Original Investigation Clinical and Genetic Features of Autosomal Dominant Alport Syndrome: A Cohort Study Monica Furlano, Victor Martínez, Marc Pybus, Yolanda Arce, Jaume Crespí, María del Prado Venegas, Gemma Bullich, Andrea Domingo, Nadia Ayasreh, Silvia Benito, Laura Lorente, Patricia Ruíz, Vanesa Lopez Gonzalez, Rosa Arlandis, Elisa Cabello, Ferran Torres, Lluis Guirado, Elisabet Ars,* and Roser Torra* Visual Abstract online Rationale & Objective: Alport syndrome is a across sex (P = 0.8), causative genes (P = 0.6), common genetic kidney disease accounting for or type of variant (P = 0.9). Microhematuria was Complete author and article approximately 2% of patients receiving kidney the most common kidney manifestation (92.1%), information provided before references. replacement therapy (KRT). It is caused by and extrarenal features were rare. Findings on pathogenic variants in the gene COL4A3, kidney biopsies ranged from normal to focal Correspondence to E. Ars COL4A4, or COL4A5. The aim of this study was segmental glomerulosclerosis. The slope of esti- (ears@fundacio-puigvert. es) or R. Torra (rtorra@ to evaluate the clinical and genetic spectrum of mated glomerular filtration rate change fundacio-puigvert.es) patients with autosomal dominant Alport was −1.46 (−1.66 to −1.26) mL/min/1.73 m2 per syndrome (ADAS). year for the overall group, with no significant dif- *E.A. and R.T. contributed equally to this work. ferences between ADAS genes (P = 0.2). Study Design: Retrospective cohort study. Am J Kidney Dis. Setting & Participants: 82 families (252 pa- Limitations: The relatively small size of this series 78(4):560-570. Published tients) with ADAS were studied. Clinical, genetic, from a single country, potentially limiting online April 7, 2021. laboratory, and pathology data were collected. generalizability. doi: 10.1053/ j.ajkd.2021.02.326 Observations: A pathogenic DNA variant in Conclusions: Patients with ADAS have a wide spectrum of clinical presentations, ranging from © 2021 The Authors. COL4A3 was identified in 107 patients (35 Published by Elsevier Inc. families), whereas 133 harbored a pathogenic asymptomatic to kidney failure, a pattern not on behalf of the National variant in COL4A4 (43 families). Digenic/com- clearly related to the causative gene or type of Kidney Foundation, Inc. This plex inheritance was observed in 12 patients. variant. The diversity of ADAS phenotypes is an open access article Overall, the median kidney survival was 67 (95% contributes to its underdiagnosis in clinical under the CC BY-NC-ND CI, 58-73) years, without significant differences practice. license (http:// creativecommons.org/ licenses/by-nc-nd/4.0/). A lport syndrome was the first inherited kidney disease for which the genetic bases were identified.1 It ac- counts for at least 1%-2% of all cases of patients under- always the case; (2) thin basement membrane disease is a histopathologic finding associated with other histologic abnormalities in a minor, but not insignificant, percentage going kidney replacement therapy (KRT).2,3 of patients; and (3) kidney disease may develop in “carriers Pathogenic variants in the COL4A3, COL4A4, and COL4A5 of ARAS,” meaning they are not only carriers but patients genes cause defective synthesis of the α3, α4, and α5 chains themselves. The term autosomal dominant Alport syn- of collagen IV, preventing correct assembly of the drome (ADAS) has historically been reserved for patients glomerular basement membrane (GBM) collagen network with heterozygous disease-causing variants in COL4A3 or and causing Alport syndrome.4,5 Classical Alport syndrome COL4A4 with kidney failure,10,11 but there is increasing is characterized by hematuria with progressive proteinuric agreement that any patient harboring a heterozygous kidney disease, GBM abnormalities, hearing loss, and ocular disease-causing variant in COL4A3 or COL4A4 should be abnormalities.4,6,7 This full-blown presentation is typically considered as having ADAS.12,13,20–22 found in patients with autosomal recessive Alport syndrome In recent years, the use of next-generation sequencing (ARAS) and male patients with X-linked Alport syndrome. has allowed increased identification of disease-causing Women with X-linked Alport syndrome show a variable variants in COL4A3 and COL4A4. These have proven to be phenotype due to the X inactivation phenomenon.8 Patients behind 40%-60% cases of familial hematuria23 and most with heterozygous disease-causing variants in COL4A3 or familial cases of focal segmental glomerulosclerosis COL4A4 display a wide spectrum of manifestations, ranging (FSGS), but they are also found in sporadic FSGS or he- from asymptomatic to presentation with hematuria alone or maturia and chronic kidney disease (CKD).24–27 Recently, with proteinuria and subsequent kidney failure in addition