RESEARCH ARTICLE
Genotype–Phenotype Correlations in 17 ChinesePatients With Autosomal Recessive Alport SyndromeYanqin Zhang,1 FangWang,1 Jie Ding,1* Hongwen Zhang,1 Dan Zhao,1 Lixia Yu,1 Huijie Xiao,1 Yong Yao,1
Xuhui Zhong,1 and Suxia Wang2
1Department of Pediatrics, Peking University First Hospital, Beijing, P.R. China2Department of Electron Microscopy, Peking University First Hospital, Beijing, P.R. China
Manuscript Received: 6 February 2012; Manuscript Accepted: 19 May 2012
Autosomal recessive Alport syndrome (ARAS) results from
mutations in the COL4A3 or COL4A4 gene. We analyzed the
genotype and phenotype of 17 unrelated Chinese patients with
ARAS. Clinical data were reviewed. All coding exons of COL4A3
and COL4A4 genes were PCR-amplified and sequenced from
genomic DNA. We identified pathologic mutations in all
patients, giving a mutation detection rate of 100%, with 82%
in COL4A3 gene and 18% in COL4A4 gene. Sixteen novel
mutations in COL4A3 gene and four novel mutations in
COL4A4 gene were identified. Furthermore, a previously
reported in-frame deletion mutation (40_63del24) in exon 1
of the COL4A3 gene was found in four patients in our study.
A single 40_63del24mutation inCOL4A3 seems to result inmild
or no renal manifestations, whereas the homozygous state of
40_63del24 in COL4A3 gene or compound heterozygous muta-
tionof 40_63del24plus anothernonsenseor frameshiftmutation
in COL4A3 gene seems to result severe ARAS with hearing loss.
Half of the probands’ parents had hematuria with or without
mild proteinuria. Therefore, we recommend that ARAS be
considered when a patient has a positive family history of
hematuria, and screening for COL4A3 mutations firstly may
be an efficient strategy for molecular diagnosis of ARAS.
� 2012 Wiley Periodicals, Inc.
Key words: autosomal recessive Alport syndrome; mutation;
genotype–phenotype correlation; type IV collagen
INTRODUCTION
Alport syndrome (AS) is a hereditary nephritis characterized by
hematuria, progressive renal failure, and specific ultrastructural
lesions of the glomerular basement membrane (GBM). The
X-linked form of the disease (XLAS), accounting for 85% of all
cases, results frommutations in the type IV collagen a5 chain gene(COL4A5), and the rarer autosomal modes of inheritance, includ-
ing autosomal recessive AS (ARAS) and autosomal dominant AS
(ADAS), are caused by mutations in COL4A3 or COL4A4 gene,
encoding a3 (IV) and a4 (IV) chains, respectively. Immunohis-
tochemical analysis of a (IV) chains in renal and skin biopsy
specimens can distinguish ARAS from XLAS [Heidet and Gubler,
2009]. Namely, patients with ARAS generally have no a3 (IV),
a4 (IV), and a5 (IV) chains in the GBM with normal staining of
a5 (IV) chain in the Bowman’s capsular basement membrane
(BCBM), part of the tubular basement membrane (TBM) and
the epidermal basement membrane (EBM). However, most
patientswithXLASdemonstrate not onlynegative (male)ormosaic
(female) immunostaining ofa3 (IV),a4 (IV), anda5 (IV) chains inGBM, BCBM, and TBM, but also negative (male) or mosaic
(female) immunostaining of a5 (IV) chain in the EBM.
Mutations in COL4A3/4 genes have been described in ARAS,
ADAS, and thin basement membrane nephropathy (TBMN),
which demonstrates phenotypic resemblance to collagen IV
disorders [Badenas et al., 2002; Longo et al., 2002]. Since some
heterozygous COL4A3 and COL4A4mutations have been found in
both ARAS and TBMN [Lemmink et al., 1996; Buzza et al., 2001;
Vega et al., 2003; Wang et al., 2004], TBMN may represent the
carrier state ofARAS.However, how the heterozygousmutations in
COL4A3/4 genes cause both ARAS and TBMN remains unclear.
Grant sponsor: National Nature Science Foundation; Grant numbers:
30400482, 81070545, 39970775; Grant sponsor: Beijing Nature Science
Foundation; Grant number: 7102148; Grant sponsor: National ‘‘Eleventh
Five-Year’’ Science and Technology Support Project; Grant number:
2006BAI05A07.
Yanqin Zhang and Fang Wang contributed equally to this work.
*Correspondence to:
Jie Ding, M.D., Ph.D., Department of Pediatrics, Peking University First
Hospital, No. 1, Xi An Men Da Jie, Beijing 100034, P.R. China.
