The Alu-Rich Genomic Architecture of SPAST Predisposes to Diverse and Functionally Distinct Disease-Associated CNV Alleles

Abstract

Intragenic copy-number variants (CNVs) contribute to the allelic spectrum of both Mendelian and complex disorders. Although pathogenic deletions and duplications in SPAST (mutations in which cause autosomal-dominant spastic paraplegia 4 [SPG4]) have been described, their origins and molecular consequences remain obscure. We mapped breakpoint junctions of 54 SPAST CNVs at nucleotide resolution. Diverse combinations of exons are deleted or duplicated, highlighting the importance of particular exons for spastin function. Of the 54 CNVs, 38 (70%) appear to be mediated by an Alu-based mechanism, suggesting that the Alu-rich genomic architecture of SPAST renders this locus susceptible to various genome rearrangements. Analysis of breakpoint Alus further informs a model of Alu-mediated CNV formation characterized by small CNV size and potential involvement of mechanisms other than homologous recombination. Twelve deletions (22%) overlap part of SPAST and a portion of a nearby, directly oriented gene, predicting novel chimeric genes in these subjects' genomes. cDNA from a subject with a SPAST final exon deletion contained multiple SPAST:SLC30A6 fusion transcripts, indicating that SPAST CNVs can have transcriptional effects beyond the gene itself. SLC30A6 has been implicated in Alzheimer disease, so these fusion gene data could explain a report of spastic paraplegia and dementia cosegregating in a family with deletion of the final exon of SPAST. Our findings provide evidence that the Alu genomic architecture of SPAST predisposes to diverse CNV alleles with distinct transcriptional--and possibly phenotypic--consequences. Moreover, we provide further mechanistic insights into Alu-mediated copy-number change that are extendable to other loci.

A polymorphic Alu insertion that mediates distinct disease-associated deletions

“…On the basis of the degree of homology for the predicted breakpoints, one deletion would have occurred by nonhomologous end joining, whereas the other would resemble the majority of previously resolved rearrangements by being based on non-allelic homologous recombination between corresponding parts of Alus. 19 In summary, our findings add to the spectrum of mutational mechanisms responsible for SPAST deletions, confirm the pivotal role played by Alus, and reveal that Alu insertion-associated deletions may form by two temporally separated mutational steps.…”

“…18 Indeed, the majority of deletions previously resolved at sequence level involve Alus present in the introns, the 3′UTR, and the neighboring intergenic regions. 19 The present study reveals that SPAST is still actively targeted by Alu retrotransposition. We show that an AluYb8, which was newly inserted into the intronic sequence, would have been involved in the two distinct deletions.…”

Unbalanced translocations arise from diverse mutational mechanisms including chromothripsis

“…Fusion genes are common in chromosome rearrangements in leukemia but are rarely reported in germline rearrangements (Backx et al 2011;Rippey et al 2013;Boone et al 2014;van Heesch et al 2014;Newman et al 2015). In the 51 simple translocations with 102 sequenced or fine-mapped breakpoints, 44 (43%) of the breakpoints lie in a gene.…”

Alu elements and DNA double-strand break repair

“…These types of complex chimeric Alu/Alu recombination products have been seen to occur in natural gene mutations as well. 12 One of the most surprising findings with our new system was that the rate of genomic deletions in our reporter increased specifically within the 15-30% Alu element sequence divergence range. 16 This was not due to any increase in Alu/Alu recombination, but instead resulted from NHEJ between sequences primarily found within the Alu elements (see schematic in Fig.…”