NDT Advance Access originally published online on December 7, 2005
Nephrology Dialysis Transplantation 2006 21(3):665-671; doi:10.1093/ndt/gfi312
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© The Author [2005]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
Original Articles: Clinical Nephrology
Autosomal recessive Alport syndrome: an in-depth clinical and molecular analysis of five families
1 Medical Genetics, Department of Molecular Biology, University of Siena, 2 Clinica Pediatrica "G. e D. De Marchi", University of Milano, 3 Centro Ricerche Cliniche Malattie Rare, Villa Camozzi Ranica, Bergamo and 4 Dipartimento di Medicina Pediatrica, Ospedale Regionale Pediatrico Giovanni XXIII, Bari, Italy, 5 Simon Winter Institute for Human Genetics, Bnai-Zion Medical Center, Technion-Rappaport Faculty of Medicine, Haifa, Israel and 6 Northern Regional Genetic Service, Auckland Hospital, New Zealand
Correspondence and offprint requests to: Alessandra Renieri, MD, PhD, Associate Professor, Medical Genetics, University of Siena, Policlinico "S.Maria alle Scotte", V.le Bracci 2, 53100 Siena, Italy. Email: renieri{at}unisi.it
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Background. Alport syndrome (ATS) is a progressive inherited nephropathy characterized by irregular thinning, thickening and splitting of the glomerular basement membrane (GBM) often associated with hearing loss and ocular symptoms. ATS has been shown to be caused by COL4A5 mutations in its X-linked form and by COL4A3 and COL4A4 mutations in its autosomal forms.
Methods. Five families with a suspicion of ATS were investigated both from a clinical and molecular point of view. COL4A3 and COL4A4 genes were analysed by DHPLC. Automated sequencing was performed to identify the underlying mutation.
Results. Molecular analysis indicated that in all 5 cases the correct diagnosis was autosomal recessive ATS. In three families in which parental consanguinity clearly pinpointed to autosomal recessive ATS, we found COL4A4 homozygous mutations in two of them and COL4A3 homozygous mutation in the other one. In the remaining two families a differential diagnosis including X-linked ATS, autosomal recessive ATS and thin basement membrane nephropathy was considered. The molecular analysis demonstrated that the probands were genetic compounds for two different mutations in the COL4A4 gene pinpointing to the correct diagnosis of autosomal recessive ATS.
Conclusions. A clinical evaluation of probands and their relatives of the five families carrying mutations in either the COL4A3 or the COL4A4 gene was carried out to underline the natural history of the autosomal recessive ATS. In addition, this paper stresses the complexity of the clinics and genetics of ATS and how a correct diagnosis is based on a combination of: (i) an in-depth clinical investigation; (ii) a detailed formal genetic analysis; (iii) a correct technical choice of the gene to be investigated; (iv) a correct technical choice of the family member to be included in the mutational screening. A correct diagnosis is the basis for an appropriate genetic counselling dealing with both the correct prognosis and the accurate recurrence risk for the patients and family members.
Keywords: autosomal recessive ATS; collagen IV genes; DHPLC; inherited nephropathy; phenotypic variability
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Introduction |
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Alport syndrome (ATS) is a hereditary disease of glomerular basement membrane (GBM). It is clinically characterized by recurrent or persistent haematuria, proteinuria, development of renal failure, high-tone sensorineural deafness and ocular defects affecting the lens and the fundus. It is mainly transmitted as an X-linked semi-dominant trait caused by mutations in the COL4A5 gene (OMIM #305010). In this form males are more severely affected than females in the majority of pedigrees [1,2]. The existence of a pure autosomal dominant form, associated with COL4A3 and COL4A4 genes, has been questioned for decades (OMIM #104200). Only recently, some pedigrees in which genetic and clinical evaluation were suggestive of autosomal dominant ATS were reported [3–5]. In general, autosomal dominant ATS families have a relatively mild phenotype, indicated by a slower rate of progression to end-stage renal failure (ESRD) than most patients with X-linked ATS [6].
