Research ArticleA Novel COL4A5 Mutation Identified ina Chinese Han Family Using Exome Sequencing

Xiaofei Xiu12 Jinzhong Yuan3 Xiong Deng1 Jingjing Xiao4 Hongbo Xu1

Zhaoyang Zeng5 Liping Guan4 Fengping Xu4 and Sheng Deng12

1 Center for Experimental Medicine and Department of Neurology TheThird Xiangya Hospital Central South UniversityChangsha Hunan 410013 China

2Department of Pharmacy Xiangya Hospital Central South University Xiangya Road 87 Kaifu District ChangshaHunan 410008 China

3Department of Nephrology TheThird Xiangya Hospital Central South University Changsha Hunan 410013 China4 BGI-Shenzhen Shenzhen Guangdong 518083 China5 Cancer Research Institute Xiangya Medical School of Central South University Changsha Hunan 410008 China

Correspondence should be addressed to Sheng Deng dengsxysinacom

Received 14 May 2014 Accepted 20 June 2014 Published 6 July 2014

Academic Editor Yi Guo

Copyright copy 2014 Xiaofei Xiu et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Alport syndrome (AS) is a monogenic disease of the basement membrane (BM) resulting in progressive renal failure due toglomerulonephropathy variable sensorineural hearing loss and ocular anomalies It is caused by mutations in the collagen typeIV alpha-3 gene (COL4A3) the collagen type IV alpha-4 gene (COL4A4) and the collagen type IV alpha-5 gene (COL4A5) whichencodes type IV collagen 1205723 1205724 and 1205725 chains respectively To explore the disease-related gene in a four-generation Chinese Hanpedigree of AS exome sequencing was conducted on the proband and a novel deletion mutation c499delC (pPro167Gln119891119904lowast36)in the COL4A5 gene was identified This mutation absent in 1000 genomes project HapMap dbSNP132 YH1 databases and 100normal controls cosegregated with patients in the family Neither sensorineural hearing loss nor typical COL4A5-related ocularabnormalities (dot-and-fleck retinopathy anterior lenticonus and the rare posterior polymorphous corneal dystrophy)were presentin patients of this familyThe phenotypes of patients in this AS family were characterized by early onset-age and rapidly developinginto end-stage renal disease (ESRD) Our discovery broadens the mutation spectrum in the COL4A5 gene associated with ASwhich may also shed new light on genetic counseling for AS

1 Introduction

Alport syndrome (AS) is a monogenic disease of the base-ment membrane (BM) resulting in progressive renal failuredue to glomerulonephropathy variable sensorineural hearingloss and ocular anomalies It is caused by defects of type IVcollagen which is themajor structural component of BM andnecessary for BM maintenance [1] Type IV collagen com-prises six 120572 chains (1205721ndash1205726) encoded by the collagen type IValpha-1 gene (COL4A1) to the collagen type IV alpha-6 gene(COL4A6) respectively These six 120572 chains share a com-mon primary structure an approximately 25-residue ldquo7Srdquodomain at the amino terminus a collagenous domain ofapproximately 1400 Gly-X-Y repeats and an approximately

230-residue noncollagenous (NC1) domain at the carboxylterminus [2] AS is caused by mutations in the collagen typeIV alpha-3 gene (COL4A3) the collagen type IV alpha-4 gene(COL4A4) and the collagen type IV alpha-5 gene (COL4A5)encoding type IV collagen 1205723 1205724 and 1205725 chains respectively[3] The estimated gene mutation frequency is 15000ndash110000 [4] Three inheritance patterns of AS have beenreported themost commonX-linked inheritance (mutationsin the COL4A5 gene sim85) the less common autosomalrecessive inheritance (mutations in theCOL4A3 gene and theCOL4A4 gene sim15) and the rare autosomal dominantinheritance [5] Genotype-phenotype correlations of AS havebeen extensively described Patients may present with awide spectrum of phenotypes ranging from benign familial

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 186048 5 pageshttpdxdoiorg1011552014186048

2 BioMed Research International

hematuria (BFH) or thin basement membrane nephropathy(TBMN) to end-stage renal disease (ESRD) resulting fromvarious mutations though the COL4A5-related BFH andTBMN were considered to be the milder subtypes of AS [5ndash7]

The purpose of our study is to explore the disease-relatedgene in a four-generation Chinese Han pedigree of ASExome sequencing is a powerful and cost-effective tool foruncovering the genetic basis of diseases [8 9] Conventionalmutation screening by Sanger sequencing is time consumingand expensive due to genetic heterogeneity of AS and largesize of those three genes (COL4A3 COL4A4 and COL4A5)Therefore we detected the proband of the family using exomesequencing to identify the gene responsible for this diseaseA novel deletion mutation c499delC (pPro167Gln119891119904lowast36) inthe COL4A5 gene was identified and it cosegregated with thedisease in the family Our data broaden the genotypic spec-trum of COL4A5mutations associated with AS

2 Materials and Methods

21 Subjects A pedigree consisting of 10 individuals across 4generations of Chinese Han family was enrolled in this study(Figure 1) Peripheral blood samples were collected from 6members of this family including 4 patients Peripheral bloodsamples were also collected from 100 unrelated ethnicallymatched normal controls (malefemale 5050 age 406 plusmn 84years) All participants underwent clinical evaluation audi-tory and typical COL4A5-related ophthalmological examina-tions (dot-and-fleck retinopathy anterior lenticonus and therare posterior polymorphous corneal dystrophy) The proto-col of this study was approved by the Ethics Committee oftheThird Xiangya Hospital Central South University and allparticipants signed informed consent

22 Clinical Data All family members underwent urinalysisand renal function evaluation Members with no more thantrace amount of hematuria or proteinuria and normal renalultrasound examination were considered normal [10] Kid-ney biopsy was performed for the proband Global andsegmental sclerosis and mesangial expansion were identifiedby light microscopy Electron microscopy revealed irreg-ular thickening and splitting of the glomerular basementmembranes (GBMs) Immunofluorescence and electronmicroscopy detected no immunoglobulin A (Ig A) deposi-tion None of the family members showed any evidence ofauditory typical COL4A5-related ophthalmological (dot-and-fleck retinopathy anterior lenticonus and posteriorpolymorphous corneal dystrophy) or platelet abnormalitiesor leiomyomatosis

23 Exome Capture Genomic DNA was isolated fromperipheral blood leukocytes by standard phenol-chloroformextraction method [11] Three micrograms (120583g) of genomicDNAwas used to construct the exome library GenomicDNAof the proband was sheared by sonication and hybridized tothe Nimblegen SeqCap EZ Library for enrichment accordingto the manufacturerrsquos protocol Enriched exome fragments

2

21

1NM

3M

1 2NM

1NM

NN

NN3 4

I

II

III

IV

Figure 1 Pedigree of the family with X-linked Alport syndrome Nnormal M COL4A5 c499delC (pPro167Gln119891119904lowast36) mutation

were sequenced on the HiSeq 2000 platform (Illumina SanDiego CA USA) to get paired-end reads with read length of90 bp A mean exome coverage of 8165times was obtained toaccurately call variants at 9941 of the targeted exome [1213]

24 Read Mapping and Variant Analysis The sequence readswere aligned to human genome reference obtained fromUCSC database (httpgenomeucscedu) version hg19(build 371) using the program SOAP aligner Single nucl-otide polymorphisms (SNPs) were called using SOAPsnp setwith the default parameters after the duplicated reads(produced mainly in the PCR step) were deleted [14] Shortinsertions or deletions (indels) altering coding sequence orsplicing sites were also identified by GATK We filtered can-didate SNPs with the following criterion SNP quality ge20sequencing depth ge4 the estimated copy number le2 and thedistance between two SNPs gt5 (the quality score is a Phredscore generated by the program SOAPsnp103 and qualityscore 20 represents 99 accuracy of a base call) [6] Can-didate mutations were filtered against databases includingthe single nucleotide polymorphism database (dbSNP132httpwwwncbinlmnihgovprojectsSNPsnp summarycgi) 1000 genomes data (1000 genomes release 20100804)HapMap (2010-08 phase II + III) and YanHuang1 (YH1)project and synonymous substitutions Potential disease-causing variants were evaluated by SIFT prediction(httpsiftjcviorg) Sanger sequencing was employed toverify the identified potential disease-causing variants withABI3500 sequencer (Applied Biosystems Foster City CAUSA) Sequences of the primers were as follows 51015840-TGA-ATCTTCAGATCATTTTTCTGG-31015840 and 51015840-GAGGGA-TTGTTGTAATCTTCTGG-31015840

3 Results

We performed exome sequencing of the proband (III 1Figure 1) in a Chinese Han family with AS We generated814 billion bases of 90-bp paired-end read sequence forthe patient Among the 814 billion bases 788 billion (9681)passed the quality assessment 737 billion (9353) aligned

BioMed Research International 3

T C A C T G C C A G G A C C A

T C A C T G C A G G A C C A A

c499delC

(a)

T C A C T G C A G G A C C A

c499delC

(b)

Figure 2 Sequencing analysis of COL4A5 c499delC (pPro167Gln119891119904lowast36) mutation The arrow shows site of the novel c499delC(pPro167Gln119891119904lowast36) deletion mutation in the COL4A5 gene (a) Heterozygous mutation carrier (III 1) (b) Hemizygous mutation carrier(III 3)

to the human reference sequence and 360 billion bases(4885) mapped to the targeted bases with a mean coverageof 8165-fold 105963 genetic variants including 14723 non-synonymous variants were identified in either the codingregions or the splice sites A prioritization schemewas appliedto identify the pathogenic mutation in the patient similar torecent studies [6 15] We excluded known variants identifiedin 1000 genomes project HapMap dbSNP132 and YH1Applying the above strategy we reduced the number ofcandidate genes by more than 9033

