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NDT Advance Access originally published online on February 3, 2007
Nephrology Dialysis Transplantation 2007 22(5):1338-1346; doi:10.1093/ndt/gfl793
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© The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

A novel Cys1638Tyr NC1 domain substitution in {alpha}5(IV) collagen causes Alport syndrome with late onset renal failure without hearing loss or eye abnormalities

Jane C. Wilson1, Han-Seung Yoon1, Robert J. Walker2 and Michael R. Eccles1

1Department of Pathology and 2Department of Medical and Surgical Sciences, University of Otago, P.O. Box 913, Dunedin, New Zealand

Correspondence and offprint requests to: Michael Eccles, Department of Pathology, University of Otago, P.O. Box 913, Dunedin, New Zealand. Email: michael.eccles{at}stonebow.otago.ac.nz



   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Background. Mutations in the type IV collagen gene, COL4A5, are associated with Alport syndrome, characterized by ultrastructural abnormalities of the glomerular basement membrane (GBM), with or without progressive loss of renal function, characteristic ophthalmic signs and/or high tone sensorineural deafness. More than 300 sequence variants in type IV collagen have been identified, including alterations in the non-collagenous NC1 domain.

Methods. We performed linkage analysis and sequencing to identify the mutation in a New Zealand family with Alport glomerulonephritis and late onset renal failure without hearing loss or eye abnormalities.

Results. We report a novel c.4913G>A (p.Cys1638Tyr) alteration in the NC1 domain of COL4A5, identified in a moderately large family, eight of whom were confirmed by renal biopsy to have renal abnormalities. Only three of eight mutant male members of the pedigree progressed to end-stage renal failure. The remaining five mutant males exhibit either chronic renal disease at age 36, 46 and 72, or as yet show no renal disease at ages 39 and 39. Extra-renal manifestations such as sensorineural deafness or ocular changes were absent from all family members carrying the mutation.

Conclusion. This variant is the first reported to affect the tenth of 12 cysteine residues in the NC1 domain. We conclude that the cysteine to tyrosine substitution in the NC1 domain of the {alpha}5(IV) collagen chain in this family leads to a mild form of Alport syndrome, including absence of extra-renal features.

Keywords: alport syndrome; COL4A5; mild phenotype; mutation analysis



   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The glomerular basement membrane (GBM) comprises a specialized extracellular matrix that plays a crucial role in the purification of blood plasma by the kidney. The major constituent of the GBM is type IV collagen, which together with laminin, nidogen and sulfated proteoglycans maintains the filtration barrier and provides the substrata and signals necessary for proper renal cell function [1–4].

The type IV collagen family is comprised of six homologous {alpha}-chains designated {alpha}1(IV)–{alpha}6(IV) encoded for by the COL4A1-6 genes, respectively [4]. Each {alpha}-chain consists of a collagenous domain of ~1400 residues of Gly-Xaa-Yaa repeats flanked by two non-collagenous domains: a 7S domain at the amino terminal and a carboxyl NC1 domain. Three {alpha} chains assemble into triple-helical molecules called protomers that then assemble into supramolecular networks by the association of four protomers at the N-terminus, forming a 7S tetramer, and the dimerization of two protomers at the C-terminus, forming an NC1 hexamer [3]. To date, only three different types of collagen protomers have been identified; {alpha}1.{alpha}1.{alpha}2(IV), {alpha}3.{alpha}4.{alpha}5(IV) and {alpha}5.{alpha}5.{alpha}6(IV) [1]. In the GBM, the {alpha}1.{alpha}1.{alpha}2(IV) network predominates during early nephrogenesis but is replaced by the {alpha}3.{alpha}4.{alpha}5(IV) network in the mature glomerulus. Alterations in any of the COL4A3, COL4A4 and COL4A5 genes may cause Alport syndrome, a hereditary nephropathy characterized by haematuria in childhood followed by a slow progressive loss of renal function and ultrastructural abnormalities of the GBM [5]. Alport syndrome is characterized by ‘basket-weave’ changes in the GBM on electron microscopy along with one or more of the following criteria: a positive family history of haematuria with or without renal failure; diagnostic ophthalmic signs and high tone sensorineural deafness [6]. Immunohistochemical studies show that mutations in the COL4A5 gene, which cause the X-linked form of Alport syndrome, frequently result in the loss of all three of the {alpha}3(IV), {alpha}4(IV) and {alpha}5(IV) chains in the GBM [7]. Thus, the absence of a functionally normal {alpha}5(IV) chain can disrupt assembly of the triple-helical protomer, and frequently leads to loss of the entire {alpha}3.{alpha}4.{alpha}5(IV) network in the GBM.