Autosomal recessive inheritance (OMIM #203780) due to COL4A3 and COL4A4 mutations accounts for approximately 15% of ATS cases [7–11]. Autosomal recessive transmission is suggested by the presence of one of the following features: (i) severe early disease in both females and males; (ii) absence of severe symptoms in parents (they may be completely asymptomatic or may have isolated microhaematuria); (iii) parental consanguinity.
In this study we present five families with autosomal recessive transmission and mutations in either the COL4A3 or the COL4A4 gene. A clinical evaluation of probands and their relatives was carried out to underline the natural history of the autosomal recessive form.
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Clinical analysis
Urinalysis and renal function evaluation, were performed in patients and all family members. Audiological and ophthalmic examinations were performed in several patients and family members. Whenever indicated, a renal biopsy was performed and it was always evaluated using electron microscopy.
Mutation analysis
Blood samples were collected after a written informed consent of affected and healthy family members. Genomic DNA was isolated from peripheral blood leukocytes of the patients according to the standard methods [12]. In those families without parental consanguinity (families 1 and 2) mutation analysis was performed in probands (#2074 and 2650). In families with parental consanguinity (families 3, 4 and 5) mutation analysis was performed in one parent (#2245, 2777, 2456). This choice was due to the increased sensitivity of the denaturing high performance liquid chromatography (DHPLC) analysis in detecting heterozygous mutation. All the COL4A3 and COL4A4 exons were amplified using primers and PCR conditions already described [5]. Mutation analysis was performed by DHPLC using the Transgenomic WAVETM (Transgenomic, San Jose, CA, USA) [4]. PCR products were denatured at 95°C, re-annealed at 65°C for 1 min and cooled at 4°C to generate heteroduplexes. The optimal column temperature for fragments analysis was calculated using the WaveMaker Software (Transgenomic, San Jose, CA, USA). DHPLC analysis was performed at the melting temperature of 54.3°C for exon 3, 55°C for exon 21 (COL4A4), 59°C for exon 25 (COL4A4), 60.6°C for exon 28 (COL4A4), 61.3°C for exon 30 and finally 59°C for exon 23 (COL4A3) gene. Direct sequencing of the purified PCR products was carried out in both directions (PE Big dye terminator cycle sequencing kit) on an ABI310 Automated Sequencer and analysed with the Sequencer software.
Segregation analysis was performed by direct sequencing in families 1, 2, 4, 5 and by RsaI restriction analysis in family 3. In addition, in family 1 segregation analysis for polymorphism status was performed using the HaeIII restriction enzyme.
Statisical analysis of Table 3 was accomplished by using the 
2 2 x 2 method.
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Family 1
The index case is a 9-year-old boy born to unrelated healthy Italian parents (Figure 1a). He presented with microhaematuria and episodic macrohaematuria since the age of 18 months and proteinuria since the age of 8 years. Electron microscopy of renal biopsy, performed at the age of 4, showed splitting, thickening and thinning of the GBM, suggesting a diagnosis of ATS. He had normal renal function, normal audiogram and no ocular signs of the disease. Both the father (48 years old) and the mother (38 years old) showed isolated microhaematuria. The maternal grandfather had episodes of macrohaematuria, and at the age of 73 he had only microhaematuria.
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males, 
females, 
pregnancy. Filled grey symbols are individuals with isolated microhaematuria. Filled black symbols indicate individuals with microhaematuria plus macrohaematuria or hypoacusia or renal failure. White symbols indicate individuals without clinical signs of the disease. An oblique bar indicates a deceased individual. The arrows indicate the index patients. The presence of mutations are indicated below each symbol as follows: – ,wild type allele; +, mutated allele. The type of mutation is indicated in brackets: m = missense; s = splice site mutation; f = frame-shift mutation. The mutated genes are indicated below each pedigree.Proband's DHPLC analysis of exons 21 and 25 of COL4A4 showed additional heteroduplex peaks (Figure 2a). Sequencing analysis resulted in the identification of two COL4A4 mutations (Figure 2a). One, inherited from the mother, in exon 21 was predicted to cause a glycine substitution in the collagenous domain of the protein (c.1433G>A, p.G478E). Another, inherited from the father, affected the penultimate nucleotide of exon 25 and was predicted to change the exonic splice site consensus (AG>GG) between exon–intron 25, resulting in a splice site mutation (c.1986A>G). Exon 21 sequence analysis of proband and maternal branch revealed in addition to the pathogenic mutation c.1433G>A, a previously reported polymorphism c.1444C>T, in heterozygous state in the proband and grandfather (C/T) and in homozygous state (T/T) in the mother [5]. In the DHPLC analysis the different allelic combination of the polymorphism masked the presence of the mutation in the mother (Figure 2a).