E-mail: [email protected]
Article first published online in Wiley Online Library
(wileyonlinelibrary.com): 00 Month 2012
DOI 10.1002/ajmg.a.35528
How to Cite this Article:Zhang Y, Wang F, Ding J, Zhang H, Zhao D,
Yu L, Xiao H, Yao Y, Zhong X, Wang S. 2012.
Genotype–phenotype correlations in 17
Chinese patients with autosomal recessive
Alport syndrome.
Am J Med Genet Part A .
� 2012 Wiley Periodicals, Inc. 1
To date, among 242 patients suspected with ARAS (mostly from
Europe) reported, 57% have mutations in COL4A3 or COL4A4
[Lemmink et al., 1994; Mochizuki et al., 1994; Ding et al., 1995;
Kalluri et al., 1995; Knebelmann et al., 1995; Boye et al., 1998;
Nomura et al., 1998; Torra et al., 1999; Heidet et al., 2001; Dagher
et al., 2002; Gross et al., 2003; Vega et al., 2003; Nagel et al., 2005;
Longo et al., 2006; Hou et al., 2007; Rana et al., 2007; Cook et al.,
2008]. The single reported Chinese family with ARAS had muta-
tions in COL4A3 [Hou et al., 2007].
In this article, we characterize the molecular spectrum in 17
unrelated Chinese patients with ARAS, delineate their phenotypes
and discuss the genotype–phenotype correlations.
MATERIALS AND METHODS
PatientsIn this study, we collected all ARAS patients from our hospital’s
hereditary renal diseases registry, begun in 1999. ARAS was diag-
nosed according to the main and minor diagnostic criteria. The
main diagnostic criteria were hematuria plus either ultrastructural
changes of the GBM suggestive for AS or immunohistochemical
changes typical of ARAS. Theminordiagnostic criteria included (1)
parental consanguinity; (2) normal distribution of a5 (IV) chain
in EBM; and (3) negative family history of hematuria or chronic
renal failure. A patient fulfilled the main diagnostic criteria and at
least one of the three minor diagnostic criteria was considered as
ARAS. Seventeen unrelated Chinese ARAS patients (two of them
were from consanguineous families) were collected. Controls were
healthy blood donors.
The Ethical Committee of Peking University First Hospital
approved the project, and informed consent was obtained from
the patients and their family members.
Clinical InvestigationsComprehensive clinical data, including renal and extrarenal man-
ifestations, GBM ultrastructural changes, immunohistochemical
changes of a (IV) chains in renal and skin tissues, and family
history, were collected and analyzed. Type IV collagen in basement
membranes were stained by murine monoclonal antibodies
(Wieslab, Lund, Sweden) against NC domain of a1 (IV), a3(IV), and a5 (IV) chains.
Identification of Mutations in COL4A3 and COL4A4GenesGenomic DNA from peripheral blood leukocytes (PBL) was first
analyzed for COL4A3 mutations. If no mutation was identified in
COL4A3 gene, the COL4A4 gene was analyzed. All coding exons of
COL4A3 and COL4A4 genes were PCR-amplified and sequenced
from genomicDNA. Primer sequences were designed by primer 3.0
and the list of primers is available on request. The PCR reactions of
all coding exons of COL4A3 and COL4A4 genes were performed in
a volume of 25 ml consisting of 12.5 ml 2� Taq PCR Master Mix
(Tiangen Biotech, Beijing, China), 1ml 5mmol/L forward primer
and reverse primer, respectively, and 1ml 100 ng/ml genomic DNA.
The amplifications were carried out under touchdown PCR with
the condition of annealing temperature from 64�C to 58�C,descending 1�C every two cycles, annealing at 58�C for 26 cycles.
Direct sequencing was performed using standard methods
(SinoGenoMax, Beijing, China). By using reverse transcription-
polymerase reaction (RT-PCR) and direct sequencing, the entire
coding region of COL4A3mRNA, extracted from the Epstein Barr
virus transfected PBL, was analyzed in five patients.
RESULTS
Clinical FeaturesHematuria was detected in all patients. Proteinuria was detected in
88% of patients at age 1 month to 27 years (median 6 years), and
nephrotic-level proteinuria (>3.5 g/24hr for adults; >0.05g/kg/24hr
for children) was detected in 41% of patients at age 20 months to
27 years (median 11 years).Of 16 patients with information of renal
function, only two males (aged 16 and 27 years, respectively) had
increased blood urea nitrogen and serum creatinine, but not high
enough to diagnose ESRD. Sensorineural hearing loss was found in
58% of patients (7/12) at age 5.5–27 years (median 11 years), and
the other five probands had normal audiograms. Of 10 patients
with ophthalmologic examination, none of them had anterior
lenticonus, and only one had unilateral cataract at age 27 years
(Table I).
