Spectrum of clinical features and type IV collagen a-chain distribution in Chinese patients with Alport syndrome

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1 Nephrol Dial Transplant (2006) 21: doi: /ndt/gfl394 Advance Access publication 29 August 2006 Original Article Spectrum of clinical features and type IV collagen a-chain distribution in Chinese patients with Alport syndrome Gong Wei, Liu Zhihong, Chen Huiping, Zeng Caihong, Chen Zhaohong and Li Leishi Research Institute of Nephrology, Jinling Hospital, Nanjing University School of Medicine, Nanjing, China Abstract Background. Alport syndrome (AS) is a clinically and genetically heterogeneous nephropathy. The goal of the present study is to delineate clinical characteristics and the distribution of type IV collagen chains in Chinese AS patients and to identify any a(iv)-chain expression and clinical phenotype correlation. Methods. A total of 126 biopsy-proven patients meeting immunofluorescence criteria for the diagnosis of AS were investigated retrospectively. Results. Microscope haematuria associated with proteinuria was observed as the initial symptom in 77.8% of the patients; 59.8% showed hearing impairment and 22.9% had ocular abnormalities. Renal biopsies from 118 patients revealed mesangial proliferative glomerulonephritis (61.9%) and focal and segmental sclerosis glomerulonephritis (37.3%). Ten different distribution patterns for the type IV collagen a-chains were found in the kidney; six of these are presented here for the first time. Based on renal immunofluorescence findings, 113 patients (89.7%) were classified as X-linked dominant inherited AS (XLAS) and 13 (10.3%) as autosomal recessive AS (ARAS). The XLAS group was divided into typical and non-typical subgroups according to the expression patterns for the a3(iv)-chain. Clinical phenotypes were more severe in XLAS patients than in ARAS patients and the prognosis was poorer in typical XLAS patients than non-typical XLAS patients. Conclusion. In China, the incidence of XLAS is 89.7% and 10.3% for ARAS. Chinese patients with AS have various distribution patterns of type IV collagen a-chains. The distribution pattern of type IV collagen a-chains in the kidney may correspond to the severity of the clinical phenotype. Keywords: Alport syndrome (AS); indirect immunofluorescence; phenotype; type IV collagen Correspondence and offprint requests to: Liu Zhihong, MD, Research Institute of Nephrology, Jinling Hospital, 305 East Zhong Shan Road, Nanjing, , China. zhihong@21cn.net Introduction Alport syndrome (AS) is a hereditary disease of basement membranes that manifests clinically as a progressive nephropathy variably associated with sensorineural deafness and ocular abnormalities [1]. AS results from a defect in the genes encoding for type IV collagen a-chain isoforms, a result that leads to a lack of development in the normal network typically formed by type IV collagen [2 3]. Six type IV collagen genes have been cloned, characterized and localized in pairs to three chromosomes. Mutations in the COL4A5 gene, coding for the a5 chain of type IV collagen, are responsible for the X-linked dominant form of AS [4 5] whereas the COL4A3 and COL4A4 genes, located on chromosome 2, are involved in the more rare autosomal recessive forms of the disease [6 8]. AS is clinically heterogeneous with a wide variability in the rate of progression to end-stage renal disease (ESRD), the type of glomerular basement membrane (GBM) changes, the presence or absence of deafness and other extrarenal manifestations such as ocular changes and diffuse oesophageal leiomyomatosis [9]. To date, a wide variety of mutations have been described in X-linked, autosomal or sporadic cases of AS [4 5,10 15]. Because of the high diversity in mutations, a large number of families whose members have AS, have been analysed to evaluate genotype phenotype correlations. Certain correlations between mutation and phenotype have been established [16]. In patients with nonsense or missense mutations, reading-frame shifts or large deletions, renal failure and sensorineural deafness generally develop by 30 years of age. In patients with splice-variants, exon-skipping mutations or glycine missense mutations in the collagen helix, health usually begins to deteriorate after 30 years of age, and these patients have mild or late-onset deafness [16 17]. The availability of monospecific antibodies against each of the six type IV collagen a-chains has made it possible to characterize the changes in type IV collagen expression that occur as a result of mutations in the ß The Author [2006]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please journals.permissions@oxfordjournals.org

