J Am Soc Nephrol 14: 2603–2610, 2003 X-Linked Alport Syndrome: Natural History and Genotype- Phenotype Correlations in Girls and Women Belonging to 195 Families: A “European Community Alport Syndrome Concerted Action” Study

JEAN PHILIPPE JAIS,* BERTRAND KNEBELMANN,‡ IANNIS GIATRAS,† MARIO DE MARCHI,§ GIANFRANCO RIZZONI,§ ALESSANDRA RENIERI,§ MANFRED WEBER,ʈ OLIVER GROSS,ʈ KAI-OLAF NETZER,ʈ FRANCES FLINTER,¶ YVES PIRSON,# KARIN DAHAN,# JO¨ RGEN WIESLANDER,** ULF PERSSON,** KARL TRYGGVASON,†† PAULA MARTIN,†† JENS MICHAEL HERTZ,‡‡ CORNELIS SCHRO¨ DER,*** MAREK SANAK,ʈʈ MARIA FERNANDA CARVALHO,¶¶ JUAN SAUS,## CORINNE ANTIGNAC,† HUBERT SMEETS,§§ and MARIE CLAIRE GUBLER† *Biostatistique et Informatique Médicale and †INSERM U574 and ‡U507, Hôpital Necker Enfants Malades, Université René Descartes, Paris, France; §The Italian Multicenter Study on Alport syndrome (Coordinators M. de Marchi, G. Rizzonim, and A. Sessa), Italy; ʈHospital Köln Merheim, Cologne, Germany; ¶Guy’s Hospital, University of London, England; #Université Catholique de Louvain, Belgium; **University Hospital, Lund, and ††Karolinska Institute, Stockholm, Sweden; ‡‡Aarhus University Hospital, Denmark; §§ Department of Genetics and Cell Biology, University of Maastricht, The Netherland; ʈʈJagiellonian University Medical School, Krakow, Poland; ¶¶Curry Cabral Hospital, Lisbonne, Portugal; and ##Fundación Valenciana de Investigaciones Biomédicas Instituto de Investigaciones Citológicas, Valencia, Spain, ***Department of Pediatric Nephrology, Wilhelmina Children’s University Hospital, Utrecht, the Netherlands.

Abstract. Alport syndrome (AS) is a type IV collagen heredi- in 95% of carriers and consistently absent in the others. Pro- tary disease characterized by progressive hematuric nephritis, teinuria, hearing loss, and ocular defects developed in 75%, hearing loss, and ocular changes. Mutations in the COL4A5 28%, and 15%, respectively. The probability of developing collagen gene are responsible for the more common X-linked end-stage renal disease or deafness before the age of 40 yr was dominant form of the disease characterized by much less 12% and 10%, respectively, in girls and women versus 90 and severe disease in girls and women. A “European Community 80%, respectively, in boys and men. The risk of progression to Alport Syndrome Concerted Action” (ECASCA) group was end-stage renal disease appears to increase after the age of 60 established to delineate the Alport syndrome phenotype in each yr in women. Because of the absence of genotype-phenotype gender and to determine genotype-phenotype correlations in a correlation and the large intrafamilial phenotypic heterogene- large number of families. Data concerning 329 families, 250 of ity, early prognosis of the disease in X-linked Alport syndrome them with an X-linked transmission, were collected. Charac- carriers remains moot. Risk factors for developing renal failure teristics of heterozygous girls and women belonging to the 195 have been identified: the occurrence and progressive increase families with proven COL4A5 mutation are compared with in proteinuria, and the development of a hearing defect. those of hemizygous boys and men. Hematuria was observed

