www.nature.com/ejhg MEETING REPORT OPEN The 2019 and 2021 International Workshops on Alport Syndrome Sergio Daga1,2, Jie Ding3, Constantinos Deltas 4, Judy Savige5, Beata S. Lipska-Ziętkiewicz 6, Julia Hoefele 7, Frances Flinter8, Daniel P. Gale9,10, Marina Aksenova 11, Hirofumi Kai12, Laura Perin13,14, Moumita Barua15, Roser Torra16, Jeff H. Miner 17, Laura Massella18, Danica Galešić Ljubanović19, Rachel Lennon 20, Andrè B. Weinstock21, Bertrand Knebelmann22, Agne Cerkauskaite23,24, Susie Gear25, Oliver Gross26, A. Neil Turner 27, Margherita Baldassarri1,2,28, Anna Maria Pinto 28 and ✉ Alessandra Renieri 1,2,28 © The Author(s) 2022 In 1927 Arthur Cecil Alport, a South African physician, described a British family with an inherited form of kidney disease that affected males more severely than females and was sometimes associated with hearing loss. In 1961, the eponymous name Alport syndrome was adopted. In the late twentieth century three genes responsible for the disease were discovered: COL4A3, COL4A4, and COL4A5 encoding for the α3, α4, α5 polypeptide chains of type IV collagen, respectively. These chains assemble to form heterotrimers of type IV collagen in the glomerular basement membrane. Scientists, clinicians, patient representatives and their 1234567890();,: families, and pharma companies attended the 2019 International Workshop on Alport Syndrome, held in Siena, Italy, from October 22 to 26, and the 2021 online Workshop from November 30 to December 4. The main topics included: disease re-naming, acknowledging the need to identify an appropriate term able to reflect considerable clinical variability; a strategy for increasing the molecular diagnostic rate; genotype-phenotype correlation from monogenic to digenic forms; new therapeutics and new therapeutic approaches; and gene therapy using gene editing. The exceptional collaborative climate that was established in the magical medieval setting of Siena continued in the online workshop of 2021. Conditions were established for collaborations between leading experts in the sector, including patients and drug companies, with the aim of identifying a cure for Alport syndrome. European Journal of Human Genetics; https://doi.org/10.1038/s41431-022-01075-0 INTRODUCTION GBM disruption leads to microscopic haematuria, proteinuria Alport syndrome (AS) is a clinically heterogeneous nephropathy and progressive fibrosis, advancing to end stage kidney disease caused by pathogenic variants in collagen IV genes (COL4A3, (ESKD) in many cases. Extra-renal manifestations include cochlear COL4A4, COL4A5) with a prevalence of 1:5.000 [1]. The collagen (bilateral high-frequency sensorineural hearing loss) and ocular α3α4α5(IV) heterotrimer represents an essential constituent of abnormalities (anterior lenticonus and perimacular flecks), which the mature glomerular basement membrane (GBM) and is only often contribute to the clinical phenotype. Established therapies synthesised and secreted by podocytes in the kidney [2]. include the use of renin–angiotensin–aldosterone inhibitors 1 Medical Genetics, University of Siena, Siena, Italy. 2Med Biotech Hub and Competence Center, Department of Medical Biotechnologies, University of Siena, Siena, Italy. 3Peking University First Hospital, Beijing, China. 4Biobank.cy Center of Excellence in Biobanking and Biomedical Research and University of Cyprus Medical School, Nicosia, Cyprus. 5 Department of Medicine, Melbourne and Northern Health, The University of Melbourne, Parkville, VIC 3050, Australia. 6Rare Diseases Centre, Clinical Genetics Unit, Department of Biology and Medical Genetics, Medical University of Gdańsk, Gdansk, Poland. 7Institute of Human Genetics, Klinikum rechts der Isar, Technical University of Munich, School of Medicine, Munich, Germany. 8Department of Clinical Genetics, Guys’ and St Thomas’ NHS Foundation Trust, London, UK. 9Department of Renal Medicine, University College London, London, UK. 10Rare Renal Disease Registry, UK Renal Registry, Bristol, UK. 11Y. Veltischev Research and Clinical Institute for Pediatrics at the Pirogov Russian National Research Medical University, Taldomskaya Street, 2, Moscow 125412, Russia. 12Department of Molecular Medicine, Kumamoto University, Kumamoto, Japan. 13GOFARR Laboratory for Organ Regenerative Research and Cell Therapeutics in Urology, Saban Research Institute, Division of Urology, Children’s Hospital Los Angeles, Los Angeles, CA, USA. 14Department of Urology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA. 15Toronto General Hospital, Toronto General Research Institute, University of Toronto, Toronto, ON, Canada. 16Inherited Kidney Diseases, Nephrology Department, Fundació Puigvert, IIB-Sant Pau, Medicine Department, Universitat Autónoma de Barcelona, Barcelona, Spain. 17Division of Nephrology, Washington University School of Medicine, St. Louis, MO 63110, USA. 18Division of Nephrology, Department of Pediatric Subspecialties, Bambino Gesù Children’s Hospital, IRCCS, Rome, Italy. 19University of Zagreb School of Medicine, Department of Pathology and Department of Nephropathology and Electron Microscopy Dubrava University Hospital, Zagreb, Croatia. 20Wellcome Centre for Cell-Matrix Research, Division of Cell-Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology Medicine and Health, The University of Manchester, Manchester Academic Health Science Centre, Manchester, UK. 21Alport Syndrome Foundation, Phoenix, AZ, USA. 22Nephrology Department, Reference Center for Inherited Kidney Diseases (MARHEA), APHP, Necker Hospital, Paris University, Paris, France. 23Faculty of Medicine, Vilnius University, Vilnius, Lithuania. 24Vilnius University Hospital Santaros Klinikos, Vilnius, Lithuania. 25Alport UK, Tetbury, UK. 26Department of Nephrology and Rheumatology, University Medicine Goettingen, Gottingen, Germany. 27Centre for Inflammation, University of Edinburgh, Edinburgh, UK. 28Genetica Medica, Azienda Ospedaliero-Universitaria Senese, Siena, Italy. ✉email: alessandra.renieri@unisi.it Received: 19 December 2021 Revised: 4 February 2022 Accepted: 14 February 2022 S. Daga et al. 2 (RAAS) as standard of care, which delay the progression to ESKD. variants in about one in 100 [20]. XL and AR AS are still rare For progressive forms that result in ESKD, the only options are diseases, but the “Alport spectrum” including the XL, AR, AD and dialysis and kidney transplantation, the latter being restricted by double heterozygous (digenic) forms is now the commonest cause the availability of compatible donors, access inequity in develop- of inherited kidney disease, and the second commonest cause of ing nations and by the necessity for life-long immunosuppressive inherited kidney failure. treatment. Interestingly, a recent update of the LOVD database has The 2019 International Workshop on AS was held at the demonstrated that the mean age at kidney failure for males with University of Siena, Italy, from October 22–24, while the 2021 X-linked disease is now 29 years, whereas it was 25 years in 2016. Workshop was held online due to COVID-19 pandemic. This might be because of the beneficial effects of widespread use These meetings were organised through the concerted efforts of RAAS inhibitors, or as a consequence that the disease-causing of patient advocacy groups plus physicians, scientists, and variants detected recently are milder (because families with early researchers from around the world. The Workshops focused on onset kidney failure and more “severe” variants have already been the latest findings in the field of basic science, the importance of diagnosed). considering the involvement of other organs such as eyes and Three types of more recently identified disease-causing ears in AS pathology, and on new therapeutic perspectives. The COL4A3–5 variants were discussed in detail: hypomorphic, digenic most powerful aspect of these Workshops was their unique and modifying variants. opportunity to provide a collaborative forum for participants from Hypomorphic