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Family 2
The proband is a 13-year-old Italian female who presented with microhaematuria since the age of 4 (Figure 1b). At present, her renal function as well as audiological and ophthalmic investigation are normal. Electron microscopy of renal biopsy performed at the age of 5 showed thinning of basement membrane pinpointing to a diagnosis of thin basement membrane disease. Her elder sister, aged 17, had a very similar clinical evolution. At present, she has only microhaematuria and proteinuria. The father is healthy. The mother has microhaematuria and proteinuria. A maternal uncle has isolated microhaematuria.
DHPLC analysis of the proband revealed an abnormal elution profile in both exon 3 and 30 of COL4A4 gene (Figure 2b). In exon 30 she had a missense mutation inherited from the healthy father changing glycine 864 (c.2590G>A, p.G864R). In exon 3 she had a missense mutation inherited from the microhaematuric mother introducing an abnormal cysteine residue, c.104A>G, p.Y35C. The same pattern of genetic compound was found in the affected elder sister (Figure 1b and 2b).
Family 3
The proband is a 28-year-old male born to healthy consanguineous parents of Iraqi-Jewish origin (Figure 1c). Microhaematuria appeared at the age of 1 year, and proteinuria at the age of 5. A probable diagnosis of ATS was postulated, as his younger sister showed severe nephropathy since the age of 1 year. At the age of 10 he was diagnosed with progressive bilateral sensorineural hearing loss, and at the age of 15 he underwent kidney transplantation. At the age of 12 he had a diagnosis of type III Spinal Muscular Atrophy. He showed progressive muscular weakness and a positive EMG. Muscle biopsy was not performed because of lack of compliance. At a recent eye examination at the age of 32 he was noted to have dot-and-fleck retinopathy with no signs of anterior lenticonus. He is married to a non-related healthy female, who is now in the middle of her first pregnancy.
The proband's younger sister died at the age of 14 of end-stage nephropathy. She showed severe nephropathy since the age of 1 year and she was known also to have mild bilateral sensorineural hearing loss. The father showed isolated microhaematuria. The mother and the other three elder sibs had no signs of microhaematuria, and all of his sibs, children (9 individuals) are healthy. All family members had normal audiograms. There are no other individuals in the large family of both parents known to have kidney disorder or hearing loss.
DHPLC analysis of exon 23, of COL4A3 in the father showed an additional heteroduplex peak (Figure 2c). Sequencing analysis resulted in the identification of a COL4A3 splice site mutation predicted to cause a premature truncation of the protein (c.1504 + 2T>A). This mutation was also present in heterozygous state in the mother, in the brother and the two sisters, and it was present in homozygous state in the index case (Figure 1c and 2c).
Family 4
The proband is a 30-year-old woman born to second cousin Italian parents (Figure 1d). She presented with microhaematuria and episodes of macrohaematuria since the age of 10. At 21 she developed proteinuria (0.3 g/24 h). She reached chronic renal failure at the age of 24 (blood creatinine = 3.4 mg/dL) and ESRD at the age of 26. She was successfully transplanted at the age of 29. At present her audiological and ophthalmic investigations are still normal. A renal biopsy performed at the age of 24 showed irregular thickness of GBM suggesting a diagnosis of ATS. A younger sister of 27 years, presented with microhaematuria and proteinuria (0.3 g/24 h) at the age of 22. At present, she has chronic renal failure (serum creatinine = 1.5 mg/dL). A renal biopsy performed at the age of 22 showed thinning and splitting of GBM suggesting a diagnosis of ATS. The mother has isolated microhaematuria. The father had a renal carcinoma at the age of 49 for which he underwent surgical excision of the left kidney. Before the nephrectomy he had normal renal function. At present, at the age of 58 he has chronic renal failure. The elder brother, aged 33, presents rare red blood cells in urinalysis, the younger brother, aged 26, is healthy.