Alport syndrome (AS) is characterized by progressive hema- changes of the glomerular basement membrane (GBM), fre- turic nephritis with ultrastructural and immunohistochemical quently associated with sensorineural hearing loss (1–8). It is caused by defects in type IV collagen, a major structural component of basement membranes. Six type IV collagen Received May 13, 2003. Accepted July 10, 2003. genes have been cloned and characterized, localized in pairs on Correspondance to Dr. Marie Claire Gubler, INSERM U 574, Hôpital Necker Enfants Malades, 149 rue de Sèvres, 75743 Paris Cedex 15, France. Phone: three chromosomes (9). The COL4A3 and COL4A4 genes, on 33-1-47-83-90-16; Fax: 33-1-44-49-02-90; E-mail: [email protected] chromosome 2, are involved in the rarer autosomal recessive 1046-6673/1410-2603 forms of the disease (10–14), whereas mutations in the Journal of the American Society of Nephrology COL4A5 gene coding for the ␣5 chain of type IV collagen are Copyright © 2003 by the American Society of Nephrology responsible for the more frequent X-linked dominant form of DOI: 10.1097/01.ASN.0000090034.71205.74 AS (15,16). X-linked AS is clinically heterogeneous with a 2604 Journal of the American Society of Nephrology J Am Soc Nephrol 14: 2603–2610, 2003 wide variability in the rate of progression to end-stage renal Results disease (ESRD), the type of GBM changes, the presence or the Analysis of the pedigrees of the 195 X-linked families with absence of deafness, and other extrarenal manifestations, such identified COL4A5 mutation showed that 506 girls and women as ocular changes or diffuse esophageal leiomyomatosis. How- were obligatory carriers for the disease. Information was ob- ever, whereas progression to renal failure is constant in boys tained for 349 carriers but was not complete for all of them, and men, the course of renal involvement is much more vari- probably because of the low rate of presentation as a result of able in girls and women, remaining mild in the majority of the frequently benign course of their disease. them. Since the identification of the first COL4A5 mutations (17), more than 300 different mutations have been reported by different groups in Europe, Asia, and the United States (re- Expression of the Disease in Girls and Women viewed in [15,16,18–27]). Because of the high diversity of Age at diagnosis varied from1dto57yr.Importantly, mutations, a large number of families whose members have AS hematuria was detected before or at the age of 2 yr in 13 have to be analyzed to evaluate genotype-phenotype patients. Microscopic hematuria was found in 309 (95.5%) of correlations. 323 patients. Among the 14 without documented hematuria, 8 Thanks to the formation of the “European Community Al- (all of them more than 25 yr old) were completely asymptom- port Syndrome Concerted Action” (ECASCA) group and the atic carriers, 1 had isolated proteinuria, 2 had chronic renal recruitment of 195 families with a characterized COL4A5 failure, and 3 had diffuse leiomyomatosis with normal urinal- mutation, we recently described the natural history of X-linked ysis. Proteinuria developed in 176 of the 234 patients tested. AS in boys and men and established genotype-phenotype cor- Hearing loss was observed in 28% of the patients, excep- relations (28). Here, we analyze the clinical and pathologic tionally before the age of 20 yr. Ocular changes were detected expression of the disease in heterozygous girls and women in 15% of patients. It consisted of asymptomatic maculopathy belonging to these 195 families and attempt to correlate phe- in most of them. Anterior lenticonus was observed in only notype and genotype. To our knowledge, this is the first large- three patients. scale study of the natural history of X-linked AS in girls and Ultrastructure of the GBM was examined in 28 girls and women with proven COL4A5 mutation. It confirms the vari- women. Thick GBM was observed in 3 patients and irregular able clinical course of the disease previously observed in alternation of thick and thin segments in 17. Diffuse GBM smaller series of AS girls and women and documents the high thinning was seen in six patients who underwent biopsy at the rates of proteinuria and ESRD in heterozygous girls and age of 8 to 21 yr. The GBM was also thin in two related male women, especially in women over 40 yr. subjects (14 and 26 yr old, respectively) from two different families. Alternating thick and thin segments were observed in three related women, 21 to 40 yr old, belonging to three Material and Methods different families and in one related 22-yr-old man. The GBM Inclusion Criteria was normal in the two remaining patients, aged 39 and 52 yr at Inclusion criteria have been described previously (28). Briefly, examination. The 39-yr-old woman had diffuse esophageal inclusion of families was based on the classical criteria of AS: positive leiomyomatosis with no renal symptoms; the other belonged to family history of hematuria with or without progression to ESRD; a family in which an affected male subject had typical alter- progressive sensorineural hearing loss; characteristic ocular changes ations of the GBM. The GBM distribution of the ␣3(IV) chain, (lenticonus and/or maculopathy); and characteristic ultrastructural studied in nine patients, was normal in two and discontinuous changes of the GBM. Two additional signs were considered as new in seven. diagnostic criteria: diffuse esophageal leiomyomatosis involving the Follow-up data were available in 288 female patients. At tracheobronchial tree and the female genitalia, and abnormal GBM their last examination, 34 patients (12%) had chronic renal distribution of the ␣3, ␣4, and ␣5 chains of type IV collagen (3,8). failure, and 51 patients (18%) had reached ESRD. It occurred Data from a total of 329 AS-associated families were collected, and early, between the ages of 19 and 30 yr in 14 patients (28%), the 195 families with an identified COL4A5 mutation were selected between the ages of 31 and 40 yr in 16 patients (31%), and for the study. after 41 yr in 21 patients (41%). Renal transplantation has been performed in 13 patients, and posttransplantation anti-GBM glomerulonephritis occurred in none. Patient Database and Statistical Analyses A database was developed with ORACLE 6.09 software as de- scribed previously (28). Statistical analyses were performed by SAS Comparison between Male and Female Patients software, version 8.01, and Sϩ software, version 3.4. Curves of the As shown on Figure 1, 70% of men at the age of 30 and cumulative probability of occurrence of events (age at ESRD, age at detection of hearing loss) were computed according to the Kaplan- nearly 90% of them at age 40 had reached ESRD. In contrast, Meier method. For the statistical comparisons between curves, we nearly all female patients had functioning kidneys at age 30, used robust tests based on the Cox proportional-hazard model that and only 12% had reached ESRD at 40. Similarly, the risk of take into account the intrafamilial correlations (29). The relationship developing deafness before the age of 40 yr is about 90% in between two types of events was also assessed by considering one as male patients versus only 10% in girls and women (Figure 2). a time-dependent covariant of the other (30). However, after the age of 60, 30% to 40% of the cohort of J Am Soc Nephrol 14: 2603–2610, 2003 X-Linked Alport Syndrome in Girls and Women 2605

