The aetiology of breast and ovarian cancer (BC/OC) is multi-factorial. At present, the involvement of base excision repair (BER) glycosylases (MUTYH and OGG1) in BC/OC predisposition is controversial. The present study investigated whether germline mutation status and mRNA expression of two BER genes,
Breast cancer (BC) is the second most common cancer worldwide (
The risk of BC and/or ovarian cancer (OC) is increased in carriers of deleterious mutations in
In light of the above, it can be assumed that genetic variability related to low expression in enzymes that protect cellular DNA from oxidative damage, causing genetic instability, may favor the onset of BC and OC.
In the present study we investigated
The study was conducted on a series of BC/OC unrelated Italian patients previously analyzed for BRCA1/2 as a public health service between 2000 and 2006 from Medical Genetic Service of University ‘G. d'Annunzio’ of Chieti (
Nucleic acid extraction from peripheral blood mononuclear cells (PBMCs) and synthesis of complementary DNA (cDNA) from 1.5 µg of total RNA were performed as previously described (
The coding sequence and intron-exon borders of
Primer sequences for
To estimate the frequency of novel mutations we examined 240 chromosomes from control individuals, from the same geographical area, with no personal history of BC/OC and colorectal cancer (CC). All mutations were confirmed by sequencing of independent PCRs. The nomenclature of sequence variants follows the guidelines proposed by the Human Genome Variation Society (HGVS).
ClinVar-NCBI database (
The levels of
Continuous variables were summarized as mean and standard deviation (SD), or median and interquartile range (IQR). Categorical variables were summarized as frequency and percentages. The Student's t-test for unpaired data was performed to test the difference between the means of age at sampling in different groups. The Mann-Whitney U tests performed to evaluate differences in gene expression levels among groups (51 cases and 43 controls without cancer family history). To assess for a possible correlation between the three genes, Spearman's ρ correlation coefficient was evaluated. All P-values are two-sided and a P-value of <0.05 was considered significant. All analyses were performed using SPSS (version 20) software.
Coding regions and flanking introns of
Overall, germline mutational analysis identified 11 variants in
In particular,
Notably, 2 missense (Pro18Leu and Gly25Asp) mutations and 1 intronic variant (c.36+11C>T) were identified in the same case (B66), a patient affected by breast and thyroid cancer, who referred family history for BC and colon cancer (CC) (
Analysis of
Additional
Gene expression by qRT-PCR was analyzed in 51 patients (mean age, 48.60±12.88) and in 50 age and sex matched control individuals (mean age, 48.11±7.35). Demographic and clinical characteristics of patients undergoing gene expression analysis are shown in
Forty-seven patients reported BC and 4 patients OC, 28 (54.9%) had a first-degree family member with BC/OC. Twenty-six patients (51%) at the time of sampling were in menopause and 13 (25.5%) were smokers (
We evaluated the association among
The presence of
BC family history (either direct or indirect) did not influence the expression of the three genes; while the direct family history of cancer, other than BC, was significantly associated with the increased expression of
The Mann-Whitney U tests was performed to compare differences in gene expression levels between groups of controls without family history of cancer (n=43) and cases (n=51). BER genes showed lower expression levels in cases [0.58 (0.32-1.72); 0.93 (0.48-4.36)] than controls without cancer family history [1.04 (0.50-1.88); 1.91 (0.82-3.09)]; in particular, this difference resulted significant for
The results indicated that
Interestingly,
In summary, we observed a significant and positive correlation between gene expression of
In this study we aimed to evaluate the involvement of two BER glycosylases (OGG1 and MUTYH) in the predisposition to breast and OC. We also investigated the correlation among
Since BRCA1 is involved in oxidative stress regulation and BER after oxidative damage (
In this series we identified: 4 novel variants and one of them (in
It is interesting to note that the three novel missense mutations were found in three out of eight OC/BOC patients with OC family history (
The BC, as extra-colic manifestation, occurred in 18% of female patients affected by MUTYH-associated polyposis (MAP) (
The presence of
The time lag between diagnosis of cancer and sampling, menopausal status, and cigarette smoking did not influence the median expression of
It is shown that OGG1 is involved in the acute and systemic inflammatory response that may favor carcinogenesis (
These data, obtained from the mRNA analysis on PBMC, are very interesting and deserves further studies also on the MUTYH role in the sphere of immune functions, since the loss of this gene appears to be associated with immunosuppression and impairment of the inflammatory response (
In this study the expression of
Wild-type BRCA1 expression suppresses basal and H2O2-induced ROS production in breast and ovarian cell models (
This study highlights that germline
Relationship among mutational spectrum, gene expression and demographic characteristics, could improve the genetic diagnosis performing predictive testing of at-risk individuals belonging to families with reduced mRNA expression regardless of presence of mutation. Finally, an accurate evaluation of the reduced expression of
Not applicable.
The present study was supported by the Italian Ministry of Instruction, University and Research (grant nos. AT-Ricerca 2017Battista, AT-Ricerca2018Aceto and AT-Ricerca2018Curia).
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
CM designed the present study, performed mutation screening, RT-qPCR analyses and wrote the manuscript. MDN performed statistical calculations. SV prepared samples and performed genetic analysis. AC and MCC critically reviewed the manuscript. LS, EC, PDG and PB collected and evaluated the clinical data. GMA designed and coordinated the present study, reviewed all genetic and clinical data and drafted the manuscript. All authors read and approved the final manuscript.
All participants provided their written informed consent after verbal counselling. The study was approved by the Ethics Committee of the University ‘G. d'Annunzio’ of Chieti.
Not applicable.
The authors declare that they have no competing interests.
