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.

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 α5 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′ 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′ end of exon 33 (Figs. 1D, S1 and S2).

Figure 1

Deletion-induced exonization of AluY leading to Alport syndrome. (A) PCR amplifications of DNA (left panel) and cDNA (right panel) samples from a control (C), patient (P) and his mother (M). S, size marker; fragment sizes are shown in nts. DNA was amplified by primers 5′-AGTTTTCTGGTTGACATCTTA and 5′-ATAAGTCACTTTTCATGCTAT; cDNA was amplified by primers 5′-CAACCTGGTTTACATGGAAT and 5′-TCCAGGCAAACCCTGATAACC. (B) Sequence chromatogram of patient's DNA (upper panel) and cDNA (lower panel). (C, D) Schematic representation of the genomic deletion (C) and AluY exonization (D). Exons are shown as boxes, introns as horizontal lines, canonical (black) and aberrant (red) splicing by dotted lines above the primary transcripts. Sequence of the new 3′ splice is shown at the bottom, forward slash denotes the new intron–exon boundary. Location of the stop codon is shown by an asterisk.

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 single-strandedness observed for exonizing Alus than for nonexonizing Alus (Schwartz et al. 2009). However, the 3′ 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′ 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).

Table 1

Comparison of sequence features of the new Alu exon and exon 33

	%T	%C	%A	%G	EIE density1	3′ splice site score2	Free energy3
Deleted exon 33	18.0	24.0	26.7	31.3	660	75.96	−0.37
New Alu exon	25.5	29.1	22.0	23.4	324	82.18	−0.27

Density of exon identity elements (EIEs) (Zhang et al. 2008) was computed as described (Divina et al. 2009).

Shapiro–Senapathy score was calculated by an online tool at http://ibis.tau.ac.il/ssat/SpliceSiteFrame.htm.

kcal/mol and nt at 37°C.

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′ splice-site consensus of the new exon across species (TAG/A). This Alu element was absent in New World monkeys (Platyrrhini), indicating that the insertion took place ∼40–25 million years ago and that the 5' breakpoint (/) of the 367-deletion in the patient was in the target-site duplication sequence GGATTAAGCATTAAT/TTTTTT. Thus, the ancient Alu insertion was a prerequisite not only for the exonization event but also for the genomic deletion found in the family.

Alu exonization has facilitated gene regulation through alternative splicing during primate evolution and contributed to the expansion of proteomic interactions in humans (Makalowski et al. 1994; Sorek et al. 2002; Krull et al. 2005), however, only a very limited number of exonization mechanisms has been described, both for existing exons (Lev-Maor et al. 2003; Sorek et al. 2004) and disease-associated events (Meili et al. 2009; Vorechovsky 2010). This case demonstrates that a disease-causing Alu exonization can result from deletions not involving any transposed elements and reveals key sequence features promoting activation of the Alu exon with a poor splicing enhancer/silencer ratio, expanding the range of mutational mechanisms that introduce the most common human repeats in the mRNA.