Detection of COL4A5 gene mutations in Chinese patients with Alport's syndrome

Xiaoxia Pan, Jingyin Yan, Hong Ren, Wen Zhang, Hao Shi, Haijin Yu, Chaohui Wang, Cuilan Hao, Xiaonong Chen and Nan Chen

Department of Nephrology, Ruijin Hospital, Shanghai Second Medical University, Shanghai, People's Republic of China

Correspondence and offprint requests to: Nan Chen, Department of Nephrology, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, People's Republic of China. Email: chennan_rj{at}yahoo.com.cn



   Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. Mutations in the COL4A5 gene, encoding the {alpha}5 chain of type IV collagen, are responsible for X-linked Alport's syndrome (XLAS), a progressive nephropathy characterized by glomerular basement membrane abnormalities and usually associated with progressive hearing loss and ocular lesions.

Methods. In this study, we analysed all 51 exons of the COL4A5 gene in 20 Chinese patients with XLAS or suspected XLAS from 16 families by using polymerase chain reaction (PCR)–denaturing gradient gel electrophoresis (DGGE) DNA sequencing.

Results. Five gene mutations identified in five families were considered to be pathogenic, including one nonsense mutation in exon 1 (266C->T, Gln22Term), two missense mutations in exons 31 (2757G->T, Gly852Val) and 43 (4142C->T, Pro1314Ser), and two splice site mutations in introns 1 and 25 just next to the 3' end of their respective exons (283+1G->T, 2150+1G->T). According to GenBank, these five mutations have not been reported previously. All male patients have typical clinical manifestations and pathological findings that closely correspond to the effects of the mutations. Furthermore, seven gene polymorphisms were detected in introns 18 and 10 and exons 20, 27, 29, 39 and 46. Only the substitution in intron 18 (1234+25G->A) had a gene frequency significantly higher in patients than in normal individuals.

Conclusion. Our study demonstrated the critical role of COL4A5 gene mutations in the pathogenesis of XLAS. The linkage of the polymorphism to AS is still unknown.

Keywords: Alport's syndrome; COL4A5gene; gene polymorphism; type IV collagen



   Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
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Alport's syndrome (AS) is a progressive hereditary nephritis clinically characterized by haematuria and progressive renal failure with or without sensorineural deafness and ocular lesions. In addition, electron microscopy shows abnormalities in the glomerular basement membrane (GBM) consisting of diffuse thinning, thickening or splitting of its structure [13]. AS is genetically heterogenous [4]. Its most common form, X-linked AS (XLAS), estimated to affect 1 in 5000 males worldwide, is caused by mutations in the X-chromosomal COL4A5 gene, the gene encoding the {alpha}5 chain of type IV collagen [3,5,6]. It comprises ~250 kb of genomic DNA and contains 51 exons [7]. To date, > 300 different mutations in the COL4A5 gene have been reported in Europe, the USA, Japan and elsewhere [6,817], but few in China. The aim of this study was to investigate the characteristic features of mutations of the COL4A5 gene in Chinese patients with AS.



   Materials and methods
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 Materials and methods
 Results
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Patients and controls
From our department, 20 Chinese AS patients (10 males and 10 females) belonging to 16 families were enrolled in this study. The diagnosis of AS was suspected based on the existence of the following two criteria: (i) haematuria or proteinuria, or both, progressive renal failure with or without sensorineural hearing loss or ocular lesions; and (ii) an obvious family history; and if a patient also had one of the two following abnormalities: (i) characteristic ultrastructual changes of the GBM; and (ii) the characteristic change of the immunofluorescence staining of {alpha} chains of type IV collagen in the renal and epithelial basement membranes. Overall, five patients fulfilled four of the criteria, 10 fulfilled three of the criteria and five only the first two criteria. Of the 20 patients, all had microscopic haematuria (five macroscopic haematuria). Proteinuria was present in 17 patients, among them two with nephrotic syndrome. Of the 20 patients, 10 (seven males, three females) had chronic renal failure at an average age of 29.7 years; 13 patients developed sensorineural deafness; and three had anterior lenticonus. We examined tissues from nine patients by electron microscopy. Diffuse thinning and thickening of the GBM was observed in all renal specimens, and GBM lamellation was found in six. Immuno-histochemical study of type IV collagen chains in epithelial basement membranes (EBMs) or GBM, or in both, was performed on 18 patients [using monoclonal anti-{alpha}1,3,5(IV) chain antibodies, Wieslab Co., Sweden]. The absence of the {alpha}5(IV) chain in the EBM was observed in six male patients, and concomitant absence of {alpha}3,5(IV) chains in the GBM was observed in four. Discontinuous expression of {alpha}3,5(IV) chains was observed in five female patients. In seven patients, the expression of type IV collagen chains was normal.

