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ORIGINAL ARTICLE
Natural history of genetically proven autosomal recessiveAlport syndrome
Masafumi Oka & Kandai Nozu & Hiroshi Kaito & Xue Jun Fu & Koichi Nakanishi &Yuya Hashimura & Naoya Morisada & Kunimasa Yan & Masafumi Matsuo &
Norishige Yoshikawa & Igor Vorechovsky & Kazumoto Iijima
Received: 12 January 2014 /Revised: 18 February 2014 /Accepted: 21 February 2014# IPNA 2014
AbstractBackground Autosomal recessive Alport syndrome (ARAS)is a rare hereditary disease caused by homozygous or com-pound heterozygous mutations in either the COL4A3 orCOL4A4 genes. Failure to diagnose ARAS cases is common,even if detailed clinical and pathological examinations arecarried out. As the mutation detection rate for ARAS isunsatisfactory, we sought to develop more reliable diagnosticmethods and provide a better description of the clinicopatho-logical characteristics of this disorder.Methods A retrospective analysis of 30 genetically diagnosedpatients with ARAS in 24 pedigrees was conducted. Themutation detection strategy comprised three steps: (1) geno-mic DNA analysis using polymerase chain reaction (PCR)and direct sequencing; (2) mRNA analysis using reversetranscription (RT)-PCR to detect RNA processing abnormal-ities; (3) semi-quantitative PCR using capillary electrophore-sis to detect large heterozygous deletions.
Results Using the three-step analysis, we identified homozy-gous or compound heterozygous mutations in all patients.Interestingly, 20 % of our ARAS patients showed normalexpression of α5 in kidney tissue. The median age of devel-oping end-stage renal disease was 21 years.Conclusions The strategy described in this study improves thediagnosis for ARAS families. Although immunohistochemi-cal analysis of α5 can provide diagnostic information, normaldistribution does not exclude the diagnosis of ARAS.
Keywords Autosomal recessive Alport syndrome .
COL4A3 .COL4A4 . Type IV collagen alpha5
Introduction
Alport syndrome is a hereditary disorder that generally runs aprogressive course. It usually presents in children as hematuriaand proteinuria associated with neurosensory deafness andprogresses to end-stage renal disease (ESRD). Alport syn-drome is genetically heterogeneous and is mainly transmittedas an X-linked trait (85 %). Autosomal recessive Alportsyndrome (ARAS) and autosomal dominant modes of inher-itance are much less prevalent [1, 2].
X-linked Alport syndrome is caused by mutations in theCOL4A5 gene, which encodes the type IV collagen α5 chain[α5(IV)], and autosomal recessive and autosomal dominantforms are caused by mutations in the COL4A3 or COL4A4genes, which encode type IV collagen α3 [α3(IV)] or α4[α4(IV)] chains. Both homozygous and compound heterozy-gous mutations in either COL4A3 or COL4A4 are found inARAS. Electron microscopy examination of the glomerularbasement membrane typically shows thinning and thickeningas well as lamellation, usually called the “basket-weave”change. Staining of type IV collagen tissue typically revealsthat α3(IV), α4(IV) and α5(IV) are missing from the
This study was presented in part at Kidney Week 2013.
Electronic supplementary material The online version of this article(doi:10.1007/s00467-014-2797-4) contains supplementary material,which is available to authorized users.
M. Oka :K. Nozu (*) :H. Kaito :X. J. Fu :Y. Hashimura :N. Morisada :M. Matsuo :K. IijimaDepartment of Pediatrics, Kobe University Graduate School ofMedicine, 7-5-1 Kusunoki-cho, Chuo, Kobe, Hyogo 6500017, Japane-mail: [email protected]
K. Nakanishi :N. YoshikawaDepartment of Pediatrics, Wakayama Medical College, Wakayama,Japan
K. YanDepartment of Pediatrics, Kyorin University School of Medicine,Mitaka, Tokyo, Japan
I. VorechovskyFaculty of Medicine, University of Southampton, Southampton, UK
Pediatr NephrolDOI 10.1007/s00467-014-2797-4
glomerular basement membrane but that the α5(IV) chainpersists in Bowman’s capsule, distal tubules and the epidermalbasement membrane [2]. Although ARAS is thought to be assevere as male X-linked Alport syndrome, large-scale studiesexamining the clinical course of the disease are rare, and themutation detection rates reported in previous reports areunsatisfactory.
