Milder clinical aspects of X-linked Alport syndrome in men positive for the collagen IV α5 chain

X-linked Alport syndrome is caused by mutations in the COL4A5 gene encoding the type IV collagen α5 chain (α5(IV)). Complete absence of α5(IV) in the renal basal membrane is considered a pathological characteristic in male patients; however, positive α5(IV) staining has been found in over 20% of patients. We retrospectively studied 52 genetically diagnosed male X-linked Alport syndrome patients to evaluate differences in clinical characteristics and renal outcomes between 15 α5(IV)-positive and 37 α5(IV)-negative patients. Thirteen patients in the α5(IV)-positive group had non-truncating mutations consisting of nine missense mutations, three in-frame deletions, and one splice-site mutation resulting in small in-frame deletions of transcripts. The remaining two showed somatic mutations with mosaicism. Missense mutations in the α5(IV)-positive group were more likely to be located before exon 25 compared with missense mutations in the α5(IV)-negative group. Furthermore, urinary protein levels were significantly lower and the age at onset of end-stage renal disease was significantly higher in the positive group than in the negative group. These results help to clarify the milder clinical manifestations and molecular characteristics of male X-linked Alport syndrome patients expressing the α5(IV) chain. X-linked Alport syndrome is caused by mutations in the COL4A5 gene encoding the type IV collagen α5 chain (α5(IV)). Complete absence of α5(IV) in the renal basal membrane is considered a pathological characteristic in male patients; however, positive α5(IV) staining has been found in over 20% of patients. We retrospectively studied 52 genetically diagnosed male X-linked Alport syndrome patients to evaluate differences in clinical characteristics and renal outcomes between 15 α5(IV)-positive and 37 α5(IV)-negative patients. Thirteen patients in the α5(IV)-positive group had non-truncating mutations consisting of nine missense mutations, three in-frame deletions, and one splice-site mutation resulting in small in-frame deletions of transcripts. The remaining two showed somatic mutations with mosaicism. Missense mutations in the α5(IV)-positive group were more likely to be located before exon 25 compared with missense mutations in the α5(IV)-negative group. Furthermore, urinary protein levels were significantly lower and the age at onset of end-stage renal disease was significantly higher in the positive group than in the negative group. These results help to clarify the milder clinical manifestations and molecular characteristics of male X-linked Alport syndrome patients expressing the α5(IV) chain. Alport syndrome (AS) is a hereditary disorder of type IV collagen, characterized by chronic kidney disease progressing to end-stage renal disease (ESRD), sensorineural hearing loss, and ocular abnormalities.1Kashtan C.E. Alport syndrome and thin glomerular basement membrane disease.J Am Soc Nephrol. 1998; 9: 1736-1750Crossref PubMed Google Scholar Approximately 85% of AS patients show X-linked inheritance (XLAS), and mutations in the COL4A5 gene, which encodes the type IV collagen α5 (α5(IV)) chain, can be detected.1Kashtan C.E. Alport syndrome and thin glomerular basement membrane disease.J Am Soc Nephrol. 1998; 9: 1736-1750Crossref PubMed Google Scholar Disease-causing mutations in COL4A5 result in abnormal α5(IV) expression and typically in complete absence of α5(IV) in the glomerular basement membrane (GBM) and Bowman’s capsule (BC). However, a previous review suggested that 20% of male XLAS sufferers showed complete or partial staining for this collagen chain,2Kashtan C.E. Alport syndrome and thin basement membrane nephropathy.Gene Reviews. 