A novel deletion mutation c499delC(pPro167Gln119891119904lowast36) was identified in exon 9 of the COL4A5gene in the probandThis mutation results in premature stopcodon and a truncated protein The same mutation was sub-sequently verified in all four affected family members (II 1III 1 III 3 and IV 1 Figure 1) while being absent in un-affected members and 100 ethnically matched normalcontrols by Sanger sequencing (Figure 2) It is also absent in1000 genomes project HapMap YanHuang1 (YH1) projectand dbSNP The mutation is located in the Gly-X-Y repeatsThe pPro167 is a highly conserved amino acid residue amongdifferent species from chicken to human suggesting itsstructural and functional importance (Figure 3) This mut-ation was predicted to affect the protein features and bedisease causing (predicted by httpwwwmutationtasterorg)SIFT prediction also showed a damaging effect with aconfidence score of 0858 (httpsiftbiia-staredusgwwwSIFT indels2html)

4 Discussion

AS is a clinically and genetically heterogeneous disease andseverity of this disease is usually equal between males andfemales in the autosomal recessive form (autosomal recessiveAS ARAS) while greater in males with X-linked form (X-linked AS XLAS) XLAS is caused by mutations in theCOL4A5 gene with an approximately prevalence of 110000

Homo sapiensChimpanzeeRhesus monkeyDogCattleHorseSheepMouseChicken

PGSIIMSSLPGPKGNPGYP

PGSIIMSSLPGPKGNPGYP

PGSIIMSSLPGPKGNPGYP

PGSIIMSSLPGPKGNPGYP

PGSIIMSSLPGPKGNPGYP

PGSIIMSPLPGPKGNQGYP

PGSIIMSSLPGPKGNPGYP

PGSIIMSSLPGPKGDPGYP

AGEIITSLLPGQKGDQGFP

pPro167

Figure 3 Conservation analysis of COL4A5 pPro167 amino acidresidue

[5] and it accounts for 40ndash45 of female patients with AS[16] Female patients with XLAS have a variable and generallymild clinical course with 12 reaching ERSD by the age of40 years and about 30 by the age of 60 years in Europeancohorts [17] While male patients are more severe thanfemales with 70 of affected males developing into ESRDbefore the age of 30 years (juvenile form) the remaining30 are progressing toward ESRD after the age of 30 years(rare adult form) [18] Furthermore hearing loss and ocularabnormality happened in 90 and 35 of male patientsrespectively [10]

In our family four patients presented with heterogeneousclinical phenotypes of glomerulopathy while none of themshowed any clinical features of either sensorineural hear-ing loss or typical COL4A5-related ocular abnormalities ACOL4A5 c499delC (pPro167Gln119891119904lowast36) mutation in exon 9cosegregating with the disease was identified The deletionmutation leads to a truncated protein and is absent in 1000genomes project HapMap YanHuang1 (YH1) project dbSNPand 100 normal controls Our clinical and genetic data alsosupport an X-linked inheritance form of AS in this family

4 BioMed Research International

Table 1 Clinical and genetic data of 4 COL4A5 c499delC (pPro167Glnfslowast36) mutation carriers

Subject II 1 III 1 III 3 IV 1Gender F F M FAge (year) 42 24 22 6Onset-age (year) 13 20 3 3Genotype Heterozygote Heterozygote Hemizygote HeterozygoteRenal function ESRD at 36 years Normal Normal NormalMicroscopic hematuria Yes Yes Yes YesGross hematuria Yes No Yes NoProteinuria Yes No Yes NoUremia Yes No No NoAudiological examination Normal Normal Normal NormalOphthalmic examination⋆ No No No No⋆Dot-and-fleck retinopathy anterior lenticonus and posterior polymorphous corneal dystrophy COL4A5 the collagen type IV alpha-5 gene F female Mmale ESRD end-stage renal disease

The COL4A5 gene is located at Xq22 and contains 51exons encoding type IV collagen 1205725 chain [6] Type IV colla-gen 1205725 chain contains 1685 amino acid residues which con-sist of a 26-residue signal peptide a 1430-residue collagenousdomain starting with a 14-residue noncollagenous sequencea Gly-X-Y-repeat sequence interrupted at 22 locations anda 229-residue carboxyl-terminal NC1 domain [19] To date688 COL4A5mutations have been identified according to theHuman Gene Mutation Database (httpwwwhgmdorg)including missense nonsense deletion splicing mutationand complex rearrangements [17 20] without identificationof any mutation hot spot Genotype-phenotype correlationsbetweenCOL4A5mutations and XLAS have been extensivelydescribed For genotype-phenotype correlation purposestypical XLAS is classified into three types (1) severe type withERSD at sim20 years (juvenile-onset ESRD) 80 of hearingloss and 40 of ocular lesions caused by large rearrange-ments premature stop frameshift donor splice and muta-tions in the NC1 domain (2) moderate-severe type withESRD at sim26 years caused by non-Gly-X-Y-missense Gly-X-Y mutations in 21ndash47 exons (3) moderate type with ESRD atsim30 years (late-onset ESRD) 70 of hearing loss and lt30ocular lesions caused by Gly-X-Y mutations in 1ndash20 exons[21 22] Four patients of our family showed no clinical fea-tures of either sensorineural hearing loss or typical COL4A5-related ocular abnormalities Though our family is not largea moderate type of XLAS is considered due to the mutationlocated in COL4A5 exon 9 and the late-onset ESRD (ESRD at36 years II 1 Table 1) More severe clinical phenotypes andearlier onset-age were observed in male patient of this family(III 3) consistent with previous reports [6]

Mutations in genes encoding 120572 chain of type IV collagencould lead to dysfunction of BM and then lead to thedevelopment of human disease in the eye kidney ear and soforth [1] Once the 1205725 chain is missing the formation of thenormal 120572312057241205725 (IV) protomer is disrupted in BM of glom-erulus ear eye and lung which could lead to structural andfunctional defects [23] This is supported by the immunohis-tochemical finding of frequent loss of 1205723 1205724 and 1205725 signalsin the GBM of XLAS patients [24 25] The cause of clinicalheterogeneity of XLAS such as difference in age of disease

onset disease severity and disease progression may be mul-tifactorial including random X chromosome inactivationethnic background and environment factors

Animal models with genetic deficiency may provideprobabilities to reveal the pathogenesis and treatment of AS[26] Two Col4a5 truncation mutations have been identifiedin dogs (Samoyed and Navasota dogs) with clinical featuresof proteinuria and progressive kidney disease leading toterminal failure [27] Intriguingly a deletion in Col4a5resulting in disruption of the Gly-X-Y repeats similar tohuman pPro167Gln119891119904lowast36mutation was observed in a familyof mixed-breed dogs with an inherited nephropathy thatexhibits the clinical immunohistochemical pathologicaland ultrastructural features of human XLAS and the trun-cated peptide chain may probably prevent extracellularassembly in type IV collagen networks [28] Further studieson the Col4a5 genetic-deficient AS animal models will pro-vide new insight into mechanism research diagnosis andtarget therapy of AS in human

5 Conclusions

In our study we identified a novel deletion mutationc499delC (pPro167Gln119891119904lowast36) in the COL4A5 gene whichmay be responsible for AS in this family Our study showedthat exome sequencing is a fast sensitive and relativelylow-cost method to identify gene(s) responsible for AS Thediscovery broadens the genotypic spectrum of COL4A5mutations associatedwithAS and has implications for geneticdiagnosis therapy and genetic counseling of this family

Conflict of Interests

The authors declare that there is no conflict of interests in thispaper

Acknowledgments

The authors thank the participating individuals for theircooperation and their efforts in collecting the genetic infor-mation and DNA specimens This work was supported by

BioMed Research International 5

National Natural Science Foundation of China (8110133981001476) Natural Science Foundation of Hunan ProvinceChina (10JJ4020 10JJ5029) Construction Fund for Key Sub-jects of theThird XiangyaHospital Central South UniversityChina Postgraduates Innovative Pilot Scheme of HunanProvince (7138000008) China the Fundamental ResearchFunds for the Central Universities of Central South Univer-sity (2014zzts360) and Students Innovative Pilot Scheme ofCentral South University (YC12417) China

References

[1] T van Agtmael and L Bruckner-Tuderman ldquoBasement mem-branes and human diseaserdquo Cell and Tissue Research vol 339no 1 pp 167ndash188 2010

[2] C Arrondel G Deschenes Y le Meur et al ldquoA large tandemduplication within the COL4A5 gene is responsible for the highprevalence of Alport syndrome in French Polynesiardquo KidneyInternational vol 65 no 6 pp 2030ndash2040 2004

[3] J H Suh and J HMiner ldquoThe glomerular basementmembraneas a barrier to albuminrdquo Nature Reviews Nephrology vol 9 no8 pp 470ndash477 2013

[4] Y Pirson ldquoMaking the diagnosis of Alportrsquos syndromerdquo KidneyInternational vol 56 no 2 pp 760ndash775 1999

[5] J Kruegel D Rubel and O Gross ldquoAlport syndromemdashinsightsfrom basic and clinical researchrdquo Nature Reviews Nephrologyvol 9 no 3 pp 170ndash178 2013

[6] Y Guo J Yuan andH Liang ldquoIdentification of a novelCOL4A5mutation in a Chinese family with X-linked Alport syndromeusing exome sequencingrdquoMolecular Biology Reports 2014

[7] B G Hudson K Tryggvason M Sundaramoorthy and E GNeilson ldquoAlportrsquos syndrome Goodpasturersquos syndrome and typeIV collagenrdquoTheNew England Journal of Medicine vol 348 no25 pp 2543ndash2556 2003

[8] M J Bamshad S BNgAW Bighamet al ldquoExome sequencingas a tool for Mendelian disease gene discoveryrdquo Nature ReviewsGenetics vol 12 no 11 pp 745ndash755 2011

[9] C Ku D N Cooper C Polychronakos N Naidoo MWu andR Soong ldquoExome sequencing Dual role as a discovery anddiagnostic toolrdquo Annals of Neurology vol 71 no 1 pp 5ndash142012

