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

Abstract

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.

Figure 1.

Graphical representation of the pedigree according to Bennet et al. (14) indicating the observed mtDNA haplogroups. Analyzed tissues are indicated above the circles and squares: ‘bc’: buccal cells, ‘bl’: blood, ‘bo’: bone, ‘in’: intestinal tissue.

Figure 2.

Bar charts representing the observed mtDNA CR sequencing results from single PBMCs (Peripheral Blood Mononuclear Cells) and thrombocytes (both separated by single cell FACS), single hair roots, single hair shafts and ρ° cells, as well as single clones generated from buccal cells, blood, bone and intestinal tissue samples. The height of the bar relates to the number of observations, whereas the numbers above the plots indicate the absolute count of analyzed cells or clones. ‘M’: mixed sequence of both the hg V mitotype (V) and the hg U4c1 mitotype (U), ‘n.d.’: not detected.

Figure 3.

Characterization of the mtDNA insertion (Mega-NUMT) on chromosome 14. (A) Metaphase FISH generated with the Metasystems (ISIS) software show the Mega-NUMT on chromosome 14 (green, FITC signal, arrows). Centromeric FISH probe for chromosomes 14/22 was applied as a control (red, Rhodamin signal). Accordingly, this FISH probe shows specific signals on both chromosomes 14 and co-hybridization signal in the centromeric part of both chromosomes 22. (B and C) Detailed results per chromosome using an overlay of the green and red color channels, the fluorescence intensity profile along the chromosome, and the inverted DAPI image. (B) Derivative chromosome 14 containing the Mega-NUMT insertion at 14q31. (C) Homologue chromosome 14 without the NUMT insertion.

Figure 4.

Estimated number of mtDNA building blocks of the chromosome 14 Mega-NUMT. The ostensible mtDNA copy number per NUMT was estimated by droplet digital PCR (45.00 ± 5.27; n = 20) and real-time PCR quantification (56.16 ± 4.35; n = 76) in ρ° cell DNA extracts. The results obtained for fibroblasts of an unrelated individual (control) demonstrated efficacy of ρ° cell culture. Information contained in here summarizes data on four (ddPCR) or two (qPCR) mtDNA and one nDNA quantification target sequences and four different concentrations of ethidium bromide (0, 50, 75 and 100 ng/ml) applied to ρ° cell cultures. The lower/upper hinges of the boxes indicate the first/third quartiles (Q1/Q3), horizontal lines within boxes denote the median and the whiskers extend from Q1/Q3 to the minimum/maximum values in the dataset or, in the presence of outliers, from Q1/Q3 to the upper/lower fence of the distribution (i.e. Q1 – 1.5 × IQR, Q3 + 1.5 × IQR; IQR = Q3 – Q1). Potential outliers are shown as individual black dots below/above whiskers and the arithmetic mean is indicated by a white dot with black border. -EtBr: standard cell culture conditions without addition of ethidium bromide; +EtBr: ρ° cell culture with ethidium bromide (combined data for 50, 75 and 100 ng/ml culture medium); qPCR: real-time quantitative PCR results; ddPCR: droplet digital PCR results.