COL4A3/COL4A4 Mutations Producing Focal Segmental Glomerulosclerosis and Renal Failure in Thin...

COL4A3/COL4A4 Mutations Producing FocalSegmental Glomerulosclerosis and Renal Failure inThin Basement Membrane Nephropathy

Konstantinos Voskarides,* Loukas Damianou,† Vassos Neocleous,‡ Ioanna Zouvani,§

Stalo Christodoulidou,† Valsamakis Hadjiconstantinou,† Kyriacos Ioannou,�

Yiannis Athanasiou,� Charalampos Patsias,¶ Efstathios Alexopoulos,** Alkis Pierides,�

Kyriacos Kyriacou,‡ and Constantinos Deltas*‡

*Department of Biological Sciences, University of Cyprus, ‡Cyprus Institute of Neurology and Genetics, andDepartments of §Histopathology and �Nephrology, Nicosia General Hospital, Nicosia, Cyprus; †Department ofNephrology, Evangelismos Hospital, Athens, Greece; ¶Department of Nephrology, Larnaca General Hospital,Larnaca, Cyprus; and **Department of Nephrology, Aristotle University of Thessalloniki, Greece

ABSTRACTMutations in the COL4A3/COL4A4 genes of type IV collagen have been found in �40% of cases of thin basementmembrane nephropathy, which is characterized by microscopic hematuria and is classically thought to causeproteinuria and chronic renal failure rarely. Here we report our observations of 116 subjects from 13 Cypriotfamilies clinically affected with thin basement membrane nephropathy. These families first came to our attentionbecause they segregated microscopic hematuria, mild proteinuria, and variable degrees of renal impairment, buta dual diagnosis of focal segmental glomerulosclerosis (FSGS) and thin basement membrane nephropathy wasmade in 20 biopsied cases. Molecular studies identified founder mutations in both COL4A3 and COL4A4 genesin 10 families. None of 82 heterozygous patients had any extrarenal manifestations, supporting the diagnosis ofthin basement membrane nephropathy. During follow-up of up to three decades, 31 of these 82 patients (37.8%)developed chronic renal failure and 16 (19.5%) reached end-stage renal disease. Mutations G1334E and G871Cwere detected in seven and three families, respectively, and were probably introduced by founders. We concludethat these particular COL4A3/COL4A4 mutations either predispose some patients to FSGS and chronic renalfailure, or that thin basement membrane nephropathy sometimes coexists with another genetic modifier that isresponsible for FSGS and progressive renal failure. The findings presented here do not justify the labelling of thinbasement membrane nephropathy as a benign condition with excellent prognosis.

J Am Soc Nephrol 18: 3004–3016, 2007. doi: 10.1681/ASN.2007040444

The phenotypic heterogeneity of inherited conditionsthatarecausedbymutations ineither theCOL4A3ortheCOL4A4 gene on chromosome locus 2q36–37(COL4A3 [MIM120070] and COL4A4 [MIM120131];http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db �OMIM) extends from familial benign isolated micro-scopic hematuria as a result of thin basement membranenephropathy (TBMN), which is largely asymptomatic,to nephrotic syndrome and end-stage renal failure withsymptoms that are consistent with autosomal recessiveAlportsyndromewithprogressivehemor-rhagicnephri-tis, chronic kidney failure, sensori-neural deafness, andocular findings. Owing to their similar although hetero-geneous genetic background, it has been suggested thatthey be referred to as COL4A3/COL4A4 nephropathies.

The two genes are located head to head and code for thelong�3 and�4 collagen chains of type IV collagen that isthe main structural component of glomerular basement

Received April 13, 2007. Accepted July 2, 2007.

Published online ahead of print. Publication date available atwww.jasn.org.

L.D.’s current affiliation is Limassol General Hospital, Limassol, Cyprus.A.P.’s current affiliation is Ippokrateion Private Hospital, Nicosia, Cyprus.

Correspondence: Prof. Constantinos Deltas, Department of Bio-logical Sciences, University of Cyprus, Kallipoleos 75, 1678 Nico-sia, Cyprus. Phone: 00-357-22-892882; Fax: 00-357-22-892881;E-mail: [email protected]

Copyright © 2007 by the American Society of Nephrology

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3004 ISSN : 1046-6673/1811-3004 J Am Soc Nephrol 18: 3004–3016, 2007

membranes (GBM) (for review, see Kashtan1 and Tryggvason andPatrakka2).

The autosomal recessive form of Alport Syndrome, whichaccounts for 14% of all Alport cases, was initially linked togenes COL4A3 and COL4A4 through linkage analysis and thenby identification of causative mutations.3 The majority of Al-port cases, 85%, are accounted for by mutations in theX-linked form with mutations in COL4A5.1 A small propor-tion of patients, on the order of 1%, are also accounted for bymutations in either the COL4A3 or the COL4A4 gene, exceptthat these present in heterozygosity and are described as auto-somal dominant Alport syndrome (ADAS).4 –7 ADAS is de-scribed as a milder form of the disease with later age at onsetand with not all classical symptoms.

