European Journal of Human Genetics (2021) 29:1186–1197 https://doi.org/10.1038/s41431-021-00858-1 MEETING REPORT Consensus statement on standards and guidelines for the molecular diagnostics of Alport syndrome: refining the ACMG criteria Judy Savige 1 Helen Storey 2 Elizabeth Watson3 Jens Michael Hertz4 Constantinos Deltas 5 ● ● ● ● ● Alessandra Renieri 6 Francesca Mari7 Pascale Hilbert 7 Pavlina Plevova8 Peter Byers9 ● ● ● ● ● Agne Cerkauskaite10 Martin Gregory11 Rimante Cerkauskiene12 Danica Galesic Ljubanovic13 ● ● ● ● Francesca Becherucci14 Carmela Errichiello14 Laura Massella15 Valeria Aiello16 Rachel Lennon17 ● ● ● ● ● Louise Hopkinson 17 Ania Koziell18 Adrian Lungu19 Hansjorg Martin Rothe 20 Julia Hoefele 21 ● ● ● ● ● Miriam Zacchia22 Tamara Nikuseva Martic23 Asheeta Gupta24 Albertien van Eerde25 Susie Gear26 ● ● ● ● ● Samuela Landini 27 Viviana Palazzo28 Laith al-Rabadi29 Kathleen Claes 30 Anniek Corveleyn31 ● ● ● ● ● Evelien Van Hoof31 Micheel van Geel32 Maggie Williams33 Emma Ashton34 Hendica Belge35 Elisabeth Ars36 ● ● ● ● ● ● Agnieszka Bierzynska37 Concetta Gangemi38 Beata S. Lipska-Ziętkiewicz39 ● ● Received: 16 July 2020 / Revised: 13 February 2021 / Accepted: 2 March 2021 / Published online: 15 April 2021 © The Author(s) 2021. This article is published with open access 1234567890();,: 1234567890();,: 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. Introduction in 5,000 [3, 4]. This is consistent with Alport syndrome being the second commonest cause of inherited kidney Estimates of the prevalence of Alport syndrome vary from failure after autosomal dominant Polycystic Kidney Dis- one in 5,000 to one in 53,000 of the population [1, 2], but ease. It also means that many variants are undetected and the frequent finding of likely pathogenic COL4A5 variants that even apparently normal individuals have pathogenic in normal reference datasets and cohorts with renal failure variants in the COL4A3–COL4A5 genes. suggests that X-linked disease affects closer to closer to one Inheritance of Alport syndrome is X-linked (OMIM301050), autosomal recessive (OMIM 203780), digenic (typically with a pathogenic variant in each of Supplementary information The online version contains COL4A3 and COL4A4 [5]) or autosomal dominant (OMIM supplementary material available at https://doi.org/10.1038/s41431- 104200, or thin basement membrane nephropathy, due to 021-00858-1. heterozygous COL4A3 or COL4A4 variants [6]). The typical * Judy Savige clinical features of Alport syndrome are persistent micro- jasavige@unimelb.edu.au scopic haematuria, end-stage kidney failure, and often a Extended author information available on the last page of the article family history of haematuria or renal failure [7]. Hearing loss Consensus statement on standards and guidelines for the molecular diagnostics of Alport syndrome:. . . 1187 and non-nephrotic range proteinuria are common. Ocular syndrome’, ‘nephrotic range proteinuria’, ‘steroid-resistant abnormalities such as lenticonus with abnormal lens protru- nephrotic syndrome’) or with the renal biopsy finding of sion, or fleck retinopathy may be present [7]. Clinical features focal and segmental glomerulosclerosis (FSGS)[11, 12]. overlap between boys and men with X-linked disease, and in FSGS is one of the commonest causes of glomerular disease boys and girls, men and women with recessive or digenic resulting in progressive renal failure, and pathogenic inheritance [5, 8]. Many women with X-linked Alport syn- COL4A3–COL4A5 variants are the commonest cause of drome, and males and females with heterozygous COL4A3 or adult-onset FSGS ranging from 5% to 20% of cases with COL4A4 variants, or sometimes digenic COL4A3 and sporadic or familial disease respectively [11, 12]. In such COL4A4 variants, have only microscopic haematuria [5, 8, 9]. cases the glomerular basement membrane does not neces- In February 2020, a group of 47 medical and scientific sarily have the lamellation typical of Alport syndrome and specialists from three continents, all with an interest in the COL4A3–COL4A5 variants may be associated with an older molecular diagnostics Alport syndrome (the ‘Alport Variant age at end-stage kidney failure [13]. Pathogenic variants in Collaborative’), met in London to review the current recom- any of the COL4A3–COL4A5 genes are also found in up to mendations for genetic testing for Alport syndrome and to 10% of individuals with renal failure where the cause was further refine the American College of Medical Genetics/ not known [3]. In addition families with IgA glomer- Association of Molecular Pathologists (ACMG/AMP) criteria ulonephritis not uncommonly have mutations in the for evaluating variants in the COL4A3–COL4A5 genes that COL4A3–COL4A5 genes [14]. Finally there are reports of have been published previously [8, 10]. The