Identification of a new mutation in the {alpha}4(IV) collagen gene in a family with autosomal dominant Alport syndrome and hypercholesterolaemia

Milco Ciccarese1,2,, Domenica Casu4, Fung Ki Wong1, Rossana Faedda3, Sivonne Arvidsson1, Giancarlo Tonolo2, Holger Luthman1 and Andrea Satta3

1 Department of Molecular Medicine and CMM, Karolinska Institutet, Stockholm, Sweden, 2 Servizio Diabetologia, Dipartimento Struttura Clinica Medica-Patologia Medica, University of Sassari, Sassari, Italy 3 Istituto di Patologia Medica, University of Sassari, Sassari, Italy and 4 Nephrology Unit, Alghero, Italy



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Alport syndrome (AS) is a hereditary disease of the glomerular basement membrane in the kidney characterized by progressive renal failure, sensorineural deafness, and/or ocular abnormalities. In contrast to the well-known X-linked phenotype, very little is known about the autosomal dominant form. Rare autosomal forms of AS have been described with mutations in COL4A3 and COL4A4 at chromosome region 2q35-q37, but there have been no descriptions of dominant forms due to a mutation in COL4A4.

Methods. We describe a Sardinian family with a classical AS-phenotype plus hypercholesterolaemia, a clinical feature also present in Fechtner syndrome (FS), a disease that segregates as an autosomal dominant trait.

Results. A suggestive linkage (LOD=2.7) between AS and the COL4A3/A4 locus at 2q35-q37 was identified. Other candidate collagen genes encoding basement membrane collagen (COL4A1/A2 and COL4A5/A6) were excluded by linkage analysis (13q33-q34 and Xq22), or by sequence (COL4A3). DNA sequence analysis of the COL4A4 gene revealed that the Lys325Asn mutation was present in all affected family members, but was absent in all unaffected members and in a random sample of the Sardinian population. A clear indication of a gene-dosage effect was seen in the most severely affected family member, since she carried the mutation in the homozygous form.

Conclusions. These data confirm the importance of collagen 4A4 as a component in the structural integrity of the glomerular basement membrane and confirm the phenotypic and genetic heterogeneity of collagen disorders.

Keywords: collagen; haematuria; hypercholesterolaemia



   Introduction
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Alport syndrome (AS) is a hereditary nephropathy characterized by persistent haematuria that may evolve to end-stage renal failure (ESRF). This syndrome is associated with sensorineural hearing loss, ocular abnormalities (anterior lenticonus, macular or peripheral flecks), and ultrastructural abnormalities of the glomerular basement membrane (GBM) [1]. The ultrastructural alteration of the GBM, which is irregularly thickened and attenuated, is considered specific for the disease. In a few cases, AS has been associated with leiomyomatosis [2,3]. AS displays considerable phenotypic and genetic heterogeneity [4]. In addition to the X-linked form [5], more than 15% of all AS segregates as an autosomal trait and several mutations in COL4A3 (MIM 120070) and COL4A4 (MIM 120131) on chromosome 2q35-q37 have been described in families with autosomal recessive AS [68].

Although our understanding of AS has improved in recent years, little is known about the autosomal dominant form (MIM 104200). Linkage to the COL4A3/A4 genes has been reported in one family with autosomal dominant AS [9], and a splice site mutation in the COL4A3 gene has been detected [10]. Moreover, a positive linkage to chromosome 22q11-q13 has been reported for an autosomal dominant form that resembles AS in its clinical characteristics, haematological abnormalities, and associated hypercholesterolaemia (Fechtner syndrome) [11].

In this study, we describe a Sardinian family with autosomal dominant segregation of the classical features of AS (haematuria, deafness, ultrastructural alteration of the GBM) plus hypercholesterolaemia. Genetic linkage was initially tested with loci for obvious candidate genes for AS and markers selected for the COL4A3/COL4A4 locus provided a suggestive indication of linkage. This led us to investigate, in detail, these two collagen genes on chromosome 2q35-q37.



   Subjects and methods
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 Subjects and methods
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 Discussion
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Selection criteria
To select families for AS, we used the clinical criteria described previously [1]. The general family history was given by individuals III:1, III:2, and III:4. Each participating individual had a physical and ophthalmologic examination, a hearing test, a complete screening of kidney and liver functions, a serum lipid profile, and a blood count. Information from hospital records was obtained from the Alghero Nephrology Unit. A member was considered affected only if microscopic haematuria was detected on at least three occasions after exclusion of urinary infection. The study was approved by the Ethics Committee at the University of Sassari, and all subjects gave informed oral consent to participation in the study.

