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Detection of Ultra-Rare Mitochondrial Mutations in Breast Stem Cells by Duplex Sequencing.

Ahn EH, Hirohata K, Kohrn BF, Fox EJ, Chang CC, Loeb LA - PLoS ONE (2015)

Bottom Line: The overall rare mutation frequency is significantly lower in stem cells than in the corresponding non-stem cells.Four mutations found within the MT-ND5 gene (m.12684G>A, m.12705C>T, m.13095T>C, m.13105A>G) are present in all groups of stem and non-stem cells.Two mutations (m.8567T>C, m.10547C>G) are found only in non-stem cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Washington, Seattle, Washington, United States of America; Institute of Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
Long-lived adult stem cells could accumulate non-repaired DNA damage or mutations that increase the risk of tumor formation. To date, studies on mutations in stem cells have concentrated on clonal (homoplasmic) mutations and have not focused on rarely occurring stochastic mutations that may accumulate during stem cell dormancy. A major challenge in investigating these rare mutations is that conventional next generation sequencing (NGS) methods have high error rates. We have established a new method termed Duplex Sequencing (DS), which detects mutations with unprecedented accuracy. We present a comprehensive analysis of mitochondrial DNA mutations in human breast normal stem cells and non-stem cells using DS. The vast majority of mutations occur at low frequency and are not detectable by NGS. The most prevalent point mutation types are the C>T/G>A and A>G/T>C transitions. The mutations exhibit a strand bias with higher prevalence of G>A, T>C, and A>C mutations on the light strand of the mitochondrial genome. The overall rare mutation frequency is significantly lower in stem cells than in the corresponding non-stem cells. We have identified common and unique non-homoplasmic mutations between non-stem and stem cells that include new mutations which have not been reported previously. Four mutations found within the MT-ND5 gene (m.12684G>A, m.12705C>T, m.13095T>C, m.13105A>G) are present in all groups of stem and non-stem cells. Two mutations (m.8567T>C, m.10547C>G) are found only in non-stem cells. This first genome-wide analysis of mitochondrial DNA mutations may aid in characterizing human breast normal epithelial cells and serve as a reference for cancer stem cell mutation profiles.

No MeSH data available.


Related in: MedlinePlus

Frequencies of each type of rare mutations in the whole mtDNA.Types of rare point mutations and insertions and deletions (INDELs) in the whole mtDNA were determined using DS. Data are from human breast normal epithelial cells (non-stem vs. stem) developed from women (ID #11, #30, and #31). Error bars represent the Wilson Score 95% confidence intervals. Significant differences in mutation frequencies between the two groups are indicated (p <0.005 (**) by the 2-sample test for equality of proportions with continuity correction).
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pone.0136216.g002: Frequencies of each type of rare mutations in the whole mtDNA.Types of rare point mutations and insertions and deletions (INDELs) in the whole mtDNA were determined using DS. Data are from human breast normal epithelial cells (non-stem vs. stem) developed from women (ID #11, #30, and #31). Error bars represent the Wilson Score 95% confidence intervals. Significant differences in mutation frequencies between the two groups are indicated (p <0.005 (**) by the 2-sample test for equality of proportions with continuity correction).

Mentions: Duplex Sequencing enables the identification of 12 point mutation (substitution) types as well as insertions and deletions (INDELs). The A>G/T>C and C>T/G>A transitions are the most prevalent types of rare mutations (Fig 2A–2F). INDELs are found infrequently.


Detection of Ultra-Rare Mitochondrial Mutations in Breast Stem Cells by Duplex Sequencing.

Ahn EH, Hirohata K, Kohrn BF, Fox EJ, Chang CC, Loeb LA - PLoS ONE (2015)

Frequencies of each type of rare mutations in the whole mtDNA.Types of rare point mutations and insertions and deletions (INDELs) in the whole mtDNA were determined using DS. Data are from human breast normal epithelial cells (non-stem vs. stem) developed from women (ID #11, #30, and #31). Error bars represent the Wilson Score 95% confidence intervals. Significant differences in mutation frequencies between the two groups are indicated (p <0.005 (**) by the 2-sample test for equality of proportions with continuity correction).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4549069&req=5

pone.0136216.g002: Frequencies of each type of rare mutations in the whole mtDNA.Types of rare point mutations and insertions and deletions (INDELs) in the whole mtDNA were determined using DS. Data are from human breast normal epithelial cells (non-stem vs. stem) developed from women (ID #11, #30, and #31). Error bars represent the Wilson Score 95% confidence intervals. Significant differences in mutation frequencies between the two groups are indicated (p <0.005 (**) by the 2-sample test for equality of proportions with continuity correction).
Mentions: Duplex Sequencing enables the identification of 12 point mutation (substitution) types as well as insertions and deletions (INDELs). The A>G/T>C and C>T/G>A transitions are the most prevalent types of rare mutations (Fig 2A–2F). INDELs are found infrequently.

Bottom Line: The overall rare mutation frequency is significantly lower in stem cells than in the corresponding non-stem cells.Four mutations found within the MT-ND5 gene (m.12684G>A, m.12705C>T, m.13095T>C, m.13105A>G) are present in all groups of stem and non-stem cells.Two mutations (m.8567T>C, m.10547C>G) are found only in non-stem cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Washington, Seattle, Washington, United States of America; Institute of Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
Long-lived adult stem cells could accumulate non-repaired DNA damage or mutations that increase the risk of tumor formation. To date, studies on mutations in stem cells have concentrated on clonal (homoplasmic) mutations and have not focused on rarely occurring stochastic mutations that may accumulate during stem cell dormancy. A major challenge in investigating these rare mutations is that conventional next generation sequencing (NGS) methods have high error rates. We have established a new method termed Duplex Sequencing (DS), which detects mutations with unprecedented accuracy. We present a comprehensive analysis of mitochondrial DNA mutations in human breast normal stem cells and non-stem cells using DS. The vast majority of mutations occur at low frequency and are not detectable by NGS. The most prevalent point mutation types are the C>T/G>A and A>G/T>C transitions. The mutations exhibit a strand bias with higher prevalence of G>A, T>C, and A>C mutations on the light strand of the mitochondrial genome. The overall rare mutation frequency is significantly lower in stem cells than in the corresponding non-stem cells. We have identified common and unique non-homoplasmic mutations between non-stem and stem cells that include new mutations which have not been reported previously. Four mutations found within the MT-ND5 gene (m.12684G>A, m.12705C>T, m.13095T>C, m.13105A>G) are present in all groups of stem and non-stem cells. Two mutations (m.8567T>C, m.10547C>G) are found only in non-stem cells. This first genome-wide analysis of mitochondrial DNA mutations may aid in characterizing human breast normal epithelial cells and serve as a reference for cancer stem cell mutation profiles.

No MeSH data available.


Related in: MedlinePlus