Polymorphic variations in the FANCA gene in high‐risk non‐BRCA1/2 breast cancer individuals from the French Canadian population

Nadhir Litim; Yvan Labrie; Sylvie Desjardins; Geneviève Ouellette; Karine Plourde; Pascal Belleau; INHERIT BRCAs; Francine Durocher

doi:10.1016/j.molonc.2012.08.002

. 2012 Sep 11;7(1):85–100. doi: 10.1016/j.molonc.2012.08.002

Polymorphic variations in the FANCA gene in high‐risk non‐BRCA1/2 breast cancer individuals from the French Canadian population

Nadhir Litim ^1,^†, Yvan Labrie ^1,^†, Sylvie Desjardins ¹, Geneviève Ouellette ¹, Karine Plourde ¹, Pascal Belleau ¹; INHERIT BRCAs^1,^‡, Francine Durocher ^1,^✉

PMCID: PMC5528405 PMID: 23021409

Abstract

The majority of genes associated with breast cancer susceptibility, including BRCA1 and BRCA2 genes, are involved in DNA repair mechanisms. Moreover, among the genes recently associated with an increased susceptibility to breast cancer, four are Fanconi Anemia (FA) genes: FANCD1/BRCA2, FANCJ/BACH1/BRIP1, FANCN/PALB2 and FANCO/RAD51C. FANCA is implicated in DNA repair and has been shown to interact directly with BRCA1. It has been proposed that the formation of FANCA/G (dependent upon the phosphorylation of FANCA) and FANCB/L sub‐complexes altogether with FANCM, represent the initial step for DNA repair activation and subsequent formation of other sub‐complexes leading to ubiquitination of FANCD2 and FANCI. As only approximately 25% of inherited breast cancers are attributable to BRCA1/2 mutations, FANCA therefore becomes an attractive candidate for breast cancer susceptibility. We thus analyzed FANCA gene in 97 high‐risk French Canadian non‐BRCA1/2 breast cancer individuals by direct sequencing as well as in 95 healthy control individuals from the same population. Among a total of 85 sequence variants found in either or both series, 28 are coding variants and 19 of them are missense variations leading to amino acid change. Three of the amino acid changes, namely Thr561Met, Cys625Ser and particularly Ser1088Phe, which has been previously reported to be associated with FA, are predicted to be damaging by the SIFT and PolyPhen softwares. cDNA amplification revealed significant expression of 4 alternative splicing events (insertion of an intronic portion of intron 10, and the skipping of exons 11, 30 and 31). In silico analyzes of relevant genomic variants have been performed in order to identify potential variations involved in the expression of these spliced transcripts. Sequence variants in FANCA could therefore be potential spoilers of the Fanconi‐BRCA pathway and as a result, they could in turn have an impact in non‐BRCA1/2 breast cancer families.

Keywords: Breast cancer susceptibility, FANCA variants, Fanconi anemia, Alternative splicing

1. Introduction

Pathogenic germline mutations in BRCA1, BRCA2, TP53, ATM, CHEK2, BRIP1, PALB2 and RAD51C genes have been associated with an increased breast cancer risk and, together, are found in less than 25% of breast cancer families showing a clear pattern of inheritance (Stratton and Rahman, 2008; Wang, 2007; Moldovan and D'Andrea, 2009). Thus, it is clear that other susceptibility alleles remain to be identified to explain the increased susceptibility in the remnant high‐risk families. As the number and characteristics of such alleles are undetermined, a focussed candidate gene approach based on genes closely interacting with the known susceptibility genes (in particular BRCA1 and BRCA2) constitutes a study design of choice to identify rare moderate‐penetrance susceptibility alleles.

It is noteworthy that of the genes recently associated with an increased susceptibility to breast/ovarian cancer, four are Fanconi Anemia (FA) genes: FANCD1/BRCA2, FANCJ/BACH1/BRIP1, FANCN/PALB2, and FANCO/RAD51C (Somyajit et al., 2010; Cantor et al., 2001, 2004; Seal et al., 2006; Rahman et al., 2007; Erkko et al., 2007; Pang et al., 2011; Zheng et al., 2010; Meindl et al., 2010), therefore connecting FA proteins to homologous recombination repair (HR).

FANCA is the most frequently mutated gene in FA, representing 60–70% of the cases. FANCA is highly polymorphic, with over 350 unique mutations reported (Fanconi Mutation Database; http://www.rockefeller.edu/fanconi/mutate), including large genomic deletions mediated by the unusually high density of ALU repetitions found in its genomic sequence (Levran et al., 2005). For FANCA, an association of sequence alterations or altered expression have been suggested in some instances of ovarian cancer and leukemia (Thompson et al., 2005; Lensch et al., 2003; Tischkowitz et al., 2008). In addition, homozygous mutations of FANCA were also associated with esophageal cancer in Iranian population (Akbari et al., 2011).

FA proteins not only interact with each other but also work in a network of processes implicated in the maintenance of genome integrity during DNA replication and following some types of DNA damage. When activated, the FANCI/D2 complex associates with chromatin and colocalizes in DNA damage‐induced S‐phase foci with DNA repair response proteins such as BRCA1, FANCD1/BRCA2, FANCJ, RAD51, PCNA and NBS1 to form the complex II (Wang, 2007; Raschle et al., 2008; Knipscheer et al., 2009).

It has been proposed that the formation of FANCA/G (dependent upon the phosphorylation of FANCA) and FANCB/L subcomplexes altogether with FANCM, represents the initial step for DNA repair activation and for regulating the nuclear accumulation of FANCL, and therefore the subsequent formation of other subcomplexes and ubiquitination of FANCD2 and FANCI (Medhurst et al., 2006). It is worth mentioning that FANCA protein has been also shown to interact with ERCC4 (Sridharan et al., 2003), FANCC (Reuter et al., 2000), FANCF (de Winter et al., 2000), FANCE (Medhurst et al., 2001), BRCA1 (Folias et al., 2002) and SMARCA4 (Otsuki et al., 2001).

As a direct and constitutive interaction between FANCA and BRCA1 has been shown to occur in the cell (Folias et al., 2002), and the close connection between the FANC and BRCA1/2 proteins in DNA repair, we screened the proximal promoter, the coding sequence and intron–exon boundaries of the FANCA gene in a cohort of 97 BRCA1/2‐negative (BRCAX) breast cancer families from the French Canadian population as well as 95 healthy controls from the same origin for sequence variations or splicing variants that could modulate breast cancer risk.

2. Material and methods

2.1. Ascertainment of families and DNA purification

Recruitment of high‐risk French Canadian breast/ovarian families (i.e. families in which multiple cases of breast/ovarian cancer are present in close relatives – 3 cases in 1st or 4 cases in 2nd degree relatives – or with strong evidence of a familial component) was part of the major interdisciplinary research program INHERIT BRCAs further explained elsewhere, in which a major component was to identify and characterize BRCA1/2 mutations (Simard et al., 2007). A subset of 97 high‐risk French Canadian breast/ovarian cancer families was drawn from the initial study based on the absence of detectable BRCA1/2 mutation (so‐called BRCAX) and constituted the cohort used for another study specifically aiming at the identification of other susceptibility loci/genes to breast cancer. This latter study obtained ethics approvals from all participating institutions, and each participant, knowing their inconclusive BRCA1/2 test results signed a specific informed consent, had to be at least 18 years of age and mentally capable. One individual affected with breast cancer per family was selected for analysis, with a selection preference for the youngest subject available in the family. The ascertainment criteria of recruitment and BRCA1/2 status analysis have been described previously (Antoniou et al., 2006; Durocher et al., 2006). In all instances, diagnosis of breast cancer was confirmed by pathology reports.

A cohort of 95 healthy unrelated individuals from the French Canadian population was also included in the study. They were obtained from Dr Damian Labuda at the Centre de Cancérologie Charles Bruneau, Hôpital Ste‐Justine, Montreal, Canada. The individuals who provided these samples were recruited on a non‐nominative basis, in the framework of long‐term studies aiming the characterization of the genetic variability in human populations, approved by the Institutional Ethic Review Board. Blood samples were taken for each participant. Details of the clinical procedures and BRCA1/2 testing methodology are described elsewhere (Simard et al., 2007; Desjardins et al., 2008, 2009). Genomic DNA extraction of the 97 BRCAX breast cancer cases as well as 95 healthy individuals has been performed as previously described (Durocher et al., 2006).

Additional control genotyping data coming from the French Canadian population were extracted either from the CARTaGENE database (n = 140) or from the asthma familial collection of Saguenay–Lac‐Saint‐Jean (NorthEastern region of the province of Quebec in Canada) (n = 254 unrelated subjects with asthma frequency similar to general population, i.e. 10%). To assess whether the frequencies of the rare significant DNA variants (n = 4, namely c.‐1C/A (NA), c.894‐8A/G (rs1164881), c.1627‐32T/C (rs17226337) and c.3348 + 18A/G (rs1800347)) detected in our study might be caused by population stratification, these variant were genotyped in a second French Canadian cohort comprising 192 subjects selected at random from the CRCHUQ glaucoma DNA bank (Vincent Raymond).