Patients and family members
The pedigree of the Sardinian family is shown in Figure 1Go, and their clinical characteristics are described in Table 1Go. The proband (IV:2) is a female of 31 years with severe bilateral high frequency sensorineural deafness, who presented with haematuria at 8 years of age. At 20 years of age hypercholesterolaemia was diagnosed (total cholesterol (T. Chol.) was 8.5 mM/l), and upon renal biopsy she was diagnosed with AS. Haemodialysis was initiated 9 years after diagnosis. Her brother (IV:3) is 26-years-old and presented with haematuria at 5 years of age. At the time of this study, he had normal renal function and hearing, but persistent haematuria and cholesterol values at the upper limit of age-adjusted normal cholesterol values (T. Chol. was 5.6 mM/l and LDL-Cholesterol (LDL-Chol.) was 3.5 mM/l). The brother (IV:1), and the cousins (IV:4, IV:5, IV:6) showed no signs of microhaematuria or deafness; they had normal renal function, and T. Chol. concentrations were lower than 5 mM/l. The father (III:1) presented with haematuria at 40 years of age. When he was 53, he was diagnosed with severe hypercholesterolaemia (T. Chol. was 10.5 mM/l), severe bilateral high frequency sensorineural deafness, and he developed ESRF at 66 years of age. The mother (III:2) presented with haematuria at 40 years of age, and was diagnosed with AS by renal biopsy (Figure 2Go). She had persistent haematuria, normal renal function, and mild hypercholesterolaemia (T. Chol. was 5.7 mM/l, with LDL Chol. of 4.2 mM/l). According to hospital records, individual III:3 was affected with AS, but declined to participate in the study. Individual III:4 was diagnosed with AS at 54 years of age and had T. Chol. of 7.7 mM/l; haemodialysis was initiated 6 years later. Individuals III:2 and III:4 presented with moderate bilateral high-frequency sensorineural deafness.



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Fig. 1. Pedigree of the family with autosomal dominant Alport syndrome. The combined haplotypes are indicated for markers D2S1649-D2S434-D2S351-COL4A4 Lys325Asn mutation (K/N)-D2S401-D2S1363-D2S338, located on chromosome 2 region 2q35-q37. The left haplotypes are from the fathers and the right from mothers. The haplotype co-segregating with the disease-phenotype is boxed. Filled symbols denote affected individuals; females are indicated by circles and males are indicated by squares. Double lines indicate consanguineous matings. Roman numerals denote generations and Arabic numerals identify position within the generation. The asterisks above symbols denote individuals included in the genetic analysis.

 

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Table 1. Clinical features of the 9 family members analyzed

 


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Fig. 2. Renal biopsy electron micrograph from individual III:2 demonstrating the irregular and thickened glomerular basement membrane.

 
None of the family members showed any signs of leiomyomatosis, ocular abnormality, altered liver function, macrothrombocytopenia, or polymorphonuclear inclusion body, and no member had a nephrotic syndrome that could explain the hypercholesterolaemia.

The grandfathers (II:2, II:3) had experienced a long history of renal disease prior to their death at 52 years. Both died from suspected myocardial infarction. Individual I:1 died of unknown causes at 35 years of age (medical records are not available). Family members (I:2, II:1, and II:4) died when they were over 80. Individuals II:1, II:4 and III:5 had a negative history of haematuria and no renal disease.

Linkage analysis
Genomic DNA was purified from peripheral lymphocytes and 5 ng was PCR amplified in 10 µl containing 0.15 µM of each primer 0.2 mM dNTP, 1xDynazyme Buffer (10 mM Tris-HCl, pH 8.8, 1.5 mM MgCl2, 150 mM KCl, and 0.1% Triton X-100) and 0.2 U of DynaZyme. PCR cycles were as follows: 95°C for 5 min, followed by 35 cycles consisting of 95°C for 30 s, annealing at primer-specific temperatures (53°C, 55°C, 58°C) for 75 s; and 72°C for 15 s, followed by a final extension at 72°C for 10 min. We used 31 fluorescent microsatellite markers, selected from published maps [12] to cover the q-arms of chromosomes 2, 13, and X. To verify the initial linkage to chromosome 2, two additional markers were chosen (D2S351 and D2S401) that flank the COL4A3 and COL4A4 genes [9]. The PCR products were separated on 4% polyacrylamide gels on an ABI377 sequencer and genotypes were identified using the ABI Genescan/Genotyper software (Perkin Elmer, CA, USA). After analysis of the phenotypic data, linkage was calculated under a dominant model with a penetrance of 0.9, no phenocopies, and assuming a disease gene frequency of 0.001, using the GENEHUNTER software (Cambridge, UK) [13]. Allele frequencies for the microsatellite markers used in the analysis were taken from data collected in the Sardinian population [14].

