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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

Non-homoplasmic mutation frequency in the whole mtDNA.Point mutations in the whole mtDNA were determined using DS. Data are from human breast normal epithelial cells (non-stem (N) vs. stem) developed from women (ID #11, #30, and #31). (A-B) The cutoffs of mutation frequency (% clonality) used for rare, low-heteroplasmic, high-heteroplasmic, and homoplasmic mutations are: 0−0.5%, >0.5−20%, >20−<95%, and 95−100%, respectively. (A) The distribution (%) of rare, low-heteroplasmic, high-heteroplasmic, and homoplasmic mutations is calculated as numbers of corresponding specific-clonality range mutations per numbers of total (0–100% clonality) mutations. (B-C) Error bars represent the Wilson Score 95% confidence intervals. (B) Significant differences in rare mutation frequencies between non-stem and stem cells from two women (ID #11 and #30) are indicated (p <0.05 (*) by the 2-sample test for equality of proportions with continuity correction).
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pone.0136216.g001: Non-homoplasmic mutation frequency in the whole mtDNA.Point mutations in the whole mtDNA were determined using DS. Data are from human breast normal epithelial cells (non-stem (N) vs. stem) developed from women (ID #11, #30, and #31). (A-B) The cutoffs of mutation frequency (% clonality) used for rare, low-heteroplasmic, high-heteroplasmic, and homoplasmic mutations are: 0−0.5%, >0.5−20%, >20−<95%, and 95−100%, respectively. (A) The distribution (%) of rare, low-heteroplasmic, high-heteroplasmic, and homoplasmic mutations is calculated as numbers of corresponding specific-clonality range mutations per numbers of total (0–100% clonality) mutations. (B-C) Error bars represent the Wilson Score 95% confidence intervals. (B) Significant differences in rare mutation frequencies between non-stem and stem cells from two women (ID #11 and #30) are indicated (p <0.05 (*) by the 2-sample test for equality of proportions with continuity correction).

Mentions: Most point mutations are found at less than 0.5% clonality (Fig 1A) and thus would not be accurately scored by conventional NGS. However, these rare variants are accurately detected by Duplex Sequencing. In two (women ID #11 and #30) of the three paired groups of normal cells, the frequencies of rare mutations in stem cells is significantly lower than in those of non-stem cells (Fig 1B: p-values 0.015 and 0.037 for ID #11 and #30, respectively). In contrast, there is no significant difference between the frequencies of mutations greater than 0.5% (Fig 1C).


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)

Non-homoplasmic mutation frequency in the whole mtDNA.Point mutations in the whole mtDNA were determined using DS. Data are from human breast normal epithelial cells (non-stem (N) vs. stem) developed from women (ID #11, #30, and #31). (A-B) The cutoffs of mutation frequency (% clonality) used for rare, low-heteroplasmic, high-heteroplasmic, and homoplasmic mutations are: 0−0.5%, >0.5−20%, >20−<95%, and 95−100%, respectively. (A) The distribution (%) of rare, low-heteroplasmic, high-heteroplasmic, and homoplasmic mutations is calculated as numbers of corresponding specific-clonality range mutations per numbers of total (0–100% clonality) mutations. (B-C) Error bars represent the Wilson Score 95% confidence intervals. (B) Significant differences in rare mutation frequencies between non-stem and stem cells from two women (ID #11 and #30) are indicated (p <0.05 (*) 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.g001: Non-homoplasmic mutation frequency in the whole mtDNA.Point mutations in the whole mtDNA were determined using DS. Data are from human breast normal epithelial cells (non-stem (N) vs. stem) developed from women (ID #11, #30, and #31). (A-B) The cutoffs of mutation frequency (% clonality) used for rare, low-heteroplasmic, high-heteroplasmic, and homoplasmic mutations are: 0−0.5%, >0.5−20%, >20−<95%, and 95−100%, respectively. (A) The distribution (%) of rare, low-heteroplasmic, high-heteroplasmic, and homoplasmic mutations is calculated as numbers of corresponding specific-clonality range mutations per numbers of total (0–100% clonality) mutations. (B-C) Error bars represent the Wilson Score 95% confidence intervals. (B) Significant differences in rare mutation frequencies between non-stem and stem cells from two women (ID #11 and #30) are indicated (p <0.05 (*) by the 2-sample test for equality of proportions with continuity correction).
Mentions: Most point mutations are found at less than 0.5% clonality (Fig 1A) and thus would not be accurately scored by conventional NGS. However, these rare variants are accurately detected by Duplex Sequencing. In two (women ID #11 and #30) of the three paired groups of normal cells, the frequencies of rare mutations in stem cells is significantly lower than in those of non-stem cells (Fig 1B: p-values 0.015 and 0.037 for ID #11 and #30, respectively). In contrast, there is no significant difference between the frequencies of mutations greater than 0.5% (Fig 1C).

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