group comprised COL4A3–COL4A5 variants being associated with renal adult (n = 12) and paediatrics (n = 6) nephrologists, histo- cysts where autosomal dominant polycystic kidney disease pathologists (n = 2), geneticists (n = 17), laboratory scientists has been excluded [15, 16]. (n = 4), researchers (n = 2), industry scientists (n = 3) and a Thus, not only individuals with the typical features of Alport representative of the Alport UK Patient Support group syndrome should undergo genetic testing, but also those with (n = 1). Attendees had submitted difficult-to-evaluate proteinuria or FSGS, with familial IgA glomerulonephritis or COL4A3–COL4A5 variants, which were precirculated, and with end-stage kidney failure where there is no obvious cause they spent two sessions at the meeting examining these var- (Table 1). For individuals with proteinuria or FSGS, or end- iants, and discussing the evidence for pathogenicity. Their stage kidney failure, the presence of haematuria or a family conclusions have been summarised into the following history of haematuria, and in the case of IgA disease, a family guidelines for genetic testing for Alport syndrome for use by history of haematuria, all make the detection of a pathogenic molecular testing laboratories. A draft of these guidelines was COL4A3–COL4A5 variant more likely. However the meeting then sent to all attendees, modified based on their comments, considered there was currently insufficient evidence to and the final document approved by the group prior to sub- recommend that individuals with cystic kidney disease should mission. Decisions about individual variants are ongoing with also be tested for COL4A3–COL4A5 variants. further clinical data being sought in order to reach at least two-thirds consensus on the assertions. Genetic testing of the COL4A3–COL4A5 genes Broadening the phenotype Finding a COL4A3–COL4A5 variant that affects the struc- Increasingly, pathogenic variants in COL4A3–COL4A5 are ture or function of a collagen IV α chain confirms the found in individuals with proteinuria (‘nephrotic diagnosis of Alport syndrome, and indicates that other Table 1 Recommendations for genetic testing for Alport syndrome. Clinical spectrum The typical clinical features of Alport syndrome are persistent haematuria, sometimes with progressive renal failure, together with a family history of haematuria or renal failure. Proteinuria, a hearing loss or a lamellated or thinned glomerular basement membrane may be present. Other phenotypes associated with pathogenic variants in the COL4A3–COL4A5 genes and hence Alport syndrome include persistent proteinuria, steroid-resistant nephrotic syndrome and focal and segmental glomerulosclerosis (FSGS); familial IgA glomerulonephritis; and end-stage kidney failure of unknown cause. Pathogenic COL4A3–COL4A5 variants are the commonest genetic abnormality found in all these phenotypes. Genetic testing The most accurate test for the detection of a causative pathogenic variant in the COL4A3–COL4A5 genes is comprehensive parallel genetic testing of the entire coding sequences of all three COL4A5, COL4A3 and COL4A4 genes. Where Alport syndrome is suspected, genetic testing for diagnosis should take precedence over even a renal biopsy. Where a causative variant is demonstrated, a renal biopsy may not be necessary. The COL4A3–COL4A5 genes should also be included in multigene Massively Parallel Sequencing renal disease panels for the extended Alport phenotypes. The Alport syndrome- specific modifications of the ACMG standards and guidelines for the Interpretation of Sequence Variants apply. 1188 J. Savige et al. family members should be investigated. Genetic testing also nonsense in 10%, canonical splice sites in 10% and fra- identifies the mode of inheritance, and which family meshifts in 20% (databases.lovd.nl/shared/genes/COL4A5). members who are at risk of being affected. End-stage kid- About half the pathogenic COL4A3–COL4A5 variants ney failure is very likely in hemizygous COL4A5 males found in diagnostic laboartories are novel. There appear to (90% by the age of 40 years) and individuals with biallelic be some changes in the types of pathogenic variants found defects in COL4A3 or COL4A4, but much less common in in the last years. Fewer large deletions have been reported females with a heterozygous COL4A5 variant (15–30% by possibly because MPS detects these less well, or because 60 years) and a person with a heterozygous pathogenic the more severe disease that these people have means that variant in COL4A3 or COL4A4 [17]. The nature of indivi- they have already been studied. In addition, more non- dual variants also indicates the likelihood of early-onset canonical splice site variants, and non-Gly substitutions in kidney failure and extra-renal features such as ocular the