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Commentary |
From the Department of Medicine, Renal Division, Washington University School of Medicine, St. Louis, Missouri
The 1990s have been an exciting and productive decade for the molecular dissection of the etiology of Alport syndrome. Alport syndrome is a hereditary glomerulonephritis accompanied usually by sensorineural deafness, frequently by ocular abnormalities, and rarely by diffuse leiomyomatosis (DL), which is characterized by benign nodular smooth muscle tumors of esophagus, tracheo-bronchial tree, and genital tract. The primary mode of inheritance of Alport syndrome is X-linked-dominant, though there are also autosomal-recessive and autosomal-dominant forms, and its overall prevalence is estimated to be 1 in 5000. The only treatments for the nephropathy at end stage are dialysis and renal transplantation.1-6
Alport syndrome is a basement membrane disease involving type IV
collagen. Collagen IV is a major component of all basement membranes.
It is composed of 
chains that trimerize to form long triple helical
protomers. Protomers are secreted by cells and associate with each
other in the extracellular matrix to form a chicken-wire-like
network.7
This serves as the scaffold for assembly of the
basement membrane, which also contains laminin, entactin/nidogen, and
sulfated proteoglycans.8,9
There are six genetically distinct collagen IV chains,
1(IV)-6(IV). The collagen 1(IV) and 2(IV) chains are the
classical chains and are essentially ubiquitous in basement membranes.
Mutations in COL4A1 and COL4A2 have not
been found in mammals and would likely be embryonically lethal. In
contrast, the underlying genetic defect in Alport syndrome is a
mutation in any one of three genes encoding what have been termed novel
type IV collagen chains. These chains have a restricted tissue
distribution. Importantly, they are all major components of the
glomerular basement membrane (GBM), which is characteristically
thinned, thickened, and split in Alport syndrome. X-linked Alport
syndrome is caused by mutations in the collagen 5(IV) chain gene
COL4A5, and mutations in COL4A3 and
COL4A4, which are linked head-to-head on chromosome 2,
are responsible for the autosomal forms
of the disease.10-12
A molecular hallmark of the severe forms of Alport syndrome is that
mutations affecting only one of the
COL4A3-COL4A5 genes result in the absence
all three gene products from the GBM. This has been used as
circumstantial evidence to suggest a model in which the 3-5(IV)
chains coassemble in a manner that requires all three
chains.13
In this model, the nonmutated genes would be
transcribed and translated normally, but the chains they encode would
be degraded on failing to assemble due to the absence of a normal third
chain. This model is attractive, because it is consistent with the
inherent trimeric structure of collagen IV protomers. Alternatively,
the defect in assembly could be at the level of protomer:protomer
interactions in the extracellular matrix.
Good evidence for transcriptional down-regulation of the nonmutated
genes in a canine model of Alport syndrome has been presented. These
data show that the lack of collagen 5(IV) protein due to
COL4A5 mutation was associated with a decrease in
3(IV) and 4(IV) steady-state mRNA levels.14
This
could explain in part the absence of the 3(IV) and 4(IV) chains
in mutant dog kidney basement membranes. However, RNA studies in humans
and in the two mouse models of Alport syndrome did not find such a
down-regulation in steady-state mRNA levels and thus do not support
such a transcriptional mechanism.15-17
The collagen 6(IV) chain is unique in that it has a restricted
tissue distribution but is not deposited in the GBM. It is found in
basement membranes associated with Bowman's capsule, epidermis, and a
subset of smooth muscle cells.18,19
Consistent with its
absence from GBM, mutations that affect only COL4A6 have
not been found in Alport patients.20
However,
COL4A6 is located on the X chromosome head-to-head with
COL4A5 and some COL4A5 deletion mutations
that cause Alport syndrome extend into
COL4A6.21-23
Thus, the 5' ends of both
genes are affected. Cases of Alport syndrome associated with diffuse
leiomyomatosis always fall into this category, but the extent of the
deletion into COL4A6 is limited to the alternative exons
1 and 1', intron 1, exon 2, and part of the very large intron 2.
Interestingly, if the deletion extends into exon 3, then diffuse
leiomyomatosis is not observed.22,23
This leads to the question of how and why some deletions that affect
COL4A6 result in diffuse leiomyomatosis, whereas the
most extensive ones do not. It has been hypothesized that the more
restricted deletions may allow production of a truncated 6(IV)
protein in smooth muscle that might be capable of aberrant signaling
and lead to the observed benign tumors.22
However, no
stable integration of any 6(IV) protein into esophageal tumor
basement membranes from appropriate patients was
observed.24
Another possibility is that there is a gene,
which may or may not encode a protein, embedded in the large second
intron of COL4A6 that is somehow transformed into a
dominant promoter of smooth muscle cell proliferation by the deletions
that cause Alport syndrome with diffuse leiomyomatosis.22
Further studies of the ~140-kb second intron of COL4A6
will be necessary to test this hypothesis.
