Detection of mutational frequency and related methods

Abstract

Methods are provided herein for measuring the mutational frequency of a DNA molecule in cells, for example stem cells or hematopoietic cells such as CD34 + cells or granulocytes. The method includes sequencing corresponding regions of mtDNA from a set of hematopoietic cells, or a set of clonal populations of hematopoietic cells, and comparing the sequence of the corresponding regions of mtDNA from the cells, or clonal populations of cells. The method also includes the comparison of mtDNA sequences with genomic DNA sequences. Also provided are methods for screening for an agent that has a mutagenic effect on a cell. The method includes contacting, or treating, clonal populations of cells with an agent and comparing the sequence of the mtDNA obtained from the treated clonal populations of cells, with the sequence of the corresponding region of mtDNA obtained from a control clonal populations of cells.

1 . A method of screening for an agent that increases the mutational frequency of a hematopoietic cell, comprising: 
 contacting isolated hematopoietic cells with the agent to produce treated hematopoietic cells, wherein the isolated hematopoietic cells each contain at least one mitochondrion comprising mitochondrial DNA;    determining the mutational frequency of the treated hematopoietic cells using a method of measuring a mutational frequency of a mitochondrial DNA sequence, wherein measuring comprises: 
 separately sequencing the same regions of the mitochondrial DNA from individual, non-clonally expanded treated hematopoietic cells or from individual, clonal populations of the treated hematopoietic cells; and  
   determining a proportion of the treated hematopoietic cells exhibiting mitochondrial DNA heterogeneity within the sequenced regions of the mitochondrial DNA, wherein the proportion corresponds to the mutational frequency of the mitochondrial DNA sequence, thereby determining the mutational frequency of the treated hematopoietic cells; and    comparing the mutational frequency of the treated hematopoietic cells to a mutational frequency of hematopoietic cells that were not contacted with the agent, wherein an increase in the mutational frequency of the treated hematopoietic cells, compared to the mutational frequency of hematopoietic cells that were not contacted with the agent, indicates that the agent increases the mutational frequency of the hematopoietic cell, thereby screening for the agent that increases the mutational frequency of the hematopoietic cell.    
     
     
         2 . The method of  claim 1 , further comprising: 
 expanding individual treated hematopoietic cells into individual clonal populations of treated hematopoietic cells;    extracting mitochondrial DNA from each of the clonal populations of treated hematopoietic cells; and    determining a proportion of the clonal populations of treated hematopoietic cells exhibiting mitochondrial DNA heterogeneity within the sequenced regions of the mitochondrial DNA, wherein the proportion corresponds to the mutational frequency of the mitochondrial DNA sequence.    
     
     
         3 . The method of  claim 1 , wherein the agent comprises a small molecule, chemical compound, a radioisotope, a protein, a peptide, or a peptidomimetic.  
     
     
         4 . The method of  claim 3 , wherein the chemical compound comprises a chemotherapeutic drug.  
     
     
         5 . The method of  claim 1 , wherein isolated hematopoietic cells comprise CD34 +  cells, granulocytes, monocytes or macrophages.  
     
     
         6 . The method of  claim 1 , wherein the hematopoietic cells are isolated from bone marrow, peripheral blood, or umbilical cord blood.  
     
     
         7 . The method of  claim 1 , wherein the mutation comprises a point mutation, a polymorphism, a frame-shift mutation, a missense mutation, a nonsense mutation, a silent mutation, or a deletion mutation.  
     
     
         8 . The method of  claim 1 , wherein the mutation is in a homopolymeric C tract of a mitochondrial DNA control region or in a gene in a mitochondrial DNA coding region.