2 Type IV collagen a-chain distribution in Chinese Alport syndrome patients 3147 COL4A3, COL4A4 or COL4A5 genes. These changes are diagnostically useful; indeed, X-linked dominant inherited AS (XLAS) and autosomal recessive AS (ARAS) may be distinguishable through an immunohistochemical analysis of renal biopsy specimens [18]. Information regarding the relationship of specific type IV collagen a-chain expression to phenotype is still being accumulated. Since gene analysis is too expensive and time-consuming to be extensively applied in clinical settings, it is necessary to first clarify the relationship between type IV collagen a-chain expression and specific phenotype in AS patients as an effort to guide a specific approach to clinical therapy. In this study, we determined the immunolocalization of the a3 (IV)-, a5 (IV)-chain distribution in the basement membranes of 126 patients with clinical- and biopsy-proven AS. As it is a valuable cohort that can serve comparisons with other published cohorts from Europe and Asia, our specific goal was to analyse the clinical features and pathological findings of Chinese AS patients and to investigate the relationship between type IV collagen a chain distribution and the phenotype in these patients. Methods Patients A total of 151 biopsy-proven patients (111 males and 40 females) with AS were recruited in this study. All these patients met the Gregory s criteria of AS [19], briefly, family history of nephritis of unexpected haematuria in a first degree relative of the index case or in a male relative linked through any numbers of females; persistent haematuria without evidence of another possibly inherited nephropathy; bilateral sennsorineural hearing loss in the Hz range; a mutation in COL4A3, 4 or 5; immunohitochemical evidence of complete or partial lack of the Alport epitope in glomerular, or epidermal basement membranes or both; widespread GBM ultrastructural abnormalities; ocular lesions; gradual progression to ESRD in the index case of at least two family members and macrothrombocytopenia or granulocytic inclusions and diffuse leiomyomatosis of oesophagus or female genitalia or both. In the following study, 126 patients (96 males and 30 females) were selected. Twenty-five patients with AS (15 males and 10 females) were not included, as their renal type IV collagen a-chain staining patterns appeared normal, and this study s aim is to investigate the relationship between abnormal type IV collagen a chain distribution and the phenotype in AS patients. creatinine (SCr), 24-h urine protein excretion and quantitative urine sedimentation] and extrarenal manifestations (including hearing loss and ocular changes) were also collected. Patients were instructed to begin the 24-h collection immediately after the first voiding in the morning, and to collect their urine for 24 h, including a final voiding at the completion of the 24-h period. Urinary protein was detected by the Biuret method (normal value <0.4 g/day). Renal pathology Light microscopy. A total of 143 patients underwent a percutaneous renal biopsy. Renal specimens were fixed in 10% neutrally buffered formalin and embedded in paraffin using routine procedures. Sections (2 mm in thickness) were stained with haematoxylin/eosin (HE), periodic acid- Schiff reagent (PAS), silver methenamine and Ledewig s trichrome. Electron microscopy. The GBM structure was evaluated using electron microscopy (EM) specimens that had been routinely prepared from renal biopsies for EM studies. Briefly, small renal tissue fragments were fixed in a cool 3.75% phosphate buffered glutaraldehyde solution (0.2 M, ph 7.2), post-fixed in 1% osmium tetroxide and embedded in Epon. For each case of the 143 patients, ultra-thin sections were observed and microphotographs of GBM were obtained using EM. Immunohistochemistry Monoclonal antibodies to the a3-chain (MAB3) and the a5-chain (MAB5) of type IV collagen were purchased from Wielisa. Fluorescence isothiocyanate (FITC)- conjugated rabbit against mouse IgG antibody was purchased from DAKO. Kidney and cutaneous tissues were snap-frozen in dry ice, cut into 4 mm sections, air-dried, fixed in cool acetone for ten minutes and rinsed with PBS (0.01 mol/lpbs, ph 7.4). MAB3 and MAB5 were used at a working dilution of 1:50. After 120-min incubation at room temperature, FITC-conjugated rabbit against mouse IgG secondary antibodies were added. Prior to the anti-a5 (IV) antibody incubation, tissues were denatured with 6 mol/l urea in 0.1 mol/l glycine/hcl buffer (ph 3.5) to unmask any hidden epitopes of the Col-IV a5-chain [20]. Approval from the ethics committee was obtained for Nanjing University, and patients gave their written informed consent before entering the study. Clinical data Clinical data consisted of gender, clinical onset age, course of disease and initial symptoms. The clinical onset was defined as the first recognition of AS symptom, including haematuria, proteinuria and hearing loss. Parameters of renal involvement [including blood urea nitrogen (BUN), serum Statistical analysis Statistical analyses were performed using SPSS software (Version 11.0). Data were expressed as mean SD and analysed using the 2 -test and paired Student s t-test. Statistical significance was assumed for P < 0.05, and very high significance levels were defined as P < 0.01.

