CASE REPORT

Siblings with Alport’s syndrome showing unique stainingpatterns for a5(IV) and a6(IV) chains of collagen type IV

Takayuki Tsuji • Yoshihide Fujigaki •

Masanori Sakakima • Yoshikazu Sado •

Akira Hishida

Received: 23 April 2009 / Accepted: 15 January 2010 / Published online: 9 March 2010

� Japanese Society of Nephrology 2010

Abstract Here we report two brothers with electron-

microscopically diagnosed Alport’s syndrome (AS) who

showed normal staining patterns for the a1(IV)–a4(IV)

chains of collagen type IV, but abnormal expression of the

a5(IV) and a6(IV) chains. Both patients had microscopic

hematuria and mild proteinuria from around 10 years old,

and had renal biopsies at 23 (older) and 26 (younger) years

old due to increased proteinuria (0.5–0.8 g/day) with nor-

mal renal function. A skin biopsy of the patients’ mother

showed similar abnormal staining patterns for the a5(IV)

and a6(IV) chains in the skin basement membranes. Both

of them showed slow progression of renal dysfunction and

no extrarenal manifestations. The existences of incomplete

a3,a4,a5(IV) molecules in the glomerular basement mem-

brane (GBM) and inadequately formed a5,a5,a6(IV) mol-

ecules are suggested for these patients. A missense

mutation of the COL4A5 gene may present in this family

as possible X-linked inheritance and a mild form of AS.

Keywords Alport’s syndrome � Collagen type IV �a5 � a6

Introduction

Alport’s syndrome (AS) is a genetically heterogeneous

hereditary nephritis characterized by progressive renal

failure, high-tone sensorineural deafness and ocular lesions

[1, 2]. The pathogenesis of AS has been linked to abnor-

malities of collagen type IV, which is a major structural

component of basement membrane [2].

Six a(IV) chains have been identified, encoded by the

genes COL4A1 to COL4A6. These chains can form only

three sets of triple helical molecules called protomers, which

are designated a1.a1.a2, a3.a4.a5 and a5.a5.a6. These pro-

tomers create collagenous networks [3, 4]. In mammalian

normal kidney, the a3.a4.a5–a3.a4.a5 and the a1.a1.a2–

a1.a1.a2 networks are distributed in the glomerular basement

membrane (GBM) and some tubular basement membranes

(TBMs), and the a1.a1.a2–a5.a5.a6 and the a1.a1.a2–

a1.a1.a2 networks are distributed in Bowman’s capsule

basement membrane (BCBM) and some TBMs [3, 5].

In AS, a variety of mutations in the COL4A3, COL4A4

or COL4A5 genes and various clinical phenotypes have

been reported [6–9]. Mutations in the COL4A5 gene are

responsible for X-linked AS, which accounts for 85% of

AS familial cases [10]. 15% of AS families show autoso-

mal inheritance of the disease, with 14% recessive and 1%

dominant, both of which are caused by homozygous or

heterozygous mutations in the COL4A3 or COL4A4 col-

lagen genes [11]. These mutations lead to a morphologi-

cally abnormal GBM. Thus, immunohistochemical

examination reveals negative or mosaic staining of the

a3(IV), a4(IV) or a5(IV) chains in the GBM.

Here we report two brothers who were diagnosed with

AS by electron microscopy and showed unique immuno-

histochemical staining patterns of the a5(IV) and a6(IV)

chains in renal basement membranes.

T. Tsuji (&) � Y. Fujigaki � M. Sakakima � A. Hishida

First Department of Medicine,

Hamamatsu University

School of Medicine, 1-20-1 Handayama,

Higashi-ku, Hamamatsu, Shizuoka 431-3192, Japan

e-mail: tsu@hama-med.ac.jp

Y. Sado

Division of Immunology,

Shigei Medical Research Institute,

Okayama, Japan

123

Clin Exp Nephrol (2010) 14:283–287

DOI 10.1007/s10157-010-0265-4

Patient report

Two brothers were admitted to our hospital in 1999 (older,

23 years old) and 2004 (younger, 26 years old) due to

increased proteinuria (0.5–0.8 g/day) and persistent

microscopic hematuria. They had presented microscopic

hematuria and mild proteinuria from around 10 years old.