[10] B Becknell G A Zender R Houston et al ldquoNovel X-linkedglomerulopathy is associated with a COL4A5 missense muta-tion in a non-collagenous interruptionrdquo Kidney Internationalvol 79 no 1 pp 120ndash127 2011

[11] X Xiu Z Song K Gao et al ldquoGenetic analysis of the FBXO48gene in Chinese Han patients with Parkinson diseaserdquo Neuro-science Letters vol 541 pp 224ndash226 2013

[12] Z Chen J Wang B Tang et al ldquoUsing next-generation sequ-encing as a genetic diagnostic tool in rare autosomal recessiveneurologicMendelian disordersrdquoNeurobiology of Aging vol 34no 10 pp 2442e11ndash2442e17 2013

[13] Y Guo L Yuan J Yi et al ldquoIdentification of aGJA3mutation ina Chinese family with congenital nuclear cataract using exomesequencingrdquo Indian Journal of Biochemistry amp Biophysics vol50 no 4 pp 253ndash258 2013

[14] R Li Y Li K Kristiansen and J Wang ldquoSOAP short oligonu-cleotide alignment programrdquo Bioinformatics vol 24 no 5 pp713ndash714 2008

[15] C Gilissen H H Arts A Hoischen et al ldquoExome sequencingidentifies WDR35 variants involved in Sensenbrenner syn-dromerdquo The American Journal of Human Genetics vol 87 no3 pp 418ndash423 2010

[16] O Gross and C E Kashtan ldquoTreatment of Alport syndromebeyond animal modelsrdquo Kidney International vol 76 no 6 pp599ndash603 2009

[17] L Heidet and M Gubler ldquoThe renal lesions of Alport syn-dromerdquo Journal of the American Society of Nephrology vol 20no 6 pp 1210ndash1215 2009

[18] R Artuso C Fallerini L Dosa et al ldquoAdvances in Alport syn-drome diagnosis using next-generation sequencingrdquo EuropeanJournal of Human Genetics vol 20 no 1 pp 50ndash57 2012

[19] J Zhou J M Hertz A Leinonen and K Tryggvason ldquoCom-plete amino acid sequence of the human 1205725(IV) collagen chainand identification of a single-base mutation in exon 23 con-verting glycine 521 in the collagenous domain to cysteine in anAlport syndrome patientrdquo The Journal of Biological Chemistryvol 267 no 18 pp 12475ndash12481 1992

[20] Q Wang F Liu Y Xing et al ldquoMutation c359 363delGTATTi-nsATAC in the COL4A5 Causes alport syndrome in a Chinesefamilyrdquo Gene vol 512 no 2 pp 482ndash485 2013

[21] M Slajpah B Gorinsek G Berginc et al ldquoSixteen novelmutations identified in COL4A3 COL4A4 and COL4A5 genesin Slovenian families with Alport syndrome and benign familialhematuriardquo Kidney International vol 71 no 12 pp 1287ndash12952007

[22] P Demosthenous K Voskarides K Stylianou et al ldquoX-linkedAlport syndrome inHellenic families phenotypic heterogeneityand mutations near interruptions of the collagen domain inCOL4A5rdquo Clinical Genetics vol 81 no 3 pp 240ndash248 2012

[23] A F Zehnder J C Adams P A Santi et al ldquoX-linked Alportsyndrome in Hellenic families phenotypic heterogeneity andmutations near interruptions of the collagen domain inCOL4A5rdquo Archives of Otolaryngology-Head and Neck Surgeryvol 131 no 11 pp 1007ndash1013 2005

[24] Y M Chen and J H Miner ldquoGlomerular basement membraneand related glomerular diseaserdquoTranslational Research vol 160no 4 pp 291ndash297 2012

[25] J C Wilson H Yoon R J Walker and M R Eccles ldquoA novelCys1638Tyr NC1 domain substitution in 1205725(IV) collagen causesAlport syndrome with late onset renal failure without hearingloss or eye abnormalitiesrdquo Nephrology Dialysis Transplantationvol 22 no 5 pp 1338ndash1346 2007

[26] O Gross B Beirowski M Koepke et al ldquoPreemptive ramipriltherapy delays renal failure and reduces renal fibrosis inCOL4A3-knockout mice with Alport syndromerdquo Kidney Inter-national vol 63 no 2 pp 438ndash446 2003

[27] G E Lees ldquoKidney diseases caused by glomerular basementmembrane type IV collagen defects in dogsrdquo Journal of Veteri-nary Emergency and Critical Care vol 23 no 2 pp 184ndash1932013

[28] M L Cox G E Lees C E Kashtan andK EMurphy ldquoGeneticcause of X-linked Alport syndrome in a family of domesticdogsrdquoMammalian Genome vol 14 no 6 pp 396ndash403 2003

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Microbiology

2 BioMed Research International

hematuria (BFH) or thin basement membrane nephropathy(TBMN) to end-stage renal disease (ESRD) resulting fromvarious mutations though the COL4A5-related BFH andTBMN were considered to be the milder subtypes of AS [5ndash7]

The purpose of our study is to explore the disease-relatedgene in a four-generation Chinese Han pedigree of ASExome sequencing is a powerful and cost-effective tool foruncovering the genetic basis of diseases [8 9] Conventionalmutation screening by Sanger sequencing is time consumingand expensive due to genetic heterogeneity of AS and largesize of those three genes (COL4A3 COL4A4 and COL4A5)Therefore we detected the proband of the family using exomesequencing to identify the gene responsible for this diseaseA novel deletion mutation c499delC (pPro167Gln119891119904lowast36) inthe COL4A5 gene was identified and it cosegregated with thedisease in the family Our data broaden the genotypic spec-trum of COL4A5mutations associated with AS

2 Materials and Methods

21 Subjects A pedigree consisting of 10 individuals across 4generations of Chinese Han family was enrolled in this study(Figure 1) Peripheral blood samples were collected from 6members of this family including 4 patients Peripheral bloodsamples were also collected from 100 unrelated ethnicallymatched normal controls (malefemale 5050 age 406 plusmn 84years) All participants underwent clinical evaluation audi-tory and typical COL4A5-related ophthalmological examina-tions (dot-and-fleck retinopathy anterior lenticonus and therare posterior polymorphous corneal dystrophy) The proto-col of this study was approved by the Ethics Committee oftheThird Xiangya Hospital Central South University and allparticipants signed informed consent

22 Clinical Data All family members underwent urinalysisand renal function evaluation Members with no more thantrace amount of hematuria or proteinuria and normal renalultrasound examination were considered normal [10] Kid-ney biopsy was performed for the proband Global andsegmental sclerosis and mesangial expansion were identifiedby light microscopy Electron microscopy revealed irreg-ular thickening and splitting of the glomerular basementmembranes (GBMs) Immunofluorescence and electronmicroscopy detected no immunoglobulin A (Ig A) deposi-tion None of the family members showed any evidence ofauditory typical COL4A5-related ophthalmological (dot-and-fleck retinopathy anterior lenticonus and posteriorpolymorphous corneal dystrophy) or platelet abnormalitiesor leiomyomatosis

23 Exome Capture Genomic DNA was isolated fromperipheral blood leukocytes by standard phenol-chloroformextraction method [11] Three micrograms (120583g) of genomicDNAwas used to construct the exome library GenomicDNAof the proband was sheared by sonication and hybridized tothe Nimblegen SeqCap EZ Library for enrichment accordingto the manufacturerrsquos protocol Enriched exome fragments

2

21

1NM

3M

1 2NM

1NM

NN

NN3 4

I

II

III

IV

Figure 1 Pedigree of the family with X-linked Alport syndrome Nnormal M COL4A5 c499delC (pPro167Gln119891119904lowast36) mutation

were sequenced on the HiSeq 2000 platform (Illumina SanDiego CA USA) to get paired-end reads with read length of90 bp A mean exome coverage of 8165times was obtained toaccurately call variants at 9941 of the targeted exome [1213]

24 Read Mapping and Variant Analysis The sequence readswere aligned to human genome reference obtained fromUCSC database (httpgenomeucscedu) version hg19(build 371) using the program SOAP aligner Single nucl-otide polymorphisms (SNPs) were called using SOAPsnp setwith the default parameters after the duplicated reads(produced mainly in the PCR step) were deleted [14] Shortinsertions or deletions (indels) altering coding sequence orsplicing sites were also identified by GATK We filtered can-didate SNPs with the following criterion SNP quality ge20sequencing depth ge4 the estimated copy number le2 and thedistance between two SNPs gt5 (the quality score is a Phredscore generated by the program SOAPsnp103 and qualityscore 20 represents 99 accuracy of a base call) [6] Can-didate mutations were filtered against databases includingthe single nucleotide polymorphism database (dbSNP132httpwwwncbinlmnihgovprojectsSNPsnp summarycgi) 1000 genomes data (1000 genomes release 20100804)HapMap (2010-08 phase II + III) and YanHuang1 (YH1)project and synonymous substitutions Potential disease-causing variants were evaluated by SIFT prediction(httpsiftjcviorg) Sanger sequencing was employed toverify the identified potential disease-causing variants withABI3500 sequencer (Applied Biosystems Foster City CAUSA) Sequences of the primers were as follows 51015840-TGA-ATCTTCAGATCATTTTTCTGG-31015840 and 51015840-GAGGGA-TTGTTGTAATCTTCTGG-31015840

3 Results

We performed exome sequencing of the proband (III 1Figure 1) in a Chinese Han family with AS We generated814 billion bases of 90-bp paired-end read sequence forthe patient Among the 814 billion bases 788 billion (9681)passed the quality assessment 737 billion (9353) aligned

BioMed Research International 3

T C A C T G C C A G G A C C A

T C A C T G C A G G A C C A A

c499delC

(a)

T C A C T G C A G G A C C A

c499delC

(b)