3 3148 G. Wei et al. Results Clinical manifestations of patients with Alport Syndrome Generic data. The present study includes 126 patients and the ratio of male to female was 3.2:1. Age of clinical onset ranged from 1 to 48 years old and 62.7% were before 20 years of age. There was an obvious difference in the age of clinical onset between the two genders (P < 0.01); the average age of clinical onset for males was years; for females, years. Microscopic haematuria associated with protein uria was observed as the initial symptom in 69% of the patients. Gross haematuria was the initial symptom in 15.9% of the patients. Patients who suffered from hearing loss as an initial symptom were all male (n ¼ 9). Renal damage. Microscope haematuria associated with non-nephrotic proteinuria (defined as 24-h urine protein excretion <3.5 g) was the most common manifestation (77.8%). Nephrotic proteinuria (defined as 24-h urine protein excretion >3.5 g) was seen in 22.2% of the patients, and 88.9% of them were male patients (p < 0.01). More than one-third of the patients (37.3%) had been in early renal failure [defined as SCr>1.24 mg/dl (110 mmol/l)] when diagnosed with AS. Pathological changes of renal tissue. In 118 renal biopsies, findings by light microscopy mostly revealed mesangial proliferative glomerulonephritis (61.9%) and focal and segmental sclerosis glomerulonephritis (37.3%). All of the patients (100%) showed thickening, multilayering and fibrosis of Bowman s capsule. In periodic acid-silver methenamine (PASM)-Masson staining, 44.4% showed changes of the peripheral loop, such as thickening, multilayering, double contour and chain-like. Interstitial foam cells of tuft or sheet distribution were found in 69.5% of the biopsies. Most patients (82.2%) had thickening and atrophy of the tubular basement membrane. All the patients examined by EM showed altered GBM lesions. The characteristic lesions of the GBM were thinning and/or thickening, splitting or basket weaving of the lamina densa. Hearing loss. A total of 112 patients were given audiograms and, 67 of them (59.8%) were evaluated as abnormal. Of the 67 patients, 94% had high frequency sensorineural hearing loss. This occurred in 67.4% of the males at an average age of years and in 34.6% of the females at an average age of years. Male patients tend to suffer more than females from hearing loss (P < 0.01) as well as earlier (P < 0.01) in terms of age clinical onset age than the female patients. Ocular abnormalities Ocular defects were found in 27 of the 118 patients (22.9%), including anterior lenticonus, cataract and pigmentary changes in the perimacular region. The classic anterior lenticonus was an infrequent symptom in ocular defects with an occurrence of 14.8%. The characteristics of type IV collagen a3-, a5-chains distribution Distribution of type IV collagen a3-, a5-chains in renal tissue. Renal biopsy tissue was incubated with type IV collagen a-chain antibodies and indirect immunofluoresecence was used to detect distribution patterns. Ten different patterns were observed for type IV collagen a chain distribution in the renal tissue (Table 1, Figure 1). Patterns for the type IV collagen a chain distribution in groups A, B, C and D have been reported previously [21 25]. From groups E through J, the patterns of type IV collagen a chain distribution are reported here for the first time. The most characteristic feature of these patterns is the normal expression of the a3 (IV)-chain in the GBM and distal tubular basement membrane (TBM). Groups A and C examined by EM showed diffuse, moderate to severe basket weaving of the lamina densa, with variable thinning and/or thickening of the GBM. Group B and groups D through J showed segmental, mild to moderate basket weaving of the lamina densa, with variable thinning and/or thickening of the GBM (Figure 