COL4A5-specific mutations lead to the absence of
collagen 6(IV) in renal and epidermal basement
membranes,18,19,25
suggesting that the 6(IV) chain
cannot assemble into these basement membranes without the 5(IV)
chain. One important issue that has not been addressed is the status of
collagen 6(IV) protein in the smooth muscle basement membranes of
such Alport patients, who do not have deletions extending into
COL4A6 and who do not develop leiomyomata. The formal
possibility exists that, despite its absence from kidney and skin
basement membranes, these patients maintain a somewhat normal
complement of 6(IV) protein in their smooth muscle basement
membranes. This might play some role in preventing overproliferation of
smooth muscle cells. However, if true, then it would be difficult to
explain the absence of tumors when COL4A6 deletions
extend into exon 3. Nevertheless, whether
COL4A5-specific mutations lead to an absence of 6(IV)
in smooth muscle basement membranes is certainly worth investigating.
The article by Zheng et al26
published in this issue of
The Americal Journal of Pathology finally addresses this
and other important points using a canine model of X-linked Alport
syndrome. Paul Thorner and colleagues have previously studied this
family of Samoyed dogs in depth; they have identified a single base
non-sense mutation in COL4A5 and have shown that the
affected dogs exhibit many of the characteristics observed in human
Alport syndrome.27-30
In this issue of the
Journal they report the cloning and sequencing of
DNA adjacent to the 5' end of canine COL4A5 and show
that dog has a COL4A6 gene with many similarities to the
human gene, including the tightly linked, head-to-head arrangement with
COL4A5. This is the first cross-species comparison of
this region, and it shows that although exon 1 is very conserved
between human and dog, exon 1' is not. The authors rightly question the
functionality of this exon in dog. Indeed, by Northern blot analysis,
they show that COL4A6 mRNAs from bladder smooth muscle
contain exon 1 but not exon 1'. The authors use immunohistochemistry to
show that the collagen 6(IV) chain is present in bladder smooth
muscle basement membranes from a normal dog but is completely absent
from the COL4A5 mutant dog smooth muscle. Moreover,
despite the absence of 6(IV) protein, 6(IV) mRNA levels in
bladder smooth muscle are nearly normal. Finally, leiomyomata have
never been observed in this family of dogs.
These results reveal important new information regarding the biology of
type IV collagen and the etiology of Alport syndrome with diffuse
leiomyomatosis. First, a point mutation in COL4A5 is
sufficient to prevent incorporation of the collagen 6(IV) chain into
smooth muscle basement membranes, independent of a reduction in
6(IV) mRNA levels. This provides further evidence for requisite
coassembly of the 5 and 6(IV) chains, in agreement with the
observed absence of 6(IV) from renal and epidermal basement
membranes in Alport patients with COL4A5-specific
mutations.18,19,25
However, it contrasts with the
transcriptional mechanisms previously proposed as negative regulators
of expression of the 3 and 4(IV) chains in COL4A5
mutant dog kidney.14
Second, the mere absence of 6(IV) from dog smooth muscle is not
sufficient to cause diffuse leiomyomatosis. By analogy, based on these
studies of dog, human Alport patients with COL4A5
mutations would lack the 6(IV) chain in smooth muscle, but only
those with the additional appropriate COL4A6 deletions
would develop leiomyomata. Thus, these deletions are likely affecting
something other than expression of 6(IV) and its incorporation into
basement membranes. Determining what this something really is will
solve an important mystery and could force revisions in our
understanding of gene structure, regulation of cell proliferation, and
development of tumors and perhaps cancer.
Footnotes
Address reprint requests to Dr. Jeffrey H. Miner, Department of Medicine, Renal Division, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail: minerj{at}thalamus.wustl.edu
Accepted for publication March 11, 1999.
References
3 (COL4A3) gene in autosomal recessive Alport syndrome. Hum Mol Genet 1994, 3:1269-12733(IV) and 4(IV) collagen genes in autosomal recessive Alport syndrome. Nat Genet 1994, 8:77-82[Medline]
3, 4, and 5 chains of collagen type IV: evidence from a canine model of X-linked nephritis with a COL4A5 gene mutation. J Biol Chem 1996, 271:13821-138283 and 4 chain mRNA in X-linked Alport syndrome. J Am Soc Nephrol 1996, 7:938-945[Abstract]
3(IV): implications for Alport syndrome. J Cell Biol 1996, 135:1403-14131(IV), 5(IV), and 6(IV) collagen chains in normal human adult and fetal tissues and in kidneys from X-linked Alport syndrome patients. J Clin Invest 1995, 96:1948-1957
5(IV) and 6(IV) collagen genes in inherited smooth muscle tumors. Science 1993, 261:1167-11695(IV) and 6(IV) collagen genes in Alport syndrome and in Alport syndrome associated with smooth muscle tumours. Hum Mol Genet 1995, 4:99-108
6(IV) collagen in kidney and skin of X-linked Alport syndrome patients. Pediatr Nephrol 1996, 10:742-744[Medline]
6(IV) chain of collagen type IV in Alport syndrome is related to a failure at the protein assembly level and does not result in diffuse leiomyomatosis. Am J Pathol 1999, 154:1883-18915 chain of collagen type IV. Proc Natl Acad Sci USA 1994, 91:3989-3993This article has been cited by other articles:
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  R. Kalluri and D. Cosgrove Assembly of Type IV Collagen. INSIGHTS FROM alpha 3(IV) COLLAGEN-DEFICIENT MICE J. Biol. Chem., April 21, 2000; 275(17): 12719 - 12724. [Abstract] [Full Text] [PDF]
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