collagenous and carboxy NC1 domains have been abnormalities [18, 19]. Hearing loss is common with X- reported. linked and recessive disease [19]. Targeted diagnostics can be used to identify other affected family members, to ensure that an affected person does not act as a kidney donor, and Definition of pathogenicity in preimplantation genetic diagnosis [8]. Where a variant is identified in a person referred with FSGS for testing, the The definition of pathogenicity has become less clear for finding of a causative pathogenic variant in one of the sequence variants in COL4A3–COL4A5. Previous meetings COL4A3–COL4A5 genes means that treatment with corti- have concluded that the demonstration of a pathogenic costeroids or immunosuppressants is not usually useful variant was the best evidence for the diagnosis of Alport [4, 20, 21], and that this type of FSGS does not typically syndrome [8, 10]. Despite the introduction of the standards recur after renal transplantation. and guidelines for the interpretation of sequence variants MPS usually identifies at least 80% of disease-causing (ACMG/AMP criteria [24]) there is sometimes insufficient variants in COL4A3–COL4A5 where Alport syndrome is data for a conclusion of pathogenicity. The association with suspected clinically [22] because of microscopic haematuria haematuria or kidney failure, and a family history of hae- or progressive kidney failure, together with a positive maturia or renal failure may be the strongest evidence for family history of haematuria or renal failure. Where a the diagnosis of Alport syndrome. Microscopic haematuria causative variant or variants are not found, it is important to is found in 95% of those with a pathogenic COL4A5 variant ensure that the genes are sufficiently ‘covered’ by the [8], and at least 67% with a pathogenic heterozygous sequencing technique. Other explanations for the inability COL4A3 or COL4A4 variant [6]. End-stage kidney to detect pathogenic variants are the large genomic imbal- failure occurs in all males, but many fewer females with a ances encompassing one or more exons (such as deletions, pathogenic COL4A5 variant [9]. The situation is more duplications or inversions) that MPS detects less well; a complicated in recessive Alport syndrome by the require- location deep within an intron or regulatory element or ment for two COL4A3 or COL4A4 variants in trans [8]. affecting a non-canonical splice site (for example, 5 Renal failure is less common, but recognised increasingly, nucleotides from the intron-exon boundary); there being with pathogenic heterozygous COL4A3 or COL4A4 insufficient evidence for pathogenicity and the variant being variants [17]. assessed as a Variant of Uncertain Significance (VUS); the The manual application of the ACMG/AMP guidelines is variant affecting a processing enzyme or chaperone rather time-consuming, subject to error and yields inconsistent than a COL4 gene, as occurs in about 10% of cases of results [25]. The tools themselves may be flawed [26]. Several osteogenesis imperfecta; [23] and rarely because of mosai- web-based tools, such as Alamut (www.interactive-biosoft- cism. Sometimes the disease is simply not Alport syndrome. ware.com/alamut-visual/), Varsome (www.varsome.com) Phenocopies of Alport syndrome where there is pre- [27] and the ClinGen pathogenicity calculator (calculator. dominantly haematuria include Fechtner syndrome clinicalgenome.org) [25], may help in classifying variants by (OMIM155100), fibronectin glomerulopathy (OMIM summarising the results for ACMG/AMP criteria such as the 601894), Nail-Patella syndrome (OMIM 161200), Heredi- computational criteria (Polyphen2, SIFT, Mutation taster, tary Angiopathy, Nephropathy and muscle Cramps (OMIM conservation scores, and occurrence in gnomAD and other 611773), Dense deposit disease (OMIM 134370) and reference databases) in one site [24]. The ACMG/AMP CFHR5 deficiency (OMIM 614809). However increasingly recognise that “those working in specific disease groups pathogenic variants are found in genes affected in protei- should continue to develop more focused guidance regarding nuric diseases. the classification of variants in specific genes“, and thus the More pathogenic variants are known for COL4A5 than Chandos House meeting assessed the ACMG criteria in the for COL4A3 and COL4A4, and these are missense in 60%, context of Alport syndrome and the COL4A3- COL4A5 genes Consensus statement on standards and guidelines for the molecular diagnostics of Alport syndrome:. . . 1189 Table 2 Standards and guidelines for the interpretation PVS1 Null variants (nonsense, frameshift, canonical ±1 or 2 splice site) are pathogenic for Alport of sequence variants in syndrome. COL4A3–COL4A5 in Alport These account for more than 20% of the pathogenic variants in COL4A3–COL4A5. syndrome (modified ACMG/ PS3 Well-established (robust and reproducible) in vitro or in vivo functional studies supportive of a AMP criteria). 