2). Distribution of type IV collagen a3-, a5-chains in cutaneous tissue. Out of the 118 patients who were diagnosed with AS by renal biopsy, 63 also had a cutaneous biopsy (Table 1, Figure 3). Eight other patients had the cutaneous biopsy only as they were all at the uraemia stage and could not undergo a renal biopsy. Among them, six male patients showed the absence of the a5-chain from the epidermal basement membrane (EBM). The two female patients showed a discontinuous staining of the a5 chain from the EBM. Since most males with XLAS do not show a5 (IV) expression in the EBM and female heterozygotes frequently display mosaicism of a5 (IV) staining in the EBM [18], these eight patients were diagnosed as XLAS. Evaluation of subtypes of Alport syndrome Immunohistochemistry characteristics of the type IV collagen a3-, a5-chains in AS renal tissue may aid in subtypes of AS [21 26]. Distribution of type IV collagen a3-, a5-chains in the XLAS group. The abnormalities seen in the collagen distribution of groups A, B and D displayed patterns as previously reported for type IV collagen expression in XLAS patients [22, 24 25]. Thus, the patients in these groups were classified as XLAS. Patients from groups A, E and H J, all accepted both renal and cutaneous biopsies, and all did not show type IV collagen a5-chain expression in the EBM. Male patients from groups D, E and G-J and

4 Type IV collagen a-chain distribution in Chinese Alport syndrome patients 3149 Table 1. Distribution of type IV collagen a chains in basement membranes in patients with Alport syndrome Group Renal tissue Skin tissue Subtype Familiar/ sporadic case Discontinuous (male/female) Absent (male/female) n Male/female a3 chain a5 chain n Male/female Normal (male/female) GBM dtbm GBM dtbm BC A 41 41/ /0 0 18(18/0) 0 XLAS 26/15 B 3 0/3 M M M M M 1 0/ (0/1) XLAS 0/3 C 13 9/4 þ þ 4 3/1 4 (3/1) 0 0 ARAS 7/6 D 7 5/2 þ 3 2/ (2/1) XLAS 5/2 E 8 8/0 þ þ 3 3/0 3 (3/0) 0 0 XLAS 8/0 F 3 0/3 þ þ M M M 3 0/3 2 (0/2) 0 1(0/1) XLAS 2/1 G 3 3/0 þ þ þ þ 3 3/0 1 (1/0) 0 2(2/0) XLAS 3/0 H 6 3/3 þ þ þ þ 5 3/2 0 2(2/0) 3(1/2) XLAS 3/3 I 22 12/10 þ þ þ þ 14 6/8 6 (2/4) 2(2/0) 6(2/4) XLAS 11/11 J 12 9/3 þ þ 9 6/3 3 (1/2) 1(1/0) 5(4/1) XLAS 9/3 GBM, glomerular basement membrane; dtbm, distal tubuler basement membrane; BC, Bowman s capsules;, absent; þ, normal; M, mosaic. female patients from groups B, D, F and H J showed discontinuous staining for the type IV collagen a5 chain in the EBM. Thus, groups E J also displayed characteristics of XLAS. To summarize, there were 113 XLAS patients (groups A, B and D J) in our study, with an occurrence rate of 89.7%. The distribution of type IV collagen a3 a5-chains in the ARAS group. Group C displayed patterns as previously reported for type IV collagen expression in ARAS patients [21]. Thus group C patients were classified as ARAS, with an occurrence rate of 10.3%. Clinical manifestations of AS patients in subtypes Clinical onset age. No distinct difference between genders or between XLAS and ARAS groups was observed as far as clinical onset age is concerned. In the XLAS group, the initial clinical onset age for male patients was earlier than for female patients (P < 0.01; Table 2). There did not appear to be any difference in clinical onset age between the genders in the ARAS group. Renal damage, hearing loss and ocular abnormalities. There was no significant difference in the occurrence of renal failure, hearing loss and ocular abnormalities between the ARAS or XLAS groups. For patients from the ARAS group, the initial age of renal failure was earlier than those in the XLAS group (P < 0.05). There did not appear to be a difference between the XLAS and ARAS groups when comparing the initial age for hearing loss and ocular abnormalities. In the XLAS group, male patients had a higher occurrence of renal failure and