2.2. RNA isolation from immortalized cell lines and cDNA synthesis

Lymphocytes were isolated and immortalized from 7 to 9 ml of blood samples using the Epstein–Barr Virus (EBV) in 15% RPMI media as previously described (Durocher et al., 2006). Total RNA was extracted from EBV‐transformed β‐lymphoblastoid cell lines and seven breast cancer cell lines obtained from the American Type Culture Collection (ATCC) including, two estrogen receptor (ER)‐negative breast cancer cell lines (BT‐20 and MDA ‐231), five ER + breast cancer cell lines (SUM‐40, CAMA‐1, MCF7, T47D and SKBR3) as well as the MCF‐10A human epithelial cell line which was used as control, using TRI REAGENT^® (Molecular Research Center, inc., Cincinnati, OH, USA) according to the manufacturer's instructions. The purified RNA was stored at −80 °C until use. Following RNA extraction, reverse transcription of 5 μg of RNA was performed as previously described (Durocher et al., 2006).

2.3. PCR amplification, mutation analysis and variant characterization

The coding and 5′UTR (comprising the proximal promoter region) sequences, including intron‐exon boundaries of the FANCA gene (NM_000135.2) was amplified and sequenced using primers listed in Supplemental Table 1. Sequencing of all 43 exons was performed on breast cancer cases and control individuals using the Big Dye 3.1 chemistry and loaded on an ABI3730XL automated sequencer according to the manufacturer's instructions (Applied Biosystems, Foster City, USA). Analysis of sequence data was done using the Staden package. Allelic frequency was evaluated in both series by means of a Chi² test. p‐values less than 0.05 were considered as significant. Protein and nucleotide sequence alignments with other species were performed using data extracted from the National Center for Biotechnology Information (NCBI) and UCSC databases.

2.4. In silico analyzes

The SIFT and PolyPhen web‐based softwares were used to predict the effect of amino acid substitution on protein structure (Ng and Henikoff, 2003; Sunyaev et al., 2001; Ramensky et al., 2002). Assessment of the putative deleterious effect of FANCA missense substitutions was also performed with the ALIGN‐GVGD algorithm (Tavtigian et al., 2006; Mathe et al., 2006) using a full‐length alignment of validated and predicted FANCA sequences (produced by aligning human FANCA exons to the genomic sequences) selected from the NCBI and Ensembl databases. The alignment was made using T‐Coffee (Notredame et al., 2000) and was followed by minor handmade adjustments.

The prediction of the effect of a given variant on exonic and intronic splicing modulators was assessed using the Human Splicing Finder (HSF) (Desmet et al., 2009) which contains all available matrices from auxiliary sequence prediction softwares such as ESEfinder 3.0 program (Cartegni et al., 2003; Smith et al., 2006) for binding sites of the 9G8, Tra2‐β and hnRNP A1 proteins. The putative impact of intronic variants on splice consensus sites was evaluated using Splice Site Prediction programs using Neural Networks (SSPNN) (Reese et al., 1997) with default parameters.

2.5. Alternative splicing analysis

Screening for evaluating alternative splicing events (ASEs) occurring in FANCA gene was performed by PCR amplification on cDNA on a subset of 10 breast cancer cases using the primers described below. Following screening of the whole cDNA sequence, targeted ASE amplification of cDNA regions covering exons 6–15 and 26–33 was performed on cDNA from BRCAX individuals and human cell lines using the following primers: Exons 6–15, Forward primer: 5′‐cattgtgagcctgcaagagctg‐3′ and Reverse primer: 5′‐cttcttgctgcagccatggtag‐3′; Exons 26–33, Forward primer: 5′‐agcctcctgacctgtaggacga‐3′ and Reverse primer: 5′‐gaagaactgctcgcatctggc‐3′.

2.5.1. QRT‐PCR assays

QRT‐PCR assays were performed on StepOnePlus™ Real‐Time PCR Systems. For all the assays, a reaction mixture was prepared in a final volume of 20 μl with Fast SYBR^® Green Master Mix (Applied Biosystems) which included 500 nM of each primer for FANCA wild‐type and alternative transcript variants. cDNA samples were reverse‐transcribed from total RNA. To perform fluorescent‐based real‐time PCR, the amount of cDNA used for quantitation was 20 ng of total RNA coming from the immortalized cell lines of the carriers of the variants, as well as ten wild type individuals. The primer sequences were; FANCA wild‐type exon 10–11: forward primer: 5′ cacggttgatgtactgcagagaa 3′, reverse primer: 5′ actgaacactccgaaccagca 3′; FANCA deletion of the exon 11 splice transcript: forward primer: 5′ cacggttgatgtactgcagagaa 3′, reverse primer: 5′ ctgaacagcatcagatgcagc 3′; FANCA insertion of the intronic fragment 10A splice transcript forward primer: 5′ cacggttgatgtactgcagagaa 3′, reverse primer: 5′ tcctcctcacgcacgttatcg 3′; FANCA wild‐type exon 30–31: forward primer: 5′ tccgagaggtgttgaaagagg 3′, reverse primer: 5′ ggtcataactccttgagctttgg 3′; FANCA deletion of the exon 30 splice transcript forward primer: 5′ tccgagaggtgttgaaagagg 3′, reverse primer: 5′ ggtcataactccttgagcgttca 3′; FANCA deletion of the exon 31 splice transcript forward primer: 5′ tccgagaggtgttgaaagagg 3′, reverse primer: 5′ ggtcagctaccatctcctttgg 3′. Expression analyzes were then carried out using the ΔΔCt method.

2.5.2. Polysome analysis

12x10⁷ cells were suspended in 3 volumes of RNAse‐free lysis buffer (250 mM sucrose solution, Cycloheximide 10 mg/ml, KCl 25 mM, MgCl2 5 mM, tris HCl 50 mM, DTT 2 mM) containing NP40 (0.5%), Roche protease tablet and phosphatase inhibitor 1×. The cells were homogenized in the solution and incubated 10 min on ice. The nuclei were pelleted by centrifugation at 750 g for 10 min at 4 °C. The supernatant was then centrifuged at 12,000 g for 10 min to pellet the mitochondria. The supernatant was loaded onto linear gradient of 15–45% sucrose (W/W) and centrifuged at 38,000 rpm for 2 h at 4 °C in Bekman SW‐41 Ti rotor. As a control, equivalent supernatants were prepared and centrifuged in sucrose gradient in buffers in which MgCl₂ was replaced by 10 mM EDTA. The absorbance of the gradients at 260 nm was determined and the gradients were fractionated manually. RNA extraction: SDS was added to a final concentration of 2% and proteinase K to a final concentration of 100 μg/ml to each sucrose gradient fraction for 1 h at 37 °C. An equal volume of phenol:chloroform (1:1) was then added to each fraction. The sucrose gradient fractions were concentrated by two precipitations with ethanol and dissolved in DEPC water.

2.6. Electronic databases

ESE Finder: http://rulai.cshl.edu/cgi‐bin/tools/ESE3/esefinder.cgi

Human Splicing Finder (HSF): http://www.umd.be/HSF

Splice Site Prediction Program using Neural Networks (SSPNN): (http://www.fruitfly.org/seq_tools/splice.html)

UCSC Genome Bioinformatics: http://genome.ucsc.edu/

SIFT: http://blocks.fhcrc.org/sift/SIFT.html

PolyPhen: http://genetics.bwh.harvard.edu/pph/

3. Results

Sequencing of FANCA genomic and complementary DNA of 97 BRCAX breast cancer subjects from high risk French Canadian breast/ovarian cancer families and 95 healthy controls led to the identification of sequence variants and spliced isoforms. In silico approaches provided significant knowledge concerning the potential impact of these sequence variants into splicing of exonic regions of transcripts and consequently into the potential variations involved in the expression of these spliced transcripts. The impact of a given variant in non‐BRCA1/2 breast cancer families and the potential disruption of the Fanconi‐BRCA pathway could thus justify this screening strategy.

3.1. Variant characterization

In order to perform the mutational analysis of FANCA, all 43 exons and adjacent intronic sequences were sequenced in our case and control datasets. In the 97 BRCAX breast cancer patients (1 per family) and 95 healthy individuals studied, we identified 85 sequence variants (Table 1). Among the twenty‐eight exonic variants identified, ten were found only in cases, while 5 others are found exclusively in healthy individuals. A high proportion (10 out of 28) of the exonic sequence variants were not reported in public databases or relevant publications. The c.796A/G (Thr266Ala) and c.1501G/A (Gly501Ser) are the coding variants whose corresponding frequencies show a significant difference of MAF between cases and controls with p‐values of 0.048 and 0.003, respectively. These variants are over‐represented in controls and both non‐synonymous substitutions show a high MAF >20% (Table 1). It is important to mention that to further support the frequency of the variants identified in our cohort, additional genotype data from two independent cohorts, both from the French Canadian control populations, were obtained (n = 254 and n = 140, respectively), for a total of 24 sequence variants. In addition, the four variants displaying a significant p‐value were genotyped in an additional distinct cohort of 192 control individuals, also of French Canadian origin. We were able to extract information on a total of 28 SNPs displaying a reasonable frequency for a total of 586 new control individuals.