1993–2013Google Scholar although the genetic and clinical backgrounds of male XLAS patients presenting with such atypical immunohistological findings have not yet been elucidated. The aim of this study was to clarify the genetic and clinical backgrounds of XLAS patients with positive expression of α5(IV), and to determine if expression of this chain correlated with renal phenotype in men with XLAS. A total of 139 patients were referred to our hospital for mutational analysis between January 2006 and January 2011. Among these, 63 female patients were excluded from this study. Of the remaining 76 male patients, 62 were genetically defined as male XLAS sufferers. α5(IV) Staining was not carried out in 10 patients, and 52 men with α5(IV) expression data were therefore included in this study (Figure 1). Thirty-seven (71%) patients showed no α5(IV) staining in kidney tissue (negative group), whereas the remaining 15 (29%) showed positive staining for α5(IV) (positive group). Thirteen patients in the positive group showed normal α5(IV) distribution, although some showed reduced expression levels, and two showed a mosaic expression pattern (Supplementary Table 2 online). The characteristics of the two groups are summarized in Table 1. Although the ages at kidney biopsy and mutational analysis were similar in both groups, the time between age at biopsy and age at mutational analysis was significantly shorter in the positive group. This could be because atypical cases require genetic diagnosis, and doctors may tend to request mutational analysis sooner in these patients than in typical cases. The shorter duration of follow-up in positive patients could help to explain the smaller number of patients developing ESRD. Download .xls (.03 MB) Help with xls files Supplementary InformationTable 1Clinical and laboratory data and treatments in α5(IV)-negative and α5(IV)-positive patientsα5(IV)-Negative group (n=37)α5(IV)-Positive group (n=15)P-valueAge at analysis (years)14.0±9.212.8±7.00.88Age at kidney biopsy (years)8.0±6.210.5±6.60.13Time between kidney biopsy and mutational analysis (years)5.8±7.52.3±3.00.041Age at the detection of urinary protein (years)4.0±3.26.3±4.80.036Urinary protein/creatinine ratio (g/g Cr)aValues for patients with ESRD were excluded from this analysis.1.7±2.40.78±1.00.027Estimated GFR (ml/min per 1.73m2)111.2±31.6122.9±20.60.43Number of patients developing ESRD510.66Hearing loss (%)bHearing test was not conducted in one patient in the positive group.54.300.0002Ocular abnormality (%)cOphthalmologic exams were not conducted in five patients in the positive group.15.66.70.65No medication34ACEI/ARB only2911CyA only00ACEI/ARB +CyA50Abbreviations: ACEI, angiotensin converting enzyme inhibitor; ARB, angiotensin receptor blocker; CyA, cyclosporine A; ESRD, end-stage renal disease; GFR, glomerular filtration rate; α5(IV), type IV collagen α5.a Values for patients with ESRD were excluded from this analysis.b Hearing test was not conducted in one patient in the positive group.c Ophthalmologic exams were not conducted in five patients in the positive group. Open table in a new tab Abbreviations: ACEI, angiotensin converting enzyme inhibitor; ARB, angiotensin receptor blocker; CyA, cyclosporine A; ESRD, end-stage renal disease; GFR, glomerular filtration rate; α5(IV), type IV collagen α5. Five (13.5%) patients developed ESRD in the negative group, compared with one (6.7%) in the positive group. Three patients in the negative group and four in the positive group received no medications. Twenty-nine in the negative group and 11 in the positive group received angiotensin-converting enzyme inhibitors and/or angiotensin receptor blockers, whereas five in the negative group received angiotensin-converting enzyme inhibitor/angiotensin receptor blocker and cyclosporin treatment. There were no significant differences in medications between the two groups (P=0.091). All mutations detected in the positive and negative groups are shown in Supplementary Tables 1 and 2 online, respectively. Regarding COL4A5 genotype, 23 (62%) of the 37 patients in the negative group had truncating mutations, including nonsense (n=5), insertion (n=7), deletion (n=6), and splice-site mutations (n=5). All these insertion, deletion, and splice-site mutations led to out-of-frame mutations. In contrast, no patients in the positive group had truncating mutations. Mutations in the positive group included missense (n=9), large in-frame deletion (n=1, exons 