Evidence for multi-copy Mega-NUMTs in the human genome.

The maternal mode of mitochondrial DNA (mtDNA) inheritance is central to human genetics. Recently, evidence for bi-parental inheritance of mtDNA was claimed for individuals of three pedigrees that suffered mitochondrial disorders. We sequenced mtDNA using both direct Sanger and Massively Parallel Sequencing in several tissues of eleven maternally related and other affiliated healthy individuals of a family pedigree and observed mixed mitotypes in eight individuals. Cells without nuclear DNA, i.e. thrombocytes and hair shafts, only showed the mitotype of haplogroup (hg) V. Skin biopsies were prepared to generate ρ° cells void of mtDNA, sequencing of which resulted in a hg U4c1 mitotype. The position of the Mega-NUMT sequence was determined by fluorescence in situ hybridization and two different quantitative PCR assays were used to determine the number of contributing mtDNA copies. Thus, evidence for the presence of repetitive, full mitogenome Mega-NUMTs matching haplogroup U4c1 in various tissues of eight maternally related individuals was provided. Multi-copy Mega-NUMTs mimic mixtures of mtDNA that cannot be experimentally avoided and thus may appear in diverse fields of mtDNA research and diagnostics. We demonstrate that hair shaft mtDNA sequencing provides a simple but reliable approach to exclude NUMTs as source of misleading results.

A novel and effective separation method for single mitochondria analysis.

To investigate the set of mtDNA molecules contained in small biological structures, powerful techniques for separation are required. We tested flow cytometry (FCM(1)), laser capture microdissection (LCM(2)) and a method using optical tweezers (OT(3)) in combination with a 1µ-Ibidi-Slide with regard to their ability to deposit single mitochondrial particles. The success of separation was determined by real-time quantitative PCR (qPCR(4)) and sequencing analysis. OT revealed the highest potential for the separation and deposition of single mitochondrial particles. The study presents a novel setup for effective separation of single mitochondrial particles, which is crucial for the analysis of single mitochondria.

Developing equine mtDNA profiling for forensic application.

Horse mtDNA profiling can be useful in forensic work investigating degraded samples, hair shafts or highly dilute samples. Degraded DNA often does not allow sequencing of fragments longer than 200 nucleotides. In this study we therefore search for the most discriminatory sections within the hypervariable horse mtDNA control region. Among a random sample of 39 horses, 32 different sequences were identified in a stretch of 921 nucleotides. The sequences were assigned to the published mtDNA types A-G, and to a newly labelled minor type H. The random match probability within the analysed samples is 3.61%, and the average pairwise sequence difference is 15 nucleotides. In a "sliding window" analysis of 200-nucleotide sections of the mtDNA control region, we find that the known repetitive central motif divides the mtDNA control region into a highly diverse segment and a markedly less discriminatory segment.

Pulmonary arteriovenous malformation causing sudden death due to spontaneous hemothorax.

A sudden death due to hemothorax caused by spontaneous rupture of a congenital pulmonary arteriovenous malformation (AVM) is reported. A 44-year-old woman died unexpectedly with chest pain and dyspnea. The post-mortem examination revealed a massive right-sided hemothorax arising from a subpleural AVM of the upper lobe. There were multiple telangiectases in the tongue and the tonsils, as typically associated with Osler-Weber-Rendu disease (hereditary hemorrhagic telangiectasia, HHT). The post-mortem molecular genetic analysis proved the presence of a disease-causing mutation in the endoglin gene constituting a predisposition for pulmonary AVMs. According to the literature, almost half of the AVMs in the lung are seen in HHT patients. Based on the presented case and the relevant literature, the article addresses the forensic aspects of fatal hemothorax and the importance of detecting the source of bleeding.