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Multigene panel analysis identified germline mutations of DNA repair genes in breast and ovarian cancer.

Hirotsu Y, Nakagomi H, Sakamoto I, Amemiya K, Oyama T, Mochizuki H, Omata M - Mol Genet Genomic Med (2015)

Bottom Line: These subjects included 11 BRCA1/2 mutation-positive cases and 144 negative cases.Of these, three patients (1.9%) had pathogenic mutations in ATM, MRE11A, or MSH6, all of which have a central role in DNA repair and the mismatch repair pathway.The MSH6 splice-site mutation (IVS6+1G>T) was predicted to be pathogenic, as demonstrated by in vitro and immunohistochemical analyses.

View Article: PubMed Central - PubMed

Affiliation: Genome Analysis Center, Yamanashi Prefectural Central Hospital 1-1-1 Fujimi, Kofu, Yamanashi, 400-8506, Japan.

ABSTRACT
Approximately 5-10% of all breast and/or ovarian cancer cases are considered as inherited. BRCA1 and BRCA2 tumor suppressor genes account for a high penetrance of hereditary cases, but familial cases without mutations in these genes can also occur. Despite their low penetrance, other hereditary cancer-related genes are known to be associated with breast and ovarian cancer risk. However, the extent to which these genes prevail in breast and ovarian cancer remains to be elucidated. To estimate the frequency of mutations in these predisposition genes, we analyzed the germline mutations of 25 hereditary cancer-related genes in 155 patients using targeted next-generation sequencing. These subjects included 11 BRCA1/2 mutation-positive cases and 144 negative cases. Of these, three patients (1.9%) had pathogenic mutations in ATM, MRE11A, or MSH6, all of which have a central role in DNA repair and the mismatch repair pathway. The MSH6 splice-site mutation (IVS6+1G>T) was predicted to be pathogenic, as demonstrated by in vitro and immunohistochemical analyses. These results suggested deficiencies in cellular DNA repair functions result in the development of breast and ovarian cancer.

No MeSH data available.


Related in: MedlinePlus

MSH6 splice-site mutation is pathogenic. (A) Schematic of MSH6 transcript. The arrows indicate the primers used in this analysis, located in exon (Ex) 5 and exon 7. (B) Reverse transcriptase polymerase chain reaction analysis of RNA from peripheral blood of a control (Con) and patient (Pt) with ovarian and endometrial cancer harboring the MSH6 IVS6+1G>T splice-site mutation. Gel electrophoresis showing PCR fragments (∼356 bp in length). DNA marker (M) was loaded in the left lane. ACTB was used as an internal control. (C) Representative images of MSI profiles. Ovarian tumor showing microsatellite instability for BAT25, BAT26, and D2S123. C, control; T, tumor tissue; IVS, intervening sequence; MSI, microsatellite instability.
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fig03: MSH6 splice-site mutation is pathogenic. (A) Schematic of MSH6 transcript. The arrows indicate the primers used in this analysis, located in exon (Ex) 5 and exon 7. (B) Reverse transcriptase polymerase chain reaction analysis of RNA from peripheral blood of a control (Con) and patient (Pt) with ovarian and endometrial cancer harboring the MSH6 IVS6+1G>T splice-site mutation. Gel electrophoresis showing PCR fragments (∼356 bp in length). DNA marker (M) was loaded in the left lane. ACTB was used as an internal control. (C) Representative images of MSI profiles. Ovarian tumor showing microsatellite instability for BAT25, BAT26, and D2S123. C, control; T, tumor tissue; IVS, intervening sequence; MSI, microsatellite instability.

Mentions: The splice-site mutation in MSH6 (IVS6+1G>T) disrupts the canonical GT dinucleotide at the 5′ splice site and has not been reported previously to our knowledge. In addition, loss of MSH6 promotes tumorigenesis in colorectal cancer is well known, but not in ovarian cancer. To examine whether this G to T substitution influences transcriptional processing, we synthesized MSH6 complementary DNA (cDNA) from peripheral blood RNA and performed PCR with primers flanking exon 6 (Fig.3A). The PCR product sizes were ∼356 bp in length, which represented the normal transcript of exon 5–7 expressed in both controls and patients (Fig.3B). However, MSH6 mRNA expression levels were apparently reduced in patients compared with controls (Fig.3B). These results suggested the splice-site mutation introduced premature termination codons and subsequently mRNA was degraded by nonsense-mediated decay. We next performed MSI analysis and immunohistochemistry staining for MSH6. This revealed that the tumors had MSI-high and lacked MSH6 protein expression (Figs.3C, 4). These results showed the MSH6 IVS6+1G>T variant disrupted mismatch-repair function and promoted tumorigenesis.