Groopman et al found that, in a large adult cohort of pa- to hematuria. There is a lot of controversy as to how to tients with CKD, COL4A3, COL4A4, and COL4A5 were the designate the disease.9–16 In cases in which there is only most frequent causative genes of monogenic CKD after the hematuria, the terms “familial benign hematuria” and “thin polycystic kidney disease genes PKD1 and PKD2.28 Although basement membrane disease” have been used.17–19 How- some attempts have been made to provide insights into the ever, these terms now seem outdated because: (1) familial clinical features of patients with heterozygous disease- benign hematuria implies a very mild disease, which is not causing variants in COL4A3 and COL4A4, most studies 560 AJKD Vol 78 | Iss 4 | October 2021 Furlano et al birth, age at diagnosis of kidney disease, age-related PLAIN-LANGUAGE SUMMARY decrease in kidney function, repeated measurements of Autosomal dominant Alport syndrome is among the urinary protein-creatinine ratio, need for KRT, age at the most underdiagnosed inherited kidney diseases and is time of KRT initiation, and description of genetic variants. not commonly diagnosed by most nephrologists. Results of light microscopy of kidney biopsy specimens Compared with X-linked Alport syndrome or the were classified into 4 groups: normal, FSGS, expansion of autosomal recessive form, this entity is much less se- the mesangial matrix with positive immunofluorescence vere: most patients have hematuria but only a minority staining (unspecific immunoglobulin M and C3 deposits), will experience chronic kidney disease or extrarenal and expansion of the mesangial matrix with negative involvement. The relatively low prevalence of this immunofluorescence staining. If electron microscopy (EM) was available, the GBM findings were recorded. condition, the paucity of clinical features in some pa- Kidney function was assessed by the CKD Epidemiology tients, and a prominent role of environmental/genetic Collaboration (CKD-EPI) creatinine equation for calcu- modifiers may account for its infrequent diagnosis. lating estimated glomerular filtration rate (eGFR) in in- Raising awareness of autosomal dominant Alport syn- dividuals with at least 3 creatinine values 3 years apart. Age drome will foster its diagnosis and reduce the number at the time of KRT initiation was defined as the age at of patients in whom kidney failure develops without an which the patient had a CKD-EPI–calculated etiologic diagnosis. eGFR <10 mL/min/1.73 m2 or began KRT. Age at diag- nosis of hearing loss, type of hearing loss, and ocular le- sions were recorded. have included a low number of patients and/or have been subject to bias because patients were selected from a pre- Genetic Testing viously defined phenotype, such as isolated hematuria or Genetic testing was performed in index cases by next- FSGS, resulting in very mild or very severe phenotypes, generation sequencing of a kidney disease gene panel respectively.27,29 There is also evidence for digenic in- containing COL4A3, COL4A4, and COL4A5 genes as previ- heritance (understood as patients carrying pathogenic ously reported.31 Filtering of the variants was performed variants in different COL4A genes) in collagen type IV by minor allele frequency <0.0005 in the Genome Ag- nephropathies, which, together with modifying genetic or gregation Database and in our in-house database. Variant environmental factors, further complicates the disease annotation and impact prediction were performed with phenotype.30 the SnpEff 4.3 program. Missense variants were further In this study, we provide an in-depth analysis of clinical evaluated using several pathogenic prediction algorithms features and types of variants in a large cohort of families included in the dbNSFP v4.0 missense variant database and with heterozygous disease-causing variants in COL4A3 and using the VarSome tool. Splicing variants were evaluated COL4A4. using the Human Splicing Finder. All candidate disease- causing variants identified were checked for reports in the ClinVar database and Leiden Open Variation Database. Methods Validation and segregation analysis of the candidate disease- Patients causing variants were performed by Sanger sequencing or A total of 82 families (252 patients) were referred from by multiplex ligation-dependent probe amplification anal- Spanish hospitals between 2000 and 2019, and all had an ysis. Variants were classified using the guidelines of the index case with a heterozygous disease-causing variant in American College of Medical Genetics and Genomics COL4A3 or COL4A4, which was the inclusion criterion. (ACMG),32 and those variants classified as pathogenic or Genetic tests were performed in these families in the case likely pathogenic were considered causative of disease of: (1) families with autosomal dominant