Their father was healthy, whereas their mother had been on

hemodialysis from the age of 41 years because of chronic

renal failure due to mesangial proliferative glomerulone-

phritis (no immunofluorescent and no electron microscopic

examinations were performed) without sensorineural

deafness or ocular lesions. Their maternal grandfather died

of chronic renal failure at 50 years old. Physical examin-

ations of the brothers revealed that they were in good

general condition and had normal blood pressure. Neither

of them had sensorineural deafness nor ocular lesions.

Laboratory tests of the older and younger brothers without

any medication showed the following results: 0.77 and

0.77 mg/dl of serum creatinine, 165 and 140 l/day of cre-

atinine clearance, 6.0 and 7.4 g/dl of total protein, 4.0 and

4.5 g/dl of albumin and 167 and 180 mg/dl of total cho-

lesterol, 2? and 2? of urinary occult blood, and urinary

protein excretion was 0.9 and 0.8 g/day, respectively.

Their renal biopsies revealed mild-to-moderate mesan-

gial proliferative glomerulonephritis on light microscopy.

There was no significant deposition of immunoreactants

(IgG, IgA, IgM, C1q, C3 and C4) upon immunofluorescent

examinations. Electron microscopy showed irregular

thickening of the GBM with splitting, fragmentation of the

lamina densa, and remnants, indicating AS in both patients

(Fig. 1). The GBM abnormalities in the older patient were

localized, while those in the younger patient were diffuse

and were associated with extensive thinning (Fig. 1).

Apparent ultrastructural abnormalities could not be found

in the BCBM and TBM. An immunofluorescence study kit

for AS (Cosmo Bio, Tokyo, Japan) showed normal staining

patterns for a2(IV) and a5(IV) in both patients.

The distribution of the a1(IV)–a6(IV) chains was further

examined by immunohistochemistry. The immunoperoxi-

dase method was applied on 10% formalin-fixed paraffin

sections of both patients. Rat monoclonal antibodies that

recognized the six different a(IV) chains of human type IV

collagen were used [12]. Antibodies H11, H22, H31, H43

and H63, specific for the a1(IV), a2(IV), a3(IV), a4(IV)

and a6(IV) chains, respectively, were raised against the

synthetic peptides of the human a(IV) noncollagenous

(NC) domain. Antibody H53, specific for the a5(IV) chain,

Fig. 1 Electron micrographs of

the glomerular capillary walls

from the older (a, b) and the

younger (c, d) brothers, showing

a irregular thickening of the

GBM with splitting, b, dfragmentation of the lamina

densa and remnants, and cextensive thinning of the GBM

284 Clin Exp Nephrol (2010) 14:283–287

123

was raised against that of the human a5(IV) helical domain

[13]. Antigen retrieval was performed with autoclave

heating according to the method reported by Naito et al.

[13]. The sections were heated at 110–121�C for 12 min to

evaluate the GBM, the BCBM, the TBM and skin base-

ment membranes. Renal biopsy specimens obtained from a

patient with interstitial nephritis in our hospital were used

at the same time as a positive control. Ten and 21 glomeruli

were evaluated in the older and younger brothers, respec-

tively. All of the examined glomeruli showed the same

expression pattern for the immunostaining of a(IV) chains.

Immunostainings of the a1(IV) and a2(IV) chains were

intense in the GBM, the mesangial matrix, the BCBM and

the TBM. The a3(IV), a4(IV) and a5(IV) chains in the

GBM were clearly stained after heating at 121�C. The

a3(IV) and a4(IV) chains in the TBM were partially

stained after heating at 121�C. However, the a5(IV) and

a6(IV) chains in the BCBM and the TBM were not stained

after heating at 121�C. Even after heating at 115 and

118�C, they were not stained in the BCBM and the TBM

(Fig. 2), whereas those of the positive control were clearly

stained (not shown). Immunohistochemistry of the a1(IV)–

a4(IV) chains indicated normal staining patterns for the

a1(IV)–a4(IV) chains, but abnormal expression of the

a5(IV) and a6(IV) chains in the kidneys of both patients

(Fig. 2; Table 1). A skin biopsy was performed on their

mother at the age of 54. Immunohistochemistry of the

a(IV) chains in cutaneous tissues showed that the staining

of the a2(IV) chains was intense. However, the signal

intensities of the staining for a5(IV) and a6(IV) chains

were weak in the skin basement membranes (Fig. 3) when

compared with those in the normal control (not shown).

