We have tested a simple procedure, disease association by locus stratification, for identifying breast cancer patients with pathogenetic allelic variants at several candidate loci. The strategy was based on the assumption of epistatic interactions of the candidates. We analyzed 66 independent cases from sib pairs affected with breast cancer that had previously been collected during an investigation of pathogenetic-allele-sharing at the HRAS1 mini-satellite locus. An exon 24 polymorphism of ATM, substituting arginine for proline was associated with breast cancer in these cases with an overall odds ratio of 4.5 (95% confidence interval, 1.2-20.5, nominal p = 0.02, 2-tail Fisher exact test). In the presence of a rare HRAS1 allele, the odds ratio increased to 6.9 (95% CI, 1.2-38.3, p = 0.03). Thus, our procedure identified at least one allelic variant of ATM associated with breast cancer, and indicated that the ATM locus may interact with HRAS1.

Background Drug therapy yields different results depending on its recipient population. Cisplatin, a commonly used chemotherapeutic agent, causes different levels of resistance and side effects for different patients, but the mechanism(s) are presently unknown. It has been assumed that this variation is a consequence of differences in nuclear (n) DNA, epigenetics, or some external factor(s). There is accumulating evidence that an individual’s mitochondrial (mt) DNA may play a role in their response to medications. Variations within mtDNA can be observed, and an individual’s mtDNA can be categorized into haplogroups that are defined by accumulations of single nucleotide polymorphisms (SNPs) representing different ethnic populations. Methods The present study was conducted on transmitochondrial cytoplasmic hybrids (cybrids) that possess different maternal-origin haplogroup mtDNA from African (L), Hispanic [A+B], or Asian (D) backgrounds. Cybrids were created by fusing Rho0 ARPE-19 cells (lacking mtDNA) with platelets, which contain numerous mitochondria but no nuclei. These cybrid cells were cultured to passage five, treated with cisplatin, incubated for 48 h, then analyzed for cell metabolic activity (tetrazolium dye (MTT) assay), mitochondrial membrane potential (JC-1 assay), cytotoxicity (lactate dehydrogenase (LDH) assay), and gene expression levels for ALK, BRCA1, EGFR, and ERBB2/HER2. Results Results indicated that untreated cybrids with varying mtDNA haplogroups had similar relative metabolic activity before cisplatin treatment. When treated with cisplatin, (1) the decline in metabolic activity was greatest in L (27.4%, p < 0.012) < D (24.86%, p = 0.0001) and [A+B] cybrids (24.67%, p = 0.0285) compared to untreated cybrids; (2) mitochondrial membrane potential remained unchanged in all cybrids (3) LDH production varied between cybrids (L >[A+B], p = 0.0270). (4) The expression levels decreased for ALK in L (p < 0.0001) and [A+B] (p = 0.0001) cybrids but not in D cybrids (p = 0.285); and decreased for EGFR in [A+B] cybrids (p = 0.0246) compared to untreated cybrids. Conclusion Our findings suggest that an individual’s mtDNA background may be associated with variations in their response to cisplatin treatment, thereby affecting the efficiency and the severity of side effects from the treatment.

No abstract

No abstract

Purpose Mitochondrial (mt) DNA damage is associated with age-related macular degeneration (AMD) and other human aging diseases. This study was designed to quantify and characterize mtDNA low-frequency heteroplasmy single nucleotide polymorphisms (SNPs) of three different tissues isolated from AMD subjects using Next Generation Sequencing (NGS) technology. Methods DNA was extracted from neural retina, [RPE+choroid] and blood from three deceased age-related macular degeneration (AMD) subjects. Entire mitochondrial genomes were analyzed for low-frequency heteroplasmy SNPs using NGS technology that independently sequenced both mtDNA strands. This deep sequencing method (average sequencing depth of 30,000; range 1,000–100,000) can accurately differentiate low-frequency heteroplasmy SNPs from DNA modification artifacts. Twenty-three ‘hot-spot’ heteroplasmy mtDNA SNPs were analyzed in 222 additional blood samples. Results Germline homoplasmy SNPs that defined mtDNA haplogroups were consistent in the three tissues of each subject. Analyses of SNPs with <40% heteroplasmy revealed the blood had significantly greater numbers of heteroplasmy SNPs than retina alone (p≤0.05) or retina+choroid combined (p = 0.008). Twenty-three ‘hot-spot’ mtDNA heteroplasmy SNPs were present, with three being non-synonymous (amino acid change). Four ‘hot-spot’ heteroplasmy SNPs (m.1120C>T, m.1284T>C, m.1556C>T, m.7256C>T) were found in additional samples (n = 222). Five heteroplasmy SNPs (m.4104A>G, m.5320C>T, m.5471G>A, m.5474A>G, m.5498A>G) declined with age. Two heteroplasmy SNPs (m.13095T>C, m.13105A>G) increased in AMD compared to Normal samples. In the heteroplasmy SNPs, very few transversion mutations (purine to pyrimidine or vice versa, associated with oxidative damage) were found and the majority were transition changes (purine to purine or pyrimidine to pyrimidine, associated with replication errors). Conclusion Within an individual, the blood, retina and [RPE+choroid] contained identical homoplasmy SNPs representing inherited germline mtDNA haplogroup. NGS methodology showed significantly more mtDNA heteroplasmy SNPs in blood compared to retina and [RPE+choroid], suggesting the latter tissues have substantial protection. Significantly higher heteroplasmy levels of m.13095T>C and m.13105A>G may represent potential AMD biomarkers. Finally, high levels of transition mutations suggest that accumulation of heteroplasmic SNPs may occur through replication errors rather than oxidative damage.