SVA retrotransposon insertion-associated deletion represents a novel mutational mechanism underlying large genomic copy number changes with non-recurrent breakpoints

Abstract

Background: Genomic disorders are caused by copy number changes that may exhibit recurrent breakpoints processed by nonallelic homologous recombination. However, region-specific disease-associated copy number changes have also been observed which exhibit non-recurrent breakpoints. The mechanisms underlying these non-recurrent copy number changes have not yet been fully elucidated.

Results: We analyze large NF1 deletions with non-recurrent breakpoints as a model to investigate the full spectrum of causative mechanisms, and observe that they are mediated by various DNA double strand break repair mechanisms, as well as aberrant replication. Further, two of the 17 NF1 deletions with non-recurrent breakpoints, identified in unrelated patients, occur in association with the concomitant insertion of SINE/variable number of tandem repeats/Alu (SVA) retrotransposons at the deletion breakpoints. The respective breakpoints are refractory to analysis by standard breakpoint-spanning PCRs and are only identified by means of optimized PCR protocols designed to amplify across GC-rich sequences. The SVA elements are integrated within SUZ12P intron 8 in both patients, and were mediated by target-primed reverse transcription of SVA mRNA intermediates derived from retrotranspositionally active source elements. Both SVA insertions occurred during early postzygotic development and are uniquely associated with large deletions of 1 Mb and 867 kb, respectively, at the insertion sites.

Conclusions: Since active SVA elements are abundant in the human genome and the retrotranspositional activity of many SVA source elements is high, SVA insertion-associated large genomic deletions encompassing many hundreds of kilobases could constitute a novel and as yet under-appreciated mechanism underlying large-scale copy number changes in the human genome.

Figure 1

Location of the breakpoints of the 17 atypical NF1 deletions. At the top is a schematic representation of the NF1 gene and its flanking regions. The relative positions of the genes located within this region are denoted by horizontal black bars. Below, the extents of the 17 NF1 deletions analyzed are indicated by horizontal bars. The centromeric breakpoints of the deletions depicted by red bars are located within SUZ12P. None of these deletions had telomeric breakpoints located within SUZ12. The deletions depicted by blue bars exhibit breakpoints located within NF1-REPa. Two deletions (grey bars) extended beyond the region indicated here in a centromeric direction (indicated by dotted lines). The patient identification numbers are given on the left. cen, centromere; tel, telomere.

Figure 2

Structure of the SVA elements inserted at the NF1 deletion breakpoints and their source elements. (A) The SVA_F1 element H10_1 spans 4,039 bp and is the likely source element of the SVA copy that inserted within SUZ12P intron 8 in the grandmother of patient DA-77. Starting at its 5′ end, H10_1 comprises a target site duplication (TSD), a transduced sequence (5′TD) and a full-length AluSc from chromosome 9p13.3, a transduced partial exon 1 of MAST2, an Alu-like region, a variable number of tandem repeats (VNTR) region, a SINE-R, a polyA₍₁₇₎ tract, the second TSD, an AluSp element, a second polyA₍₁₇₎ tract and a non-repetitive, unique sequence resulting from a 3′ transduction that harbors two polyadenylation signals (AATAAA). The size of each region is given in basepairs. (B) A copy of the source element H10_1 integrated within SUZ12P intron 8 in the grandmother of patient DA-77. The SVA insertion was associated with a deletion of approximately 1 Mb. The inserted SVA spans 1.7 kb and is 5′ truncated. (C) Structure of the putative source SVA element H6_1084, which spans 2,691 bp and belongs to the SVA_F subfamily. A copy of H6_1084 is presumed to have integrated within SUZ12P intron 8 in patient ASB4-55. Full-length H6_1084 has the following structure starting from the 5′ end: a TSD, a 5′TD from chromosome 12p11.21, a CCCTCT₍₄₎ repeat, a 343-bp Alu-like region, a GC-rich VNTR region, a SINE-R element, two polyadenylation signals, a polyA₍₁₁₎ tract and the second TSD. The length of each region is indicated in basepairs. (D) Structure of the 5′ truncated copy of H6_1084 that has integrated within SUZ12P intron 8 in patient ASB4-55. The SVA insertion was associated with an atypical NF1 deletion of 867 kb. The SVA integration sites within SUZ12P intron 8 demarcate the centromeric breakpoints of the atypical NF1 deletions.

Figure 3

Locations of the telomeric breakpoints identified in the 17 atypical NF1 deletions. A schematic representation of the genes located within the region is given on top. The extent of each of the 17 atypical NF1 deletions is indicated by a red bar. The centromeric breakpoints of these deletions differ from each other and are not indicated on this schema. The numbering of the breakpoint locations is according to the human GRCh37/hg19 assembly. Five deletions exhibited breakpoints that were located within a 32.6 kb region (demarcated by a grey box). tel, telomeric direction.

Figure 4

Centromeric breakpoint positions of the 11 atypical NF1 deletions with centromeric breakpoints located within SUZ12P. The exon-intron structure of SUZ12P is indicated as well as the numbering of the exons presented as vertical black lines. The extent of each of the 11 atypical NF1 deletions is shown by red bars. The telomeric breakpoints of these deletions differ from each other and are not indicated on this schema. The numbering of the centromeric breakpoint locations is given according to the human GRCh37/hg19 assembly. tel, telomeric direction.

Figure 5

Putative mechanism underlying the large atypical NF1 deletions identified in patient ASB4-55 and the grandmother of patient DA-77. The deletions were associated with the insertion of an SVA element mediated by the LINE 1 protein machinery via target-primed reverse transcription. (A) SUZ12P intron 8 is indicated in lilac whereas the telomeric part of the NF1 region is shown in green. The dotted lines indicate the approximately 1-Mb distance between these two regions. The SVA insertion within SUZ12P intron 8 is likely to have been initiated by the L1 endonuclease (L1-EN), which will have introduced a nick at the consensus cleavage site 5′-CTTT/A-3′. (B) Next, the SVA mRNA annealed to the T-overhang by means of its polyA-tail. Then, the L1 reverse transcriptase used the SVA mRNA as a template for reverse transcription to synthesize the SVA cDNA (blue). Second strand cleavage by the L1-EN occurred upstream of the first-strand cleavage site. Independently, a double strand break (DSB) occurred in the telomeric region of 17q11.2. (C,D) After dissociation of the SVA mRNA, the integration process was not finalized by recombinational repair using the downstream SUZ12P intron 8 sequence. Instead, the DNA ends were ligated by NHEJ to the open-ended DNA sequence located within the telomeric 17q11.2 region, between the RAB11FIP4 and COPRS genes, which resulted in the deletion of the intervening sequence and hence the occurrence of the atypical NF1 deletion (D).