2–8, 384-bp deletion), small in-frame deletions (n=2, 9-bp and 36-bp deletions), splice-site mutation (n=1, leading to exon 9, 81-bp skipping), and somatic mosaic mutations (n=2; Table 2). Twenty (38%) patients in this study had missense mutations in COL4A5; 11 in the negative and 9 in the positive groups. All the missense mutations in this study resulted in glycine substitutions. The positions of the missense mutations are shown in Figure 2. Receiver operating characteristic curve analysis of patients with missense mutations showed a cutoff value at exon 25 in both groups (sensitivity 0.778, specificity 0.8, Figure 2). Seven patients in the positive group and two in the negative group had missense mutations in the exons between 1 and 25, whereas two and nine patients, respectively, had mutations between exons 26 and 51 (P=0.02). Missense mutations in the positive group thus showed a significant tendency to be located before exon 25.Table 2Mutation types in α5(IV)-negative and α5(IV)-positive patientsα5(IV)-Negative groupα5(IV)-Positive groupGenotype(n=37)(n=15)TotalMissense11920Nonsense505Insertion7 (0)0 (0)7Deletion6 (0)3 (3)9Splice site8 (3)1 (1)9Somatic mosaicism02aBoth patients showed α5(IV) expression with mosaic pattern.2Total371552Truncating mutation23062%0%Abbreviation: α5(IV), type IV collagen α5.Number of patients with in-frame mutation are shown in parenthesis.a Both patients showed α5(IV) expression with mosaic pattern. Open table in a new tab Abbreviation: α5(IV), type IV collagen α5. Number of patients with in-frame mutation are shown in parenthesis. Regarding phenotypes, the age at first proteinuria detection was significantly younger (4.0±3.2 and 6.3±4.8, respectively; P=0.036, Table 1) and the urinary protein/creatinine ratio at the time of mutational analysis was significantly higher in the negative group than in the positive group (1.7±2.4g/g Cr and 0.78±1.0g/g Cr, respectively; P=0.027, Table 1), although the ages at mutational analysis were similar in both groups. Only six patients developed ESRD, because of their relatively young age. We therefore compared the age at onset of ESRD in patients and their affected male family members. Five patients and 9 affected male family members in the negative group, and 2 patients and 13 affected male family members in the positive group developed ESRD (Supplementary Table 1 and 2 online). The age at onset of ESRD was significantly lower in the negative group than in the positive group (24.00±14.2 and 37.53±16.30 years, respectively; P=0.018, Figure 3). Two patients in the positive group demonstrated somatic mosaicism; both showed a mosaic pattern of α5(IV) expression in the GBM and BC, one of which we have recently reported on elsewhere.3Krol R.P. Nozu K. Nakanishi K. et al.Somatic mosaicism for a mutation of the COL4A5 gene is a cause of mild phenotype male Alport syndrome.Nephrol Dial Transplant. 2008; 23: 2525-2530Crossref PubMed Scopus (24) Google Scholar Previous reports have suggested a probability of hearing loss of 50% by age 15 in men with X-linked AS.4Jais J.P. Knebelmann B. Giatras I. et al.X-linked Alport syndrome: natural history in 195 families and genotype- phenotype correlations in males.J Am Soc Nephrol. 2000; 11: 649-657Crossref PubMed Google Scholar Our results showed that 54.3% of patients in the negative group developed hearing loss, with a median age of 13 years; however, no patients in the positive group developed hearing loss (Table 1 and Figure 4). It has also been reported that about 15% of X-linked AS men exhibit anterior lenticonus or other eye lesions.4Jais J.P. Knebelmann B. Giatras I. et al.X-linked Alport syndrome: natural history in 195 families and genotype- phenotype correlations in males.J Am Soc Nephrol. 