Table 1.

Sequence variations in FANCA gene and genotype frequencies in familial breast cancer cases and controls.

SNP	SNP ID^a (dbSNP ID)	Location	Amino acid change	Series (N = )	Hom	Het	Rare hom	MAF^b	p‐value^c	Other cohorts
1	c.‐42‐100ins13 (rs36233534)	Promoter	‐	Cases (95)	59	31	5	0.216	0.146
1	c.‐42‐100ins13 (rs36233534)	Promoter	‐	Controls (93)	48	38	7	0.280
2	c.‐42‐76 G/C (NA)	Promoter	‐	Cases (95)	95	0	0	0	0.311
2	c.‐42‐76 G/C (NA)	Promoter	‐	Controls (93)	92	1	0	0.005
3	c.‐1C/A (NA)	5' UTR	‐	Cases (96)	92	4	0	0.021	0.047
3	c.‐1C/A (NA)	5' UTR	‐	Controls (93)	93	0	0	0		0.009^f
4	c.17 T/A (rs1800282)	Exon 1	Val6Asp	Cases (96)	84	12	0	0.063	0.888
4	c.17 T/A (rs1800282)	Exon 1	Val6Asp	Controls (93)	82	11	0	0.059
5	c.343 G/A (NA)	Exon 4	Gly115Arg	Cases (97)	96	1	0	0.005	0.326
5	c.343 G/A (NA)	Exon 4	Gly115Arg	Controls (93)	93	0	0	0
6	c.427‐59A/G (rs2074963)	Intron 4	‐	Cases (97)	89	8	0	0.041	0.296
6	c.427‐59A/G (rs2074963)	Intron 4	‐	Controls (93)	81	12	0	0.065		0.042^d
7	c.694A/C (rs61757384)	Exon 7	Arg232Arg	Cases (96)	94	2	0	0.010	0.157
7	c.694A/C (rs61757384)	Exon 7	Arg232Arg	Controls (95)	95	0	0	0
8	c.695 G/A (NA)	Exon 7	Arg232Lys	Cases (96)	96	0	0	0	0.314
8	c.695 G/A (NA)	Exon 7	Arg232Lys	Controls (95)	94	1	0	0.005
9	c.710‐12A/G (rs1800286)	Intron 7	‐	Cases (96)	30	45	21	0.453	0.570
9	c.710‐12A/G (rs1800286)	Intron 7	‐	Controls (91)	32	42	17	0.418		0.417^d, 0.389^e
10	c.796A/G (rs7190823)	Exon 9	Thr266Ala	Cases (97)	51	38	8	0.278	0.048
10	c.796A/G (rs7190823)	Exon 9	Thr266Ala	Controls (94)	36	43	15	0.388		0.329^d, 0.357^e
11	c.894‐8A/G (rs1164881)	Intron 10	‐	Cases (97)	95	2	0	0.010	0.006
11	c.894‐8A/G (rs1164881)	Intron 10	‐	Controls (90)	79	11	0	0.061		0.059^f
12	c.1083 + 120 G/A (rs17226159)	Intron 12	‐	Cases (96)	69	23	4	0.161	0.381
12	c.1083 + 120 G/A (rs17226159)	Intron 12	‐	Controls (93)	72	20	1	0.118
13	c.1083 + 142 G/A (NA)	Intron 12	‐	Cases (96)	94	2	0	0.010	0.159
13	c.1083 + 142 G/A (NA)	Intron 12	‐	Controls (94)	94	0	0	0
14	c.1083 + 195C/T (NA)	Intron 12	‐	Cases (96)	93	3	0	0.016	0.317
14	c.1083 + 195C/T (NA)	Intron 12	‐	Controls (94)	94	1	0	0.005
15	c.1083 + 222 G/C (rs6500453)	Intron 12	‐	Cases (96)	46	38	12	0.323	0.270
15	c.1083 + 222 G/C (rs6500453)	Intron 12	‐	Controls (95)	38	42	15	0.379		0.425^d
16	c.1084‐49 G/C (rs1800287)	Intron 12	‐	Cases (96)	50	38	8	0.281	0.094
16	c.1084‐49 G/C (rs1800287)	Intron 12	‐	Controls (95)	38	43	14	0.374		0.425^d
17	c.1084‐30A/G (rs6500452)	Intron 12	‐	Cases (96)	60	32	4	0.208	0.070
17	c.1084‐30A/G (rs6500452)	Intron 12	‐	Controls (95)	47	39	9	0.300		0.241^d, 0.271^e
18	c.1143 G/T (rs1800331)	Exon 13	Thr381Thr	Cases (97)	88	9	0	0.046	0.338
18	c.1143 G/T (rs1800331)	Exon 13	Thr381Thr	Controls (95)	82	13	0	0.068		0.042^d
19	c.1225 + 12A/G (NA)	Intron 13	‐	Cases (97)	96	1	0	0.005	0.321
19	c.1225 + 12A/G (NA)	Intron 13	‐	Controls (95)	95	0	0	0
20	c.1225 + 151C/T (rs6500451)	Intron 13	‐	Cases (96)	83	13	0	0.068	0.379
20	c.1225 + 151C/T (rs6500451)	Intron 13	‐	Controls (95)	86	9	0	0.047
21	c.1226‐80 T/C (rs6500450)	Intron 13	‐	Cases (96)	53	35	8	0.266	0.050
21	c.1226‐80 T/C (rs6500450)	Intron 13	‐	Controls (95)	39	41	15	0.374		0.458^d
22	c.1226‐75C/G (NA)	Intron 13	‐	Cases (96)	96	0	0	0	0.313
22	c.1226‐75C/G (NA)	Intron 13	‐	Controls (95)	94	1	0	0.005
23	c.1226‐20A/G (rs1800330)	Intron 13	‐	Cases (97)	57	34	6	0.237	0.151
23	c.1226‐20A/G (rs1800330)	Intron 13	‐	Controls (95)	46	39	10	0.311		0.383^d
24	c.1235C/T (rs11646374)	Exon 14	Ala412Val	Cases (97)	88	9	0	0.046	0.457
24	c.1235C/T (rs11646374)	Exon 14	Ala412Val	Controls (95)	83	12	0	0.063		0.053^d, 0.058^e
25	c.1501 G/A (rs2239359)	Exon 16	Gly501Ser	Cases (97)	55	28	14	0.289	0.003
25	c.1501 G/A (rs2239359)	Exon 16	Gly501Ser	Controls (94)	33	42	19	0.426		0.362^d
26	c.1627‐32 T/C (rs17226337)	Intron 17	‐	Cases (97)	93	4	0	0.021	0.049
26	c.1627‐32 T/C (rs17226337)	Intron 17	‐	Controls (92)	92	0	0	0		0.025^f
27	c.1627‐31C/G (NA)	Intron 17	‐	Cases (97)	96	1	0	0.005	0.530
27	c.1627‐31C/G (NA)	Intron 17	‐	Controls (92)	90	2	0	0.011
28	c.1679C/T (NA)	Exon 18	Thr561Met	Cases (97)	94	3	0	0.015	0.086
28	c.1679C/T (NA)	Exon 18	Thr561Met	Controls (94)	94	0	0	0
29	c.1715 + 82 T/C (rs1800335)	Intron 18	‐	Cases (97)	50	39	8	0.284	0.135
29	c.1715 + 82 T/C (rs1800335)	Intron 18	‐	Controls (91)	37	40	14	0.374
30	c.1776 + 64 T/C (rs2302162)	Intron 19	‐	Cases (97)	88	9	0	0.046	0.660
30	c.1776 + 64 T/C (rs2302162)	Intron 19	‐	Controls (89)	79	10	0	0.056
31	c.1826 + 15 T/C (rs1800337)	Intron 20	‐	Cases (97)	50	39	8	0.284	0.068
31	c.1826 + 15 T/C (rs1800337)	Intron 20	‐	Controls (89)	34	40	15	0.393		0.425^d
32	c.1826 + 29insGT (rs1799742)	Intron 20	‐	Cases (97)	88	9	0	0.046	0.365
32	c.1826 + 29insGT (rs1799742)	Intron 20	‐	Controls (89)	77	12	0	0.067
33	c.1830A/G (rs1800338)	Exon 21	Ala610Ala	Cases (91)	88	3	0	0.016	0.081
33	c.1830A/G (rs1800338)	Exon 21	Ala610Ala	Controls (91)	91	0	0	0
34	c.1874 G/C (NA)	Exon 21	Cys625Ser	Cases (91)	91	0	0	0	0.316
34	c.1874 G/C (NA)	Exon 21	Cys625Ser	Controls (91)	90	1	0	0.005
35	c.1927C/G (rs17232910)	Exon 22	Pro643Ala	Cases (97)	90	7	0	0.036	0.192
35	c.1927C/G (rs17232910)	Exon 22	Pro643Ala	Controls (93)	81	12	0	0.065
36	c.2014 + 42 G/T (rs1800339)	Intron 22	‐	Cases (97)	89	8	0	0.041	0.208
36	c.2014 + 42 G/T (rs1800339)	Intron 22	‐	Controls (93)	80	13	0	0.070		0.05^d
37	c.2151 G/T (rs1131660)	Exon 23	Met717Ile	Cases (97)	87	10	0	0.052	0.128
37	c.2151 G/T (rs1131660)	Exon 23	Met717Ile	Controls (90)	86	4	0	0.022
38	c.2151 + 8 T/C (rs1800340)	Intron 23	‐	Cases (97)	56	35	6	0.242	0.130
38	c.2151 + 8 T/C (rs1800340)	Intron 23	‐	Controls (90)	42	38	10	0.322
39	c.2222 + 73A/G (rs1800341)	Intron 24	‐	Cases (93)	81	12	0	0.065	0.612
39	c.2222 + 73A/G (rs1800341)	Intron 24	‐	Controls (86)	77	9	0	0.052
40	c.2222 + 100A/G (rs886950)	Intron 24	‐	Cases (93)	48	38	7	0.280	0.143
40	c.2222 + 100A/G (rs886950)	Intron 24	‐	Controls (86)	35	37	14	0.378		0.433^d
41	c.2222 + 107 T/C (rs886951)	Intron 24	‐	Cases (93)	48	38	7	0.280	0.143
41	c.2222 + 107 T/C (rs886951)	Intron 24	‐	Controls (86)	35	37	14	0.378
42	c.2223‐113C/T (rs886952)	Intron 24	‐	Cases (94)	48	40	6	0.277	0.121