Figure 2 Sequencing analysis of COL4A5 c499delC (pPro167Gln119891119904lowast36) mutation The arrow shows site of the novel c499delC(pPro167Gln119891119904lowast36) deletion mutation in the COL4A5 gene (a) Heterozygous mutation carrier (III 1) (b) Hemizygous mutation carrier(III 3)

to the human reference sequence and 360 billion bases(4885) mapped to the targeted bases with a mean coverageof 8165-fold 105963 genetic variants including 14723 non-synonymous variants were identified in either the codingregions or the splice sites A prioritization schemewas appliedto identify the pathogenic mutation in the patient similar torecent studies [6 15] We excluded known variants identifiedin 1000 genomes project HapMap dbSNP132 and YH1Applying the above strategy we reduced the number ofcandidate genes by more than 9033

A novel deletion mutation c499delC(pPro167Gln119891119904lowast36) was identified in exon 9 of the COL4A5gene in the probandThis mutation results in premature stopcodon and a truncated protein The same mutation was sub-sequently verified in all four affected family members (II 1III 1 III 3 and IV 1 Figure 1) while being absent in un-affected members and 100 ethnically matched normalcontrols by Sanger sequencing (Figure 2) It is also absent in1000 genomes project HapMap YanHuang1 (YH1) projectand dbSNP The mutation is located in the Gly-X-Y repeatsThe pPro167 is a highly conserved amino acid residue amongdifferent species from chicken to human suggesting itsstructural and functional importance (Figure 3) This mut-ation was predicted to affect the protein features and bedisease causing (predicted by httpwwwmutationtasterorg)SIFT prediction also showed a damaging effect with aconfidence score of 0858 (httpsiftbiia-staredusgwwwSIFT indels2html)

4 Discussion

AS is a clinically and genetically heterogeneous disease andseverity of this disease is usually equal between males andfemales in the autosomal recessive form (autosomal recessiveAS ARAS) while greater in males with X-linked form (X-linked AS XLAS) XLAS is caused by mutations in theCOL4A5 gene with an approximately prevalence of 110000

Homo sapiensChimpanzeeRhesus monkeyDogCattleHorseSheepMouseChicken

PGSIIMSSLPGPKGNPGYP

PGSIIMSSLPGPKGNPGYP

PGSIIMSSLPGPKGNPGYP

PGSIIMSSLPGPKGNPGYP

PGSIIMSSLPGPKGNPGYP

PGSIIMSPLPGPKGNQGYP

PGSIIMSSLPGPKGNPGYP

PGSIIMSSLPGPKGDPGYP

AGEIITSLLPGQKGDQGFP

pPro167

Figure 3 Conservation analysis of COL4A5 pPro167 amino acidresidue

[5] and it accounts for 40ndash45 of female patients with AS[16] Female patients with XLAS have a variable and generallymild clinical course with 12 reaching ERSD by the age of40 years and about 30 by the age of 60 years in Europeancohorts [17] While male patients are more severe thanfemales with 70 of affected males developing into ESRDbefore the age of 30 years (juvenile form) the remaining30 are progressing toward ESRD after the age of 30 years(rare adult form) [18] Furthermore hearing loss and ocularabnormality happened in 90 and 35 of male patientsrespectively [10]

In our family four patients presented with heterogeneousclinical phenotypes of glomerulopathy while none of themshowed any clinical features of either sensorineural hear-ing loss or typical COL4A5-related ocular abnormalities ACOL4A5 c499delC (pPro167Gln119891119904lowast36) mutation in exon 9cosegregating with the disease was identified The deletionmutation leads to a truncated protein and is absent in 1000genomes project HapMap YanHuang1 (YH1) project dbSNPand 100 normal controls Our clinical and genetic data alsosupport an X-linked inheritance form of AS in this family

4 BioMed Research International

Table 1 Clinical and genetic data of 4 COL4A5 c499delC (pPro167Glnfslowast36) mutation carriers

Subject II 1 III 1 III 3 IV 1Gender F F M FAge (year) 42 24 22 6Onset-age (year) 13 20 3 3Genotype Heterozygote Heterozygote Hemizygote HeterozygoteRenal function ESRD at 36 years Normal Normal NormalMicroscopic hematuria Yes Yes Yes YesGross hematuria Yes No Yes NoProteinuria Yes No Yes NoUremia Yes No No NoAudiological examination Normal Normal Normal NormalOphthalmic examination⋆ No No No No⋆Dot-and-fleck retinopathy anterior lenticonus and posterior polymorphous corneal dystrophy COL4A5 the collagen type IV alpha-5 gene F female Mmale ESRD end-stage renal disease

The COL4A5 gene is located at Xq22 and contains 51exons encoding type IV collagen 1205725 chain [6] Type IV colla-gen 1205725 chain contains 1685 amino acid residues which con-sist of a 26-residue signal peptide a 1430-residue collagenousdomain starting with a 14-residue noncollagenous sequencea Gly-X-Y-repeat sequence interrupted at 22 locations anda 229-residue carboxyl-terminal NC1 domain [19] To date688 COL4A5mutations have been identified according to theHuman Gene Mutation Database (httpwwwhgmdorg)including missense nonsense deletion splicing mutationand complex rearrangements [17 20] without identificationof any mutation hot spot Genotype-phenotype correlationsbetweenCOL4A5mutations and XLAS have been extensivelydescribed For genotype-phenotype correlation purposestypical XLAS is classified into three types (1) severe type withERSD at sim20 years (juvenile-onset ESRD) 80 of hearingloss and 40 of ocular lesions caused by large rearrange-ments premature stop frameshift donor splice and muta-tions in the NC1 domain (2) moderate-severe type withESRD at sim26 years caused by non-Gly-X-Y-missense Gly-X-Y mutations in 21ndash47 exons (3) moderate type with ESRD atsim30 years (late-onset ESRD) 70 of hearing loss and lt30ocular lesions caused by Gly-X-Y mutations in 1ndash20 exons[21 22] Four patients of our family showed no clinical fea-tures of either sensorineural hearing loss or typical COL4A5-related ocular abnormalities Though our family is not largea moderate type of XLAS is considered due to the mutationlocated in COL4A5 exon 9 and the late-onset ESRD (ESRD at36 years II 1 Table 1) More severe clinical phenotypes andearlier onset-age were observed in male patient of this family(III 3) consistent with previous reports [6]

Mutations in genes encoding 120572 chain of type IV collagencould lead to dysfunction of BM and then lead to thedevelopment of human disease in the eye kidney ear and soforth [1] Once the 1205725 chain is missing the formation of thenormal 120572312057241205725 (IV) protomer is disrupted in BM of glom-erulus ear eye and lung which could lead to structural andfunctional defects [23] This is supported by the immunohis-tochemical finding of frequent loss of 1205723 1205724 and 1205725 signalsin the GBM of XLAS patients [24 25] The cause of clinicalheterogeneity of XLAS such as difference in age of disease

onset disease severity and disease progression may be mul-tifactorial including random X chromosome inactivationethnic background and environment factors

Animal models with genetic deficiency may provideprobabilities to reveal the pathogenesis and treatment of AS[26] Two Col4a5 truncation mutations have been identifiedin dogs (Samoyed and Navasota dogs) with clinical featuresof proteinuria and progressive kidney disease leading toterminal failure [27] Intriguingly a deletion in Col4a5resulting in disruption of the Gly-X-Y repeats similar tohuman pPro167Gln119891119904lowast36mutation was observed in a familyof mixed-breed dogs with an inherited nephropathy thatexhibits the clinical immunohistochemical pathologicaland ultrastructural features of human XLAS and the trun-cated peptide chain may probably prevent extracellularassembly in type IV collagen networks [28] Further studieson the Col4a5 genetic-deficient AS animal models will pro-vide new insight into mechanism research diagnosis andtarget therapy of AS in human

5 Conclusions

In our study we identified a novel deletion mutationc499delC (pPro167Gln119891119904lowast36) in the COL4A5 gene whichmay be responsible for AS in this family Our study showedthat exome sequencing is a fast sensitive and relativelylow-cost method to identify gene(s) responsible for AS Thediscovery broadens the genotypic spectrum of COL4A5mutations associatedwithAS and has implications for geneticdiagnosis therapy and genetic counseling of this family

Conflict of Interests

The authors declare that there is no conflict of interests in thispaper

Acknowledgments

The authors thank the participating individuals for theircooperation and their efforts in collecting the genetic infor-mation and DNA specimens This work was supported by

BioMed Research International 5

National Natural Science Foundation of China (8110133981001476) Natural Science Foundation of Hunan ProvinceChina (10JJ4020 10JJ5029) Construction Fund for Key Sub-jects of theThird XiangyaHospital Central South UniversityChina Postgraduates Innovative Pilot Scheme of HunanProvince (7138000008) China the Fundamental ResearchFunds for the Central Universities of Central South Univer-sity (2014zzts360) and Students Innovative Pilot Scheme ofCentral South University (YC12417) China

References

[1] T van Agtmael and L Bruckner-Tuderman ldquoBasement mem-branes and human diseaserdquo Cell and Tissue Research vol 339no 1 pp 167ndash188 2010

[2] C Arrondel G Deschenes Y le Meur et al ldquoA large tandemduplication within the COL4A5 gene is responsible for the highprevalence of Alport syndrome in French Polynesiardquo KidneyInternational vol 65 no 6 pp 2030ndash2040 2004

[3] J H Suh and J HMiner ldquoThe glomerular basementmembraneas a barrier to albuminrdquo Nature Reviews Nephrology vol 9 no8 pp 470ndash477 2013

[4] Y Pirson ldquoMaking the diagnosis of Alportrsquos syndromerdquo KidneyInternational vol 56 no 2 pp 760ndash775 1999

[5] J Kruegel D Rubel and O Gross ldquoAlport syndromemdashinsightsfrom basic and clinical researchrdquo Nature Reviews Nephrologyvol 9 no 3 pp 170ndash178 2013

[6] Y Guo J Yuan andH Liang ldquoIdentification of a novelCOL4A5mutation in a Chinese family with X-linked Alport syndromeusing exome sequencingrdquoMolecular Biology Reports 2014

[7] B G Hudson K Tryggvason M Sundaramoorthy and E GNeilson ldquoAlportrsquos syndrome Goodpasturersquos syndrome and typeIV collagenrdquoTheNew England Journal of Medicine vol 348 no25 pp 2543ndash2556 2003