To improve current knowledge on ARAS we conducted aretrospective study in which we characterized the clinical andpathological aspects of 30 genetically proven ARAS patientsfrom 24 families. This study is one of the largest of this raredisease performed to date. Using a three-step mutation detec-tion analysis, we identified homozygous or compound hetero-zygous mutations in COL4A3 or COL4A4 in all patients. In arecently published study we reported that 29 % of our patientswith X-linked Alport syndrome were positive for expressionof α5(IV) [3]. In the present article we discuss the distributionof α5(IV) in the glomerular basement membrane in ARASpatients. Storey et al. [4] very recently reported that patientswith a truncating mutation on at least one allele tend to showearly onset of renal failure (age <30 years) compared withpatients without truncatingmutations. This result led us to alsoexamine genotype–phenotype correlations in our patients.
Subjects and methods
Patients
Patients who satisfied one or more of the following selectioncriteria were enrolled in this retrospective study:
1) Patients with clinically proven proteinuria and hematuriaor ESRD, renal pathology showing the basket-weavechange or a thin glomerular basement membrane byelectron microscopy and α5(IV) expression only atBowman’s capsule;
2) Siblings of patients with genetically proven ARAS whohad proteinuria and hematuria or ESRD;
3) Patients with proteinuria and hematuria or ESRD whoserenal pathology showed the basket-weave change or athin glomerular basement membrane by electron micros-copy, and normal α5(IV) expression at the glomerularbasement membrane; these patients were first screenedfor COL4A5 mutations. If the results were negative,screening for the COL4A3 or COL4A4 genes followed;
4) Patients with proteinuria and hematuria or ESRD whoserenal pathology showed the basket-weave change or athin glomerular basement membrane by electron micros-copy but α5(IV) staining was not present. If a patient’sfamily included a severely affected female patient (4a) ora parent showed consanguinity (4b), they were also in-cluded in the study (see Table 1).
Most patients were followed in various local hospitals inJapan. All clinical, laboratory and pathological data werecollected when the request for mutational analysis was accept-ed. Estimated glomerular filtration rates (eGFR) were mea-sured based on the data in the respective patient’s medicalhistory and were calculated using the Schwartz formula forpatients aged ≤19 years and using the Cockcroft–Gault for-mula for patients aged ≥20 years [5–7]. In Japan, hearingscreening by audiometry is available for children aged 6–8,10, 12, 14 and 15 years, and information on age at detection ofhearing loss is thus very reliable. We asked each doctor toconduct an ophthalmologic test once the patient had agreed tomutational analysis.
Protein expression analysis for kidney and skin specimens
The immunofluorescence method used in this study hasbeen described previously [8]. Rat monoclonal antibodiesfor α2(IV) and α5(IV) chains were used (H22 and H52;Shigei Medical Research Institute, Okayama, Japan). Im-ages of kidney α5(IV) staining were sent to us forevaluation of staining patterns and assessed by the sameperson (KN).
Mutation analysis
The genetic analysis consisted of a three-step strategy.
Step 1: Polymerase chain reaction (PCR) and direct sequenc-ing analyses were performed for the COL4A3 andCOL4A4 genes.
Genomic DNAwas isolated from peripheral bloodleucocytes using a QuickGene-Mini80 (FujifilmCorp., Tokyo, Japan) according to the manufacturer’sinstructions. All exons and exon–intron boundaries ofCOL4A3 and COL4A4 were amplified by PCR usingprimer pairs described previously [9]. PCR productswere purified and subjected to direct sequencing.
Step 2: If homozygous or compound heterozygousmutationsinCOL4A3 andCOL4A4were not detected in Step 1,mRNA analysis using reverse transcription (RT)-PCR was performed.
Briefly, RNAwas isolated from blood leucocytesand/or urine sediments using TRIzol (Invitrogen,Carlsbad, CA) and was reverse-transcribed intocDNA using random hexamers and the SuperscriptIII kit (Invitrogen) [10, 11]. cDNAwas amplified bynested PCR. PCR products were purified and sub-jected to direct sequencing. PCR primers are avail-able on request.
Step 3: If mutation detection failed in Steps 1 and 2, weperformed semiquantitative PCR using capillaryelectrophoresis to detect large deletions.