42	c.2223‐113C/T (rs886952)	Intron 24	‐	Controls (86)	34	37	15	0.390		0.425^d
43	c.2316 + 67 G/A (rs62989960)	Intron 25	‐	Cases (94)	78	15	1	0.090	0.193
43	c.2316 + 67 G/A (rs62989960)	Intron 25	‐	Controls (87)	78	9	0	0.052		0.025^d
44	c.2426 G/A (rs7195066)	Exon 26	Gly809Asp	Cases (94)	52	36	6	0.255	0.209
44	c.2426 G/A (rs7195066)	Exon 26	Gly809Asp	Controls (87)	40	36	11	0.333		0.273^d, 0.302^e
45	c.2574C/G (rs17233141)	Exon 27	Ser858Arg	Cases (97)	97	0	0	0	0.078
45	c.2574C/G (rs17233141)	Exon 27	Ser858Arg	Controls (95)	92	3	0	0.016
46	c.2589C/A (rs72807571)	Exon 27	Gly863Gly	Cases (97)	96	1	0	0.005	0.321
46	c.2589C/A (rs72807571)	Exon 27	Gly863Gly	Controls (95)	95	0	0	0
47	c.2779‐7 T/C (rs17233253)	Intron 28	‐	Cases (96)	87	9	0	0.047	0.548
47	c.2779‐7 T/C (rs17233253)	Intron 28	‐	Controls (91)	80	11	0	0.060
48	c.2852 + 137 T/C (rs12933317)	Intron 29	‐	Cases (96)	87	9	0	0.047	0.531
48	c.2852 + 137 T/C (rs12933317)	Intron 29	‐	Controls (90)	79	11	0	0.061
49	c.2852 + 314A/T (rs78904586)	Intron 29	‐	Cases (97)	79	16	1	0.094	0.284
49	c.2852 + 314A/T (rs78904586)	Intron 29	‐	Controls (80)	60	18	2	0.138
50	c.2859C/G (NA)	Exon 30	Asp953Glu	Cases (97)	97	0	0	0	0.311
50	c.2859C/G (NA)	Exon 30	Asp953Glu	Controls (95)	94	1	0	0.005
51	c.2901C/T (rs17226980)	Exon 30	Ser967Ser	Cases (97)	89	8	0	0,041	0.440
51	c.2901C/T (rs17226980)	Exon 30	Ser967Ser	Controls (95)	84	11	0	0.058
52	c.3067‐23 G/A (rs17227057)	Intron 31	‐	Cases (96)	87	9	0	0.047	0.601
52	c.3067‐23 G/A (rs17227057)	Intron 31	‐	Controls (94)	83	11	0	0.059
53	c.3067‐4 T/C (rs17227064)	Intron 31	‐	Cases (96)	87	9	0	0.047	0.601
53	c.3067‐4 T/C (rs17227064)	Intron 31	‐	Controls (94)	83	11	0	0.059
54	c.3069 G/T (NA)	Exon 32	Glu1023Asp	Cases (96)	95	1	0	0.005	0.321
54	c.3069 G/T (NA)	Exon 32	Glu1023Asp	Controls (94)	94	0	0	0
55	c.3239 + 32indel19 (NA)	Intron 32	‐	Cases (96)	96	0	0	0	0.311
55	c.3239 + 32indel19 (NA)	Intron 32	‐	Controls (94)	93	1	0	0.005
56	c.3240‐42 G/A (rs1800345)	Intron 32	‐	Cases (97)	62	31	4	0.201	0.045
56	c.3240‐42 G/A (rs1800345)	Intron 32	‐	Controls (91)	45	36	10	0.308
57	c.3263C/T (rs17233497)	Exon 33	Ser1088Phe	Cases (97)	88	9	0	0.046	0.584
57	c.3263C/T (rs17233497)	Exon 33	Ser1088Phe	Controls (94)	83	11	0	0.059
58	c.3348 + 18A/G (rs1800347)	Intron 33	‐	Cases (97)	82	15	0	0.077	0.022
58	c.3348 + 18A/G (rs1800347)	Intron 33	‐	Controls (94)	89	4	1	0.032		0.04^f
59	c.3348 + 25C/T (NA)	Intron 33	‐	Cases (97)	97	0	0	0	0.308
59	c.3348 + 25C/T (NA)	Intron 33	‐	Controls (94)	93	1	0	0.005
60	c.3348 + 29C/T (rs1800348)	Intron 33	‐	Cases (97)	95	2	0	0.010	0.579
60	c.3348 + 29C/T (rs1800348)	Intron 33	‐	Controls (94)	93	1	0	0.005
61	c.3348 + 40 T/A (NA)	Intron 33	‐	Cases (97)	95	2	0	0.010	0.579
61	c.3348 + 40 T/A (NA)	Intron 33	‐	Controls (94)	93	1	0	0.005
62	c.3408 + 21C/G (NA)	Intron 34	‐	Cases (97)	96	1	0	0.005	0.324
62	c.3408 + 21C/G (NA)	Intron 34	‐	Controls (94)	94	0	0	0
63	c.3408 + 45 G/A (rs1800355)	Intron 34	‐	Cases (96)	87	9	0	0.047	0.456
63	c.3408 + 45 G/A (rs1800355)	Intron 34	‐	Controls (94)	82	12	0	0.064		0.042^d
64	c.3427C/G (rs61753269)	Exon 35	Leu1143Val	Cases (96)	95	1	0	0.005	0.321
64	c.3427C/G (rs61753269)	Exon 35	Leu1143Val	Controls (94)	94	0	0	0
65	c.3513 + 62C/T (rs1800356)	Intron 35	‐	Cases (96)	82	13	1	0.078	0.538
65	c.3513 + 62C/T (rs1800356)	Intron 35	‐	Controls (95)	84	10	1	0.063
66	c.3654A/G (rs1800358)	Exon 37	Pro1218Pro	Cases (97)	82	15	0	0.077	0.888
66	c.3654A/G (rs1800358)	Exon 37	Pro1218Pro	Controls (95)	81	14	0	0.074
67	c.3711C/G (NA)	Exon 37	Val1237Val	Cases (97)	97	0	0	0	0.311
67	c.3711C/G (NA)	Exon 37	Val1237Val	Controls (95)	94	1	0	0.005
68	c.3765 + 37 G/A (rs34420680)	Intron 37	‐	Cases (97)	88	9	0	0.046	0.602
68	c.3765 + 37 G/A (rs34420680)	Intron 37	‐	Controls (95)	84	11	0	0.058
69	c.3765 + 41 G/A (NA)	Intron 37	‐	Cases (97)	97	0	0	0	0.311
69	c.3765 + 41 G/A (NA)	Intron 37	‐	Controls (94)	94	1	0	0.005
70	c.3765 + 61A/G (NA)	Intron 37	‐	Cases (97)	97	0	0	0	0.311
70	c.3765 + 61A/G (NA)	Intron 37	‐	Controls (94)	94	1	0	0.005
71	c.3807 G/C (rs11649210)	Exon 38	Leu1269Leu	Cases (96)	84	12	0	0.063	0.631
71	c.3807 G/C (rs11649210)	Exon 38	Leu1269Leu	Controls (94)	80	14	0	0.074		0.042^d
72	c.3828 + 81 G/T (rs11649162)	Intron 38	‐	Cases (96)	88	8	0	0.042	0.319
72	c.3828 + 81 G/T (rs11649162)	Intron 38	‐	Controls (94)	82	12	0	0.064		0.042^d
73	c.3828 + 251A/G (rs17227347)	Intron 38	‐	Cases (94)	88	6	0	0.032	0.148
73	c.3828 + 251A/G (rs17227347)	Intron 38	‐	Controls (94)	92	2	0	0.011
74	c.3828 + 295C/T (NA)	Intron 38	‐	Cases (95)	94	1	0	0.005	0.319
74	c.3828 + 295C/T (NA)	Intron 38	‐	Controls (94)	94	0	0	0
75	c.3828 + 313 G/A (rs17233734)	Intron 38	‐	Cases (95)	95	0	0	0	0.313
75	c.3828 + 313 G/A (rs17233734)	Intron 38	‐	Controls (94)	93	1	0	0.005
76	c.3829‐306 G/A (rs55927037)	Intron 38	‐	Cases (95)	94		0	0.005	0.994
76	c.3829‐306 G/A (rs55927037)	Intron 38	‐	Controls (94)	93	1	0	0.005
77	c.3829‐225C/T (rs11648689)	Intron 38	‐	Cases (95)	88	7	0	0.037	0.310
77	c.3829‐225C/T (rs11648689)	Intron 38	‐	Controls (94)	83	11	0	0.059
78	c.3829‐107A/T (rs11644967)	Intron 38	‐	Cases (95)	83	12	0	0.063	0.673
78	c.3829‐107A/T (rs11644967)	Intron 38	‐	Controls (95)	81	14	0	0.074		0.042^d
79	c.3935‐16C/T (rs1061646)	Intron 39	‐	Cases (95)	59	32	4	0.211	0.082
79	c.3935‐16C/T (rs1061646)	Intron 39	‐	Controls (91)	45	36	10	0.308
80	c.3981C/T (NA)	Exon 40	His1327His	Cases (97)	96	1	0	0.005	0.324
80	c.3981C/T (NA)	Exon 40	His1327His	Controls (94)	94	0	0	0		0.4^d
81	c.3982A/G (rs9282681)	Exon 40	Thr1328Ala	Cases (97)	89	8	0	0.041	0.747
81	c.3982A/G (rs9282681)	Exon 40	Thr1328Ala	Controls (94)	85	9	0	0.048		0.05^d
82	c.4010 + 92 T/C (rs9282682)	Intron 40	‐	Cases (97)	83	14	0	0.072	0.953
82	c.4010 + 92 T/C (rs9282682)	Intron 40	‐	Controls (92)	79	13	0	0.071
83	c.4055C/A (NA)	Exon 41	Ala1352Asp	Cases (97)	96	1	0	0.005	0.329
83	c.4055C/A (NA)	Exon 41	Ala1352Asp	Controls (92)	92	0	0	0
84	c.4167 + 46C/T (NA)	Intron 41	‐	Cases (97)	94	3	0	0.015	0.093
84	c.4167 + 46C/T (NA)	Intron 41	‐	Controls (90)	90	0	0	0
85	c.4260 + 29 T/C (rs1800359)	Intron 42	‐	Cases (97)	30	46	21	0.454	0.407
85	c.4260 + 29 T/C (rs1800359)	Intron 42	‐	Controls (90)	33	38	19	0.422		0.431^d, 0.4^e