Over 300 mutations in COL4A5 have been reported, all causally linked with Alport syndrome. Indeed, considerable allelic heterogeneity has been observed, and recently, a number of researchers have attempted to link genotypes in Alport syndrome to phenotypes [8–10]. Gross and colleagues [8] have proposed a classification linking phenotype and genotype into three categories:

  1. Phenotypically severe Alport syndrome. Genotypic alterations in this category in COL4A5 include major gene rearrangements, premature stop codons, frameshift mutations and donor splice site alterations. The phenotype is characterized by early onset of end-stage renal failure (ESRF) at about 20 years of age and significant extra-renal manifestations including 80% with sensorineural hearing loss and 40% with ocular lesions.
  2. Moderately severe Alport syndrome. The genotype in this group is characterized by non-glycine-XY missense alterations, in-frame deletions/insertions, acceptor splice site changes and glycine-XY substitutions involving exons 21–47. This type is associated with ESRF appearing in the mid-twenties with about 65% of individuals exhibiting hearing loss and 30% ocular defects.
  3. Moderate Alport syndrome. The genotype is glycine-XY substitutions involving exons 1–20. The phenotype appears to be milder with a later onset of ESRF at about 30 years of age, including a significant number of individuals with sensorineural hearing loss (70%) and ocular lesions (30%).

It should be noted that on average 80–90% of males with mutations of COL4A5 in Alport syndrome progress to ESRF by the age of 40 [9], and while hearing loss or ocular abnormalities may be delayed or infrequent in some families, or indistinguishable from age-related changes, it is exceptional for extra-renal features to be absent from very many affected members of a family with Alport syndrome.

This report describes a unique cysteine to tyrosine substitution in the NC1 domain of COL4A5 in a New Zealand family exhibiting Alport syndrome with late onset renal failure, without hearing loss or eye abnormalities. Only three of eight mutant male family members progressed to ESRF in their twenties. The remaining five mutant male members remain with either chronic renal disease, or as yet show no renal disease. None of the family members were shown to have extra-renal abnormalities.



   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Acknowledgements
 References
 
Collection of pedigree and clinical ascertainment
Investigation of the family was approved by the Otago Ethics Committee, and all family members who took part in subsequent investigations gave written informed consent for their participation. Most family members, including all individuals identified with renal disease, had a comprehensive review by a consultant ophthalmologist and an ENT surgeon. Ocular examination included visual acuity and intraocular pressure measurements, and examination for anterior lenticonus, cataract, or optic disc or retinal abnormalities. Investigation for sensorineural hearing loss was carried out using standard audiometry tests.

