ALPORT SYNDROME: ABNORMALITIES OF TYPE IV COLLAGEN GENES AND PROTEINS

INTRODUCTION

Alport syndrome is an inherited disorder of basement membranes, arising from mutations in type IV collagen, the major collagenous constituent of these structures. While the clinical picture is dominated by the renal manifestations of these mutations, significant abnormalities of the eye and the cochlea are observed in many patients. Multidisciplinary research efforts carried out over the past 30 years have revealed much about the pathogenesis of AS, and contributed a great deal of information about the composition of normal basement membranes.

Genetics and Biochemistry of Type IV Collagen

Basement membranes are composed of several major and minor glycoprotein constituents. Type IV collagen is present ubiquitously in basement membranes, where it is the major collagenous component. Type IV collagen molecules secreted by endothelial and epithelial cells self-associate into polygonal networks, which interact with laminin networks, as well as with entactin, proteoglycans, and other glycoproteins, to form basement membranes. The ability of the GBM to filter enormous amounts of water and solutes, while allowing minimal passage of protein, appears to depend in part upon its type IV collagen composition. Proteinuria is an important feature of AS in both humans and animals, and appears to arise, through mechanisms that remain obscure, from the abnormal type IV collagen composition of the Alport GBM.

Type IV Collagen α Chains

Each type IV collagen molecule is a trimer composed of three α chains. Six genetically distinct type IV collagen chains have been identified, and the genes encoding these chains have been cloned and sequenced. The major structural features of α(IV) chains include a collagenous domain of about 1400 residues containing the repetitive triplet sequence glycine (Gly)-X- Y, in which X and Y represent a variety of other amino acids; a carboxyterminal noncollagenous (NCl) domain of about 230 residues; and a noncollagenous aminoterminal sequence of 15–20 residues [[1]]. Approximately 20 interruptions of the collagenous triplet sequence are present in the collagenous domain. The NCl domains each contain 12 completely conserved cysteine residues, which participate in intrachain and interchain disulfide bonds.

Type IV collagen chains form trimers through associations between their carboxyterminal NCl domains, associated with folding of the collagenous domains into triple helices. Variable residues within the NCl domains may dictate which chains can interact with each other. Type IV collagen triple helices form networks through several types of intermolecular interaction. These include end-to-end linkages between the carboxyterminal domains of two type IV collagen triple helices, covalent interactions between four triple helices at their aminoterminal ends, and lateral associations between triple helices via binding of the carboxyterminal domains to sites along the collagenous region of the triple helix [2-6]. These various linkages between type IV collagen molecules produce a nonfibrillar polygonal assembly that serves as a scaffolding for the deposition of other matrix glycoproteins and for the attachment of cells.

Type IV Collagen Genes

The six type IV collagen genes are arranged in pairs on three different chromosomes. Comparison of the peptide sequences of the six α(IV) chains allows grouping into two classes: αl-like, including the αl,α3 and α5(IV) chains; and α2-like, consisting of the α2, α4 and α6(IV) chains [[1]]. The type IV collagen genes are arranged so that each (l-like gene is paired with an α2-like gene. The human αl and α2(IV) chains are encoded by the genes COLAAl and COLAA2, respectively, on chromosome 13 [[7]]. COL4A3 and COL4A4 encode the α3 and α4 chains of type IV collagen, respectively, and are located on chromosome 2q36 [[8]]. The α5 and α6(IV) chains are respectively encoded by the COL4A5 and COLAA6 genes on the long arm of the X chromosome ([[9]] [[10]] [[11]]). The 5′ ends of each gene pair are adjacent to each other, separated by sequences of varying length containing motifs involved in the regulation of transcriptional activity, and the genes are transcribed in opposite directions.