inherited (Table S1). Digenic/complex inheritance pattern was microhematuria and proteinuria and/or CKD or (2) index considered present when 2 disease-causing variants were patients with an abnormal GBM. Only 1 family with iso- identified in COL4A3 and COL4A4 in the index case. lated microhematuria was included. A genetic diagnosis Variants were classified into the following categories: was made in this family to rule out X-linked Alport syn- in-frame insertion and deletion (indel) <5 amino acids, in- drome in the context of mother-to-son transmission. frame indel >5 amino acids, frame-shift indel (also The study was approved by the Fundaci o Puigvert referred to herein as a truncating indel because the Institutional Review Board, and all participants signed their frameshift variants we observed were predicted to lead to a informed consent to participate in the study. truncated protein), missense, nonsense, and splicing. To study whether the type of disease-causing variant corre- Clinical Data lated with age at the time of KRT initiation, variants were Clinical data from patients and deceased family members classified into different groups (the 6 categories listed were retrospectively obtained from medical records and above or various combinations or subdivisions of these were recorded in a database that included sex, date of groups) according to their predicted severity. AJKD Vol 78 | Iss 4 | October 2021 561 Furlano et al Statistical Analysis were from families with ADAS. None of the patients For descriptive statistics, continuous variables are pre- without hematuria had exhibited proteinuria, but 2 had sented as mean ± standard deviation or median (inter- CKD glomerular filtration rate category 2 (CKD G2; ie, 60- quartile range [IQR]) according to their adherence to the 89 mL/min/1.73 m2) at 76 and 61 years of age, seem- Gaussian distribution, and categorical data are presented as ingly due to reasons other than their COL4A disease- frequencies and percentages. A Fisher exact test was used causing variant. to compare categorical variables, whereas the Mann- Data on proteinuria were available for 241 patients, of Whitney test was used to compare continuous variables whom 157 (65.2%) had proteinuria. None of the patients between sex and COL4A disease-causing variant. without proteinuria needed KRT (only 1 patient without The survival function as well as the median (95% CI) proteinuria presented with an eGFR <45 mL/min/ time to KRT initiation was estimated by the Kaplan-Meier 1.73 m2; this person had CKD G3b and was 70 years of age method. We used the date of birth as the starting point, with a history of nephrectomy), but, among those with and the last point of the survival analysis was the age at proteinuria, 61 of 157 (38.9%) received KRT. Data on the KRT initiation or the last available observation. No patient use of renin-angiotensin-aldosterone system (RAAS) in- died before reaching kidney failure (ie, no competing hibitors were not properly recorded. risks), and the number of at-risk patients is listed by time Overall kidney survival was 67 (95% CI, 58-76) years, point in the figures. We used the time-varying proteinuria whereas, for patients with proteinuria, it was 58 (95% status to assess the impact of proteinuria on KRT. Group CI, 56-57) years (Fig 1A). The presence of proteinuria comparisons and hazard ratios with 95% CIs were calcu- significantly increased the risk of KRT in the time-varying lated by means of the Cox model using the family as a survival analysis (hazard ratio, 5.57 [95% CI, 2.68- cluster to account for intragroup correlation. Kidney 11.60]; P < 0.001; Fig 1B). In the whole group, 61 pa- function was assessed using a slope analysis by means of tients received KRT (24.2%) at mean and median ages of mixed models for repeated measurements, including time 54.0 ± 12.2 and 55 (IQR, 49-58) years, respectively. Six (age or time from diagnosis), participant nested within patients needed KRT before 41 years of age. The slope of family, and, when applicable, genetic data and the time- change in eGFR, which was −1.46 (95% CI, −1.66 varying proteinuria status over time. The intrafamilial to −1.26) mL/min/1.73 m2 per year for the overall variability with regard to age at initiation of KRT was group (Fig 2A), showed no significant differences be- illustrated using a graphical procedure by plotting the tween COL4A3 and COL4A4 (P = 0.2). The slopes of present age of patients not receiving KRT and the age at the change in eGFR for patients with versus without pro- start of KRT for patients who received a transplant or teinuria were −1.78 and −0.97 mL/min/1.73 m2 per dialysis per family. The analysis was performed using SAS year (difference, 0.80 [95% CI, 0.45–1.18]; P < 0.001; software (version 9.4; SAS Institute Inc), and the level of Fig 2B). A large intrafamilial