2000; 11: 649-657Crossref PubMed Google Scholar However, there was no significant difference in ocular abnormalities between the two groups in this study (15.6% in the negative group and 6.7% in the positive group; P=0.65; Table 1). This study provides the first report of the genetic and clinical backgrounds of male XLAS patients with atypical immunohistological findings of α5(IV) in the kidney. The results show valuable and novel information on the renal outcome and genetic background of these patients based on immunostaining patterns. We determined the genotypes of patients with immunohistochemical evidence of α5(IV) expression, and confirmed that this staining pattern was associated with a milder renal course. We demonstrated that 29% of male XLAS patients were positive for α5(IV) staining; 60% of them had missense mutations, 27% had an in-frame deletions, and 13% had somatic mosaic mutations. In contrast, all patients with truncating mutations showed negative staining for α5(IV). Several groups have described genotype–phenotype correlations in XLAS.5Jais J.P. Knebelmann B. Giatras I. et al.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.J Am Soc Nephrol. 2003; 14: 2603-2610Crossref PubMed Scopus (314) Google Scholar Large rearrangements and all mutations that change the reading frame of the gene were associated with severe types of AS,6Gubler M.C. Inherited diseases of the glomerular basement membrane.Nat Clin Pract Nephrol. 2008; 4: 24-37Crossref PubMed Scopus (92) Google Scholar whereas patients with in-frame mutations tended to have relatively mild phenotypes.4Jais J.P. Knebelmann B. Giatras I. et al.X-linked Alport syndrome: natural history in 195 families and genotype- phenotype correlations in males.J Am Soc Nephrol. 2000; 11: 649-657Crossref PubMed Google Scholar,6Gubler M.C. Inherited diseases of the glomerular basement membrane.Nat Clin Pract Nephrol. 2008; 4: 24-37Crossref PubMed Scopus (92) Google Scholar,7Gross O. Netzer K.O. Lambrecht R. et al.Meta-analysis of genotype-phenotype correlation in X-linked Alport syndrome: impact on clinical counselling.Nephrol Dial Transplant. 2002; 17: 1218-1227Crossref PubMed Scopus (194) Google Scholar Jais et al.4Jais J.P. Knebelmann B. Giatras I. et al.X-linked Alport syndrome: natural history in 195 families and genotype- phenotype correlations in males.J Am Soc Nephrol. 2000; 11: 649-657Crossref PubMed Google Scholar reported normal GBM incorporation of defective α5(IV) and the related α3(IV) and α4(IV) chains in two patients, indicating that normal GBM expression of α5(IV) did not preclude a diagnosis of XLAS. Mazzucco et al.8Mazzucco G. Barsotti P. Muda A.O. et al.Ultrastructural and immunohistochemical findings in Alport's syndrome: a study of 108 patients from 97 Italian families with particular emphasis on COL4A5 gene mutation correlations.J Am Soc Nephrol. 1998; 9: 1023-1031PubMed Google Scholar also reported three female XLAS patients with normal α3(IV) and α5(IV) staining patterns and proven COL4A5 mutations, including two patients with small in-frame mutations and one with a missense mutation. In addition, two patients showed positive α3(IV) staining patterns, despite negative staining for α5(IV) chain, and these authors hypothesized that very low levels of α5(IV) expression that were undetectable by immunohistochemical study may still be sufficient to allow the correct folding of the triple helix comprising the α3-α4-α5 chains.9Wongtrakul P. Shayakul C. Parichatikanond P. et al.Immunohistochemical study for the diagnosis of Alport's syndrome.J Med Assoc Thai. 2006; 89: S171-S181PubMed Google Scholar Massella et al.10Massella L. Gangemi C. Giannakakis K. et al.Prognostic value of glomerular collagen IV immunofluorescence studies in male patients with X-linked Alport syndrome.Clin J Am Soc Nephrol. 2013; 8: 749-755Crossref PubMed Scopus (14) Google Scholar recently reported that 3 out of 22 patients (14%) showed α5(IV) positivity (one diffuse and two segmental), and 5 of 22 patients (23%) showed diffuse α3(IV) staining. Type IV collagen, which is a component of the GBM, is a triple helix composed of three α chains. We hypothesized that some missense and in-frame mutations might affect the structure of this triple helix, but its rate of degradation is low. The α3-α4-α5(IV) triple helix network in GBM may thus sometimes be present in reduced amounts, and low, rather than absent expression levels may lead to the milder phenotype of XLAS.11Kashtan C.E. Alport syndromes: phenotypic heterogeneity of progressive hereditary nephritis.Pediatr Nephrol. 