[8] M J Bamshad S BNgAW Bighamet al ldquoExome sequencingas a tool for Mendelian disease gene discoveryrdquo Nature ReviewsGenetics vol 12 no 11 pp 745ndash755 2011

[9] C Ku D N Cooper C Polychronakos N Naidoo MWu andR Soong ldquoExome sequencing Dual role as a discovery anddiagnostic toolrdquo Annals of Neurology vol 71 no 1 pp 5ndash142012

[10] B Becknell G A Zender R Houston et al ldquoNovel X-linkedglomerulopathy is associated with a COL4A5 missense muta-tion in a non-collagenous interruptionrdquo Kidney Internationalvol 79 no 1 pp 120ndash127 2011

[11] X Xiu Z Song K Gao et al ldquoGenetic analysis of the FBXO48gene in Chinese Han patients with Parkinson diseaserdquo Neuro-science Letters vol 541 pp 224ndash226 2013

[12] Z Chen J Wang B Tang et al ldquoUsing next-generation sequ-encing as a genetic diagnostic tool in rare autosomal recessiveneurologicMendelian disordersrdquoNeurobiology of Aging vol 34no 10 pp 2442e11ndash2442e17 2013

[13] Y Guo L Yuan J Yi et al ldquoIdentification of aGJA3mutation ina Chinese family with congenital nuclear cataract using exomesequencingrdquo Indian Journal of Biochemistry amp Biophysics vol50 no 4 pp 253ndash258 2013

[14] R Li Y Li K Kristiansen and J Wang ldquoSOAP short oligonu-cleotide alignment programrdquo Bioinformatics vol 24 no 5 pp713ndash714 2008

[15] C Gilissen H H Arts A Hoischen et al ldquoExome sequencingidentifies WDR35 variants involved in Sensenbrenner syn-dromerdquo The American Journal of Human Genetics vol 87 no3 pp 418ndash423 2010

[16] O Gross and C E Kashtan ldquoTreatment of Alport syndromebeyond animal modelsrdquo Kidney International vol 76 no 6 pp599ndash603 2009

[17] L Heidet and M Gubler ldquoThe renal lesions of Alport syn-dromerdquo Journal of the American Society of Nephrology vol 20no 6 pp 1210ndash1215 2009

[18] R Artuso C Fallerini L Dosa et al ldquoAdvances in Alport syn-drome diagnosis using next-generation sequencingrdquo EuropeanJournal of Human Genetics vol 20 no 1 pp 50ndash57 2012

[19] J Zhou J M Hertz A Leinonen and K Tryggvason ldquoCom-plete amino acid sequence of the human 1205725(IV) collagen chainand identification of a single-base mutation in exon 23 con-verting glycine 521 in the collagenous domain to cysteine in anAlport syndrome patientrdquo The Journal of Biological Chemistryvol 267 no 18 pp 12475ndash12481 1992

[20] Q Wang F Liu Y Xing et al ldquoMutation c359 363delGTATTi-nsATAC in the COL4A5 Causes alport syndrome in a Chinesefamilyrdquo Gene vol 512 no 2 pp 482ndash485 2013

[21] M Slajpah B Gorinsek G Berginc et al ldquoSixteen novelmutations identified in COL4A3 COL4A4 and COL4A5 genesin Slovenian families with Alport syndrome and benign familialhematuriardquo Kidney International vol 71 no 12 pp 1287ndash12952007

[22] P Demosthenous K Voskarides K Stylianou et al ldquoX-linkedAlport syndrome inHellenic families phenotypic heterogeneityand mutations near interruptions of the collagen domain inCOL4A5rdquo Clinical Genetics vol 81 no 3 pp 240ndash248 2012

[23] A F Zehnder J C Adams P A Santi et al ldquoX-linked Alportsyndrome in Hellenic families phenotypic heterogeneity andmutations near interruptions of the collagen domain inCOL4A5rdquo Archives of Otolaryngology-Head and Neck Surgeryvol 131 no 11 pp 1007ndash1013 2005

[24] Y M Chen and J H Miner ldquoGlomerular basement membraneand related glomerular diseaserdquoTranslational Research vol 160no 4 pp 291ndash297 2012

[25] J C Wilson H Yoon R J Walker and M R Eccles ldquoA novelCys1638Tyr NC1 domain substitution in 1205725(IV) collagen causesAlport syndrome with late onset renal failure without hearingloss or eye abnormalitiesrdquo Nephrology Dialysis Transplantationvol 22 no 5 pp 1338ndash1346 2007

[26] O Gross B Beirowski M Koepke et al ldquoPreemptive ramipriltherapy delays renal failure and reduces renal fibrosis inCOL4A3-knockout mice with Alport syndromerdquo Kidney Inter-national vol 63 no 2 pp 438ndash446 2003

[27] G E Lees ldquoKidney diseases caused by glomerular basementmembrane type IV collagen defects in dogsrdquo Journal of Veteri-nary Emergency and Critical Care vol 23 no 2 pp 184ndash1932013

[28] M L Cox G E Lees C E Kashtan andK EMurphy ldquoGeneticcause of X-linked Alport syndrome in a family of domesticdogsrdquoMammalian Genome vol 14 no 6 pp 396ndash403 2003

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 3

T C A C T G C C A G G A C C A

T C A C T G C A G G A C C A A

c499delC

(a)

T C A C T G C A G G A C C A

c499delC

(b)

Figure 2 Sequencing analysis of COL4A5 c499delC (pPro167Gln119891119904lowast36) mutation The arrow shows site of the novel c499delC(pPro167Gln119891119904lowast36) deletion mutation in the COL4A5 gene (a) Heterozygous mutation carrier (III 1) (b) Hemizygous mutation carrier(III 3)

to the human reference sequence and 360 billion bases(4885) mapped to the targeted bases with a mean coverageof 8165-fold 105963 genetic variants including 14723 non-synonymous variants were identified in either the codingregions or the splice sites A prioritization schemewas appliedto identify the pathogenic mutation in the patient similar torecent studies [6 15] We excluded known variants identifiedin 1000 genomes project HapMap dbSNP132 and YH1Applying the above strategy we reduced the number ofcandidate genes by more than 9033

A novel deletion mutation c499delC(pPro167Gln119891119904lowast36) was identified in exon 9 of the COL4A5gene in the probandThis mutation results in premature stopcodon and a truncated protein The same mutation was sub-sequently verified in all four affected family members (II 1III 1 III 3 and IV 1 Figure 1) while being absent in un-affected members and 100 ethnically matched normalcontrols by Sanger sequencing (Figure 2) It is also absent in1000 genomes project HapMap YanHuang1 (YH1) projectand dbSNP The mutation is located in the Gly-X-Y repeatsThe pPro167 is a highly conserved amino acid residue amongdifferent species from chicken to human suggesting itsstructural and functional importance (Figure 3) This mut-ation was predicted to affect the protein features and bedisease causing (predicted by httpwwwmutationtasterorg)SIFT prediction also showed a damaging effect with aconfidence score of 0858 (httpsiftbiia-staredusgwwwSIFT indels2html)

4 Discussion

AS is a clinically and genetically heterogeneous disease andseverity of this disease is usually equal between males andfemales in the autosomal recessive form (autosomal recessiveAS ARAS) while greater in males with X-linked form (X-linked AS XLAS) XLAS is caused by mutations in theCOL4A5 gene with an approximately prevalence of 110000

Homo sapiensChimpanzeeRhesus monkeyDogCattleHorseSheepMouseChicken

PGSIIMSSLPGPKGNPGYP

PGSIIMSSLPGPKGNPGYP

PGSIIMSSLPGPKGNPGYP

PGSIIMSSLPGPKGNPGYP

PGSIIMSSLPGPKGNPGYP

PGSIIMSPLPGPKGNQGYP

PGSIIMSSLPGPKGNPGYP

PGSIIMSSLPGPKGDPGYP

AGEIITSLLPGQKGDQGFP

pPro167

Figure 3 Conservation analysis of COL4A5 pPro167 amino acidresidue

[5] and it accounts for 40ndash45 of female patients with AS[16] Female patients with XLAS have a variable and generallymild clinical course with 12 reaching ERSD by the age of40 years and about 30 by the age of 60 years in Europeancohorts [17] While male patients are more severe thanfemales with 70 of affected males developing into ESRDbefore the age of 30 years (juvenile form) the remaining30 are progressing toward ESRD after the age of 30 years(rare adult form) [18] Furthermore hearing loss and ocularabnormality happened in 90 and 35 of male patientsrespectively [10]

In our family four patients presented with heterogeneousclinical phenotypes of glomerulopathy while none of themshowed any clinical features of either sensorineural hear-ing loss or typical COL4A5-related ocular abnormalities ACOL4A5 c499delC (pPro167Gln119891119904lowast36) mutation in exon 9cosegregating with the disease was identified The deletionmutation leads to a truncated protein and is absent in 1000genomes project HapMap YanHuang1 (YH1) project dbSNPand 100 normal controls Our clinical and genetic data alsosupport an X-linked inheritance form of AS in this family

4 BioMed Research International

Table 1 Clinical and genetic data of 4 COL4A5 c499delC (pPro167Glnfslowast36) mutation carriers

Subject II 1 III 1 III 3 IV 1Gender F F M FAge (year) 42 24 22 6Onset-age (year) 13 20 3 3Genotype Heterozygote Heterozygote Hemizygote HeterozygoteRenal function ESRD at 36 years Normal Normal NormalMicroscopic hematuria Yes Yes Yes YesGross hematuria Yes No Yes NoProteinuria Yes No Yes NoUremia Yes No No NoAudiological examination Normal Normal Normal NormalOphthalmic examination⋆ No No No No⋆Dot-and-fleck retinopathy anterior lenticonus and posterior polymorphous corneal dystrophy COL4A5 the collagen type IV alpha-5 gene F female Mmale ESRD end-stage renal disease