Electronmicroscopy was performed in 15 patients. Lamellation,
thickening and thinning ofGBMwere observed in 14 patients at age
20 months to 27 years (median 6 years). Diffuse thinning with
segmental thickening of GBM and splitting of the lamina densa
occurred in one patient (Patient 16) at age 6 years. Immunohis-
tochemical analysis showed 16 patients with an absence of a3 (IV)and a5 (IV) chains in the GBM and normal staining of a5 (IV)
chain in the BCBM and part of the TBM, only one patient (Patient
16) showed the normal expression ofa3 (IV) anda5 (IV) chains inrenal tissue. All 13 patients who had skin biopsies showed positive
staining of a5 (IV) chain in the EBM.
Clinical data of 17 family members, including 13 parents and 4
unrelated siblings were obtained. Four parents presented with
hematuria, two presented with hematuria and proteinuria, and
the other seven had a normal urinalysis. In two families, neither
consanguineous, both parents had abnormal urinalysis. Data of
renal function were not available for any parent, and kidney biopsy
was not performed in six parents with abnormal urinalyses. All four
siblings had hematuria and proteinuria. The elder sister of Patient 9
diedwith uremia at the age of 14, and the elder brother of Patient 17
had sensorineural hearing loss and normal renal function at the age
of 10. Data of renal function were not available for the other two
siblings.
COL4A3 MutationsEighteen pathologic COL4A3 mutations were identified in 14 of
the 17 patients, a mutation detection rate of 82% (Table II).
According to the Human Gene Mutation database, 16 (89%)
of these mutations were novel. Four patients had homozygous
mutation, and 10 compound heterozygousmutations. In addition,
five different polymorphisms were detected, of which four were
novel.
2 AMERICAN JOURNAL OF MEDICAL GENETICS PART A
COL4A4 MutationsFive functionally significant COL4A4mutations were identified in
three patients who were not found to carry a pathogenic COL4A3
mutation (Table II). Four (80%) of these mutations were novel.
Two patients had compound heterozygousmutations, and one had
only one mutation.
Correlations Between Genotypes and PhenotypesAn in-frame deletion (40_63del24) in exon 1 of the COL4A3 was
found in four patients in our study. Patient 4 was a compound
heterozygote forG836E and the deletion inCOL4A3, Patient 5was a
compound heterozygote for R791X from his mother and the
deletion from his father, Patient 15 was a compound heterozygote
for 713_714insC and the deletion in COL4A3, and Patient 6 was a
homozygote for the deletion. The age at first feature identified in
these patients was 5.6 years, 1 month, 1 year, and 2.5 years,
respectively. Patients 5, 6, and 15 had hearing loss since age 6,
11, and 8.5, respectively, and Patient 5 had increased blood urea
nitrogen and serum creatinine at age 16 years. All parents who
carriedheterozygous 40_63del24mutationhadno renal symptoms.
Data on renal function were available for 17 patients from 16
families. Only two male patients (Patients 2 and 5 above) had
increased blood urea nitrogen and serum creatinine. Patient 2, as
reported previously [Hou et al., 2007] was a 27-year-old male born
to consanguineous parents. Hearing loss appeared at age 14 years
and unilateral cataract at age 21 years. At age 27, he presented with
microhematuria, nephrotic-level proteinuria, increased blood urea
nitrogen and serum creatinine, and hypertension (150/100mmHg).
He had a G1242D homozygous mutation.
The pure-tone audiograms of 12 unrelated probands with
COL4A3 gene mutations documented, and hearing loss in seven
(58%), six of them had compound heterozygous or homozygous
mutations which were predicted to produce a truncated a3 (IV)
chain, and the remaining patient carried the G1242D homozygous
mutation.
Both clinical data andmutation analysis were available for some
family members of probands. The younger brother of Patient 7,
affected with a COL4A3 homozygous mutation (1323_1324insA),
presented with hematuria and proteinuria, and the elder brother
of Patient 17, affected with two different COL4A4 mutations
(G728E and IVS22þ 1G>C), manifested with microhematuria,
proteinuria (1.4 g/24 hr), normal renal function and hearing loss at
age 10 years. Of seven parents with a heterozygous COL4A3
mutation, twohad isolatedhematuria, andfivewere asymptomatic.
Of three parents with a heterozygous COL4A4 mutation, two
presented with hematuria and proteinuria, and one with isolated
hematuria.