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Exonic variants are displayed in bold characters.

SNP ID are indicated according to the nomenclature guidelines of the Human Genome Variation Society (RefSeq NM_000135.2 corresponding to transcript variant 1). The first base from the ATG codon is counted as +1. dbSNP ID is indicated according to build 129, NA indicating a SNP not found in the database.

MAF: Minor allele frequency.

p‐Value of the common homozygotes versus heterozygotes and rare homozygotes.

254 controls from the asthma familial collection of Saguenay‐Lac‐Saint‐Jean (NorthEastern region of the province of Quebec in Canada).

140 controls coming from French Canadian population (CARTaGENE).

192 controls from French Canadian population selected at random from the CRCHUQ glaucoma DNA bank (Vincent Raymond).

As indicated in Table 1, in all but one instance (rs17226337), the MAF observed in the other control cohorts are similar to those calculated in our initial cohort. This confirms that the frequency of the variants included in Table 1 is a reliable representation of the frequency observed in the French Canadian population not affected with breast cancer. As for the four SNPs displaying a p‐value <0.05, the c.1627‐32T/C (rs17226337) variant which was further genotyped in 192 individuals from the French Canadian population provided by Dr. Vincent Raymond, presents a MAF (0.025) similar to what is seen in the breast cancer series, suggesting that this variant is unlikely to have an effect on breast cancer risk in the French Canadian population.

Intronic analyzes were performed on sequences adjacent to the transcribed DNA portions and the resulting proximal variants are also presented in Table 1. Among these intronic variants, a significant difference in genotype frequencies was observed for a variant located in intron 10 (c.894‐8A/G) that was under‐represented in the case dataset (AA vs. AG + GG 0.151 95% CI 0.0133–0.702; p = 0.00634) and for another variant namely, c.3348 + 18A/G found in intron 33 which was over‐represented in cases (AA vs. AG + GG 3.256 95% CI 1.133–9.358; p = 0.02206). Borderline significance is estimated for the three following intronic variants: c.1226‐80T/C, c.1627‐32T/C and c.3240‐42G/A.

3.2. In silico and splicing analyzes

The two variants located in the proximal promoter region (less than 150 bp from the transcriptional start site) were also evaluated for their potential influence on transcription factor binding sites using the MatInspector program. The 13 bp duplication sequence (c.‐42‐87ins13) is associated with the appearance of four motifs that are not present on the reference sequence: a motif for the TATA box binding protein (TBP), an estrogen response element (ERE), a motif for the vertebrate steroidogenic factor SF1 and a second motif for the cAMP‐responsive element binding protein (CREB). Interestingly the c.‐42‐76G/C variant could potentially create a binding site for p53 and a motif for a chorion‐specific transcription factor with a GCM DNA binding domain (GCMF) (data not shown).

As shown in Table 2, the Thr561, Cys625, Glu1023, Leu1143 and Ala1352 amino acid show a high degree of conservation in lower species, suggesting that these amino acids are under strong functional constraint or may have a specific role on protein conformation, while the other residues leading to amino acid changes are not well conserved in distant species. Analysis of amino acid changes was performed in an attempt to predict the functional consequences of FANCA missense variants using the PolyPhen and SIFT softwares as well as the ALIGN‐GVGD algorithm implementing an extension of the Grantham difference. As displayed in Table 3, both the SIFT and PolyPhen softwares predicted the Thr561Met, Cys625Ser and Ser1088Phe changes to be damaging for protein conformation and function. Following analysis with the Align‐GVGD program, among the variants found in our case dataset, only 3 have a grade above C0. The Thr561Met variant, found in three cases, falls within the C65 grade which is the most likely deleterious grade. The Glu1023Asp variant (one case) has an intermediate probability to be damaging and the Ala1352Asp variant (one case) falls within the low probability class C15. As for the variants found in healthy control individuals, only the Cys625Ser rare variant is predicted to be deleterious (grade C65). Therefore, the Thr561Met and Cys625Ser amino acid changes are predicted to be damaging for protein function by all three programs and most of the changes leading to a potential deleterious effect identified by at least one of the programs used, are located in important domains or binding sites such as the BRCA1 binding site region (aa 1–589) or the Leucine zipper domain (aa 1069–1090).

Table 2.

Non‐synonymous sequence variants detected in human FANCA protein and residues found in orthologues.