Lemmink et al.8 were the first to describe mutations in theCOL4A4 gene in cases of benign familial hematuria. Soon after,various other groups9 –18 presented patients with a spectrum ofsymptoms and different diagnoses that were attributed to mu-tations in either of the two genes. Of particular interest is thatseveral mutations in heterozygosity can lead to isolated micro-scopic hematuria that is histologically characterized by TBMN,whereas others can occasionally lead to more severe symptomsand ADAS. TBMN is the most common cause of persistentmicroscopic hematuria in children and adults; other commoncauses include IgA nephropathy and Alport syndrome.19 –21

TBMN has a frequency of �1% in the general population,most times described as an inherited, nonprogressive disor-der.2,22 Clinically, the critical question is how to discriminate ina young patient between true benign familial hematuria causedby nonprogressive TBMN and ADAS, which could presentwith microscopic hematuria and thin basement membranes atits early stages, only to progress later to proteinuria and severeglomerular disease that nearly always leads to ESRD.19

After the first link between TBMN with familial hematuriaand COL4A3/COL4A4 mutations was made a decade ago, sev-eral groups8 –18 in different countries investigated and pub-lished cases with disparate clinical presentation. In approxi-mately 40% of families with TBMN, the phenotype has beenshown to segregate with COL4A3/COL4A4, whereas the restcould be attributed to spontaneous new mutations, preventingclear-cut linkage analysis results, or incomplete penetrance ormutations in other, as-yet-unknown genes. A predominantfeature was the significant allelic and phenotypic heterogeneityin the presence also of incomplete penetrance.2,20,23,24 Of im-portance is also that TBMN in several reports (reference 21 andreferences therein) was shown to be associated with other glo-merulopathies, such as IgA nephropathy, FSGS, minimal-change disease, mesangioproliferative glomerulonephritis,and others.

In this study, we describe a cohort of Greek-Cypriot pa-tients with renal disease of variable severity, characterized inadolescence and early adult life by familial microscopic hema-turia and TBMN in all cases in which renal biopsies were per-formed. A striking common feature in a significant percentageof affected older members was the presence of establishedchronic renal failure (CRF) or even ESRD, which on renal bi-opsy was due to advanced segmental glomerulosclerosis. Thesepatients did not develop any visual or auditory deficits; how-ever, in subsequent years, they developed proteinuria, ne-phrotic syndrome, and progressive CRF; the histologic picture

of FSGS is an important association that has only infrequentlybeen described in the past.25–27

RESULTS

Light and Electron Microscopy Observations on RenalBiopsies Revealed FSGS and TBMNThe common histologic picture of FSGS in 20 renal biopsies of13 families that segregated microscopic hematuria and CRFwas the initial main finding that prompted us to investigatethem clinically and molecularly. In patients with establishedCRF, there were sclerosed glomeruli up to 50%, and the re-maining glomeruli showed focal segmental lesions of sclerosisthat were characterized by increased matrix, causing oblitera-tion of the capillary lumina, and by hyaline material deposi-tion. Distribution of lesions within the tuft was variable, affect-ing both peripheral and perihilar segments. Sclerosedsegments formed adhesions to Bowman’s capsule (Figure 1A).

Figure 1. (A) Light microscope showing a glomerulus with FSGS.This biopsy is from a patient from family CY5303, carrying muta-tion 3854delG in COL4A4, performed at age 40. (B) Electronmicrograph showing an increase in mesangial matrix (M), as wellas partial occlusion of capillary lumina. Thinning of GBM thatmeasure 250 nm is also seen (arrows). Note the marked fusion ofpodocytes (arrowheads). This biopsy is from a patient also offamily CY5303, performed at age 47. N, nucleus; L, lumina; RBC,red blood cell. Magnifications: �250 in A; �9000 in B.

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J Am Soc Nephrol 18: 3004–3016, 2007 COL4A3/COL4A4 Mutations in TBMN/FSGS 3005

These changes were much milder in patients who underwentbiopsy early, when they had normal serum creatinine and onlyshowed microscopic hematuria with minimal proteinuria. Insome glomeruli, periglomerular fibrosis was evident. Immu-nofluorescence results were essentially negative. None of ninebiopsies that also were submitted for electron microscopy(EM) showed electron-dense deposits. All EM biopsy speci-mens showed mild to moderate segmental increase in mesan-gial matrix with variable folding and collapse of GBM. Themain ultrastructural features included thinning of GBM andeffacement of the podocytes foot processes (Figure 1B). Theaverage thickness of the GBM did not exceed 250 nm, whereasthe thinning of the GBM varied from extensive to focal areas ofthinning. In these attenuated GBM regions, the GBM segmentwidths did not exceed 150 nm. None of the biopsies showedsplitting or lamellation. In one patient of family CY5303, theGBM thickness had an average measurement of 300 nm, whichis borderline normal.28

DNA Linkage Analysis around Candidate LociRevealed Strong Linkage to 2q36–37, the Locus forCOL4A3/COL4A4Starting with a possible diagnosis of familial FSGS, we initiallysuspected a primary form of autosomal dominant FSGS, althoughwe did not exclude the possibility that FSGS was of secondaryetiology. Taking into consideration also the uniform existence offamilial hematuria, we proceeded with linkage analysis usingpolymorphic markers at four relevant loci: 19q13.1 for ACTN4