Pediatr Nephrol
This step involved semi-quantitative PCR ampli-fication using capillary electrophoresis that was de-signed to separate PCR products based on their size-to-charge ratio in the interior of a small capillaryfilled with an electrolyte. This method has recentlybeen reported [12, 13]. For quantification of theamplified products, 1 μL of each reaction mixturemixed with 5 μL of loading buffer solution contain-ing size markers (15 and 1500 bp) was analyzedusing capillary electrophoresis (Agilent 2001Bioanalyzer with DNA 1000 Lab Chips; AgilentTechnologies, Palo Alto, CA). Each PCR productwas quantified by measuring the peak area andcalculating the ratio of COL4A3 or COL4A4 PCRproducts of the patient against a normal control. Areduction in the ratio to about 0.5 indicated thepresence of a heterozygous deletion.
Statistical analyses
Statistical analyses were performed using JMP (JMP ver. 8package for Windows; SAS Institute, Cary, NC). Graphs ofthe occurrence of events (age at ESRD, age at detection ofhearing loss) were computed according to the Kaplan–Meiermethod.
Ethical considerations
All procedures were reviewed and approved by the Institu-tional Review Board of Kobe University School of Medicine.Informed consent was obtained from all patients or theirparents.
Results
Clinical features
Thirty genetically proven ARAS patients from 24 familieswho met the selection criteria were enrolled in the study.The distribution of these patients with respect to the inclusioncriteria is shown in Table 1, and the clinical data and selectedlaboratory findings are summarized in Table 2. Hematuria andproteinuria were detected in all cases. The mean age of thepatients at the genetic diagnosis of ARASwas 17.7±8.1 years.Thirteen patients developed ESRD (43 %). The probabilitiesfor the development of ESRD and hearing loss are shown inFig. 1a. The median age of developing ESRD was 21 years.Sensorineural hearing loss was detected in 12 patients (40 %),and the median age of developing hearing loss was 20 years.Of the 30 patients three showed ocular lesions (10 %): one
developed retinal degeneration, one had a perimacular fleckand the remaining patient had band keratopathy.
Pathological findings
Electron microscopy studies on 25 patients revealed thebasket-weave change in 23 patients and thin basement mem-branes in two (aged 2 and 6 years) (Table 2).
Immunohistochemical staining ofα5(IV) was conducted in20 patients, of whom 16 showed an absence of α5(IV) in theglomerular basement membrane and only staining ofBowman’s capsule, which is a typical staining pattern forARAS. Four patients showed normal expression of α5(IV)(20%). Immunohistochemical staining forα3(IV) andα4(IV)expression revealed that two patients of these four patientsshowing normal expression of α5(IV) also showed normalexpression of α3(IV) and α4(IV). However, two patientsshowing α5(IV) expression in Bowman’s capsule only didnot show α3(IV) and α4(IV) expression. Also, the skin biop-sies of two patients showed positive staining for α5(IV) in theepidermal basement membrane.
Family history
Of the 48 parents, 32 did not show any urinary abnormality, 14had hematuria with no proteinuria and one showed hematuriaand mild proteinuria. Data were not available for one parent.
Mutation detection
Detected mutations are shown in Table 3. Of the total 24families with ARAS, 17 had mutations in the COL4A3 geneand seven had mutations in theCOL4A4 gene. Four mutationsin the COL4A3 gene were homozygous mutations, with threepresent in consanguineous families. The remaining 13 patientshad compound heterozygous mutations. For the COLA4A4gene, one patient had a homozygous mutation, consistent withobserved consanguinity in the family, and six had compoundheterozygous mutations.