COL4A4 and COL4A3 gene sequencing
We sequenced the 48 exons of the COL4A4 gene and the 52 exons of the COL4A3 gene, using the following PCR conditions (with primers as published) [6,8,15]: genomic DNA (50 ng) was PCR amplified in 50 µl containing 0.15 µM of each primer, 0.2 mM dNTP, 1xDynazyme buffer, 2.5 MgCl2, and 1 U of DynaZyme. PCR cycles were as follows: 95°C for 5 min, followed by 35 cycles consisting of 95°C for 15 s, annealing at 55°C for 30 s, and 72°C for 30 s, ended by a final extension at 72°C for 10 min. The PCR products were purified and their sequences determined with the BigDye Terminator sequencing chemistry (Perkin Elmer, CA, USA), and the products were separated on an ABI377 DNA sequencer.



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 Results
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Linkage analysis
A suggestive linkage (LOD=2.7) between AS and chromosome 2q35-q37 was detected using 11 markers across the region (Figure 3Go). The analysis of the most likely haplotype demonstrated that a common haplotype co-segregated with the disease; it was not present in the four unaffected individuals in the family (Figure 1Go). The most severely affected individual (IV:2) inherited two copies of the affected haplotype, one from each parent. Family member IV:2 also had a crossover in one of her haplotypes, between markers D2S434 and D2S351. Together, these observations argue that the disease-causing mutation is located telomeric of D2S351. The COL4A4/COL4A3 locus is located telomeric of D2S351 [9]. Linkage to the other basement-type (type 4) collagen genes was excluded: COL4A1 and COL4A2 on chromosome 13q33-q34, and COL4A5 and COL4A6 on chromosome Xq22-q23.



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Fig. 3. Multipoint linkage analysis and location of microsatellite markers on chromosome 2q35-q37. To the left, the end of the q-arm of chromosome 2 is shown with the markers in the middle. The right part displays the LOD-scores for the different markers.

 

Mutation analysis
Genomic sequence of COL4A4 [8] revealed a heterozygous G to T substitution at position 1183 (last nucleotide of exon 16) in affected individuals III:1, III:2, III:4, and IV:3. The lysine codon AAG at position 325 was changed into AAT, encoding asparagine (Figures 4aGo). The mutation was detected as a homozygous substitution in the most severely affected individual (IV:2) (Figures 4bGo), and was not detected in the unaffected members of the family (IV:1, IV:4, IV:5, IV:6), nor in 50 unrelated controls of Sardinian origin. Moreover, all COL4A4 genes analysed in this family had a homozygous transition from C to T at position 4415, resulting in a substitution of proline into serine. This genetic variant has been described before as an amino acid polymorphism not related to disease [8]. The sequence of COL4A3 was determined, and no DNA sequence variants were identified, either in the 52 exons, or in the conserved exon-intron borders.



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Fig. 4. DNA sequence of the COL4A4 gene around codon 325 in exon 16. (a) Sequence generated from family member IV:3 (identical sequences were obtained from III:1, III:2, and III:4). (b) Sequence generated from family member IV:2, who was homozygous for the G to T substitution changing the AAG-codon for lysine to AAT (asparagine). The arrows indicate the position of the G to T substitution.

 



   Discussion
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 Abstract
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 Subjects and methods
 Results
 Discussion
 References
 
We report here, for the first time, a new mutation in the COL4A4 gene found in a family with an autosomal dominant form of AS. The dominant mode of inheritance in this family is supported by the male-to-male transmission of the disease-phenotype for three generations along with the observed shared extended haplotype around the COL4A3/COL4A4 locus. Autosomal dominant AS is a rare form of the disease, and very little is known about the phenotype and genotype of this condition.