SNPa	SNP IDb	Amino acid change	Macaca mulatta	Gallus gallus	Mus musculus	Canis lupus familiaris	Loxodonta africana	Monodelphis domestica	Xenopus ropicalis
4	c.17 T/A	Val6Asp	N/A	N/A	Ala	Thr	Ala	Ser	N/A
5	c.343 G/A	Gly115Arg	N/A	Lys	Lys	Gln	Arg	Gln	N/A
8	c.695 G/A	Arg232Lys	N/A	Gly	Glu	Gln	Arg	Gly	N/A
10	c.796A/G	Thr266Ala	N/A	Cys	Ala	Ala	N/A	Cys	N/A
24	c.1235C/T	Ala412Val	N/A	Ser	Ala	Thr	Ala	Ala	N/A
25	c.1501 G/A	Gly501Ser	N/A	Thr	Ser	Ser	Glu	Val	Pro
28	c.1679C/T	Thr561Met	N/A	Thr	Thr	Thr	Thr	Thr	Thr
34	c.1874 G/C	Cys625Ser	N/A	Cys	Cys	Cys	Cys	Cys	N/A
35	c.1927C/G	Pro643Ala	N/A	N/A	Ala	Ala	Pro	Thr	N/A
37	c.2151 G/T	Met717Ile	N/A	N/A	Ala	Gln	Glu	Lys	N/A
44	c.2426 G/A	Gly809Asp	N/A	N/A	Ser	Val	Ala	N/A	N/A
45	c.2574C/G	Ser858Arg	N/A	N/A	Asn	Ser	Gly	Asn	N/A
46	c.2859C/G	Asp953Glu	N/A	N/A	Asp	Asp	Asp	Tyr	N/A
54	c.3069 G/T	Glu1023Asp	Glu	Glu	Glu	Glu	Glu	Glu	N/A
57	c.3263C/T	Ser1088Phe	Ser	N/A	Ser	Ser	Thr	Ser	N/A
64	c.3427C/G	Leu1143Val	Leu	Leu	Leu	Ser	Leu	Leu	Val
81	c.3982A/G	Thr1328Ala	Thr	N/A	Thr	Ile	Ile	Ile	Leu
83	c.4055C/A	Ala1352Asp	Ala	N/A	Ala	Ala	Ala	Ala	Asp

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N/A: no corresponding residue found in this species.

According to Table 1.

According to the nomenclature of the Human Genome Variation Society.

Table 3.

Non‐synonymous amino acid changes identified in FANCA protein and prediction of the substitution on protein function using SIFT, PolyPhen and Align GVGD softwares.

SNPa	Amino acid change	Domain or binding sites (bs)	SIFT	PolyPhen	Align GVGD
4	Val6Asp	CENP‐E and BRCA1 bs	Tolerated	Possibly damaging	Class C0
5	Gly115Arg	CENP‐E and BRCA1 bs	Tolerated	Benign	Class C0
8	Arg232Lys	CENP‐E and BRCA1 bs	Tolerated	Benign	Class C0
10	Thr266Ala	CENP‐E and BRCA1 bs	Tolerated	Benign	Class C0
24	Ala412Val	BRCA1 bs	Tolerated	Benign	Class C0
25	Gly501Ser	BRCA1 bs	Tolerated	Possibly damaging	Class C0
28	Thr561Met	BRCA1 bs	Not tolerated	Probably damaging	Class C65
34	Cys625Ser	None	Not tolerated	Probably damaging	Class C65
35	Pro643Ala	None	Tolerated	Benign	Class C0
37	Met717Ile	None	Tolerated	Benign	Class C0
44	Gly809Asp	None	Tolerated	Benign	Class C0
45	Ser858Arg	None	Tolerated	Probably damaging	Class C0
46	Asp953Glu	None	Tolerated	Benign	Class C0
54	Glu1023Asp	None	Tolerated	Benign	Class C15
57	Ser1088Phe	Leucine zipper domain	Not tolerated	Possibly damaging	Class C0
64	Leu1143Val	BRG1 bs	Tolerated	Benign	Class C0
81	Thr1328Ala	BRG1 bs	Tolerated	Benign	Class C0
83	Ala1352Asp	BRG1 bs	Not tolerated	Benign	Class C15

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According to Table 1.

Analysis of FANCA cDNA was performed in breast cancer cases and highlighted the expression of four distinct alternative splicing events (ASEs) (Figure 1). These ASEs are observed in the FANCA genomic regions of exons 10–11 and 30–31 (BU616925, CN404731, AK301168, and BI908441). The first alternative spliced isoform (designed FANCAins10A) involved the insertion of 128 bp of intronic sequence located between exons 10 and 11 and is expected to result in a premature termination of FANCA open reading frame leading to a protein of 297 aa. The other ASE identified in this region, FANCAΔ11, involves the skipping of exon 11 (113 bp), which is also expected to produce a truncated protein of 299 aa. It should be noted that both proteins lack the major C‐terminal part of the protein which contains particularly a part of the BRCA1 binding site as well as the whole leucine zipper domain and BRG1 binding site region (Figure 1B). As for the exonic 30–31 region, the deletion of exon 30 (FANCAΔ30) is an in frame deletion of 129 bp, while the FANCAΔ31 involves the skipping of exon 31 (85 bp) and could potentially lead to a putative protein of 996 amino acids lacking the BRG1 binding site and the leucine zipper domain (Figure 1C).

FANCA interaction domains, and FANCA splicing. (A) Schematic representation of FANCA interaction domains based on the literature. (B) FANCA exon structure and schematic representation of the putative proteins of the two splicing variants located in the genomic region of exons 9–12, that could be detected via cDNA analyzes: FANCAins10a and FANCAΔ11. (C) FANCA exon structure and schematic representation of the putative proteins of the two splicing variants located in the genomic region of exons 29–32, that could be detected via cDNA analyzes: FANCAΔ30 and FANCAΔ31.

Quantitative PCR amplification of the four ASEs described above (FANCAins10A, FANCAΔ11, FANCAΔ30 and FANCAΔ31), performed in eight human breast cancer cell lines (Figure 2) as well as in BRCAX individuals are displayed in Supplemental Figure 1. q‐PCR amplification using oligonucleotides specific to each spliced form in the cell lines shows modest expression of FANCAΔ11, FANCAΔ30 and FANCAΔ31 mRNA. However, the variant FANCAins10A exhibits a high mRNA expression level (Figure 2). Relative expression levels of FANCAins10A spliced form are highest in SUM140 cell line and lowest in BT‐20 and MDA‐231 but no significant variation is observed according to estrogen receptor or differentiation status.

Expression levels of FANCA Δ11, ins10A, Δ30 and Δ31 spliced forms in cell lines as measured by quantitative real‐time PCR experiments. Relative expression levels of FANCA Δ11, ins10A, Δ30 and Δ31 spliced forms were calculated as E(ΔCt wild‐type allele –ΔCt splice form) in eight breast cancer cell lines where E is primer efficiency.

3.3. Polysome analysis of FANCA ASEs mRNAs

Given that we could not assume that the spliced forms described above were not subject to mRNA regulation through nonsense mediated decay (NMD), additional experiments have been performed. A series of cell lines from breast cancer patients, in which the alternative form of interest was observed, were treated with puromycin (half of the cells were treated and half were not), an agent known to inhibit NMD. RT‐PCR was performed on FANCAins10A, FANCAΔ11, FANCAΔ30 and FANCAΔ31 using specific oligonucleotides to each spliced form, and clearly these forms do not seem to be subject to NMD (data not shown). To further determine whether FANCA splicing variant mRNAs are efficiently translated, polysome profiles were performed in the breast cancer cell line T‐47D and mRNA levels were measured using splice variant‐specific qRT‐PCR primers. Efficiently translated transcripts are associated with polysomes, whereas those degraded by NMD are generally associated with monosomes because they are degraded during the pioneering round of translation, before loading of additional ribosomes. Figure 3 shows the resulting profile with Δ11, ins10A, Δ30 and Δ31 mRNA which are most abundant in polysome fractions 11 to 13 suggesting there are efficiently translated. These spliced mRNAs are thus not degraded by NMD and are associated with translating ribosomes, suggesting efficient production of Δ11, ins10A, Δ30 and Δ31 proteins in T‐47D cell line. Interestingly, ribosomal analysis shows that the variant carrying the insertion of the intronic fragment exhibit the highest level of mRNA which is in concordance with the results obtained with quantification of the variants in the different cell lines.

Polysome analysis of FANCA ASEs mRNAs. Fractionation of monosomes and polysomes (with or without EDTA) as measured by A260. q‐RT PCR of FANCA Δ11, ins10A, Δ30 and Δ31 spliced forms was performed on T‐47D cells and relative expression values for each fraction were calculated by the equation R = (E)(Ctref−Ct), where Ctref is the average cycle threshold of all the fractions for that variant.

Following identification of ASEs expressed in BRCAX individuals and cancer cell lines, the putative impact of the relevant FANCA periexonic and exonic variants (i.e. located in the vicinity of the ASE genomic regions) on mRNA splicing was assessed by in silico methods using the HSF web program which allows the identification of enhancer and silencer splicing sites as well as branch point sequences (Table 4). The acceptor and donor motif sequences of exons 10, 10A and 11 have relatively good motif scores, all being over 0.80. Moreover the exon 10A possesses putative branch point sequences located 80 and 65 bp upstream of the beginning of exon 10A. The only relevant variant located in intron 10 in the proximity of the acceptor site sequence of exon 11, c.894‐8A/G, creates new sites for SF2/ASF (IgM‐BRCA1) binding as well as new enhancer EIE and silencer motif 2 binding sites. As for the region containing exons 30–31, the wild type acceptor and donor motif sequences of exons 30 and 31 demonstrate efficient splicing site scores. Two variants located in exon 30, c.2859C/G and c.2901C/T are predicted to have significant effect on binding capacity of several splicing factors. Particularly, c.2859C/G disrupts motifs for SC35, PESE octamer as well as for enhancer EIE and 9G8. This variant also creates silencer motifs 1 and 2. As for exon 31, the two relevant variants c.2982‐102G/C and c.2982‐73G/A, located in intron 30 upstream of exon 31, have been evaluated. The c.2982‐102G/C variant deletes several consensus motifs such as those for PESE octamer, enhancer EIE and 9G8 as well as silencer motifs 1 and IIE and hnRNP A1 protein (Table 4).

