSP00000385021	FANCA	DNA repair protein that may operate in a postreplication repair or a cell cycle checkpoint function. May be involved in interstrand DNA cross-link repair and in the maintenance of normal chromosome stability.
				ENSP00000418960	FANCL	Mediates monoubiquitination of FANCD2, a key step in the DNA damage pathway. Also mediates monoubiquitination of FANCI.
Patient 3	5202	Transcriptional misregulation in cancer	3	ENSP00000278616	ATM	Acting as a DNA damage sensor. regulating DNA damage response mechanism. Also plays a role in pre-B cell allelic exclusion。
				ENSP00000337088	CEBPA	Transcription factor that coordinates proliferation arrest and the differentiation of myeloid progenitors, adipocytes, hepatocytes, and cells of the lung and the placenta.
				ENSP00000427514	MEN1	A complex that specifically methylates 'Lys-4' of histone H3 (H3K4). Functions as a transcriptional regulator.

Data availability

All data generated or analysed during this study are included in this published article