2000; 14: 502-512Crossref PubMed Scopus (46) Google Scholar The apparent discrepancies in α5(IV) positivity between the current and previous reports may be the result of the use of different antibodies with different sensitivities, associated with the methods of antibody production.12Sado Y. Kagawa M. Kishiro Y. et al.Establishment by the rat lymph node method of epitope-defined monoclonal antibodies recognizing the six different alpha chains of human type IV collagen.Histochem Cell Biol. 1995; 104: 267-275Crossref PubMed Scopus (205) Google Scholar, 13Naito I. Kawai S. Nomura S. et al.Relationship between COL4A5 gene mutation and distribution of type IV collagen in male X-linked Alport syndrome. Japanese Alport Network.Kidney Int. 1996; 50: 304-311Abstract Full Text PDF PubMed Scopus (76) Google Scholar, 14Kagawa M. Kishiro Y. Naito I. et al.Epitope-defined monoclonal antibodies against type-IV collagen for diagnosis of Alport's syndrome.Nephrol Dial Transplant. 1997; 12: 1238-1241Crossref PubMed Scopus (55) Google Scholar These factors should be taken into account when interpreting the results of these studies. We also examined the correlation between mutation positions and staining patterns in patients with missense mutations. Missense mutations located in exons 1–25 were more common in α5(IV)-positive patients. The three α-chains comprising the triple helix of type IV collagen consist of triple helical protomers with different compositions.15Timpl R. Structure and biological activity of basement membrane proteins.Eur J Biochem. 1989; 180: 487-502Crossref PubMed Scopus (811) Google Scholar Each protomer has a 7S triple helical domain at the N-terminal, a collagenous domain in the middle of the molecule of Gly-X-Y repeats, and a non-collagenous trimer (NC1) at the C-terminal. The repetitive Gly sequence in the collagenous domain is required for proper assembly of the collagen triple helix and the amino-acid residues in the X–Y positions are located on the outside of the triple helix.16Kawai S. Nomura S. Harano T. et al.The COL4A5 gene in Japanese Alport syndrome patients: spectrum of mutations of all exons. The Japanese Alport Network.Kidney Int. 1996; 49: 814-822Abstract Full Text PDF PubMed Scopus (56) Google Scholar The NC1 domain has an important role in heterotrimer formation,6Gubler M.C. Inherited diseases of the glomerular basement membrane.Nat Clin Pract Nephrol. 2008; 4: 24-37Crossref PubMed Scopus (92) Google Scholar,17Hudson B.G. The molecular basis of Goodpasture and Alport syndromes: beacons for the discovery of the collagen IV family.J Am Soc Nephrol. 2004; 15: 2514-2527Crossref PubMed Scopus (143) Google Scholar because the zipper-like folding mechanism of the triple helix of type IV collagen is believed to start from the C-terminal end.7Gross O. Netzer K.O. Lambrecht R. et al.Meta-analysis of genotype-phenotype correlation in X-linked Alport syndrome: impact on clinical counselling.Nephrol Dial Transplant. 2002; 17: 1218-1227Crossref PubMed Scopus (194) Google Scholar,18Bekheirnia M.R. Reed B. Gregory M.C. et al.Genotype-phenotype correlation in X-linked Alport syndrome.J Am Soc Nephrol. 2010; 21: 876-883Crossref PubMed Scopus (163) Google Scholar,19Boutaud A. Borza D.B. Bondar O. et al.Type IV collagen of the glomerular basement membrane. Evidence that the chain specificity of network assembly is encoded by the noncollagenous NC1 domains.J Biol Chem. 2000; 275: 30716-30724Crossref PubMed Scopus (178) Google Scholar We used receiver operating characteristic analysis to evaluate the distance from the NC domain of the missense mutations affecting α5(IV) expression, and found a cutoff point at exon 25 that distinguished between the two groups; mutations in the positive group were significantly more likely to be located before exon 25. A previous study analyzed the effect of mutation position on disease severity by comparing 98 glycine-substituting missense mutations between two groups with mutations located in exons 1–20 and 21–47 of COL4A5, respectively. They found that patients with mutations in exons 1–21 had less severe disease in terms of ESRD.7Gross O. Netzer K.O. Lambrecht R. et al.Meta-analysis of genotype-phenotype correlation in X-linked Alport syndrome: impact on clinical counselling.Nephrol Dial Transplant. 