1995; 5:1714-1717). ABSTRACT. Autosomal recessive. Alport syndrome can arise ... Alport syndrome. Sensorineural deafness developed during adolescence, and ..... Turner. N, Mason. PJ,. Brown. R, et at.: Molecular cloning of the human.
Autosomal Recessive Alport Syndrome: COL4A3 Gene in a Woman With Alport Posttransplant Antiglomerular Basement Jie
Ding,
Joel
Stitzel,
Phillip
Berry,
Edith
Hawkins,
J, Ding, J. Stitzel, CE. Kashtan, Department of Pediatrics. Division of Pediatric Nephrology, University of Mmnesota. Minneapolis. MN P. Berry, Department of Pediatrics, University sas for Medical Sciences, Little Rock, AR E. Hawkins, Department Hospital, Houston, TX (J. Am.
Soc.
Nephrol.
of Pathology,
Texas
of ArkanChildren’s
1995;
recessive
Alport
syndrome
any family history of renal at age 8 showed immunoglobulin
can
arise
from
disease. Renal A nephrop-
athy and Alport syndrome. Sensorineural deafness developed during adolescence, and the patient’s renal disease progressed to terminal renal failure by age 20. She received a living related donor renal allograft basement months
at
age 20 and developed antiglomerular membrane nephritis of the allograft 8 after transplantation. Amplification and seof exon 5 of COL4A3 (counting from the 3’
quencing end of the gene) revealed a 7-base-pair deletion, producing a shift of the reading frame and the creation of a premature heterozygous for the
stop codon. Each parent was normal and mutant exon 5
sequences. This mutation in COL4A3 would result in the loss of 222 amino acids from the carboxy-terminal noncollagenous domain of the a3(IV) chain. The mutant other
chain would be Type IV collagen
1
ReceIved
2
Correspondence
Department MN 55455.
5eptember of Pediatrics,
unable to form trimers c chains. In addition,
27, 1994. Accepted
to Dr. CE.
Kashtan,
November
University
Box 491 UMHC,
515 Delaware
with the
22, 1994.
of Minnesota
104&6673/0509-1 714$03.00/0 Journal of the American Society of Nephrology Copyright © 1995 by the American society of Nephrology
1714
mutant which
E. Kashtan2 chain resides
would lack the Goodpasture in the carboxy-terminal
enous domain ofthe epitope may underly
a3(IV) chain. The absence of this the subsequent development of
anti-glomerular allograft.
basement
membrane
Key Words:
syndrome, nephritis
COL4A3,
Alport membrane
epitope, noncollag-
nephritis
in the
antiglomerular
base-
A
5:1714-1717)
a mutation in either ofthe genes COL4A3 and COL4A4 on chromosome 2, which encode, respectively, the a3 and a4 chains of Type IV collagen. This report describes a mutation in COL4A3 in a girl who presented at age 5 with hematuria and proteinuria, lacking biopsy
Clifford
ment
ABSTRACT Autosomal
and
Mutation in the Syndrome and Membrane
Street,
Medical
School,
SE, Minneapolis,
lport syndrome is an inherited, progressive gbmerulopathy associated with distinctive abnormalities of glomerular basement membranes (GBM), frequently accompanied by sensorineural hearing loss and ocular lesions ( 1 ). In most kindreds with Alport syndrome, the disorder is transmitted as an X-linked dominant trait, and numerous mutations in the Xchromosomal gene COL4A5, which encodes the a5 chain of Type IV (basement membrane) collagen, have been described (2). However, pedigree analyses have suggested that there is a subset of Alport kmndreds In which the disease is transmitted as an autosomal recessive trait, and in some of these kindreds, the linkage of the disease to the COL4A5 gene has been excluded (3,4). COL4A3 and COL4A4 encode the a3 and a4 chains of Type IV collagen, respectively, and are located on chromosome 2 (5-7). Recently, mutations in COL4A3 were reported in two Alport kindreds and mutations in COL4A4 were found in two other Alport kindreds, conclusively demonstrating the existence of an autosomal recessive form of Alport syndrome (8). This report concerns a woman with Alport syndrome who developed crescentic gbomerubonephritis in her renal albograft associated with circulating and bound antibodies to GBM, i.e. , posttransplant anti-GBM nephritis. This rare complication of renal transplantation In patients with Alport syndrome occurs almost entirely, but not exclusively, in male patients (9). It has been postulated that some male patients with Alport syndrome fail to establish immunologic tolerance for a normal GBM component, presumably the a5 chain of Type IV collagen, because they are hemizygous for defects in COL4A5 that preclude the synthesis The
of an immunogenic failure of a female
portion of Alport patient
a5(IV) to
immunologic tolerance for a normal GBM could be explained by preferential inactivation
Volume
5
-
( 10,
1 1).