Immunohistochemistry
Immunohistochemical studies on paraffin-embedded kidney biopsy sections were carried out using an autoclave antigen retrieval method and rat monoclonal antibodies H11, H22, H31, H43, H53 and H63 specific for the {alpha}1(IV), {alpha}2(IV), {alpha}3(IV), {alpha}4(IV), {alpha}5(IV), and {alpha}6(IV), respectively, as previously described [11]. Due to limited sample availability, sections were heated at the temperature that was found to be optimal for antigen retrieval in the GBM. Technical difficulties prevented adequate results for {alpha}6(IV) being obtained. Sections (4 µm) on 3-amino propyl triethoxysilone (APES) coated slides were deparaffinized and rehydrated. Antigen retrieval was carried out by autoclaving for 6 min in 0.2N HCl solution using a small autoclave (LabClave, Mercer, New Zealand) set at 125°C. Endogenous peroxidase activity was quenched using 3% H2O2 (in dH2O) for 10 min at room temperature. After 30 min incubation with ImmunoCoat, 1X BSA Immunoassay MasterMix (ImmSolv, USA) blocking serum, sections were incubated with each monoclonal antibody at room temperature for 1 h, followed by incubation with a biotinylated anti-rat IgG (BA-4000, Vector Laboratories, USA) for 30 min at room temperature. Staining was then carried out using the Vectastain ABC universal kit (Vector Laboratories, USA) followed by incubation with DAB substrate (Sigma, St Louis, MO). Sections were dehydrated, and mounted using DPX resin (Aldrich, St Louis, USA).

DNA isolation and genotyping
Genomic DNA was isolated from EDTA whole blood using the QIAamp system, as described by the manufacturer (Qiagen GmbH, Germany). Highly polymorphic microsatellite markers were selected from the Genethon Human Genetic Linkage Map [12] and primers either purchased from Research Genetics (MapPairs) or custom-made using published primer sequences. PCR was carried out using standard techniques with a radiolabelled primer, as described [12,13]. Amplification products were separated by electrophoresis on a 6% polyacrylamide gel and visualized after appropriate exposure to X-Omat AR film (Eastman Kodak, USA). Alleles were scored manually. Linkage analysis was carried out using the MLINK program of the LINKAGE package [14] assuming X-linked inheritance, equal allele frequency, 100% penetrance in males, 50% penetrance in females and a disease gene frequency of 0.0001.

Sequence analysis of COL4A5
Intronic primer pairs for each of the 51 exons making up the COL4A5 transcript (Genbank accession number NM_000495 [GenBank] ) and the entire promoter region between COL4A5 and COL4A6 [15] were designed from the genomic sequence and used for PCR and sequencing. DNA from the proband was amplified, purified using a PCR Product Pre-Sequencing Kit (USB Corporation, USA) and sequenced using ABI©BigDye Terminator v3.0 chemistry on an ABI 377 DNA sequencer (Applied Biosystems, USA). The c.4913G>A nucleotide substitution was analysed in other family members and control individuals by PCR amplification of exon 50 (primers: 5'-ctg ggc ctg ttc ctt cac-3' and 5'-ttg ttg agg ata aac cat cc-3') using the following PCR programme: 2 min at 94°C, followed by 10 cycles of 94°C for 48 s, 57°C for 48 s, 72°C for 48 s, then 25 cycles of 94°C for 48 s, 57°C for 48 s, 72°C for 48 s. Following amplification, PCR products were digested with MaeIII restriction enzyme (Roche GmbH, Germany) for 1 h at 55°C and electrophoresed on a 1% agarose gel.



   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Acknowledgements
 References
 
Clinical presentation
The initial clinical identification of this family occurred when two sisters (IV26 and IV28) presented to the clinic to be considered as potential live kidney donors for their sons (V29 and V35, respectively) who had ESRF (Tables 1 and 2). Both women were found to have significant proteinuria and hypertension and they proceeded to a renal biopsy. A strong family history of hypertension was identified as well as familial renal disease. Previous generations had died of predominantly cardiovascular events. Subsequent clinical review of the family identified a number of additional family members with renal disease (Table 2). Extra-renal examinations were negative for sensorineural hearing loss, and except for mild myopia, eye examinations were negative in all individuals tested.


