Variable numbers of tandem repeats in Plasmodium falciparum genes

Abstract

Genome variation studies in Plasmodium falciparum have focused on SNPs and, more recently, large-scale copy number polymorphisms and ectopic rearrangements. Here, we examine another source of variation: variable number tandem repeats (VNTRs). Interspersed low complexity features, including the well-studied P. falciparum microsatellite sequences, are commonly classified as VNTRs; however, this study is focused on longer coding VNTR polymorphisms, a small class of copy number variations. Selection against frameshift mutation is a main constraint on tandem repeats (TRs) in coding regions, while limited propagation of TRs longer than 975 nt total length is a minor restriction in coding regions. Comparative analysis of three P. falciparum genomes reveals that more than 9% of all P. falciparum ORFs harbor VNTRs, much more than has been reported for any other species. Moreover, genotyping of VNTR loci in a drug-selected line, progeny of a genetic cross, and 334 field isolates demonstrates broad variability in these sequences. Functional enrichment analysis of ORFs harboring VNTRs identifies stress and DNA damage responses along with chromatin modification activities, suggesting an influence on genome mutability and functional variation. Analysis of the repeat units and their flanking regions in both P. falciparum and Plasmodium reichenowi sequences implicates a replication slippage mechanism in the generation of TRs from an initially unrepeated sequence. VNTRs can contribute to rapid adaptation by localized sequence duplication. They also can confound SNP-typing microarrays or mapping short-sequence reads and therefore must be accounted for in such analyses.

Fig. 1

Positions of TRs across the 3D7 genome. Each circle depicts one TR and its genomic location on the 14 chromosomes. Circle diameter indicates repeat unit size as shown in the legend. Red circles indicate VNTRs identified in gene coding regions

Fig. 2

Selection against TRs that cause frameshift mutations. A very high percentage of TRs in exons were 3× repeats. The blue portions of the bars represent the proportion of TRs in each group that are 3× repeats. The black text indicates the number of TRs in each group; the white, underlined text indicates the percentage of 3× repeats for each group. A chi-square test was used to compare the categorical observations (** P < 0.01; **** P < 0.0001)

Fig. 3

Spatial distribution of TRs in genes. The observed spatial distribution of TRs in genes compared to the expected frequency from a random distribution shows a greater frequency of TRs in the midsegment and 5′ segment than expected, and lower frequency observed in the 3′ segment than expected

Fig. 4

Slipped strand mispairing can generate sequence duplication. A sequence overhang may cause slipped strand mispairing to occur allowing sequence duplication. a No tandemly repeated sequence is present; however, two stretches of non-adjacent identical sequence are present (blue segments). b The overhang in the flanking region can become mispaired with the start region of the repeat unit. This can cause a bulge in the DNA in either the template or nascent strand. c DNA synthesis will lead to duplication or deletion. Duplication will facilitate further mispairing, increasing the likelihood of greater repeat variability

Fig. 5

TRs and sequence overhangs in P. falciparum and P. reichenowi. Several VNTR loci from P. falciparum and P. reichenowi illustrate how sequence overhangs may initiate TR repeat generation or sequence deletion. a PFA0175w is a variable locus in P. falciparum where a 24 bp repeat with a 10 bp overhang in the flanking region is present. In P. reichenowi, the sequence is not repeated and the 10 bp overhang contains three SNPs that reduce sequence identity to the potential repeat motif, eliminating the chance for replication slippage to tandemly duplicate the sequence. b MAL8P1.151 contains an 18 bp repeat with a 9 bp overhang. In the homologous P. reichenowi sequence, the 18 bp sequence is not repeated and there are two SNPs in the overhang. c PFF0325c contains an unrepeated 33 bp sequence tduhat is flanked by a 15 bp sequence overhang. In P. reichenowi, the 33 bp sequence is absent, and the flanking 15 bp overhang is all that remains