three in-frame deletions (analyzed with missense mutations), they were expected to result in the synthesis of a truncated ␣5 chain. Seventy-five were missense mutations, 59 of them lead- ing to glycine substitution, and 29 were splice-site mutations. These mutations have been described in detail previously (28)

Correlations between Genotypes and Phenotypes In female patients, some differences in the probability of developing ESRD were observed according to the type of mutation, with the lowest rate of progression for missense mutations (Figure 3), but in contrast to what has been observed in boys and men, none of the differences were statistically significant. Moreover, a large intrafamilial variability for the occurrence and the age at ESRD was observed, whatever the Figure 1. Probability of end-stage renal disease (ESRD) in 315 boys type of mutation. In 12 families (two large rearrangements; and men and 288 girls and women with COL4A5 mutation. In the third three insertion, deletion, or nonsense; three splice sites; and curve, girls and women with incomplete clinical data were added four missense mutations), one woman had progressed rapidly because they were not in ESRD at last follow-up. to ESRD before the age of 31 yr. Analysis of these families showed that in four of them, severe renal disease was also observed in one relative female (ESRD at 30, 30, 33, and 36 yr, respectively), but renal function was normal in 15 other carri- ers (aged 13 to 83 yr). In family 4, ESRD occurred in two women at ages 54 and 59 yr, respectively, but renal function was normal in two others aged 21 and 61 yr. In the other families, no ESRD was observed in the six related carriers aged 40 to 82 yr. No correlation could be established with the severity of the renal disease in boys and men (Figure 4). For example, in family 10, one woman with frameshift mutation developed ESRD at 28 yr, and the disease was also severe in related boys and men who developed ESRD between 13 and 25 yr of age. Conversely, in family 8, with missense mutation, 10 related men of a woman who experienced ESRD at 27 yr of age Figure 2. Probability of hearing loss in 144 boys and men and 151 developed ESRD at various ages between 22 and 76 yr, and at girls and women with COL4A5 mutation. more than 50 yr in five of them. Twenty-eight percent of female patients developed hearing female patients regularly followed have developed ESRD, and 20% have hearing loss. In an attempt to correct the bias linked to the loss to follow-up of approximately half of the patients—probably those with a benign course of the disease—we performed a parallel analysis taking in account 61 additional girls and women for which partial information was available (Figure 1). The derived curve probably gives more precise information on the actual risk for girls and women with X-linked AS of developing severe complications. However, in this analysis, we could not include data of 157 obligatory carriers appearing in the genealogical trees, for whom birth date was not provided and for whom no information at all was available.

