Saudi Journal of Kidney Diseases and Transplantation

: 2008  |  Volume : 19  |  Issue : 5  |  Page : 767--774

Clinical and Genetic Mapping of X Chromosome in the X-linked Dominant Inherited Alport's syndrome

Yong Dai1, Yuanshuai Huang2, Xiaolei He1, Shayan Wang1, Ruifang Huang1, Min Tang2, Chenxiao Hu1,  
1 Shenzhen People’s Hospital, Guangdong Province, China; The Second Clinical Medical College of Jinan University, Chongqing, China
2 Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education, Chongqing Medical University, Chongqing, China

Correspondence Address:
Yong Dai
The Clinical Medical Research Center of Shenzhen People’s Hospital, 1017# North Road of Easy Door, Shenzhen, Guangdong Province, 518020


To study the hereditary mode and clinical characteristics and detect mutations of gene COL4A5 encoding type IV collagen a5 chain among family members of an X-linked dominant inherited Alport«SQ»s syndrome (AS) family of China, we studied all of 38 family members of whom 2 volunteers underwent renal biopsy. Genomic DNA from all members of the AS family was characterized. All of 51 exons of COL4A5 gene were amplified by polymerase chain reaction (PCR) with the primers synthesized according to the published flanking intervening sequences. PCR products were further analyzed by agarose gel electrophoresis and single strand conformation polymorphism (SSCP) analysis. The study subjects revealing polymorphism by SSCP analysis were directly sequenced. Suspected exons were analyzed with reverse sequencing. Six males and 9 females of the family were diagnosed to have AS by clinical manifestations, family history and/or renal biopsy. Four patients died of end-stage renal disease (ESRD), and 1 patient received kidney transplantation. In the rest of the family members renal function remained normal, however, 22 (58%) revealed hematuria, 11/22 (59%) of them also had proteinuria. The hearing loss was detected in 6 (16%) and ocular lesion in 20 (53%) of family members. By PCR-SSCP analysis, 17 PCR products were identified with different mobility of single strand DNA in volunteers and 9 suspected mutations were revealed with DNA sequencing analysis, but all of which could not be proven by bidirectional sequencing analysis. We conclude that the incidence of hematuria and ophthalmopathy is higher in the X-linked dominant inherited AS in this Chinese family, while some patients have isolated hematuria. Bidirectional sequence analysis should be taken to identify mutations of certain genes. No mutations were found on the region of exons of gene COL4A5.

How to cite this article:
Dai Y, Huang Y, He X, Wang S, Huang R, Tang M, Hu C. Clinical and Genetic Mapping of X Chromosome in the X-linked Dominant Inherited Alport's syndrome.Saudi J Kidney Dis Transpl 2008;19:767-774

How to cite this URL:
Dai Y, Huang Y, He X, Wang S, Huang R, Tang M, Hu C. Clinical and Genetic Mapping of X Chromosome in the X-linked Dominant Inherited Alport's syndrome. Saudi J Kidney Dis Transpl [serial online] 2008 [cited 2021 Sep 22 ];19:767-774
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Full Text


Alport's 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 a­chain isoforms necessary for proper development of the basement membranes. [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],[7],[8]

AS is clinically heterogeneous with a wide variability in the rate of progression to end­stage renal disease (ESRD), the type of glome­rular basement membrane (GBM) changes, the presence or absence of deafness and other extra-renal manifestations such as ocular changes and diffuse esophageal leiomyomatosis. [9]

To date, a wide variety of mutations have been described in X-linked, autosomal or spo­radic cases of AS. [4],[5],[10],[11],[12],[13],[14],[15] Because of the high diversity in mutations, a large number of fa­milies whose members have AS, have been analyzed 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 a­chains has made it possible to characterize the changes in type IV collagen expression that occur as a result of mutations in the 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.

According to a study of 126 biopsy-proven AS patients in China; [19] the incidence of X­linked dominant inherited AS was 89.7% and 10.3% for autosomal recessive AS. Chinese patients with AS have various distribution pa­tterns of type IV collagen a-chains. The distribution pattern of type IV collagen a­chains in the kidney may correspond to the severity of the clinical phenotype.

