ATM germline mutations
In the present study, we have used the DGGE method to screen the complete
ATM ORF to obtain insight in the
ATM missense mutation spectrum in (contralateral) breast cancer patients. With DGGE we were able to confirm all the previously identified truncating mutations. A subset of the CBC patients described in this study had been screened in the past for
ATM truncating mutations with the Protein Truncating Test, revealing seven
ATM truncating mutations (including a non-sense mutation and small insertions and deletions; generating stop codons within a previously functional protein coding sequence causing premature termination of translation of the protein) [
10]. Among all 443-breast cancer patients that were tested in this study with DGGE we detected a large number of
ATM silent mutations (presumed neutral polymorphisms, data not shown and excluded from all analyses) and missense mutations (causing an amino acid substitution in the coded protein, most common ones; i.e.
D1853N, not included in further analysis).
ATM missense mutation spectrum
In our study cohort we have detected 35 distinct
ATM missense variants and 6 distinct truncating mutations. Several of the detected missense variants have been reported in the ATM database as being detected in A-T patients/or as polymorphisms (Table
1). None of the missense variants identified in this study are known as pathogenic A-T causing missense mutations. Seventeen of the missense variants have not been reported previously. Eleven missense variants were exclusively found in the CBC group and 10 exclusively in the UBC group. Whether this distinction in the spectrum indicates an association between particular variants and bilateral breast cancer risk cannot be concluded from the small numbers obtained in this study population. The
ATM protein has several functional domains and the identified missense variants are located throughout the ORF. Potential functional implications of the newly identified unclassified variants remain to be established.
Table 1
ATM missense variant and truncating mutation spectrum in contralateral and unilateral breast cancer patients
37C>T | R13C | | 1 | |
146C>G | S49C | 5 | 5 | database |
162T>C | Y54H | 2 | 1 | |
378A>T | D126E | | 1 | database |
1009C>T | R337C | | 1 | Novel |
1132A>G | S377G | | 1 | Novel |
1229T>G | V410A | 2 | 1 | |
1810C>T | P604S | 1 | | database |
2119T>C | S707P | 7 | 8 | database |
2276G>A | S759N | | | Novel |
2336T>C | M779T | | 1 | Novel |
2414G>A | R805Q | 2 | | Novel |
2572T>C | F858L | 4 | 3 | database |
2650C>T | P884S | 1 | | |
2650C>T | P884S | 1 | | Novel |
2614C>T | P872S | | | |
3161C>G | P1054R | 8 | 13 | database |
3925G>A | A1309T | 1 | 1 | |
4138C>T | H1380Y | 1 | | database |
4258C>T | L1420F | 5 | 4 | database |
4324T>C | Y1442H | | 2 | Novel |
4362A>C | K1454N | | 1 | database |
4477C>G | L1493V | 1 | | Novel |
4664T>A | L1555H | | 1 | Novel |
4722G>T | L1574F | 1 | | Novel |
5044G>T | D1682Y | 1 | | database |
5071A>C | S1691R | 2 | 2 | database |
5557G>A
b
|
D1853N
|
35
|
49
| database |
5558A>T | D1853V | 3 | 1 | database |
5741A>G | D1914G | | | Novel |
6067G>A | G2023R | 1 | | database |
6820G>A | A2274T | 1 | | database |
6919C>T | L2307F | 1 | | |
7446G>A | M2482I | 1 | | Novel |
7874A>G | D2625G | | 1 | Novel |
8659C>G | H2887D | | 1 | Novel |
Truncating mutations |
IVS10-6T>G | 419X | 1 | 2 | |
1563delAG | 521X | | 1 | database |
1660delA | 554X | 1 | | Novel |
IVS14 + 2T>G | del 601-633 | 1 | | database |
2572insT | F858X | | 1 | Novel |
3115A>T | R1039X | 1 | | Novel |
Despite the fact that
ATM plays a role in breast cancer risk, the role of most distinct
ATM missense variants remains unclear. Some studies tried to predict the relevance of each particular mutation on basis of co-segregation with breast cancer in families, the location in a functional domain or interference with the splicing machinery. Only a few studies present functional analysis that are necessary to assess the biological impact of unidentified variants found frequently in
ATM [
18‐
20].
