Consensus statement on standards and guidelines for the molecular diagnostics of Alport syndrome: refining the ACMG criteria

Abstract

The recent Chandos House meeting of the Alport Variant Collaborative extended the indications for screening for pathogenic variants in the COL4A5, COL4A3 and COL4A4 genes beyond the classical Alport phenotype (haematuria, renal failure; family history of haematuria or renal failure) to include persistent proteinuria, steroid-resistant nephrotic syndrome, focal and segmental glomerulosclerosis (FSGS), familial IgA glomerulonephritis and end-stage kidney failure without an obvious cause. The meeting refined the ACMG criteria for variant assessment for the Alport genes (COL4A3–5). It identified ‘mutational hotspots’ (PM1) in the collagen IV α5, α3 and α4 chains including position 1 Glycine residues in the Gly-X-Y repeats in the intermediate collagenous domains; and Cysteine residues in the carboxy non-collagenous domain (PP3). It considered that ‘well-established’ functional assays (PS3, BS3) were still mainly research tools but sequencing and minigene assays were commonly used to confirm splicing variants. It was not possible to define the Minor Allele Frequency (MAF) threshold above which variants were considered Benign (BA1, BS1), because of the different modes of inheritances of Alport syndrome, and the occurrence of hypomorphic variants (often Glycine adjacent to a non-collagenous interruption) and local founder effects. Heterozygous COL4A3 and COL4A4 variants were common ‘incidental’ findings also present in normal reference databases. The recognition and interpretation of hypomorphic variants in the COL4A3–COL4A5 genes remains a challenge.

Last Nucleotide Substitutions of COL4A5 Exons Cause Aberrant Splicing

“…[15][16][17] For this reason, most Gly missense variants in the COL4A5 triple-helical region are caused by Gly substitutions regardless of the position of the single-base substitutions, and male patients with XLAS with these pathogenic variants have mild phenotypes. 18,19 Nevertheless, single-base substitutions, especially located at the last nucleotide position of each exon, sometimes affect splicing patterns. [20][21][22][23] In fact, in the COL4A5 gene, several single-base substitutions at the last nucleotide position in each exon have been reported and interpreted as missense variants in the Human Gene Mutation Database Q2 (Cardiff, United Kingdom).…”

“…Genomic DNA of all wild-type (WT) samples and 8 patient samples (numbers [Nos.] 1, 4,10,11,13,16,18,19) in our cohort were extracted from whole blood using QuickGene DNA Whole Blood Kit S (Kurabo Industrial Ltd., Osaka, Japan) or QuickGene-Auto S DNA Blood Kit (Kurabo Industrial Ltd.), in accordance with the manufacturer's instructions. Fragment primers were designed to contain 1 exon and >100-base pair upstream and downstream flanking introns to this exon (Supplementary Table S1).…”

“…37 Infusion cloning for 8 variants (Nos. 1,4,10,11,13,16,18,19) and all WT samples was reacted using Infusion HD Cloning Kit (Takara Bio Inc., Tokyo, Japan) according to the manufacturer's instructions. For the reported 12 variants in Human Gene Mutation Database (Nos.…”

Molecular Basis, Diagnostic Challenges and Therapeutic Approaches of Alport Syndrome: A Primer for Clinicians

“…Collagen chains are assembled in the endoplasmic reticulum and secreted to the extracellular space [ 52 ]. Assembly starts from the C-terminal NC1 end [ 53 ] and depends on the formation of disulfide bonds between cysteine residues [ 52 , 54 ] and the stabilization by sulfilimine bonds [ 50 , 55 ] and chloride ions [ 53 , 56 ].…”

“…However, it was recently described that mutations in COL4A genes explain 38% of cases of familial FSGS and 3% of sporadic FSGS [ 75 ]. Skin biopsy could also be useful in cases of XLAS, although it is currently an obsolete technique [ 54 ]. Histological techniques have been used for decades for the diagnosis of AS and innovations are ongoing.…”