1 U.O. di Nefrologia, Dialisi e Trapianto Renale, Ospedale S. Orsola, Bologna, 2 Dipartimento di Medicina Sperimentale e Patologia, Università La Sapienza, Rome, 3 Genetica Medica, Dipartimento di Biologia Molecolare, Università di Siena, Siena, 4 CMID, Osp. L. Einaudi e U.O. Nefrologia e Immunologia C.I.O.V e Dipartimento Medicina e Oncologia Sperimentale, Università di Torino, Turin, 5 Divisione di Nefrologia e Dialisi, Ospedale Regina Margherita, Turin, 6 Dipartimento di Pediatria, Università di Catania, 7 Divisione di Nefrologia e Dialisi, Viterbo, 8 U.O. di Nefrologia, Dialisi e Trapianto, Ospedale Careggi, Florence 9 Divisione di Nefrologia, Dialisi e Trapianto, Policlinico, Bari, and 10 Nephrology and Dialysis Unit-Umberto I Hospital - V. Conca 71-60020, Ancona, Italy.
Correspondence and offprint requests to: Giovanni M. Frascà, MD, Nephrology, Dialysis and Renal Transplantation Unit, St Orsola University Hospital, V. Massarenti 9, 40137 Bologna, Italy. E-mail: frasca{at}orsola-malpighi.med.unibo.it
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Abstract |
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Methods. Patients were investigated as follows: (a) clinical picture and family investigation; (b) renal biopsy findings; (c) immunohistochemical study of renal tissue for collagen IV -chains; (d) pedigree reconstruction and molecular investigations in genes encoding type IV collagen chains, when DNA samples were available; and (e) follow-up data.
Results. Renal biopsy analysis revealed no light microscopy changes in 27 patients and minimal abnormalities in the remainder. Global glomerular sclerosis was found in seven cases and superimposed mesangial immunoglobulin-A deposits in four. Normal staining of GBM for (IV) chains was observed in all but one patient, where
5(IV) was absent and molecular investigation revealed a COL4A5 mutation. Five out of 25 cases had a mutation in the COL4A3/COL4A4 genes. Eight out of 38 patients followed up for 12240 months (21%) showed signs of disease progression or hypertension.
Conclusions. This study confirms that a considerable proportion of patients with TBMD have a type IV collagen disorder and that this lesion is not always benign. Thus, families should be investigated carefully whenever possible and patients and affected relatives should be examined periodically for signs of disease progression.
Keywords: Alport's syndrome; benign familial haematuria; COL4A3; COL4A4; mutations; thin basement membrane disease
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Introduction |
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The lack of sensorineural deafness in 15% of ATS patients and the absence of a family history of nephritis in 10% of cases contribute to the difficulty in establishing a correct diagnosis. In addition, family investigations may sometimes be difficult or even impossible in clinical practice. Immunohistological study of renal tissue may be helpful in distinguishing between ATS and TBMD, since the latter does not show any abnormality in type IV collagen chain distribution in the GBM [1].
In recent years, several genetic studies have demonstrated heterozygous mutations of COL4A3/COL4A4 genes in patients with TBMD; thus, suggesting that they may be carriers of autosomal recessive ATS [26], although linkage analysis did not always reveal an association between TBMD and COL4A3 or COL4A4 genes [7,8]. In rare instances, heterozygous mutations in the same genes lead to autosomal dominant ATS [9].
Some studies have shown that renal function may decline in some patients with TBMD during prolonged follow-up and blood pressure may increase in 35% of affected individuals [10], suggesting that the disease may be less benign than believed previously. Thus, at present, there is evidence that TBMD is a type IV collagen disorder in a sizable proportion of patients and that some cases may experience a progression of the disease.
The present paper reports on a multicentre study designed by the Renal Immunopathology Group of the Italian Society of Nephrology to investigate a selected group of patients without clinical evidence of ATS, where a TBMD was diagnosed by ultrastructural examination of renal biopsy. The aim of the study was to verify the prevalence of type IV collagen abnormalities in this cohort and their clinical course.
