Detection of Splicing Abnormalities and Genotype-Phenotype Correlation in X-linked Alport Syndrome

Abstract

Background: X-linked Alport syndrome (XLAS) is a progressive hereditary nephropathy caused by mutations in the COL4A5 gene. Genotype-phenotype correlation in male XLAS is relatively well established; relative to truncating mutations, nontruncating mutations exhibit milder phenotypes. However, transcript comparison between XLAS cases with splicing abnormalities that result in a premature stop codon and those with nontruncating splicing abnormalities has not been reported, mainly because transcript analysis is not routinely conducted in patients with XLAS.

Methods: We examined transcript expression for all patients with suspected splicing abnormalities who were treated at one hospital between January of 2006 and July of 2017. Additionally, we recruited 46 males from 29 families with splicing abnormalities to examine genotype-phenotype correlation in patients with truncating (n=21, from 14 families) and nontruncating (n=25, from 15 families) mutations at the transcript level.

Results: We detected 41 XLAS families with abnormal splicing patterns and described novel XLAS atypical splicing patterns (n=14) other than exon skipping caused by point mutations in the splice consensus sequence. The median age for developing ESRD was 20 years (95% confidence interval, 14 to 23 years) among patients with truncating mutations and 29 years (95% confidence interval, 25 to 40 years) among patients with nontruncating mutations (P=0.001).

Conclusions: We report unpredictable atypical splicing in the COL4A5 gene in male patients with XLAS and reveal that renal prognosis differs significantly for patients with truncating versus nontruncating splicing abnormalities. Our results suggest that splicing modulation should be explored as a therapy for XLAS with truncating mutations.

Keywords: X-linked Alport syndrome; genotype-phenotype correlation; renal prognosis; splicing abnormalities; transcript analysis.

Graphical abstract

Figure 1.

Mutations and their consequences (the same figures are shown in Supplemental Figure 2 with larger scales). Upper panels show schemas of aberrant splicing (red lines). Normal splicing is indicated by black lines. The original and new splice sites and flanking sequences are shown below. Patients’ flanking genomic DNA and cDNA sequences are shown in the lower panels. (A) Patient ID A196. IVS23–1 G>A eliminated the splice acceptor site of intron 23 to activate a new splice site, one nucleotide downstream. (B) Patient ID A333. IVS49+1 G>A disrupted the splicing donor site of intron 49, resulting in an intron 49 insertion, which creates a transcript with a 345-bp insertion. (C) Patient ID A424. IVS 6–1 G>A altered the splice acceptor site of intron 6 one nucleotide downstream, which creates a transcript with a 1-bp deletion. (D) Patient ID A231, A258, A298. IVS35–4 A>G altered the splice acceptor site of intron 35 three nucleotides upstream, which creates a transcript with a 3-bp insertion. (E) Patient ID A247. IVS 12+5 G>A disrupted the splice donor site of intron 12, resulting in exon 12 skipping, which creates a transcript with a 42-bp deletion. (F) Patient ID A299. IVS29+3 A>G disrupted the splice donor site of intron 29, resulting in exon 29 skipping, which creates a transcript with a 151-bp deletion. (G) Patient ID A323. IVS40–9 C>G altered the splice acceptor site of intron 40 nine nucleotides upstream, which creates a transcript with a 9-bp insertion. (H) Patient ID A371. IVS 18+3_6 del AAGT disrupted the splice donor site of intron 18, resulting in exon 18 skipping, which creates a transcript with a 42-bp deletion. (I) Patient ID A384. IVS48–11A>G altered the splice acceptor site of intron 48 ten nucleotides upstream, which creates a transcript with a 10-bp insertion. (J) Patient ID A402. IVS27+4 del T disrupted the splice donor site of intron 27, resulting in exon 27 skipping, which creates a transcript with a 105-bp deletion. (K) Patients ID A452. IVS29+5 G>A disrupted the splice donor site of intron 29, resulting in exon 29 skipping, which creates a transcript with a 151-bp deletion. (L) Patient ID A329. IVS21–367 C>T produced a new splice donor site, resulting in a cryptic exon activation between exons 21 and 22 and creating a transcript with a 93-bp insertion. (M) Patient ID A375. Mutation in last nucleotide of exon 25, C1948 G>T, disrupted the splice donor site of intron 25, resulting in exon 25 skipping, which creates a transcript with a 169-bp deletion. (N) Patient ID A422. Mutation of the second nucleotide of exon 10, C548 dup G, disrupted the splicing acceptor site of intron 9, resulting in exon 10 skipping, which creates a transcript with a 63-bp deletion. gDNA, genomic DNA.

Figure 1.

Mutations and their consequences (the same figures are shown in Supplemental Figure 2 with larger scales). Upper panels show schemas of aberrant splicing (red lines). Normal splicing is indicated by black lines. The original and new splice sites and flanking sequences are shown below. Patients’ flanking genomic DNA and cDNA sequences are shown in the lower panels. (A) Patient ID A196. IVS23–1 G>A eliminated the splice acceptor site of intron 23 to activate a new splice site, one nucleotide downstream. (B) Patient ID A333. IVS49+1 G>A disrupted the splicing donor site of intron 49, resulting in an intron 49 insertion, which creates a transcript with a 345-bp insertion. (C) Patient ID A424. IVS 6–1 G>A altered the splice acceptor site of intron 6 one nucleotide downstream, which creates a transcript with a 1-bp deletion. (D) Patient ID A231, A258, A298. IVS35–4 A>G altered the splice acceptor site of intron 35 three nucleotides upstream, which creates a transcript with a 3-bp insertion. (E) Patient ID A247. IVS 12+5 G>A disrupted the splice donor site of intron 12, resulting in exon 12 skipping, which creates a transcript with a 42-bp deletion. (F) Patient ID A299. IVS29+3 A>G disrupted the splice donor site of intron 29, resulting in exon 29 skipping, which creates a transcript with a 151-bp deletion. (G) Patient ID A323. IVS40–9 C>G altered the splice acceptor site of intron 40 nine nucleotides upstream, which creates a transcript with a 9-bp insertion. (H) Patient ID A371. IVS 18+3_6 del AAGT disrupted the splice donor site of intron 18, resulting in exon 18 skipping, which creates a transcript with a 42-bp deletion. (I) Patient ID A384. IVS48–11A>G altered the splice acceptor site of intron 48 ten nucleotides upstream, which creates a transcript with a 10-bp insertion. (J) Patient ID A402. IVS27+4 del T disrupted the splice donor site of intron 27, resulting in exon 27 skipping, which creates a transcript with a 105-bp deletion. (K) Patients ID A452. IVS29+5 G>A disrupted the splice donor site of intron 29, resulting in exon 29 skipping, which creates a transcript with a 151-bp deletion. (L) Patient ID A329. IVS21–367 C>T produced a new splice donor site, resulting in a cryptic exon activation between exons 21 and 22 and creating a transcript with a 93-bp insertion. (M) Patient ID A375. Mutation in last nucleotide of exon 25, C1948 G>T, disrupted the splice donor site of intron 25, resulting in exon 25 skipping, which creates a transcript with a 169-bp deletion. (N) Patient ID A422. Mutation of the second nucleotide of exon 10, C548 dup G, disrupted the splicing acceptor site of intron 9, resulting in exon 10 skipping, which creates a transcript with a 63-bp deletion. gDNA, genomic DNA.

Figure 2.

Probability of developing ESRD according to mutation types. Solid line indicates patients with truncating splicing mutations (n=21). The median age for developing ESRD was 20 years. Dash-dots indicate nontruncating splicing mutations (n=25). The median age for developing ESRD was 29 years.