gene (�-actinin 4),29,30 11q22 for TRPC6,31,32 6p12.3 (CD2AP),33

and 2q36–37 (COL4A3/COL4A4) (Table 1). Suggestive linkagewas found for two families at 19q13.1 and another family at11q22, whereas none linked to 6p12.3. For families linking to19q13.1, mutation screening was performed for ACTN4 and alsofor NEPH3 and WTIP, which are important genes expressed inpodocytes and mapped in proximity to ACTN4.34,35 No muta-tions segregating with the disease were identified. One large fam-ily, CY5308, did not show linkage to any of the tested candidateloci. It is interesting that one family, CY5306, provided a positivelogarithm of odds (LOD) score suggestive of linkage at two loci,19q13.1 and 2q36–37. Five other families provided positive link-age for locus 2q36–37, which harbors the autosomal genes fortype IV collagen of the basement membranes, COL4A3/COL4A4.The maximum total LOD score at locus 2q36–37 for these sixfamilies was 5.8 at recombination fraction � � 0.06 (markerD2S1363, penetrance 0.7). Four haplotypes were constructed withmarkers D2S1363, D2S159, D2S401, and D2S439, which co-seg-regated with the disease in all six families. Haplotype K was com-mon in four families, haplotype C was found in one family, andhaplotype Ky was found also in one family (data not shown).Subsequently, haplotype K was documented in three additionalfamilies and haplotype Ky in two additional small families.

Mutation Screening and DNA Sequencing IdentifiedThree Mutations in 10 of the 13 FamiliesGenetic screening with SURVEYOR endonuclease (Trans-genomic, Cheshire, United Kingdom) and DNA sequencing of

Table 1. Presentation of 13 families and characteristics of patientsa

Family

No of Patientsb

No. of

Family

Members

with

DNA

Sample

Biopsy

Results

(n)

Positive

LinkageMutation/s MC

MC

(Clinical

Data

Available)

MC with

Negative

Urine

Findingsc

MH

Onlyc

MH �

Proteinuria,

No CRFc

CRF or

ESRDcESRDc

Total Male Female

CY5301 22 11 11 49 FSGS (3), EM ND 2q36 G1334E (COL4A3) 21 21 1 9 4 7 5

CY5303 8 4 4 19 FSGS-TGBM (3) 2q36 3854delG (COL4A4) 12 12 4 5 1 2 1

CY5304 8 4 4 32 FSGS-TGBM (1) 2q36 G1334E (COL4A3) 7 7 1 2 1 3 2

CY5306 5 3 2 13 FSGS (1), EM ND 19q13 2q36 G871C (COL4A3) 5 5 0 3 1 1 1


CY5308 19 10 9 75 FSGS (1), EM ND Negative – – – – – – – –

CY5309 7 4 3 21 FSGS (1), FSGS-TGBM (2) 19q13 Not found – – – – – – –

CY5312 4 2 2 20 FSGS (1), EM ND 11q22 Not found – – – – – – –


CY5314 6 3 3 12 FSGS-TGBM (1) ND G1334E (COL4A3) 6 6 0 4 1 1 0

CY5323 2 2 0 4 FSGS (1), EM ND ND G871C (COL4A3) 2 2 0 1 0 1 0

CY5326 1 1 0 1 TGBM � Alport (1)d ND G1334E (COL4A3) 1 1 0 0 1 0 0

CY4201/1637 12 5 7 28 Alport (2), FSGS (1), EM ND ND G1334E (COL4A3) –

G871C (COL4A3)

17 12 0 3 0 7 3

Sum 116 59/116 57/116 373 22 95 84/95 6/82 33/82 12/82 31/82 16/82

% 50.9 49.1 88.4 7.3 40.2 14.6 37.8 19.5

aMC, mutation carrier; MH, microscopic hematuria; ND, not done; TGBM, thin GBM.bPatients for whom we have DNA sample.cThe 82 MC for whom we have clinical data. The two compound heterozygous patients of family 4201/1637 are excluded.dThe biopsy was done on the mother of the patient we examined. She had developed Alport syndrome at advanced age, documented by EM and in thepresence of deafness.


3006 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 3004–3016, 2007

COL4A3/COL4A4 revealed the same substitution of A nucleo-tide for G at position 4001, converting glycine 1334 to gluta-mate (G1334E) in seven families, in whom the disease co-seg-regated with haplotype K (Figure 2). It is widely known that inthe collagenous domain of all collagens, the glycines are indis-pensable, because they are the smallest amino acids that canaccommodate themselves where the three chains meet to formthe triple helix. The common haplotype and same mutation inseven families raises the possibility for a founder effect.36 –38

Mutation G1334E had been previously reported in a Cypriotpatient.39

In three families who carried haplotype Ky, a substitution ofa G nucleotide at position 2611 by T resulted in conversion ofglycine 871 to cysteine (Gly871Cys; Figure 2), also strengthen-ing the hypothesis of a founder ancestor. In family CY4201/CY1637 that segregates classic autosomal Alport syndromeand microscopic hematuria, both of these mutations werefound (Figure 2C). The parents are heterozygous, whereastheir son and one daughter inherited both mutations and de-veloped classic Alport syndrome. It is interesting that themother, who is heterozygous for G1334E, also developed CRFin her late 50s after donating one of her kidneys to her son.Testing of the mutations on extended pedigrees confirmedtheir co-segregation with the disease phenotype. In addition,none of 56 or 141 unrelated healthy individuals of the generalpopulation carried mutation G871C or G1334E, respectively.Several polymorphisms were also detected (Table 2). Quickand efficient examination of samples for both mutations wasperformed by restriction enzyme digestion (Figure 2A).