variability with regard to significance was established at the 0.05 level (2-sided). age at initiation of KRT was also observed (Fig 3). The slope of the increase in urinary protein-creatinine ratio was 11.34 (95% CI, 1.95–20.72) mg/g for each year of Results life. Clinical Features Overview Hearing Loss Of the cohort of 252 patients, there were 142 women Among 131 patients with audiometry data recorded, (56.3%), and the mean age at diagnosis was 33.6 ± 17.1 35.9% showed hearing loss, but only 11 patients (8.4%) years. No significant differences in kidney survival ac- showed high-tone bilateral sensorineural hearing loss. cording to sex were identified (P = 0.8; Fig S1; 14 patients Only 1 patient with sensorineural hearing loss received were excluded from kidney survival analysis because of KRT (at 67 years of age; Table S2). digenic/complex inheritance or lack of kidney function data). Thirty-four individuals were relatives of patients Ocular Abnormalities with ARAS. Some ADAS families had the same heterozy- Seventy-five patients underwent a thorough ophthalmo- gous disease-causing variant as ARAS families and showed logic examination, and, even though 23% of them had a mild phenotype, an exception being ADAS family ALP- abnormal findings, only 2 had anomalies possibly related 281 (who had the same variant as family ALP-189), in to Alport syndrome: one had recurrent corneal erosions which several individuals were undergoing KRT (this individual did wear contact lenses) and another had (Table S2). corneal dystrophy (Table S2). Kidney Disease Pathologic Changes Hematuria was present in 92.1% of patients (232 of 252). Forty-nine of 157 patients with proteinuria underwent a Of 20 patients without hematuria, 16 belonged to families kidney biopsy, whereas no patient with hematuria alone in which someone had been diagnosed with ARAS and 4 had a kidney biopsy (Table S2). Based on histology 562 AJKD Vol 78 | Iss 4 | October 2021 Furlano et al Figure 1. Probability of reaching KRT. (A) Kaplan-Meier plot for the overall cohort. (B) Cox survival function with proteinuria status as time-varying covariate (hazard ratio for incident KRT, 5.57 [95% CI, 2.68-11.60] in those with vs without proteinuria; P < 0.001). (Table S3), patients were classified as follows: normal by EM was performed in 20 specimens, disclosing thinning light microscopy and with negative immunostaining with or without splitting and lamellation of the GBM in all (Fig S2A), 32.7%; FSGS (Fig S2B), 32.7%; expansion of patients and podocyte effacement in some. The mean age at the mesangial matrix with unspecific positive immuno- the time of kidney biopsy was 36.6 ± 12.6 (median, 35 staining (Fig S2C), 18.4%; and expansion of the [IQR, 26–51]) years, and mean eGFR was 79.1 ± 28.9 mL/ mesangial matrix with negative immunofluorescence min/1.73 m2. Magnitude of proteinuria at the time of staining (Fig S2D), 16.3%. Of those with FSGS, 75% kidney biopsy ranged from 0.10 to 6.8 (mean, 1.5 ± 1.65) needed KRT. g/d, with 81% having proteinuria >300 mg/d. AJKD Vol 78 | Iss 4 | October 2021 563 Furlano et al Figure 2. eGFR decrease trajectories of patients with heterozygous disease-causing variants in COL4A3 and COL4A4. The x axis shows the age of the patients, and the y axis shows eGFR in mL/min/1.73 m2. The dots are 2 or more eGFR data derived from creat- inine measurement; the black line is the estimated trajectory. Results are shown for the overall cohort (A) and (B) using proteinuria status as time-varying covariate (slope difference, −0.81 [−1.18 to −0.45]; P < 0.001). Genetic Testing disease-causing variants but were not available for genetic Genetic testing was performed in 216 patients; the testing. A heterozygous disease-causing variant in COL4A3 remaining 36 had a history of kidney disease compatible was identified in 35 families (95 patients genetically with Alport syndrome and family members with identified analyzed and 12 inferred), and a disease-causing variant 564 AJKD Vol 78 | Iss 4 | October 2021 Furlano et al Figure 3. Intrafamilial variability of ADAS. Circles indicate present age of patients who are not undergoing KRT, and red symbols represent age at the start of KRT for transplant or dialysis recipients. in COL4A4 was identified in 43 families (109 patients Genotype–Phenotype Correlation genetically analyzed and 24 inferred) (Tables S1-S2). Although there was not a statistically significant difference Digenic/complex inheritance was identified in 12 patients in kidney survival when comparing causative genes (Table S2). (P = 0.6), the median age at KRT was 64 (95% CI, 57-71) Missense variants were the most common type of years for COL4A3, versus 69 (95% CI, 58-81) years for disease-causing variant (Fig 4). The amino acid distribu- COL4A4 (Fig 5A). Hence, to evaluate the remaining tion among the missense