2002; 17: 1218-1227Crossref PubMed Scopus (194) Google Scholar Dividing our patients into the same categories in terms of mutation locations showed that patients with missense mutations located in exons 1–21 were more likely to be positive (P=0.05). The results of this study suggest that mutations located between exons 1 and 25 may lead to a less critical disruption of triple helix-forming process. Naito et al.13Naito I. Kawai S. Nomura S. et al.Relationship between COL4A5 gene mutation and distribution of type IV collagen in male X-linked Alport syndrome. Japanese Alport Network.Kidney Int. 1996; 50: 304-311Abstract Full Text PDF PubMed Scopus (76) Google Scholar reported that a point mutation, such as a Gly substitution, within the collagenous domain had no effect on the construction of the NC1 domain. Predicted minimal changes in protein structure cause late onset of ESRD. Our study indicated that positivity was related to less severe effects on urinary protein levels and older age at onset of ESRD. We also demonstrated the incidence of somatic mosaic mutations in male XLAS patients with mild phenotype. We previously reported a somatic mosaic mutation in COL4A5 in a male XLAS sufferer,3Krol R.P. Nozu K. Nakanishi K. et al.Somatic mosaicism for a mutation of the COL4A5 gene is a cause of mild phenotype male Alport syndrome.Nephrol Dial Transplant. 2008; 23: 2525-2530Crossref PubMed Scopus (24) Google Scholar and the current report showed two patients with this pattern, including a previously reported case, both of whom showed relatively mild phenotypes. This suggests that somatic mosaic mutations should be considered in male XLAS patients with mild phenotypes and mosaic α5(IV) expression. None of the α5(IV)-positive patients developed hearing loss in this study, compared with more than half of the negative-group patients (54%). There was no difference in the incidence of ocular lesions between the two groups, although this could have been because of the relatively small number of patients in this study. These results suggest that α5(IV)-positive patients exhibit milder renal and cochlear phenotypes. This study had several limitations related to its retrospective nature and the small number of patients who developed ESRD. A previous study reported a median renal survival rate of 25 years.4Jais J.P. Knebelmann B. Giatras I. et al.X-linked Alport syndrome: natural history in 195 families and genotype- phenotype correlations in males.J Am Soc Nephrol. 2000; 11: 649-657Crossref PubMed Google Scholar Patients in this study were too young to permit differences in clinical severity to be detected by estimated glomerular filtration rate. In addition, the sample size in this study was relatively small, and further studies with more patients should be conducted to confirm the current findings. In conclusion, male XLAS patients with positive α5(IV) chain expression had milder clinical manifestations than those with no α5(IV) expression. All α5(IV)-positive patients had non-truncating or somatic mosaic mutations. Furthermore, the location of missense mutations was related to differences in α5(IV) expression. All procedures were reviewed and approved by the Institutional Review Board of Kobe University School of Medicine, and consent for the study was obtained from the patients or their parents. Clinical and laboratory findings for patients with XLAS were obtained from their medical records. Patients were referred to our hospital for clinical evaluation or genetic analysis. Most of the patients were followed in various local hospitals in Japan. DNA and data sheets were sent to our lab after acceptance of the request for mutational analysis. All patients in this study were identified with disease-causing mutations in the COL4A5 gene and satisfied at least one of the following criteria: (1) male patients with proteinuria and hematuria or ESRD, whose renal