hearing loss than female patients (P < 0.05). Also, the initial age for hearing loss was earlier in males than in female patients (P < 0.05, Tables 3 and 4). Different clinical phenotype of patients with X-linked Alport syndrome Although all of the genotypes from groups A, B and D J were classified as XLAS, the distribution of the type IV collagen a3-, a5-chains of these patients was inconsistent. These differences may reflect different gene mutations of type IV collagen and could, therefore, affect clinical manifestations and prognosis. Based on the distribution patterns of the type IV collagen a3- a5-chains in these patients, we made further comparisons by grouping the patients into various classifications. According to their distribution of type IV collagen a3- a5-chains (Table 1), group A patients were designated as typical male XLAS; group B as typical female XLAS. The distribution pattern of the type IV collagen a5-chain in the skin of the typical XLAS group was absent in male patients and mosaic in female patients. Therefore, patients from groups A and B, as well as the eight patients that underwent

5 3150 G. Wei et al. Fig. 1. Immunohistochemical distribution of type IV collagen a3 and a5 chains in patient and control kidneys (400). Control: anti-a3(iv) antibodies stained the GBM and dtbm; anti-a5(iv) antibodies stained the GBM, dtbm and BC. Group A: anti-a3(iv) antibodies show an absence of GBM and dtbm staining; anti-a5(iv) antibodies indicate an absence of GBM, dtbm and BC staining. Group B: anti-a3(iv) antibodies mosaic stained the GBM and dtbm; anti-a5(iv) antibodies mosaic stained the GBM, dtbm and BC. Group C: anti-a3(iv) antibodies show an absence of GBM and dtbm staining; anti-a5(iv) antibodies show an absence of GBM staining. Group D: anti-a3(iv) antibodies indicate an absence of GBM and dtbm staining; anti-a5(iv) antibodies show an absence of GBM and dtbm staining. Group E: anti-a3(iv) antibodies stained the GBM and dtbm; anti-a5(iv) antibodies revealed an absence of GBM, dtbm and BC staining. Group F: anti-a3(iv) antibodies stained the GBM and dtbm; anti-a5(iv) antibodies mosaic stained the GBM, dtbm and BC. Group G: anti-a3(iv) antibodies stained the GBM and dtbm; absence of GBM staining is shown with anti-a5(iv) antibodies. Group H: anti-a3(iv) antibodies stained the GBM and dtbm; anti-a5(iv) antibodies show an absence of dtbm staining. Group I: anti-a3(iv) antibodies stained the GBM and dtbm; absence of BC staining is shown with anti-a5(iv) antibodies. Group J: absence of dtbm staining is shown with anti-a3(iv) antibodies; anti-a5(iv) antibodies show an absence of dtbm and BC staining. a single cutaneous biopsy, were defined as a typical XLAS group. There were 52 patients (47 males and 5 females) in this group. At the same time, groups D J were classified as non-typical XLAS groups. Sixty-one patients (40 males and 21 females) were included in this group. Initial age. The initial age of clinical onset in patients from the typical XLAS group was earlier than in the non-typical XLAS group (P < 0.01; Table 5). The age of clinical onset in the male patients from the typical XLAS group was earlier than the age observed in the males from the non-typical XLAS group (P < 0.01). Renal damage. There was no difference between the two groups in the occurrence of renal failure. In the non-typical group, male patients had a higher occurrence of renal failure and hearing loss than female patients (P < 0.05). The onset age of renal failure in the typical XLAS group was earlier than in the non-typical XLAS group (P < 0.01). The onset age of renal failure for the male patients was earlier in the typical XLAS group than in the non-typical XLAS group (P < 0.01). In both the groups, the onset age of renal failure in male patients was earlier than in female patients (P < 0.05, Tables 6 and 7).