Table 1 Patient inclusion criteria
Inclusion criteria Number ofpatients
(1) α5 staining: BC only 16
(2) Siblings of genetically proven ARAS 2
(3) α5 staining: GBM and BC; COL4A5 screening:negative
4
(4a) Patients with severely affected female family member 4
(4b) Patients with consanguineous parents 4
BC, Bowman’s capsule; ARAS, autosomal recessive Alport syndrome;GBM, glomerular basement membrane
Pediatr Nephrol
Table2
Clin
icalandpathologicalfindings
Patient
IDSex
Age
(years)
ESRD[age
(years)at
diagnosis]
Hearing
loss
(age
atdetection)
Ocularlesion
Serum
creatin
ine
(µmol/l)
Serum
albumin
(g/dl)
Urinary
protein
creatin
ineratio
eGFR
(ml/m
in/1.73m
2)
Electron
microscopy
findings
Alpha-5
[α5(IV
)]staining
Skin
Alpha-3,-4
[α3(IV
),[α4(IV
)]staining
85Female
28–
––
0.64
3.5
2.9
120.8
BWC
GBM,B
CND
Normal
94Female
17–
––
13.9
0.24
72.8
BWC
GBM,B
CND
ND
108
Male
16–
––
0.71
4.4
1.2
128.5
BWC
BC
ND
ND
114
Male
20–
Severe
(18years)
–2.24
3.1
1.56
50.2
BWC
BC
ND
ND
115
Female
19–
Severe
(6years)
–1.06
3.6
0.12
69BWC
BC
Normal
ND
125–1
Female
23–
–Retinalregeneratio
n1.2
3.6
0.46
39.2
BWC
BC
ND
ND
125–2
Male
2222
––
7.5(ESR
D)
4.2
2.4
7.1
BWC
ND
ND
ND
125–3
Male
11–
––
0.4
4.2
0.14
127.4
ND
ND
ND
ND
130–1
Female
1811
––
ESR
DBWC
ND
ND
ND
130–2
Female
1615
–Perim
acular
fleck
ESR
DND
ND
Normal
ND
137–1
Female
2726
––
ESR
DBWC
ND
ND
ND
137–2
Female
2513
Mild
(20years)
–ESR
DBWC
ND
ND
ND
143
Female
2–
––
0.24
4.1
0.57
111.1
TBM
BC
ND
ND
145
Female
17–
––
0.56
3.6
0.42
107.1
BWC
GBM,B
CND
ND
155–1
Male
3619
Severe(5
years)
–ESR
DBWC
ND
ND
ND
155–2
Female
3321
Severe(5
years)
Band-keratopathy
ESR
DND
ND
ND
ND
156
Female
8–
––
0.29
40.43
131
BWC
BC
ND
ND
165
Male
6–
––
0.2
4.2
0.3
139.7
TBM
BC
ND
ND
166
Female
2018
––
ESR
DBWC
BC
ND
ND
167
Female
21–
Severe
(10years)
–0.41
2.6
2.1
140.7
BWC
BC
ND
ND
168
Male
2019
Mild
(13years)
–ESR
DBWC
BC
ND
ND
169
Male
19–
Mild
(19years)
–0.79
3.4
6.3
117.4
BWC
BC
ND
Negative
170
Female
7–
––
0.33
3.7
0.8
120.1
BWC
GBM,B
CND
Normal
171–1
Female
1811
Mild
(3years)
–ESR
DBWC
ND
ND
ND
171–2
Male
169
Mild
(1years)
–ESR
DND
ND
ND
ND
172
Male
1614
Mild
(16years)
–ESR
DBWC
BC
ND
Negative
173
Male
2525
Mild
(20years)
–ESR
DBWC
BC
ND
ND
174
Male
3–
––
0.32
4.1
0.53
102.6
BWC
BC
ND
ND
204
Male
9–
––
0.44
3.6
1.1
111.1
BWC
BC
ND
ND
218
Male
13–
––
0.77
4.3
0.17
98.2
BWC
BC
ND
ND
ESR
D,E
nd-stage
renald
isease;eGFR
:estim
ated
glom
erular
filtrationrate;E
M,electronmicroscopicfindings;B
WC,basket-weave
changes;TBM,thinbasalm
embrane;ND,not
determ
ined
Pediatr Nephrol
Although most mutations were found during Step 1 of ourmutation detection pipeline, six DNA alternations from fivefamilies were not identified by standard PCR and direct
sequencing of each exon and exon–intron boundaries. Thesepatients are described in more detail in the Electronic Supple-mentary Material (ESM).
Nineteen of 24 familial cases had homozygous or com-pound heterozygous mutations detected by standard PCR anddirect sequencing (Step 1), and the mutations of a further fivefamilies were detected using the remaining mutation detectiontechniques. Three mutations influenced RNA processing,namely, a putative branch site mutation (ESM Fig. 1), acryptic splice site activation derived from Alu retrotransposon(ESM Fig. 2) and a small intron deletion resulting in exonskipping (ESM Fig. 3). In two patients we proceeded to Step3, which revealed a large heterozygous deletion mutationencompassing exons 43 and 44 in one patient (ESM Fig. 4)and a large heterozygous deletion of exons 8–25 in the other(ESM Fig. 5).
All patients, with the except of the two whose parents’DNAwas not available (parents of patients ID 156 and 204),showed mutations in trans (mutation 1 is on the paternal alleleand Mutation 2 is on the maternal allele in Table 3).