In this study, we used PCR-SSCP, sequence analysis and other methods to study the mu­tation of virulence gene of a large X-linked dominant inherited AS family of China, to explore the mutation pattern and search for new mutation type. The results may help to improve the gene diagnosis, prevention and treatment of AS and also lead to the develop­ment of gene therapy of AS.

 Patients and Methods

In this study, we describe a large Chinese family with progressive hemorrhagic nephro­pathy and sensorineural hearing loss. 38 mem­bers of this family were studied [Figure 1]. Two volunteers underwent renal biopsy that was evaluated by histopahological electronic microscopic examination. Each person was subjected to a physical examination. A quali­tative urinalysis for hematuria and proteinuria was followed by quantitative urinalysis and renal function tests. Hematological tests, oph­thalmologic examinations, hearing test, choles­terol monitoring and genetic analysis were also performed on all studied subjects.

The criteria for the diagnoses of AS were recommend by Flinter, [20] (fulfill three of the following four criteria) positive family history of hematuria or chronic renal failure; electron microscopic evidence of AS on renal biopsy; progressive high-tone sensorineural deafness; and AS characteristic eye signs. Family mem­bers were considered affected, only if he/she had microscopic hematuria after exclusion of urinary infection and renal tumor.

All participants signed an informed consent according to the recommendations of the Kid­ney Disease Study Institute of the Chinese Academy of Medical Sciences ethical review committees, which approved the study.

Family survey and diagnostic tests were per­formed on all 38 family members. DNA samples were extracted from peripheral blood leuko­cytes (QIAGEN Kit) from each of the definite AS patients.

All of 51 exons of COL4A5 gene were am­plified by polymerase chain reaction (PCR) with the primers synthesized according to the published flanking intervening sequences.

The PCR amplification reactions were carried out in a reaction volume of 50 µL with 0.4~0.8 µg of genomic template DNA, 1 µmol/L of pri­mers, 400 µmol/L of D-nucleoside 5'-triphos­phate (dNTP), concentration of MgCl2 varied for different exons, and 2.5U Taq DNA-poly­merase (MBI, Fermentas). The PCR conditions for each primer pair are described in [Table 1] for exons of COL4A5, respectively.

At the first 5 cycles, samples were denatured for one minute at 94°C, followed by 30-second annealing at 68°C, 66°C, 64°C, 62°C, 60°C respectively and 5 minutes extension at 72°C. After 30 cycles samples were denatured for one minute at 94°C, 30-second annealing at 39.1~54.7° C [Table 1], and 45–second exten­sion at 72°C. Before performing DNA se­quencing, the length of the PCR fragments was checked using a standard 2% agarose gel.

All COL4A5 coding exons were amplified by PCR using primers selected with the PRIMER 5.0 program. The 3' ends of PCR primers were located between 15 and 26 bp from the exon boundary. PCR products were screened by single strand conformation polymorphism (SSCP) analysis using Hoefer Automated Gel Stainer and silver staining (Pharmacia Biotech, Hoefer Automated Gel Stainer). Samples with diffe­rent mobility were further tested by direct automated sequencing (Applied Biosystems, 377 DNA sequence analyzer). By PCR, we amplified all exons that demonstrated band shifts, and products were used for sequencing.


Pedigree analysis

This pedigree consisted of four generations in­cluding 45 members (24 males and 21 females females and no spouse included except I1). Seven members had died 4 of who died of end stage renal disease (ESRD). 38 members were still living. One patient had kidney trans­plantation. Six males and 9 females met clini­cal criteria for the diagnoses of AS. Another 4 candidates had only hematuria/proteinuria and no progressive high-tone sensorineural deafness or ocular characteristics. This family showed a serial passage pattern. Patients were scattered in each generation with more affected females and males were more symptomatic.

Children (both gender) of an AS mother were likely to be affected also. Daughters of an AS father were more likely to be affected than the sons. The heredity pattern thus followed the X­linked dominant inherited AS pedigree [Figure 1].

Clinical Features

Among eleven positive patients alive: one was hypertensive; four had dysaudia; ten had visual disturbance (naked visual acuity was lower than 0.4); ten had hematuria and 7 had proteinuria. Only one patient developed renal failure and had kidney transplantation.