COL4A5 and COL4A5/COL4A6 Mutation Distribution in Families Figure 3. Probability of end-stage renal disease in 288 female patients COL4A5 mutations consisted of 38 large deletions of the according to the type of COL4A5 mutation. Rear indicates rearrange- COL4A5 gene (also involving the COL4A6 gene in 12) and ment; mut, mutation; d, deletion; i, insertion; and n, nonsense muta- 157 small mutations. Among the small deletions, 31 were tion. Three small in-frame deletions were analyzed with missense deletions, 9 insertions, and 12 nonsense mutations; except for mutations. 2606 Journal of the American Society of Nephrology J Am Soc Nephrol 14: 2603–2610, 2003

when tested, for the ␣4 (IV) and ␣5 (IV) chains) was discon- tinuous in seven girls and women with different types of COL4A5 mutations. Conversely, it was normal in one female subject with glycine substitution and another with in-frame small deletion (Table 2).

Prognostic Features The renal prognosis of AS in women is unpredictable at an early age. During the course of evolution, the occurrence of proteinuria has an ominous prognosis (Figure 6) (P Ͻ 0.001). The risk for developing ESRD is also higher in girls and women with, rather than without, hearing loss (Figure 7) (P ϭ 0.02). Among the three patients with lenticonus, one developed Figure 4. Age at end-stage renal disease (ESRD) of the female ESRD at 24 yr, whereas the two others had normal renal patients (solid symbols) who progressed to renal failure in 42 families. function at 63 and 52 yr, respectively. Diffuse leiomyomatosis Comparison with age at ESRD of related boys and men (open is not specifically associated with progression of the renal symbols) disease. Discussion loss; no significant correlation was observed with the various We have previously reported the natural history of the dis- types of mutation (Figure 5) or the occurrence of hearing loss ease in male patients belonging to 195 X-linked AS families in related boys and men. Ten patients with AS had diffuse with identified COL4A5 mutation (28). Here, we focus our esophageal leiomyomatosis. In all of them, a deletion removing report on the expression of the disease in related girls and the 5' end of both the COL4A5 and the COL4A6 genes, with women in an attempt to evaluate the actuarial risk of develop- a breakpoint located in the second intron of COL4A6, was ing renal failure and deafness and to establish genotype phe- identified. None of these patients had developed ESRD at the notype correlations. This is the first large-scale study of the age of 23 to 53 yr, and three had normal urinalysis findings. natural history of X-linked AS in girls and women with proven However, two of them were proteinuric at 23 and 26 yr, COL4A5 mutation. It confirms the variable clinical course of respectively. Five patients died of leiomyomatosis complica- the disease previously observed in smaller series of AS girls tions or superimposed cancers between the ages of 39 and 53 and women and documents the high rates of proteinuria and yr. ESRD in heterozygous girls and women, especially in women Abnormal thick or alternatively thick and thin GBM was over 40 yr. observed in 19 patients with various types of mutation (Table Hematuria, usually microscopic, is the cardinal feature of the 1). Two female patients (one with a large COL4A5-COL4A6 disease (2,31) and was the presenting symptom in nearly all deletion and DEL, and the other with a missense mutation) had girls and women. During the course of the disease, it was normal GBM. Thin GBM was predominantly seen in patients present in 96% of girls and women. This result is in agreement with missense mutation. But overall, no significant correlation with the study by Flinter et al. (4) and Dagher et al. (32) who could be established between the type of mutation and the found hematuria in 100% of female carriers when repeated GBM ultrastructural changes. GBM staining for ␣3 (IV) (and, urinalysis was performed. Proteinuria developed in 75% of girls and women. Hearing loss, usually occurring after 30 to 40 yr of age, was observed in 28% of patients, and ocular changes, mostly macular flecks, were detected in 15%. Ten patients had diffuse esophageal leiomyomatosis; as previously shown, there is no correlation between the presence of leiomyomatosis and the severity of the renal disease (33). Ultrastructural changes of the GBM were found in 26 of 28 patients and consisted of typical thick and split or alternatively thick and thin GBM in 19 patients. Interestingly, in six pa- tients, the GBM was diffusely thin, a lesion not specific of benign familial hematuria. GBM immunohistochemical stain- ing for the ␣3-␣5(IV) chains, performed in nine patients, was discontinuous in seven. This specific anomaly was observed in patients with missense or frameshift mutation, but normal expression of the chains was also seen in two patients with Figure 5. Probability of hearing loss in 150 female patients according deletion or missense mutation. to the type of COL4A5 mutation. Rear indicates rearrangement; mut, The prognosis of X-linked AS is usually regarded as favor- mutation; d, deletion; i, insertion; and n, nonsense mutation. able in girls and women (2,31,33). Actually, 51 girls and J Am Soc Nephrol 14: 2603–2610, 2003 X-Linked Alport Syndrome in Girls and Women 2607