Family CY5303 had haplotype C (data not shown) at locus2q36 –37, and DNA sequencing revealed a single nucleotidedeletion, 3854delG, which results in frameshift at Ser1217 andintroduces a stop codon at amino acid residue 1287. Mutation3854delG did not create or abolish a known restriction recog-nition site; therefore, a new recognition site was engineered inby designing a modified 5� primer for PCR amplification. Thismutation was previously reported in one patient.40

Molecular diagnosis of all family members revealed thatsix individuals, representing 7.3% of the 82 individuals forwhom we had detailed clinical data, had inherited the mu-tation but were healthy, with no signs of hematuria on morethan one urine examination, indicating incomplete pen-etrance of 92.7% (Table 1). In addition, three patients, twowith documented microscopic hematuria and one with doc-umented TBMN on biopsy, did not carry the relevant mu-tation, obviously representing phenocopies (Figure 3), mostprobably because their clinical condition had a differentcause.

For a comprehensive view of our patient cohort, we dividedthem into four age groups and classified them according tosymptom severity (Figure 4). Evidently, on long-term follow-up, an increasing proportion of mutation carriers progress toCRF. When we confined the observation to patients who wereolder than 51 yr, 28 (80%) of 35 had developed CRF or ESRD,

and 16 (46%) of 35 had progressed to ESRD, a finding notconforming to the term of benign familial hematuria.

DISCUSSION

We studied families and patients of the Greek-Cypriot com-munity with a population of 646,400. We presented for the firsttime detailed clinical and molecular data pertaining toCOL4A3/COL4A4 mutations, a field that in recent years hasattracted much attention, perhaps because it has been docu-mented that such mutations in homozygosity are responsiblefor the rare autosomal recessive Alport syndrome phenotype,and in heterozygosity for the more common phenotype of be-nign familial hematuria with TBMN.

Heterozygous COL4A3/COL4A4 Mutations as a Causeof TBMN Associated with FSGSIn 20 renal biopsies from patients who were aged 32 to 63 yrand belonged to 13 unrelated families with familial hematuria,there was FSGS in the presence of TBMN. In 10 (77%) of thesefamilies, mutations were found in the COL4A3/COL4A4 locus.All families had interesting clinical and genetic features with aspectrum of symptoms ranging from totally asymptomatic iso-lated microscopic hematuria to proteinuria and CRF caused byFSGS, relatively frequently leading to ESRD. There was evi-dence for phenocopies; three patients presented with hematu-ria without having inherited the respective mutation. Therewas also proof of reduced penetrance of 92.7%; six of 82 indi-viduals who carried a mutation were healthy.

All 15 patients who underwent biopsy and belonged to the10 families who segregated familial hematuria and TBMN inthe presence of COL4A3/COL4A4 mutations also had FSGS(Table 1). This association of FSGS with TBMN is a strikingfinding that allows the hypothesis that either FSGS is a second-ary development of the COL4-linked TBMN or the result of aco-inherited glomerulopathy that is actually responsible forthe progressive nature of the disease, a likelihood that also hasbeen suggested by others.27 Our data do not allow us to distin-guish with certainty between these two situations. Norby andCosio27 suggested that all patients with suspected TBMN andheavy proteinuria or renal insufficiency should undergo a renalbiopsy examination. Nieuwhof et al.25 published the results ofa prospective study with a 12-yr follow-up of 19 patients withTBMN and microscopic (18 of 19) or macroscopic (1 of 19)hematuria. They were the first to note clearly the associationbetween TBMN and late-onset renal impairment on long-termfollow-up in elderly patients. In six first-degree relatives ofthese 19 patients, ESRD was established, prompting the au-thors to conclude that TBMN predisposes to premature glo-merular obsolescence that may lead to late-onset renal insuffi-ciency. Sue et al.,41 in a retrospective review of 658 nativekidney biopsies, concluded that TBMN on its own is a benigncondition not contributing to the final renal outcome, which isdetermined only by another, coexisting glomerulopathy. To a



Figure 2. Molecular analysis of mutations G871C (right) and G1334E (left) in COL4A3 that were found in families carrying haplotype Ky andhaplotype K, respectively. Family CY4201/CY1637 carried both haplotypes and both mutations (MW, 100-bp molecular weight marker). (A)Mutation G1334E created a new restriction site recognition for enzyme Hpy188III, and mutation G871C created a new recognition site forenzyme HpyCH4III. PCR-amplified products were digested and electrophoresed on 3% agarose gels. Samples 1, 3, 4, and 5 on the left andsamples 2, 3, and 4 on the right carry the respective mutations. (B) Electropherograms from automatic sequencer CEQ2000 showing normalsamples at the bottom and heterozygous samples on the top. In the sequence, the wild-type nucleotide is in bold. (C) Part of the largepedigree for family CY4201/1637, showing the heterozygous parents who carry each of the mutations, whereas two of the children (filledsymbols) are compound heterozygotes for both and developed Alport syndrome. Shown are the haplotypes K and Ky as they were phasedon the basis of larger number of family members. It is interesting that the mother, who is heterozygous for G1334E, also developed CRF inher 50s and received a transplant at 62, long after she had donated one kidney to her son.



similar conclusion had arrived Nogueira et al.,26 who had pre-sented eight patients with TBMN, five of whom had heavyproteinuria and nephrotic syndrome as a consequence of asso-ciated FSGS.

van Paassen et al.,42 in their comprehensive elegant work,recognized that in a series of 22 patients who originally hadbeen classified as having primary FSGS with microscopic he-maturia, 50% had secondary FSGS as a result of TBMN,

thereby admitting that FSGS can develop on the genetic back-ground of TBMN. On the contrary, Haas43 reported that noneof 37 biopsies of patients who had TBMN and presented withpersistent or recurrent hematuria showed FSGS, whereas inseven of 10 patients who had FSGS and were incidentally dis-covered to have TBMN, their FSGS was likely of primary na-ture.