Moreover, staining of the a5(IV) chains showed an

apparent intermittent pattern, whereas the signal intensity

of the staining for a6(IV) chains was too weak to assess a

staining pattern (Fig. 3). The findings suggested abnormal

expression of the a5(IV) and a6(IV) chains in the mother’s

skin basement membranes, possibly because of the similar

abnormalities of the a(IV) chains to those in her sons’ renal

tissues.

Since they were diagnosed as having AS, treatment with

losartan, dipyridamole and simvastatin was started in the

older patient, and candesartan and dilazep hydrochloride in

the younger patient. Recent (December 2008) laboratory

tests on the older and younger brothers gave the following

results: 1.17 and 0.89 mg/dl of serum creatinine, 6.8 and

7.3 g/dl of total protein, 4.6 and 4.6 g/dl of albumin, 190

and 191 mg/dl of total cholesterol, 3? and 3? of urinary

occult blood, and urinary protein excretion was 0.93 and

0.34 g/g creatinine, respectively. They showed no signs of

sensorineural deafness and ocular lesions, and their renal

functions have remained relatively stable, suggesting that

they have a mild form of AS.

Discussion

We described two brothers with a mild form of AS that was

diagnosed by electron microscopy (Fig. 1). Immunohisto-

chemical examination showed normal staining patterns for

the a1(IV)–a4(IV) chains, but abnormal expressions of the

a5(IV) and a6(IV) chains in the BCBM and TBM; the

GBM showed a normal staining pattern (Fig. 2; Table 1).

A skin biopsy of the patients’ mother, who is undergoing

hemodialysis, revealed a similar abnormal pattern for the

a(IV) chains in her cutaneous basement membranes

(Fig. 3).

The six a(IV) chains are known to be encoded by six

genes, COL4A1 to COL4A6, that are arranged as head-to-

head pairs (COL4A1–COL4A2, COL4A3–COL4A4 and

COL4A5–COL4A6), and are localized on chromosomes

13q34, 2q35-37 and Xq22, respectively [3]. These chains

form three sets of triple-helical molecules called protom-

ers: a1,a1,a2(IV), a3,a4,a5(IV) and a5,a5,a6(IV) [3]. In

the GBM, the a1,a1,a2(IV) network that develops during

early nephrogenesis is replaced by an a3,a4,a5(IV) net-

work in the mature glomerulus [14]. Mutations in

COL4A3, COL4A4 and COL4A5 observed in AS patients

produce post-translational defects in the a3(IV), a4(IV) and

a5(IV) chains, major structural components of the GBM.

Heterozygous mutations in COL4A5 are reported to be

responsible for the major X-linked AS [15–17]. More than

300 mutations in COL4A5 were reported. Recently, a

number of researchers have attempted to link genotypes in

AS to phenotypes. Gross et al. [18] have reported a

genotype–phenotype correlation in German X-linked AS

families. They concluded that knowledge of the COL4A5

mutation provides significant information on the progress

of renal and extrarenal manifestations. Homozygous or

compound heterozygous mutations in COL4A3 or

COL4A4 are responsible for autosomal recessive AS.

Autosomal dominant AS caused by heterozygous COL4A3

and COL4A4 mutations is rare.

Naito et al. [19] previously reported on patients with low

a5(IV) chains expression who had missense mutations in

the COL4A5 gene. One patient in their report showed

atypical expression of a5(IV) chain positivity in the GBM

and the BCBM and a6(IV) chain positivity only in the

BCBM. This patient possessed a missense mutation in the

COL4A5 gene that may lead to this atypical collagen

expression pattern. Other patients also showed higher

expression of a5(IV) chains than a6(IV) chains. In addi-

tion, Naito et al. [20] also reported a family with X-linked

AS may have a missense mutation in the COL4A5 gene,

resulting in abnormal staining patterns for a3(IV), a4(IV)

and a5(IV) chains in the GBM and normal staining patterns

for a5(IV) and a6(IV) chains in the BCBM and the skin.