DHPLC analysis of exon 28 of COL4A4 gene of mother's DNA revealed the presence of a heterozygous missense mutation, c.2374G>A, p.G792R (Figure 2d). Father and elder brother carried the same mutation. Proband and affected sister were homozygous for the mutation (Figure 2d).
Family 5
The proband is a 49-year-old women from New Zealand (Figure 1e). Her parents are first cousins. She underwent a renal transplant at 16 years and developed deafness at 46 years. She has two healthy younger sisters aged 40 and 42 years. Her mother is reportedly healthy. Her father died, but the age and cause of his death are unknown. The proband has a 30-year-old daughter without clinical signs.
We decided to perform DHPLC analysis on the mother's DNA sample, and its elution profile showed an additional peak in exon 28, of COL4A4 gene (Figure 2e). The sequence analysis revealed the c.2279–2280insG mutation in heterozygous status in the mother's DNA (Figure 2e). This mutation has a clear pathogenic role since it creates a shift of the reading frame leading to a truncated protein. The c.2279–2280insG was found in homozygous status in the proband's DNA and in heterozygous status both in the two sisters and in her daughter (Figures 1e and 2e).
The main clinical and genetic features of the 7 homozygous patients belonging to the five families described above are summarized in Table 1 (age range 9–49; mean age 24.7). Table 2 shows the same data concerning the 16 heterozygous relatives of the index cases of the five families (age range 28–71; mean age 49.4).
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Discussion |
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In families 3, 4 and 5 the hypothesis of autosomal recessive form of ATS was suggested by parents, consanguinity and the early ESRD (15, 26 and 16 years, respectively). The progressive bilateral sensorineural hearing loss found in the probands of families 3 and 5 and the results of renal biopsy performed in the proband of family 4 provided additional support for the diagnosis. The identification of underlying homozygous mutations (in COL4A4 or in COL4A3) confirmed the clinical suspicion and enabled correct genetic counselling. In particular in family 3, the proband and his wife were reassured for having a very low recurrence risk in their offspring of autosomal recessive ATS (since the wife had not any renal sign). In addition, during genetic counselling the couple was informed that the phenotype of their offspring might range from normal (like proband's mother) to microhaematuria (like proband's father) with a low, but still increased risk of renal failure.
In families 1 and 2 the clinical suspicion of autosomal recessive ATS was less clear. In family 1, ATS was suspected on the basis of GBM alterations at renal biopsy. The absence of consanguinity and the young age of the proband (9 years) made it difficult to distinguish between the autosomal and the X-linked forms of ATS. The father could have had microhaematuria due to different process, and the clinical features of the mother (macrohaematuria) could be in accord with X-linked ATS. Unfortunately, combined immunohistochemical analysis of skin (
5 chain) and kidney (3, 4, 5 chains) was not performed. This analysis would be able to settle a distinction between X-linked and recessive autosomal forms [13]. In this context the clinical investigation of the maternal grandfather, who at the age of 73 exhibited only macrohaematuria, represented an additional clinical hint suggestive of autosomal recessive ATS. The identification of mutations in the COL4A4 gene allowed to firmly establish that the proband has autosomal recessive ATS.