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  		Table 1. Renal disease identified prior to screening the family

 

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  		Table 2. Renal disease or carrier status identified after screening the family

 
Proteinuria and hypertension were present in multiple members of the family (Tables 1 and 2). The most severely affected males (individuals IV3, V29, V35) presented with a progressive chronic glomerulonephritis, and received renal transplants at age 41, 26 and 28, respectively. They had not been previously screened for renal disease. Three affected males remain with chronic kidney disease currently aged 36, 46 and 72 (IV5, V31 and IV39), and two mutant males (both currently aged 39) do not have chronic kidney disease or ESRF (V24 and V42). Subsequent screening of family members identified a number of asymptomatic female carriers, several of whom developed haematuria or proteinuria alone (IV24, IV26, IV28, V40, IV36, IV47, V44). Only one female developed ESRF (III2). Unfortunately, a biopsy was not undertaken at the time she presented. Hypertensive and proteinuric individuals have been treated with an ACE inhibitor, although this will not necessarily prevent ESRF from occurring in mutant males. Individuals IV26, IV28 and IV34 did not have haematuria, despite carrier status.

Renal histology, electron microscopy and immunohistochemistry
Eight members of the family have had a renal biopsy. Biopsies from the male family members with ESRF were consistent with chronic glomerulonephritis (Figure 1, Table 1). Areas of mild mesangial proliferation were also observed in some carrier females (Table 2). Immunofluorescence was non-contributory. Electron microscopy of a renal biopsy from an affected male (V42) demonstrated the classical basket-weave pattern or splitting of the basement membrane characteristic of Alport syndrome, despite no evidence of renal disease (Figure 2A). A renal biopsy from a carrier female (IV28) with proteinuria, hypertension and normal renal function demonstrated focal areas of irregular thickening and thinning of the GBM (Figure 2B), which was also present in some regions in affected males (data not shown). Immunohistochemistry for the {alpha}1–{alpha}5 type IV collagens in kidney biopsies from affected and carrier individuals showed that in individuals IV26, V31, V35 and V42 the GBMs were positive for {alpha}1–{alpha}5 type IV collagens, as exemplified in Figure 2C. Due to limited sample availability, not all biopsied family members were analysed, and antigen retrieval was only optimized for the GBM.


Figure 1
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  		Fig. 1. Kidney histology of affected family members. (A) Glomerulus from kidney biopsy of individual V35 shows mild mesangial matrix expansion, glomerulosclerosis and Bowman's capsule fibrosis. (B) Glomerulus from individual V42 shows mesangial-proliferative glomerulonephritis with mesangial matrix expansion, and Bowman's capsule fibrosis. H&E stain, magnification bar in B shows 50 µm.

 

Figure 2
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  		Fig. 2. Transmission electron microscopy and immunohistochemistry. (A, B) Electron micrographs of individual V42 show sections of thickened GBM with a ‘basket-weave’ appearance, characteristic of Alport syndrome (asterisks). Original magnification 15 500x. (C) Electron micrograph of individual IV28 shows regions in the GBM that are relatively thin, and regions with irregular thickening of the GBM (arrowheads). Original magnification 10 500x. (D) Immunohistochemistry using a monoclonal antibody against {alpha}V collagen in a kidney biopsy from patient V31 shows clear immunoreactivity in the GBM. A negative control with a secondary antibody showed no immunoreactivity (data not shown).

 
Identification of a novel sequence variant in COL4A5
The extended family pedigree is shown in Figure 3. The predominance of the phenotype in males and the lack of male-to-male transmission were consistent with an X-linked dominant pattern of inheritance. Segregation analysis of chromosome X microsatellite markers [12] in 16 family members revealed three markers (DXS6789, DXS8096, DXS1210) that formed a haplotype (1-1-4) co-segregating with the disorder (data not shown). Two-point analyses between the disease gene and each marker gave strong evidence of linkage with Lod scores at {theta} = 0.00 ranging from 3.14 to 3.59 (not shown). The linked region encompassed COL4A5, the gene mutated in Alport syndrome [16].