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Subjects and methods |
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Electron microscopy examination of renal biopsies was carried out in three pathology departments using the same methods for specimen processing. GBM measurement was performed according to Das et al. [11]. The three laboratories had their own standards for a normal GBM thickness to be used as a control measurement. Briefly, the average values were 360±35 nm for adult males and 325±42 nm for adult females; therefore, a basement membrane in adults was defined as thin when it was, on average, <200 nm in thickness. For the purpose of the present study, the electron microscopy findings were reviewed by one author (A.O.M.).
Criteria for inclusion were: (i) morphological diagnosis of TBMD by light microscopy, immunofluorescence and electron microscopy examination of renal biopsy; (ii) absence of sensorineural hearing deficit by audiometry at the time of renal biopsy; and (iii) absence of ocular abnormalities.
The 51 patients included in the study were 33 women and 18 men (female:male ratio = 1.8) aged 851 years (mean: 28 years) at the time of renal biopsy, belonging to 47 unrelated families. In addition, 273 family members were investigated for the presence of renal failure, hearing deficit and persistent haematuria without urinary tract infection or abnormalities at ultrasound scan.
Follow-up data were available for 38 patients for 12240 months after histological diagnosis.
Immunohistochemistry
Monoclonal antibodies against 1(IV),
3(IV) and
5(IV) chains were purchased from Wislab (Wislab AB, Ideon, Sweden).
Immunohistochemical studies were performed using an indirect immunofluorescence method on frozen specimens routinely prepared for immunofluorescence on kidney biopsies and stored at 80°C. Cryostat sections (5 µm thick) were incubated in the primary antibodies against 1(IV) and
5(IV) at a working dilution of 1:20 in 0.5% bovine serum albumin in phosphate-buffered saline (PBS/BSA). Additional sections from each case were denatured by exposure to 6 M urea in 0.1 M glycine HCl buffer (pH 3.5) to unmask the hidden hepitope of
5(IV) chain and incubated in the anti-
5(IV) antibody at a final dilution of 1:10 in PBS/BSA. As the secondary antibody, fluorescein isothiocyanate-conjugated rabbit antibody against mouse immunoglobulins was used, at a working dilution of 1:20 in PBS/BSA.
Standard immunohistochemical controls were performed, replacing the primary antibodies with non-immune mouse serum or unrelated primary antibodies.
Genetic analysis
Family history investigation was performed carefully in each case by reconstruction of the pedigree for at least three generations.
On the basis of the clinical evaluation and of the pedigree analysis to establish the transmission pattern within a family, COL4A3/COL4A4 or COL4A5 gene mutation analysis was performed after informed consent by the patients. In particular, we analysed the COL4A5 gene in four families out of 30 familial cases in which the clinical features and the transmission pattern were compatible with X-linked ATS. Molecular genetic analysis was carried out after informed consent in 25 out of 51 patients where DNA samples were available. They included seven sporadic cases in which the genetic analysis was performed to rule out a possible de novo mutation and 18 familial cases.
For the COL4A5 gene, both Southern blot analysis and single-strand conformational polymorphisms (SSCP) were performed as described previously [12,13].
COL4A3 and COL4A4 analysis was performed by a combination of SSCP and denaturing high-performance liquid chromatography techniques [9].
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Results |
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Immunohistochemistry
In all cases, the presence of 1(IV) and
3(IV) chains was revealed diffusely in the glomeruli. As expected,
1(IV) was seen in the mesangium and, although less intense, along the GBM, while
3(IV) was limited to the GBM. Staining for
5(IV) was also evident in all but one case (B1), where the signal was absent, suggesting a diagnosis of ATS, which was confirmed subsequently by genetic analysis (see below).
Family study
Family investigation was not possible in two patients who had been adopted. Among the remainder, 30 patients (60%) had one or more relatives with persistent haematuria without urinary tract infection or abnormalities at ultrasound scan. In nine families (17.6%) a member with CRF was found: no information was available for one of them who had died many years before; in a second family the relative with CRF had a long history of renal stone and urinary tract infection; and in a third family CRF was reported in the father of a 23-year-old female patient where monolateral nephrectomy had been carried out several years earlier due to renal neoplasm. In the remaining four families the relatives with CRF had a clinical history suggestive of a nephritic disorder.