The COL4A5 gene is located at Xq22 and contains 51exons encoding type IV collagen 1205725 chain [6] Type IV colla-gen 1205725 chain contains 1685 amino acid residues which con-sist of a 26-residue signal peptide a 1430-residue collagenousdomain starting with a 14-residue noncollagenous sequencea Gly-X-Y-repeat sequence interrupted at 22 locations anda 229-residue carboxyl-terminal NC1 domain [19] To date688 COL4A5mutations have been identified according to theHuman Gene Mutation Database (httpwwwhgmdorg)including missense nonsense deletion splicing mutationand complex rearrangements [17 20] without identificationof any mutation hot spot Genotype-phenotype correlationsbetweenCOL4A5mutations and XLAS have been extensivelydescribed For genotype-phenotype correlation purposestypical XLAS is classified into three types (1) severe type withERSD at sim20 years (juvenile-onset ESRD) 80 of hearingloss and 40 of ocular lesions caused by large rearrange-ments premature stop frameshift donor splice and muta-tions in the NC1 domain (2) moderate-severe type withESRD at sim26 years caused by non-Gly-X-Y-missense Gly-X-Y mutations in 21ndash47 exons (3) moderate type with ESRD atsim30 years (late-onset ESRD) 70 of hearing loss and lt30ocular lesions caused by Gly-X-Y mutations in 1ndash20 exons[21 22] Four patients of our family showed no clinical fea-tures of either sensorineural hearing loss or typical COL4A5-related ocular abnormalities Though our family is not largea moderate type of XLAS is considered due to the mutationlocated in COL4A5 exon 9 and the late-onset ESRD (ESRD at36 years II 1 Table 1) More severe clinical phenotypes andearlier onset-age were observed in male patient of this family(III 3) consistent with previous reports [6]

Mutations in genes encoding 120572 chain of type IV collagencould lead to dysfunction of BM and then lead to thedevelopment of human disease in the eye kidney ear and soforth [1] Once the 1205725 chain is missing the formation of thenormal 120572312057241205725 (IV) protomer is disrupted in BM of glom-erulus ear eye and lung which could lead to structural andfunctional defects [23] This is supported by the immunohis-tochemical finding of frequent loss of 1205723 1205724 and 1205725 signalsin the GBM of XLAS patients [24 25] The cause of clinicalheterogeneity of XLAS such as difference in age of disease

onset disease severity and disease progression may be mul-tifactorial including random X chromosome inactivationethnic background and environment factors

Animal models with genetic deficiency may provideprobabilities to reveal the pathogenesis and treatment of AS[26] Two Col4a5 truncation mutations have been identifiedin dogs (Samoyed and Navasota dogs) with clinical featuresof proteinuria and progressive kidney disease leading toterminal failure [27] Intriguingly a deletion in Col4a5resulting in disruption of the Gly-X-Y repeats similar tohuman pPro167Gln119891119904lowast36mutation was observed in a familyof mixed-breed dogs with an inherited nephropathy thatexhibits the clinical immunohistochemical pathologicaland ultrastructural features of human XLAS and the trun-cated peptide chain may probably prevent extracellularassembly in type IV collagen networks [28] Further studieson the Col4a5 genetic-deficient AS animal models will pro-vide new insight into mechanism research diagnosis andtarget therapy of AS in human

5 Conclusions

In our study we identified a novel deletion mutationc499delC (pPro167Gln119891119904lowast36) in the COL4A5 gene whichmay be responsible for AS in this family Our study showedthat exome sequencing is a fast sensitive and relativelylow-cost method to identify gene(s) responsible for AS Thediscovery broadens the genotypic spectrum of COL4A5mutations associatedwithAS and has implications for geneticdiagnosis therapy and genetic counseling of this family

Conflict of Interests

The authors declare that there is no conflict of interests in thispaper

Acknowledgments

The authors thank the participating individuals for theircooperation and their efforts in collecting the genetic infor-mation and DNA specimens This work was supported by

BioMed Research International 5

National Natural Science Foundation of China (8110133981001476) Natural Science Foundation of Hunan ProvinceChina (10JJ4020 10JJ5029) Construction Fund for Key Sub-jects of theThird XiangyaHospital Central South UniversityChina Postgraduates Innovative Pilot Scheme of HunanProvince (7138000008) China the Fundamental ResearchFunds for the Central Universities of Central South Univer-sity (2014zzts360) and Students Innovative Pilot Scheme ofCentral South University (YC12417) China

References

[1] T van Agtmael and L Bruckner-Tuderman ldquoBasement mem-branes and human diseaserdquo Cell and Tissue Research vol 339no 1 pp 167ndash188 2010

[2] C Arrondel G Deschenes Y le Meur et al ldquoA large tandemduplication within the COL4A5 gene is responsible for the highprevalence of Alport syndrome in French Polynesiardquo KidneyInternational vol 65 no 6 pp 2030ndash2040 2004

[3] J H Suh and J HMiner ldquoThe glomerular basementmembraneas a barrier to albuminrdquo Nature Reviews Nephrology vol 9 no8 pp 470ndash477 2013

[4] Y Pirson ldquoMaking the diagnosis of Alportrsquos syndromerdquo KidneyInternational vol 56 no 2 pp 760ndash775 1999

[5] J Kruegel D Rubel and O Gross ldquoAlport syndromemdashinsightsfrom basic and clinical researchrdquo Nature Reviews Nephrologyvol 9 no 3 pp 170ndash178 2013

[6] Y Guo J Yuan andH Liang ldquoIdentification of a novelCOL4A5mutation in a Chinese family with X-linked Alport syndromeusing exome sequencingrdquoMolecular Biology Reports 2014

[7] B G Hudson K Tryggvason M Sundaramoorthy and E GNeilson ldquoAlportrsquos syndrome Goodpasturersquos syndrome and typeIV collagenrdquoTheNew England Journal of Medicine vol 348 no25 pp 2543ndash2556 2003

[8] M J Bamshad S BNgAW Bighamet al ldquoExome sequencingas a tool for Mendelian disease gene discoveryrdquo Nature ReviewsGenetics vol 12 no 11 pp 745ndash755 2011

[9] C Ku D N Cooper C Polychronakos N Naidoo MWu andR Soong ldquoExome sequencing Dual role as a discovery anddiagnostic toolrdquo Annals of Neurology vol 71 no 1 pp 5ndash142012

[10] B Becknell G A Zender R Houston et al ldquoNovel X-linkedglomerulopathy is associated with a COL4A5 missense muta-tion in a non-collagenous interruptionrdquo Kidney Internationalvol 79 no 1 pp 120ndash127 2011

[11] X Xiu Z Song K Gao et al ldquoGenetic analysis of the FBXO48gene in Chinese Han patients with Parkinson diseaserdquo Neuro-science Letters vol 541 pp 224ndash226 2013

[12] Z Chen J Wang B Tang et al ldquoUsing next-generation sequ-encing as a genetic diagnostic tool in rare autosomal recessiveneurologicMendelian disordersrdquoNeurobiology of Aging vol 34no 10 pp 2442e11ndash2442e17 2013

[13] Y Guo L Yuan J Yi et al ldquoIdentification of aGJA3mutation ina Chinese family with congenital nuclear cataract using exomesequencingrdquo Indian Journal of Biochemistry amp Biophysics vol50 no 4 pp 253ndash258 2013

[14] R Li Y Li K Kristiansen and J Wang ldquoSOAP short oligonu-cleotide alignment programrdquo Bioinformatics vol 24 no 5 pp713ndash714 2008

[15] C Gilissen H H Arts A Hoischen et al ldquoExome sequencingidentifies WDR35 variants involved in Sensenbrenner syn-dromerdquo The American Journal of Human Genetics vol 87 no3 pp 418ndash423 2010

[16] O Gross and C E Kashtan ldquoTreatment of Alport syndromebeyond animal modelsrdquo Kidney International vol 76 no 6 pp599ndash603 2009

[17] L Heidet and M Gubler ldquoThe renal lesions of Alport syn-dromerdquo Journal of the American Society of Nephrology vol 20no 6 pp 1210ndash1215 2009

[18] R Artuso C Fallerini L Dosa et al ldquoAdvances in Alport syn-drome diagnosis using next-generation sequencingrdquo EuropeanJournal of Human Genetics vol 20 no 1 pp 50ndash57 2012

[19] J Zhou J M Hertz A Leinonen and K Tryggvason ldquoCom-plete amino acid sequence of the human 1205725(IV) collagen chainand identification of a single-base mutation in exon 23 con-verting glycine 521 in the collagenous domain to cysteine in anAlport syndrome patientrdquo The Journal of Biological Chemistryvol 267 no 18 pp 12475ndash12481 1992

[20] Q Wang F Liu Y Xing et al ldquoMutation c359 363delGTATTi-nsATAC in the COL4A5 Causes alport syndrome in a Chinesefamilyrdquo Gene vol 512 no 2 pp 482ndash485 2013

[21] M Slajpah B Gorinsek G Berginc et al ldquoSixteen novelmutations identified in COL4A3 COL4A4 and COL4A5 genesin Slovenian families with Alport syndrome and benign familialhematuriardquo Kidney International vol 71 no 12 pp 1287ndash12952007

[22] P Demosthenous K Voskarides K Stylianou et al ldquoX-linkedAlport syndrome inHellenic families phenotypic heterogeneityand mutations near interruptions of the collagen domain inCOL4A5rdquo Clinical Genetics vol 81 no 3 pp 240ndash248 2012

[23] A F Zehnder J C Adams P A Santi et al ldquoX-linked Alportsyndrome in Hellenic families phenotypic heterogeneity andmutations near interruptions of the collagen domain inCOL4A5rdquo Archives of Otolaryngology-Head and Neck Surgeryvol 131 no 11 pp 1007ndash1013 2005

[24] Y M Chen and J H Miner ldquoGlomerular basement membraneand related glomerular diseaserdquoTranslational Research vol 160no 4 pp 291ndash297 2012