DISCUSSION
There are no large studies of the clinical history of ARAS, so present
study helps to characterize the clinical features of Chinese ARAS
patients. All patients presented with hematuria, and 88% showed
with proteinuria. None of this series of patients developed ESRD,
and only two patients (aged 16 and 27 years, respectively) had
increased blood urea nitrogen and serum creatinine. Other series
TABLE I. Clinical Features of Patients With Autosomal Recessive Alport Syndrome
Patient Gender
Age atdiagnosis/onsetage (years) Consanguinity
Nephrotic-levelproteinuriaa
Renalfunctiona
Hearinglossa
Ocularlesionsa
Familyhistory ofhematuria
1 M 4.6/3.8 � � N ND ND þ2 M 27/14 þ þ Decreased þ þ �3 F 7.5/2 � þ N þ � �4b F 6/5.6 � � N ND ND �5 M 16/0.1 � þ Decreased þ � �6 M 11/2.5 � þ N þ � �7 F 14/14 � þ N þ ND þ8 F 3/3 � þ N � � �9 F 11/11 þ � N � � þ10 M 3/2.5 � � N ND ND þ11c M 1.7/0.6 � þ N ND ND þ12 F 4/2 � � N � � �13 M 12/11 � � N þ � �14 F 4/4 � � N ND ND �15 M 8.5/1 � � N þ � þ16 F 6/1 � � N � � �17 M 7/5.5 � � N � ND þM, male; F, female; N, normal; ND, not determined.aDetermined at the age of diagnosis.bThe proteinuria at last follow-up of patient 4 (aged twelve) was 1.05 g/24 hr with ARB (Losartan) therapy.cThe proteinuria at last follow-up of patient 11 (aged three) was 1.31 g/24 hr with ACEI (Captopril) therapy.
ZHANG ET AL. 3
TABLE
II.CO
L4A3
andCO
L4A4
MutationsandVariants
Identified*
Mutationor
variant
Nucleotide
change
Predictedeffect
onprotein
Location
Region
Mutation
Status
Patient
Frequency
in
controls
Refs.
PathogenicmutationsinCOL4A3
Missense
Gly1207Arga
c.3619G>C
p.Gly1207Arg
Exon
42
CDComphet
10,16
0/50
Thisstudy
Gly1415Arga
c.4243G>C
p.Gly1415Arg
Exon
47
CDH
90/50
Thisstudy
Gly836Glua
c.2507G>A
p.Gly836Glu
Exon
32
CDComphet
40/50
Thisstudy
Gly1242Asp
c.3725G>A
p.Gly1242Asp
Exon
42
CDComphet,H
12,2
0/50
Hou
etal.[2007]
Nonsense
Lys1411Xa
c.4231A>T
p.Lys1411
Exon
47
CDComphet
8ND
Thisstudy
Arg791X
c.2371C>T
p.Arg791
Exon
30
CDComphet
5ND
Thisstudy
Ser425Xa
c.1274C>A
p.Ser425
Exon
21
CDComphet
14
ND
Thisstudy
Deletion
40_63dela
c.40_63del
p.Leu14-Leu21del
Exon
17S
Comphet,H
4,5,6,15
ND
Longo
etal.[2002];
Vega
etal.[2003]
2043_2050del
c.2043_2050del
p.Cys682fs
Exon
28
CDComphet
13
ND
Thisstudy
3392delG
c.3392delG
p.Gly1130fs
Exon
39
CDComphet
14
ND
Thisstudy
Insertion
1323_1324insA
c.1323_1324insA
p.Val442fs
Exon
22
CDH,Comphet
7,3
ND
Thisstudy
713_714insC
c.713_714insC
p.Pro240fs
Exon
13
CDComphet
15
ND
Thisstudy
Splicing
IVS50þ2T>Ab
c.4755þ2T>A
p.Phe1586fs
Intron
50
NC1
Comphet
10
ND
Thisstudy
IVS24þ5G>Tc
c.1575þ5G>T
p.Arg503fs
Intron
24
CDComphet
8ND
Thisstudy
IVS43þ5G>Cd
c.3882þ5G>C
Aberrant
splicing
Intron
43
CDComphet
13
0/50
Thisstudy
IVS11þ2T>C
c.645þ2T>C
Aberrant
splicing
Intron
11
CDComphet
12
ND
Thisstudy
IVS46þ1G>A
c.4153þ1G>A
Aberrant
splicing
Intron
46
CDComphet
3ND
Thisstudy
IVS48-17_18insA
dc.4463-17_4463-18insA
Aberrant
splicing
Intron
48
—Comphet
16
0/50
Thisstudy
Polymorphicvariants
inCOL4A3
Gly43Arg
c.127G>C
p.Gly43Arg
Exon
2CD
H10
ND
Heidet
etal.[2001]
Glu269Lys
c.805G>A
p.Glu269Lys
Exon
14
CDh
11
3/10
Thisstudy
766-13G>A
c.766-13G>A