Molecular genetics
COL4A3 and COL4A4 analysis. Mutation analysis of COL4A3 and COL4A4 genes was performed in 25 patients. Among these 25 cases, 15 were familial, nine were sporadic and one patient was adopted. Mutations in the COL4A4 gene were identified in three families, while a polymorphic variant of the COL4A4 gene (c.27175 A>T) was found in two patients. In two families a COL4A3 mutation was identified.
A clearly pathogenetic COL4A4 mutation was found in patient F1 (p.G864W) (Figure 1A). The proband underwent renal biopsy at the age of 51 for persistent microscopic haematuria detected since the age of 40, with a diagnosis of TBMD. Three years later he had normal blood pressure and the glomerular filtration rate (GFR) was at the lower limit of the normal range (creatinine clearance: 73 ml/min/1.73 m2). His 26-year-old daughter had had persistent microhaematuria since the age of 17 and experienced a single episode of macroscopic haematuria. Again, at renal biopsy a diffuse GBM thinning was found by electron microscopy.
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In the third family (patient T9) the COL4A4 mutation p.G481S was identified. The same mutation was also present in the mother (Figure 1C). The proband was an 8-year-old girl who underwent renal biopsy for an episode of macroscopic haematuria with persistent microhaematuria. Microscopic haematuria was also detected in her mother.
A COL4A3 mutation was found in two cases. Patient C4 (Figure 1D) had a heterozygous deletion of 9 bp in exon 38 (c.3321del9), which leads to an in-frame deletion of a tripeptide in the collagenous domain of the protein. Family investigation was not possible, since she had been adopted. Extensive clinical and molecular description of this case is reported elsewhere as case DIT [9].
We found a heterozygous missense mutation (p.G1198S), inherited from the father, in a second female patient C7 (Figure 1E). She had persistent microscopic haematuria detected for the first time by chance when she was 8 years old. Her father had microhaematuria, but his renal function was unknown, and her paternal grandmother, who had died some years earlier, was reported as having impaired renal function. Patient C7 underwent renal biopsy at the age of 24. Ultrastructural examination of renal tissue showed a diffuse thinning of GBM (120 nm) without immunological deposits at immunofluorescence. No glomerular sclerosis was seen by light microscopy. Eight years later, urinalysis showed microscopic haematuria along with a small amount of protein (300 mg/day) and GFR was within the normal range (serum creatinine: 1 mg/dl; creatinine clearance: 80 ml/min/1.73 m2). Blood pressure was normal.
COL4A5 analysis. Among the 30 familial cases, we observed a transmission pattern suggestive of X-linked ATS in four families. Moreover, the accurate clinical analysis revealed the progression to renal failure in some of the relatives. We thus selected these families to perform mutation analysis of the COL4A5 gene. Direct mutation analysis of COL4A5 revealed the presence of a COL4A5 mutation in one case (patient B1). The pedigree analysis showed the presence of a maternal uncle with progressive nephropathy. The presence of this affected relative was unknown at the time of renal biopsy. The patient showed an exon 17 deletion in the COL4A5 gene. Extension of the pedigree revealed that this patient was related to the family previously described as DEL [12].
Follow-up
Follow-up data were available for 38 patients followed for 12240 months. All of them had persistent microscopic haematuria at urinalysis; urinary protein excretion was significantly increased in one patient, where proteinuria was already found when renal biopsy was carried out, and glomerular sclerosis had been observed. Two patients had abnormal serum creatinine (2.8 mg/dl after 112 months and 3 mg/dl after 60 months, respectively) associated with arterial hypertension. One of them was the patient with the COL4A5 mutation and X-linked ATS. The other one had a superimposed IgA nephropathy. Five additional patients had elevated blood pressure (12132 months after biopsy). Four of them were treated with angiotensin-converting enzyme inhibitors and one with ß-blockers.
Among patients with a COL4A3 or COL4A4 heterozygous mutation, only one developed arterial hypertension and moderate proteinuria (0.7 g/day) at follow-up (range: 1296 months) and was treated with angiotensin-converting enzyme inhibitors.