Type IV Collagen Distribution in Basement Membranes

Expression of the various type IV collagen chains in human and animal tissues has been studied using monoclonal and affinity-purified polyclonal antibodies ([[12]] [[13]] [[14]] [[15]] [[16]] [[16]] [[17]] [[18]] [[19]]). The α1 (IV) and α2(IV) chains are normally present in all basement membranes; the α3(IV) -α6(IV) chains exhibit a more restricted distribution. For example, in the kidney the αl(IV) and α2(IV) chains are found in the glomerular mesangium, GBM, Bowman's capsule, all tubular basement membranes, and all vascular basement membranes. The α3(IV), α4(IV) and α5(IV) chains are present in GBM, Bowman's capsule and the basement membranes of distal and collecting tubules, but absent from mesangium and vascular basement membranes, while the α6(IV) chain is found only in Bowman's capsule and distal and collecting tubule basement membranes. Analogous situations exist in other organs, such as the eye and the inner ear, in which the α1(IV) and α2(IV) chains are found in all basement membranes, while only selected basement membranes contain the α3(IV) -α6(IV) chains. The distribution of type IV collagen is frequently altered in kidney disease, but the nature of the alteration varies according to the chain involved. For example, in diabetic nephropathy, the thickening GBM expresses increasing amounts of α3(IV), α4(IV) and α5(IV) chains, while the α1(IV) and α2(IV) chains disappear from the GBM [[20]]. The new GBM laid down by podocytes in membranous nephropathy contains α3(IV), α4(IV) and α5(IV) chains, but not α1(IV) and α2(IV) chains (21). These findings, along with the changes observed in Alport syndrome (as discussed below) suggest that the α3(IV), α4(IV) and α5(IV) chains comprise a basement membrane collagen network that is distinct from the network formed by α1(IV) and α2(IV) chains. Some basement membranes, such as the basement membrane under the epidermis, normally contain α1(IV) and α2(IV) chains, as well as α5(IV) and α6(IV) chains, but do not express the α3(IV) and α4(IV) chains ([[18]] [[19]] [[22]]). These basement membranes appear to contain two type IV collagen networks, one composed of α1(IV) and α2(IV) chains, and the other consisting of α5(IV) and α6(IV) chains.

Expression of Type IV Collagen in Alport Basement Membranes

Several studies in the 1980s established that the native kidneys of male Alport patients failed to bind anti-GBM antibodies in sera from patients with Goodpasture syndrome or from Alport patients with post-transplant anti-GBM nephritis ([[23]] [[24]] [[25]] [[26]] [[27]] [[28]]). These studies, combined with the landmark studies that localized the Goodpasture epitope to type IV collagen ([[29]] [[30]] [[31]]), helped to confirm the pathogenetic link between AS and type IV collagen originally hypothesized by Spear [[32]]. The availability of mono specific antibodies against each of the six type IV collagen α chains has made it possible to characterize the changes in type IV collagen expression that occur in patients with Alport syndrome ([[11]] [[19]] [[22]] [[33]] [[34]] [[35]] [[36]] [[37]]). These diagnostically useful changes have been extensively illustrated in recent reviews([[36]] [[38]] [[39]]).

Normally, the α3(IV), α4(IV) and α5(IV) chains are highly expressed in basement membranes that are demonstrably or potentially involved in Alport syndrome: glomerular basement membrane, anterior lens capsule, Descemet's membrane, Bruch's membrane, and several basement membranes of the cochlea including the basilar membrane and the basement membranes of the stria vascularis, spiral limbus and spiral prominence ([[12]] [[13]] [[14]] [[15]] [[16]] [[17]] [[18]] [[19]] [[40]]). In males with X-linked Alport syndrome (XLAS), the GBM, distal TBM and Bowman's capsules usually fail to stain for the α3(IV), α4(IV) and α5(IV) chains, but expression of the α1(IV) and α2(IV) chains is preserved and, in fact, increased. Basement membranes of some males with XLAS exhibit normal, or reduced but positive, staining for the α3(IV), α4(IV) and α5(IV) chains. The α6(IV) chain is not expressed in Bowman's capsule or distal TBM of XLAS males whose basement membranes lack α5(IV) expression. Women who are heterozygous for XLAS mutations frequently exhibit mosaicism of GBM expression of the α3(IV), α4(IV) and α5(IV) chains, while expression of the α1(IV) and α2(IV) chains is preserved. Epidermal basement membranes (EBM) normally express the α1(IV), α2(IV), α5(IV) and α6(IV) chains, but not the α3(IV) or α4(IV) chains. Most males with XLAS show no EBM expression of α5(IV) or α6(IV), while female heterozygotes frequently display mosaicism. Lens capsules of some males with XLAS do not express the α3(IV), α4(IV) or α5(IV) chains, while expression of these chains appears normal in other patients [[41]]. The expression of type IV collagen chains in the cochleae of human Alport patients has not been studied.

In patients with autosomal recessive Alport syndrome (ARAS), GBMs usually show no expression of the α3(IV), α4(IV) or α5(IV) chains, but α5(IV) and α6(IV) are expressed in Bowman's capsule, distal TBM and EBM [[34]]. Therefore, XLAS and ARAS may be distinguishable by immunohistochemical analysis of renal biopsy specimens. The expression of type IV collagen chains in basement membranes of patients with autosomal dominant Alport syndrome (ADAS) has not been characterized.

