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Kidney International Vol 47 (1995), pp. 1199-1204
RAPID COMMUNICATION
A COL4A3 gene mutation and post-transplant anti-a3 (IV)
collagen alio antibodies in Alport syndrome
R a g h u K a l l u r i , 1 L .P . v a n d e n H e u v e l , H .J .M . S m e e ts , C .H . S c h r o d e r , H .H . Lem m ink,
A riel B o u t a u d , E ric
G.
N e il so n ,
and B illy G.
H u d so n
Department oj BiochemisUy and Molecular Biology, University o f Kansas Medical Center, Kansas City, Kansas, USA; Department o f Pediatrics and
H um an Geneticsy University Hospital Nijmegen, Nijmegent The Netherlands; Penn Center for Molecular Studies o f Kidney Diseases, University o f
Pennsylvania Medical School, Philadelphia, Pennsylvania, USA
A COL4A3 gene mutation and post-transplant anti-a3(IV) collagen
alloantibodies in Alport syndrome. The X-Iinked Alport syndrome is
associated with mutations and deletions in COL4A5 gene, one of six genes
which constitute the ^-chains of type IV collagen in basement membranes.
The autosomal recessive form of Alport syndrome is characterized by
mutations and deletions in the COL4A3 and COL4A4 genes. A fraction of
Alport patients who undergo renal transplantation develop anti-glomerular basement membrane (GBM) nephritis, which results in loss of the
renal allograft function. Recently, the target for alloantibodies from an
X-linked Alport patient with complete COL4A5 gene deletion was
determined to be the a3 chain of type IV collagen. The present study
characterized the post-transplant alloantibodies from an autosomal reces­
sive Alport patient with anti-GBM glomerulonephritis and a COL4A3
gene mutation which predicted a loss of 85% of the a3(IV) NCI domain.
The specificity of these new antibodies were studied using glomerular
basement membrane constituents and recombinant type IV collagen
domains. The results establish the target for the alloantibodies, from an
autosomal recessive Alport patient with COL4A3 deletion as principally
the cy3(IV) collagen chain, similar to the post-transplant alloantibodies
from X-linked Alport patients with COL4A5 gene deletions, The absence
of cy3(IV) chain in the GBM of patients with both these forms of Alport
syndrome, due either to a failure of synthesis or a failure of assembly,
presumably leads to a loss of immunologic tolerance for the a3(IV) NCI
domain in transplanted allografts.
Alport syndrome is a progressive hereditary kidney disease
characterized by hematuria, sensorineural hearing loss, and ocular
lesions with structural defects in GBM [1-3]. The disease is
primarily X chromosome-linked, but autosomal forms of inheri­
tance are also known [4]. The X-linked syndrome is associated
with mutations and deletions in COL4A5 gene, which encodes the
a5(IV) chain, one of six genetically distinct type IV collagen gene
products [3, 5-7]. The rare autosomal forms of Alport syndrome
are associated with recessive mutations in the COL4A3 and
COL4A4 genes which encode the a3(IV) and a4(IV) chains,
respectively [8, 9].
Anti-GBM disease appears in 5 to 10% of Alport patients who
1 Present address: Penn Center for Molecular Studies of Kidney Dis­
eases, University of Pennsylvania Medical School, Philadelphia, PA, USA.
Received for publication December 6, 1994
and in revised form January 9, 1995
Accepted for publication January 9, 1995
© 1995 by the International Society of Nephrology
receive a kidney transplant following the development of renal
failure. In the recent years, insight into the structural nature of the
defect in Alport GBM has been determined by establishing the
target of anti-GBM alloantibodies produced by these patients
[10»14]. Alloantibodies from seven post-transplant Alport pa­
tients, for example, were found reactive to the NCI domain of
type IV collagen [9-14]. Three of these antisera, under closer
scrutiny, did not bind to Alport GBM and were found reactive to
the «3(IV) chain [11]. Recently, post-transplant anti-GBM alloantibodies harvested from a X-linked Alport patient with complete
COL4A5 gene deletion was characterized [15], These alloantibod­
ies were also specifically targeted to the a3(IV) chain [15]. This
study suggested a pivotal role for the «5 (IV) chain in the secretion
or assembly of type IV collagen co-expressing the a3(IV) moiety.
The present study characterizes the target antigen for the
post-transplant alloantibodies from an autosomal recessive Alport
patient with COL4A3 gene deletion.
b
Methods
Patient histoty
The case history of family VB is described elsewhere [8,9], The
affected female had hematuria from age 4, and typical ultrastructural lesions of Alport syndrome on electron microscopy of a renal
biopsy and sensorineural deafness. Renal function deteriorated
gradually until hemodialysis was started at age 9. She received a
renal allograft at age 10 and developed anti-GBM nephritis six
months later. Her brother has hematuria, deafness, and deterio­
rating renal function. The parents have no hematuria, proteinuria
or deafness. There is no known consanguinity, but the parents and
their known ancestors originate from the same small village in the
Netherlands. The affected female in family VB has a deletion of
last 198 amino acids of the a3(IV) chain. This was predicted to
result in a chain termination after 33 amino acids of the NCI
domain.
GBM antigens and analytic assays
The preparation of the GBM constituents, fibronectin, laminin,
heparan sulfate proteoglycan, entactin, 7 S domain of type IV
collagen, pepsin solubilized triple helical fragments of type IV
collagen and NCI domains of type IV collagen a-chains was
described previously [16]. Recombinant human type IV collagen
a-chains (al-aS) were expressed in E. coli and purified as before
[17].