establish
component of the
Number
9
-
X
1995
Ding
chromosome carrying the normal recessive mutations in an autosomal GBM component, such as COL4A3 fore, herein
we for
CASE
examined a mutation
COL4A5 allele or by gene encoding a or COL4A4. There-
DNA from the in the COL4A3
patient gene.
described
DESCRIPTION
H.P. is a 22-yr-old woman who was referred to Texas Children’s Hospital for the evaluation of hematuria and proteinuria at the age of5 yr, 10 months. She had normal complement levels and normal renal function but was mildly hypertensive. Renal biopsy revealed diffuse mesangial deposition of immunogbobulin A (IgA), compatible with a diagnosis of IgA nephropathy. By the age of 7 yr. 5 months, she had developed nephrotic-range proteinuria (4 g/24 h). Her renal function declined gradually between 8 and 19 yr of age, culminating in ESRD and the initiation of chronic peritoneal dialysis at age 20. She also developed bilateral sensorineural deafness during her adolescence. H.P. received a living related donor kidney from her mother and was maintained on immunosuppression with prednisone, azathioprine, and cyclosporine. The albograft functioned well for 6 months, but her serum creatinine then began a gradual rise. A renal allograft biopsy performed 8 months after transplantation showed a mild interstitial lymphocytic infiltrate compatible with acute cellular rejection. Intravenous methylprednisone was administered for 3 days, without improvement in graft function. This was followed by a 10-day course of humoral antilymphocyte therapy with OKT3 (Orthocbone#{174}), during which the serum creatinine rose from 4.4 to 1 1 .6 mg/dL. A second renal albograft biopsy showed no evidence of rejection but did show crescentic glomerubonephritis and strong linear deposition of IgG along the GBM, compatible with anti-GBM disease. A test for circulating anti-GBM antibodies was positive. A review of the electron micrographs of the native kidney biopsy showed diffuse GBM thickening and multilamellation, as well as focal GBM thinning, findings typical of Alport syndrome (Figure 1). Renal albograft function did not recover, and the patient has returned to dialysis. H.P.’s Neither known tamed
family history is negative for renal disease. parent has hematuria, and they are not to be consanguinous. To date, urinalyses obon her two siblings have been normal.
METHODS PCR Amplification Genomic white Exons
DNA
of Exons was
extracted
blood cells according to 1 to 5 ofCOL4A3 (counted
were amplified directly elsewhere ( 1 3). Primers were (Table
derived from 1). Polymerase
in a total
volume
facturer
Journal
et al
the
of
the
American
from
the
patient’s
standard from the
peripheral
procedures 3’ end of the
(12). gene)
from genomic DNA as described for the amplification of these exons respective intronic chain reactions
of 100
(Perkin-Elmer,
1 to 5 of COL4A3
flanking (PCR) were
,,L as recommended Norwalk,
Society
CT).
of
sequences performed
by the The
Nephrology
reactions
manucon-
Figure 1 Electron micrograph The GBM is thickened and reduplication of the lamina areas between the duplicated
of H.P’s native renal biopsy. shows marked splitting and densa, with electron-lucent strands.
.
sisted
of
controls through
1 g
of genomic
as template 35 cycles as
and
denaturing
and
annealing
for for
DNA
and follows: 1 .5
30
50
mm,
slow
s, and
BRL,
Gaithersburg,
Sequencing The
products were (BIO1O1 , performed with thefmo! Madison, WI) according some modifications for
through
at 20
sequencing
purified
over
slow
45
heating
carried to 94#{176}C s to
over
55#{176}C 30
to
products were electrophoa 1 -klbobase DNA marker
cycles.