The abnormalities of type IV collagen expression observed in XLAS and ARAS patients indicate that a mutation affecting one of the chains involved in the putative α3- α4-α5(IV) network can prevent basement membrane expression not only of that chain but of the other two chains as well, and that a mutation involving the α5(IV) chain can interfere with basement membrane expression of α6(IV). The mechanisms that produce these effects remain under investigation. It is likely that least some mutations interfere in various ways with the formation of trimeric type IV collagen molecules, leading to degradation of normal chains that have been prevented from forming trimers, or that have formed abnormal trimers. This kind of process accounts for abnormal type I collagen deposition in bone in osteogenesis imperfecta [[42]]. In some instances, a mutation at one of the type IV collagen loci may result in reduced transcription of other type IV collagen genes, or may accelerate degradation of rnRNA transcribed from these genes. Thomer and colleagues [[43]] found that kidneys of male dogs with Samoyed hereditary nephropathy, a canine form of AS that arises from a COL4A5 mutation, contained levels of mRNA level for the α3(IV) and α4(IV) chains that were substantially less than the levels found in kidneys of unaffected males. However, Nakanishi et al [[44]] found no differences in mRNA levels for α3(IV) and α4(IV) chains, as measured by competitive RT -PCR, in kidneys of men with XLAS, when compared with normal male kidneys. Similarly, we have observed that dermal fibroblasts of males with XLAS express levels of α6(IV) mRNA that are comparable to levels expressed in normal fibroblasts [[45]]. In transgenic mice with COL4A3 mutations, renal mRNA levels for the α4(IV) and α5(IV) chains are not different from levels in normal mice([[46]] [[47]]).

In summary, it appears that several distinct networks of type IV collagen may exist in basement membranes: a ubiquitous network composed of the αl(IV) and α2(IV) chains, and other networks composed of α3(IV), α4(IV) chains and α5(IV) chains, or of α5(IV) and α6(IV) chains. For example, GBM appears to include separate αl/α2(1V) and α3/α4/α5(IV) networks, while epidermal basement membranes seem to contain separate networks of αl/α2(IV) chains and α5/α6(IV) chains. Several biochemical studies of extracted basement membranes have recently lent support to this “separate networks” hypothesis ([[48]] [[49]] [[50]]). It appears very likely that these networks have different capabilities and that the α3-α4-α5(IV) network performs functions in GBM, ocular basement membranes and cochlear basement membranes that cannot be adequately performed by αl and α2(IV) chains. The Alport phenotype(s) results from the absence of the α3-α4-α5(IV) network from these basement membranes, or from the presence of a network that is functionally deficient.

Molecular Genetics of Alport Syndrome

There are three genetic varieties of AS: X-linked (XLAS), which results from mutations in the COL4A5 gene and accounts for about 80% of patients; autosomal recessive (ARAS), due to mutations in either COL4A3 or COL4A4 and responsible for about 15% of patients; and autosomal dominant (ADAS), making up the remainder. The locus for at least some families with ADAS appears to be COL4A3 or, perhaps, COL4A4 (51).

X-Linked Alport syndrome (XLAS)

The genetics of Alport syndrome was recently reviewed by Lemmink and colleagues [[52]]. This report includes 176 reported mutations in the COL4A5 gene in families with XLAS. These mutations are distributed throughout the gene, with no apparent hot spots. With few exceptions, each family carries a unique mutation. Approximately 10 to 15% of COL4A5 mutations are de novo, having occurred in the gamete of a parent. Thirty-eight of the 176 mutations (21%) consist of deletions of substantial portions of the gene. The actual incidence of major rearrangements of COL4A5 in XLAS families is 5-15%, if families in which a deletion has been excluded but the precise mutation has not been identified are included ([[53]] [[54]] [[55]]). All families in which XLAS cosegregates with diffuse leiomyomatosis (see below) exhibit large deletions that span the adjacent 5^′ ends of the COL4A5 and COL4A6 genes ([[56]] [[57]]). The deletions involve varying lengths of COL4A5, but the COL4A6 breakpoint is always located in the second intron of the gene ([[58]] [[59]]). Leiomyomatosis does not occur in patients with deletions of COL4A5 and COL4A6 that extend beyond intron 2 of COL4A6. Mutations of COL4A6 alone do not appear to cause AS, consistent with the absence of the α6(IV) chain from normal GBM (57,60).

