A sensitive scanning technology for low frequency nuclear point mutations in human genomic DNA

Knowledge of the kinds and numbers of nuclear point mutations in human tissues is essential to the understanding of the mutation mechanisms underlying genetic diseases. However, nuclear point mutant fractions in normal humans are so low that few methods exist to measure them. We have now developed a means to scan for point mutations in 100 bp nuclear single copy sequences at mutant fractions as low as 10^–6. Beginning with about 10⁸ human cells we first enrich for the desired nuclear sequence 10 000-fold from the genomic DNA by sequence-specific hybridization coupled with a biotin–streptavidin capture system. We next enrich for rare mutant sequences 100-fold against the wild-type sequence by wide bore constant denaturant capillary electrophoresis (CDCE). The mutant-enriched sample is subsequently amplified by high fidelity PCR using fluorescein-labeled primers. Amplified mutant sequences are further enriched via two rounds of CDCE coupled with high fidelity PCR. Individual mutants, seen as distinct peaks on CDCE, are then isolated and sequenced. We have tested this approach by measuring N-methyl-N ′-nitro-N-nitrosoguanidine (MNNG)-induced point mutations in a 121 bp sequence of the adenomatous polyposis coli gene (APC) in human lymphoblastoid MT1 cells. Twelve different MNNG-induced GC→AT transitions were reproducibly observed in MNNG-treated cells at mutant fractions between 2 × 10^–6 and 9 × 10^–6. The sensitivity of this approach was limited by the fidelity of Pfu DNA polymerase, which created 14 different GC→TA transversions at a mutant fraction equivalent to ~10^–6 in the original samples. The approach described herein should be general for all DNA sequences suitable for CDCE analysis. Its sensitivity and capacity would permit detection of stem cell mutations in tissue sectors consisting of ~10⁸ cells.     

Population density of the Réunion Grey White-eye Zosterops borbonicus within the summit ecosystems of Réunion,Mascarene Islands

Assessing population density is crucial for studying the ecology and evolutionary biology of species as well as for conservation purposes. Here we used point count methods to infer population density in a single-island endemic passerine bird, the Réunion Grey White-eye Zosterops borbonicus, that displays striking evidence of differentiation at a small spatial scale. Population density was estimated at 5.17 birds ha^?1 (CL 4.85–5.50), a value somewhat higher than previously believed. This estimation provides the first detailed estimation of bird population density in the vulnerable summit ecosystems of Réunion and will possibly allow a better understanding of the evolutionary causes of this plumage colour variation.     

Clustering of mutant mitochondrial DNA copies suggests stem cells are common in human bronchial epithelium

Tissue maintenance stem cells, as opposed to transition and/or terminal cells in the epithelium, are possible progenitor cells for human tumors, but little is known about their frequency in human tissues. It occurred to us that the colonies of mutants that should be created when a stem cell mutates and transmits the rare mutation to its descendent transition and terminal cells should, given a quantitative mutation assay, define the average number of cells in a maintenance turnover unit and permit calculation of stem cell number. To test this concept we used a combination of high fidelity PCR and constant denaturant capillary electrophoresis to enumerate mitochondrial point mutations and define their number and distribution among multiple small samples of approximately one million cells containing about 400 million copies of mitochondrial DNA. The bulk of the data were best explained by a model in which most stem cells, defined here as long-lived cells, give rise to colonies of approximately 8-128 cells. In addition, we found that about 1.5% of colonies contained hundreds or even thousands of homoplasmic mutant cells. These expanded turnover units suggest the bronchial epithelium may contain large clusters of cells with mutations, and possibly phenotypic alterations as well.     

Distributions of five common point mutants in the human tracheal-bronchial epithelium

The mutations C742T, G746T, G747T in the TP53 gene and G35T in the KRAS gene have been repeatedly found in sectors of human tumors by direct DNA sequencing. The mutation G508A in the HPRT1 gene has been repeatedly found among peripheral T lymphocytes by clonal expansion under selective conditions. To discover if these mutations also occur frequently in normal tissues from which tumors arise, we have developed and validated allele-specific mismatch amplification mutation assays (MAMA) for each mutation. Reconstruction experiments demonstrated linearity in the range of 9-3000 mutant alleles among 3 x 10(6) wild-type alleles. The cumulative distributions of all negative controls established robust detection limits (P<0.05) of 34-125 mutants per 10(6) copies assayed depending on the mutation. One hundred and seventy-seven micro-anatomical samples of approximately (0.5-6)x10(6) tracheal-bronchial epithelial cells from nine non-smokers were assayed representing en toto the equivalent of approximately 1.6 human bronchial trees to the fifth bifurcation. Statistically significant mutant copy numbers were found in 257 of 463 assays. Clusters of mutant copies ranged from 10 to 1000 in 239/257 positive samples. As all five point mutations were detected at mutant fractions of >10(-5) in two or more lungs, we infer that they are mutational hotspots generated in lung epithelial stem cells. As the cancer-associated mutations did not differ in cluster size distribution from the HPRT1 mutation, we infer that none of the mutations conferred a growth advantage to somatic heterozygous clusters or maintenance turnover units. Specific mutants appeared in very large copy numbers, 1000-35,000, in 18/257 positive assays. Various hypotheses to account for the observed cluster size distributions are offered.     