In our cohort, in a total of 82 patients from 10 families

Table 2. Mutations and polymorphisms found in the studied genesa

Type of Variant/Gene Nucleotide Change Exon Reference/Database

MutationCOL4A3

G871C 2611 G�T 32 This studyG1334E 4001 G�A 45 39

COL4A43854delG (frameshift at Ser1217, Stop at 1287) 39 40

PolymorphismCOL4A3

IVS5 �73C/T Intron 5 Ensembl databaseP141L 422 C/T 7 12

E162G 485 A/G 9 39

IVS15 �30G/A Intron 15 Ensembl databaseIVS17 �80T/C Intron 17 This studyK834R 2501 A/G 32 15

IVS41 �110T/G Intron 41 This studyIVS42 �66C/T Intron 42 This studyQ1495R 4484 A/G 49 12

IVS50 �67delA Intron 50 Ensembl databaseCOL4A4

P482S 1444 C/T 21 11

G545A 1634 G/C 23 40

L1004P 3011 T/C 33 40

G1198G 3594 G/A 39 11

K1228K 3684 G/A 39 11

IVS40 �9G/C Intron 40 Ensembl databaseIVS41 �34T/C Intron 41 Ensembl databaseM1327V 3979 A/G 42 40

P1360P 4080 A/G 42 11

IVS43 �36G/A Intron 43 Ensembl databaseP1403S 4207 C/T 44 40

IVS44 �24C/T Intron 44 Ensembl databaseV1516V 4548 A/G 47 11

ACTN4P179P 537 G/A 5 Ensembl database

TRPC6IVS3 �100G/A Intron 3 Ensembl databaseN561N 1683 T/C 6 Ensembl databaseIVS10 �138C/T Intron 10 Ensembl databaseIVS12 �20–22delCTT Intron 12 NCBI database

NEPH3�170 508 G/A 4 Ensembl database�351� 1053 G/T 8 Ensembl databaseV353M 1057 G/A 9 Ensembl database

WTIPIVS4 �8G/C Intron 4 This studyIVS5 �25C/T Intron 5 Ensembl

aNucleotide number 1 is the A of the first ATG codon of translation. NCBI, National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/)



with detailed clinical information, 31 (38%) patients hadprogressed to CRF and 16 (19.5%) had progressed to ESRDthat required renal replacement therapy (Table 1). Confin-ing the statistics to patients who were older than 51 yr, 28

(80%) of 35 had developed CRF or ESRD and 16 (46%) of 35had progressed to ESRD (Figure 4), representing the highestpercentages of patients who had TBMN and progressed toFSGS and ESRD, to our knowledge.19 These high figures are

Figure 3. CY5301 is one of our largest pedigrees carrying mutation G1334E. Carriers of this same mutation present with a spectrumof symptoms ranging from microscopic hematuria in the presence of TBMN to ESRD in five patients (see also Table 1). Samples wereavailable from individuals in generations III and IV. It is interesting that we noticed that individual III4 represents phenocopy, becauseshe has biopsy-proven TBMN in the presence of microscopic hematuria and in the absence of the mutation. Individual IV25 carries theG1334E mutation but is asymptomatic (reduced penetrance). Monozygotic twins IV9 and IV10, who were shown to carry the mutation,exhibited identical phenotype. Some individuals of the youngest generation who seem healthy have not been examined clinically ormolecularly.



not the norm in existing literature, where most times TBMNis described as a benign condition with excellent prognosis.Another likely hypothesis is that the mutations presentedhere are more pathogenic and predispose a subset of pa-tients to severe phenotype. Perhaps future work could focuson detailed examination of patients carrying the same mu-tation to evaluate carefully the intrafamilial variability andassess the impact of the mutation on pathology. Anotherapproach is to create transgenic mice with this and anothermutation and assess their long-term phenotypes; however,many similar mutations substituting the essential glycineresidues have been described by others,24 without notingsuch high percentage of adverse outcome.

Kashtan, in his review,1 asked the rhetorical question ofwhether TBMN and benign familial hematuria should be con-sidered synonymous, and he provided the answer: “Probablynot.” Our work here enhances this propensity; in fact, our datasuggest that an ever-increasing percentage of COL4A3/COL4A4 mutation carriers may shift to the group of patientswith CRF by advanced age (Figure 4). It is reasonable to hy-pothesize that those who progress to CRF do so under theinfluence of yet-unknown modifier genes that on the back-ground of TBMN predispose to FSGS and CRF, although onecannot exclude that the modifier gene effect creates an age-dependent penetrance, as suggested by the results in Figure 4.Another explanation for not seeing this adverse outcome moreoften in many patients with TBMN could be the short fol-low-up of these patients, considering that in most cases thedisease course takes more than two decades to cause protein-uria and detectable renal impairment. One advantage in oursetup is our small communities, the frequent and systematicfollow-up, and the controlled delivery of services by a smallteam of specialists.