disease-causing variants in which genotype-phenotype correlations, we considered COL4A3 there was substitution of glycine (91.9%) was as follows: and COL4A4 variants together. When comparing patients 35.2% arginine, 29.6% valine, 20% glutamate, 9.6% with truncating and nontruncating variants, no significant serine, 1.6% aspartate, and 4% cysteine. The percentage of differences regarding kidney survival were observed patients with truncating disease-causing variants was 31%. (P = 0.5; Fig 5B). Additionally, kidney survival did not vary by other classifications based on the predicted severity of the variant type (Figs S3-S5). Of 6 patients starting KRT before 41 years of age, 3 had a missense disease-causing variant involving substitution of glycine, 1 had a splicing disease-causing variant, and 2 had an indel disease-causing variant: one in-frame indel involving more than 5 amino acids, and the other a truncating indel due to a frameshift variant. Patients with a digenic/complex inheritance were not included in the survival analysis because of the low number of patients but are described in Table S2. Whereas some of them had an early phenotype, others had very mild disease. Discussion The present article provides clinical and genetic informa- tion for a Spanish cohort of 252 patients with disease- causing variants in COL4A3 and COL4A4. In recent years, the increased number of disease-causing variants identified in COL4A3, COL4A4, and COL4A5 has led to an expansion in our understanding of the phenotypic Figure 4. Percentage of patients according to the type of variant spectrum of AS14,26,33,34 and has increased the estimated in COL4A3 and COL4A4. Abbreviations: aa, amino acid; indel, prevalence of ADAS from being “very rare” to accounting insertion and deletion. for at least 20% of cases of Alport syndrome.20,21,35 The AJKD Vol 78 | Iss 4 | October 2021 565 Furlano et al Figure 5. Probability of reaching KRT (A) in patients with heterozygous disease-causing variants in COL4A3 or COL4A4 (P = 0.6) and (B) comparing truncating versus nontruncating disease-causing variants (P = 0.5). Truncating disease-causing variants included 14 nonsense variants, 23 frameshift indels resulting in premature termination, and 36 splicing variants. Analysis limited to individuals available to assess kidney survival. scientific community can choose to keep defining Alport option becomes accepted, nephrologists, genetic coun- syndrome as a full-blown syndrome6 or redefine it based selors, and patients will need to be educated on the wide on genetic findings.12–14,36,37 The latter implies that pa- spectrum of the disease.38 Also, the number of people tients may be diagnosed with Alport syndrome even when carrying one of these variant alleles may be high, and they they have only microhematuria or even when they are may be identified frequently by next-generation asymptomatic as a result of incomplete penetrance. If this sequencing.28 A precise genetic diagnosis of Alport 566 AJKD Vol 78 | Iss 4 | October 2021 Furlano et al syndrome prevents changes in the clinical diagnosis based all mechanisms of the disease. In the present study, 65.2% on the clinical features that occur during life or the of patients exhibited proteinuria and 38.9% of these assignment of diverse diagnoses to different members of a needed KRT. CKD developed in only 1 patient without single family. In this discussion, we will use the term ADAS proteinuria, a 70-year-old patient with a single kidney, for patients with disease-causing variants in COL4A3 or confirming the natural history of ADAS (microhematuria, COL4A4, while accepting that there is as yet no interna- proteinuria, and CKD). Nephrotic-range proteinuria tional consensus. developed in only 3 patients. In one of them (DNA 17- The present cohort of patients with ADAS is, to our 142), immunologic test results were negative and kidney knowledge, the largest published to date. Furthermore, biopsy revealed unspecific deposits of immunoglobulin M based on the inclusion of patients with very diverse with expansion of the mesangial matrix. Another (DNA severity of the disease, there may be less ascertainment bias 14-216) presented with CKD G5 and kidney biopsy was than in other series in which patients were recruited from not feasible, and the third patient (DNA 16-133) had familial hematuria, FSGS, or CKD cohorts.21,27,28 The only 1 kidney and a kidney biopsy was decided against. present data support previous studies in which progression Because of the rarity of this condition and because other to KRT has been observed in some patients whereas others glomerular diseases may coexist, we recommend kidney remain with isolated hematuria throughout their life, thus biopsy be performed, even with proven disease-causing showing huge inter- and intrafamilial variability. Median variants in the