Fetal-juvenile origins of point mutations in the adult human tracheal-bronchial epithelium: absence of detectable effects of age, gender or smoking status

Allele-specific mismatch amplification mutation assays (MAMA) of anatomically distinct sectors of the upper bronchial tracts of nine nonsmokers revealed many numerically dispersed clusters of the point mutations C742T, G746T, G747T of the TP53 gene, G35T of the KRAS gene and G508A of the HPRT1 gene. Assays of these five mutations in six smokers have yielded quantitatively similar results. One hundred and eighty four micro-anatomical sectors of 0.5-6x10(6) tracheal-bronchial epithelial cells represented en toto the equivalent of approximately 1.7 human smokers' bronchial trees to the fifth bifurcation. Statistically significant mutant copy numbers above the 95% upper confidence limits of historical background controls were found in 198 of 425 sector assays. No significant differences (P=0.1) for negative sector fractions, mutant fractions, distributions of mutant cluster size or anatomical positions were observed for smoking status, gender or age (38-76 year). Based on the modal cluster size of mitochondrial point mutants, the size of the adult bronchial epithelial maintenance turnover unit was estimated to be about 32 cells. When data from all 15 lungs were combined the log2 of nuclear mutant cluster size plotted against log2 of the number of clusters of a given cluster size displayed a slope of approximately 1.1 over a range of cluster sizes from approximately 2(6) to 2(15) mutant copies. A parsimonious interpretation of these nuclear and previously reported data for lung epithelial mitochondrial point mutant clusters is that they arose from mutations in stem cells at a high but constant rate per stem cell doubling during at least ten stem cell doublings of the later fetal-juvenile period. The upper and lower decile range of summed point mutant fractions among lungs was about 7.5-fold, suggesting an important source of stratification in the population with regard to risk of tumor initiation.     

Detection and frequency estimation of rare variants in pools of genomic DNA from large populations using mutational spectrometry

DNA variants underlying the inheritance of risk for common diseases are expected to have a wide range of population allele frequencies. The detection and scoring of the rare alleles (at frequencies of <0.01) presents significant practical problems, including the requirement for large sample sizes and the limitations inherent in current methodologies for allele discrimination. In the present report, we have applied mutational spectrometry based on constant denaturing capillary electrophoresis (CDCE) to DNA pools from large populations in order to improve the prospects of testing the role of rare variants in common diseases on a large scale. We conducted a pilot study of the cytotoxic T lymphocyte-associated antigen-4 gene (CTLA4) in type 1 diabetes (T1D). A total of 1228 bp, comprising 98% of the CTLA4 coding sequence, all adjacent intronic mRNA splice sites, and a 3′ UTR sequence were scanned for unknown point mutations in pools of genomic DNA from a control population of 10,464 young American adults and two T1D populations, one American (1799 individuals) and one from the United Kingdom (2102 individuals). The data suggest that it is unlikely that rare variants in the scanned regions of CTLA4 represent a significant proportion of T1D risk and illustrate that CDCE-based mutational spectrometry of DNA pools offers a feasible and cost-effective means of testing the role of rare variants in susceptibility to common diseases.     

Mismatch repair deficient human cells: spontaneous and MNNG-induced mutational spectra in the HPRT gene

We have determined both the spontaneous and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)-induced mutational spectra in the HPRT gene of human cells (MT1) defective in the mismatch repair gene hMSH6 (GTBP). Eight of nine exons and nine of sixteen intronic flanking sequences were scanned, encompassing >900 bp of the HPRT gene. Mutant hotspots were detected and separated by differences in their melting temperatures using constant denaturant capillary electrophoresis (CDCE) or denaturing gradient gel electrophoresis (DGGE).

A key finding of this work is that a high proportion of all HPRT inactivating mutations is represented by a small number of hotspots distributed over the exons and mRNA splice sites. Thirteen spontaneous hotspots and sixteen MNNG-induced hotspots accounted for 55% and 48% of all 6TG^R point mutations, respectively. MNNG-induced hotspots were predominantly G:C→A:T transitions. The spontaneous spectrum of cells deficient in hMSH6 contained transversions (A:T→T:A, G:C→T:A, A:T→C:G), transitions (A:T→G:C), a plus-one insertion, and a minus-one deletion. Curiously, G:C→A:T transitions, which dominate human germinal and somatic point mutations were absent from the spontaneous hMSH6 spectra.     

Analysis of mutational spectra by denaturing capillary electrophoresis

The point mutational spectrum over nearly any 75- to 250-bp DNA sequence isolated from cells, tissues or large populations may be discovered using denaturing capillary electrophoresis (DCE). A modification of the standard DCE method that uses cycling temperature (e.g., +/-5 degrees C), CyDCE, permits optimal resolution of mutant sequences using computer-defined target sequences without preliminary optimization experiments. The protocol consists of three steps: computer design of target sequence including polymerase chain reaction (PCR) primers, high-fidelity DNA amplification by PCR and mutant sequence separation by CyDCE and takes about 6 h. DCE and CyDCE have been used to define quantitative point mutational spectra relating to errors of DNA polymerases, human cells in development and carcinogenesis, common gene-disease associations and microbial populations. Detection limits are about 5 x 10(-3) (mutants copies/total copies) but can be as low as 10(-6) (mutants copies/total copies) when DCE is used in combination with fraction collection for mutant enrichment. No other technological approach for unknown mutant detection and enumeration offers the sensitivity, generality and efficiency of the approach described herein.