6 Type IV collagen a-chain distribution in Chinese Alport syndrome patients 3151 Fig. 2. Immunohistochemical distribution of type IV collagen a5-chain in skin. (A) Anti-a5(IV) antibodies reveal linear staining in normal control EBM. (B) Anti-a5(IV) antibodies show discontinuous staining of the EBM in a XLAS female patient. (C) Staining of the EBM was not observed with anti-a5(iv) antibodies in an XLAS male patient. (400) Hearing loss. Patients of the typical XLAS group had a higher occurrence (P < 0.05) and earlier initial age (P < 0.01) of hearing loss than those in the non-typical XLAS group. In the typical XLAS group, male patients were more inclined to be involved with hearing loss than the female patients (P < 0.01). The occurrence of hearing loss of the male patients was higher in the typical XLAS group than non-typical group (P < 0.05). There was an obvious difference between the male patients from these two groups in terms of the initial age of hearing loss (P < 0.01, Tables 6 and 7). Ocular abnormalities. The occurrence of ocular abnormalities was higher in the typical XLAS group than in the non-typical XLAS group (P < 0.01). The occurrence of ocular abnormalities was higher in both the male and female patients of the typical XLAS group than in the non-typical XLAS group (P < 0.01). The initial age of ocular abnormalities was earlier in the typical XLAS group than in the non-typical XLAS group (P < 0.01). There was an obvious difference between the male patients from these two groups Fig. 3. Electron micrographs of the GBM from patients with AS showing diffuse, moderate to severe basket weaving of the lamina densa (groups A and C) and segmental, mild to moderate basket weaving of the lamina densa (groups B and D). Table 2. Comparison of onset age between patients with different inheritance modes Years XLAS group ARAS group Male (n) * (87) (9) Female (n) (26) (4) Total (n) (113) (13) Values are expressed as mean SD. P < 0.01 vs XLAS female patients. in the initial age of ocular abnormalities (P < 0.01, Tables 6 and 7). Discussion In the present study, we report on the clinical features and pathological findings in a large cohort of subjects with AS in China. The most common initial symptom of Chinese AS patients was microscope haematuria associated with proteinuria. Jais et al. [17] showed that simple microscope haematuria was an initial symptom of European patients. Pajari H et al. [27] showed that, in Finland, the first sign of the disease was most often haematuria, but sometimes proteinuria or hearing loss. Examination in late disease stage might be used as an explanation for this difference from the European patients, but we also found that some patients had proteinuria as their initial symptom without haematuria [28 29]. The occurrence of hearing loss and ocular defects in Chinese AS patients (59.8% with hearing impairment and 22.9% with ocular abnormalities) was higher than European patients (28% hearing impairment and 15% ocular abnormalities) [9] but lower than Indian patients (82.5% hearing impairment and 66.7% ocular abnormalities) [30]. Whether or not there is any difference between races or if perhaps some special

7 3152 G. Wei et al. Table 3. Comparisons in the occurrence of renal failure, hearing loss and ocular abnormalities between patients with different inheritance modes Renal failure Hearing loss Ocular abnormalities n % n % n % XLAS group Male 39/ * 53/ * 22/ Female 5/ / / Total 44/ / / ARAS group Male 2/ / / Female 1/ / /4 0 Total 3/ / / *P < 0.05 vs XLAS female patients. Table 4. Age comparisons for renal failure, hearing loss and ocular abnormalities between patients with different inheritance modes Renal failure Hearing loss Ocular abnormalities n (years) n (years) n (years) XLAS group Male # Female Total * ARAS group Male Female Total Values are expressed as mean SD. *P < 0.05 vs ARAS group; # P < 0.05 vs ARAS male patients; P < 0.05 vs XLAS female patients. Table 5. Age comparison at onset between patients in typical and non-typical XLAS groups Years Typical Non-typical Male (n) (47)* (40) Female (n) (5) (21) Total (n) (52)** (61) Values are expressed as mean SD. *P < 0.01 vs male patients in non-typical XLAS group; **P < 0.01 vs non-typical XLAS group. gene mutation causes the corresponding symptoms needs to be studied further. Consistent with the Western report [31 32], our light microscopy findings of renal biopsies mostly revealed mesangial proliferative glomerulonephritis and focal and segmental sclerosis glomerulonephritis. Most of the patients showed thickening, multilayering and fibrosis of the Bowman s capsule.interstitial foam cells were commonly found. GBM abnormalities observed by electron microscopy were specific with thickening, splitting and fragmentation of the lamina densa. Based on the immunohistochemistry characteristics of type IV collagen a-chains in different AS genotype groups, the inherited types of 126 patients