Table 4.

In silico analysis of FANCA genomic regions and variants potentially involved in the expression of FANCAins10a, FANCAΔ11, FANCAΔ30 and FANCAΔ31 spliced transcripts.

Exon or variant	Location	Motif	Reference score (WT)	Variant score	Potential effect on exon inclusion
Region of exons 10–11
Exon 10 wt	–	Acceptor sitea	90.1	–	Positive
Exon 10 wt	–	Donor sitea	86.4	–	Positive
Exon 10A	–	Acceptor sitea	81.3	–	Positive
	–	Donor sitea	86.9	–	Positive
	Exon 10A‐80	Branch point sitea	90.8	–	Positive
	Exon 10A‐65	Branch point sitea	90.7	–	Positive
Exon 11	–	Acceptor sitea	86.6	–	Positive
Exon 11	–	Donor sitea	88.5	–	Positive
c.894‐8A/G	Intron 10	Potential donor sitea	n.d.	66.6	Not determined
		SF2/ASF (IgM‐BRCA1)a	n.d.	73.1	Positive (exon 11)
		Enhancer EIEb	No value	New site	Positive (exon 11)
		Silencer Motif 2c	n.d.	New site (72.3)	Negative (exon 11)
		Silencer IIEb	No value	Site broken	Positive (exon 11)
		Silencer IIEb	No value	New site	Negative (exon 11)

Region of exons 30–31
Exon 30	–	Acceptor sitea	83.3	–	Positive
Exon 30	–	Donor sitea	91.8	–	Positive
c.2859 C/G	Exon 30	Potential acceptor sitea	n.d.	71	Not determined
		SC35a	85	Site broken	Negative (exon 30)
		Enhancer PESE octamerd	35.8	Site broken	Negative (exon 30)
		Enhancer EIEb	No value	Site broken	Negative (exon 30)
		Enhancer 9G8a	59.5	Site broken	Negative (exon 30)
		Silencer Motif 1c	n.d.	New site (69.7)	Negative (exon 30)
		Silencer Motif 2c	n.d.	New site (60.2)	Negative (exon 30)
c.2901 C/T	Exon 30	SRp55a	n.d.	New site (76.1)	Positive (exon 30)
		Enhancer PESE octamerd	27.8	Site broken	Negative (exon 30)
		Silencer IIEb	No value	New site	Negative (exon 30)
Exon 31	–	Acceptor sitea	92.0	–	Positive
Exon 31	–	Donor sitea	98.8	–	Positive
c.2982‐102 G/C	Intron 30	Branch point sitea	72.2	77.2	Positive (exon 31)
		SRp40a	n.d.	New site (85.7)	Positive (exon 31)
		Enhancer PESE octamersd	41.1	Site broken	Negative (exon 31)
		Enhancer EIEb	No value	Site broken	Negative (exon 31)
		Enhancer 9G8a	59.5	Site broken	Negative (exon 31)
		Silencer Motif 3c	–	New site (70.0)	Negative (exon 31)
		Silencer Motif 1c	65.8	site broken	Positive (exon 31)
		Silencer IIEb	No value	Site broken	Positive (exon 31)
		Silencer hnRNP A1a	73.3	Site broken	Positive (exon 31)
c.2982‐73 G/A	Intron 30	Branch point sitea	75.7	71.8	Negative (exon 31)
		SF2/ASF (IgM‐BRCA1) a	84.5	71.5	Negative (exon 31)
		Enhancer PESE Octamerd	n.d.	New site (57.6)	Positive (exon 31)
		Enhancer EIEb	No value	New site	Positive (exon 31)
		Enhancer 9G8a	71.1	Site broken	Negative (exon 31)
		Silencer Motif 1c	74.5	Site broken	Positive (exon 31)
		Silencer Motif 2c	66.3	Site broken	Positive (exon 31)
		Silencer hnRNP A1a	78.6	Site broken	Positive (exon 31)

Open in a new tab

n.d.: Not detected.

EIE: exon‐identity elements.

IIE: Intron‐identity elements.

Based on Human Splicing Finder matrices.

Zhang C et al., 2008, PNAS 105:5797.

Sironi M et al., 2004, Nucleic acids research 32:1783.

Zhang and Chasin 2004, Genes Dev 18:1241.

To investigate the potential association between the variants c.2901C/T (rs17226980), c.2982‐102G/C (rs12931267) and c.2982‐73G/A and the expression of the FANCAΔ30 and FANCAΔ31, real‐time PCR was performed using RNA samples from five heterozygotes for c.2901C/T, two heterozygotes for c.2982‐102G/C, one heterozygote for c.2982‐73G/A and ten wild‐type individuals (Supplemental Figure 1). The presence of the Δ30 and Δ31 spliced transcripts were detected in all individuals, including wild‐type individuals, supporting the fact that the expression of these variants are not associated with none of these polymorphisms. A similar experiment was performed to analyze the assessment between the c.894‐8A/G (rs1164881) variation and the expression of the FANCAΔ11 and FANCAins10A (data not shown) and no significant correlation was observed.

4. Discussion

Over the last decade, several evidences linked the two major breast cancer susceptibility genes BRCA1 and BRCA2 to an emerging network of proteins implicated in DNA repair, and whose bi‐allelic mutations cause Fanconi Anemia (Wang, 2007). Mono‐allelic mutations, like those found in BRCA1 and BRCA2/FANCD1, have been described for FANCJ, FANCN and FANCO in breast cancer susceptibility (Somyajit et al., 2010; Cantor et al., 2001, 2004; Seal et al., 2006; Rahman et al., 2007; Erkko et al., 2007; Pang et al., 2011; Zheng et al., 2010; Meindl et al., 2010), and a recent study conducted with 944 family members being part of the International Fanconi Anemia Registry revealed an increased risk of breast cancer among grandmothers carriers of FANCC mutations (Berwick et al., 2007). However, apart from these genes, the involvement of the other FANC genes in breast cancer susceptibility remains unclear. Indeed, epidemiological studies focusing on heterozygous mutation carriers have yielded conflicting results (Tischkowitz et al., 2008; Mathew, 2006). Using variant screening and discovery on both genomic and cDNA material of the FANCA gene in a cohort of 97 familial breast cancer cases without BRCA1 or BRCA2 mutations as well as among 95 healthy unrelated controls from the same population, we found that: 1) re‐sequencing did not identify any deleterious mutations, 2) some variants, and particularly the Gly501Ser non‐synonymous change, are associated with a protective effect against breast cancer risk in our cohort and, 3) the FANCA gene is subject to multiple alternative splicing events expression. Although extensive promoter screening was beyond the scope of the current study, two variants, including the unreported c.‐42‐76G/C, were found in the proximal region of the transcriptional starting site. The 13 bp duplication (c.‐42‐87ins13) has been previously associated with a protective effect in ovarian cancer (OR = 0.72; 95% CI, 0.53–0.99), while no significant effect was seen in breast cancer patients (Thompson et al., 2005). Both promoter variants identified in this study are of interest as even single base changes in promoter sequences can alter regulation of gene expression and contribute to tumorigenesis, particularly if this could affect transcription factor binding site for proteins such as TBP, estrogen receptor, SF1 and CREB as predicted for the c.‐42‐87ins13 variant (Hasselbach et al., 2005; Najafi and Jangravi, 2010; Zhu et al., 2001; Bond et al., 2004).

Interestingly, among the 85 sequence variants identified in our case and control datasets, the variant showing the most significant p‐value (p = 0.003) is the c.1501G/A (Gly501Ser) non‐synonymous change. This variant is over‐represented in our control dataset, which is suggestive of a protective effect of the A allele in our cohort. To date, the Gly501Ser variant of FANCA has been recently associated with an increased risk of cervical intraepithelial neoplasia grade 3 cancer, with a risk of 1.7 fold (95% CI 1.1–2.6 fold) for the GG genotype when compared to the AA genotype (Wang et al., 2009). Another significant over‐representation in the healthy individuals was observed for the c.894‐8A/G variation (p = 0.006). As predicted by in silico analysis, this variant creates a new potential donor site and affects binding site score for SF2/ASF, Enhancer EIE and few silencer proteins. Moreover this variant is located 8 nucleotides upstream of exon 11, which is skipped in the FANCAΔ11 mRNA. Therefore this variant could potentially be involved in the expression of this splicing event. As for the variant c.3348 + 18A/G showing a significant difference of frequency between both series (p = 0.022), it creates a new donor site (score = 72.1), a new motif 2 silencer site, and abolishes a EIE site. Among the variants showing a borderline significance, both c.‐1C/A and c.1627‐32T/C are observed in the case series only. Moreover these variants are carried by the same four individuals likely indicating that both variants are being part of the same allele. The unreported c.‐1C/A variation is located in a crucial consensus sequence involved in the initiation of translation. The C nucleotides at position −1 and −2 do not need to be conserved, but contribute to the overall strength (Kozak, 1986). The c.2574C/G variant (Ser858Arg), known as the Indian mutation and located in exon 27, has been previously reported as a disease‐causing mutation in Fanconi Anemia (Tamary et al., 2000; Wijker et al., 1999). However this Ser858Arg change is found exclusively in our control dataset, therefore suggesting that this variant at the heterozygous state is non‐pathogenic in breast cancer predisposition.