Genotype–phenotype correlations
Truncating mutations were present on both alleles in threepatients and on one allele in eight patients. Nineteen patientshad non-truncating mutations on both alleles (Table 3). Wecompared renal severity and hearing loss probability betweentwo groups of patients, namely, those having truncating mu-tations on at least one allele (Group T, n=11) and thosewithout truncating mutations (Group N, n=19). No significantdifferences were detected in ESRD probability and hearingloss probability between the two groups (P=0.40 and 0.42,respectively) (Fig. 1b,c).
We conducted an intra-familial validation of the five fam-ilies with more than two affected members. In four of thesefamilies, two affected members from each family had alreadydeveloped ESRD at the ages of 11 and 15, 26 and 13, 19 and21, and 11 and 9, respectively (patient ID 130, 137, 155 and171, respectively). These data indicate that intra-familial va-lidity was not common in the ARAS families. There werethree patients with two truncating mutations; one was 19 yearsold and her eGFR was 69 ml/min/1.73 m2, and the other twodeveloped ESRD at the ages of 19 and 21 years, respectively.Our total dataset showed that our patients with ARAS devel-oped ESRD at the median age of 21 years in ARAS, suggest-ing that the presence of two truncating mutations did not affectthe severity of ARAS.
Discussion
To the best of the authors’ knowledge, this is one of the largeststudies conducted to date which has examined the clinical
Fig. 1 a Probability of end-stage renal disease (ESRD) and hearing loss(HL). The median ages for developing ESRD and HL were 21 and20 years, respectively. b Probability of ESRD according to the type ofmutations.Group T patients with truncatingmutations on at least one allele,Group N patients without truncating mutations. There was no significantdifference between two groups (P=0.40). c Probability of HL according tothe type of mutations. Group T Patients with truncating mutations on atleast one allele, Group N patients without truncating mutations. There wasno significant difference between two groups (P=0.42).
Pediatr Nephrol
Table3
Mutationalanalysisandfamily
histories
Patient
IDGene
Mutation1
Mutation2
Num
berof
truncating
mutations
Father
Mother
Consanguinity
Positio
nNucleotidechange
Aminoacid
change
Positio
nNucleotidechange
Aminoacid
change
85COL4
A4
Exon34
c.3151G>C
p.G1051R
Exon29
c.2510G>C
p.G837A
0OB(G
1051R)
–
94COL4
A3
Intron
2c.162–2A
>G
Exon3(90bp)skip
Exon51
c.4793
T>G
p.L1598R
0–
–
108
COL4
A3
Intron
45c.4028–27A
>G
Exon46
(126
bp)skip
Exon33
c.2698_2714del
17bp
deletio
n
(frameshift)
1OB(IVS4
6-27A>G)
–
114
COL4
A3
Exon38
(H)
c.3266G>A
p.G1089D
0–
–
115
COL4
A3
Exon26
c.1843insC
1-bp
insertion
(frameshift)
Exon42
c.3687delG
fram
eshift
2–
–
125
COL4
A3
Exon48
c.4354A>T
p.S1
452C
Exon30
c.2330G>A
p.G777D
0ND
–
130
COL4
A3
Exon51
c.4793
T>G
p.L1598R
Exon30
c.2330G>A
p.G777D
0–
pro+OB+(G
777D
)
137
COL4
A3
Exon51
c.4928G>A
p.R1643K
Exon1
c.40_63del
24bp
deletio
n0
––
143
COL4
A3
Exon28
c.2125G>T
p.G709X
Exon51
c.4793
T>G
p.L1598R
1–
OB(L1598R)
145
COL4
A4
Exon36
(H)
c.3307G>A
p.G1103R
0–
–+
155
COL4
A3
Intron
48(H
)c.4463–523C>G
Crypticexon
(139
bp)in
intron
48with
stop
codon
2–
–+
156
COL4
A4
Exon30
c.2608G>C
p.G870R
Exon39
c.3687insA
fram
eshift
1–
–
165
COL4
A3
Exon13
c.689G
>A
p.G230D
Intron
24c.1476–20_6del15bp
Exon25
(183
bp)skip
0OB
OB
166
COL4
A3
Exon19
c.1060G>T
p.G354X
Exon26
c.1855G>A
p.G619R
1–
OB(G
619R
)
167
COL4
A3
Exon50
c.4708
T>C
p.C1570R
Exon1
c.40_63del
24bp
deletio
n0
––
168
COL4
A3
Exon26
(H)
c.1918G>A
p.G640R
0OB(G
640R
)OB(G
640R
)+
169
COL4
A3
Exon51
c.4793
T>G
p.L1598R
Exon43–44
c.3752–511_3955+576del
Exon43–44
(204
bp)
deletion
0–
–
170
COL4
A3
Exon22
c.1354G>A
p.G452R
Exon43
c.3821insG
1bp
insertion
(frameshift)
1OB(G
452R
)–
171
COL4
A3
Exon1(H
)c.40_63del
24-bpdeletion
0–
–+
172
COL4
A4
Exon39
c.3612_3621del
10-bpdeletion
(frameshift)
Exon27
c.2084G>A
p.G695D
1OB(c.3612_3621del10bp)
OB(G
695D
)
173
COL4
A3
Exon51
c.4793
T>G
p.L1598R
Exon40
c.3464
T>G
p.G1155D
0–
–
174
COL4
A4
Exon24
c.1733G>T
p.G578V
Exon45
c.4241_4254del14bp
fram
eshift
1–
OB(c.4241_4254del14bp)
204
COL4
A4
Exon27
c.2084G>A
p.G695D
Exon46
c.4469G>C
p.G1490A
0–
–
218
COL4
A4
Exon32
c.2878G>A
p.G960R
Exon8–25
c.559–491_1460–
808delinsPolyT
Exon8–25
(1498bp)
deletion
1OB(c.2878G
>A)
OB(Exon8–25
deletio
n)
Pediatr Nephrol