Mutation Screening, DNA Sequencing, and FounderEffectsIt is worth commenting that the strategy used for mutationscreening of these multiexon large genes, with the systematicuse of SURVEYOR enzyme accompanied by direct automatedDNA sequencing, is very reliable and effective and may be con-sidered for use in routine clinical diagnostic set-up. Themethod is suitable for larger DNA fragments (we applied it tofragments of nearly 800 bp), and it allows accurate localizationof the mismatch upon electrophoresis. The future develop-ment of an automated method for mutation screening willprove to be instrumental in cases in which a kidney biopsy isnot possible, and the noninvasive molecular diagnosis may re-place renal biopsy and EM for the diagnosis of TBMN. Theavailability of fast and reliable molecular testing will be evenmore helpful in distinguishing between IgA nephropathy andTBMN or Alport syndrome in early stages, and it will be ofparamount importance in cases of potential living-related kid-ney donors.

Finally, mutations G1334E and G871C on common haplo-types in seven and three families, respectively, suggest foundereffects, something not uncommon among the Greek-Cypriotpopulation. There are two good examples for dominant dis-eases. One is the PKD2 mutation R742X in a family with thetype 2 variant of polycystic kidney disease, with �80 affectedindividuals in Lythrodontas village.37 The second refers tomedullary cystic kidney disease 1, for which we reported on sixlarge families in whom the disease segregates with a commonMCKD1 gene haplotype. All patients are from the area of Pafosnear the west coast, geographically clustered in a triangle ofthree neighboring villages.44 For a recessive disorder, a classicexample is the CFTR mutation F508del, with a carrier fre-quency of 1:14 in the village of Athienou, near Nicosia, the

Figure 4. Diagrammatic representation of 76 patients who are heterozygous for a COL4A3/COL4A4 mutation (the two compoundheterozygous patients of family CY4201/1637 are excluded). They are classified in four age intervals on the basis of three clinicalsymptoms, namely microscopic hematuria, proteinuria, and CRF. In age interval 11 to 30, 18 patients; 31 to 50, 23 patients; 51 to 70,28 patients; 71 to 90, seven patients. Note that 80% (28 of 35) of patients aged 51 to 90 yr have progressed to CRF or even ESRD. Thesix patients who carried a mutation but were asymptomatic were not included in this representation.



capital36 (for an extensive description of founder effects inCyprus, see Deltas38).

Many publications emphasize that TBMN co-segregatingwith heterozygous COL4A3/COL4A4 mutations is a cause ofbenign familial hematuria with excellent prognosis. Our ex-perience with 82 patients from 10 families was that 38% ofpatients of all ages developed CRF and 19.5% progressed toESRD, thereby suggesting that the term “benign” is a mis-nomer, at least in our population. That 15 biopsies withFSGS were from families with familial hematuria, TBMN,and heterozygous COL4A3/COL4A4 mutations allows thehypothesis of a causal relationship. This is also supported bysimilar findings in a COL4A3�/� knockout murine modelthat develops TBMN coinciding with focal glomeruloscle-rosis and other renal pathology.45 In addition, the presenceof phenotypic heterogeneity allows the hypothesis that ge-netic modifiers and/or a co-inherited glomerulopathy maycontribute to the disease course. One way to deal with thesignificant variable expressivity in carriers of such muta-tions might be to refer to them collectively as COL4A3/COL4A4 nephropathies, thereby treating them under a uni-fying molecular terminology.23

CONCISE METHODS

PatientsPatients and healthy individuals belonged to 13 families from Cyprus

who had no documented relationship. A distant, unknown relation-

ship to one or more common ancestors cannot be excluded; in fact, it

is highly suggested on the basis of the molecular findings. All families

were initially selected through a proband because of FSGS in their

renal biopsies performed after the development of proteinuria and/or

mild renal failure, in the presence of microscopic hematuria. Addi-

tional family members were recruited on the basis of the observation

of familial microscopic hematuria as well as other renal symptoms,

such as proteinuria and/or renal failure. Under certain circumstances

(Table 1), several patients from the same family underwent biopsy.

Patients with clinical or histologic evidence of systemic lupus ery-

thematosus, vasculitis, collagen diseases, and drug consumption were

excluded. Patients with HIV or hepatitis B or C infection were also

excluded. CRF was defined as an elevated serum creatinine �1.5 mg/

100 ml. In total, 20 biopsies were performed percutaneously at ages

that ranged from 32 to 63 yr (mean age 45.5 yr). In one family, a renal

biopsy was available on the mother of the patient (family CY5326;

Table 1). In family CY4201/1637, two additional biopsies were done

in two young siblings who proved to be compound heterozygotes with

mutations in the COL4A3. They both developed early-onset Alport

syndrome with sensorineural deafness and underwent kidney trans-

plantation. Their mother carried mutation G1334E, and after donat-

ing one of her kidneys, she also gradually developed end-stage renal

failure and received a transplant at age 60. Her husband and his sib-

lings carried mutation G871C, showed microscopic hematuria, and

developed various degrees of CRF. The symptoms of the patients in

our cohort varied significantly, ranging from asymptomatic isolated

microscopic hematuria to additional proteinuria, up to CRF and

ESRD that required hemodialysis or kidney transplantation, perhaps

justifying the diagnosis of ADAS, although with an incomplete spec-

trum of symptoms.

