Repeatome Analysis of Plasma Circulating DNA in Patients with Cardiovascular Disease: Variation with Cell-Free DNA Integrity/Length and Clinical Parameters

Abstract

Repetitive DNA represents over 50% of the human genome and is an abundant component of circulating cell-free DNA (cfDNA). We previously showed that cfDNA levels and integrity can predict survival in elderly patients with cardiovascular disease. Here, we aimed to clarify whether a low-pass next-generation sequencing (NGS) approach can characterize the repeat content of cfDNA. Considering the bimodal distribution of cfDNA fragment lengths, we examined the occurrence of repetitive DNA subfamilies separately in dinucleosomal (>250 bp) and mononucleosomal (≤250 bp) cfDNA sequences from 24 patients admitted for heart failure. An increase in the relative abundance of Alu repetitive elements was observed in the longer fraction, while alpha satellites were enriched in the mononucleosomal fraction. The relative abundance of Alu, ALR, and L1HS DNA in the dinucleosomal fraction correlated with different prognostic biomarkers, and Alu DNA was negatively associated with the presence of chronic kidney disease comorbidity. These results, together with the observed inverse correlation between Alu DNA abundance and cfDNA integrity, suggest that the composition of plasma cfDNA could be determined by multiple mechanisms in different physio-pathological conditions. In conclusion, low-pass NGS is an inexpensive method to analyze the cfDNA repeat landscape and identify new cardiovascular disease biomarkers.

Keywords: Alu elements; CKD; cardiovascular disease; cfDNA; circulating biomarkers; eGFR; hearth failure; low-pass NGS; repetitive DNA.

Figure 1

Graphical representation of the bimodal distribution of cfDNA. (A) Length distribution of NGS library reads of an example cfDNA (sample 2). (B) Automated electrophoresis of the same cfDNA sample conducted by high-sensitivity D1000 screen tapes on TapeStation System 4200. The two peaks represent the abundance of mononucleosomal (≤250 bp) and dinucleosomal (>250 bp) cfDNA fragments. Graphical representation of the length distribution of the other cfDNA samples and NGS libraries are shown in Figure S2.

Figure 2

Representation of repetitive and non-repetitive elements in cell-free DNA and in GRCh38. The pie charts illustrate the overall percentage distribution of cfDNA sequencing reads mapped to the full list of 1355 human and ancestral repetitive subfamilies by RepBase Update (A) or mapped to the human reference genome (GRCh38) and overlapping (100% overlap of mapped sequence) with a RepeatMasker annotation (B).

Figure 3

Representation of repetitive element subfamilies in cell-free DNA and in GRCh38. (A) The pie chart illustrates the overall distribution of cfDNA 100 bp fragment sequences mapped to specific DNA repeat categories (families or groups of repeat subfamilies). The total (100%) represents all the sequences mapped to the whole list of 1355 human-specific and human-ancestral consensus sequences obtained by the RepBase Update database. For the same repeat categories/subfamilies, the pie chart in (B) shows the percentage of sequence of the human genome reference annotated with repeat elements of the considered category/subfamily. In this case, 100% represents the total of the genome reference sequence that has been annotated with repetitive DNA elements.

Figure 4

(A) Differential distribution of major repetitive DNA classes between mononucleosomal and dinucleosomal cfDNA fragments. The heatmap was obtained by mapping reads to the RepBase reduced list. The Z score is calculated as Count per million (CPM). Average linkage and Spearman Rank correlation were used for representation. (B) Mean difference (±S.E. of mean) in CPM of major repetitive DNA classes between mononucleosomal and dinucleosomal cfDNA sequences. Asterisks indicate differences significantly different from 0 (paired sample t-test), after Bonferroni correction: * = p < 0.01; ** = p < 0.001.

Figure 5

Bioinformatics pipeline. The flowcharts summarize the bioinformatic analyses performed using CLC Genomics Workbench software, version 7. The analyses were designed to (i) quantify repetitive elements in cfDNA by counting the number of reads mapped against the reference genome annotated by RepeatMasker (shown in blue); (ii) characterize the composition of repeat families and subfamilies in cfDNA by aligning reads to RepBase full list of repeat consensus sequences (orange); and (iii) assess the distribution of repetitive elements across short and long cfDNA fragments. The latter was achieved by first performing a de novo assembly to generate a reduced RepBase dataset, followed by separate mapping of mono- and dinucleosomal reads against these custom reference sequences (green).