with AS have been defined. A total of 113 patients (89.7%) were diagnosed with XLAS and 13 patients (10.3%) with ARAS. In the present study, the XLAS and ARAS patients revealed different characteristics in terms of clinical manifestations. The XLAS patients showed obvious differences between genders. Male patients tended to have an earlier onset age and higher percentages for renal insufficiency than the female patients. In the ARAS patients, there was no gender preference evident. The reason is likely that the XLAS heterozygous female patients have a normal COL4A5 allele. XLAS patients had more severe clinical manifestations than the ARAS group. All the patients who had renal insufficiency as the initial symptom were XLAS patients. The percentage of XLAS patients with renal insufficiency, hearing loss and ocular abnormalities was also higher than ARAS patients. The interstitial foam cells were excessive in XLAS patients. AS has various distribution patterns for the type IV collagen a-chains. The distribution pattern and degree of expression of type IV collagen a-chains in the kidney may correspond to the severity of the clinical phenotype; for different gene mutations, location of type IV collagen resulted in different gene productions. In this study, we found obvious differences in the severity of the disease and ultrastructure changes between the typical and non-typical XLAS patients

8 Type IV collagen a-chain distribution in Chinese Alport syndrome patients 3153 Table 6. Comparison for the occurrence of renal failure, hearing loss and ocular abnormalities between patients in typical and non-typical XLAS groups Renal failure Hearing loss Ocular abnormalities n % n % n % Typical Male 21/ / ** # 17/ x Female 2/ /3 0 2/ xx Total 23/ /44 75 ## 19/ ## Non-typical Male 18/ * 20/ / Female 3/ / /19 0 Total 21/ / / *P < 0.05 vs non-typical XLAS female group; **P < 0.01 vs typical XLAS female group; # P < 0.05 vs non-typical XLAS male group; ## P < 0.01 vs non-typical XLAS group; x P < 0.01 vs non-typical XLAS male group; xx P < 0.01 vs non-typical XLAS female group. Table 7. Comparison of age of time of renal failure, hearing loss and ocular abnormalities between patients in typical and non-typical XLAS groups Renal failure Hearing loss Ocular abnormalities n (years) n (years) n (years) Typical Male ** x ** ** Female / Total xx xx xx Non-typical Male # Female / Total Values are expressed as mean SD. **P < 0.01 vs male patients in non-typical XLAS group; x P < 0.05 vs female patients in typical XLAS group; xx P < 0.01 vs non-typical XLAS group; # P < 0.05 vs female patients in non-typical XLAS group. who were grouped based on the different distribution pattern of type IV collagen a-chains. The typical XLAS patient had a more severe prognosis and clinical manifestations than the nontypical patients. The onset age of renal insufficiency and hearing loss in typical XLAS patients was earlier than in non-typical XLAS patients. The occurrence of ocular abnormalities was distinctly higher in typical XLAS patients. Group A, as the typical XLAS patients, showed more severe GBM lesions in EM than the non-typical XLAS patients. Also compared with the typical XLAS patients, discontinuous rather than uniform lack of staining of EBM for a5-chain occurred in non-typical XLAS patients. Previous research indicated that the absence of the type IV collagen a3-chain in glomerular basement membranes showed more severe renal pathological changes in AS patients [16,33]. In the present study, the main difference observed between typical and nontypical XLAS patients, as far as the absence of any of the type IV collagen a-chains, was whether or not they had normal expression of the type IV collagen a3-chain. AS gene mutation sites may directly influence clinical manifestations in patients [32], a finding that reveals different sites and different degrees of gene mutation corresponds to different distribution patterns of type IV collagen a-chains. Indeed, we found that different distributions of type IV collagen a chains corresponded to different clinical manifestations. Immunolocalization analysis for the type IV collagen a chains is a simple technique with a straightforward interpretation that can be accomplished on frozen kidney specimens or even on a simple cutaneous biopsy. This type of analysis could be very useful in not only diagnosing AS but also enabling researchers to determine the mode of transmission of AS. To exclude the misclassification by using this technique, gene diagnosis is a useful approach. But unfortunately, due to the large size of AS associated genes and the high number of polymorphisms, mutation screening is not currently available for performing molecular diagnosis of AS [34]. Conflict of interest statement. None declared. References 1. Alport AC. Hereditary familial congenital haemorrhagic nephritis. Br Med J 1927; 1:

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