Other reported types of COL4A5 mutation include missense mutations causing amino acid substitutions (35%), mutations resulting in premature stop codons (nonsense mutations, small deletions or insertions, splice site mutations –39%), and small, in-frame deletions. The great majority of amino acid substitutions occur in the collagenous domain of α5(IV) and involve replacement of glycine residues ([[52]] [[54]] [[61]]). Such mutations are thought to interfere with the normal folding of the mutant α5(IV) chain into triple helices with other type IV collagen α chains. Glycine lacks a side chain, making it the least bulky of amino acids, and is small enough to allow three glycine residues to fit into the interior of a tightly wound triple helix. The presence of a bulkier amino acid in a glycine position presumably creates a kink or an unfolding in the triple helix. Glycine substitutions account for the majority of mutations in genetic diseases of collagen [[62]]. Abnormally folded collagen triple helices exhibit increased susceptibility to proteolytic degradation ([[42]] [[63]]). The position of the substituted glycine, or the substituting amino acid itself, may influence the effect of the mutation on triple helical folding, and ultimately the impact of the mutation on the severity of the clinical phenotype.

A minority of amino acid substitutions in the α5(IV) chain involve critical residues in the carboxyterminal noncollagenous (NC1) domain, such as one of the 12 conserved cysteine moieties. The loss of one of these cysteines would eliminate a disulfide bond, which could interfere with the formation of triple helices, or with the construction of networks involving α5(IV) chains.

Autosomal recessive Alport syndrome (ARAS)

To date, mutations causing ARAS have been found in the COL4A3 gene in six patients ([[64]] [[65]] [[66]]) and in the COL4A4 gene in 12 patients ([[67]] [[68]]). Some of these patients are homozygous for their mutations, some are compound heterozygotes, and some are heterozygotes in whom only one of the mutant alleles has been identified. As with COL4A5, there appear to be no mutation hot spots in COL4A3 or COL4A4.

Autosomal dominant Alport syndrome (ADAS)

As noted above, ADAS has been mapped in a single family to chromosome 2, in proximity to COL4A3 and COL4A4 [[51]]. A specific mutation of either gene in this family has yet to be reported.

Benign familial hematuria (BFH)

A heterozygous missense mutation in COL4A4 has been identified in a family with autosomal dominant transmission of hematuria, in the absence of renal failure [[69]]. The observation that carriers of a single mutant COL4A4 allele may have hematuria provides further evidence that COL4A4 defects are involved in at least some families with BFH [[68]], although other alleles may be involved in other families [[70]].

Genotype-phenotype Correlations

The two major elements of the Alport phenotype are renal disease and deafness. Families with XLAS can be divided into two groups on the basis of the timing of end-stage renal disease (ESRD) in affected males [[71]]. In families with the juvenile form of XLAS, the mean age of onset of ESRD in affected males is 30 years or less, while in families with the adult form of the disease the mean age of onset of ESRD is greater than 30 years. Male patients with COL4A5 deletions and other null mutations of COL4A5 consistently exhibit juvenile ESRD, associated with deafness. Most of the missense and splicing mutations of COL4A5 described thus far are also associated with juvenile ESRD and deafness. Several missense mutations of COL4A5 have been associated with adult ESRD and late development of deafness([[52]] [[72]]).

Among females who are heterozygous for a COL4A5 mutation, about 15% or so develop renal insufficiency and deafness by middle age. The true incidence of renal failure in elderly heterozygotes is unknown. Less than 10% of heterozygotes are asymptomatic, i.e. they cannot be shown to have hematuria or any hearing deficit. The majority of female carriers have asymptomatic microhematuria and normal hearing. The severity of the Alport phenotype in a female carrier probably depends on the extent of inactivation of the X chromosome carrying the normal COL4A5 allele [[73]], as well as the specific nature of the mutation.

There is evidence to suggest that COL4A5 mutations that are associated with preservation of GBM expression of α3(IV), α4(IV) and α5(IV) chains often result in a less severe phenotype ([[37]] [[74]] [[75]]). Information about the effects of individual COL4A5 mutations on basement membrane expression of these chains, and the relationship of chain expression to phenotype, is still being accumulated.

Patients with ARAS consistently exhibit juvenile ESRD and deafness, regardless of gender ([[64]] [[65]] [[66]] [[68]]). Patients with ADAS appear to exhibit a slower rate of progression to ESRD than most patients with XLAS (76).