The sequence analysis of the COL4A4 gene revealed a Lys325Asn mutation, and a number of observations indicate a causative function of this mutation. First, this mutation co-segregated completely with the disease in all affected family members affecting males as well as females. In addition, the most severely affected individual (IV:2) carries a homozygous Lys325Asn mutation and has a crossover centromeric of the COL4A4 gene. Second, the mutant allele was not found in any unaffected individuals in the family nor in 50 unrelated individuals from the Sardinian population. Third, we were able to rule out the COL4A3 gene, which has been implicated in several cases of AS transmitted with an autosomal recessive or dominant mode of inheritance [6,7,10,15]. Fourth, the sequence of COL4A4 exon 16 shows a strong sequence conservation between human and mouse proteins [8]. Fifth, a similar mutation was observed in the Schmid metaphyseal dysplasias [16], another collagen disease.

Although a Lys664Asn mutation in COL4A5 gene detected in Alport patients has been considered as non-pathogenic [5], and most of the mutations in AS involve substitution of a glycine residue [17], our data indicate that the Lys325Asn mutation is responsible for AS in this family.

Splicing defects often cause genetic diseases in humans. In our study, we found a G to T mutation at the last nucleotide of exon 16 in COL4A4. The G at this position is highly conserved and is found in 80% in the last position of all mammalian exons [18]. The importance of the G in this position for correct splicing has been reported in several diseases [19]. Moreover, mutations of the last nucleotide in an exon have been described for the COL4A5 and COL4A3 genes, resulting in the removal of the entire exon or in abnormal splice products [10,20,21]. Finally, from the phenotypic point of view, a clear indication of a gene-dosage effect was seen in the most severely affected family member, since she carried the mutation in the homozygous form.

The phenotype observed in this family is very similar to the X-linked form. Although individual IV:3 carries the Lys325Asn mutation, he is not deaf but his young age should be taken into consideration. In AS, increasing age has been shown to be associated with renal function alterations and deafness [22,23]. The age difference in the affected individuals may also explain the minor phenotypic differences (e.g. renal function, deafness) observed between this family and an autosomal dominant AS family that was recently described [9].

From a phenotypic point of view, the most interesting finding in this family was the co-segregation of AS with hypercholesterolaemia, which was particularly severe in individuals III:1, III:4, and in the homozygous individual IV:2. This clinical feature has been described in another well-defined syndrome, called Fechtner syndrome (FS). This disease presents all features of classic AS, and includes haematological abnormalities, impaired liver function, and hypercholesterolaemia. FS has been mapped to chromosome 22q11-q13, and its molecular basis has been established [11,24]. In addition, a role for COL4A1/2 and COL4A3/4 was excluded by linkage. The main phenotypic differences between this AS family and the FS family described by Toren et al. [11], seems to be the absence in our family of haematological abnormalities and lack of impaired liver function. Considering the relative genetic isolation of the Sardinian population, the most likely explanation of the observed association would be the co-segregation in our family of a yet unidentified gene or genes causing the hypercholesterolaemia phenotype. The phenotypic and genetic heterogeneity of FS may also explain the differences observed between these two families. It is therefore tempting to speculate that links exist between mutated basal collagen membranes and hypercholesterolaemia.

Other extremely rare autosomal dominant Alport-like disorders (e.g. Charcot-Marie-Tooth syndrome) have been excluded in this family by physical and ophthalmologic examinations [23]. Based on family history, clinical investigation and mutation analysis, we have confirmed the extreme phenotypic and genetic heterogeneity of collagen disorders. Mutations in COL4A4 gene can produce a wide phenotype variety, ranging from benign haematuria to the most severe forms of AS.



   Acknowledgments
 
We greatly appreciate the excellent assistance of Ms Majalena Granqvist, and Ms Marianne Olsson. This work was supported by grants from the Swedish Medical Research Council, Swedish Diabetes Association, Swedish Strategic Funds, and NovoNordisk. Milco Ciccarese and Giancarlo Tonolo are partly supported by a CNR (Italy)—MFR (Sweden) Exchange Grant.



   Notes
 
Correspondence and offprint requests to: Milco Ciccarese, Servizio Diabetologia, Istituto Clinica Medica, Department of Internal Medicine, University of Sassari, V.le S. Pietro 8, I-07100 Sassari, Italy. Email: milco.ciccarese{at}tiscalinet.it Back



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 2.12.00
Revision received 20. 4.01.