Multigene panel analysis identified germline mutations of DNA repair genes in breast and ovarian cancer.

Hirotsu Y, Nakagomi H, Sakamoto I, Amemiya K, Oyama T, Mochizuki H, Omata M - Mol Genet Genomic Med (2015)

MSH6 splice-site mutation is pathogenic. (A) Schematic of MSH6 transcript. The arrows indicate the primers used in this analysis, located in exon (Ex) 5 and exon 7. (B) Reverse transcriptase polymerase chain reaction analysis of RNA from peripheral blood of a control (Con) and patient (Pt) with ovarian and endometrial cancer harboring the MSH6 IVS6+1G>T splice-site mutation. Gel electrophoresis showing PCR fragments (∼356 bp in length). DNA marker (M) was loaded in the left lane. ACTB was used as an internal control. (C) Representative images of MSI profiles. Ovarian tumor showing microsatellite instability for BAT25, BAT26, and D2S123. C, control; T, tumor tissue; IVS, intervening sequence; MSI, microsatellite instability.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4585454&req=5

fig03: MSH6 splice-site mutation is pathogenic. (A) Schematic of MSH6 transcript. The arrows indicate the primers used in this analysis, located in exon (Ex) 5 and exon 7. (B) Reverse transcriptase polymerase chain reaction analysis of RNA from peripheral blood of a control (Con) and patient (Pt) with ovarian and endometrial cancer harboring the MSH6 IVS6+1G>T splice-site mutation. Gel electrophoresis showing PCR fragments (∼356 bp in length). DNA marker (M) was loaded in the left lane. ACTB was used as an internal control. (C) Representative images of MSI profiles. Ovarian tumor showing microsatellite instability for BAT25, BAT26, and D2S123. C, control; T, tumor tissue; IVS, intervening sequence; MSI, microsatellite instability.
Mentions: The splice-site mutation in MSH6 (IVS6+1G>T) disrupts the canonical GT dinucleotide at the 5′ splice site and has not been reported previously to our knowledge. In addition, loss of MSH6 promotes tumorigenesis in colorectal cancer is well known, but not in ovarian cancer. To examine whether this G to T substitution influences transcriptional processing, we synthesized MSH6 complementary DNA (cDNA) from peripheral blood RNA and performed PCR with primers flanking exon 6 (Fig.3A). The PCR product sizes were ∼356 bp in length, which represented the normal transcript of exon 5–7 expressed in both controls and patients (Fig.3B). However, MSH6 mRNA expression levels were apparently reduced in patients compared with controls (Fig.3B). These results suggested the splice-site mutation introduced premature termination codons and subsequently mRNA was degraded by nonsense-mediated decay. We next performed MSI analysis and immunohistochemistry staining for MSH6. This revealed that the tumors had MSI-high and lacked MSH6 protein expression (Figs.3C, 4). These results showed the MSH6 IVS6+1G>T variant disrupted mismatch-repair function and promoted tumorigenesis.

Bottom Line: These subjects included 11 BRCA1/2 mutation-positive cases and 144 negative cases.Of these, three patients (1.9%) had pathogenic mutations in ATM, MRE11A, or MSH6, all of which have a central role in DNA repair and the mismatch repair pathway.The MSH6 splice-site mutation (IVS6+1G>T) was predicted to be pathogenic, as demonstrated by in vitro and immunohistochemical analyses.

View Article: PubMed Central - PubMed

Affiliation: Genome Analysis Center, Yamanashi Prefectural Central Hospital 1-1-1 Fujimi, Kofu, Yamanashi, 400-8506, Japan.

ABSTRACT
Approximately 5-10% of all breast and/or ovarian cancer cases are considered as inherited. BRCA1 and BRCA2 tumor suppressor genes account for a high penetrance of hereditary cases, but familial cases without mutations in these genes can also occur. Despite their low penetrance, other hereditary cancer-related genes are known to be associated with breast and ovarian cancer risk. However, the extent to which these genes prevail in breast and ovarian cancer remains to be elucidated. To estimate the frequency of mutations in these predisposition genes, we analyzed the germline mutations of 25 hereditary cancer-related genes in 155 patients using targeted next-generation sequencing. These subjects included 11 BRCA1/2 mutation-positive cases and 144 negative cases. Of these, three patients (1.9%) had pathogenic mutations in ATM, MRE11A, or MSH6, all of which have a central role in DNA repair and the mismatch repair pathway. The MSH6 splice-site mutation (IVS6+1G>T) was predicted to be pathogenic, as demonstrated by in vitro and immunohistochemical analyses. These results suggested deficiencies in cellular DNA repair functions result in the development of breast and ovarian cancer.

No MeSH data available.


Related in: MedlinePlus