collagen type IV genes, if the patient ex- kidney survival in the present cohort was 67 (95% CI, 58- hibits sudden nephrotic-range proteinuria. There is an 76) years, which is longer than in the X-linked and increasing body of evidence supporting the efficacy of recessive forms of Alport syndrome. The name Alport RAAS inhibitors in delaying CKD in Alport syndrome,13,43 syndrome suggests severity and early age at KRT initiation, which could not be assessed in the present study because but, as evidenced in this series, the autosomal dominant of a lack of adequate retrospective data on this matter. form is much milder. This is in line with most Large series on the use of RAAS inhibitors in ADAS are autosomal dominant kidney diseases compared with X- lacking, although, per good clinical practice, patients with linked or autosomal recessive forms. any degree of proteinuria should be receiving treatment Digenic/complex inheritance has been proposed as an with RAAS inhibitors. explanation for severe phenotypes, but none of the patients As reported in all series, the pathologic changes in the who needed KRT before 41 years of age in the present present cohort were unspecific. In early stages, thinning of cohort showed digenic/complex inheritance. The large GBM on EM is the only finding, although no patient with inter- and intrafamilial variability suggests a role for ge- only microhematuria underwent a biopsy. FSGS develops netic modifiers and epigenetic or environmental factors.39 over time, and its presence depends on the stage of the The usual late age of onset of the disease, together with disease at which the biopsy is performed. Even in patients the finding of COL4A3 and COL4A4 disease-causing variants with a certain degree of proteinuria, 32.7% of kidney bi- in a substantial percentage of patients with CKD,28,34 may opsy results were normal on light microscopy, whereas suggest that anomalies in the triple helix of collagen IV unspecific changes were identified in the remainder. may be sufficient to cause severe kidney damage in some Twenty patients underwent EM analysis, which showed patients, but it cannot be ruled out that COL4A3 and thinning with or without splitting and lamellation of the COL4A4 disease-causing variants represent a harmful ge- GBM, as well as podocyte effacement in some cases. As EM netic background for other kidney conditions such as hy- is not usually performed in clinical practice, it is clear from pertensive or diabetic kidney disease. these findings that Alport syndrome will hardly ever be Hematuria is the most prevalent feature in this study, suspected based on a kidney biopsy with only light mi- but, in 7.9% of patients, it was repeatedly absent; most of croscopy studies. Consequently, when suspicion of Alport these patients were relatives of patients with ARAS, as syndrome arises, at present, it seems more reasonable to supported by the literature.22,40,41 This implies an request a genetic test than a kidney biopsy.10 incomplete penetrance, which is not uncommon in genetic Most families had private variants, although 14 different kidney diseases.14,42 There is longstanding evidence that disease-causing variants were found in 2 or more unrelated the same disease-causing variant can cause very different families. No differences in kidney survival according to the phenotypes.19,22 causative gene were observed (although there was a In ADAS, clinical findings beyond hematuria are highly nonsignificant tendency toward better kidney survival in variable and age-dependent, with proteinuria being the patients with COL4A3 variants), and, among the 61 patients next most frequent finding. Although many patients may who received KRT, the proportions with COL4A3 and have only hematuria during their lifetime, some exhibit COL4A4 variants were similar. In contrast to the previously proteinuria, the reported frequency of which varies among reported genotype–phenotype correlation in male patients studies. The pathogenesis of proteinuria is not well un- with X-linked Alport syndrome and patients with derstood, but altered permselectivity of the GBM, ARAS44–46 or ADAS,27 kidney survival did not correlate abnormal cell-matrix interactions, and defective trafficking with type of variant in the present cohort. Discrepancies of GBM matrix components by the podocyte are probably in genotype-phenotype correlations may be due to a AJKD Vol 78 | Iss 4 | October 2021 567 Furlano et al relatively small cohort size, as demonstrated in other Article Information autosomal dominant diseases such as autosomal dominant polycystic kidney disease, in which large series have Authors’ Full Names and Academic Degrees: M onica Furlano, MD, PhD, Victor Martínez, MD, PhD, Marc Pybus, PhD, Yolanda succeeded in showing excellent genotype-phenotype Arce, MD, Jaume Crespí, MD, PhD, María del