Immunofluorescence, Light Microscopy, and EMRenal biopsy samples were processed for light microscopy and EM

and for direct immunofluorescence. For light microscopy, paraffin

sections were stained with hematoxylin and eosin, periodic acid-

Schiff, silver methenamine, and Masson’s trichrome. For immuno-

fluorescence, cryostat sections were incubated with a panel of FITC-

linked mouse antibodies against human IgA, IgG, IgM, C1q, C3, and

fibrin. For EM, specimens were processed as described previously28

and photographed in a JEM-1010 transmission electron microscope

(JEOL 1010, Tokyo, Japan). The diagnosis of TBMN was made at the

ultrastructural level as described previously46 and considering the

thickness of the GBM to be 300 to 400 nm in normal adults.

DNA Linkage AnalysisAll individuals were informed and gave their consent for participa-

tion. DNA from peripheral blood of 373 individuals in total, 116 of

whom were classified as affected on the basis of clinical criteria, was

isolated by one of two methods: Either a salting-out procedure47 or

the Qiagen kit (Qiagen, Hilden, Germany). DNA linkage analysis at

locus 2q36 –37 that contains the COL4A3/COL4A4 genes was per-

formed with polymorphic microsatellite markers D2S1363-0.8, Mb-

D2S159-0.43, Mb-D2S401-0.92, and Mb-D2S439 (Ensemble Data-

base; http://www.ensembl.org/Homo_sapiens/index.html), and the

results were processed for two-point linkage analysis using the

MLINK program of the LINKAGE package of programs.48 Statistical

analysis and LOD score estimation were performed using disease pen-

etrance varying from 0.5 to 1.0. Linkage analysis was also performed

for loci 19q13, 11q22, and 6p12.3, which contain genes, mutations in

which are responsible for primary FSGS.29 –33

Mutation Screening and DNA SequencingAll exons of candidate genes were screened for mutations using the

SURVEYOR endonuclease, which cleaves double-stranded DNA at

positions of mismatches.49 For this, the PCR-amplified genomic se-

quences were subjected to denaturation by heating to 95°C for 5 min.

Then the DNA was allowed to renature by cooling to 65°C for 30 min

and 25°C for 30 min. The renatured PCR-amplified DNA were incu-

bated with 0.5 �l of SURVEYOR enzyme at 42°C for 20 min and then

examined by electrophoresis on high-resolution Eurobio 3:1 agarose

gels, 3%. When cleavage was evident, DNA sequencing was per-

formed using a kit for Dye Terminator Cycle DNA sequencing from

Beckman Coulter and fractionated on an automatic DNA sequencer

(CEQ2000XL; Beckman Coulter, Fullerton, CA). The oligonucleotide

primers were designed to include single or multiple exonic coding

sequences,39,40 as well as at least 60 bp of splice junctions and intronic

regions (Primer_3 software; http://www-genome.wi.mit.edu/cgi-bin/

primer/primer3_www.cgi) (Tables 3 and 4).

Mutation G871C in COL4A3 created a new recognition site for

restriction enzyme HpyCH4III, and mutation G1334E created a rec-

ognition site for enzyme Hpy188III (New England Biolabs, Boston,



MA). These enzymes were used for easy examination of additional

family members and for testing a number of healthy individuals in the

general population to ascertain whether they are pathogenic muta-

tions or neutral variants. Mutation 3854delG in COL4A4 was tested

by amplification and digestion of the relevant sequence in exon 39.

Amplification was with the modified forward primer 3854delG-F:

ggaatacctgggctaaaaggggagagaggagaccctcg and the reverse primer

3854delG-R: tctggaggaatgtgggaccagagagtggcagaatagaag. The forward

primer was modified by substitution of the penultimate nucleotide of

G by C (italics) so that upon amplification of the mutant sequence, a

restriction recognition site for XhoI is created.

ACKNOWLEDGMENTS

This work was funded mainly through a grant by the Cyprus Research

Promotion Foundation (�� X/0505/02) and partly by the Cyprus

Ministry of Health, The Cyprus Kidney Association (scholarship to

Table 3. Oligonucleotide primers and conditions for PCR amplification and DNA sequencing of COL4A3 exons

Exon Sense (5�33�) Antisense (5�33�)PCR AnnealingTemperature

(oC)

PCR ProductSize (bp)