[25] J C Wilson H Yoon R J Walker and M R Eccles ldquoA novelCys1638Tyr NC1 domain substitution in 1205725(IV) collagen causesAlport syndrome with late onset renal failure without hearingloss or eye abnormalitiesrdquo Nephrology Dialysis Transplantationvol 22 no 5 pp 1338ndash1346 2007

[26] O Gross B Beirowski M Koepke et al ldquoPreemptive ramipriltherapy delays renal failure and reduces renal fibrosis inCOL4A3-knockout mice with Alport syndromerdquo Kidney Inter-national vol 63 no 2 pp 438ndash446 2003

[27] G E Lees ldquoKidney diseases caused by glomerular basementmembrane type IV collagen defects in dogsrdquo Journal of Veteri-nary Emergency and Critical Care vol 23 no 2 pp 184ndash1932013

[28] M L Cox G E Lees C E Kashtan andK EMurphy ldquoGeneticcause of X-linked Alport syndrome in a family of domesticdogsrdquoMammalian Genome vol 14 no 6 pp 396ndash403 2003

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 BioMed Research International

Table 1 Clinical and genetic data of 4 COL4A5 c499delC (pPro167Glnfslowast36) mutation carriers

Subject II 1 III 1 III 3 IV 1Gender F F M FAge (year) 42 24 22 6Onset-age (year) 13 20 3 3Genotype Heterozygote Heterozygote Hemizygote HeterozygoteRenal function ESRD at 36 years Normal Normal NormalMicroscopic hematuria Yes Yes Yes YesGross hematuria Yes No Yes NoProteinuria Yes No Yes NoUremia Yes No No NoAudiological examination Normal Normal Normal NormalOphthalmic examination⋆ No No No No⋆Dot-and-fleck retinopathy anterior lenticonus and posterior polymorphous corneal dystrophy COL4A5 the collagen type IV alpha-5 gene F female Mmale ESRD end-stage renal disease

The COL4A5 gene is located at Xq22 and contains 51exons encoding type IV collagen 1205725 chain [6] Type IV colla-gen 1205725 chain contains 1685 amino acid residues which con-sist of a 26-residue signal peptide a 1430-residue collagenousdomain starting with a 14-residue noncollagenous sequencea Gly-X-Y-repeat sequence interrupted at 22 locations anda 229-residue carboxyl-terminal NC1 domain [19] To date688 COL4A5mutations have been identified according to theHuman Gene Mutation Database (httpwwwhgmdorg)including missense nonsense deletion splicing mutationand complex rearrangements [17 20] without identificationof any mutation hot spot Genotype-phenotype correlationsbetweenCOL4A5mutations and XLAS have been extensivelydescribed For genotype-phenotype correlation purposestypical XLAS is classified into three types (1) severe type withERSD at sim20 years (juvenile-onset ESRD) 80 of hearingloss and 40 of ocular lesions caused by large rearrange-ments premature stop frameshift donor splice and muta-tions in the NC1 domain (2) moderate-severe type withESRD at sim26 years caused by non-Gly-X-Y-missense Gly-X-Y mutations in 21ndash47 exons (3) moderate type with ESRD atsim30 years (late-onset ESRD) 70 of hearing loss and lt30ocular lesions caused by Gly-X-Y mutations in 1ndash20 exons[21 22] Four patients of our family showed no clinical fea-tures of either sensorineural hearing loss or typical COL4A5-related ocular abnormalities Though our family is not largea moderate type of XLAS is considered due to the mutationlocated in COL4A5 exon 9 and the late-onset ESRD (ESRD at36 years II 1 Table 1) More severe clinical phenotypes andearlier onset-age were observed in male patient of this family(III 3) consistent with previous reports [6]

Mutations in genes encoding 120572 chain of type IV collagencould lead to dysfunction of BM and then lead to thedevelopment of human disease in the eye kidney ear and soforth [1] Once the 1205725 chain is missing the formation of thenormal 120572312057241205725 (IV) protomer is disrupted in BM of glom-erulus ear eye and lung which could lead to structural andfunctional defects [23] This is supported by the immunohis-tochemical finding of frequent loss of 1205723 1205724 and 1205725 signalsin the GBM of XLAS patients [24 25] The cause of clinicalheterogeneity of XLAS such as difference in age of disease

onset disease severity and disease progression may be mul-tifactorial including random X chromosome inactivationethnic background and environment factors

Animal models with genetic deficiency may provideprobabilities to reveal the pathogenesis and treatment of AS[26] Two Col4a5 truncation mutations have been identifiedin dogs (Samoyed and Navasota dogs) with clinical featuresof proteinuria and progressive kidney disease leading toterminal failure [27] Intriguingly a deletion in Col4a5resulting in disruption of the Gly-X-Y repeats similar tohuman pPro167Gln119891119904lowast36mutation was observed in a familyof mixed-breed dogs with an inherited nephropathy thatexhibits the clinical immunohistochemical pathologicaland ultrastructural features of human XLAS and the trun-cated peptide chain may probably prevent extracellularassembly in type IV collagen networks [28] Further studieson the Col4a5 genetic-deficient AS animal models will pro-vide new insight into mechanism research diagnosis andtarget therapy of AS in human

5 Conclusions

In our study we identified a novel deletion mutationc499delC (pPro167Gln119891119904lowast36) in the COL4A5 gene whichmay be responsible for AS in this family Our study showedthat exome sequencing is a fast sensitive and relativelylow-cost method to identify gene(s) responsible for AS Thediscovery broadens the genotypic spectrum of COL4A5mutations associatedwithAS and has implications for geneticdiagnosis therapy and genetic counseling of this family

Conflict of Interests

The authors declare that there is no conflict of interests in thispaper

Acknowledgments

The authors thank the participating individuals for theircooperation and their efforts in collecting the genetic infor-mation and DNA specimens This work was supported by

BioMed Research International 5

National Natural Science Foundation of China (8110133981001476) Natural Science Foundation of Hunan ProvinceChina (10JJ4020 10JJ5029) Construction Fund for Key Sub-jects of theThird XiangyaHospital Central South UniversityChina Postgraduates Innovative Pilot Scheme of HunanProvince (7138000008) China the Fundamental ResearchFunds for the Central Universities of Central South Univer-sity (2014zzts360) and Students Innovative Pilot Scheme ofCentral South University (YC12417) China

References

[1] T van Agtmael and L Bruckner-Tuderman ldquoBasement mem-branes and human diseaserdquo Cell and Tissue Research vol 339no 1 pp 167ndash188 2010

[2] C Arrondel G Deschenes Y le Meur et al ldquoA large tandemduplication within the COL4A5 gene is responsible for the highprevalence of Alport syndrome in French Polynesiardquo KidneyInternational vol 65 no 6 pp 2030ndash2040 2004

[3] J H Suh and J HMiner ldquoThe glomerular basementmembraneas a barrier to albuminrdquo Nature Reviews Nephrology vol 9 no8 pp 470ndash477 2013

[4] Y Pirson ldquoMaking the diagnosis of Alportrsquos syndromerdquo KidneyInternational vol 56 no 2 pp 760ndash775 1999

[5] J Kruegel D Rubel and O Gross ldquoAlport syndromemdashinsightsfrom basic and clinical researchrdquo Nature Reviews Nephrologyvol 9 no 3 pp 170ndash178 2013

[6] Y Guo J Yuan andH Liang ldquoIdentification of a novelCOL4A5mutation in a Chinese family with X-linked Alport syndromeusing exome sequencingrdquoMolecular Biology Reports 2014

[7] B G Hudson K Tryggvason M Sundaramoorthy and E GNeilson ldquoAlportrsquos syndrome Goodpasturersquos syndrome and typeIV collagenrdquoTheNew England Journal of Medicine vol 348 no25 pp 2543ndash2556 2003

[8] M J Bamshad S BNgAW Bighamet al ldquoExome sequencingas a tool for Mendelian disease gene discoveryrdquo Nature ReviewsGenetics vol 12 no 11 pp 745ndash755 2011

[9] C Ku D N Cooper C Polychronakos N Naidoo MWu andR Soong ldquoExome sequencing Dual role as a discovery anddiagnostic toolrdquo Annals of Neurology vol 71 no 1 pp 5ndash142012

[10] B Becknell G A Zender R Houston et al ldquoNovel X-linkedglomerulopathy is associated with a COL4A5 missense muta-tion in a non-collagenous interruptionrdquo Kidney Internationalvol 79 no 1 pp 120ndash127 2011

[11] X Xiu Z Song K Gao et al ldquoGenetic analysis of the FBXO48gene in Chinese Han patients with Parkinson diseaserdquo Neuro-science Letters vol 541 pp 224ndash226 2013

[12] Z Chen J Wang B Tang et al ldquoUsing next-generation sequ-encing as a genetic diagnostic tool in rare autosomal recessiveneurologicMendelian disordersrdquoNeurobiology of Aging vol 34no 10 pp 2442e11ndash2442e17 2013

[13] Y Guo L Yuan J Yi et al ldquoIdentification of aGJA3mutation ina Chinese family with congenital nuclear cataract using exomesequencingrdquo Indian Journal of Biochemistry amp Biophysics vol50 no 4 pp 253ndash258 2013

[14] R Li Y Li K Kristiansen and J Wang ldquoSOAP short oligonu-cleotide alignment programrdquo Bioinformatics vol 24 no 5 pp713ndash714 2008

[15] C Gilissen H H Arts A Hoischen et al ldquoExome sequencingidentifies WDR35 variants involved in Sensenbrenner syn-dromerdquo The American Journal of Human Genetics vol 87 no3 pp 418ndash423 2010

[16] O Gross and C E Kashtan ldquoTreatment of Alport syndromebeyond animal modelsrdquo Kidney International vol 76 no 6 pp599ndash603 2009

[17] L Heidet and M Gubler ldquoThe renal lesions of Alport syn-dromerdquo Journal of the American Society of Nephrology vol 20no 6 pp 1210ndash1215 2009

