An original phylogenetic approach identified mitochondrial haplogroup T1a1 as inversely associated with breast cancer risk in BRCA2 mutation carriers

Introduction: Individuals carrying pathogenic mutations in the BRCA1 and BRCA2 genes have a high lifetime risk of breast cancer. BRCA1 and BRCA2 are involved in DNA double-strand break repair, DNA alterations that can be caused by exposure to reactive oxygen species, a main source of which are mitochondria. Mitochondrial genome variations affect electron transport chain efficiency and reactive oxygen species production. Individuals with different mitochondrial haplogroups differ in their metabolism and sensitivity to oxidative stress. Variability in mitochondrial genetic background can alter reactive oxygen species production, leading to cancer risk. In the present study, we tested the hypothesis that mitochondrial haplogroups modify breast cancer risk in BRCA1/2 mutation carriers.

Methods: We genotyped 22,214 (11,421 affected, 10,793 unaffected) mutation carriers belonging to the Consortium of Investigators of Modifiers of BRCA1/2 for 129 mitochondrial polymorphisms using the iCOGS array. Haplogroup inference and association detection were performed using a phylogenetic approach. ALTree was applied to explore the reference mitochondrial evolutionary tree and detect subclades enriched in affected or unaffected individuals.

Results: We discovered that subclade T1a1 was depleted in affected BRCA2 mutation carriers compared with the rest of clade T (hazard ratio (HR) = 0.55; 95% confidence interval (CI), 0.34 to 0.88; P = 0.01). Compared with the most frequent haplogroup in the general population (that is, H and T clades), the T1a1 haplogroup has a HR of 0.62 (95% CI, 0.40 to 0.95; P = 0.03). We also identified three potential susceptibility loci, including G13708A/rs28359178, which has demonstrated an inverse association with familial breast cancer risk.

Conclusions: This study illustrates how original approaches such as the phylogeny-based method we used can empower classical molecular epidemiological studies aimed at identifying association or risk modification effects.

Prevalence of the prothrombin G20210A mutation in the Irish populations:use of a novel polymerase chain reaction approach

The prothrombin G20210A polymorphism is associated with a threefold-increased risk of venous thrombosis. There is considerable variation in the reported prevalence of this polymorphism within normal populations, ranging from 0 to 6.5%. The prevalence within the Irish population has not been determined. A restriction fragment length polymorphism (RFLP)-based assay is commonly used for the detection of the prothrombin 20210A allele. This assay does not include a control restriction digest fragment and, consequently, failure of the enzyme activity or lack of addition of enzyme to the sample cannot be distinguished from wild-type prothrombin. We developed a RFLP-based assay, which incorporates an invariant digest site, resulting in the generation of a control digest fragment. Furthermore, we developed a nested polymerase chain reaction (PCR) method for the amplification and digestion of poor-quality or low-concentration DNA. In the Irish population studied, five of 385 (1.29%) were heterozygous and one patient was homozygous for the prothrombin 20210A polymorphism. This is the first reported data on an Irish or Celtic population and suggests that the allele frequency is similar to Anglo-Saxon populations. The nested PCR method successfully amplified and digested 100/100 (100%) of the archived samples; none of these samples could be analyzed by the standard single-round PCR method. In conclusion, nested PCR should be considered in the analysis of archived samples. Single-round PCR is appropriate for recently collected samples; however, an invariant control digest site should be incorporated in RFLP-based assays to validate the integrity of the digestion enzyme and limit the risk of false-negative results.