Prado Venegas, MD, correlation whereas smaller ones ruled out any genotype- PhD, Gemma Bullich, PhD, Andrea Domingo, MS, Nadia Ayasreh, phenotype correlation.47,48 MD, PhD, Silvia Benito, MD, Laura Lorente, BS, Patricia Ruíz, BS, Extrarenal features were cardinal for the diagnosis of Vanesa Lopez Gonzalez, PhD, Rosa Arlandis, MD, Elisa Cabello, Alport syndrome before the genomic era; a proteinuric MD, Ferran Torres, MD, PhD, Lluis Guirado, MD, PhD, Elisabet Ars, PhD, and Roser Torra, MD, PhD. kidney disease with hearing loss was highly suggestive of Alport syndrome, whereas, at present, patients are usually Authors’ Affiliations: Inherited Kidney Diseases, Nephrology Department (MF, RT), Molecular Biology Laboratory (MP, AD, LL, diagnosed on the basis of genetic testing and do not all PR, EA), Department of Pathology (YA), and Nephrology show the full-blown syndrome. Extrarenal involvement Department (NA, SB, LG), Fundaci o Puigvert, Instituto de seems to be infrequent in ADAS22,23,27,49; only 9% of Investigaciones Biom edicas Sant Pau, Universitat Aut onoma de patients in the present cohort had sensorineural hearing Barcelona, Red de Investigaci on Renal, Instituto de Investigaci on loss, and only 2 had ophthalmologic findings compatible Carlos III; Departments of Ophthalmology (JC) and Otolaryngology-Head and Neck Surgery (MdPV), Hospital de Sant with (though not pathognomonic for) Alport syndrome. Pau i la Santa Creu, Instituto de Investigaciones Biomedicas Sant This highlights the need not to attribute to Alport syn- Pau, Universitat Aut onoma de Barcelona; Centre Nacional drome those common eye or ear abnormalities that are d’An alisi Gen omica, Centre for Genomic Regulation, Barcelona usually related to age. Institute of Science and Technology (GB); Biostatistics Unit, In summary, the diagnostic process for ADAS is very Faculty of Medicine, Universitat Aut onoma de Barcelona (FT); Medical Statistics Core Facility, Institut d’Investigacions challenging because of its wide spectrum, which makes Biom ediques August Pi i Sunyer, Hospital Clinic de Barcelona ADAS a very underdiagnosed genetic kidney disease. Based (FT), Barcelona; Nephrology Department (VM) and Genetics on all the available evidence, we can infer that ADAS may Laboratory (VLG), Hospital Clínico Universitario Virgen de la be behind many cases of CKD of unknown cause, espe- Arrixaca, Murcia; Nephrology Department, Hospital General cially if there are affected relatives and/or presence of Universitario de Castellon, Castellon de la Plana (EC); and Nephrology Department, Hospital General de la Palma, Islas hematuria. A proper diagnosis of ADAS will allow genetic Canarias (RA), Spain. counseling for families, avoidance of kidney biopsy, and Address for Correspondence: Roser Torra, MD, PhD, Inherited treatment advice. Given the extremely wide phenotypic Kidney Diseases, Nephrology Department Fundaci o Puigvert, expression of the disease, predictors of rapid progression Cartagena 340-350, 08025 Barcelona, Spain (email: rtorra@ for ADAS are absolutely needed to facilitate appropriate fundacio-puigvert.es) or Elisabet Ars, PhD, Molecular Biology prescription of approved drugs and recruitment of patients Laboratory, Fundaci o Puigvert, Cartagena 340-350, 08025 Barcelona, Spain (email: ears@fundacio-puigvert.es). for clinical trials. Authors’ Contributions: Research idea and study design: MF, EA, RT; data analysis/interpretation: MF, MP, YA, JC, MPV, GB, AD, NA, LL, PR, EA, RT; statistical analysis: FT; patient acquisition: Supplementary Material MF, VM, JC, MPV, NA, SB, VLG, RA, EC, LG, EA, RT; supervision Supplementary File (PDF) or mentorship: EA, RT. EA and RT contributed equally to this work. Figure S1: Probability of incident KRT by sex. Each author contributed important intellectual content during manuscript drafting or revision and agrees to be personally Figure S2: Light microscopy images of the 4 lesion types found in accountable for the individual’s own contributions and to ensure kidney biopsies of ADAS. that questions pertaining to the accuracy or integrity of any portion Figure S3: Probability of incident KRT comparing (1) insertions, of the work, even one in which the author was not directly deletions, nonsense, splicing, and missense variants involving sub- involved, are appropriately investigated and resolved, including stitution of glycine by arginine, glutamate, or aspartate versus (2) with documentation in the literature if appropriate. missense variants involving substitution of glycine by an amino acid Support: This study was funded by the Instituto de Salud Carlos III/ other than arginine, glutamate, or aspartate. Fondo Europeo de Desarrollo Regional (FEDER) funds, RETIC REDINREN RD16/0009 FIS FEDER FUNDS (PI15/01824, PI16/ Figure S4: Probability of incident KRT comparing (1) nontruncating 