Intron
13
—h
11
1/5
Thisstudy
1030-18G>A
c.1030-18G>A
Intron
18
—h
15
1/5
Thisstudy
Met1209Ile
c.3627G>A
p.Met1209Ile
Exon
42
CDh
1/5
Thisstudy
PathogenicmutationsinCOL4A4
Missense
Gly728Glu
c.2183G>A
p.Gly728Glu
Exon
28
CDComphet
17
0/50
Thisstudy
Gly1069Glu
c.3206G>A
p.Gly1069Glu
Exon
34
CDComphet
10/50
Thisstudy
Pro1572Leu
c.4715C>T
p.Pro1572Leu
Exon
47
NC1
h11
ND
Boyeet
al.[1998]
Splicing
IVS22þ1G>C
c.1623þ1G>C
Aberrant
splicing
Intron
22
CDComphet
17
ND
Thisstudy
IVS28þ1G>T
c.2383þ1G>T
Aberrant
splicing
Intron
28
CDComphet
1ND
Thisstudy
H,homozygousmutation;h,heterozygous
mutation;Comphet,compoundheterozygous
mutation;7S,the7Sdomainof
a3(IV)
chain;CD,thecollagenousdom
ainof
a3(IV)
chain;NC1,theNCdomainof
a3(IV)
chain;ND,not
detected.
*Mutationsnam
edfollowingtheguidelines
oftheHum
anGenom
eVariationSociety(http://www.hgvs.org/mutnomen).Positionsof
genom
icDNAandam
inoacidwerenum
beredaccordingto
thereference
sequences(NM_000091.4,NP_000082.2)
forCOL4A3
and(NM_000092.4,NP_000083.3)forCOL4A4
usingthefirstcodingATGof
exon
1as
initiation
codon.
aIdenticalCOL4A3
mutationsweredetected
both
atmRNAlevelandgenom
icDNAlevel.
bThemutationIVS50þ2T>Awas
detected
from
DNAspecimen,andtheinsertionof
29bp
ofIVS50was
foundfrom
RNAspecimen.
cThemutationIVS24þ5G>Twas
detected
from
DNAspecimen,andtheloss
ofupstream
exon
24was
foundfrom
RNAspecimen.
dAccordingto
theRESCUE-ESEWebserver
(http://www.genes.mit.edu/burgelab/rescue-ese/),themutationaffected
anexonicsplicer
enhancer(ESE)site.
4 AMERICAN JOURNAL OF MEDICAL GENETICS PART A
found a frequency of ESRD or renal failure was 73% in ARAS
patients [Dagher et al., 2001; Heidet et al., 2001]. Compared to
those studies [Dagher et al., 2001; Heidet et al., 2001], hearing loss
and ocular abnormalities were detected less frequently in our series
(77–91% vs. 58% for hearing loss, and 61–91% vs. 10% in eye
lesions). One possible explanation for the lower rates of ESRD,
hearing loss and ophthalmologic lesions in our series, is that all but
one patient was under age 18 years.
Because COL4A3 and COL4A4 genes are very large, containing
52 and 48 exons, respectively, and there is no mutational hot spots,
genetic diagnosis of ARAS seems better from mRNA than from
DNA. However, it is difficult to obtain COL4A3 and COL4A4
mRNA from target tissues. Meanwhile, in our experience,
COL4A3 and COL4A4 variants detected by analysis of cDNA
generated from Epstein Barr virus-transfected lymphocytes were
not always consistent with the genomic DNA level (data not
shown). There are two patients with inconsistencies between
RNA and DNA detection. In Patient 5, there was no mutation
detected in the RNA specimen, but compound heterozygousmuta-
tions were found in his DNA. In Patient 14, one heterozygous
mutation was detected in RNA, while this mutation plus another
heterozygous mutation were detected in her DNA specimen. Con-
sequently, the entire COL4A3 and COL4A4 coding sequences of all
17 unrelated patients were analyzed by PCR and direct sequencing
of genomic DNA. Mutations were detected in all patients, 82% of
them with mutations in COL4A3 gene and 18% in COL4A4 gene.