In
CA).
agarose
Direct
gels
with
sequencing
the was
DNA Sequencing System (Promega, to the manufacturer’s protocol, with end-labeling of primers and exten-
order
5 from
exon 5 primers of the initial
from
Vista,
reactions: 42#{176}Cfor 20
exon
internal products
normal
Analysis Kit
sion-termination annealing
or
MD).
PCR
MERmaid
subjects
each primer over 1 mm
cooling
finally,
72#{176}C and extension for 45 s. PCR resed on a 1 .5% agarose gel with (Gibco
from
pmol of slow heating
PCR
denaturation s, and extension to
obtain
parental (Table as
at 94#{176}Cfor at 72#{176}Cfor sufficient
DNA,
1 ) was templates.
material
nested performed,
PCR with
20 20
s, s, for
with the
RESULTS Agarose electrophoresis of the exons revealed that exon 5 from slightly smaller than the normal Exons 1 to 4 appeared normal in examined further. The nucleotide patient’s exon 5 and the normal compared (Figure 3). The patient’s 7-base-pair deletion, which produces reading frame, resulting in a missense codons would
followed result in
by a the loss
premature of 222
amplified COL4A3 the patient was exon 5 (Figure 2). size and were not sequences of the exon 5 were then sequence shows a a shift of the sequence of 76 stop
amino
codon. This acids from the
1715
COL4A3
Mutation
TABLE 1 Primers .
in Autosomal
Recessive
used to amplify
exons
Alport
Syndrome
1 to 5 of COL4A3
Exon
5,
3’
1
5’CCCCCCGmGGITITITIAAGTA3’
5’ACAGCATGTTCTGTCATrACmGTrC3’
2
5’CAATGGACAGAGTG1TrA1TCAG3’
5’TCATCAGA1TAAGC1TGATGGTGA3’
3
5’AGAAAGTGGCAATGCCGCCATAGTC3’
5’CTCACCATGATGAATGAAAATCCT3’
4
5’CTGAAAGTGCTATACTCAGTCTGATG3’
5’TCCCATFGTAAAACTAGGGGA1TGGAT3’
5
5’TATGTrGCAACATTrAGAATGTGT3’
5’AGCATAACTGGTAACTGGGACTGGG3’
5’GGAAAACGTGGAGACAGTGGATC3’
5’TCCGTGGGCTCG1TGA1TI3’
5 (Nested
PCR)
A
A
C
0
B
T 0
A Ser CC AC
I
3’
I
C
AC
T
G C*Arg
-I
0
C*HiS Val
A
CC C
C A
Thr
Figure 2. Agarose gel electrophoresis of PCP-amplifled exon 5 of COL4A3. M, 1-kllobase DNA marker; 1 , exon 5 amplified from genomic DNA of patient normal genomic DNA.
H.P.; 2, exon
5 amplifIed
from
NC 1 domain of a3(1V) ( 1 4). In DNA from each parent, analysis of the nested PCR products revealed two sequences, one corresponding to the normal exon 5 sequence and the other showing the 7-base-pair deletion found In the patient (Figure 4).
DISCUSSION This report describes a new mutation in COL4A3 in a patient with Alport syndrome. In this patient, a 7-base-pair deletion In exon 5, counting from the 3’ end of the gene, results in a shift of the reading frame as well as a premature stop codon. Because the five exons at the 3’ end of COL4A3 encode the carboxyterminal noncollagenous (NC 1 ) domain of a3(IV), this portion of the protein would not be synthesized (14). Type IV collagen molecules are heterotrimers formed by the folding of three a chains, Initiated by Interaclions between their NC1 domains (15). The absence of the NC 1 domain would chain from participating mers and from incorporation a3(IV) chain appears to a4(IV) and a5(IV) chains, also a5(IV)
1716
disrupt into
prevent the mutant a3(IV) in the formation of heterotriinto GBM. Because the form heterotrimers with the mutations in a3(IV) might
the normal incorporation the GBM (2, 16, 17). Native
of a4(lV) and kidney tissue
Thr
C T
TPhe
TPhe
5’
5’
\T \
A
C
Figure 3. Partial nucleotide sequence of exon 5 of COL4A3 from H.P. and comparison with normal sequence. (A) Sequence of DNA from patient H.P.; the derived amino acid sequence is shown to the right of the nucleotide sequence. The deletion breakpoint is indicated by the arrow. (B) Normal DNA sequence; the deleted nucleotides are starred.