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Collagen IV NC1 domains
Fig. 2. Identification GBM constituent(s) that hind to the Alport alloanti­
bodies. The various GBM constituents were F, fibronectin, L, Iaminin,
HSP, heparan sulfate proteoglycan, E, entactin, 7 S, 7 S domain of type IV
collagen, pepsin solubilized TIT, triple helical fragments of type IV
collagen and dimers and monomers of NCI domains of type IV collagen
a-chains. The dilution of alloantibodies was 1:500. The control serum did
not bind to any of the GBM constituents.
upon incubation with the circulating alloantibodies (Fig. IB).
These results suggest that additional binding sites for the alloantisera in the transplanted kidney are accessible in vitro compared
to in vivo and that there is a structural difference within the GBM
of the Alport and transplanted kidney.
(D1-dimers) and a3, «4, a5 and a6 NCI dimers (D2-dimers). At
the NCI hexamer level, the alloantibodies bound strongly to the
54 kDa dimers, a 28 kDa monomer, and very faintly to a 26 kDa
monomer (data not shown), as visualized by one dimensional
immunoblotting. To further evaluate this binding in terms of type
IV collagen a-chains, we performed two-dimensional gel electro­
phoresis and immunoblotting using bovine NCI hexamer. The
bovine NCI hexamer was used since all the spots originating from
the six a-chains have been identified based on their pi and
molecular weight by two-dimensional gel electrophoresis (Identi­
fication of a l-a 5 spots was previously reported by Gun war et al
[16]. The monomeric and dimeric spots originating from the
a6(IV) was recently identified. R. Kalluri, J. Zhou, and B.G.
Hudson, unpublished data.) The alloantibodies reacted specifi­
cally to the a3(IV) NCI dimers and monomers, identical to the
spots that bind to anti-a3(IV) chain specific antibodies as previ­
ously described [16] (Fig. 3). No reactivity was found with the
al(IV ), a2(IV), a4(IV), a5(IV) and aó(IV) NCI dimers and
monomers. Additionally, the alloantibodies did not react to the al
4* a2(IV) NCI dimers and monomers, as evaluated by one
dimensional immunoblotting (data not shown). These results
establish the target for the alloantibodies as the a3(IV) chain of
bovine type IV collagen.
Inhibition ELISA
Type IV collagen a-chain specific antibodies were used to
perform inhibition ELISA using bovine NCI hexamer as the
antigen. The NCI hexamer was allowed to bind with one of the six
a-chain specific antibodies and subsequently followed with the
Alport alloantibodies. The anti-a3(IV) chain specific antibodies
inhibited the alloantibodies binding (Figs. 4 A and B). All the
other antibodies did not inhibit the alloantibodies binding to the
NCI hexamer. These results establish the alloantibodies as antia3(IV)NCl antibodies.
Specificity o f the alloantibodies to the recombinant human type IV
collagen N C I domains
,
Specificity o f alloantibodies to GBM constituents and bovine type
IV collagen domains
The specificity of post-transplant anti-GBM alloantibodies from
an autosomal recessive Alport patient with COL4A3 deletion was
determined using bovine GBM constituents and bovine NCI
domains of the a-chains of type IV collagen. The GBM constit­
uents used were: fibronectin, Iaminin, heparan sulfate proteogly­
can and entactin. Bovine type IV collagen domains used were: 7 S
domain of type IV collagen, pepsin solubilized triple helical
fragments of type IV collagen and dimeric and monomeric NCI
domains of type IV collagen a-chains. The alloantibodies reacted
specifically to the a3(IV) NCI containing dimers and a3(IV) NCI
monomers (Fig. 2).
The alloantibodies were further analyzed for their capacity to
bind recombinant human type IV collagen NCI domains (a l to
a5). The alloantibodies reacted very strongly to the recombinant
a3(IV) NCI (Fig. 5) and to a very minor degree with the
recombinant a5(lV)NCl domain (1:200 dilution of alloantisera,
lost at 1:500). The weak reactivity with the a5(IV)NCl may be a
cross-reactivity phenomenon, due to the high homology between
the NCI domains [24]. Alternatively, the weak binding may be
specific reactivity only observed with human a5(IV)NCl se­
quence; repeating the gel with half the amount of target NCI
domains eliminated evidence of weak binding while preserving the
strong reactivity to a3. These results establish the principal target
for post-transplant anti-GBM nephritis in an Alport patient with
COL4A3 gene deletion as the a3 chain of type IV collagen.
Discussion
Specificity o f the alloantibodies to bovine type IV collagen N C I
domains by one - and two-dimensional gel electrophoresis
and immunoblotting
The alloantibodies from the affected patient were analyzed by
one- and two-dimensional gel electrophoresis and immunoblot­
ting with the bovine NCI hexamer, bovine a l + a2 NCI dimers
In several previous studies, the target for X-linked Alport
alloantibodies was identified as the NCI domain of type IV
collagen [10-14]. Hudson et al have shown the target for three
post-transplant alloantibodies as the a3(IV) chain [1.1]. Kleppel et
al implicated the a5(IV) chain as a target for alloantibodies in
their patients [25, 26]. Their assumption of a5 chain reactivity was
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1204
Kalluri et al: Gene Mutation in Alport’s syndrome
and c*4(IV) collagen genes in autosomal recessive Alport syndrome.
Nature Genet (in press)
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AF: Posttransplant anti-glomeular basement membrane nephritis in
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1990
11. H u d s o n BG, K a l l u r i R, G u n w a r S, W e b e r M, B a l l e st e r F,
H u d s o n JK, N o e l k e n ME, S a r r a s M, R ic h a r d s o n W R , S aus J:
The pathogenesis o f Alport syndrome involves type IV collagen
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