Both the Thr561Met and Cys625Ser changes located within or in the vicinity of the BRCA1 binding site are predicted to be deleterious by all three programs used. Although Thr561Met is found exclusively in three cases, both variants did not show any significant difference in MAF between both cohorts. The Cys625Ser variant has been identified and predicted as pathogenic in the Spanish FA population but this variant was not characterized regarding its effect on protein function (Castella et al., 2011).

Several splicing variants of FANCA gene have been identified and partially characterized in the last decade and result from the presence of specific genomic variants. For instance, the c.1567‐20A/G (intron 16), c.2278 + 83C/G (intron 28) and c.2222 + 166A/G (intron 24) are known to affect splicing of the FANCA protein (Savino et al., 2003; Bouchlaka et al., 2003). Several other spliced variants have been reported in the Japanese population (Tachibana et al., 1999; Yagasaki et al., 2004). The significant expression of four splicing variants have been detected in our BRCAX breast cancer individuals and all variants namely FANCAins10A (BU616925), FANCAΔ11 (CN404731), FANCAΔ30 (AK301168) and FANCAΔ31 (BI908441), have been reported in NCBI EST databases but have not been characterized. Of these, only the FANCAΔ30 splice variant results in an in‐frame deletion of 43 amino acids located outside known domains, therefore the significance of this deletion is unknown. FANCAins10A, FANCAΔ11 and FANCAΔ30 lead to truncated proteins lacking the BRG‐1 binding domain, while the FANCAins10A and FANCAΔ11 truncated proteins have also an excised BRCA‐binding domain. Moreover several putative nuclear export sequences located in the region of aa 518, 860 and 1013, are missing in these spliced proteins (Ferrer et al., 2005). Thus, this suggests that interactions with BRCA1 (Folias et al., 2002), the chromatin remodeling BRG1 protein (Otsuki et al., 2001) and with other known protein partners might be affected. No clear polymorphism/mutation seem to be involved in the expression of these spliced transcripts which are not significantly degraded by the NMD mechanism, and are most likely transcribed efficiently as strongly suggested by the polysomal experiments. However, we can not exclude deeper intronic variations affecting intronic splicing enhancers/silencers or creating new cryptic splice sites. Moreover, as reported for some genes related to specific diseases (Ricketts et al., 1987; Naylor et al., 1993; Dietz et al., 1993), the skipping of exons could maintain transcription and translation of a partially functional protein and thus moderate the disease phenotype.

Large intragenic and exonic deletions have been identified in FA patients of different populations such as Afrikaners, Spanish or other European populations (Wijker et al., 1999; Tachibana et al., 1999; Morgan et al., 1999; Tipping et al., 2001; Callén et al., 2004; Centra et al., 1998; Levran et al., 1998; Nakamura et al., 1999). Previous studies have shown that deletions account for 40% of FANCA mutations (Morgan et al., 1999). Indeed it is known that the majority of deletions occurred by recombination between two ALU repeats located in cis as reported for many other genes (Morgan et al., 1999; Centra et al., 1998; Levran et al., 1998). As demonstrated by Morgan et al (Morgan et al., 1999), recombination hotspots within the FANCA gene seem to be located in the genomic regions of introns 17 and 31, which reflect the similar regions of LD block recombination identified in our dataset (data not shown). Regarding the involvement of FANCA deletions in breast cancer susceptibility, a novel heterozygous deletion, removing the promoter and 12 exons of the FANCA gene, was recently identified in one Finnish breast cancer family (Solyum et al., 2011). However, as reported previously (Seal et al., 2003), MLPA analyzes did not identify any large genomic or exonic deletions within the FANCA gene in our French Canadian breast cancer patients.

5. Conclusion

This is the first comprehensive mutation screening of FANCA gene in the French Canadian population. Although no deleterious mutation was found, we identify 24 novel polymorphisms not reported in databases including 7 missense variations, as well as four alternative splicing events. It will also be interesting to determine whether inherited polymorphisms in FANCA gene resulting in more subtle defects in protein expression or function, can contribute to increased cancer risk or to variable tumor responses to conventional therapies.

Supporting information

The following are the supplementary data related to this article:

Supplementary data

Click here for additional data file.^{(17.7KB, pdf)}

Supplementary data

Click here for additional data file.^{(53.4KB, pdf)}

Acknowledgments

The authors would like to thank all individuals and families who participated in this study. We thank M. Tranchant, Dr. M. Dumont, and G. Leblanc of the Cancer Genomics Laboratory for sample management and mutation screening. We also thank M. Ouellet, C E Bénard and A.M. Moisan for skillful technical help, Dr. Fabienne Lesueur (IARC, Lyon) for the help with GV‐GD analysis and Dr. D. Labuda and C. Moreau at the Centre de Cancérologie Charles Bruneau of Ste‐Justine Hospital for help with control DNA samples. We also thank Catherine Laprise (UQAC) for providing genotyping information and Vincent Raymond (CRCHUQ) for providing DNA samples on additional controls from the French Canadian population. The authors also acknowledge the work of Professor Bartha Maria Knoppers and her colleagues regarding ELSI issues, as well as the ethics committees of all participating institutions.

Financial support: This work was supported by the Canadian Institutes of Health Research for the “CIHR Team in Familial Risks of Breast Cancer” program and by the Canadian Breast Cancer Research Alliance, the Fond de la Recherche en Santé du Québec (FRSQ)/Réseau de Médecine Génétique Appliquée (RMGA), the CURE Foundation and Fanconi Canada. N.L. and S.D. hold studentships from Fondation René Bussières and Fondation Desjardins (S.D.), and F.D. held a chercheur‐boursier from the Fonds de la Recherche en Santé du Québec (FRSQ).

Appendix A Supplementary data 1.

1.1.

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.molonc.2012.08.002.

Appendix B 1.

1.1.

Other members of INHERIT BRCAs involved in clinical aspects of this study:

Paul Bessette: Service de gynécologie, Centre Hospitalier Universitaire de Sherbrooke, Fleurimont, QC, J1H 5N4, Canada.

Jocelyne Chiquette: Clinique des maladies du sein Deschênes‐Fabia, Hôpital du Saint‐Sacrement, Québec, QC, G1S 4L8, Canada.

Rachel Laframboise: Service de médecine génétique, CHUQ, Pavillon CHUL, Québec, QC, G1V 4G2, Canada.

Jean Lépine: Centre Hospitalier régional de Rimouski, Rimouski, QC, G5L 5T1, Canada.

Bernard Lespérance, Roxane Pichette: Service d'hémato‐oncologie, Hôpital du Sacré‐Cœur, Montréal, QC, H4J 1C5, Canada.

Marie Plante: Service de gynécologie, CHUQ, L'Hôtel‐Dieu de Québec, Québec, QC, G1R 2J6, Canada.

Litim Nadhir, Labrie Yvan, Desjardins Sylvie, Ouellette Geneviève, Plourde Karine, Belleau Pascal, INHERIT BRCAs , Durocher Francine, (2013), Polymorphic variations in the FANCA gene in high‐risk non‐BRCA1/2 breast cancer individuals from the French Canadian population, Molecular Oncology, 7, doi: 10.1016/j.molonc.2012.08.002.

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PERMALINK

Polymorphic variations in the FANCA gene in high‐risk non‐BRCA1/2 breast cancer individuals from the French Canadian population

Nadhir Litim

Yvan Labrie

Sylvie Desjardins

Geneviève Ouellette

Karine Plourde

Pascal Belleau

Francine Durocher

Abstract

1. Introduction

2. Material and methods

2.1. Ascertainment of families and DNA purification

2.2. RNA isolation from immortalized cell lines and cDNA synthesis

2.3. PCR amplification, mutation analysis and variant characterization

2.4. In silico analyzes

2.5. Alternative splicing analysis

2.5.1. QRT‐PCR assays

2.5.2. Polysome analysis

2.6. Electronic databases

3. Results

3.1. Variant characterization

Table 1.

3.2. In silico and splicing analyzes

Table 2.

Table 3.

Figure 1.

Figure 2.

3.3. Polysome analysis of FANCA ASEs mRNAs

Figure 3.

Table 4.

4. Discussion

5. Conclusion

Supporting information

Acknowledgments

Appendix A Supplementary data 1.

1.1.

Appendix B 1.

1.1.

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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