01998, PI18/00362, PI19/01633), and the Catalan Government disease-causing missense variants involving a glycine substitution (AGAUR 2017/SGR-00676). None of the funders had any role in versus (2) nontruncating disease-causing missense variants not study design, data collection, analysis, reporting or the decision to involving a glycine substitution versus (3) truncating disease-causing submit for publication. variants. Financial Disclosure: The authors declare that they have no Figure S5: Probability of incident KRT comparing the type of variant: relevant financial interests. missense, nonsense, splicing, frameshift indel, in-frame indel >5 Acknowledgements: The authors would like to thank all the amino acids, and in-frame indel <5 amino acids. patients, geneticists, and nephrologists who provided DNA Table S1: Variant classification according to ACMG guidelines. samples and collected clinical data: Dr Madrid (Hospital Sant Joan de D eu, Barcelona), Dr Guill en (Hospital Clínico Universitario Table S2: Clinical and genetic data of patients with disease-causing Virgen de la Arrixaca, Murcia), Dr Espinosa (Hospital General variants in COL4A3 and COL4A4. Universitario Reina Sofía, C ordoba), Dr M endez-P erez (Hospital Table S3: Patient characteristics stratified by type of lesion on kid- Perpetuo Socorro), Dr Martorell (Hospital Sant Joan de D eu de ney biopsy. Esplugues), Drs García Carro and Agraz (Hospital Vall d’Hebr on, 568 AJKD Vol 78 | Iss 4 | October 2021 Furlano et al Barcelona), Drs Rodríguez-Pe~ na, Lopez-Gonz alez, and Pi~nero thin basement membrane nephropathy. J Am Soc Nephrol. (Hospital Clínico Universitario Virgen de la Arrixaca, Murcia), Dr 2013;24(3):364-375. Alegre de Montaner (Fundaci on Jim enez Díaz, Madrid), Dr Barros 11. Savige J, Ariani F, Mari F, et al. Expert consensus guidelines for Freiria (Hospital Josep Trueta, Girona), Dr Sans (Hospital del Mar, the genetic diagnosis of Alport syndrome. Pediatr Nephrol. Barcelona), Dr Miquel (Hospital Universitario de Canarias), Dr 2019;34(7):1175-1189. Segarra (Hospital General de Lleida), Drs Garin Elkoro and 12. Kashtan CE, Ding J, Garosi G, et al. Alport syndrome: a unified Beristain Mendizabal (Hospital Universitario Txagorritxu, Vitoria classification of genetic disorders of collagen IV α345: a po- Gasteiz), Dr Gonzalez Rodríguez (Hospital General Universitario sition paper of the Alport Syndrome Classification Working Santa Lucía de Cartagena, Murcia), Dr Javier Naranjo (Hospital Group. Kidney Int. 2018;93(5):1045-1051. Universitario Puerta del Mar), Dr Quintanilla Mata (Complejo 13. Torra R, Furlano M. New therapeutic options for Alport syn- Hospitalario Universitario de Albacete), and Dr García Agudo drome. Nephrol Dial Transplant. 2019;34(8):1272-1279. (Hospital General La Mancha Centro, Alc azar de San Juan). We 14. Stokman MF, Renkema KY, Giles RH, Schaefer F, Knoers N, also acknowledge Ricard Pellejero from Fundaci o Puigvert for his bibliographic assistance and Ignasi J. Gich Saladich from Hospital Van Eerde AM. The expanding phenotypic spectra of kidney de Sant Pau i la Santa Creu for his assistance in statistical diseases: insights from genetic studies. Nat Rev Nephrol. analysis. We thank IIB Sant Pau Biobank for providing some of 2016;12(8):472-483. the samples. 15. Deltas C, Savva I, Voskarides K, Papazachariou L, Pierides A. Carriers of autosomal recessive Alport syndrome with thin Prior Presentation: Part of this work was presented as an oral basement membrane nephropathy presenting as focal communication at the annual meeting of the European Renal Association 54th ERA-EDTA Congress, June 3-6, 2017, Madrid, segmental glomerulosclerosis in later life. Nephron. Spain; at the International Workshop on Alport Syndrome in 2015;130(4):271-280. Glasgow, 2017, and at the XLVIIth National Congress of 16. Gross O, Kashtan CE, Rheault MN, et al. 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J Nephrol. 2003;16(2):314-316. 1471. 570 AJKD Vol 78 | Iss 4 | October 2021 Furlano et al Clinical and Genetic Features of Autosomal Dominant Alport Syndrome: A Case Series Settings g & Participants Observations Results Retrospective ccohort study 92.1% had Extrarenal microhematuria features rare n = 252 patients pa 3 38.9% of patients with n = 82 fam families Overall kidney survival: proteinuria reached Spanish hospitals 67 (58–76) years KRT (61/157) No patients without No genotype-phenotype proteinuria correlation was observed Heterozygous ozygous disease-ca disease-causing developed CKD variants t iin COL4A3 & COL4A4 CO CONCLUSION: ADAS patients present a wide spectrum of symptoms, regardless of the CON affected gene or disease-causing variants, making the diagnosis very challenging. Furlano M, Martínez V, Pybus M, et al (2021) @AJKDonline | DOI: 10.1053/j.ajkd.2020.02.326 AJKD Vol 78 | Iss 4 | October 2021 570.e1