Table 2.

Standards and guidelines for the interpretation of sequence variants in COL4A3–COL4A5 in Alport syndrome (modified ACMG/AMP criteria).

PVS1	Null variants (nonsense, frameshift, canonical ±1 or 2 splice site) are pathogenic for Alport syndrome. These account for more than 20% of the pathogenic variants in COL4A3–COL4A5.
PS3	Well-established (robust and reproducible) in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product. Due to their complexity, functional assays are mostly used for research and not their diagnostic utility. The findings must be specific for the variant being tested and not simply be true for the gene.
PS4	The prevalence of the variant in affected individuals is increased compared with controls. Pathogenic variants in the Alport genes are not uncommon in reference databases because of gender-specific (COL4A5) or age-dependent penetrance as well as variable expressivity (such as the lack of haematuria in ~30% of pathogenic COL4A3 and COL4A4 heterozygous variants).
PM1	Located in a mutational hotspots or critical and well-established domains without benign variation. Most Glycine residues in the collagenous domain of the collagen IV α5, α3 and α4 chains should be recognised as critical residues equivalent to a functional domain. The Cysteines in the carboxy NC domain are also critical.
PM2	Absent from controls (or at extremely low frequency if recessive) in gnomAD, ESP, 1000 Genomes or ExAC. Monoallelic pathogenic variants in COL4A5 affect at least one in 5000 of the population, and heterozygous pathogenic COL4A3 and COL4A4 variants one in 100, which means some pathogenic variants are present in large reference databases of normals.
PM5	Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before. Glycines can be substituted with 8 other amino acids or with a stop codon. There are many examples of multiple substitutions at the same Glycine residue in the collagenous domain.
PP2	Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease. The collagen IV α5, α3 and α4 chains are highly conserved from H sapiens (humans) to X tropicalis (frogs), especially the Glycine residues in the collagenous domain and many residues in the carboxy NC domains
PP4	Patients’ phenotype or family history is highly specific for a disease with a single genetic aetiology. Applicable in families with history of microscopic haematuria, hearing loss and renal failure. At least 80% of individuals with inherited haematuria can be demonstrated to have a pathogenic variant in one or more of the COL4A3–COL4A5 genes.
BA1	The allele frequency of a variant is above 5% in normal variant databases. This threshold is also appropriate for benign variants in the Alport genes. It is not possible to further refine the threshold because of the abundance of hypomorphic variants in all the Alport genes in normal variant databases, and the different modes of inheritance of Alport syndrome
BP1	Missense variant in a gene for which primarily truncating variants are known to cause disease. This is not relevant for the Alport genes where both missense and nonsense variants cause disease.
BP2	Observed in trans with a pathogenic variant for a fully penetrant domain gene/disorder; or observed in cis with a pathogenic variant in any inheritance pattern. These criteria do not exclude a pathogenic variant in the Alport genes, where variants in cis or trans may be pathogenic and worsen the disease phenotype.
BP3	In-frame deletions/insertions in a repetitive region without a known function. This is not relevant for the Alport genes.
BP5	Variant found in a case with an alternate molecular basis of disease. Variants in the Alport genes occur commonly in individuals with other inherited renal diseases and at least sometimes worsen disease severity.