This single COL4A5 mutation could lead to the atypical

Clin Exp Nephrol (2010) 14:283–287 285

123

expression patterns for both the a5(IV) and the a6(IV)

chains, though in theory our patients could have both the

COL4A5 and the COL4A6 mutations. Furthermore,

mutations in the COL4A6 gene have not been reported to

lead to AS. Nevertheless, to our knowledge, our patients

are the first cases in which a5(IV) and a6(IV) chain

staining was faint only in the BCBM and the TBM but

normal in the GBM. However, it is impossible to evaluate

the exact strength of the expression of a(IV) chains on

paraffin-embedded tissues [13].

Patients with missense mutations of the COL4A5 gene

usually show adult onset AS. Our patients have presented

mild clinical features, probably because their a5(IV) chains

in the GBM are not completely deficient. If our patients

have the missense mutation in the COL4A5 gene, their

mild phenotype might be explained by this missense

mutation. Genetic analysis is needed to clarify which gene

abnormality is responsible for the disease in this family.

Table 1 A summary of type IV collagen chain staining in the kidney

Mes mat GBM BCBM TBM

a1 ? ? ? ?

a2 ? ? ? ?

a3 - ? - ±

a4 - ? - ±

a5 - ? -a -a

a6 - - -a -a

Mes mat mesangial matrix, GBM glomerular basement membrane,

BCBM Bowman’s capsular basement membrane, TBM tubular base-

ment membranea Abnormal findings in the present family

1

2

3

4

* *5

6

A B

* *

Fig. 2 Immunohistochemical staining for the a1(IV)–a6(IV) chains

on paraffin-embedded renal biopsy sections in the older (a) and the

younger (b) brothers. Staining patterns for the a1(IV)–a4(IV) chains

are normal. Staining for the a5(IV) chains is intense in the GBM, but

faint in the Bowman’s capsular basement membrane (BCBM) and the

tubular basement membrane (TBM) (asterisk). Staining for the a6(IV)

chains is faint in the BCBM and the TBM (asterisk) (9400)

2

5

6

Fig. 3 Immunohistochemical staining for the a2(IV), a5(IV) and

a6(IV) chains in paraffin-embedded skin biopsy sections from the

mother. Staining for the a2(IV) chains is intense. Staining for the

a5(IV) chains and the a6(IV) chains are weak in the skin basement

membranes (9200)

286 Clin Exp Nephrol (2010) 14:283–287

123

Since benign familial hematuria, thin basement mem-

brane disease, autosomal dominant AS and autosomal

recessive AS caused by mutations of the COL4A3 and

COL4A4 genes show various severities, ranging from

isolated hematuria to renal failure, Torra et al. [21] termed

these renal diseases ‘‘collagen type IV (a3–a4) nephropa-

thy.’’ Although our cases showed that the GBM abnor-

malities correlate with AS, the abnormal immunostaining

patterns for a5(IV) and a6(IV) chains in the renal basement

membranes were not compatible with those of ‘‘collagen

type IV (a3–a4) nephropathy.’’

In conclusion, we presented two brothers with a mild

form of AS that was diagnosed by electron microscopy.

The expressions of their a5(IV) and a6(IV) chains by

immunohistochemical analysis showed unique patterns in

renal basement membranes. These findings suggest that

there are cases with a missense mutation of the COL4A5

gene that leads to a mild form of AS.

References

1. Alport AC. Hereditary familial congenital haemorrhagic nephri-

tis. Br Med J. 1927;1:504–6.

2. Flinter FA, Cameron JS, Chantler C, Houston I, Bobrow M.

Genetics of classic Alport’s syndrome. Lancet. 1988;8618:

1005–7.

3. Hudson BG, Tryggvason K, Sundaramoorthy M, Neilson EG.

Alport’s syndrome, Goodpasture’s syndrome, and type IV col-

lagen. N Engl J Med. 2003;348:2543–56.

4. Timple R, Wiedemann H, van Delen V, Furthmayr H, Kuhn K. A

network model for the organization of type IV collagen mole-

cules in basement membranes. Eur J Biochem. 1981;120:203–11.

5. Sado Y, Kagawa M, Naito I, Ueki Y, Seki T, Momota R, et al.

Organization and expression of basement membrane collagen IV

genes and their roles in human disorders. J Biochem (Tokyo).