phenotypes and pathological characteristics of ARAS accom-panied by a step-wise and comprehensive mutation analysischaracterizing each disease allele at the nucleotide level. Allprevious reports describing ARAS are summarized in Table 4[4, 14–29]. Heidet et al. studied 60 patients with ARAS from45 families and detected mutations on both alleles in 53 % ofthe 45 families [18]. In that study, 44 patients had progressedto ESRD at a median age of 21.8 years, and hearing loss wasdetected in 27 of the 35 patients who underwent hearing tests.More recently, Storey et al. [4] reported on 40 ARAS patients.These researchers detected mutations on both alleles in the 40ARAS patients (20 %) from among the 205 patients referredfor genetic testing, including not only typical ARAS cases butalso cases with a thin basement membrane which could be acarrier state of ARAS. The median age at ESRD was22.5 years, and 23 of 35 (66 %) patients and 10 of 18(56 %) patients had hearing loss and ocular lesions, respec-tively. The median age of patients developing ESRD in ourstudy was 21.0 years, suggesting that renal symptoms inARAS might be more severe than those in X-linked Alportsyndrome [30]. In males with X-linked Alport syndrome, theprobability of hearing loss by the age of 15 years is 50 % [30].Our results suggest that, in contrast to renal symptoms, co-chlear hearing loss in ARASmay not be as severe as that in X-linked Alport syndrome, in which the median age for thedevelopment of hearing loss was 20 years with a frequencyof 40 %. These data are very reliable because due to thehearing screening system for school children in Japan. About15% ofmales with X-linked Alport syndrome exhibit anteriorlenticonus or other eye lesions [30]. In our study, three of 30patients (10 %) showed eye lesions, a somewhat lower fre-quency. Our data also show a relatively rare incidence ofhearing loss and ocular lesions, which may be due to theinclusion of younger patients in our study (average age17 years) compared with previous studies [4, 18]. Storeyet al. [4] reported that the median ages for detecting hearingloss and ophthalmologic findings among their patients withARAS were 33.82 and 37.4 years, respectively. Other studieshave also reported relatively older median ages for detectingthese abnormalities for the first time (Table 4). It is possiblethat the differing results are due to tests being conducted byinexperienced physicians.
In our study, four of the 20 patients (20 %) who underwentimmunohistochemical staining of α5(IV) showed normal ex-pression of α5(IV) in the glomerular basement membrane.Two of these four patients had two missense mutations, onehad a missense mutation and a splice site mutation resulting inan in-frame deletion and one had a missense mutation and aframe-shift mutation. We attempted to uncover the geneticbackground of these four α5(IV)-positive patients, similar towhat we had done in our previous study on X-linked Alportsyndrome cases [3], but this attempt was unsuccessful due tothe small population size. Therefore, the only conclusion to be
drawn is that patients with two truncating mutations did notshow α5(IV) expression. Two patients with normal α5(IV)expression were also examined for α3(IV) and α4(IV) ex-pression, and interestingly, both showed normal expression ofthe latter in the glomerular basement membrane. Two patientswho showedα5(IV) only at Bowman’s capsule were analyzedusingα3(IV) andα4(IV) stainingm and both showed negativeexpression of the two proteins in the glomerular basementmembrane. Some of the ARAS patients reported in previousstudies also showed normal expression of α5(IV) in the glo-merular basement membrane. Gubler et al. reported that onepatient from among eight ARAS kindreds showed normalexpression of α3(IV), α4(IV) and α5(IV) and that oneshowed a peculiar pattern of distribution of these chains[31]. Zhang et al. reported that one of 17 ARAS patientsshowed a normal distribution of both α3(IV) and α5(IV)[28]. Heidet et al. reported that α5(IV) expression was posi-tive in three of their 11 patients, although they were unable todetermine whether those three patients had mutations in theCOL4A3 or COL4A4 gene [18]. Our study is the first to showthe rate of α5(IV) positivity in genetically proven ARASpatients. Thus, although immunohistochemical analysis ofthese proteins can provide diagnostic information, normaldistribution of these chains does not exclude a diagnosis ofARAS, and genetic testing is generally a more reliable tool forthe diagnosis of this condition.