Table 1. X-linked Alport syndromea

Mutation GBM Large Splice Nonsense Rearrangement Missense Site Deletion Insertion Total

Normal 1 1 0 0 2 Thin 0 5 0 1 6 Thick, or thick and thin 3 10 3 4 20

a Correlation between the type of COL4A5 mutation and the type of GBM ultrastructural changes in 28 female patients. GBM indicates glomerular basement membrane.

Table 2. Immunohistochemical data, clinical status, and COL4A5 mutation state in nine patients with examination of ␣- chain expressiona

Phenotype/Genotype GBM ␣3/␣5 (IV) Hematuria Proteinuria HT Renal Hearing Ocular Last FU (yr) (yr) (yr) Function Loss Changes GBM (yr) Mutations

ϩ 11 19 — N ϪϪThick and thin 26 Del 3BP, ex 33 ϩ 19 19 — N ϪϪ ND 27 Miss ex 27b ϩ/Ϫ 31921NϪϪ Thin 21 Miss ex 36b ϩ/Ϫ 1 1 35 N ϪϪ ND 36 Miss ex 35b ϩ/Ϫ 17 17 — N ϪϪThick and thin 47 Miss ex 26 ϩ/Ϫ 79—NϪϪ Thin 19 Miss ex 26 ϩ/Ϫ 26 26 — N ϪϪThick and thin 28 Miss ex 41b ϩ/Ϫ 14 14 — ESRD 24 ϩ AL Thick and thin 24 Del 1BP, ex 41 ϩ/Ϫ 4——NϪϪThick and thin 15 Splice site intron 15

a ϩ indicates normal distribution; ϩ/Ϫ, discontinuous distribution; GBM, glomerular basement membrane; FU, follow-up; ND, not determined; HT, hypertension; AL, anterior lenticonus; Del, deletion; Miss, missense; and ex, exon. b Four of the seven missense mutations resulted in glycine substitution.

