Alport syndrome caused by a COL4A5 deletion and exonization of an adjacent AluY

Mutation-induced activation of splice sites in intronic repetitive sequences has contributed significantly to the evolution of exon–intron structure and genetic disease. Such events have been associated with mutations within transposable elements, most frequently in mutation hot-spots of Alus. Here, we report a case of Alu exonization resulting from a 367-nt genomic COL4A5 deletion that did not encompass any recognizable transposed element, leading to the Alport syndrome. The deletion brought to proximity the 5′ splice site of COL4A5 exon 33 and a cryptic 3′ splice site in an antisense AluY copy in intron 32. The fusion exon was depleted of purines and purine-rich splicing enhancers, but had low levels of intramolecular secondary structure, was flanked by short introns and had strong 5′ and Alu-derived 3′ splice sites, apparently compensating poor composition and context of the new exon. This case demonstrates that Alu splice sites can be activated by outlying deletions, highlighting Alu versatility in shaping the exon–intron organization and expanding the spectrum of mutational mechanisms that introduce repetitive sequences in mRNAs.


Abstract
Mutation-induced activation of splice sites in intronic repetitive sequences has contributed significantly to the evolution of exon-intron structure and genetic disease. Such events have been associated with mutations within transposable elements, most frequently in mutation hot-spots of Alus. Here, we report a case of Alu exonization resulting from a 367-nt genomic COL4A5 deletion that did not encompass any recognizable transposed element, leading to the Alport syndrome. The deletion brought to proximity the 5 0 splice site of COL4A5 exon 33 and a cryptic 3 0 splice site in an antisense AluY copy in intron 32. The fusion exon was depleted of purines and purine-rich splicing enhancers, but had low levels of intramolecular secondary structure, was flanked by short introns and had strong 5 0 and Alu-derived 3 0 splice sites, apparently compensating poor composition and context of the new exon. This case demonstrates that Alu splice sites can be activated by outlying deletions, highlighting Alu versatility in shaping the exon-intron organization and expanding the spectrum of mutational mechanisms that introduce repetitive sequences in mRNAs.
The mutation was found in a 14-year-old boy who developed macrohematuria at the age of 4 months. He was diagnosed with the Alport syndrome 14 months later following a confirmatory renal biopsy that showed a typical lamellation of the glomerular basement membrane and the absence of type IV collagen a5 chain by immunohistochemistry (data not shown) carried out as described (Oka et al. 2014). The patient developed mental retardation and autism; his severe proteinuria eventually culminated in renal failure at the age of 10 and he underwent preemptive renal transplantation using a kidney from his father. He did not show any detectable hearing loss or ocular abnormalities. His mother had hematuria and mild proteinuria since early childhood.
The disease-causing deletion was found by PCR amplifications of patient's DNA across exon 33, revealing a smaller fragment in the patient and in his heterozygous mother (Fig. 1A, left panel). DNA sequencing of the new fragment showed a 367-nt deletion (COL4A5 c.2768-230_c.2904del367 at Xq22.3) encompassing most of the 150-nt exon 33 ( Fig. 1B and C). Amplicons of cDNA samples reverse transcribed from blood or urine RNA also showed a fragment with a slightly greater mobility (Fig. 1A, right panel). Sequencing of the cDNA fragment revealed the inclusion of a new exon of 141 nt, which contained a premature termination codon in an AluY-derived sequence of intron 32 ( Fig. 1B and D). The adjacent deleted sequence was devoid of any transposed elements, as determined by the most sensitive option of the RepeatMasker (http://www.repeatmasker. org/cgi-bin/WEBRepeatMasker, version 4.0.5), yet the deletion was capable of activating a distant cryptic 3 0 splice site of the new exon 128-nt upstream of the deletion breakpoint in the left arm of the AluY copy. Thus, the fusion exon was composed of an Alu-derived sequence of intron 33, 15-nt linker between the AluY and the deletion breakpoint, and the 3 0 end of exon 33 (Figs. 1D, S1 and S2).
Although cryptic exons derived from transposed elements causing genetic disease contain on average more splicing enhancers and less silencers than average human exons (Vorechovsky 2010), the new fusion exon was rich in pyrimidines and in splicing silencers and was depleted of purine-rich enhancers, the most potent exon recognition motifs. For example, the density of exon identity elements (Zhang et al. 2008) was only 68% of the average exon density and less than a half of the density calculated for the deleted exon counterpart (Table 1). This was also reflected in a lower predicted stability across the Alu portion of the new exon, consistent with a higher singlestrandedness observed for exonizing Alus than for nonexonizing Alus (Schwartz et al. 2009). However, the 3 0 splice site of the new exon was stronger than that of exon 33, most likely compensating the unfavorable nucleotide composition of the new exon (Table 1) in the context of a strong 5 0 splice site (score 95.04). Finally, the inclusion of the new exon in the COL4A5 mRNA (Fig. 1A) may have been assisted by shortening of the intron preceding exon 33 by a half and by a relatively short downstream intron, because long introns tend to associate with nonexonizing rather than exonizing Alus (Schwartz et al. 2009).
Multiple sequence alignments of available primate orthologs coupled with evolutionary reconstruction of the insertion event (Krull et al. 2005) showed the presence of this Alu in Old World monkeys and the same 3 0 splicesite consensus of the new exon across species (TAG/A).  Density of exon identity elements (EIEs) (Zhang et al. 2008) was computed as described (Divina et al. 2009).

Supporting Information
Additional Supporting Information may be found in the online version of this article: Figure S1. Alignment of antisense COL4A5 intronic Alu-Ym1 in patient ID45 and the AluYm1 consensus. v, transversions; i, transitions. The 3 0 splice-site AG is in red. Figure S2. The genomic sequence of COL4A5 across the fusion exon. Exons are in upper case, introns are in lower case. Deletion is highlighted in gray; sequence of the new fusion exon is in italics; target-site duplications are underlined.