For control, we used 100 genomic DNA samples extracted from 100 unrelated normal persons.

PCR–DGGE analysis and direct sequencing
All COL4A5 coding exons were amplified by polymerase chain reaction (PCR) using 49 flanking intronic primers. The primers were designed based on the the sequences reported by Zhou et al. [7] and Martin et al. [13] (Table 1).


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Table 1. Oligonucleotide primers and PCR–DGGE characteristics

 
PCR products were screened by denaturing gradient gel electrophoresis (DGGE) analysis. The gels were prepared with a vertical linear denaturant gradient from 30–40% (top) to 60–80% (bottom). The 100% denaturant stock solution contained 7.5% acrylamide, 40% fornamide and 7 M urea in TAE buffer and the 0% denaturing solution contained 7.5% acrylamide in TAE buffer. To prepare the gradient gel, 15 ml of each denaturant, mixed with 80 µl of ammonium persulfate and 12 µl of TEMED, was poured into the gradient maker. The PCR samples were electrophoresed at an appropriate temperature, determined according to the melting map of each PCR product analysed with Macmelt software (Biorad Co.). The characterisitcs of PCR and DGGE are shown in Table 1.

The genomic DNA from individuals with mobility shift was studied by an automated direct sequencer (Applied Biosystems).



   Results
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 Materials and methods
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Gene mutations and gene polymorphisms
We identified 12 nucleotide variants, including five gene mutations and seven gene polymorphisms (Table 2).


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Table 2. Characteristis of nucleotide variants in the COL4A5 gene

 
Five gene mutations found in seven patients from five families were considered to be pathogenic. They included one nonsense mutation, two missense mutations and two splice site alterations.

One nonsense mutation, a change from C to T of the first base of codon 22 in exon 1, resulted in a truncated protein without an NC1 domain.

One missense mutation in exon 31, a single G->T transversion, resulted in the substitution of glycine by valine at codon 852. A substitution of glycine in the COL4A5 gene is thought to be a pathogenic mutation. This change was detected in one male patient. The search by immunofluorescence microscopy for the {alpha}3,5 chains of type IV collagen within his epithelial and renal basement membranes found no staining. The other missense mutation was found in exon 43 (4142C->T, Pro1314Ser), which was identified in a male proband, and in his mother as well as his younger brother. The absence of staining with anti-{alpha}3,5(IV) chain antibodies was detected in the proband and his brother, and discontinuous deposits in his mother, indicating that this codon change was an inherited one.

Two splice site mutations were detected, one each in introns 1 and 25; a change from G to T at the first base of the splice site sequence of intron 1 (283+1) and intron 25 (2150+1), in a male and a female patient, respectively. In both, the detection patterns of the changes of {alpha}3,5(IV) chains were typical (negative in the male patient and discontinuously positive in the female).

Clearly, gene polymorphisms are seven nucleotide substitutions, because they were also found in control DNA. Of these, five were located in exons 20, 27, 29, 39 and 46; four were silent substitutions, and the remaining one was an isoleucine to serine substitution in the collagenous domain. There is not a statistically significant difference in gene frequency between the patients and normal subjects. Two other gene polymorphisms were detected in introns 10 and 18 (Table 2). In patients, the gene frequency of 1234+25G->A in intron 18 was significantly higher than in controls. In addition, Ile444Ser [10,1416], Pro783Pro [14,16], Gln1171Gln [10,1416], Asp1425Asp [1416] and 811+21T->C [11] have been reported by others. Our findings have further confirmed that these nucleotide variants are gene polymorphisms.

