In silico identification and in vitro expression analysis of breast cancer-related m⁶A-SNPs

Figures & data

Figure 1. Flow chart of study design and the main results. IBS: Iberian Population in Spain from 1000 Genomes Project.

Table 1. List of identified m⁶A-SNPs associated with BC that presented cis-eQTL effect.

m^6A-SNP	CHR^a	Position^b	Gene	Mutation type	Strand	Proteins bound	Motifs changed	m^6A ID
rs1801270	6	36,684,194	CDKN1A	Missense	+	POL2 and ZBTB33	Ascl2, ELF1 HEN1, and Sin3AK-20	m6A_ID_71975
rs9379084	6	7,231,610	RREB1	Missense	+	No	VDR	m6A_ID_72448
rs76563149	5	178,730,572	ZNF354A	5’-UTR^c	-	POL2, SIN3AK20, POL24H8, TAF1, E2F1, HAE2F1, TR4, CCNT2, HEY1, HMGN3, and ZNF263	EBF and Ets	m6A_ID_187781
rs11614913	12	53,991,815	MIR196A2	miRNA^d	+	No	HMG-IY	m6A_ID_358107

^aCHR: chromosome; ^b Assembly: GRCh38.p13; ^c 5’-UTR: 5’-untranslated region; ^d miRNA: microRNA

Table 2. Alleles and genotypes frequencies for the four m⁶A-SNPs in different populations. AFR: African; AMR: American; EAS: East Asian; EUR: European; and SAS: South Asian.

		Frequency of alleles and genotypes
m^6A-SNP	Alleles/Genotypes	AFR	AMR	EAS	EUR	SAS
rs1801270	AA + AC	0.526	0.421	0.716	0.127	0.262
	CC	0.474	0.579	0.284	0.873	0.738
	A	0.309	0.249	0.488	0.067	0.145
	C	0.691	0.751	0.512	0.933	0.885
rs9379084	AA + AG	0.012	0.216	0.284	0.247	0.202
	GG	0.988	0.784	0.716	0.753	0.798
	A	0.006	0.111	0.159	0.132	0.106
	G	0.994	0.889	0.841	0.868	0.894
rs76563149^a	TT + GT	0.156	0.412	0.357	0.508	0.593
	GG	0.844	0.588	0.643	0.492	0.407
	T	0.081	0.235	0.201	0.298	0.368
	G	0.919	0.765	0.799	0.702	0.632
rs11614913	CC + TC	0.977	0.838	0.714	0.817	0.930
	TT	0.023	0.162	0.286	0.183	0.070
	C	0.860	0.611	0.458	0.589	0.742
	T	0.140	0.389	0.542	0.411	0.258

^aThe variants for this m⁶A-SNP in the mRNA are A or C.

Figure 2. A graphical representation of the miRNA structure obtained from the UNAFold Web Server (http://www.unafold.org/mfold/applications/rna-folding-form.php) [52]. The m⁶A-SNP rs11614913 (C/U) is found inside the red circle and it may influence the m⁶A modification of the complete DRACH motif (green boxes) and incomplete one (blue box). (a) Representation of the miRNA structure with the variant C for the m⁶A-SNP rs11614913. (b) Representation of the miRNA structure with the variant U for the m⁶A-SNP rs11614913.

Table 3. Alleles and genotypes frequencies and χ² test for the patients with BC and female from the IBS population. We took values of p < 0.05 as significant (indicated in bold in the table below).

		Frequencies
m^6A-SNP/Gene	Alleles/Genotypes	Controls (n = 53)	BC patients (n = 35)	χ^2	P-value
rs1801270 CDKN1A	AA + AC	0.170	0.114	0.516	0.472
	CC	0.830	0.886
	A	0.085	0.086	<0.001	0.985
	C	0.915	0.914
rs76563149^a ZNF354A	TT + GT	0.453	0.657	3.536	0.060
	GG	0.547	0.343
	T	0.245	0.400	4.745	0.029
	G	0.755	0.600
rs11614913 MIR196A2	CC + CT	0.830	0.800	0.129	0.719
	TT	0.170	0.200
	C	0.642	0.657	0.045	0.832
	T	0.358	0.343

^aThe variants for this m⁶A-SNP in the mRNA are A or C.

Table 4. Average of relative gene expression for the m⁶A-SNP rs76563149 localized in ZNF354A gene with its standard deviation (SD) in the tumour breast tissue and its corresponding adjacent normal breast tissue and the p-values obtained for the Mann–Whitney U and Kruskal–Wallis H tests. We took p-values of <0.05 as significant, except in the Kruskal–Wallis H test, where a p-value of <0.005 was considered as significant (indicated in bold in the table below).

		Luminal A	Luminal B HER2-	Luminal B HER2+	HER2+	TNBC
Average ± SD	Normal tissue	1.016 ± 0.191	1.151 ± 0.645	1.032 ± 0.270	1.274 ± 1.161	1.037 ± 0.304
	Tumour tissue	0.924 ± 0.657	0.752 ± 0.167	0.492 ± 0.163	0.622 ± 0.468	1.156 ± 1.064
Mann–Whitney U test (p)		0.180	0.180	0.001	0.109	0.222
Kruskal–Wallis H test (p)		0.117

Figure 3. Relative expression levels of the ZNF354A gene. The fragment analysed contains the m⁶A-SNP rs76563149. Gene expression between each tumour subtype and its corresponding adjacent normal tissues: (a) TNBC. (b) Luminal A. (c) Luminal B HER2 +. (d) Luminal B HER2-. (e) HER2 +. (f) Gene expression between the different tumour subtypes. * P-value <0.05. The circles are outlier.

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Data Availability Statement

The data that support the findings of this study are available from the corresponding authors, MMP and FO, upon request.