was not available for staining with antibodies against these chains. Electron-dense mesangial deposits are not typical of the ultrastructural changes of Alport syndrome but have been found in some patients ( 18, 19). A patient with IgA nephropathy superimposed on Alport syndrome has been described ( 19), as well as a patient in whom Alport syndrome was associated with membranoproliferative glomerulonephritis Type I (20). Mochizuki and colleagues have reported mutations in COL4A3 in two kindreds with autosomal recessive Alport syndrome (8). In one family, a 5-base-pair deletion in exon 5 leading to a shift in the reading frame and a premature stop codon was found, and in the other family, the mutation consisted of conversion of a codon for arginine to a stop codon, again in exon 5. Taken together with our findings, these observations suggest that exon 5 may be a “hotspot” for mutations in COL4A3. Confirmation of this hypothesis must await description of other COL4A3 mutations. The NC 1 domaIn of a3(IV) contains the Goodpasture epitope,
i.e.
,
the
target
of
anti-GBM
Volume
autoantibodies
5
‘
Number
9
‘
1995
Ding
type W collagen to chromosome Genomics 1992:13:809-813.
2 bands
of
MOTHER ATCG
FATHER ATCG
7,
8.
C/A*
G/C*
9.
A/C* c* A*
10.
11.
Figure 4. Partial nucleotide sequences of exon 5 of COL4A3 from H.P’s mother and father, showing the transition from a single to a double sequence in each parent. The starred nucleotides denote the wild-type sequence. present in the circulation of patients with Goodpasture syndrome (anti-GBM nephritis and pulmonary hemorrhage) (2 1-23). Some patients with Alport syndrome who developed posttransplant anti-GBM nephritis were found to have anti-GBM antibodies that recognized the Goodpasture epitope ( 1 7,24). Although no sera containing anti-GBM activity were available from our patient, we can postulate that she may have generated antibodies against the NC 1 domaIn of a3(IV) present in the GBM of the renal allograft.
17.
18.
MC, Atkin CL. Alport syndrome. In: Schrier RW, Gottschalk CW, Eds. Diseases of the Kidney. 5th Ed. Boston: Littie Brown: 1993:571-591. 2. Knebelmann B, Antignac C, Gubler M-C, Grunfeld J-P: A molecular approach to inherited kidney disorders. Kidney Int 1993:44:1205-1216. Feingold J, Bois E, Chompret
A, Broyer M, Gubler MC, JP: Genetic heterogeneity of Alport syndrome. Kidney Int 1985:27:672-677. 4. Knebelmann B, Benessy F, Buemi M, Grunfeld JP, Gubler MC, Antignac C: Autosomal recessive (AR) inherGrunfeld
Mariyama cDNAs other
6.
type
localization
Journal
R, Hudson membrane a4(IV) J Biol
of the
IV M,
bovine collagens. Zheng
of the
American
K,
genes
Yang-Feng
for the
Society
Reeders
ST:
[Abstract].
J
Am
BG,
Reeders
collagen:
and
comparison
Chem
Reeders a4(IV)
and
of
24.
of Nephrology
Co-
chains
New
York:
Cold
Spring
139:401-4
10.
of 58
cases
and
Med 1981:70:493-505. Sessa A, Cioffi A, Conte phropathy with nerve
a review
of the
literature.
Am
J
F, D’Amico G: Hereditary nedeafness (Alport’s syndrome).
1974:13:404-415.
13374-13380.
with ST:
Ed.