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Discussion |
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In this setting, immunohistological evaluation of type IV collagen -chains in the kidney may be helpful [14], being an easy, rapid and inexpensive procedure that can be carried out with commercially available specific antibodies. This investigation should always be performed when a TBMD is found, to rule out cases of X-linked or autosomal recessive ATS. However, immunohistological analysis may, in some cases, give false negative results. It is known that
20% of male patients with X-linked ATS or patients with autosomal recessive ATS may show a faint or even normal staining for
3(IV) and
5(IV) [1,14]. Considering these findings, we suggest performing genetic analysis in patients with TBMD and a family history positive for haematuria and renal failure or with an unknown family history, even if immunohistological evaluation for
(IV) chains is apparently normal (Figure 2).
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All our patients with COL4A3 or COL4A4 mutations presented, in addition to haematuria, other clinical signs, such as proteinuria, macrohaematuria or hypoacusia (Figure 1). These data suggest that one is more likely to identify mutations in COL4A3 or COL4A4 genes in those patients with additional symptoms besides microhaematuria or with a positive family history for CRF and/or persistent haematuria.
Cases B1 and C7 clarify how a correct diagnosis can be reached by careful clinical investigation of relatives and appropriate molecular analysis. In particular, in case B1, X-linked ATS was suspected by family history and confirmed by immunohistochemistry and COL4A5 mutation analysis. In case C7, clinical features and biopsy findings were compatible with BFH. However, pedigree reconstruction revealed the presence of CRF in the paternal grandmother and molecular analysis identified a pathogenic mutation in the COL4A3 gene. These findings suggest considering the autosomal dominant form of ATS as a differential diagnosis with regard to BFH, since autosomal dominant ATS may have a relatively benign clinical course, characterized by a very slow progression towards renal failure [16].
Our observations confirm that TBMD may not always be benign, as pointed out already [10]. Follow-up data were available for 38 patients included in the study: eight of them showed worsening of the clinical picture or arterial hypertension.
A decline in GFR was observed in the patient with X-linked ATS. Among patients with a COL4A3 or COL4A4 heterozygous mutation, only one case showed a worsening of the clinical picture during follow-up while the other four patients did not exhibit any sign of progressive disease. Thus, molecular investigation seems of limited value in predicting renal prognosis in heterozygous carriers of COL4A3 or COL4A4 mutations, particularly when exhaustive family history is lacking (as in case C4). To better understand the genotype/phenotype correlations and in order to identify clinically and genetically specific features of TBMD and ATS, it is necessary to increase the number of patients studied. The availability of larger clinical and genetic data may allow us to improve the prognostic value of COL4A3/COL4A4 molecular analysis. This is particularly relevant when considering that shifting the predicted effect of a mutation from TBMD to recessive or dominant ATS dramatically affects genetic counselling and risk ascertainment.
In patient B12, presenting a decline in GFR, immunofluorescence investigation of the renal biopsy showed a superimposed IgA nephropathy. An association between TBMD and other glomerulonephritis, including IgA nephropathy, has been described already [18] and in families suffering from TBMD some members have been reported to develop a superimposed IgA nephropathy [19]. It is still unclear whether these cases represent a chance association of different unrelated glomerular diseases or whether a thinner GBM would facilitate the deposition of immunoglobulins. This latter hypothesis is supported by studies on the permselectivity of the GBM in patients with biopsy-proven TBMD, where a significantly increased fractional clearance of neutral molecules with Stokes radius >42 Å has been found [20]. However, whether correlated or not, the association with IgA nephropathy may alter the clinical course of these patients, turning a benign lesion into a progressive disease.
We therefore suggest also following-up symptomatic family members of patients with TBMD and considering carrying out a renal biopsy in those cases showing clinical signs of disease progression, in order to disclose a superimposed nephropathy that might benefit from pharmacological treatment.
Our study confirms that a considerable proportion of patients with TBMD carry a type IV collagen disorder and that a thin GBM should not be regarded as invariably benign, even in cases with no evident collagen IV abnormalities. As a consequence, families should be investigated carefully whenever possible and patients and affected relatives should be examined periodically for signs of disease progression.
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Acknowledgments |
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Conflict of interest statement. None declared.
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References |
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