ATM missense mutations and contralateral breast cancer
Twenty-one per cent of the patients carried at least one
ATM germline variant (missense and truncating; Table
2). Among the patients with CBC (
n = 247) we identified in total 55
ATM variants in 45 individuals (18%); 51 missense variants and 4 truncating mutations (Table
2). Eight CBC patients had multiple
ATM missense variants and 2 patients carried both a missense and a truncating
ATM mutation. In the women with UBC (
n = 196) we identified 52
ATM variants in 46 individuals (23%); 48 missense and 4 truncating mutations. Three UBC patients carried double missense and 3 patients both a truncating and a missense variant. Although it is known from the literature that
ATM missense variants might be involved in breast cancer pathogenesis, the identified 17% missense variant carriers among the CBC patients compared to the 21% missense variants among the UBC patients indicate that there is not a significantly increased risk for bilateral breast cancer among
ATM missense variant carriers, OR 0.77 (95% CI 0.48–1.24).
Table 2
ATM variant frequencies in all breast cancer patients diagnosed under age 50 and according to uni- or contralateral breast cancer
Total ATM variantsa
| | 55: 51 missense and 4 truncating | 52: 48 missense and 4 truncating |
At least one ATM variant | 91 (21%) | 45 (18%) | 46 (23%) |
At least one ATM missense variant | 85 (19%) | 43 (17%) | 42 (21%) |
Only one ATM truncating mutations | 3 | 2 | 1 |
One truncating and one missense variant | 5 | 2 | 3 |
Double missense variants | 11 | 8 | 3 |
Association with radiation treatment
Women at high risk for developing breast cancer may respond differently to radiation exposures associated with screening and treatment, than the general population. Candidate-genes like
ATM are implicated in maintenance of genome integrity. Their involvements in breast cancer susceptibility as well as their role in DNA-damage repair signalling make them excellent candidates for a role in radiation-induced breast cancer [
21]. Recently, we showed that women with a pathogenic germline mutation in a DNA repair pathway gene (e.g.
BRCA1, BRCA2, CHEK2 and
ATM) have an over 2-fold increased risk of developing radiation-associated breast cancer (manuscript under review). Therefore, we now investigated whether exposure to ionising radiation had a greater biological impact on certain
ATM genotypes than on others.
We did not detect a significantly increased risk of developing radiation-associated CBC among missense mutation carriers. Among those 169 CBC patients who had developed a second primary breast tumour following radiotherapy for their first breast tumour we identified 19.5% ATM missense variants carriers compared to 13% among those CBC patients who did not receive RT, the OR from this case-only analysis is 1.65 [95% CI (0.77–3.55) p = 0.2]. Furthermore, we have observed that 21% of the UBC patients, who received RT but did not develop a CBC carried an ATM missense variant, compared to 19.5% of the CBC patients that received RT for their first tumour [OR 0.86 (95% CI 0.52–1.43)]. These results suggest that RT is not a strong risk factor for the development of CBC among carriers of those ATM missense variants.
It has however been shown that particular alterations in the
ATM gene are associated with increased radiation sensitivity [
22‐
24]. Gutierrez-Enriquez et al. showed that lymfoblastoid cell lines carrying the
ATM variant 3161G (linked to 2572C) was associated with increased in vitro chromosomal radio-sensitivity, perhaps by interfering with
ATM function in a dominant-negative manner [
22]. We found this particular variant allele (3161G/2572C) exclusively in our CBC group exposed to radiotherapy (four times) and not in the non-RT-exposed CBC group. This finding supports the hypothesis that particular
ATM variants might play a differential role in radiation response. Although a subset of the missense variants was only detected in the RT exposed subpopulation, individual numbers were probably too small to detect a significant effect of particular mutations associated with treatment.
We observed that CBC patients with an ATM missense variant had an mean interval between the first and second breast tumour of ∼101 months, compared to 122 months for non-carriers CBC patients (p = 0.085). Interestingly, the combination of radiation treatment and a missense variant resulted in an even shorter mean interval of a 92 months in the CBC patients compared to a 136-month interval for CBC patients who neither received RT nor carried a germline variant (p = 0.029). These data suggest that carrier-ship of an ATM missense variant may accelerate the development of a second tumour and decreases the age at onset of the second breast tumour, especially in case of exposure to RT.
The suggestion of a shorter induction period of RT-associated breast cancer in patients, who carry an ATM missense mutation, while the proportion of patients with missense variants was similar in CBC and UBC cases, might be attributable to a different spectrum of mutations in those patients who developed CBC. A big challenge in such a study remains to assess which particular missense mutations have an impact on ATM function. Large association studies, as performed by the Breast Cancer Association Consortium (coordinated by Doug Easton and Paul Pharoah, Cambridge), and functional studies are clearly necessary to determine the importance of particular variants and their contribution to the breast cancer risk.