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. and made the following recommendations (Table 2) including The Chandos House meeting did not revise the weighting the identification of significant functional domains and system for the ACMG/AMP criteria for Alport variants mutational hotspots (PM1); the degree of conservation of the [24]. Nor did it did it reach consensus on who had the protein sequences in different species (PP3); the ‘well-estab- responsibility for requesting a review of Variants of lished’ functional assays that indicate pathogenicity (PS3, Uncertain Significance for a reclassification to Likely BS3); whether pathogenic variants are absent from databases Pathogenic/Pathogenic or Likely Benign/Benign and how with healthy control individuals (PM2) [28] and the threshold often this assessment should be undertaken. Many labora- in these databases above which variants are likely to be tories considered that it was the clinician’s responsibility to benign (BA1, BS1). request a VUS review based on the ongoing need for a 1190 J. Savige et al. Table 3 Location of missense variants in the collagen IV α5 chain in different variant databases. Number of missense variants Number of amino acids (% of total) ClinVar ARUP HGMD LOVD Total 1691 (100%) 487 347 430 639 Amino NC domain 41 (2%) 4 (1%) 0 7 (2%) (p = 0.34) 1 (0%) (p < 0.0001) Intermediate collagenous domain 1418 (84%) 427 (88%) 303 (87%) 368 (86%) 565 (88%) (p = 0.006) Carboxy NC domain 231 (14%) 56 (11%) 44 (13%) 55 (13%) 73 (11%) (p = 0.17) p values indicate the difference in the number of missense variants found in the region according to this database compared with the expected number based on the number of amino acids in this domain using Fisher’s exact test. Table 4 Comparison of variant databases for COL4A5. COL4A5 ClinVar ARUP HGMD LOVD Website www.ncbi.nlm.nih.gov/clinvar/ arup.utah.edu/database/ www.hgmd.cf.ac.uk www.lovd.nl/ ALPORT/ALPORT_ COL4A5 display.php Total number of 1260 807 905 (1169 for subscribers) 1595 variants Curated Submitters’ assessments recorded but a star system Submitters’ assessments Yes Submitters’ depending on number of reports and expert panel recorded assessments recorded assessment Clinical details Includes publications with clinical details Includes publications Includes publications Clinical details from available submitters plus publications Includes benign or Yes (n = 101) Yes (n = 23) Assessments only available to Yes (n = 177) likely benign subscribers variants Cost Freely available online Freely available online Publicly-available version up to 3 Freely years previously; otherwise available online professional version The databases were accessed in April-May 2020. diagnosis or the availability of new clinical information. On interruptions, are more difficult. In-silico tools used to assess the other hand, clinicians considered that laboratories were the effect of a particular amino acid substitution on the protein more aware of advances in algorithms for defining patho- structure may not take into account the character of the genicity. Finally, the ACMG/AMP recommendations for Gly-Xaa-Yaa triplets (a Glycine followed by two other amino reporting incidental findings in Clinical Exome and Gen- acids) typical of the collagen chains and as a result their pre- ome Sequencing do not include pathogenic variants in the dictions of the effect on structure may be inaccurate. COL4A3–COL4A5 genes [29] despite these being common Most nonsense variants in the COL4A3–COL4A5 genes and clinically significant. result in nonsense-mediated mRNA decay and loss of the corresponding collagen IV α chains from affected mem- branes. Because the α5 chain is part of the α3α4α5 hetero- Modification of the ACMG/AMP criteria for trimer, the α3 and α4 chains with which it normally forms the the COL4A3–COL4A5 genes and rationale heterotrimer are imperfectly incorporated or not incorporated at all [30], and there is a compensatory increase in the Types of pathogenic variants expression of the α1α1α2 network [31]. This means that males with a COL4A5 nonsense variant typically have no Missense variants are the commonest changes found in α3α4α5 (or α5α5α6) network in affected membranes. In COL4A3–COL4A5. For the collagen IV α5 chain, more mis- females with COL4A5 heterozygous nonsense variants, sense variants are found in the large intermediate collagenous however, this loss is segmental depending on lyonisation domain (84–88% depending on the database) which is more since clusters of cells produce normal amounts of the α5 chain than expected based on length (p ≤ 0.006, LOVD, Table 3) and [32]. In males and females with COL4A3 or COL4A4 