pathology showed thickening and thinning with lamellation (basket-weave changes) or thin GBM by electron microscopy and total absence of α5(IV) in the GBM and BC. (2) Male patients with proteinuria and hematuria or ESRD whose renal pathology showed basket-weave changes or thin GBM by electron microscopy and positive α5(IV) expression in the GBM and BC. The degree of urinary protein excretion was evaluated by the urinary protein/creatinine ratio. Estimated glomerular filtration rate was calculated using Schwartz’s formula20Schwartz G.J. Haycock G.B. Edelmann Jr., C.M. et al.A simple estimate of glomerular filtration rate in children derived from body length and plasma creatinine.Pediatrics. 1976; 58: 259-263PubMed Google Scholar,21Schwartz G.J. Gauthier B. A simple estimate of glomerular filtration rate in adolescent boys.J Pediatr. 1985; 106: 522-526Abstract Full Text PDF PubMed Scopus (347) Google Scholar or glomerular filtration rate-estimating equations for Japanese,22Matsuo S. Imai E. Horio M. et al.Revised equations for estimated GFR from serum creatinine in Japan.Am J Kidney Dis. 2009; 53: 982-992Abstract Full Text Full Text PDF PubMed Scopus (4531) Google Scholar for patients under and over 21 years, respectively. All clinical, laboratory, and pathological data were collected when the request for mutational analysis was accepted. Images of kidney α5(IV) staining were sent to us for evaluation of the staining patterns and assessed by the same person (KN). Estimated glomerular filtration rate was measured based on the data in the data sheets. In Japan, mass urinary proteinuria screening is available for children aged 3 years, and every year from 6 to 18 years old. Information on the age at first detection of proteinuria is thus reliable. Hearing screening by audiometry is also available for children aged 6, 7, 8, 10, 12, 14, and 15 years, and information on age at detection of hearing loss is thus also very reliable. Mutational analyses of COL4A5 were carried out using the following methods: (1) PCR and direct sequencing of genomic DNA for all exons and exon–intron boundaries; (2) reverse transcription–polymerase chain reaction of mRNA and direct sequencing to detect abnormal splicing, and (3) multiplex ligation-dependent probe amplification to detect copy number variations. Genomic DNA was isolated from peripheral blood leukocytes from patients and family members using the Quick Gene Mini 80 system (Fujifilm Corporation, Tokyo, Japan) according to the manufacturer’s instructions. For genomic DNA analysis, all specific 51 exons of COL4A5 were amplified by PCR, as described previously.23Martin P. Heiskari N. Zhou J. et al.High mutation detection rate in the COL4A5 collagen gene in suspected Alport syndrome using PCR and direct DNA sequencing.J Am Soc Nephrol. 1998; 9: 2291-2301PubMed Google Scholar The PCR-amplified products were then purified and subjected to direct sequencing using a Dye Terminator Cycle Sequencing Kit (Amersham Biosciences, Piscataway, NJ) with an automatic DNA sequencer (model ABI Prism 3130; Perkin Elmer Applied Biosystems, Foster City, CA). Total RNA was extracted from blood leukocytes and/or urine sediments. RNA from leukocytes was isolated using a Paxgene Blood RNA Kit (Qiagen, Chatsworth, CA) and was then reverse-transcribed into complementary DNA using random hexamers and the Superscript III Kit (Invitrogen, Carlsbad, CA). RNA from urine sediment was isolated as described previously.24Kaito H. Nozu K. Fu X.J. et al.Detection of a transcript abnormality in mRNA of the SLC12A3 gene extracted from urinary sediment cells of a patient with Gitelman's syndrome.Pediatr Res. 2007; 61: 502-505Crossref PubMed Scopus (15) Google Scholar Complementary DNA was amplified by nested PCR using primer pairs for COL4A5 as described previously25Inoue Y. Nishio H. Shirakawa T. et al.Detection of mutations in the COL4A5 gene in over 90% of male patients with X-linked Alport's syndrome by RT-PCR and direct sequencing.Am J Kidney Dis. 1999; 34: 854-862Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar,26Nakanishi K. Iijima K. Kuroda N. et al.Comparison of alpha5(IV) collagen chain expression in skin with disease severity in women with X-linked Alport syndrome.J Am Soc Nephrol. 