Figure 6. Probability of end-stage renal disease according to the Figure 7. Probability of end-stage renal disease according to the ϭ ϭ presence (n ϭ 119) or absence (n ϭ 43) of proteinuria presence (n 165) or absence (n 55) of hearing defect. women developed ESRD, and 55% of them reached this stage renal function in 113 female patients with X-linked AS: 15% of before the age of 40 yr, the youngest at the age of 19 yr. The them had manifested chronic renal failure at an average age of risk of developing ESRD or deafness before the age of 40 yr is 40 yr. However, we were surprised by the severity of the 12% and 10%, respectively. It is clearly different in related long-term outcome of the disease in our group of female boys and men, in whom the risk of developing ESRD and patients: in them, the probability of having ESRD by the age of deafness before the age of 40 yr is about 90% (28). Similar 60 is about 30%. This rate could be overestimated because results have been found by Flinter et al. (4), who analyzed approximately one-third of girls and women, likely to be the 2608 Journal of the American Society of Nephrology J Am Soc Nephrol 14: 2603–2610, 2003 less severely affected, were lost to follow-up, but it suggests the renal disease. This finding was not confirmed by one of us that AS in girls and women is less benign than previously (42). thought. At the clinical level, the absence of genotype-phenotype High levels of progression to ESRD have been reported in a correlations and the intrafamilial heterogeneity of the pheno- few studies, but these were probably the result of the mode of type make it impossible to predict the renal course in AS selection of patients or a referral bias (34,35). In the analysis by individual girls and women from family analysis or genetic Grünfeld et al. (34) designed to determine the predictive fac- studies. These patients have to be systematically followed on a tors of renal prognosis in girls and women with AS, 40% of longitudinal basis, and as previously shown by Grünfeld et al. patients developed ESRD before (nine patients) or after (five (34) and as demonstrated in this study, the best ominous patients) the age of 35 yr. But the 36 patients have been prognostic marker remains the occurrence and the progressive selected on the basis of persistent urinary abnormalities, and increase in proteinuria. The risk of developing ESRD is also the mode of transmission of their disease is not determined. higher in patients with, rather than without, hearing loss. These The 63% rate of progression to ESRD observed in the series of data have to be taken in account in the difficult discussion of Chugh et al. (35) is clearly the result of a referral bias in favor kidney donation by related Alport heterozygotes. It cannot be of the most symptomatic patients because only 11 of 63 pa- accepted if the presumptive donor is young, or if proteinuria, tients with AS assessed in their center were girls and women. hearing defect, hypertension, or renal insufficiency are present. On the other hand, it is interesting to note that in the Finnish Conversely, heterozygous women who have asymptomatic he- experience, the median age at ESRD was 31.1 yr (range, 20.2 maturia when older than 30 to 40 yr have a very small risk of to 39.9 yr) in the 5 of 40 female patients from 25 families who developing late renal failure. If they are strongly motivated and had a progressive renal disease, not much later than in the 20 well informed, they cannot be definitely refused as potential of 38 related boys and men who developed ESRD at a median kidney donors. age of 24.9 yr (range, 14.5 to 39 yr) (36). We tried to determine whether the clinical course of the disease depends on the genetic defect. The lowest rate of Acknowledgments progression to ESRD is associated with missense mutations, as We thank the patients, the families, and the physicians who con- in related boys and men; however, the association is not tributed to this project. The study was supported by grants from the statistically significant. Similarly, the 11 girls and women with European Communities (contracts BMH1 CT93 1052; PL94-1074) X-linked AS who developed ESRD in the recent study of Hertz and by funds from the Association Claude Bernard (MCG), the Assistance Publique-Hôpitaux de Paris (MCG), the Caisse Nationale et al. (27) bore different types of mutations from missense to d’Assurance Maladie des Travailleurs Salariés (MCG), the Dagmar frameshift, and in the series of Inoue et al. (22), the two girls Marshall Foundation (JMH), the Danish Foundation for the Advance- with heavy proteinuria at, respectively, 13 and 15 yr of age had ment of Medical Science (JMH), the Danish Medical Association missense mutation. No genotype-phenotype correlation was Research Fund (JMH), the Swedish Medical Research Council (con- detected regarding the occurrence of hearing loss. tract 16x-09487) (JW), the National Kidney Research Fund (FF), the A peculiar feature of X-linked AS is the large intrafamilial Dutch Kidney Foundation (HJMS, CS), and the Comisión Intermin- variability of the disease in girls and women. This heteroge- isterial de Ciencia y Tecnología y Plan Nacional IϩD of Spain (JS). neity was already seen in the family reported by Alport (1) and further completed by Crawfurd et al. (37): age at ESRD in the five girls and women with a progressive course varied from 12 References to 83 yr. The same variability was observed in our series, and 1. 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