PreviousDNA-based single stranded conformation polymorphism
(SSCP) analyses of COL4A3 and COL4A4 in ARAS reported by
others identified mutations in 17–71% [Boye et al., 1998; Heidet
et al., 2001; Longo et al., 2002; Vega et al., 2003]. The highmutation
detection rate in the present study may be attributed to the
sensitivity of direct sequencing and stringent criteria for selecting
patients.
In this study, we characterized the molecular spectrum in
Chinese ARAS patients. First, 82% of our patients had mutations
ofCOL4A3, which suggests that testing forCOL4A3 first may be an
efficient strategy for genetic diagnosis of ARAS. In one series of 10
ARASpatients [Vega et al., 2003], ninehadCOL4A3mutations, and
the ten had a COL4A4 homozygous mutation. In contrast, seven of
10 ARAS in two other series [Longo et al., 2006; Rana et al., 2007]
had COL4A4 mutations. Second, compared to the Human Gene
Mutation Database, splice site mutations accounted for a greater
proportion ofCOL4A3mutations in our series (33% vs. 15%), and
fewer missense or nonsense substitutions (40% vs. 60%). One
possible explanation for this discrepancy is the technical limitations
of the SSCP method. Another possible explanation is the ethnic
diversity: prior series were on European [Vega et al., 2003; Longo
et al., 2006; Rana et al., 2007]. Third, only two of our four patients
affected with homozygous mutations were from consanguineous
families, which accounted for 12% of our patients, and COL4A3 or
COL4A4 compound heterozygousmutations were detected in 71%
of our patients. Other series showed that 38–41% of ARAS patients
with mutations were from consanguineous families, and the fre-
quency ofCOL4A3orCOL4A4 compoundheterozygousmutations
ranged from 14% to 37% [Lemmink et al., 1994; Boye et al., 1998;
Heidet et al., 2001;Vega et al., 2003].This discrepancy indicates that
individuals carrying a heterozygousCOL4A3 orCOL4A4mutation
are relatively common in China. Meanwhile, about 50% of
probands’ parents, carrying a heterozygous COL4A3/4 mutation,
had hematuria with or without mild proteinuria, which suggested
that ARAS should be considered as well as TBMN when a patient
reports hematuria at least in one parent, since the two diseases have
overt different prognoses. Finally, 89% of COL4A3mutations and
80% of COL4A4 mutations identified in the present study were
novel, extending COL4A3 and COL4A4 mutational spectrum and
evidencing the allelic heterogeneity of these genes.
We detected only one single COL4A4 mutation (P1572L) in
Patient 11 born to unrelated parents. He was coincidentally found
to have microscopic haematuria and proteinuria at age 7 months.
On referred to our hospital at age 1.7 years, urinary protein
excretion was 0.86 g/24 hr, and renal functionwas normal. Electron
microscopy of renal biopsy showed typical GBM changes of AS.
Immunohistochemical analysis showednegative stainingofa3 (IV)and a5 (IV) chains in GBM, whereas normal staining of a5 (IV)
chain in the BCBM, part of the TBM and EBM. His mother had
microhaematuria and proteinuria, exhibited normal staining ofa5(IV) in EBM, and carried the same heterozygous mutation.
Urinalysis was not performed in his father. Both his maternal
grandfather and one sister of his maternal grandmother had
hematuria. A heterozygous mutation of P1572L has been seen
before [Boye et al., 1998]. Previously, 7–24% of ARAS patients
were thought vary with a single COL4A3 or COL4A4 mutation
[Boye et al., 1998;Heidet et al., 2001;Vega et al., 2003], as found also
in TBMN [Lemmink et al., 1996; Buzza et al., 2001, 2003; Gross
et al., 2003; Vega et al., 2003; Wang et al., 2004; Hou et al., 2007;
Slajpah et al., 2007].HowheterozygousCOL4A3/4mutations cause
both ARAS and TBMN remains to be established. There is only one
AS patient reported to be double heterozygous with a COL4A3
mutation and a COL4A4 mutation [Nagel et al., 2005].
An in-framedeletion (40_63del24) in exon1of theCOL4A3 gene
was found in four patients in our study, reported previously. An
ADAS family [Longo et al., 2002] with a heterozygous 40_63del24
mutation was ascertained through a 14-year-old boy with micro-
hematuria and mild proteinuria since age 4. At age 14, he had
normal renal function and no ocular abnormalities or hearing loss.
His father had microhematuria, macrohematuria since childhood,
and normal renal function at age 46. Both individuals had the
heterozygous 40_63del24 mutation. His mother had a normal
urinalysis, and no mutation was detected in her. A female with
ARAS [Vega et al., 2003], who was a compound heterozygote for
40_63del24 and a frame-shift deletion of 4316delC in COL4A3,
presented with proteinuria (4.3 g/day), a normal serum creatinine,
sensorineural hearing loss, andmacular degeneration. Her father, a
carrier of 40_63del24, showed no renal disorder. In summary, the
single 40_63del24mutation inCOL4A3 seems to result in little orno
renal dysfunction, whereas the homozygous state of 40_63del24 in
COL4A3 or compound heterozygous mutation of 40_63del24 plus
another nonsense or frameshiftmutation inCOL4A3 gene seems to
cause severe ARAS with hearing loss.
In conclusion, this study clearly demonstrates the main role of
the COL4A3 and COL4A4 genes in the pathogenesis of ARAS and
extends the mutation spectrum of COL4A3 and COL4A4 genes.
And the genotype–phenotype correlations may provide informa-
tion for a better understanding of ARAS.
ZHANG ET AL. 5
ACKNOWLEDGMENTS
Wegreatly appreciate all patients and their families for participating
in this study. We thank Professor JohnMulvihill (America) for the
editing of thismanuscript. This workwas supported by grants from
National Nature Science Foundation (30400482, 81070545, and
39970775), Beijing Nature Science Foundation (7102148), and
National ‘‘Eleventh Five-Year’’ Science and Technology Support
Project (2006BAI05A07).
REFERENCES
BadenasC,PragaM,Taz�onB,Heidet L,ArrondelC,ArmengolA,Andr�esA,Morales E, Camacho JA, Lens X, et al. 2002. Mutations in the COL4A4and COL4A3 genes cause familial benign hematuria. J Am Soc Nephrol13:1248–1254. [PubMed: 11961012].
Boye E,Mollet G, Forestier L, Cohen-Solal L, Heidet L, Cochat P, Gr€unfeldJP, Palcoux JB, Gubler MC, Antignac C. 1998. Determination of thegenomic structure of the COL4A4 gene and of novel mutations causingautosomal recessive Alport syndrome. Am J Hum Genet 63:1329–1340.[PubMed: 9792860].
BuzzaM,Wang YY, Dagher H, Babon JJ, Cotton RG, Powell H, Dowling J,Savige J. 2001. COL4A4 mutation in thin basement membrane diseasepreviously described in Alport syndrome. Kidney Int 60:480–483.[PubMed: 11473630].
Buzza M, Dagher H, Wang YY, Wilson D, Babon JJ, Cotton RG, Savige J.2003. Mutations in the COL4A4 gene in thin basement membranedisease. Kidney Int 63:447–453. [PubMed: 12631110].
CookC, FriedrichCA, Baliga R. 2008.Novel COL4A3mutations inAfricanAmerican siblings with autosomal recessive Alport syndrome. Am JKidney Dis 51:e25–e28. [PubMed: 18436078].
Dagher H, Buzza M, Colville D, Jones C, Powell H, Fassett R, Wilson D,Agar J, Savige J. 2001. A comparison of the clinical, histopathologic,and ultrastructural phenotypes in carriers of X-linked and autosomalrecessive Alport syndrome. Am J Kidney Dis 38:1217–1228. [PubMed:11728953].
Dagher H, Yan Wang Y, Fassett R, Savige J. 2002. Three novel COL4A4mutations resulting in stop codons and their clinical effects in autosomalrecessive Alport syndrome.HumMutat 20:321–322. [PubMed: 12325029].
Ding J, Stitzel J, BerryP,HawkinsE,KashtanCE. 1995.Autosomal recessiveAlport syndrome:Mutation in theCOL4A3 gene in awomanwithAlportsyndrome and posttransplant antiglomerular basement membranenephritis. J Am Soc Nephrol 5:1714–1717. [PubMed: 7780062].
Gross O, Netzer KO, Lambrecht R, Seibold S, Weber M. 2003. NovelCOL4A4 splice defect and in-framedeletion in a large consanguine familyas a genetic link between benign familial haematuria and autosomalAlport syndrome. Nephrol Dial Transplant 18:1122–1127. [PubMed:12748344].
Heidet L,GublerMC. 2009. The renal lesions ofAlport syndrome. J AmSocNephrol 20:1210–1215. [PubMed: 19470679].
Heidet L, Arrondel C, Forestier L, Cohen-Solal L, Mollet G, Gutierrez B,StavrouC,GublerMC, AntignacC. 2001. Structure of the human type IVcollagen gene COL4A3 and mutations in autosomal Alport syndrome.J Am Soc Nephrol 12:97–106. [PubMed: 11134255].
Hou P, Chen Y, Ding J, Li G, Zhang H. 2007. A novel mutation ofCOL4A3 presents a different contribution to Alport syndrome andthin basement membrane nephropathy. Am J Nephrol 27:538–544.[PubMed: 17726307].
Kalluri R, van den Heuvel LP, Smeets HJ, Schroder CH, Lemmink HH,Boutaud A, Neilson EG, Hudson BG. 1995. A COL4A3 gene mutationand post-transplant anti-alpha 3(IV) collagen alloantibodies in Alportsyndrome. Kidney Int 47:1199–1204. [PubMed: 7783419].
Knebelmann B, Forestier L, Drouot L, Quinones S, Chuet C, Benessy F,Saus J, Antignac C. 1995. Splice-mediated insertion of an Alu sequencein the COL4A3 mRNA causing autosomal recessive Alport syndrome.Hum Mol Genet 4:675–679. [PubMed: 7633417].
Lemmink HH,Mochizuki T, van den Heuvel LP, Schr€oder CH, BarrientosA,Monnens LA, vanOost BA, BrunnerHG,Reeders ST, SmeetsHJ. 1994.Mutations in the type IV collagen alpha 3 (COL4A3) gene in autosomalrecessive Alport syndrome. Hum Mol Genet 3:1269–1273. [PubMed:7987301].
Lemmink HH, Nillesen WN, Mochizuki T, Schr€oder CH, Brunner HG,van Oost BA, Monnens LA, Smeets HJ. 1996. Benign familial hematuriadue to mutation of the type IV collagen alpha4 gene. J Clin Invest 98:1114–1118. [PubMed: 8787673].
Longo I, Porcedda P, Mari F, Giachino D, Meloni I, Deplano C, Brusco A,Bosio M, Massella L, Lavoratti G, et al. 2002. COL4A3/COL4A4mutations: from familial hematuria to autosomal-dominant orrecessive Alport syndrome. Kidney Int 61:1947–1956. [PubMed:12028435].
Longo I, Scala E, Mari F, Caselli R, Pescucci C, Mencarelli MA,Speciale C, Giani M, Bresin E, Caringella DA, et al. 2006. Autosomalrecessive Alport syndrome: An in-depth clinical and molecular analysisof five families. Nephrol Dial Transplant 21:665–671. [PubMed:16338941].
Mochizuki T, LemminkHH,MariyamaM,AntignacC,GublerMC, PirsonY, Verellen-Dumoulin C, Chan B, Schr€oder CH, Smeets HJ. 1994.Identification of mutations in the alpha 3(IV) and alpha 4(IV) collagengenes in autosomal recessive Alport syndrome. Nat Genet 8:77–81.[PubMed: 7987396].
Nagel M, Nagorka S, Gross O. 2005. Novel COL4A5, COL4A4, andCOL4A3 mutations in Alport syndrome. Hum Mutat 26:60. [PubMed:5954103].
Nomura S, Naito I, Fukushima T, Tokura T, Kataoka N, Tanaka I, TanakaH, Osawa G. 1998. Molecular genetic and immunohistochemical studyof autosomal recessive Alport’s syndrome. Am J Kidney Dis 31:E4.[PubMed: 10074584].
Rana K, Tonna S, Wang YY, Sin L, Lin T, Shaw E, Mookerjee I, Savige J.2007. Nine novel COL4A3 and COL4A4 mutations and polymorphismsidentified in inherited membrane diseases. Pediatr Nephrol 22:652–657.[PubMed: 17216251].
SlajpahM, Gorinsek B, Berginc G, Vizjak A, Ferluga D, Hvala A, Meglic A,Jaksa I, Furlan P, Gregoric A, et al. 2007. Sixteen novel mutationsidentified inCOL4A3,COL4A4, andCOL4A5genes in Slovenian familieswith Alport syndrome and benign familial hematuria. Kidney Int 71:1287–1295. [PubMed: 17396119].
Torra R, Badenas C, Cof�an F, Callis L, P�erez-Oller L, Darnell A. 1999.Autosomal recessive Alport syndrome: Linkage analysis and clinicalfeatures in two families. Nephrol Dial Transplant 14:627–630. [PubMed:0193810].
Vega BT, Badenas C, Ars E, Lens X, Mil�a M, Darnell A, Torra R. 2003.Autosomal recessive Alport’s syndrome and benign familial hematuriaare collagen type IV diseases. Am J Kidney Dis 42:952–959. [PubMed:14582039].
WangYY,RanaK,Tonna S, LinT, SinL, Savige J. 2004.COL4A3mutationsand their clinical consequences in thinbasementmembranenephropathy(TBMN). Kidney Int 65:786–790. [PubMed: 14871398].
6 AMERICAN JOURNAL OF MEDICAL GENETICS PART A
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