PCR Amplification of Exon Regions in COL4A5

51 exons in COL4A5 were identified, and exons that were too short or too close to their neighbour were PCR amplified and 46 PCR products were obtained [Table 1]. The regions around these 46 exons at the 5' end of COL4A5 were sequenced from the DNA of all of the 2 propositus. The sequences flanking the exons and the primers used for PCR ampli­fication and DNA sequencing are shown in [Table 1].

SSCP Analysis

SSCP results of all the exons allowed the de­tection of multiple band shifts. The exons numbered 9, 21, 24, 25, 27, 28, 29, 31, 32, 35, 39, 40, 41, 42, 45, 48 and 50 were identified with different mobility of single strand DNA

Direct DNA Sequencing

For one-way DNA sequencing, exons num­bered 9, 28, 31, 32, 35, 40, 45, 50 and 48 were suspected to have mutations, [Table 2], how­ever, reverse DNA sequencing did not confirm this. No evidence was found to identify muta­tions in COL4A5 gene.


Alport's syndrome (AS) is an inherited prog­ressive kidney disease first described by Hurst and Guthrie almost a century ago. The main clinical symptoms are hematuria, sensorineural hearing loss, ocular lesions, and pathological changes in GBM. The criteria [20] for clinical diagnosis in patients with hematuria or renal failure require that the patients have three of following four criteria:

positive family history of hematuria/chronic renal failure;electron microscopic evidence on renal biopsy;progressive high-tone sensorineural hearing loss; andcharacteristic eye signs.

The draw back of these criteria is that patients with only kidney lesion or hearing loss may be missed. On the basis of these diagnostic cri­teria, 11 (28%) of our family members can be definitely diagnosed as AS. Hearing loss and characteristic eye signs are not obvious at birth, and may progress at highly variable rate with age. Family members III1, III4, III5 and IV2 in this Chinese family had long-time he­maturia and or proteinuria, without any evi­dence of hearing loss and eye signs. We there­fore suggest that physicians should consider any patient with hematuria as a potential AS patient if he/she also has hearing loss, GBM changes, or a family history of hematuria or renal failure.

Type IV collagen is a special type of collagen found only in basal laminae with triple-helical isoforms consisting of six genetically distinct chains, designating a1–a6 forming several com­binations of trimers. This disease has two forms, X chromosome-linked associated with muta­tions in COL4A5 gene and autosomal rece­ssive associated with mutations in COL4A3 or COL4A4 genes. The heredity manner of AS is heterogenous and X-linked dominant AS is the commonly described heredity manner. Our subjects coincided with the regularity of X­linked dominant inherited AS pedigree on the whole.

The COL4A5 gene has 51 exons and is about 160 Kb in length. Until now, numerous muta­tions in COL4A5 of AS patients have been characterized (missense mutation, deletion; small or large gene insertion or rearrangements; and even complete deletion of the gene). [21],[22] The dominant nature of COL4A5 mutations and the different clinical manifestations can be explained by the integration of the altered collagen chain in the final network, thereby disturbing normal structure and functioning.

The random distribution of these mutations, nevertheless, might result in imprecise geno­type and phenotype manifestation. We per­formed mutation screening by PCR-SSCP on all COL4A5 coding exons of 2 patients with XD AS and detected 17 samples with different mobility of single strands. This may be related to gene polymorphism, gene mutation and nonspecific products of PCR. We found 9 sus­pected mutations by one-way DNA sequen­cing, but reverse DNA sequencing results did not confirm this. Bi-directional sequencing therefore, should be performed to definitely ascertain pathogenic gene mutation. Absence of any mutation in the two AS patients su­ggests that mutations in this gene alone may not be the cause of AS. It has been shown recently that direct sequencing of COL4A5 exons in X-linked AS greatly increases the mutation detection rate. In addition, it is likely that mutations in introns [23] or regulatory ele­ments of type IV collagen genes are being overlooked.

In conclusion, AS in our Chinese pedigree was the result of X linked inheritance. Muta­tions other than in COL4A5 gene might also be responsible for the phenotype. Other studies looking at other genetic elements might be able to clearly delineate its mode of inhe­ritance.


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