het- many of which are Glycine substitutions (95% (278/294) in erozygous nonsense variants, affected cells result in less of the ARUP, arup.utah.edu/database/). Missense variants in the α3α4α5 network and membrane thinning. carboxy non-collagenous domain are also common (11–14%). Variants in COL4A5 have been collated into a number of Missense variants that are not Glycine substitutions are prob- databases (Table 4), from the literature and submissions from ably the most difficult to classify, but even some Glycine individual laboratories. Currently all databases record variant substitutions, especially those adjacent to non-collagenous assessments made by the submitting laboratories. The role of Consensus statement on standards and guidelines for the molecular diagnostics of Alport syndrome:. . . 1191 such databases in variant assertions is controversial [33, 34] α5α5α6) [42].The α3α4α5 network is the main component but they still represent useful repositories. ClinVar is the only of the glomerular basement membrane, encoded by the database that independently evaluates variants but it has no COL4A3, COL4A4 and COL4A5 genes respectively. The expert panel assessments for the COL4A3–COL4A5 variants. amino acid sequences of these chains are each highly Most laboratories also consult their own databases of variants conserved in different species, and also between the that they have assessed previously. individual α5, α3 and α4 chains (Fig. 1, Supplementary Figs. 1 and 2). Functional studies Each collagen IV α chain has an amino non- collagenous (NC) domain; an intermediate collagenous The in vitro or in vivo functional studies used in patho- domain; and a carboxy NC domain (Fig. 1). The inter- genicity assessments must be specific for the individual mediate collagenous domain has the sequence Gly-Xaa- gene variant and not for the gene as a whole. One of the Yaa where X and Y are often Proline or Hydroxyproline. commonest in vitro tests for Alport syndrome is immu- The presence of a Glycine in every third position in the nohistochemistry of a renal or skin biopsy to demonstrate Gly-Xaa-Yaa repeats is critical to triple helix formation that the collagen IV α5 chain is absent in X-linked and because Glycine is the smallest amino acid and fits within autosomal recessive Alport syndrome [35, 36]. However the helix interior. The Proline residues contribute flex- this does not confirm that a particular variant is patho- ibility. The three α3, α4 and α5 chains bind together genic but only that the individual has a collagen IV defect through disulphide bonds at the Cysteine residues and by consistent with the diagnosis of Alport syndrome. a sulfilimine bond between Lysine and Methionine resi- Among the functional studies that support a variant dues all within the carboxy NC domain [43]. There are having a damaging effect on the gene or gene product, also multiple non-collagenous interruptions in the col- in vitro trimerization assays that examine secretion into the lagenous domains that confer flexibility and facilitate extracellular space [37] correlate well with clinical variant network formation (Tables 5–7) [44]. Each interruption severity, especially in distinguishing pathogenic from non- has adjacent Glycine residues, and some incorporate a pathogenic Glycine variants in COL4A5. In addition, for Glycine. However the biochemistry is even more com- non-canonical splice sites, minigene assays or sequencing plicated because while the transcript for individual α of urinary podocyte or fibroblast mRNA can be used, but chains is read off forwards 5’ to 3’, from the amino to the are currently only available in specialised laboratories [38]. carboxy terminus, the three chains bind through dis- Increasingly, COL4A5 variants affecting non-canonical ulphide and sulphilimide bonds in the carboxy terminus splice sites, up to 11 nucleotides from the intron-exon from which the triple helix winds up ‘backwards’ [45, 46]. boundary, are identified in individuals with X-linked Alport Substitution of these critical position 1 Glycine residues syndrome. Sometimes synonymous variants within the within the Gly-Xaa-Yaa sequence distorts and disrupts coding region result in splicing changes that are also helix formation. Glycine substitutions in other inherited disease-causing. Potential splicing changes can be exam- collagen diseases are typically pathogenic, for example, in ined with on-line prediction tools such as MaxEntScan or COL1A1 in osteogenesis imperfecta [47]. The effect of SpliceSiteFinder, but eventually require confirmation with Glycine substitutions in positions 2 or 3 or within the in vitro assays. interruption is not known. The local environment is also Functional assays used in research include evaluation of important and Glycine substitutions adjacent to interrup- abnormalities