The genomic architecture of NLRP7 is Alu rich and predisposes to disease-associated large deletions

Abstract

NLRP7 is a major gene responsible for recurrent hydatidiform moles. Here, we report 11 novel NLRP7 protein truncating variants, of which five deletions of more than 1-kb. We analyzed the transcriptional consequences of four variants. We demonstrate that one large homozygous deletion removes NLRP7 transcription start site and results in the complete absence of its transcripts in a patient in good health besides her reproductive problem. This observation strengthens existing data on the requirement of NLRP7 only for female reproduction. We show that two other variants affecting the splice acceptor of exon 6 lead to its in-frame skipping while another variant affecting the splice donor site of exon 9 leads to an in-frame insertion of 54 amino acids. Our characterization of the deletion breakpoints demonstrated that most of the breakpoints occurred within Alu repeats and the deletions were most likely mediated by microhomology events. Our data define a hotspot of Alu instability and deletions in intron 5 with six different breakpoints and rearrangements. Analysis of NLRP7 genomic sequences for repetitive elements demonstrated that Alu repeats represent 48% of its intronic sequences and these repeats seem to have been inserted into the common NLRP2/7 primate ancestor before its duplication into two genes.

Figure 1

Transcriptional characterization of four NLRP7 variants using RT-PCR on RNA from EBV-transformed patient cells. Red arrows denote missing or abnormal fragments observed only in the patients. (a) RT-PCR on RNA from patient 1341 showing complete loss of NLRP7 transcripts using various combinations of primers located in exons 5–11. Primers from exons 5 and 7 of ZNF28 gene were used in combination with those of NLRP7 as positive controls for the RT-PCR. (b) RT-PCR on RNA from patient 1291 with a 9-bp deletion removing the invariant splice acceptor sites of exon 6 showing the deletion of exon 6 from the cDNA. (c) RT-PCR on RNA from patient 1200 with an invariant splice acceptor site variant showing the loss of exon 6. (d) RT-PCR on RNA from patients 1074 and 1243 (e) with the same variant affecting the invariant splice donor of exon 9. This variant leads to one common abnormal fragment of 673-bp in the two patients resulting from the insertion of 162-bp of intron 9 between exons 9 and 10. In patient 1243, we observed other faint fragments due to minor aberrant splicing, of them a fragment smaller than 673-bp is shown in this figure and is indicated by a red arrow.

Figure 2

Characterization of five large NLRP7 deletions identified in this study and schematic representation of the Alu elements at the breakpoints. The microhomology sequences surrounding the deletions are shown in capital letters and the unique sequences on each side of the microhomology sequences are shown in small letters. Red letters indicate differences between the two microhomology elements or the flanking sequences. Dashed red lines delimit the deletions. The orientation of the Alu repeats is indicated by arrows above each repeat.

Figure 3

Recapitulation of all NLRP7 large deletions and distribution of Alu elements in its intronic sequences. To date, eight Alu-repeat mediated deletions and rearrangements in NLRP7 have been described. Colored boxes refer to specific Alu subfamilies, each with a specific color. Novel and previously reported mutations are in red and black, respectively. Arrows on the top of the colored boxes indicate the orientation of the Alu elements. Six of the 16 deletions and rearrangement breakpoints occurred in intron 5 and define a hotspot of Alu instability, deletions, and rearrangements shown in a rectangle. N, stands for number; seq. stands for sequences. % of Alu seq, indicates the percentage of the length of Alu sequences in each intron divided by the total length of the sequence of each intron.

Figure 4

Alu distribution in human NLRP genes. For all genes, the genomic structures were downloaded from the UCSC Genome browser (hg19) and each included 1-kb upstream the first exon and 1-kb downstream of the last exon. The presence of Alu repeats was investigated using CENSOR (http://www.girinst.org/censor/). The percentage of Alu sequences in each gene represents the total length of its Alu sequences over the length of its genomic structure. We note that Alu elements were mostly found in introns, which is in agreement with known data about most Alu sequences.