X-Linked Alport Syndrome Caused by Splicing Mutations in COL4A5

Abstract

Background and objectives X-linked Alport syndrome is caused by mutations in the COL4A5 gene. Although many COL4A5 mutations have been detected, the mutation detection rate has been unsatisfactory. Some men with X-linked Alport syndrome show a relatively mild phenotype, but molecular basis investigations have rarely been conducted to clarify the underlying mechanism.Design, setting, participants, & measurements In total, 152 patients with X-linked Alport syndrome who were suspected of having Alport syndrome through clinical and pathologic investigations and referred to the hospital for mutational analysis between January of 2006 and January of 2013 were genetically diagnosed. Among those patients, 22 patients had suspected splice site mutations. Transcripts are routinely examined when suspected splice site mutations for abnormal transcripts are detected; 11 of them showed expected exon skipping, but others showed aberrant splicing patterns. The mutation detection strategy had two steps: (1) genomic DNA analysis using PCR and direct sequencing and (2) mRNA analysis using RT-PCR to detect RNA processing abnormalities.Results Six splicing consensus site mutations resulting in aberrant splicing patterns, one exonic mutation leading to exon skipping, and four deep intronic mutations producing cryptic splice site activation were identified. Interestingly, one case produced a cryptic splice site with a single nucleotide substitution in the deep intron that led to intronic exonization containing a stop codon; however, the patient showed a clearly milder phenotype for X-linked Alport syndrome in men with a truncating mutation. mRNA extracted from the kidney showed both normal and abnormal transcripts, with the normal transcript resulting in the milder phenotype. This novel mechanism leads to mild clinical characteristics.Conclusions This report highlights the importance of analyzing transcripts to enhance the mutation detection rate and provides insight into genotype-phenotype correlations. This approach can clarify the cause of atypically mild phenotypes in X-linked Alport syndrome.

Functional assessment of a novel COL4A5 splice region variant and immunostaining of plucked hair follicles as an alternative method of diagnosis in X-linked Alport syndrome

“…On the other hand, mild renal disease in a male, despite carrying a splice region variant that causes a truncation, has also been described before. Nozu et al demonstrated the presence of wild type and abnormal transcripts in the kidney of an affected male with a splice region variant in COL4A5 that causes a truncation [21]. Our findings also suggest that incomplete splicing abnormalities might occur in the kidney and lead to a variable phenotype.…”

“…Although other groups have reported using RNA extracted from hair follicles to assay COL4A5 expression and/or mRNA splicing by RT-PCR [21–23], we were unable to obtain enough intact RNA to determine the extent to which normal splicing of exon 25 occurs in vivo . However, if a low frequency of exon 25 retention does occur in vivo , it could partly explain the differences in disease severity among the affected males in this family (e.g., individual 3 compared to individuals 3222 and 3227), and it may also partly explain differences in phenotype between affected females (e.g., individual 3223 who has preserved renal function and her daughter, individual 3226, who has CKD stage 3).…”

“…Reduced but not absent α5(IV) protein in the GBM of males with X-linked AS has been described before. Only one of the α5(IV)-positive patients described by Hashimura et al had a splice site mutation; however, we know from Nozu et al that splice region variants can lead to expression of both normal and abnormal transcripts in the kidneys of male patients with X-linked AS [21, 28]. …”

A birth of bipartite exon by intragenic deletion

“…; Nozu et al. ), although dominant negative effects of the mutated allele cannot be excluded. Our results highlight the importance of characterizing aberrant transcripts for accurate prognosis of this patient and hereditary disorders in general, with potentially important implications for their management.…”

Somatic mosaicism and variant frequency detected by next-generation sequencing in X-linked Alport syndrome

“…22 The milder phenotype in men with XLAS is currently defined by the following five patterns: (1) missense variants in COL4A5; 4-6 (2); in-frame variants in COL4A5; 4,6 (3) somatic mosaic variants in COL4A5; 7,19-21 (4) α5(IV)-positive expression in the glomerulus; 6 and (5) aberrant splicing variants in COL4A5, leading to both normal and abnormal mRNAs. 22 In this study, we reported four cases with milder phenotypes: two with splice site variants (ID14 and 28) and two with missense variants (ID52 and 252). These variant types could contribute to a modulation of the phenotype.…”