[18] R Artuso C Fallerini L Dosa et al ldquoAdvances in Alport syn-drome diagnosis using next-generation sequencingrdquo EuropeanJournal of Human Genetics vol 20 no 1 pp 50ndash57 2012

[19] J Zhou J M Hertz A Leinonen and K Tryggvason ldquoCom-plete amino acid sequence of the human 1205725(IV) collagen chainand identification of a single-base mutation in exon 23 con-verting glycine 521 in the collagenous domain to cysteine in anAlport syndrome patientrdquo The Journal of Biological Chemistryvol 267 no 18 pp 12475ndash12481 1992

[20] Q Wang F Liu Y Xing et al ldquoMutation c359 363delGTATTi-nsATAC in the COL4A5 Causes alport syndrome in a Chinesefamilyrdquo Gene vol 512 no 2 pp 482ndash485 2013

[21] M Slajpah B Gorinsek G Berginc et al ldquoSixteen novelmutations identified in COL4A3 COL4A4 and COL4A5 genesin Slovenian families with Alport syndrome and benign familialhematuriardquo Kidney International vol 71 no 12 pp 1287ndash12952007

[22] P Demosthenous K Voskarides K Stylianou et al ldquoX-linkedAlport syndrome inHellenic families phenotypic heterogeneityand mutations near interruptions of the collagen domain inCOL4A5rdquo Clinical Genetics vol 81 no 3 pp 240ndash248 2012

[23] A F Zehnder J C Adams P A Santi et al ldquoX-linked Alportsyndrome in Hellenic families phenotypic heterogeneity andmutations near interruptions of the collagen domain inCOL4A5rdquo Archives of Otolaryngology-Head and Neck Surgeryvol 131 no 11 pp 1007ndash1013 2005

[24] Y M Chen and J H Miner ldquoGlomerular basement membraneand related glomerular diseaserdquoTranslational Research vol 160no 4 pp 291ndash297 2012

[25] J C Wilson H Yoon R J Walker and M R Eccles ldquoA novelCys1638Tyr NC1 domain substitution in 1205725(IV) collagen causesAlport syndrome with late onset renal failure without hearingloss or eye abnormalitiesrdquo Nephrology Dialysis Transplantationvol 22 no 5 pp 1338ndash1346 2007

[26] O Gross B Beirowski M Koepke et al ldquoPreemptive ramipriltherapy delays renal failure and reduces renal fibrosis inCOL4A3-knockout mice with Alport syndromerdquo Kidney Inter-national vol 63 no 2 pp 438ndash446 2003

[27] G E Lees ldquoKidney diseases caused by glomerular basementmembrane type IV collagen defects in dogsrdquo Journal of Veteri-nary Emergency and Critical Care vol 23 no 2 pp 184ndash1932013

[28] M L Cox G E Lees C E Kashtan andK EMurphy ldquoGeneticcause of X-linked Alport syndrome in a family of domesticdogsrdquoMammalian Genome vol 14 no 6 pp 396ndash403 2003

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 5

National Natural Science Foundation of China (8110133981001476) Natural Science Foundation of Hunan ProvinceChina (10JJ4020 10JJ5029) Construction Fund for Key Sub-jects of theThird XiangyaHospital Central South UniversityChina Postgraduates Innovative Pilot Scheme of HunanProvince (7138000008) China the Fundamental ResearchFunds for the Central Universities of Central South Univer-sity (2014zzts360) and Students Innovative Pilot Scheme ofCentral South University (YC12417) China

References

[1] T van Agtmael and L Bruckner-Tuderman ldquoBasement mem-branes and human diseaserdquo Cell and Tissue Research vol 339no 1 pp 167ndash188 2010

[2] C Arrondel G Deschenes Y le Meur et al ldquoA large tandemduplication within the COL4A5 gene is responsible for the highprevalence of Alport syndrome in French Polynesiardquo KidneyInternational vol 65 no 6 pp 2030ndash2040 2004

[3] J H Suh and J HMiner ldquoThe glomerular basementmembraneas a barrier to albuminrdquo Nature Reviews Nephrology vol 9 no8 pp 470ndash477 2013

[4] Y Pirson ldquoMaking the diagnosis of Alportrsquos syndromerdquo KidneyInternational vol 56 no 2 pp 760ndash775 1999

[5] J Kruegel D Rubel and O Gross ldquoAlport syndromemdashinsightsfrom basic and clinical researchrdquo Nature Reviews Nephrologyvol 9 no 3 pp 170ndash178 2013

[6] Y Guo J Yuan andH Liang ldquoIdentification of a novelCOL4A5mutation in a Chinese family with X-linked Alport syndromeusing exome sequencingrdquoMolecular Biology Reports 2014

[7] B G Hudson K Tryggvason M Sundaramoorthy and E GNeilson ldquoAlportrsquos syndrome Goodpasturersquos syndrome and typeIV collagenrdquoTheNew England Journal of Medicine vol 348 no25 pp 2543ndash2556 2003

[8] M J Bamshad S BNgAW Bighamet al ldquoExome sequencingas a tool for Mendelian disease gene discoveryrdquo Nature ReviewsGenetics vol 12 no 11 pp 745ndash755 2011

[9] C Ku D N Cooper C Polychronakos N Naidoo MWu andR Soong ldquoExome sequencing Dual role as a discovery anddiagnostic toolrdquo Annals of Neurology vol 71 no 1 pp 5ndash142012

[10] B Becknell G A Zender R Houston et al ldquoNovel X-linkedglomerulopathy is associated with a COL4A5 missense muta-tion in a non-collagenous interruptionrdquo Kidney Internationalvol 79 no 1 pp 120ndash127 2011

[11] X Xiu Z Song K Gao et al ldquoGenetic analysis of the FBXO48gene in Chinese Han patients with Parkinson diseaserdquo Neuro-science Letters vol 541 pp 224ndash226 2013

[12] Z Chen J Wang B Tang et al ldquoUsing next-generation sequ-encing as a genetic diagnostic tool in rare autosomal recessiveneurologicMendelian disordersrdquoNeurobiology of Aging vol 34no 10 pp 2442e11ndash2442e17 2013

[13] Y Guo L Yuan J Yi et al ldquoIdentification of aGJA3mutation ina Chinese family with congenital nuclear cataract using exomesequencingrdquo Indian Journal of Biochemistry amp Biophysics vol50 no 4 pp 253ndash258 2013

[14] R Li Y Li K Kristiansen and J Wang ldquoSOAP short oligonu-cleotide alignment programrdquo Bioinformatics vol 24 no 5 pp713ndash714 2008

[15] C Gilissen H H Arts A Hoischen et al ldquoExome sequencingidentifies WDR35 variants involved in Sensenbrenner syn-dromerdquo The American Journal of Human Genetics vol 87 no3 pp 418ndash423 2010

[16] O Gross and C E Kashtan ldquoTreatment of Alport syndromebeyond animal modelsrdquo Kidney International vol 76 no 6 pp599ndash603 2009

[17] L Heidet and M Gubler ldquoThe renal lesions of Alport syn-dromerdquo Journal of the American Society of Nephrology vol 20no 6 pp 1210ndash1215 2009

[18] R Artuso C Fallerini L Dosa et al ldquoAdvances in Alport syn-drome diagnosis using next-generation sequencingrdquo EuropeanJournal of Human Genetics vol 20 no 1 pp 50ndash57 2012

[19] J Zhou J M Hertz A Leinonen and K Tryggvason ldquoCom-plete amino acid sequence of the human 1205725(IV) collagen chainand identification of a single-base mutation in exon 23 con-verting glycine 521 in the collagenous domain to cysteine in anAlport syndrome patientrdquo The Journal of Biological Chemistryvol 267 no 18 pp 12475ndash12481 1992

[20] Q Wang F Liu Y Xing et al ldquoMutation c359 363delGTATTi-nsATAC in the COL4A5 Causes alport syndrome in a Chinesefamilyrdquo Gene vol 512 no 2 pp 482ndash485 2013

[21] M Slajpah B Gorinsek G Berginc et al ldquoSixteen novelmutations identified in COL4A3 COL4A4 and COL4A5 genesin Slovenian families with Alport syndrome and benign familialhematuriardquo Kidney International vol 71 no 12 pp 1287ndash12952007

[22] P Demosthenous K Voskarides K Stylianou et al ldquoX-linkedAlport syndrome inHellenic families phenotypic heterogeneityand mutations near interruptions of the collagen domain inCOL4A5rdquo Clinical Genetics vol 81 no 3 pp 240ndash248 2012

[23] A F Zehnder J C Adams P A Santi et al ldquoX-linked Alportsyndrome in Hellenic families phenotypic heterogeneity andmutations near interruptions of the collagen domain inCOL4A5rdquo Archives of Otolaryngology-Head and Neck Surgeryvol 131 no 11 pp 1007ndash1013 2005

[24] Y M Chen and J H Miner ldquoGlomerular basement membraneand related glomerular diseaserdquoTranslational Research vol 160no 4 pp 291ndash297 2012

[25] J C Wilson H Yoon R J Walker and M R Eccles ldquoA novelCys1638Tyr NC1 domain substitution in 1205725(IV) collagen causesAlport syndrome with late onset renal failure without hearingloss or eye abnormalitiesrdquo Nephrology Dialysis Transplantationvol 22 no 5 pp 1338ndash1346 2007

[26] O Gross B Beirowski M Koepke et al ldquoPreemptive ramipriltherapy delays renal failure and reduces renal fibrosis inCOL4A3-knockout mice with Alport syndromerdquo Kidney Inter-national vol 63 no 2 pp 438ndash446 2003

[27] G E Lees ldquoKidney diseases caused by glomerular basementmembrane type IV collagen defects in dogsrdquo Journal of Veteri-nary Emergency and Critical Care vol 23 no 2 pp 184ndash1932013

[28] M L Cox G E Lees C E Kashtan andK EMurphy ldquoGeneticcause of X-linked Alport syndrome in a family of domesticdogsrdquoMammalian Genome vol 14 no 6 pp 396ndash403 2003

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology