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: djnc_5855@126.com

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].

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