A, |
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Frequency n (%) | |||||||
Nucleotide change(s) | Effect | SNP | Clinical significance |
Cases (n=59) | Controls (n=120) | Word population MAF |
Mutpred value |
c.36+11C>T | - | rs2275602 | VUS | 1 (1.7) | 0 (0) | 0.01 | - |
c.53C>T | Pro18Leu | rs79777494 | VUS | 1 (1.7) | 0 (0) | <0.01 | - |
c.64G>A | Val22Met | rs3219484 | N | 2 (3.4) | - | 0.02 | - |
c.74G>A | Gly25Asp | rs75321043 | VUS | 1 (1.7) | 0 (0) | <0.01 | - |
c.157+30A>G | - | rs3219485 | N | 2 (3.4) | - | 0.01 | - |
c.504+35A>G | - | rs3219487 | N | 8 (13.6) | - | 0.06 | - |
c.701T>A | Val234Gly | - | D/Novel | 1 (1.7) | 0 (0) | - | 0.798 |
c.1014 G>C | Gln338His | rs3219489 | N | 19( |
- | 0.31 | - |
c.1171 G>T | Val390Leu | - | Novel | 1 (1.7) | 0 (0) | - | 0.335 |
c.1431G>C | Thr477Thr | rs74318065 | N | 1 (1.7) | 0 (0) | 0.01 | - |
c.1477-40C>G | - | rs3219493 | N | 5 (8.5) | - | 0.06 | - |
B, |
|||||||
Frequency n (%) | |||||||
Nucleotide change(s) | Effect | SNP | Clinical significance |
Cases (n=59) | Controls (n=120) | Word population MAF |
Mutpred value |
c.137 G>A | Arg46Gln | rs104893751 | D | 1 (1.7) | 0 (0) | <0.01 | - |
c.253G>A | Ala85Thr | rs17050550 | VUS | 1 (1.7) | 0 (0) | <0.01 | - |
c.384 G>A | Gln128Gln | - | Novel | 1 (1.7) | 0 (0) | - | - |
c.667G>A | Ala223Thr | - | Novel | 1 (1.7) | 0 (0) | - | 0.095 |
c.899G>A | Gly300Glu | rs548981683 | VUS | 1 (1.7) | 0 (0) | <0.01 | - |
c.923 G>A | Gly308Glu | rs113561019 | VUS | 1 (1.7) | 0 (0) | <0.01 | - |
c.977 C>G | Ser326Cys | rs1052133 | N | 23( |
- | 0.30 | - |
aResults based on the ClinVar-NCBI database.
bResults based on Ensembl genome browser 9. The pathogenicity of novel missense mutations was predicted using MutPred2 with a cut-off value of 0.61. D, deleterious; N, most likely neutral; VUS, variant of unknown clinical significance; SNP, single polymorphic nucleotide; MAF, minor allele frequency.
Clinical and molecular characteristics of cases carrying rare
Case code | Age range at diagnosis (years) | Cancer type | Direct BC/OC transmission | Family history | ||
---|---|---|---|---|---|---|
B12 | 30-35 | - | Gly197Glu | BC | No | - |
B17 | 25-30 | - | Gly300Glu | BC | No | - |
B28 | 40-45 | - | BC | No | - | |
B48 | 40-45 | - | OC | No | OC | |
B58 | 40-45 | - | Arg46Gln |
BC | Yes | BC, Leu |
B62 | 30-35 | - | OC | Yes | OC | |
B66 | 55-60 | c.36 + 11 C>T | - | BC/TC | No | BC, CC |
Pro18Leu | ||||||
Gly25Asp | ||||||
B75 | 45-50 | - | Ala85Thr | BC | No | BC, OC |
B105 |
45-50 | - | BC/OC | Yes | OC |
aPathogenic variants.
bPutative pathogenic variants predicted by MutPred2 software;
cBRCA1 mutation carrier (Glu1373Ter). Novel variants are in bold. BC, breast cancer; OC, ovarian cancer; TC, thyroid cancer; CC, colon cancer; Leu, leukemia.
Demographic characteristics of cases employed for gene expression analysis (n=51).
Variable | Value |
---|---|
Age at diagnosis, years (mean±SD) | 45.2±11.9 |
Age at sampling, years (mean±SD) | 48.6±12.9 |
Menopause, n (%) | 26 (51.0) |
Cigarette smoking, n (%) | 13 (25.5) |
Type of cancer, n (%) | |
BC | 47 (92.2) |
OC | 4 (7.8) |
Family history n (%) | |
BC/OC | |
Direct | 28 (54.9) |
Indirect | 10 (19.6) |
Other cancers | |
Direct | 16 (31.4) |
Indirect | 5 (9.8) |
BC, breast cancer; OC, ovarian cancer.
Comparison of median gene expression between cases and controls without a family history of cancer.
Variable | Cases (n=51) | Controls without a family history of cancer (n=43) | Mann-Whitney P-value |
---|---|---|---|
Age at sampling, mean±SD | 48.60±12.88 | 50.09±7.49 | 0.159 |
0.58 (0.32-1.72) | 1.04 (0.50-1.88) | 0.035 | |
0.93 (0.48-4.36) | 1.91 (0.82-3.09) | 0.358 | |
0.09 (0.03-0.32) | 0.14 (0.07-0.21) | 0.297 |
aStudent's t test for unpaired data. IQR, interquartile range.
Correlation among
Group | Rho di Spearman | P-value | Rho di Spearman | P-value |
---|---|---|---|---|
Cases | 0.406 | 0.004 | ||
|
0.186 | 0.200 | ||
|
0.471 | 0.001 | ||
Controls without a family history of cancer | 0.036 | 0.840 | ||
|
0.164 | 0.361 | ||
|
0.445 | 0.011 |