Figure 3
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  		Fig. 3. Family pedigree. Extended pedigree of the family showing males (squares) and females (circles) indicated by generation (I–VI) and identification number. The same identity for each individual is used in Tables 1 and 2. Black symbols indicate individuals with biopsy-confirmed GN. Black dots inside the symbol indicate obligate carriers. Those who have been confirmed by DNA testing are indicated with a cross. Grey symbols indicate individuals with clinical manifestations of renal disease and therefore presumed GN. White symbols indicate individuals without clinical signs of the disease.

 
The entire coding region of the COL4A5 gene in an affected male was sequenced and a G to A substitution at nuclotide 4913 in exon 50 was identified (Figure 4A). This substitution removes a restriction site for the enzyme Mae III. Restriction analysis with Mae III in other family members showed that the A allele was present in all affected family members (Figure 4B) and not present in 192 healthy control individuals from the New Zealand population (data not shown). The c.4913G>A nucleotide substitution is predicted to alter the amino acid at position 1638 of the {alpha}5 chain of type IV collagen chain from cysteine to tyrosine. This cysteine residue is located in the NC1 domain of the {alpha}5 chain and is one of the 12 NC1 domain cysteine residues that are completely conserved in all six type IV collagen {alpha}-chains across all species.


Figure 4
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  		Fig. 4. Nucleotide sequence alteration in COL4A5 in affected family members. (A) Nucleotide sequence from affected male family members and control DNA showing the c.4913G>A substitution (indicated with an asterisk). (B) MaeIII restriction enzyme digest of exon 50 PCR products. Lane M, 1 Kb PlusTM DNA Ladder (Invitrogen, USA); Lanes 1–2, control human genomic DNA, undigested and digested, respectively; Lanes 3–8, affected male family members, IV3 (lane 3), IV5 (lane 4), V29 (lane 5), V31 (lane 6), V35 (lane 7), V42 (lane 8); Lanes 9–15, affected and carrier female family members, IV24 (lane 9), IV26 (lane 10), IV28 (lane 11), IV31 (lane 12), IV34 (lane 13), IV36 (lane 14), V40 (lane 15). Note, IV31 and IV36 are female carriers who are represented in Figure 3A, but not represented on the pedigree in Figure 3B.

 


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Acknowledgements
 References
 
Here we report a New Zealand kindred with a novel alteration in COL4A5 involving the NC1 domain. The pattern of disease inheritance in the family was consistent with an X-linked dominant mode, with reduced penetrance. Linkage to the COL4A5 region, and identification of the NC1 domain mutation strongly implicated COL4A5 as the causative gene locus in this family. However, compared with the majority of families with Alport syndrome, this mutation conferred a very mild phenotype. Compared with other NC1 domain missense mutations, the renal and auditory phenotypes were less severe.

In general, substitution and missense mutations in the NC1 domain, as in other regions of COL4A5, lead to haematuria, proteinuria, ESRF and sensorineural hearing loss with an overwhelming predominance in males [8,17–24]. Figure S1 shows the age-related incidence in this family of ESRF or hearing loss, and the probability of ESRF or hearing loss occurring in another published Alport family with an L1649R missense mutation in COL4A5 [17]). Although there are only eight affected males in the present study, comparison of the phenotype in this family with the largest reported families with missense mutations in the NC1 domain of COL4A5 [17,18] reveals some similarities in the age of onset of end-stage renal disease, but also differences in the fraction of individuals developing renal disease, and in the percentage of individuals who develop extra-renal abnormalities. For example 40% of carrier males had not developed end-stage renal disease by age 40 in a family with an L1649R mutation in the NC1 domain, and 15% had not developed end-stage renal disease by age 40 in a family with a C1564S mutation [17,18]. In these families, approximately 30% and 10%, respectively, had not yet developed sensorineural hearing loss by age 40. In the present family, more than 60% of affected males have not yet developed ESRF, with four of these males being 39 years or older. Moreover, none of the family members have developed extra-renal abnormalities at any age, very different from the large families reported earlier [17,18]. In addition, just within the present family the overall difference in disease severity between the more-severely affected males and the less-severely affected males who carry the mutation was greater than expected for Alport syndrome patients.