1998; 9: 1433-1440PubMed Google Scholar with slight modifications (sequences available on request). The PCR-amplified products were purified and subjected to direct sequencing. Immunohistochemical analyses were performed using either frozen- or paraffin-embedded sections of kidney tissue. The immunohistochemical procedure has been described previously.12Sado Y. Kagawa M. Kishiro Y. et al.Establishment by the rat lymph node method of epitope-defined monoclonal antibodies recognizing the six different alpha chains of human type IV collagen.Histochem Cell Biol. 1995; 104: 267-275Crossref PubMed Scopus (205) Google Scholar,13Naito I. Kawai S. Nomura S. et al.Relationship between COL4A5 gene mutation and distribution of type IV collagen in male X-linked Alport syndrome. Japanese Alport Network.Kidney Int. 1996; 50: 304-311Abstract Full Text PDF PubMed Scopus (76) Google Scholar,26Nakanishi K. Iijima K. Kuroda N. et al.Comparison of alpha5(IV) collagen chain expression in skin with disease severity in women with X-linked Alport syndrome.J Am Soc Nephrol. 1998; 9: 1433-1440PubMed Google Scholar The mixture of fluorescein isothiocyanate-conjugated rat monoclonal antibody against human α5(IV) chain (H53) and Texas red-conjugated rat monoclonal antibody against human α2(IV) chain (H25) was purchased from Shigei Medical Research Institute (Okayama, Japan). Their epitopes were EAIQP at position 675–679 of the α2(IV) chain, and IDVEF at position 251–255 of the α5(IV) chain.14Kagawa M. Kishiro Y. Naito I. et al.Epitope-defined monoclonal antibodies against type-IV collagen for diagnosis of Alport's syndrome.Nephrol Dial Transplant. 1997; 12: 1238-1241Crossref PubMed Scopus (55) Google Scholar Patients showing complete negativity for α5(IV) staining were classified as the negative group; all other patients were classified as the positive group, including those with normal and mosaic expression patterns. Data were expressed as mean±s.d. All calculations were made using standard statistical software (JMP version 8 package for Windows, SAS, Cary, NC). The genetic and clinical backgrounds of patients in both groups were compared using Fisher’s exact test, Wilcoxon’s test, and receiver operating characteristic analysis. A P-value of <0.05 was considered statistically significant. We gratefully acknowledge the cooperation of the attending physicians in this study: Katsumi Ushijima, Shuji Tanda, Kunio Makita, Yushi Kaneko, Yuko Tezuka, Yoshitsugu Kaku, Ken Matsuyama, Tomoko Uehara, Tomoko Kawata, Mitsuru Nakajima, Ken Nakanishi, Ryojiro Tanaka, Katsuhiko Asanuma, Hironobu Nagasako, Eihiko Takahashi, Kazunari Kaneko, Masafumi Hasui, Keisuke Sugimoto, Kunimasa Yan, Yuji Sekine, Naoko Ito, Tetsushi Inagaki, Hajime Yamazaki, Yoshimitsu Goto, Akiko Tutsumi, Tomohiro Udagawa, Koichi Kamei, Makiko Nakayama, Shuichi Ito, Mureo Kasahara, Michiko Ando, Chieko Matsumura, Toshiyuki Imazawa, Toshiaki Suzuki, Yohei Ikezumi, Kiyoshi Hamahira, Yoshinori Araki, Kenichi Satomura, Keiko Yasuda, Yasufumi Ohtsuka, Taishi Hirano, Naonori Kumagai, Kenji Ishikura, Yuko Hamasaki, Kenichiro Miura, Takashi Sekine, Hidekazu Sugiura, Junko Arai, Kayoko Saito, Masami Yoneda, Noboru Igarashi, Taishi Nagata, Koji Nagatani, Akira Mastunaga, Ryo Kadoya, Emi Sawanobori, and Mari Saito. This study was supported by a grant from the Ministry of Health, Labour, and Welfare, Japan, for Research on Rare Intractable Diseases in Kidney, and Urinary Tract (H24-nanchitou (nan)-ippan-041 to Kazumoto Iijima) in ‘Research on Measures for Intractable Diseases’ Project and Grant-in-Aid for Scientific Research (KAKENHI) from the Ministry of Education, Culture, Sports, Science and Technology (subject ID: 25893131 to Kandai Nozu), and partly presented at Kidney Week 2012. Table S1. COL4A5 mutations and family history in α5(IV)-negative group. Table S2. COL4A5 mutations, α5 expression pattern, and family history in α5(IV)-positive group. Supplementary material is linked to the online version of the paper at http://www.nature.com/ki