in electrophoresis and thermal stability of tions, where there is more chain flexibility, appear to be collagen IV (previously common in collagen type I); less pathogenic [48, 49]. [39, 40] and mRNA quantitation [41]. Again these assays Proline substitutions in the collagen IV α5 chain are are not specific for individual gene variants unless a zeb- reported pathogenic much less often than Glycine sub- rafish, mouse or normal cell line has been engineered to stitutions (databases.lovd.nl/shared/genes/COL4A5). Pro- incorporate the variant being assessed. line confers flexibility and its replacement with another amino acid is less problematic than for Glycine. However, Critical and well-established domains and Proline residues are also found in both the integrin binding mutational hotspots sites and in the GlyProProGlyProPro binding site for gly- coprotein VI which is needed for platelet activation [50], Collagen IV differs from most other collagens in forming but no Proline substitutions have been described in these a chicken-wire network rather than fibrillar structure. The residues in the COL4A3–5 genes. COL4A1–COL4A6 genes code for the collagen IV α1–α6 Interestingly, pathogenic variants have been reported for chains. Collagen IV is a heterotrimer with three of the 6 α 10 of the 12 Cysteine residues that are critical for cross- chains producing a triple helix (α1α1α2, α3α4α5 or linking in the carboxy NC domain (databases.lovd.nl/ 1192 J. Savige et al. a Carboxy non- Intermediate collagenous domain with Gly-Xaa-Yaa repeats and 23 collagenous non-collagenous interrupons domain b c Fig. 1 Collagen IV α5 chain. a Schematic of collagen IV α5 chain conserved between different species both in the collagenous and in the with amino and carboxy non-collagenous domains, and intermediate carboxy non-collagenous domains. This sequence also includes a non- collagenous sequence with Gly-Xaa-Yaa repeats and 23 non-col- collagenous interruption as well as the carboxy terminus (both in lagenous interruptions. Gly substitutions adjacent to the interruptions boxes). c Clustal sequence demonstrating that the human collagen IV are often hypomorphic with a milder clinical phenotype. b Clustal α5, α3 and α4 chains are also conserved. sequence demonstrating that the collagen IV α5 chain is highly shared/genes/COL4A5). No substitutions have been α5, α3 and α4 chains should be recognised as critical residues described for the Lysine and Methionine residues in the NC equivalent to a functional domain. However some Glycine domain that form the sulfilimine bond. substitutions adjacent to NC interruptions represent hypo- Thus, the Chandos House meeting recommended that most morphic variants with a milder clinical phenotype [49, 51] such Glycine residues in the collagenous domain of the collagen IV as p.(Gly624Asp) in COL4A5. Clinical information is critical Consensus statement on standards and guidelines for the molecular diagnostics of Alport syndrome:. . . 1193 Table 5 Non-collagenous domains and interruptions in the collagen IV Table 6 Non-collagenous domains and interruptions in the collagen IV α5 chain [44]. α3 chain [56]. Non-collagenous Residues Residue number of Gly Interruptions Residues Residue number of Gly interruption (residues) (residues) Amino NC (1–42) MSA…FCD 30, 37 Amino -NC MKL…DCS 5, 11, 21, 31, 35 I (160–170) AKEEDIELDAK domain (1–41) II (222–223) VI I (160–167) SIIMSSLP III (245–258) IVTLTGPDNRTDLK 250 II (220–223) LNFQ IV (283–287) YGSEK 284 III (243–257) QISEQKRPIDVEFQK V (345–353) EYYDTYQEK IV (282|283) – VI (387–388) RP V (344–355) LVIPRPGTGITI 350, 352 VII (413–414) AM VI (390–393) AAVM VIII (442–445) VFRK VII (416–419) ISIP IX (476–483) CTQCPYIP VIII (442–453) PHIPPSDEICEP X (544–547) LQPE IX (479–487) DICFNCIGT 486 XI (587–589) ALS X (549–554) DILTFP XII (617–618) GY 617 XI (595–599) GITFK 595 XIII (649–655) SVSTPVP XII (625) F XIV (698–699) GI 698 XIII (657–662) QTITQP XV (745–749) AVAMP XIV (706) I XVI (810–811) IE XV (753–756) FALP XVII (848–849) RS XVI (818) I XVIII (946–951) EISHVI XVII (853–856) LDVP XIX (1060–1064) EGTRP 1061 XVIII (954–960) PMDPNLL XX (1176–1179) RAPP XIX (1070–1073) ISSI XXI (1234–1235) IP XX (1189) F XXII (1263–1264) VI XXI (1245–8) PALE XXIII KIISLP XXII (1373–78) QSIIIK (1352–1357) Carboxy -NC TSS…KRT 1467, 1485, 1492, 1500, Carboxy NC ATW…KRH 1445, 1463, 1470, 1478, domain 1506, 1510, 1513, 1560, (1439–1670) 1484, 1488, 1491, 1538, (1461–1691) 1595, 1602, 1612, 1615, 1553, 1573, 1580, 1590, 1617, 1624, 1650, 1593, 1595, 1602, 1618, 1642, 1675 1620, 1653 This table is from the original report. Since then the Ref Seq has been changed by the addition of 6 amino acids 1264–1264: G>GPTGFQG in the kidney transcript, which alters the numbering. Interruption IV is formed from two collagenous regions directly definitions or criteria for their identification. They may adjacent to one another. This results in the Gly-Xaa-Yaa-Gly-Gly-Xaa- result in haematuria only, later-onset kidney failure, or Yaa-Gly sequence and a structural kink. GBM thinning rather than lamellation in a male. Variants in the COL4A3–COL4A5 genes may also modify the patho- for the complete evaluation of variants, and where there are genic effects of genes encoding other podocyte or glo- discrepancies, the evidence from many laboratories, such as merular membrane proteins. segregation of haematuria with the variant in multiple families The COL4A5 hypomorphic variant, NM_000495.5: or within many members of the same family, may confirm a c.1871G>A substitution (rs104886142); p.(Gly624Asp) pathogenic nature. The 12 Cysteines in the carboxy NC results in late-onset kidney failure and is the commonest domain should also be considered critical domains, and any pathogenic variant causing X-linked Alport syndrome in substitutions are likely to be pathogenic. Central and Eastern Europe [51]. It has previously been considered benign [10] or a VUS. This variant is located Hypomorphic variants adjacent to a non-collagenous interruption in the Gly-X-Y sequence which reduces the effect of the Gly substitution Hypomorphic variants are increasingly described in the (Table 5). The Chandos House meeting considered that COL4A5 gene [51–53] although there are, as yet, no hypomorphic variants, such as p.(Gly624Asp) may still be 1194 J. Savige et al. Table 7 Non-collagenous domains and interruptions in the collagen IV variation/24573/). Both p.(Gly545Ala) and p.(Gly999Glu) α4 chain [57]. affect Gly residues within the collagenous domain of the Interruptions Residues Residue number of Gly collagen IV α4 chain itself. These are considered Benign** (residues) (www.ncbi.nlm.nih.gov/clinvar/variation/255015/) and with Amino NC (1–61) MWS…PEK 22, 38, 40, 45, 48, 49 Conflicting interpretation of pathogenicity* (www.ncbi. I (176–183) VFILGAVK 180 nlm.nih.gov/clinvar/variation/191312/) respectively. Thus II (235–236) GV 235 interpretation of pathogenicity is complicated, and variants III (258–269) LLVEPPDFCLYK while themselves not causing haematuria may still worsen IV (294–295) GI 294 renal failure progression. V (359–366) VTPPLPLK VI (400–401) CA Conclusions VII (429–432) DSAP VIII (457–462) VIYCSV The COL4A3–COL4A5 genes have particular characteristics IX (493–496) ACEP that must be considered in assessing variants for a patho- X (560–565) VVSRVK genic or benign nature. Most notable are the importance of XI (605–609) EDATP the position 1 Glycine substitutions in the collagenous XII (631–632) GL 631 domains, Cysteine substitutions in the carboxy NC domains XIII (666–673) ESCNVTYP and the recognition of hypomorphic variants associated XIV (716–718) EIP with a milder clinical phenotype with microscopic haema- XV (740–741) PV turia only, late-onset renal failure or GBM thinning rather XVI (763–764) AF than lamellation. Evidence is emerging for the importance XVII (828–830) CER of other non-Glycine substitutions especially in the carboxy XVIII (966–971) AIISQK NC domains but these await further evaluation. XIX (1014) R XX (1078–1081) ASHF Acknowledgements The following authors of this publication (BSLZ, RC, LM, AvE, KC, EA) are members of the European XXI (1196–1197) DV Reference Network for Rare Kidney Diseases (ERKNet)– Project ID XXII (1222–1223) PP No 739532. XXIII (1251–1257) PKDIPDP XXIV (1285–1288) DLLR Compliance with ethical standards XXV (1370–1379) ADVDDCPRIP XXVI (1404–1405) GP 1404 Conflict of interest The authors declare no competing interests. Carboxy NC PGY…KYS 1461, 1464, 1465, 1483, Publisher’s note Springer Nature remains neutral with regard to (1460–1690) 1490, 1498, 1508, 1511, jurisdictional claims in published maps and institutional affiliations. 1598, 1606, 1608, 1611, 1612, 1613, 1620, 1636, 1639 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if actionable, requiring renin-angiotensin- aldosterone block- changes were made. The images or other third party material in this ade consistent with published guidelines for other COL4A5 article are included in the article’s Creative Commons license, unless variants [8, 54]. indicated otherwise in a credit line to the material. If material is not The nature of other variants such as NM_000495.5: included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted c.2858G>T (rs78972735; p.(Gly953Val)) in COL4A5 [55], use, you will need to obtain permission directly from the copyright and NM_000092.5:c.1634G>C (rs1800516; p.(Gly545Ala)) holder. To view a copy of this license, visit http://creativecommons. and NM_000092.4(COL4A4):c.2996G>A (rs13027659 org/licenses/by/4.0/. p.(Gly999Glu)) in COL4A4 is less clear. 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Affiliations Judy Savige 1 Helen Storey 2 Elizabeth Watson3 Jens Michael Hertz4 Constantinos Deltas 5 ● ● ● ● ● Alessandra Renieri 6 Francesca Mari7 Pascale Hilbert 7 Pavlina Plevova8 Peter Byers9 ● ● ● ● ● Agne Cerkauskaite10 Martin Gregory11 Rimante Cerkauskiene12 Danica Galesic Ljubanovic13 ● ● ● ● Francesca Becherucci14 Carmela Errichiello14 Laura Massella15 Valeria Aiello16 Rachel Lennon17 ● ● ● ● ● Louise Hopkinson 17 Ania Koziell18 Adrian Lungu19 Hansjorg Martin Rothe 20 Julia Hoefele 21 ● ● ● ● ● Miriam Zacchia22 Tamara Nikuseva Martic23 Asheeta Gupta24 Albertien van Eerde25 Susie Gear26 ● ● ● ● ● Samuela Landini 27 Viviana Palazzo28 Laith al-Rabadi29 Kathleen Claes 30 Anniek Corveleyn31 ● ● ● ● ● Evelien Van Hoof31 Micheel van Geel32 Maggie Williams33 Emma Ashton34 Hendica Belge35 Elisabeth Ars36 ● ● ● ● ● ● Agnieszka Bierzynska37 Concetta Gangemi38 Beata S. Lipska-Ziętkiewicz39 ● ● 1 4 Department of Medicine (MH and NH), The University of Jens Michael Hertz, Department of Clinical Genetics, Odense Melbourne, Parkville, VIC, Australia University Hospital, Odense, Denmark 2 5 Molecular Genetics, Viapath Laboratories, Guy’s Hospital, Center of Excellence in Biobanking and Biomedical Research and London, UK Molecule Medicine Center, University of Cyprus, Nicosia, Cyprus 3 6 Elizabeth Watson, South West Genomic Laboratory Hub, North Medical Genetics, University of Siena, Siena, Italy Bristol Trust, Bristol, UK Consensus statement on standards and guidelines for the molecular diagnostics of Alport syndrome:. . . 1197 7 23 Institute de Pathologie et de Genetique ASBL, Departement de Department of Biology, School of Medicine University of Biologie Moleculaire, Gosselies, Belgium Zagreb, Zagreb, Croatia 8 24 Department of Medical Genetics, and Department of Biomedical Birmingham Children’s Hospital, Birmingham, UK Sciences, University Hospital of Ostrava, Ostrava, Czech Republic 25 Departments of Genetics and Center for Molecular Medicine, 9 Departments of Pathology and Medicine (Medical Genetics), University Medical Center, Utrecht University, Utrecht, The University of Washington, Seattle, WA, USA Netherlands 10 26 Institute of Biomedical Sciences, Faculty of Medicine, Vilnius Alport UK, Gloucester, UK University, Vilnius, Lithuania 27 Medical Genetics Unit, Department of Clinical and Experimental 11 Division of Nephrology, Department of Medicine, University of Biomedical Sciences “Mario Serio”, University of Florence, Utah Health, Salt Lake City, UT, USA Florence, Italy 12 28 Clinic of Pediatrics, Institute of Clinical Medicine, Faculty of Medical Genetics Unit, Meyer Children’s University Hospital, Medicine, Vilnius University, Vilnius, Lithuania Florence, Italy 13 29 Department of Pathology, University of Zagreb, School of Health Sciences Centre, University of UTAH, Salt Lake City, UT, Medicine, Dubrava University Hospital, Zagreb, Croatia USA 14 30 Nephrology Unit and Meyer Children’s University Hospital, Department of Nephrology and Renal Transplantation, University Firenze, Italy Hospitals Leuven, Leuven, Belgium 15 31 Division of Nephrology and Dialysis, Bambino Gesù Children’s Center for Human Genetics, University Hospitals and KU Leuven, Hospital, IRCCS, Rome, Italy Leuven, Belgium 16 32 Department of Experimental Diagnostic and Specialty Medicine Department of Clinical Genetics, Maastricht University Medical (DIMES), Nephrology, Dialysis and Renal Transplant Unit, S. Center, Maastricht, The Netherlands Orsola Hospital, University of Bologna, Bologna, Italy 33 Bristol Genetics Laboratory Pathology Sciences, Southmead 17 Wellcome Centre for Cell-Matrix Research, Division of Cell- Hospital, Bristol, UK Matrix Biology and Regenerative Medicine, School of Biological 34 Sciences, Faculty of Biology Medicine and Health, The North East Thames Regional Genetics Laboratory, Great Ormond University of Manchester, Manchester, UK Street Hospital, London, UK 35 18 School of Immunology and Microbial Sciences, Faculty of Life Department of Physiology, Radboud Institute for Molecular Life Sciences, King’s College London, London, UK Sciences, Radboud University Medical Center, Nijmegen, The Netherlands 19 Fundeni Clinical Institute, Pediatric Nephrology Department, 36 Bucharest, Romania Inherited Kidney Disorders, Fundacio Puigvert, Universitat Autonoma de Barcelona, Barcelona, Spain 20 Centre for Nephrology and Metabolic Disorders, 37 Weisswasser, Germany Bristol Renal Unit, Bristol Medical School, University of Bristol, Bristol, UK 21 Institute of Human Genetics, Technical University of Munich, 38 München, Germany Division of Nephrology and Dialysis, University Hospital of Verona, Verona, Italy 22 Nephrology Unit, University of Campania, Naples, Italy 39 Centre for Rare Diseases, and Clinical Genetics Unit, Medical University of Gdansk, Gdansk, Poland