In our recent study of patients with X-linked Alport syn-drome [3], we found that 29 % of patients showed α5(IV)positive expression. In this same study, we also observedmilder clinical phenotypes, significantly lower urinary proteinlevels, and significantly older age at onset of ESRD in theα5(IV)-positive group compared with the α5(IV)-negativegroup [3]. In the current study, the small number of patientswho tested positive for α5(IV) limited our study of correla-tions between α5(IV) positivity and its clinical phenotype.
Genetic analysis was conducted using a three-step meth-odology: (1) PCR and direct sequencing analysis of genomicDNA for all exons and exon–intron boundaries; (2) RT-PCRand direct sequencing analysis of RNA extracted from bloodleucocytes and/or urine sediments; (3) semi-quantitative PCRamplification using capillary electrophoresis. Using all steps,we detected homozygous or compound heterozygous muta-tions in all patients. Our stepwise mutation detection strategyyielded a 100 % detection rate, where 16 of the 24 familiesshowed typical pathological findings for ARAS, two werefrom consanguineous parents and two were severely affectedfemale cases. The remaining four families were first screenedfor the COL4A5 gene because they showed normal α5(IV)expression with lamellation of the glomerular basement mem-brane, and we failed to detect mutations in that gene. Wetherefore proceeded with COL4A3 and COL4A4 screeningof these families. Our stepwise approach is an improvementover previous studies which reported a detection rate of <80%
Pediatr Nephrol
Table4
Reviewof
previous
articleson
autosomalrecessiveAlportsyndrom
e(A
RAS)
Firstauthor
Methods
Totaln
umberof
ARAScases
Mutation
detection
rate
End-stage
renalfailure
Hearing
loss
Ocularlesions
Num
berof
ARAS
patients
Frequency
Median
age(years)
Num
berof
patients
Frequency
Medianage
(years)
Num
berof
patients
Frequency
Median
age(years)
DingJ[14]
gDNAsequencing
11/1(100
%)
1/1
100%
20N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
BoyeE[16]
SSC
P+gD
NAsequencing
10from
8families
8/31
(26%)
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
TorraR[25]
Linkage
analysis
5N.D.
2/5
40%
334/5
80%
17.25
0/4
0%
N.D.
HeidetL
[18]
SSC
P+gD
NAsequencing
60from
45families
22/45(49%)
44/60
73%
21.8
27/35
77%
N.D.
16/26
62%
N.D.
Longo
I[20]
SSC
P+gD
NAsequencing
302/30
(7%)
10/30
33%
24.2
15/30
50%
N.D.
6/30
20%
N.D.
SakaiK[22]
Immunohistochem
icalfindings
1N.D.
1/1
100%
301/1
100%
N.D.
N.D.
N.D.
N.D.
Gross
O[17]
gDNA+cD
NAsequencing
22/2(100
%)
0/2
0%
–2/2
100%
7.5
N.D.
N.D.
N.D.
Longo
I[19]
DHPL
C+gD
NAsequencing
7from
5families
5/5(100
%)
3/7
43%
132/5
40%
38.5
1/4
100%
28
RanaK[21]
SSC
PandgD
NAsequencing
51/5(20%)
4/5
80%
N.D.
5/5
100%
35.4
5/5
100%
35.4
ShawEA[23]
Linkage
analysis
7N.D.
6/6
100%
23.55
7/7
100%
34.43
7/7
100%
34.43
Zhang
Y[28]
gDNAsequencing
1716/17(94%)
0/15
0%
–7/12
58%
13.71
1/10
10%
27
TemmeJ[24]
Unknown
29N.D.