During protomer assembly the NC1 domains of the {alpha}3(IV), {alpha}4(IV) and {alpha}5(IV) chains specifically interact to select chains for triple-helix formation [25, 26]. In Alport syndrome, NC1 domain cysteine substitutions (Figure 5) are thought to affect the folding of the monomeric NC1 domain, preventing its participation in trimer assembly. The NC1 domain is also important for network assembly, whereby the NC1 trimers of two protomers specifically interact forming a NC1 hexamer. Variants that result in a loss of, or a defect in any of the {alpha}3(IV), {alpha}4(IV) or {alpha}5(IV) chains result in incorrect folding or assembly of the entire protomer, which may then lead to loss of the {alpha}3.{alpha}4.{alpha}5(IV) network from the GBM. However, in many instances the {alpha}3.{alpha}4.{alpha}5(IV) network is still detectable in the GBM in individuals with X-linked Alport syndrome [19]. In this family the {alpha}3(IV), {alpha}4(IV) and {alpha}5(IV) collagens were still present in the GBM, implying that the p.Cys1638Tyr alteration must allow for the correct assembly of the triple helical protomer.


Figure 5
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  		Fig. 5. Depiction of NC1 domain showing locations of cysteine sequence alterations (A) Depiction of the NC1 domain showing beta sheet domains and cysteine residues arranged linearly, and their disulfide linkages. Cysteine residue substitutions of the NC1 domain that have been previously reported are shown (^), together with the cysteine substitution identified here (* under cysteine 182; amino acid numbering in this figure is from the start of the NC1 domain). (B) Hexamer of bovine placental type IV collagen, determined to 1.5 Å (PDB code: 1T60) [29], is presented as a ribbon model. Each protein monomer is presented in a different colour. All cysteines are presented as stick-and-ball model sidechains, with bonds and atoms enlarged for clarity. All cysteines are coloured yellow with the exception of Cys 182 (pink) and Cys 176 (white, being the partner in the disulphide bond) of chain A. Cys 182 in chain A of the hexamer presented corresponds to the substituted cysteine in the affected individuals.

 
It is interesting to note that several phenotypic features observed in the mutant male members of this family with normal renal function were similar to those of familial benign haematuria, except that the GBM was not extremely thin, but instead showed focal regions of irregular GBM thickening and thinning. Familial benign haematuria, also known as thin basement membrane nephropathy (TBMN), is generally thought to involve mutations in one allele of COL4A3 or COL4A4 [27,28]. The unusually high number of males with haematuria and mutation in COL4A5 in this family who have not yet developed renal disease or sensorineural hearing loss in spite of their age, suggests that the p.Cys1638Tyr alteration predisposes to benign haematuria or chronic kidney disease, and that in about one half of males it leads to ESRF in the third to fifth decade of life.

In summary, we report a unique cysteine to tyrosine substitution in the NC1 domain of COL4A5 in a New Zealand family who presented with a phenotypically mild form of Alport syndrome, suggesting that in this family disruption of Cys1638 led to late-onset renal failure, without hearling loss or eye abnormalities.



   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
References
 
We thank Dr Merriman for providing normal DNA samples from healthy control individuals, Dr Sado, for his generous gift of the monoclonal antibodies against human type IV collagen, and our clinical colleagues including Dr John Richmond for providing clinical data. We are also indebted to Dr Jacobs for providing the ribbon model structure of {alpha}V(IV) collagen, and to Bronwyn Smaill for carrying out electron microscopy. This work was funded by the Otago Medical Research Foundation, the HealthCare Otago Charitable Trust, and the Cancer Society of New Zealand.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 

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Received for publication: 5. 4.06
Accepted in revised form: 6.12.06


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