Kleppel MM, Fan WW, Cheong HI, Michael AF: Evidence for separate networks of classical and novel basement membrane collagen: Characterization of a3(1V)-Alport antigen heterodimers. J Biol Chem 1992:267:4137414. Hudson BG, Kalluri R, Gunwar S, et aL: The pathogenesis of Alport syndrome involves type N collagen molecubes containing the a3(N) chain: Evidence from antiGBM nephritis after renal transplantation. Kidney Int 1992:42:179-187. Gubler M, Levy M, Broyer M, et at.: Alport’s syndrome: A
Nephron
The
1991:267:1253-
TL,
a3(IV)
ST:
2nd
Meroni M, Sessa A, Battini G, et aL: Alport syndrome with type I membranoproliferative gbomerulonephritis. Nephron 1993:65:479-480. 2 1 . Butkowski RJ, Langeveld JPM, Weislander J, Hamilton J, Hudson BG: Localization of the Goodpasture epitope to a novel chain of basement membrane collagen. J Blob Chem 1987:262:7874-7877, 22. Saus J, Wieslander J, Langeveld JPM, Quinones S, Hudson BG: Identification ofthe Goodpasture antigen as the a3(IV) chain of collagen N. J Biol Chem 1988:263:
Soc
isolation
Manual.
20.
23.
ofbasement
encoding
1258. Mariyama
(AS)
M, Kalluri chain
TL,
cDNA encoding the Am J Hum Genet
Harbor Laboratory Press: 1989. Ding J, Zhou J, Tryggvason K, Kashtan CE: COL4A5 deletions in three patients with Alport syndrome and post-transplant anti-GBM nephritis. J Am Soc Nephrol 1994:5: 16 1-167. Turner N, Mason PJ, Brown R, et at.: Molecular cloning of the human Goodpasture antigen demonstrates it to be the a3 chain of type N collagen. J Clin Invest 1992:89: 592-601. Weber S, Engel J, Wiedemann H, Glanville RW, Timpl R: Subunit structure and assembly of the globular domain of basement membrane collagen type N. Eur J Biochem
report
19.
syndrome
Yang-Feng
ofa partial IV collagen.
T, Mariyama M, Smeets HJM, et at.: Identffiof mutations in the a3 and cr4 type IV collagen in autosomal recessive Alport syndrome lAbJ Am Soc Nephrol 1993;4:819. Kashtan CE, Michael AF: Alport syndrome: From bedside to genome to bedside. Am J Kidney Dis 1993:22: 627-64. Kashtan C, Butkowski R, Kieppel M, First M, Michael A: Posttransplant anti-glomerular basement membrane nephrltis in related Alport males with Alport syndrome. J Lab Chin Med 1990; 116:508-515. Smeets HJM, Melenhorst JJ, Lemmink HH, et at.: Different mutations in the COL4A5 collagen gene in two patients with different features ofAlport syndrome. Kidney Int 1992:42:83-88. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning:
1984;
1 . Gregory
a4(IV)
13.
16.
REFERENCES
Alport 1993:4:263.
M,
Mochizuki
A Laboratory
15.
The authors thank Donna Floyd-Gimon for her help in obtaining blood samples from the family described in this report. The authors received support from the National Institutes of Health (A110704), American Heart Association IMinnesota Afffliate), the Variety Club, the Viking Children’s Fund. and the Children’s Kidney Disease Sodely.
5.
12.
14.
ACKNOWLEDGMENTS
in
Mariyama
q35-q37.
cation genes stracti.
T/G*
itance Nephrol
KE,
Sequence and localization human a3 chain of type 1991:49:545-554.
A/CC
3.
Morrison
et al
Neilson EG, Kalluri R, Sun MJ, et at.: Specificity of Goodpasture autoantibodies for the recombinant noncollagenous domains of human type N collagen. J Biol Chem 1993:268:8402-8405. Kalluri R, Weber M, Netzer K-O, Sun MJ, Nelson EG, Hudson BG: COL4A5 gene deletion and production of post-transplant port syndrome.
anti-cr3(N) Kidney
mt
collagen abloantibodies 1994:45:721-726.
in Al-
1717