3/29
10%
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
ArtusoR[15]
Nextg
enerationsequencing
22/2(100
%)
1/2
50%
24N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
WangY[26]
gDNAor
cDNAsequencing
15from
10families
N.D.
14/15
93%
27.2
15/15
100%
N.D.
13/15
87%
N.D.
StoreyH[4]
gDNAsequencing
4040/205
(20%)
20/32
63%
22.5
23/35
66%
33.82
10/18
56%
37.4
WebbBD[27]
Linkage
analysis
3N.D.
0/3
0%
–3/3
100%
21/3
33%
2
Crovetto
F[29]
Unknown
2N.D.
0/2
0%
–1/1
100%
110/2
0%
–
Current
study
gDNA+cD
NAsequencing
+semi-quantitativePCR
30from
24families
24/24(100
%)
13/30
43%
2112/30
40%
203/30
10%
N.D.
SSCP,Single-strand
conformationpolymorphism;D
HPL
C,enaturing
high
performance
liquidchromatography;
N.D.,notd
etermined
Pediatr Nephrol
[16, 18, 20, 32]. Zhang et al. [28] recently reported on 17ARAS cases and detected homozygous or compound hetero-zygous mutations in almost all patients except for one allele.These authors reported the absence of deep intronic mutationsor large heterozygous deletions. We believe the high detectionrate in our study is due to both our mutation detection strategyand our strict inclusion criteria.
We detected five unique deep intronic mutations that re-sulted in abnormal splicing in transcription and large hetero-zygous deletions that were not identified with standard PCRand direct sequencing methods. One affected family (patientID 155) showed a deep intronic mutation which produced anew splice site and led to the creation of cryptic exons intranscripts. This kind of mutation has been reported to bepresent in various diseases, including X-linked Alport syn-drome [33–35], and can be detected only by RT-PCR anddirect sequencing. A number of recent studies have reportedhigh sensitivity for the detection of mutations in the COL4A5gene using next generation sequencers (NGS) [15, 36–38].High-throughput sequencing technologies are intended tolower the cost of DNA sequencing and would help detectunidentified mutations, such as the somatic mosaic mutationspresent at a low frequency or deep intronic mutations. In thefuture, it may be necessary to alter Step 1 of our analysis to useNGS; however, even NGS analysis cannot detect splicingabnormalities and heterozygous large deletions which couldbe detected by Step 2 and Step 3 in the current study.
Storey et al. recently reported that patients with a truncatingmutation on at least one allele tended to show early onset ofrenal failure (age of <30 years) compared with patients with-out truncating mutations [4]. In our study, we did not observeany genotype–phenotype correlations, even when the patientswere divided into two groups similar to those of Storey et al..Further studies are therefore required.
In conclusion, th comprehensive mutation detection strate-gy reported here and its application in one of the largest cohortstudies of ARAS patients published to date contribute to ourunderstanding of the natural history of ARAS. Our resultsillustrate the importance of searching for RNA processingdefects and large deletions. Correct diagnosis is indispensablefor appropriate genetic counseling and genetic prognosis forARAS families. Normal expression of α chains in the glo-merular basement membrane does not exclude a diagnosis ofARAS. We were not able to detect genotype–phenotype cor-relations in this study.
Acknowledgements The authors gratefully acknowledge the coopera-tion of the attending physicians in this study: FusakoHashimoto, YoshimiNozu, Keiko Yasuda, Hironobu Nagasako, Eihiko Takahashi, KenjiIshikura, Naonori Kumagai, Naoko Ito, Yoshimitsu Goto, YoshitsuguKaku, Hidekazu Sugiura, Atsuya Kaimori, Tsuneki Watanabe, ShuichiIto, Aiko Nishikawa, Satsuki Daita, Yumi Furuno, Tsukasa Takemura,Yoshiyuki Namai, Taishi Hirano, Hiroaki Ueda, Yasufumi Ohtsuka,Kohei Maekawa, and Emi Sawanobori. All phases of this study were
supported by a grant from the Ministry of Health, Labour and Welfare(Japan) for Research on Rare Intractable Diseases in Kidney and UrinaryTract (H24-nanchitou (nan)-ippan-041 to Kazumoto Iijima) in the “Re-search on Measures for Intractable Diseases” Project, and a Grant-in-Aidfor Scientific Research (KAKENHI) from the Ministry of Education,Culture, Sports, Science and Technology (Subject ID: 25893131 toKandai Nozu).
Conflict of interest None.
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