The "extreme phenotype approach" applied to male breast cancer allows the identification of rare variants of ATR as potential breast cancer susceptibility alleles

In oncogenetics, some patients could be considered as "extreme phenotypes", such as those with very early onset presentation or multiple primary malignancies, unusually high numbers of cancers of the same spectrum or rare cancer types in the same parental branch. For these cases, a genetic predisposition is very likely, but classical candidate gene panel analyses often and frustratingly remains negative. In the framework of the EX2TRICAN project, exploring unresolved extreme cancer phenotypes, we applied exome sequencing on rare familial cases with male breast cancer, identifying a novel pathogenic variant of ATR (p.Leu1808*). ATR has already been suspected as being a predisposing gene to breast cancer in women. We next identified 3 additional ATR variants in a cohort of both male and female with early onset and familial breast cancers (c.7762-2A>C; c.2078+1G>A; c.1A>G). Further molecular and cellular investigations showed impacts on transcripts for variants affecting splicing sites and reduction of ATR expression and phosphorylation of the ATR substrate CHEK1. This work further demonstrates the interest of an extended genetic analysis such as exome sequencing to identify very rare variants that can play a role in cancer predisposition in extreme phenotype cancer cases unexplained by classical cancer gene panels testing

RIP-hCx36SNP<i>rs3743123</i> mice display β-cell loss and impaired glucose control over time.

<p><b>A,</b> Immunofluorescence images of islets of RIP-hCx36<i>rs3743123</i> and RIP-hCx36WT mice. In young mice, islets display a normal morphology with α cells in the periphery and β cells at the center. Aging does not alter the islets of RIP-hCx36WT mice but determines loss of organization of α and β cells in RIP-hCx36<i>rs3743123</i> mice. Scale bar 10 μm. <b>B,</b> Number of β cells per islet section in islets of RIP-hCx36WT and RIP-hCx36<i>rs3743123</i> mice. Compared to 1 month old littermates, 5 month old RIP-hCx36<i>rs3743123</i> mice display a decrease of β cells per islet section. Data show means + SEM of islets of 3–9 mice per group. <b>C,</b> Glycaemia curve of RIP-hCx36WT and RIP-hCx36<i>rs3743123</i> mice. RIP-hCx36 <i>rs3743123</i> show increase in blood glucose with aging. D, Area under the entire glycaemia curve. *P ≤ 0.05**P ≤ 0.01***P ≤ 0.001**** P ≤ 0.0001 (Student t-test with Welch’s correction).</p

SNP <i>rs3743123</i> alters the distribution of Cx36 at the cell membrane.

<p><b>A</b>, In HeLa cells stably transfected with the wild type form of Cx36 (left) the protein shows a spotted distribution (green) at the cell membrane. After transfection of the SNP <i>rs3743123</i> variant (right) the spotted distribution of the protein alternates with regions of continuous membrane staining. Scale bar, 20 μm. <b>B</b>, Freeze-fracture electron microscopy revealed polygonal and array-shaped gap junction plaques (arrow heads) in HeLa cells transfected with either the WT or variant form of Cx36 (right). Scale bar, 85 nm. <b>C,</b> Distribution of different gap junction patterns (polygonal, linear, array shaped) and <b>D,</b> numbers of particles (connexons) per plaque in HeLa cells transfected with the wild type (n = 37) and SNP <i>rs3743123</i> forms of Cx36 (n = 48). Images and mean + SEM data are from three independent clones stably expressing either the wild type or the SNP <i>rs3743123</i> form of the protein.</p

SNP <i>rs3743123</i> reduces hCx36 coupling between adjacent cells.

<p>SNP <i>rs3743123</i> reduces hCx36 coupling between adjacent cells.</p

SNP <i>rs3743123</i> modify hCx36 expression and its distribution at the beta cell membrane.

<p><b>A,</b> The volume density (Vv) of Cx36 decreases postnatally in RipβglohCx36 SNP <i>rs3743123</i> mice. <b>B,</b> The numeric density (Nv) of Cx36 is reduced with time in RIP-hCx36<i>rs3743123</i> mice. <b>C,</b> The length of the Cx36 plaques was also decreased postnatally in these mice. Data show means + SEM of islets of three mice per group. *P ≤ 0.05**P ≤ 0.01***P ≤ 0.001**** P ≤ 0.0001 versus 1 month old litters.</p

SNP <i>rs3743123</i> does not alter the stability of Cx36 mRNA.

<p>Exposure to 5 μg/ml actinomycin D, revealed comparable levels of Cx36 mRNA at different time points in 3 independent clones of HeLa cells stably transfected with either the wild type (black symbols) or the SNP <i>rs3743123</i> form of hCx36 (red symbols).</p

The "extreme phenotype approach" applied to male breast cancer allows the identification of rare variants of ATR as potential breast cancer susceptibility alleles

In oncogenetics, some patients could be considered as "extreme phenotypes", such as those with very early onset presentation or multiple primary malignancies, unusually high numbers of cancers of the same spectrum or rare cancer types in the same parental branch. For these cases, a genetic predisposition is very likely, but classical candidate gene panel analyses often and frustratingly remains negative. In the framework of the EX2TRICAN project, exploring unresolved extreme cancer phenotypes, we applied exome sequencing on rare familial cases with male breast cancer, identifying a novel pathogenic variant of ATR (p.Leu1808*). ATR has already been suspected as being a predisposing gene to breast cancer in women. We next identified 3 additional ATR variants in a cohort of both male and female with early onset and familial breast cancers (c.7762-2A>C; c.2078+1G>A; c.1A>G). Further molecular and cellular investigations showed impacts on transcripts for variants affecting splicing sites and reduction of ATR expression and phosphorylation of the ATR substrate CHEK1. This work further demonstrates the interest of an extended genetic analysis such as exome sequencing to identify very rare variants that can play a role in cancer predisposition in extreme phenotype cancer cases unexplained by classical cancer gene panels testing

Erratum to 'Predominance of healthcare-associated cases among episodes of community-onset bacteraemia due to extended-spectrum β-lactamase-producing Enterobacteriaceae' [International Journal of Antimicrobial Agents 49/1 67-73]

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