1 GACCGAGCCCTACAAAACC CTCAGCCTCGCCACTAGC 68.5 3582 GTCACCTGGCTCCTAACAGATAGT GGGAAATGGAACAACATACTGC 62 2973 TGCAAAGAGTCACCATGAAGTC GATGTGGAATTCTCTCCAGGTT 66 2964 AGCTTGGCATGTGACTGAGACT ACCACTCCTTTGACATCTACCA 66 3005 GCTATTCCCAGTTATAGTGGAGGA ATCCTGTCCTGCTTGTATTTCAGG 65 3746 GATAAGTCTTCATATCAGTCAGCCTGT CCTAAAGGAATGTTTTCAAGGGAAG 62 3727 CATGACCCAGTAGCCATAAGGAAT CTACCCCAGACTTCCCTAAAGTCAT 65 3078 GAGAGGATACACAATAGCAGAGAGG CTGAATGATTAAGGTCTGGATTATG 58 2489 CTCTGAGTACATAACTTGAAAAGCA GAAAAGAGAGACGAAGTAAAGAGAGG 58 28710 to 11 GAGGTGTTATGCTCTTCTGATTTTA CTAACAGTGACCCACATGAAGAAG 58 43912 to 13 ATTGGAACTTTGATGGGTTTAG GTAAAAGAGGTGCAGGGTCACA 60 49914 GTTAACACGAGGCACATTCATAGTT ATAGCTGCTCTGCCTGTGGAGTA 58 33815 TTCTCTCTTAGATGGCTCAGCACT GAGCTAGTTTGAGTTGGGCATCTT 65 29016 to 17 CCTGAGGTCAAGAGGAGATGAT CCTTGTACTTAGGTCCAGGGTTG 65 56018 GTAGATGGGATTCCTTACCCAGATAC GTCAAAGGGACACATAGTGACCAG 65 40319 GGGCCAAAATGAAGAAACTG CTTTCCTCACCCATGTCAATTC 55 27920 TCTGATATTTGTCTAGGTGAGAGTAGG AGCCTGGAAACACTTCTAGATCA 65 31121 GTGTTATGTACCTCTCCATTGTGC AAACCACACAGACTGATGGCTAAG 60 39822 ACCATGCTTTCTCAGTTGCAG CATCACTGACTGGTAGCTATTTGG 63 35723 GTTCTTTCTGAGGACTCAATGTAGCTT CTTCCAGTGTATTGACCCTTTTGT 60 21324 CTGTCTACTTAGAGTTGGCGTTCA AAGGCACCAAACTGGTAAGTGT 63 22225 TGAAAAGTTCTTGTCCACACTG GTTTCATTCTGCAGTTAGAGTGATG 63 39826 GATCTCAATGACAGCCTAACTGGT GAGAAGCTTCGTTAGCTCTAGAAACC 63 34827 TCGTATTTTCCGCTATCGTC AAGAGAGTCAGGGTGTTGTGTGT 63 22728 ATGAAGGAAAGTTGCTGATGTG GAACATCTTCTAAATATCCACAACAAA 63 21129 GCATCTCTAGCTGGTTGAGAGATAAG ATCTAGAGTGCTCTGCCAGATAAGTG 63 28330 to 31 CTCCATAACAGAGAGTCAATATCAGTG AGTCTCATGTCTCCTGCCCTTCT 63 69332 GTTAGTAGGGGAAAGCATTTGTGG CTATGTACAGTTGACAGAGCCACCT 64 29833 to 34 CGACCCATCTCCTAGACTAATACAGT CCTCACTGTCCTTATTTGTCAACC 64 84135 TCCCACGTAGCTGGGATTAC CTGCTGGAACACTATCAGAACAGA 64 30036 CATGCATGCAAGCAACAAGT ACACAGAAAATCTGGAGTCCTCAC 63 24137 AGCCATTCTTGGGAGAAAGC ACACCGAATCTGGGTACTTGTT 58 32138 GTGCTGGCAGATAGCAGATACTAA GATTTCAGGAGGGCTATACTCTGA 63 32339 CAGACCGTTTCAGTCACTGTTGTA CAGAAATCCAAACCTCAGGAGA 63 23040 to 41 CACTGGCAAGAAATAAATCCTCTTT ATATTCAACTGCTTTCTCTCCTTGA 63 48842 GAGTCTCAACTCAGTTTTTGTAGCTCT GGAAAGAAGAAGCAACAAGGTAACA 65.5 47743 CTCCCTGGCTGGCAATACT AACATCATGAGAATGGACTAATACAG 62 29344 GTTCTAAGTGGCCAGAGGAGTTATAG GTACTAACCCCAGCTGTCTGTACTTC 65 35445 to 46 GCACACTTCTAGTATTTGTCCTTAGAGTC GAAGTTGTATCAGCTGTTTCCAAAG 65 47747 AGGTGACTAATCATCCTAGCTTGC CATTTGATCCTTGGCCTTTG 55 39948 CCACTCTTCTCTAGGATTGCTTTC GGCCAGTGTTTTATTTGCTCTC 65 48749 to 50 GTTGTCTTTGTCCAGCTTTTGC GACCCTGGGAATCACATTTTAC 63 62151 CTCCCCTTTCTTTACTCACAGTTG AAATCATGGCTACTCGTTGACC 65 37752 ACGCCCGACCCTGTAATAAAT CAGATTAGAGACCCAGATCACAGAAC 65 397



K.V.), the University of Cyprus Research Activities 3/312, and Efsta-

thios Constantinides.

Parts of this article were presented in abstract form at the 13th

PanHellenic Conference of Nephrology; Rhodos, Greece; June 16 –19,

2004; the 38th European Human Genetics Conference; Amsterdam,

Netherlands; May 6 –9, 2006; and the 39th European Human Genetics

Conference; Nice, France; June 16 –19, 2007.

We thank the patients and their families for participating in this

study. We also thank Michalis Markou (PhD Cand, University of

Cyprus) for the installation and use of linkage analysis software and

Gregory Papagregoriou (PhD Cand, University of Cyprus) for assis-

tance with figure preparation.

DISCLOSURESNone.

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Table 4. Oligonucleotide primers and conditions for PCR amplification and DNA sequencing of COL4A4 exons

Exon Sense (5�33�) Antisense (5�33�)PCR Annealing

Temperature (oC)PCR Product

Size (bp)

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See related editorial, “The Wages of Thin,” on pages 2800–2802.



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