disease-causing variants result in a milder a wide range of backgrounds from around the world, thus phenotype, but there is no formal definition. Thus, a hypomorphic highlighting progress made since another two Workshops, 30 disease-causing COL4A5 variant may result in kidney failure in late years ago [3]. In Siena, this forum enabled a large group of AS middle age in a man, and a hypomorphic heterozygous disease- patients representing different ages, genotypes, cultures, and causing COL4A3 or COL4A4 variant may not be associated with experiences to request that their voices be received more openly microscopic haematuria, and/or lead to symptomatic disease only by the experts who study and treat AS. Specifically, they expressed if found in trans with a second, overt, disease-causing variant. their hope for a patient-centric shift in the way that AS is Typical hypomorphic disease-causing variants include many diagnosed, treated, and understood. They also raised the issue of missense changes, for example some position 1 Gly substitutions classifying AS as a spectrum disorder. The following topics were with Ala, Ser or Cys, as well as non-Gly substitutions [21]. In covered throughout the two Workshops. addition, Gly substitutions adjacent to the non-collagenous domains or non-collagenous interruptions also result in a milder phenotype. Two common less severe disease-causing variants are GENOTYPE-PHENOTYPE CORRELATION FROM MONOGENIC TO Gly624Asp in COL4A5 (which is associated with late onset kidney DIGENIC FORMS failure) [22] and Leu1474Pro in COL4A3 (which may not on its own The first day of this well-established biennial Workshop on AS is cause microscopic haematuria, but can be associated with kidney always dedicated to genetics. In the last two meetings this was failure when found together with a second disease-causing under the leadership of CD, FF, JH, BSLZ, JS and AR. The term AS COL4A3 variant in trans, or due to digenic inheritance). (ORPHA:63) encompasses a group of inherited kidney diseases Despite the Gly624Asp variant being estimated less severe, characterised by haematuria and proteinuria, progressive kidney recent evidence has highlighted cases in which the severity of the failure, hearing impairment and ocular abnormalities. Inheritance is variant is associated with clear evidence of kidney insufficiency X-linked (due to a disease-causing COL4A5 variants, (OMIM#301050, and/or ESRD. ORPHA:88917), or Autosomal Recessive (AR) (biallelic disease- Macheroux et al. [23] recently report how this variant is the causing variants in the COL4A3 or COL4A4 gene; OMIM#203780; genetic cause that reported for an unexpected severe phenotype in ORPHA:88918) [4]. In recent years, the presence of compound a X-linked family where the female proband shows microscopic heterozygous (sometimes referred to as digenic) inheritance has haematuria and proteinuria at the age of 20 and 41 years, been reported, with disease-causing variants in two of the three respectively. The microscopic haematuria was also present in the COL4A3–5 genes [5–12]. Modifying variants affecting other daughter (from 6th month of life), the son (from 22nd month of podocyte or GBM genes can worsen the phenotype [13]. life), the mother and the maternal grandniece, while the proteinuria Autosomal Dominant (AD) inheritance (with a single disease- was detected in the maternal aunt and paternal grandmother. causing variant in COL4A3 or COL4A4 OMIM#104200; Boeckhaus et al. [24] in a prospective cohort study underline ORPHA:88919) is also reported, but some prefer to avoid the how hemizygous patients for this variant have an increased risk term Alport ‘syndrome’ for this last group. There were constructive developing ESRD if they do not receive pharmacological discussions about a revised nomenclature that indicates indivi- treatment, while in patients treated early a reduced risk of reaching duals with a heterozygous disease-causing variant do not have a the endpoint is reported thanks to the nephroprotective action syndromic form of the disease, since they are much less likely to of ACEis. develop kidney failure, and do not usually develop hearing loss or Furthermore, in the EARLY PRO-TECT Alport trial it is reported ocular abnormalities [12]. Recent experience suggests that that two of eight children with the variant included in the study disease-causing COL4A3–5 variants are associated with a broad showed progress of the disease during the trial [25]. phenotypic spectrum, which is also influenced by nephroprotec- All these reports and studies illustrate the difficulties faced tive therapy [14]. These include persistent microscopic haematuria when interpreting the clinical significance of the variant which with a similar family history of microscopic haematuria, but also should be considered a milder form of the disease, but still focal segmental glomerulosclerosis (FSGS), and kidney failure [15], causing kidney failure in midlife. familial IgA glomerulonephritis [16] and possibly other types of Double heterozygous (digenic) variants are variants in the immune-mediated glomerulonephritis, as well as cystic kidney COL4A3 and COL4A4 gene. This observation is very rare and has disease [17, 18] and kidney dysplasia [19]. However, in all these generally only been detected since exome sequencing has conditions, persistent microscopic haematuria and a family history allowed the examination of multiple genes simultaneously. The or microscopic haematuria or kidney failure may be present, inheritance of COL4A3 and COL4A4 double heterozygous variants suggesting an underlying disease-causing COL4A3–5 variant. is complex, because the variants may be inherited on the same Analysis of COL4A3–5 pathogenic variants in the gnomAD chromosome (in cis) where inheritance is AD, or on different database has revealed that X-linked AS occurs in about one in chromosomes (in trans) where inheritance is AR [5, 6]. The renal 2000 individuals, and single heterozygous COL4A3 or COL4A4 outcome appears to be worse with a combination of heterozygous European Journal of Human Genetics S. Daga et al. 3 COL4A3 and COL4A4 variants than with a single variant; however, Probe Amplification and Sanger sequencing. In 2021, a consensus further studies are needed [10]. Recent advice is that these statement refining the ACMG criteria on standards and guidelines individuals should also be treated with RAAS blockade if they have for the molecular diagnostics of AS was published [32]. albuminuria or additional risk factors for progressive kidney The detection rate of disease-causing variants is higher in disease such as hypertension [26], and other first-degree family individuals with suspected X-linked AS (up to 95%) than in those members should be offered cascade genetics testing. The with a suspected heterozygous disease-causing COL4A3 or COL4A4 suitability of individuals with heterozygous variants to act as variant (where it may be only 50%). This is probably because the kidney donors continues to be contentious: they are at increased clinical features of X-linked disease are more distinctive with risk of renal disease, but those who are older, normotensive and microscopic haematuria, kidney failure, and, often, hearing loss without microalbuminuria may be highly motivated to give a and ocular abnormalities, in addition to a suggestive family kidney to a relative, particularly when there are no suitable history. On the other hand, disease-causing variants may be found alternatives. less frequently in COL4A3 and COL4A4, because these cohorts The co-inheritance of additional genetic modifiers that only include individuals with possibly other causes of persistent exert their negative effect on the background of a primary microscopic haematuria, including IgA, post-streptococcal and collagen IV variant may explain the adverse outcome seen in some C3 nephropathy. patients [27–29]. The term “modifying” variants describes the Other explanations for a reduced detection rate include association of a disease-causing variant in one of the COL4A3–5 reduced coverage of collagen IV exons (especially exons 5 and genes, with a coincidental variant in a GBM or podocyte gene [30]. 6) and the fact that large deletions (>50 bp) may be missed with Such heterozygous variants in many GBM or podocyte genes are exome sequencing in some laboratories. Although the critical not usually disease-causing on their own, either because they are nature of position 1 Gly residues is now recognised, the recessive, or they are hypomorphic and this continues to be an significance of many variants affecting the carboxy non- area of active research. collagenous domain is still unknown. There was no consensus on the strategy to address the “missing variants”. There are in silico methods to detect large deletions if DISEASE RE-NAMING, WITH THE SEARCH FOR AN these are not detected with exome sequencing, and there is now APPROPRIATE TERM THAT ENCOMPASSES THE LARGE an in vitro method for confirming collagen IV variant pathogeni- CLINICAL VARIABILITY city, but it is still a research tool. It is important to keep in mind There is an international initiative to adopt a more consistent synonymous variants (which may affect a splice site), other non- approach to naming genetic diseases: https://clinicalgenome.org. canonical splice variants, and less severe variants such as Agreement on not using the term “carriers” for COL4A5 hetero- p.Leu1474Pro in COL4A3. Some variants are difficult to call zygous females has been reached, and there is a growing conclusively as disease-causing, and Weisen Xia presented her consensus on avoiding the same term for COL4A3/A4 hetero- research on using AI to interpret pathogenicity. It is possible to zygotes. This is because there is a real risk of clinically significant check for splicing variants using a minigene assay and urinary renal impairment in heterozygous subjects [31] who should be podocytes or skin or hair roots, but splicing may be tissue specific. made aware of the necessity for regular clinical check-ups. JS Moreover, the limitation in variant detection can be explained by presented data from large-scale population sequencing initiatives, incomplete coverage of the studied region, the presence of a indicating that the prevalence of rare heterozygous COL4A3/4/5 deep intronic variant [33], variants of uncertain significance, Copy variants that are predicted to be disease-causing, and are strongly Number Variation or other types of structural variants that may associated with the presence of persistent haematuria, is as high not be detectable using panel, exome or other targeted as 0.94% in the UK population [20]. sequencing assays. This is a very high percentage and, at the same time, it implies Most laboratories represented reported that pathogenic var- that the risk of EKD is very low in individuals not ascertained by iants in other FSGS genes were uncommon when AS is suspected the presence of a personal or family history of kidney disease who clinically [34]. When a diagnosis of AS is strongly suspected harbour such variants. Many of these individuals may only have clinically, but no variant is detected following DNA analysis, clinical isolated microscopic haematuria for their entire life, or even be review may be helpful, including re-examination of a kidney completely asymptomatic. Therefore, it is necessary that the status biopsy to look for lamellation in the proband or an affected family of heterozygous subjects (especially in the absence of features member, retinal imaging and optical coherence tomography. such as proteinuria, chronic kidney disease or hypertension that Finally, genetic linkage studies can exclude a gene if DNA is suggest underlying kidney damage) is not perceived as a available from suitable relatives whose phenotype is known. devastating event. The term “Alport Syndrome” as a diagnostic If not already performed, a kidney biopsy may be necessary if label for every patient with a COL4 variant is unhelpful, as it fails to the genetic cause of kidney disease cannot be established by DNA distinguish between those at high risk of renal disease with other analysis, and/or there is an atypical clinical course in a patient with characteristic extra-renal features, and those who are at very low AS, in order to exclude other glomerular and tubulointerstitial risk of any significant problems. Alicia Byrne facilitated an kidney diseases such as IgA nephropathy (which has strong international discussion and the alternative name “Alport Spec- genetic risk factors and can occur in familial clusters) [35], trum” was preferred. If COL4A3/COL4A4 heterozygous individuals, membranous nephropathy and interstitial nephritis. Even if who are so commonly identifiable in the population, are included electron microscopy is mandatory to confirm the diagnosis of under the umbrella description of Alport spectrum, the condition AS, light microscopy plays an important role in studying the would no longer be regarded as rare. Some patients with interstitium, whose involvement could be predictive of more syndromic disease (including the extra-renal manifestations) may severe progression, and also to reveal focal and segmental still prefer the label AS. These proposals are now under glomerulosclerosis. consideration by the wider community. The results of kidney biopsy may strongly support a clinical diagnosis of AS, but do not have the advantage of identifying the underlying disease-causing variant, which is why clinicians WHAT TO DO WHEN ALPORT SYNDROME IS STRONGLY increasingly request genetic testing first when considering a SUSPECTED, BUT A PATHOGENIC VARIANT IS NOT IDENTIFIED diagnosis of AS. Four approaches are commonly used to detect COL4A3–5 variants: The presence of a glomerular disease (primary membranopro- exome sequencing, panel diagnostics, Multiple Ligation-dependent liferative GN/C3 glomerulopathy, IgA nephropathy) does not European Journal of Human Genetics S. Daga et al. 4 Fig. 1 Visual summary of current standard of care treatment recommendations and individual lifetime risk for ESRF according to the gene variant and additional risk factors. Red: patients with—classical‖ Alport syndrome and a 100% risk for early ESRF. Therapy should be initiated at diagnosis (in children 2 years and older) at very early stages of disease. Nephroprotective therapy has the potential to reduce lifetime risk for ESRF to 50% in those with less severe missense variants. Yellow: heterozygous patients with an intermediate lifetime risk of 5 to 40% for ESRF. If therapy is initiated early at microalbuminuria (or even micro-haematuria in patients with additional risk factors), nephroprotective therapy has the potential to reduce lifetime risk to 1 to 5%, only. Green: heterozygous individuals with a low risk for ESRF, which is <1%, if patients remain in a disease management programme to check for additional risk factors for their lifetime. Fig. 2 Current and possible future treatment options. Summary of current and possible future treatment options and postulated effects on (the high) cardiovascular risk of patients with Alport syndrome, which also needs to be addressed. exclude AS [16, 36, 37]. The association between AS and immune to find potential candidate compound to correct the mutant α5 glomerular diseases suggest that the abnormal GBM resulting from (IV)-dependent defect of alpha α345 (IV) trimer secretion. the presence of pathogenic COL4A3/A4/A5 variants may predispose Another strategy for confirming the pathogenicity of new to immune-mediated injury. Recently JS and Philip Harrak reported COL4A5 variants is the creation of animal or podocytes models that how often both diseases occur together is unknown because using CRISPR/Cas9 gene editing, followed by clinical evaluation or few individuals with IgA glomerulonephritis routinely undergo measurement of COL4A5 protein expression by western blot electron microscopy for GBM thinning or testing for genetic analyses and immunofluorescence assays [40]. variants. On the other hand, heterozygous COL4A3 and COL4A4 variants and IgA disease each affect ~1% of the population, so the conditions might coexist by chance in 0.01% [38]. Kohei Omachi UPDATE ON CLINICAL TRIALS (Washington University, St. Louis, USA), from the group of Recent and current clinical trial in AS were reviewed including: (1) the Jeff H. Miner, reported on ways to detect and assess the phase 3 randomised, placebo-controlled EARLY PRO-TECT Alport trial; pathogenicity of novel variants, having established a collagen (2) the phase 2 open-label, basket study AFFINITY; (3) the phase 2 alpha 345(IV) heterotrimer formation assay that is amenable to randomised, placebo-controlled HERA trial; (4) the phase 2/3 high throughput screening by using a split NanoLuc luciferase randomised, placebo-controlled CARDINAL trial; and major clinical complementarity system [39]. The assay provided ways to trials in patients with CKD (but not specific for AS), such as DAPA- determine the functionality of Gly394Ser, Gly454Ala and Gly960Arg CKD and FIDELIO-DKD. variants in COL4A4. The level of luciferase activity revealed the In summary, the EARLY PRO-TECT Alport (NCT01485978) trial in effect that those variants have on impairing NC1 and 7S domain children 2 years and older, at very early stages of AS, assembly and stability, allowing prediction of the resulting impact demonstrated the safety of Ramipril and revealed evidence of on collagen IV network and basement membrane formation. This efficacy (i.e. delaying progression of albuminuria) [41]. These assay is suitable for studying variants that affect the stability of the findings have led to an update of treatment recommendations for heterotrimer, to evaluate the pathogenicity of novel variants, and patients with AS [23] (Figs. 1 and 2). European Journal of Human Genetics S. Daga et al. 5 Figure 2 further explains current and future treatment options. levels (Statin) [44, 45]. Green circles indicate compounds, which In detail, Paricalcitol and HMG-CoA-reductase-inhibitors (Statins) positively influence both, the chronic kidney disease and the both showed pleiotropic nephroprotective effects in COL4A3−/− high cardiovascular risk in AS. Red circles indicate factors, which mice as animal models for AS [42, 43]. Both compounds are part of negatively influence both, the chronic kidney disease and the high the treatment recommendations, if indicated because of second- cardiovascular risk in AS. Again, for this reason, a healthy lifestyle ary hyperparathyroidism (Paricalcitol) or elevated cholesterol (doing sports, moderation of dietary intake of meat protein and DIRECT STRATEGIES A Gene editing in TTR Amyloidosis in B Gene editing in Alport Syndrome in Humans Animal Models Gain of function Loss of function TTR mutation COL4A5 mutation Mutated Mutated sequence sequence Cas9 Cas9 Donor DNA Normal sequence DELIVERY DELIVERY Donor-AAV Cas9-AAV Lipid nanoparticles INDIRECT STRATEGIES C D CRISPRa CRISPRi Gene Expressed Gene Silenced COL4A6 dCas9 Activator LAMA2 dCas9 Inhibitor Normal Normal sequence Promoter sequence Promoter European Journal of Human Genetics S. Daga et al. 6 Fig. 3 Gene editing approaches: comparison between direct and indirect strategies. The CRISPR/Cas9 direct approaches are targeting straightforward mutated genes (A, B). The CRISPR/Cas9 indirect approaches are based on the action of endonuclease deficient activity dCas9 fused to either transcriptional activators or repressors that either upregulate (CRISPRa) or silence (CRISPRi) genes (respectively) relevant to the pathophysiology of AS (C, D). A TTR gene mutated sequence is edited in humans by spCas9 alone. Indel (insertion/deletion) is created after the cut and the gain of function variant (the protein precipitates in aggregates dangerous for the cell) is edited in a loss of function allele. B The loss of function variant in COL4A5 gene is edited to the normal sequence, by means of spCas9, in cultured cells and animal models employing CRISPR/Cas9 approach. C The COL4A6 gene, a minor basement membrane gene, is forced by dCas9/CRISPRa to be overexpressed in podocytes, which should replace the missing α3α4α5(IV) network with the α5α5α6(IV) network; this could slow kidney disease progression. D The LAMA2 downstream gene is silenced by the dCas9/CRISPRi in glomerular cells, which should reduce laminin α2 in the GBM and slow kidney disease progression. salt, and maintaining a body mass index less than 25 kg/m2), strict further evaluation. Starting in early 2022, the mineral-corticoid blood pressure control and no smoking are part of the current receptor antagonist finerenone will be evaluated in FIONA, an treatment recommendations [26, 44]. Green letters indicate international paediatric trial in children with CKD, including AS potential new therapies being evaluated in ongoing clinical phase (Press release: https://www.media.bayer.com/baynews/baynews. 2 and 3 trials. At present (February 2022), analysis of the risk- nsf/id/Bayer-extends-clinical-development-program-finerenone- benefit ratio of Bardoxolone Methyl in patients with AS possibly Phase-III-study-children-adolescents-chronic?OpenDocument could indicate possible harm. &sessionID=1639500433). Finerenone has been approved for The AFFINITY study (NCT04573920) with the endothelin diabetic CKD and is postulated to work in an additive fashion with receptor antagonist atrasentan in patients with proteinuric SGLT2i [51]. Preclinical data in the Alport mouse model have glomerular diseases includes 20 patients with AS and an shown an aldosterone escape in Alport mice treated with ramipril, intermediate risk for fast progression. The trial is recruiting leading to progressive albuminuria and scarring, which might be currently. Preclinical data in Alport mouse models look promising; targeted by mineral-corticoid receptor antagonists [52]. however, results of the phase 2 clinical trial are not expected Shota Kaseda (Kumamoto University), from the group of before the end of 2023. Hirofumi Kai, presented the use of UBE-1099, a reversible Keap-1 The HERA study (NCT02855268) with the anti-microRNA21 inhibitor that inhibits the Keap1-Nrf2 binding inducing down- compound lademirsen is specific for AS and includes 45 patients stream Nrf2 activation [53], and evaluated the effect of UBE-1099 at high risk of fast progression. The trial is recruiting currently. in a mouse model of AS. According to transcriptome analysis the Preclinical data in Alport mouse models look promising, target UBE-1099 mechanism of improvement seems to be related to the engagement has been shown in humans; however, results of the expression of a set of genes associated with cell cycle and phase 2 clinical trial are not expected before the end of 2023. cytoskeleton. The CARDINAL study (NCT03019185) with bardoxolone methyl is specific for AS and includes 30 patients in the open-label phase 2 and about 160 in the blinded, placebo-controlled phase 3. Patients THERAPEUTIC APPROACH USING CHAPERONES in phase 2 study have finished the 2 year (104 weeks) in early 2019, Christoforos Odiatis, from the group of Constantinos Deltas, phase 3 have completed week 104 in summer 2020. According to University of Cyprus, presented data supporting the hypothesis the baseline data, phase 3 includes many patients with a low to that the administration of pharmacological repurposed chaper- intermediate risk for fast progression [46] Bardoxolone has never ones may improve clinical progression. This work was done in been tested in Alport mouse models; but there are preclinical data mice recapitulating AR AS, with the Gly1332Glu COL4A3 mutation, in other CKD mouse models, in which a bardoxolone analogue has which is a founder genetic variant amongst Cypriots. caused podocyte damage and increased albuminuria [47]. Results of the phase 2 and phase 3 clinical trial have not yet been published. In a recent meeting of the FDA Cardiovascular and GENE THERAPY APPROACHES USING GENE EDITING Renal Drugs Advisory Committee, the FDA review team did not Gene therapy is now universally recognised as a possible assess the phase 3 trial results as positive. therapeutic application for rare diseases, including AS. As first in class, the Sodium-Glucose co-Transporter-2 (SGLT2) Julian Gillmore (Centre for Amyloidosis and Acute Phase inhibitor Dapagliflozin has been approved for CKD, because of its Proteins, University College London, UK) presented recently profound nephroprotective effect and the impact on cardiovas- published work exploring a gene editing approach in humans to cular risk [48]. EMPA-REG and the ongoing EMPA-KIDNEY study treat TTR-related amyloidosis disease, due to gain-of-function point to a general nephroprotective effect in CKD [49]. However, variants (Fig. 3A) [54]. Studies in vitro have proven the ability of the very limited number of 6 patients with AS out of more than the system to match precisely the mutated locus where to direct 4000 other patients in DAPA-CKD means that it is not clear yet the Cas9 ribonucleoprotein with low off-target events. Clinical whether SGLT2i are specifically beneficial in glomerular diseases in vivo studies in a small group of patients have shown a durable such as AS. The panel agreed that SGLT2i seem to be promising knockout of TTR after the administration of a single dose. Lipid add-on therapies for AS patients with a risk of progression, and nanoparticles were used as vehicles of the Cas9 system in order to that they should be prescribed in addition to ACE inhibitors or guarantee a very rapid distribution to the liver through the hepatic ARBs where regional regulations permit. In this context, Jan artery. Furthemore, NTL-2001 was associated with only mild Boeckhaus presented the results of an observational case series in adverse events. 6 patients with (hereditary) FSGS or AS [50]. Therapy was well Sergio Daga (Medical Genetics, University of Siena, Siena, Italy), tolerated and reduced albuminuria. Interventional trials specific from the group of Aelssandra Renieri, has employed a two- for young patients with AS (including children 10 years and older) plasmid CRISPR/Cas9 genome editing approach, achieving a at early stages of disease are in preparation. Meanwhile, it is beneficial and stable correction of the COL4 loss-of-function desirable that long term outcomes in treated patients should be variants on urine-derived podocyte-lineage cells (Fig. 3B) [55, 56]. monitored (OG at gross.oliver@med.uni-goettingen.de can offer In order to translate the in-vitro application into a future in-vivo suggestions how). Although preliminary, and not targeted clinical trial, dogs and mice have been used as preclinical animal specifically to AS, these results identify SGLT2-i as a very models to investigate the safety and efficacy of the system. promising new and interesting therapeutic option, which requires Adeno-Associated-Viruses (AAVs), namely AAV9, are proposed as a European Journal of Human Genetics S. Daga et al. 7 delivery system to carry both plasmids of the CRISPR/Cas9 system Moreover, multiple studies and multiple approaches were into the affected kidney of the animals. presented during these Workshops, identifying new research Jeff H. Miner (Washington University, St. Louis, USA) described possibilities on podocytes, on renal glomerular endothelial cells two new promising approaches, namely pro-repair gene activation and on GBM, looking for a possible treatments targeted at all the (CRISPRa) to achieve gene gain of function, and pathogenic gene glomerular components involved in AS. The studies proposed deactivation or inhibition (CRISPRi) to achieve gene loss of range from diversified and innovative in vitro applications, the function. These approaches differ from the classical CRISPR/Cas9 study of organoid models, and preclinical approaches on mouse approach to directly repair the causative genetic variant of AS. and dog models, which are necessary before drugs are approved Neither of the two approaches necessitates a DNA cleavage. for clinical trials. New possible drugs need to be also considered in CRISPRa (Fig. 3C) uses a catalytically dead mutant form of Cas9 addition to the already widely used (ACE-i or ARBs). All the (dCas9), whose endonuclease activity is removed through point projects are summarised in a schematic representation in Fig. 4. mutations, fused with transcriptional activator domain to mod- ulate target gene expression. A target gene selective gRNA brings the CRISPRa complex to the genomic locus, and rather than THE VOICE OF PATIENT ADVOCACY GROUP cutting the DNA, the dCas9 complex activates the downstream The most powerful aspect of these Workshops is their unique gene expression [57]. The rationale for the application of the opportunity to provide a collaborative forum for AS patients, CRISPRa is based on the hypothesis that in AR AS, COL4A5 and caregivers, clinicians, and researchers from around the world. In COL4A6 genes are intact. Employing CRISPRa to activate COL4A6 Siena, this forum enabled a large population of AS patients gene expression in podocytes would lead to a forced expression representing diverse ages, genotypes, cultures, and experiences to followed by production and secretion of α5α5α6(IV) by podocytes, request that their voices are more openly received by the experts which will normalise the GBM’s structure and function, compared who study and treat AS. The same spirit was then held online with an altered or absent α3α4α5(IV) heterotrimer. In the CRISPRi during the second Workshop. Specifically, they hope for a patient- approach (Fig. 3D) the dCas9 is fused with a transcriptional centric shift in how AS is diagnosed, treated, and understood. repressor such that when a gRNA brings the CRISPRi complex to They also raised the potential of classifying AS as a spectrum the genomic locus, it represses the downstream gene expression disorder. instead of activating it. Since in Alport GBM, laminin subunit α2 Although advances in research and treatments to prevent the deposition is injurious to podocytes physiology and promotes loss of kidney function obviously remain the most important glomerular disease progression, inactivation of the LAMA2 gene, desire of the AS patient and caregiver community [58], research mediated by CRISPRi, in glomerular cells could slow disease efforts tend to overlook the unique psychological, sociological, progression. and interpersonal needs of individuals living with AS. Studies on Podocytes Studies on GEC Lipid accumulation miRs that modulate podocyte biology Podocyte cytoskeleton miRs studies Extracellular vesicles Lipid accumulation CRISPR/Cas9 gene editing Imaging studies CRISPRa CRISPRi Chaperones Podocyte Podocyte GBM Taken from Saurus P. Biology, 2016 Studies on GBM Proteomics Imaging Collagen and laminin turnover in-vitro and in-vivo models employed In-vivo on mice Innovative imaging In-vivo on dogs Kidney organoids Glomerulus-on-chip Test new drugs technology Fig. 4 Basic science approaches. In the upper panel are summarised the new studies and approaches on podocytes, GEC and GBM ranging from CRISPR/Cas9 application to miRs that stimulate podocyte biology, proteomics and chaperones in combination with studies on podocytes cytoskeleton. In the lower panel the downstream applications of these: imagine technology, organoids in 3D culture, glomerulus on a chip to mimic the glomerular structure in vitro, and preclinical animal model testing in dog and mice. European Journal of Human Genetics S. Daga et al. 8 Fig. 5 World map representation of AS patients distribution. World map represents the scale and scope of the ASF Facebook user community on a global scale, quantifying patients in a unique database. In the last Voice of the Patient meeting, published online by the educational tool for families to encourage conversations with their U.S. Food and Drug Administration (FDA) [58], it is reported that son/daughter about living with AS. “seventy percent of participants indicated that AS symptoms Anonymised data from the US Alport Syndrome Foundation’s interfered with their daily life at least moderately”. Their reported social media website indicate that more than 2100 individuals experience is indicative of how the majority of Alport patients from more than 70 countries use the site to collaborate, provide feel, and how much AS has a psycho-sociological impact on peer support, and interact with experts in the community. their lives. Associazione Sindrome di Alport facebook page, moderated by For example, they invited conference attendees to consider the the Italian national patient organistion ASAL Onlus, reported over negative consequences of implicit biases associated with physio- 2300 followers. The China patient organisation reported 1567 logical deafness, as well as the developmental, social, and logistical patients from 33 provinces in China are connected through complications faced by an AS patient who may appear able-bodied WeChat, China’s social media platform. Alport UK reported how but nonetheless cannot adequately perceive human speech. They Alport Warriors, their closed Facebook page, doubled in size as the asked that researchers and clinicians consider being more sensitive community went online during the pandemic and has nearly 900 in their choice of words while interacting with AS patients who members from more than 40 countries. The importance of the have hearing loss/deafness, and suggested that clinicians might international Alport Syndrome Alliance was underlined as a global offer assistive aids such as amplification in their medical offices, and network to advance treatments and knowledge. Currently particularly at medical events that feature AS-specific topics. Such operated by Alport UK, the alliance will be managed by all the aids could include providing subtitles, real-time text translation, and national associations of Alport, representing their national sign language interpretation. They also expressed families‘ need for members. Alport UK, organisers of the workshops, and ASAL help in identifying possible sources of financial and emotional Onlus will collaborate on a joint project with other Alport national support when faced with a new diagnosis of deafness/hearing loss. patient organisations to create a resource base for the Alport Families reiterated the importance of psychological support for community. This new project will accelerate collaboration across patients and families during the second Workshop. In particular, the the community of patients, clinicians, laboratory scientists and representative of the Italian Association (A.S.A.L.) described their pharma representatives. The widespread networking and expo- proposals aimed at promoting health, understood as a complex nential growth of post-conference interactions on social media system of physical, psychological and social well-being of each have incentivised international scientific and medical experts to individual, such as the Psychological Listening Desk and the prioritise further collaborative study into topics such as mental Psychological support groups. A high point of the in-person health and patient-empowerment strategies; these topics will also Workshop held in Siena occurred when the father of a child newly serve as objectives for the next International Alport Workshop, diagnosed with AS pointed across the room at patient advocate which will be in 2023, respecting the established bi-annual Sam Clarke and emotionally professed that “Sam was the answer!” cadence. In summary, there were no treatments in 2013 for Alport to the question of how he could raise his young son to believe he Syndrome, research was limited, and no pharmaceutical compa- could lead a full and productive life. This father’s fear for his son’s nies involved. 8 years on, having delivered five very successful psychological well-being was echoed in testimonies of several international ‘lock-in’ workshops, there is now a vibrant interna- patients who described overcoming struggles with isolation after tional research community evidenced by the more than 250 connecting with others who shared similar experiences. During the researchers, with 50 new research projects featured at “The 2021 2021 online Workshop, the importance of a greater involvement of Online Workshop” with more than 15 pharmaceutical or biotech young patients in these workshops was discussed in depth in order companies collaborating with the community and developing to share experiences and feelings and address the disease together. potential drug and gene therapy treatments with three being The representative of the American Association (Alport Syndrome tested in international clinical trials. Foundation) presented their colouring book that tells the story of a The global participation of the AS patients around the world is young girl with AS and her family. It was created as a gift and represented in Fig. 5. European Journal of Human Genetics S. Daga et al. 9 REFERENCES G624D variant originating in the middle ages is predominant in Central/East 1. Hertz JM, Thomassen M, Storey H, Flinter F. Clinical utility gene card for: Alport Europe and causes kidney failure in midlife. Kidney Int. 2021;99:1451–8. https:// syndrome. Eur J Hum Genet. 2015;23. https://doi.org/10.1038/ejhg.2014.254. doi.org/10.1016/j.kint.2020.10.040. 2. Abrahamson DR, Hudson BG, Stroganova L, Borza DB, St John PL. Cellular origins 23. Macheroux EP, Braunisch MC, Pucci Pegler S, Satanovskij R, Riedhammer KM, of Type IV Collagen Networks in developing Glomeruli. J Am Soc Nephrol. Günthner R, et al. The Hypomorphic Variant p.(Gly624Asp) in COL4A5 as a pos- 2009;20:1471–9. https://doi.org/10.1681/ASN.2008101086. sible cause for an unexpected severe phenotype in a family with X-linked Alport 3. Flinter F, Bobrow M. The molecular genetics of Alport syndrome: report of two syndrome. Front Pediatr. 2019;7:485. https://doi.org/10.3389/fped.2019.00485. workshops. J Med Genet. 1992;29:352–3. https://doi.org/10.1136/jmg.29.5.352. 24. Boeckhaus J, Hoefele J, Riedhammer KM, Nagel M, Beck B, Choi M, et al. Lifelong 4. Deltas C, Savva I, Voskarides K, Papazachariou L, Pierides A. Carriers of autosomal effect of therapy in young patients with the COL4A5 Alport Missense Variant p. recessive Alport Syndrome with thin basement membrane nephropathy pre- (Gly624Asp): a prospective cohort study. Nephrol Dial Transplant. 2022:gfac006. senting as focal segmental glomerulosclerosis in later life. Nephron. https://doi.org/10.1093/ndt/gfac006. 2015;130:271–80. https://doi.org/10.1159/000435789. 25. Boeckhaus J, Hoefele J, Riedhammer KM, Tönshoff B, Ehren R, Pape L, et al. 5. Mencarelli MA, Heidet L, Storey H, van Geel M, Knebelmann B, Fallerini C, et al. Precise variant interpretation, phenotype ascertainment, and genotype- Evidence of digenic inheritance in Alport syndrome. J Med Genet. phenotype correlation of children in the EARLY PRO-TECT Alport trial. Clin 2015;52:163–74. https://doi.org/10.1136/jmedgenet-2014-102822. Genet. 2021;99:143–56. https://doi.org/10.1111/cge.13861. 6. Fallerini C, Baldassarri M, Trevisson E, Morbidoni V, La Manna A, Lazzarin R, et al. 26. Kasthan CE, Gross O. Clinical practice recommendations for the diagnosis and Alport syndrome: impact of digenic inheritance in patients management. Clin management of Alport syndrome in children, adolescents, and young adults-an Genet. 2017;92:34–44. https://doi.org/10.1111/cge.12919. update for 2020. Pediatr Nephrol. 2020. https://doi.org/10.1007/s00467-020- 7. Yokota K, Nozu K, Minamikawa S, Yamamura T, Nakanishi K, Kaneda H, et al. 04819-6. Female X-linked Alport syndrome with somatic mosaicism. Clin Exp Nephrol. 27. Voskarides K, Arsali M, Athanasiou Y, Elia A, Pierides A, Delta C. Evidence that 2017;21:877–83. https://doi.org/10.1007/s10157-016-1352-y. NPHS2-R229Q predisposes to proteinuria and renal failure in familial hematuria. 8. Mastrangelo A, Giani M, Groppali E, Castorina P, Soldà G, Robusto M, et al. Pediatr Nephrol. 2012;27:675–9. https://doi.org/10.1007/s00467-011-2084-6. X-Linked Alport Syndrome in women: genotype and clinical course in 24 cases. 28. Voskarides K, Stefanou C, Pieri M, Demosthenous P, Felekkis K, Arsali M, et al. A Front Med. 2020;7:580376. https://doi.org/10.3389/fmed.2020.580376. functional variant in NEPH3 gene confers high risk of renal failure in primary 9. Zupan A, Matjašič A, Grubelnik G, Tasić V, Momirovska A. Mutations in collagen hematuric glomerulopathies. Evidence for predisposition to microalbuminuria in genes in the context of an isolated population. Genes. 2020;11:1377. https://doi. the general population. PLoS ONE. 2017;12:e0174274. https://doi.org/10.1371/ org/10.3390/genes11111377. journal.pone.0174274. 10. Furlano M, Martínez V, Pybus M, Arce Y, Crespí J, Venegas MDP, et al. Clinical and 29. Lennon R, Stuart HM, Bierzynska A, Randles MJ, Kerr B, Hillman KA, et al. Coin- genetic features of autosomal dominant Alport syndrome: a cohort study. Am J heritance of COL4A5 and MYO1E mutations accentuate the severity of kidney Kidney Dis. 2021;78:560–.e1. https://doi.org/10.1053/j.ajkd.2021.02.326. disease. Pediatr Nephrol. 2015;30:1459–65. https://doi.org/10.1007/s00467-015- 11. Zhao X, Chen C, Wei Y, Zhao G, Liu L, Wang C, et al. Novel mutations of COL4A3, 3067-9. COL4A4, and COL4A5 genes in Chinese patients with Alport Syndrome using 30. Voskarides K, Papagregoriou G, Hadjipanagi D, Petrou I, Savva I, Elia A, et al. next generation sequence technique. Mol Genet Genom Med. 2019;7:e653. COL4A5 and LAMA5 variants co-inherited in familial hematuria: digenic inheri- https://doi.org/10.1002/mgg3.653. tance or genetic modifier effect? BMC Nephrol. 2018;19:114. https://doi.org/ 12. Matthaiou APT, Deltas C. Prevalence of clinical, pathological and molecular fea- 10.1186/s12882-018-0906-5. tures of glomerular basement membrane nephropathy caused by COL4A3 or 31. Jais JP, Knebelmann B, Giatras I, De Marchi M, Rizzoni G, Renieri A, et al. X-linked COL4A4 mutations: a systematic review. Clin Kidney J. 2020;13:1025–36. https:// Alport syndrome: natural history and genotype-phenotype correlations in girls doi.org/10.1093/ckj/sfz176. and women belonging to 195 families: a “European Community Alport Syndrome 13. Savige J, Storey H, Watson E, Hertz JM, Deltas C, Renieri A, et al. Consensus Concerted Action” study. J Am Soc Nephrol. 2003;14:2603–10. https://doi.org/ statement on standards and guidelines for the molecular diagnostics of Alport 10.1097/01.asn.0000090034.71205.74. syndrome: refining the ACMG criteria. Eur J Hum Genet. 2021;29:1186–97. https:// 32. Savige J, Storey H, Watson E, Hertz JM, Deltas C, Renieri A, et al. Consensus doi.org/10.1038/s41431-021-00858-1. statement on standards and guidelines for the molecular diagnostics of Alport 14. Yamamura T, Horinouchi T, Nagano C, Omori T, Sakakibara N, Aoto Y, et al. syndrome: refining the ACMG criteria. Eur J Hum Genet. 2021;29:1186–97. https:// Genotype-phenotype correlations influence the response to angiotensin- doi.org/10.1038/s41431-021-00858-1. targeting drugs in Japanese patients with male X-linked Alport syndrome. Kid- 33. Wang X, Zhang Y, Ding J, Wang F. mRNA analysis identifies deep intronic variants ney Int. 2020;98:1605–14. https://doi.org/10.1016/j.kint.2020.06.038. causing Alport syndrome and overcomes the problem of negative results of 15. Voskarides K, Damianou L, Neocleous V, Zouvani I, Christodoulidou S, Hadjicon- exome sequencing. Sci Rep. 2021;11:18097. https://doi.org/10.1038/s41598-021- stantinou V, et al. COL4A3/COL4A4 mutations producing focal segmental glo- 97414-0. merulosclerosis and renal failure in thin basement membrane nephropathy. J Am 34. Yao T, Udwan K, John R, Rana A, Haghighi A, Xu L, et al. Integration of genetic Soc Nephrol. 2007;18:3004–16. https://doi.org/10.1681/ASN.2007040444. testing and pathology for the diagnosis of adults with FSGS. Clin J Am Soc 16. Li Y, Groopman EE, D’Agati V, Prakash S, Zhang J, Mizerska-Wasiak M, et al. Type Nephrol. 2019;14:213–23. https://doi.org/10.2215/CJN.08750718. IV collagen mutations in familial IgA nephropathy. Kidney Int Rep. 2020;5:1075–8. 35. Kiryluk K, Li Y, Scolari F, Sanna-Cherchi S, Choi M, Verbitsky M, et al. Discovery of https://doi.org/10.1016/j.ekir.2020.04.011. new risk loci for IgA nephropathy implicates genes involved in immunity against 17. Gulati A, Sevillano AM, Praga M, Gutierrez E, Alba I, Dahl NK, et al. Collagen IV intestinal pathogens. Nat Genet. 2014;46:1187–96. https://doi.org/10.1038/ng.3118. gene mutations in adults with bilateral renal cysts and CKD. Kidney Int Rep. 36. Ding Y, Tang X, Du Y, Chen H, Yu D, Zhu B, et al. Co-existence of Alport syndrome 2019;5:103–8. https://doi.org/10.1016/j.ekir.2019.09.004. and C3 glomerulonephritis in a proband with family history. Eur J Med Res. 18. Pierides A, Voskarides K, Athanasiou Y, Ioannou K, Damianou L, Arsali M, et al. 2021;26:71. https://doi.org/10.1186/s40001-021-00543-5. Clinico-pathological correlations in 127 patients in 11 large pedigrees, segre- 37. Cambier A, Robert T, Hogan J, Rabant M, Peuchmaur M, Boyer O, et al. Rare gating one of three heterozygous mutations in the COL4A3/ COL4A4 genes collagenous heterozygote variants in children with IgA nephropathy. Kidney Int associated with familial haematuria and significant late progression to protei- Rep. 2021;6:1326–35. https://doi.org/10.1016/j.ekir.2021.02.022. nuria and chronic kidney disease from focal segmental glomerulosclerosis. 38. Savige J, Harraka P. Pathogenic variants in the genes affected in Alport syndrome Nephrol Dial Transpl. 2009;24:2721–9. https://doi.org/10.1093/ndt/gfp158. (COL4A3-COL4A5) and their association with other kidney conditions: a review. 19. Groopman EE, Marasa M, Cameron-Christie S, Petrovski S, Aggarwal VS, Milo- Am J Kidney Dis. 2021;78:857–64. https://doi.org/10.1053/j.ajkd.2021.04.017. Rasouly H, et al. Diagnostic utility of exome sequencing for kidney disease. N Engl 39. Omachi K, Kamura M, Teramoto K, Kojima H, Yokota T, Kaseda S, et al. A split- J Med. 2019;380:142–51. https://doi.org/10.1056/NEJMoa1806891. luciferase-based trimer formation assay as a high-throughput screening platform 20. Gibson J, Fieldhouse R, Chan MMY, Sadeghi-Alavijeh O, Burnett L, Izzi V, et al. for therapeutics in Alport syndrome. Cell Chem Biol. 2018;25:634–643.e4. https:// Prevalence estimates of predicted pathogenic COL4A3-COL4A5 variants in a doi.org/10.1016/j.chembiol.2018.02.003. population sequencing database and their implications for Alport syndrome. J 40. Sun L, Hao S, Kuang XY, Wu Y, Huang WY. Determination of the pathogenicity of Am Soc Nephrol. 2021;32:2273–90. https://doi.org/10.1681/ASN.2020071065. a novel COL4A5 missense variant by CRISPR-Cas9 in kidney podocytes. Am J 21. Savige J, Storey H, Il Cheong H, Gyung Kang H, Park E, Hilbert P, et al. X-linked Transl Res. 2021;13:9086–94. and autosomal recessive Alport syndrome: pathogenic variant features and fur- 41. Gross O, Tönshoff B, Weber LT, Pape L, Latta K, Fehrenbach H, et al. A multicenter, ther genotype-phenotype correlations. PLoS ONE. 2016;11:e0161802. https://doi. randomized, placebo-controlled, double-blind phase 3 trial with open-arm org/10.1371/journal.pone.0161802. comparison indicates safety and efficacy of nephroprotective therapy with 22. Zurowska AM, Bielska O, Daca-Roszak P, Jankowski M, Szczepanska M, ramipril in children with Alport’s syndrome. Kidney Int. 2020;97:1275–86. https:// Roszkowska-Bjanid D, et al. Mild X-linked Alport syndrome due to the COL4A5 doi.org/10.1016/j.kint.2019.12.015. European Journal of Human Genetics S. Daga et al. 10 42. Rubel D, Stock J, Ciner A, Hiller H, Girgert R, Müller GA, et al. Antifibrotic, Foundation (IAF), Alport Syndrome Macedonia, Action for Alport (UK), Alport UK, nephroprotective effects of Paricalcitol vs. Calcitriol on top of ACE-inhibitor Alport Syndrome Foundation (USA) and ASAL ONLUS (Italy). We like to acknowledge therapy in the COL4A3-knockout mouse-model for progressive renal fibrosis. all the individuals that attended from around the world and contributed in making Nephrol Dial Transpl. 2014;29:1012–9. https://doi.org/10.1093/ndt/gft434. the Workshop a success. 43. Koepke M-L, Weber M, Schulze-Lohoff E, Beirowski B, Segerer S, Gross O. Nephroprotective effect of the HMG-CoA-reductase inhibitor cerivastatin in a mouse model of progressive renal fibrosis in Alport syndrome. Nephrol Dial AUTHOR CONTRIBUTIONS Transpl. 2007;22:1062–9. https://doi.org/10.1093/ndt/gfl810. AR was in charge of writing the report. SD writes, assembles, and integrates the 44. Kashtan CE, Ding J, Gregory M, Gross O, Heidet L, Knebelmann B, et al. Clinical different sections and provides support for figures generation. AR, JD, CD, JS, BSLZ, practice guidelines for the treatment of Alport syndrome. a statement of the JH, FF, DPG, MA, HK, MoBa, RT, LM, DGL, RL, BK, AC, OG, MaBa, and AMP write all the Alport Syndrome Research Collaborative. Pediatr Nephrol. 2013;28:5–11. https:// parts of the manuscript provide substantial contribution for integrate new insight doi.org/10.1007/s00467-012-2138-4. and harmonising the structure of the manuscript. AR and LP provide substantial 45. Gross O, Perin L, Deltas C. Alport syndrome from bench to bedside: the potential support to the basic science section. AR, JM, and HK provide substantial contributions of current treatment beyond RAAS-blockade and the horizon of future therapies. to the new therapeutics CRISPR/Cas9 section. ABW and SG were in charge of the Nephrol Dial Transplant. 2014;29:iv124-30. https://doi.org/10.1093/ndt/gfu028. patients section. SD submitted this paper. All authors have reviewed and approved 46. Chertow GM, Appel GB, Andreoli S, Bangalore S, Block GA, Chapman AB, et al. the manuscript. Study design and baseline characteristics of the CARDINAL trial: a phase 3 study of bardoxolone methyl in patients with Alport syndrome. Am J Nephrol. 2021;52:180–9. https://doi.org/10.1159/000513777. 47. Rush BM, Bondi CD, Stocker SD, Barry KM, Small SA, Ong J, et al. Genetic or FUNDING pharmacologic Nrf2 activation increases proteinuria in chronic kidney disease in AR from the University of Siena received a financial support from ATS Foundation mice. Kidney Int. 2021;99:102–16. https://doi.org/10.1016/j.kint.2020.07.036. within the project—AAV2-CRISPR/Cas9 preclinical trial on a naturally occurring ATS 48. Heerspink HJL, Stefánsson BV, Correa-Rotter R, Chertow GM, Greene T, Hou FF, dog model‖, TRAVERE Therapeutics Inc. within the project—From benchtop to et al. Dapagliflozin in patients with chronic kidney disease. N Engl J Med. preclinical trials. CRISPR/Cas9 to correct COL4A5 mutations causing Alport Syndrome 2020;383:1436–46. https://doi.org/10.1056/NEJMoa2024816. in in vitro and in vivo dog and mouse model (protocol number: 46630) and Tuscany 49. Wanner C, Inzucchi SE, Lachin JM, Fitchett D, von Eynatten M, Mattheus M, et al. Region within the project—Progetto NIKE_Bando FAS SALUTE Regione Toscana 2018. Empagliflozin and progression of kidney disease in type 2 diabetes. N Engl J Med. Work presented by CD and colleagues was funded partly by the Alport Syndrome 2016;375:323–34. https://doi.org/10.1056/NEJMoa1515920. Foundation Inc. (ASF), Pedersen Family and the Kidney Foundation of Canada (KFOC) 50. Boeckhaus J, Gross O. Sodium-glucose cotransporter-2 inhibitors in patients with Funding Programme, EU’S HORIZON 2020 Research and Innovation Programme hereditary podocytopathies, Alport syndrome, and FSGS: a case series to better under Grant Agreement No. 857122, the Republic of Cyprus, and the University of plan a large-scale study. Cells. 2021;10:1815. https://doi.org/10.3390/ Cyprus and by the—ChapERalport‖ POST-DOC/0916/0190 programme by the Cyprus cells10071815. Research and Innovation Foundation. RT research is funded by the Instituto de Salud 51. Bakris GL, Agarwal R, Anker SD, Pitt B, Ruilope LM, Rossing P, et al. Effect of Carlos III/Fondo Europeo de Desarrollo Regional (FEDER) funds, RETIC REDINREN Finerenone on Chronic Kidney Disease Outcomes in Type 2 Diabetes. N Engl J RD16/0009 FIS FEDER FUNDS (PI15/01824, PI18/00362) and Marató TV3. DGL from the Med. 2020;383:2219–29. https://doi.org/10.1056/NEJMoa2025845. University of Zagreb School of Medicine received a financial support from Croatian 52. Rubel D, Zhang Y, Sowa N, Girgert R, Gross O. Organoprotective effects of spir- Science Foundation within the project Genotype-phenotype correlation in Alport onolactone on top of ramipril therapy in a mouse model for the type IV collagen syndrome and thin glomerular basement membrane nephropathy (IP- 2014-09- disease Alport syndrome. J Clin Med. 2021;10:2958. https://doi.org/10.3390/ 2151). LP from the University of Southern California received a financial support from jcm10132958. National Institutes of Health (NIH) (1R01DK123234). 53. Kaseda S, Sannomiya Y, Horizono J, Kuwazuru J, Suico MA, Ogi S, et al. Novel Keap1-Nrf2 Protein-protein interaction inhibitor UBE-1099 ameliorates pro- gressive phenotype in Alport syndrome mouse model. Kidney 360. 2021. https:// COMPETING INTERESTS doi.org/10.34067/KID.0004572021. The authors declare no competing interests. 54. Gillmore JD, Gane E, Taubel J, Kao J, Fontana M, Maitland ML, et al. CRISPR-Cas9 in vivo gene editing for transthyretin amyloidosis. N Engl J Med. 2021;385:493–502. https://doi.org/10.1056/NEJMoa2107454. ADDITIONAL INFORMATION 55. Daga S, Baldassarri M, Lo Rizzo C, Fallerini C, Imperatore V, Longo I, et al. Urine- Correspondence and requests for materials should be addressed to Alessandra derived podocytes-lineage cells: a promising tool for precision medicine in Alport Renieri. Syndrome. Hum Mutat. 2018;39:302–14. https://doi.org/10.1002/humu.23364. 56. Daga S, Donati F, Capitani K, Croci S, Tita R, Giliberti A, et al. New frontiers to cure Reprints and permission information is available at http://www.nature.com/ Alport syndrome: COL4A3 and COL4A5 gene editing in podocyte-lineage cells. reprints Eur J Hum Genet. 2020;28:480–90. https://doi.org/10.1038/s41431-019-0537-8. 57. Omachi K, Miner JF. Comparative analysis and rational design of dCas9-VP64 Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims variants for CRISPR activation. bioRxiv. 2021. https://doi.org/10.1101/ in published maps and institutional affiliations. 2021.08.13.456279. 58. Feldman DL, Vachon G, Kasthan KE, Rheault M, Simon J, Parziale G, et al. The voice of the patient. Externally led patient-focused Drug Development Meeting on Alport Syndrome. National Kidney Foundation; 2019. 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 ACKNOWLEDGEMENTS Commons license, and indicate if changes were made. The images or other third party The organisers thank the Alport UK and Alport Syndrome Foundation for their material in this article are included in the article’s Creative Commons license, unless support. We thank Kirstie and Laura of Endless Possibilities for the graphical indicated otherwise in a credit line to the material. If material is not included in the documentation of the proceedings. The International Workshop 2019 conference article’s Creative Commons license and your intended use is not permitted by statutory centre Palazzo del Rettorato was provided free of charge by the University of Siena. regulation or exceeds the permitted use, you will need to obtain permission directly The Workshop was made possible by the generous support of Alport UK, SANOFI, from the copyright holder. To view a copy of this license, visit http://creativecommons. REATA Pharmaceutical as Event Sponsor and TRAVERE Therapeutics and Alport org/licenses/by/4.0/. Syndrome Alliance as Education Sponsor. We thank all the patients association, Alport Foundation of Australia, Alport Syndrome.ca, Alport Syndrome Parent Association (China), AIRG—France, Alport Support Group (Germany), Israel Alport © The Author(s) 2022 European Journal of Human Genetics