In family 2, the clinical suspicion of autosomal recessive ATS was even weaker. The absence of a clear evolution toward renal failure in the young proband and in her sister and the electron microscopy results of renal biopsy pointed the clinical suspicion towards a more favourable disease (thin basement membrane disease). Considering that both the above signs may be due to the young age of the proband and her sister (13 and 17, respectively), one could point to the presence of early proteinuria and make a suspicion of autosomal dominant ATS. In this case, the molecular analysis was critical in clarifying that these two young girls are instead affected by autosomal recessive ATS modifying their prognosis. Each of them carries two different mutations bringing two different alterations in the COL4A4 gene. One of them, namely p.G864R is very similar to the one already reported in a case of thin basement membrane disease, p.G864W [14]. However, in this latter case the proband has the mutation in heterozygous state, the same as the father in family 2. It is worth noting that the case reported by Frascà et al., had glomerular filtration at the lower limit of normal range as observed in the father of family 2. This observation correlates with the chronic renal failure of the father of family 2 and it suggests that even in the heterozygous state this mutation has a prognosis which is not so good as previously thought.
The state of the present study points out that an individual carrying a COL4A4 or a COL4A3 mutation in the heterozygous state may have a very different clinical outcome ranging from healthy to microhaematuria and to slow progressive renal failure (Table 2) [4,5]. We have attempted to perform a genotype– phenotype correlation in patients we have personally characterized both from a clinical and molecular point of view (Table 3) [4,5]. Although the number of patients is still low, a relative genotype–phenotype correlation does exist. Null mutations correlate with a mild outcome (healthy or microhaematuria) more than missense mutations, which in turn have a less favourable prognosis (slow progressive renal failure) (2 = 5.76) [4,5]. However, there are exceptions to this rule and starting from the type of mutation it is quite impossible to predict whether one individual will be healthy or will have microhaematuria. This is clearly demonstrated by the parents of family 3 who carry the same mutation and are discordant for the presence of microhaematuria. It is likely that other factors beside type IV collagen alteration may affect the clinical outcome.
Table 2 shows that the only heterozygous individual who developed chronic renal failure is the father of family 4 who had a nephrectomy. Although, the number of heterozygotes included in this study is limited (16 subjects), this observation may raise a question about allowing individuals who are heterozygous for ATS to be kidney donors [15–17]. The fact that this individual developed renal failure only after nephrectomy may suggest that, individuals heterozygous for COL4A3 or COL4A4 mutations should be advised not to donate kidneys, as recommended to female carriers of X-linked ATS [18].
In this study, we have confirmed that the DHPLC technique is a good tool for COL4A3 and COL4A4 analysis. The DHPLC technique appears to be a highly sensitive method with advantages in terms of flexibility, cost and time and labour sparing, compared with classical approaches of mutation scanning. However, it is important to note that sometimes the presence of polymorphism may hide the presence of a pathogenic mutation as in exon 21 in family 1 (Figure 2a). This finding is highly relevant since both COL4A3 and COL4A4 are rich in polymorphisms. The analysis at different temperatures may be required to screen polymorphic exons adequately.
In addition, the sensitivity of the DHPLC technique is high when searching for heterozygous mutations. For this reason we preferred to perform mutational screening in one of the parents of families 3, 4 and 5 in which the presence of consanguinity suggested homozygous mutation in the probands. For the same reason we chose to perform mutational screening in the proband of families 1 and 2, since the absence of consanguinity indicated the presence of mutations in the compound heterozygous state as more likely. The above choices were fruitful and a causative mutation was found in all five cases.
In conclusion, the clinics and genetics of ATS is very complex. The correct diagnosis is based on a combination of a comprehensive clinical investigation of all family members associated with a broadly formal genetic analysis of the pedigree. An appropriate technical choice of the gene to be investigated and the family member to be included in the mutational screening is necessary as well. A correct diagnosis is the basis for an appropriate genetic counselling dealing with both the correct prognosis and the accurate recurrence risk for the patients and family members.
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Acknowledgments |
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We thank Elera Marcócci for DHPLC analysis.
Conflict of interest statement. None declared.
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References |
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[Abstract/Free Full Text] - Knebelmann B, Benessy F, Buemi M et al. Autosomal recessive (AR) inheritance in Alport syndrome (AS). J Am Soc Nephrol 1993; 4: 263
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Accepted in revised form: 11.11.05
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