1998;123:767–76.

6. Jais JP, Knebelmann B, Giatras I, De Marchi M, Rizzoni G,

Renieri A, et al. X-linked Alport syndrome: natural history in 195

families and genotype-phenotype correlations in males. J Am Soc

Nephrol. 2000;11:649–57.

7. Jais JP, Knebelmann B, Giatras I, De Marchi M, Rizzoni G,

Renieri A, et al. X-linked Alport syndrome: natural history and

genotype-phenotype correlations in girls and women belonging to

195 families: a ‘‘European Community Alport Syndrome Con-

certed Action’’ study. J Am Soc Nephrol. 2003;14:2603–10.

8. Pescucci C, Mari F, Longo I, Vogiatzi P, Caselli R, Scala E, et al.

Autosomal-dominant Alport syndrome: natural history of a dis-

ease due to COL4A3 or COL4A4 gene. Kidney Int.

2004;65:1598–603.

9. Longo I, Porcedda P, Mari F, Giachino D, Meloni I, Deplano C,

et al. COL4A3/COL4A4 mutations: from familial hematuria to

autosomal-dominant or recessive Alport syndrome. Kidney Int.

2002;61:1947–56.

10. Lemmink HH, Schroder CH, Monnens LA, Smeets HJ. The

clinical spectrum of type IV collagen mutations. Hum Mutat.

1997;9:477–99.

11. van der Loop FT, Heidet L, Timmer ED, van den Bosch BJ,

Leinonen A, Antignac C, et al. Autosomal dominant Alport

syndrome caused by a COL4A3 splice site mutation. Kidney Int.

2000;58:1870–5.

12. Sado Y, Kagawa M, Kishiro Y, Sugihara K, Naito I, Seyer JM,

et al. Establishment by the rat lymph node method of epitope-

defined monoclonal antibodies recognizing the six different alpha

chains of human type IV collagen. Histochem Cell Biol.

1995;104:267–75.

13. Naito I, Ninomiya Y, Nomura S. Immunohistochemical diagnosis

of Alport’s syndrome in paraffin-embedded renal sections: anti-

gen retrieval with autoclave heating. Med Electron Microsc.

2003;36:1–7.

14. Harvey SJ, Zheng K, Sado Y, Naito I, Ninomiya Y, Jacobs RM,

et al. Role of distinct type IV collagen networks in glomerular

development and function. Kidney Int. 1998;54:1857–66.

15. Barker DF, Hostikka SL, Zhou J, Chow LT, Oliphant AR, Gerken

SC, et al. Identification of mutations in the COL4A5 collagen

gene in Alport syndrome. Science. 1990;248:1224–7.

16. Plant KE, Green PM, Vetrie D, Flinter FA. Detection of muta-

tions in COL4A5 in patients with Alport syndrome. Hum Mutat.

1999;13:124–32.

17. Knebelmann B, Breillat C, Forestier L, Arrondel C, Jacassier D,

Giatras I, et al. Spectrum of mutations in the COL4A5 collagen

gene in X-linked Alport syndrome. Am J Hum Genet.

1996;59:1221–32.

18. Gross O, Netzer KO, Lambrecht R, Seibold S, Weber M. Meta-

analysis of genotype-phenotype correlation in X-linked Alport

syndrome: impact on clinical counselling. Nephrol Dial Trans-

plant. 2002;17:1218–27.

19. Naito I, Kawai S, Nomura S, Sado Y, Osawa G. Relationship

between COL4A5 gene mutation and distribution of type IV

collagen in male X-linked Alport syndrome. Japanese Alport

Network. Kidney Int. 1996;50:304–11.

20. Naito I, Nomura S, Inoue S, Kagawa M, Matsubara T, Araki T,

et al. X-linked Alport syndrome with normal distribution of

collagen IV alpha chains in epidermal basement membrane.

Contrib Nephrol. 1997;122:134–9.

21. Torra R, Tazon-Vega B, Ars E, Balların J. Collagen type IV

(alpha3-alpha4) nephropathy: from isolated haematuria to renal

failure. Nephrol Dial Transplant. 2004;10:2429–32.

Clin Exp Nephrol (2010) 14:283–287 287

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