Phenotypes in individuals carrying COL4A5 mutations
Among seven patients (five patients, three males and two females, with XLAS; and two patients, one male and one female, with suspected XLAS) from five families, four were males and three females. DGGE shows that these males patients were homozygous, while the females were heterozygotes. Four males from three families presented with haematuria and varying amounts of proteinuria, and one had nephrotic syndrome; three of them had renal insufficiency (serum creatinine from 144 to > 500 µmol/l). Sensorineural hearing loss was found in two of them, and one had anterior lenticonus. We obtained renal specimens from three male patients. All specimens manifested variable thickening, thinning and lamellation of the GBM under electron microscopy. On the other hand, of the three heterozygote female patients, who carried one nonsense mutation, one missense mutation and one splicing mutation, two had isolated microhaematuria and one had microhaematuria and a small amount of proteinuria (0.4 g/24 h). As a whole, renal function was normal in all female patients. Only one had sensorineural hearing loss (Table 3).


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Table 3. Mutations in the COL4A5 gene and major clinical features

 


   Discussion
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 Materials and methods
 Results
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Many groups have detected COL4A5 gene mutations in patients of different ethnic origins with AS [6,818], but reports on Chinese patients with AS are rare. We studied all 51 exons of the COL4A5 gene in 20 Chinese patients with AS from 16 AS families, and discovered five gene mutations and seven gene polymorphisms.

Until now, > 300 COL4A5 gene mutations have been reported [6,818]. Most of them are small mutations, such as a single nucleotide transversion, small insertion or a deletion. No single commonly shared mutation was found. The mutations are scattered along almost the entire gene. In our study, five mutations were located in four different exons and among five unrelated families, similar to the results obtained by other researchers. To our knowledge, based on data from GenBank and the literature, none of the five gene mutations has been reported before.

Clinical and molecular analyses show that the clinical features of male XLAS patients closely correspond to the mutations (Table 3). All four male patients have typical clinical manifestations—haematuria and proteinuria, and nephrotic syndrome in one. Of the male patients, 75% had renal insufficiency between the ages of 13 and 23 years. At the same time, clinical findings in the female patients are atypical, and cannot be correlated with the mutations. The one female patient carrying the nonsense mutation only had microhaematuria and a small amount of proteinuria; her renal function was normal and she had no hearing loss or ocular lesions. The immunohistochemical examination of her type IV collagen chains was also normal. DGGE showed that all female patients were heterozygotes. The same variation between genotype and phenotype has been reported by another group [10], but its mechanism is still unknown. It may be associated with X chromosome inactivation; however, no direct evidence has verified this hypothesis. Therefore, many female patients were difficult to diagnose on the basis of clinicopathological findings. There are no obvious differences between the clinical features of patients with and without gene mutations.

Compared with normal genomic DNA, we found seven gene polymorphisms, of which five have been described before [10,1416,18]. Within the seven gene polymorphisms, with the exception of four silent changes, only the substitution in intron 18 (1234 + 25G->A) has a gene frequency significantly higher in patients than in normal persons. Therefore, Ile444Ser and 811+21T->C may have no direct influence on AS. The significance of changes within the intron is not clear at present; the correlation between the substitution of G by A at the 25th base of the splice site of intron 18 and the occurrence of AS is not definite.

We found that the 12 nucleotide variants were scattered among all 16 families; but excluding the gene polymorphisms, mutations are detected in 31.3% of families with a definite or suspected diagnosis of XLAS. Renieri et al. reported that a causative mutation was found in 60/201 (29.9%) of AS patients. Excluding the doubtful patients, the occurrence of mutations was 45% [11]. In our study, only 11 families had a certain diagnostic score. Therefore, the mutation rate in these families was 45.5%, similar to the results of previous studies by single-strand conformation polymorphism (SSCP) analysis [8,10,14,18].

Our failure to detect mutations in > 50% of families can be ascribed in part to technical limitations. PCR DNA direct sequencing may increase the detection rate. Furthermore, our analysis focused on the coding and neighbouring non-coding regions. While the COL4A5 gene is a large gene of ~250 kb, its exon size is only just over 5 kb, which indicates that most nucleotides are located in non-coding regions [7]. It is reported that ~10–16% of AS is caused by gene rearrangement and a large deletion [9,17]. DGGE analysis is a measurement used to detect small gene variations, and cannot detect large gene mutations.



   Acknowledgments
 
We thank Professor C. E. Kashtan for his generosity in donating partial anti-{alpha}3,5(IV) chain antibodies, and the molecular centre of our hospital and Professor Huang Wei for help in DGGE screening. We are grateful to the patients, families and the physicians who have contributed to this study. This study was supported by the Shanghai Educational Administration and the Shanghai Board of Health.

Conflict of interest statement. None declared.



   References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received for publication: 23. 4.03
Accepted in revised form: 20.11.03





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