Recurrent horizontal transfer identifies mitochondrial positive selection in a transmissible cancer

Abstract

Autonomous replication and segregation of mitochondrial DNA (mtDNA) creates the potential for evolutionary conflict driven by emergence of haplotypes under positive selection for 'selfish' traits, such as replicative advantage. However, few cases of this phenomenon arising within natural populations have been described. Here, we survey the frequency of mtDNA horizontal transfer within the canine transmissible venereal tumour (CTVT), a contagious cancer clone that occasionally acquires mtDNA from its hosts. Remarkably, one canine mtDNA haplotype, A1d1a, has repeatedly and recently colonised CTVT cells, recurrently replacing incumbent CTVT haplotypes. An A1d1a control region polymorphism predicted to influence transcription is fixed in the products of an A1d1a recombination event and occurs somatically on other CTVT mtDNA backgrounds. We present a model whereby 'selfish' positive selection acting on a regulatory variant drives repeated fixation of A1d1a within CTVT cells.

Fig. 1. Recurrent and recent horizontal transfer of canine mitochondrial haplotype A1d1a.

a MtDNA (left) and nuclear DNA (right) maximum likelihood phylogenetic trees for 539 CTVT tumours (coloured) and 494 dogs (black). Correspondence between equivalent CTVTs on mtDNA and nuclear trees is indicated and coloured by mtDNA donor haplotype. Trees are presented as cladograms without informative branch lengths. High resolution trees are presented in Supplementary Figs. 1 and 2. b Frequencies of 18 canine mtDNA haplotypes within a representative global CTVT host dog population (n = 495) (left), and the number of CTVT horizontal transfer (HT) events involving each haplotype in a population of 539 CTVTs (right). Donor haplotype (coloured dots) and heteroplasmic horizontal transfer events (asterisks (*) and lighter shading) are shown. Heteroplasmic tumours carry both the parental and introduced haplotype. The A1d1a heteroplasmic horizontal transfer event involves mtDNA recombination. Bars representing A1d1a are highlighted in green. c Inferred geographical locations of 19 CTVT mtDNA horizontal transfer events. Each horizontal transfer is represented by a dot coloured by donor haplotype. If all CTVTs arising from a horizontal transfer were sampled at the same location, then this was inferred as the location of the horizontal transfer. If CTVTs derived from the horizontal transfer were found in several locations, then the likely site of the horizontal transfer was inferred based on phylogenetic information^,. Heteroplasmic horizontal transfer events are indicated with an asterisk (*). d Number of somatic mtDNA mutations acquired since each horizontal transfer (HT) event. Number of CTVTs (n = 539) belonging to each HT event is indicated. Bars are split into two categories: darker colour shades represent confident somatic mutations that are polymorphic within each HT group, with error bars representing the mutation range; lighter colour shades represent variants that are fixed within each HT group, whose somatic or germline status cannot be determined (see Methods). Bars representing A1d1a HTs are highlighted in green. Donor haplotype (coloured dots) and heteroplasmy (asterisk, *) are shown.

Fig. 2. Genetic features of the A1d1a haplotype.

a Genetic variants unique to the A1d1a haplotype (and in some cases shared with the related A1d1 haplotype, Supplementary Data 5) relative to 16 other dog haplotype groups. Number of somatic occurrences on other non-A1d1a CTVT mtDNA backgrounds is indicated above each variant. Variants within protein-coding genes are annotated as synonymous (S) or non-synonymous (NS), and other variants are annotated as occurring within ribosomal RNA (rRNA) or the control region (CR). MtDNA (MT) genome coordinates are indicated. b MtDNA haplotypes detected using long-read sequencing in the two tumours (labelled tumour 1 and tumour 2) belonging to the HT3 group, in which A1d1a and CTVT_HT1 have undergone recombination, with a heteroplasmic mixture of recombination products present in each tumour. The region fixed in all haplotypes in both tumours is indicated with a dotted box. The estimated frequency of each recombinant haplotype (H) within the two tumours’ CTVT cell mtDNA population is shown. c 16660insCC sequence context and position relative to control region features. Conserved sequence blocks (CSB) 1–3 are marked, together with the light strand promoter (LSP), heavy strand promoter (HSP) and a ten-nucleotide (nt) repeat block. 16660insCC co-occurs in A1d1a with 16672C>T, a polymorphism present on several canine haplotypes.

Fig. 3. MtDNA gene transcript abundance and model.

a Mean abundance of mtDNA protein-coding gene RNA in CTVT_HT1, CTVT_HT2, CTVT_A1d1a and CTVT_HT1/HT2+insCC/insC/Rec CTVTs (n = 33). In the group of CTVT_HT1/HT2+insCC/insC/Rec CTVTs (represented in grey), two carry 16660insCC and one carries 16660insC as a somatic mutation on CTVT_HT1 or CTVT_HT2 haplotype backgrounds, and one carries 16660insCC on a CTVT_HT1/A1d1a recombinant background. Dots represent mean abundances of mtDNA protein-coding gene transcripts in each of the CTVTs from each group, diamonds indicate mean mtDNA protein-coding gene abundances for each group and bars indicate 95% confidence intervals of the mean. Asterisk (*), p = 0.015 (two-sided Mann–Whitney test; Methods). Supplementary Data 6B provides batch-corrected transcript abundance data. b A model explaining repeated CTVT capture of the A1d1a haplotype. (1) All canine mtDNA haplotypes have equal opportunity for CTVT horizontal transfer, relative to population haplotype frequency. (2) A1d1a mtDNA haplotype carries an insertion in the control region, 16660insCC, which is not present in other haplotypes, and which may have regulatory functions. (3) MtDNA protein-coding transcript abundance is decreased in tumours with the A1d1a mtDNA haplotype relative to the two most common CTVT mtDNA haplotypes. (4) After A1d1a mtDNA horizontal transfer, A1d1a becomes homoplasmic through a selfish replicative advantage.