Abnormal mRNA Splicing Effect of COL4A3 to COL4A5 Unclassified Variants

Abstract

Introduction: Genetic diagnosis of Alport syndrome (AS), which results from pathogenic variants in COL4A3, COL4A4, or COL4A5 genes, is hindered by large numbers of unclassified variants detected using next-generation sequencing (NGS). We examined the impact on splicing of variants of uncertain significance in COL4A3 to COL4A5.

Methods: Nine unrelated patients with clinical diagnosis or suspicion of AS were enrolled according to the criteria. Their clinical and genetic data were collected. Blood and urine samples were obtained from the patients and their family members. Sanger sequencing was used to confirm the 9 COL4A3 to COL4A5 unclassified variants identified by NGS. COL4A3 to COL4A5 mRNAs from urine were analyzed using targeted reverse transcription polymerase chain reaction and direct sequencing.

Results: Nine COL4A3 to COL4A5 unclassified variants were found to alter mRNAs splicing. Skipping of an exon or an exon fragment was induced by variants COL4A3 c.828+5G>A; COL4A4 c.3506-13_3528del; and COL4A5 c.451A>G (p. [Ile151Val]), c.2042-9 T>G, c.2689 G>C (p. [Glu897Gln]) and c.1033-10_1033-2delGGTAATAAA. Retention of an intron fragment was caused by variants COL4A3 c.3211-30G＞T, and COL4A5 c.4316-20T>A and c.1033-10 G>A, respectively. The 9 families in this study obtained genetic diagnosis of AS, including 3 with autosomal recessive AS and 6 with X-linked AS.

Conclusions: Our findings demonstrate that urine mRNA analysis facilitates the identification of abnormal splicing of unclassified variants in Alport genes, which provides evidence of routine use of RNA analysis to improve genetic diagnosis of AS.

Keywords: Alport syndrome; COL4A3; COL4A4; COL4A5; RNA splicing; unclassified variants.

Graphical abstract

Figure 1

Nine variants of uncertain significance (VUS) and their consequences. The upper panels show schematics of aberrant splicing (red lines). Normal splicing is indicated by black lines. The original sequences are shown below and the 9 variants in patients’ sequences are in red. Inserted sequences on transcripts are indicated by dotted box. Deleted sequences on transcripts are indicated by gray background. Flanking genomic DNA and cDNA sequences of either patients or the mothers are shown in lower panels. (a) VUS-1. c.3211-30G＞T in intron 37 of COL4A3 produced an insertion of 19-bp in abnormal transcript in patient 1. (b) VUS-2. c.828+5G>A in intron 14 of COL4A3 resulted in exon 14 skipping, which created a transcript with a 63-bp deletion in patient 2. (c) VUS-3. c.3506-13_3528del (deletion 36bp) in the intron 37-exon 38 boundary of COL4A4 resulted in exon 38 skipping, which created a transcript with a 72-bp deletion in patient 3. (d) VUS-4. c.4316-20T>A in intron 48 of COL4A5 produced an insertion of 18-bp in abnormal transcript in patient 4. (e) VUS-5. c.1033-10G>A in intron 18 of COL4A5 produced an insertion of 8-bp in abnormal transcript in patient 5 and his mother. (f) VUS-6. c.451A>G in exon 8 of COL4A5 resulted in exon 8 skipping, which created a transcript with a 27-bp deletion in patient 6. (g) VUS-7. c.1033-10_1033-2delGGTAATAAA in intron 18 of COL4A5 created a transcript with an 11-bp deletion in patient 7. (h) VUS-8. c.2042-9 T>G in intron 26 of COL4A5 resulted in exon 27 skipping, which created a transcript with a 105-bp deletion in the mother of patient 8. (i) VUS-9. c.2689 G>C in exon 32 resulted in exon 32 skipping, which created a transcript with a 90-bp deletion in the mother of patient 9 (only urine sample available).