Slowly progressive autosomal dominant Alport Syndrome due to COL4A3 splicing variant

Abstract

Alport syndrome is a rare genetic kidney disease caused by variants in the COL4A3/A4/A5 genes. It's characterised by progressive kidney failure, though therapies targeting Renin-Angiotensin System can delay its progression. Additionally, extrarenal manifestations may sometimes coexist. Recent advances in genetic analysis and the necessity to better clarify genotype-phenotype correlations in affected patients raises the importance of detecting even cryptic splicing variants, lying in both canonical and non-canonical splice sites variants such as last exonic nucleotide variants. These variants, often, do not cause an amino acid change but alter the snRNP proteins binding. We studied a big Italian family with Alport syndrome showing a clear dominant pattern of transmission with younger family members having only haematuria and older individuals presenting with End-Stage Kidney Failure (ESKF). Kidney biopsy showed the typical disease hallmarks. We deeply mined the data for SNV and CNV through exome sequencing on DNA from both peripheral blood samples and patients' podocytes-lineage cells. We identified an already reported synonymous variant, c.765G>A (p.(Thr255Thr)), in the last exonic nucleotide of exon 13 of the COL4A3 gene. Employing the patient's podocytes we demonstrated that this variant results in exon skipping leading to an in-frame deletion of 28 amino acids without leaky effect. According to the pattern of transmission, to the kidney biopsy and to the exome data analysis we provided further evidence that autosomal dominant Alport syndrome is a well-defined clinical entity. We also confirmed the pathogenicity of the synonymous COL4A3 variant for the first time demonstrating its role in a dominant pattern of transmission.

Fig. 1. Family pedigree with evident dominant segregation.

The pedigree of the family showed a typical autosomal dominant segregation of the investigated variant.

Fig. 2. Electron microscopy, Periodic acid–Schiff stain (PAS) and Acid Fuchsin Orange G (AFOG) staining on kidney biopsy sections.

A Electron microscopy, UAR, x8000. Extremely thin basal glomerular membrane (GMB) (arrow-head) with regular contour and effacement of foot processes (such lesion was widespread in the glomerular loops). Thickening and irregular appearance of GBM with fragmenting (asterisk), newly formed membrane, reduplication and focal scalloping of foot processes (arrow). These lesions were focal in the glomerular loops. B Pas, x400.Focal segmental glomerulosclerosis (FSG) with capsular adhesion (arrow). C AFOG, x400. The same lesion (FSG) in the next section (arrow). D Pas, x400 Accumulation of lipids is frequent in interstitial cells such as macrophages (asterisk).

Fig. 3. In-silico splicing variant prediction effect.

Bio-Prediction of splice variations using Alamut™ Visual Plus—Variant Annotation and Analysis Software.

Fig. 4. c.765G>A (p.(Thr255Thr)) variant pathogenic role definition by transcript analysis.

A RT-PCR was performed on the endogenous COL4A3 gene using RNA from patients-derived podocytes-lineage cells. Diagrams beside each PCR product represent the structure of detected transcripts, with arrowheads indicating the location of primers (left side). C The transcript analysis allows us to conclude about the pathogenic effect of the variant leading to the exon 13 skipping, resulting in an in-frame deletion of the entire exon and the generation of a new exonic junction between exon12 and exon 14 (B).