NDT Advance Access originally published online on March 3, 2006
Nephrology Dialysis Transplantation 2006 21(6):1582-1587; doi:10.1093/ndt/gfl051
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© The Author [2006]. 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
Tissue-specific distribution of an alternatively spliced COL4A5 isoform and non-random X chromosome inactivation reflect phenotypic variation in heterozygous X-linked Alport syndrome
1 Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, 2 Department of Nephrology, Tokyo Medical University Kasumigaura Hospital, 3-20-1 Chuo, Ami, Ibaraki 300-0332 and 3 Ibaraki Prefectural University of Health Sciences, 4669 Ami, Ami, Ibaraki 300-0394, Japan
Correspondence and offprint requests to: Yoshio Shimizu, MD, PhD, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tennodai, Tsukuba, Ibaraki 305-8575, Japan. E-mail: y-shimz{at}md.tsukuba.ac.jp
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Abstract |
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A novel type of hereditary transmission of COL4A5 in a Japanese family with X-linked Alport syndrome was detected through analysis of cDNA sequences and an X-chromosome inactivation assay. A female patient with moderately altered renal function, who was diagnosed with Alport syndrome by renal biopsy, and her mother, who was undergoing maintenance haemodialysis, showed similar tissue-specific expression of a truncated isoform of COL4A5, which was generated by alternative splicing without a splice-site mutation. Expression of the truncated isoform occurred in the renal specimen derived from the patient, but not in specimens from controls. Genomic analysis revealed two point mutations (c.4821 + 121, T>C; c.4822-151_150, ins T) in intron 49 of COL4A5 from the patient. The peripheral blood mononuclear cells of the patient and her mother showed non-random lyonization. While the females showed only renal impairment, an affected male in the same family suffered from severe renal insufficiency, visual defect and hearing disturbances. Hence, we suggest that this type of heredity COL4A5 presents with phenotypic variation in female heterozygous X-linked Alport syndrome patients.
Keywords: alport syndrome; alternatively spliced isoform; COL4A5; X-chromosome inactivation
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Introduction |
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Alport syndrome is a hereditary nephropathy characterized by a family history of haematuria, progressive renal failure, hearing disturbance and typical ocular changes [1,2]. This syndrome is caused by mutations in type IV collagen genes, which code for a major component of basement membranes [3,4]. Six genetically distinct variants (
1–6) have been identified, for which the corresponding genes are located pairwise on chromosomes X, 2 and 13. While the 1 and 2 chains of type IV collagen are ubiquitously found in basement membranes, the 3, 4 and 5 chains are restrictively distributed in the glomerulus, inner ear and eye [4]. About 80–85% of patients have the X-linked form, and most cases of X-linked Alport syndrome are caused by mutation in COL4A5 (OMIM #303630) [5]. The syndrome usually leads to terminal renal failure in males, while affected females typically display mild clinical symptoms, particularly in childhood and young adulthood. The risk of developing end-stage renal failure before the age of 40 is reported to be 12% in female patients but about 90% in male patients [6]. Similar results were obtained in a study of 113 female patients with X-linked Alport syndrome, 15% of whom had chronic renal failure at an average age of 40 years [7]. This phenomenon is thought to be caused by the presence of a normal COL4A5 allele resulting from a random pattern of X-chromosome inactivation in most heterozygotes [7,8]. We report the case of a female patient and her mother, both of whom had X-linked Alport syndrome. Tissue-specific distribution of an alternatively spliced isoform of COL4A5 and non-random lyonization were found in both mother and daughter. Analysis of these patients suggested that this type of hereditary transmission presents with phenotypic variations in female X-linked Alport syndrome.
Patient and samples
The patient was a 23-year-old female with moderate renal impairment and nephrotic syndrome. She was diagnosed with Alport syndrome based on typical glomerular basement membrane (GBM) abnormalities (lamellation shown by electron microscopy) found in a renal biopsy taken at the age of two in another hospital. This examination was performed because her mother had already been diagnosed with Alport syndrome and the patient had shown microhaematuria at birth. She had neither hearing loss nor ocular abnormalities, and she did not show clinical features of Turner syndrome. Cytogenetic analysis of her peripheral leucocytes showed a karyotype of 46, XX.
The patient's mother was 50 years old and required maintenance haemodialysis due to Alport syndrome. She had delivered our patient at the age of 27, and maintenance haemodialysis was started at the age of 37. When she was 41 years old, a cadaveric renal transplantation was performed, but she again required haemodialysis 3 years later due to chronic graft rejection. Like to her daughter, she had no abnormalities except renal impairment. Her elder brother was also affected by Alport syndrome and had undergone haemodialysis for 10 years, before subsequently dying from cerebral haemorrhage at the age of 36. During his lifetime, he had experienced both hearing and visual disturbances: he had found it hard to hear since his early teens, but he did not need a hearing aid for daily life. His visual acuity gradually worsened with time from the onset of renal failure, and bilateral cataract was later diagnosed. It was not clear whether he was affected by lenticonus.
Blood samples were obtained from the patient and her mother to extract RNA and genomic DNA. They were each given a new hairbrush, with which they collected 20–30 hair roots by combing. Two volunteers, a 23-year-old male student at our university and a 54-year-old female nurse at our university hospital, gave blood samples for the collection of normal control data. Normal hair roots were also given by the nurse. A 77-year-old male patient with renal cell carcinoma voluntarily allowed normal tissue from his resected kidney to be used as a control. The study was approved by the Institutional Ethics Review Board of the University of Tsukuba, and written informed consent was obtained from the patient and her mother.
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Methods |
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Immunofluorescence staining of renal specimens
Four-micrometer frozen sections of renal biopsy specimens were directly stained with fluorescein isothiocyanate-conjugated
5 chain of type IV collagen or Texas Red-conjugated anti-2 chain of type IV collagen monoclonal antibodies (Shigei Medical Research Institute, Okayama, Japan). The alternatively spliced variant of COL4A5 in the renal specimens was also detected by immunofluorescence, using the antipeptide antibody described subsequently as the first antibody, diluted normal goat serum as blocking agent, and Alexa488-conjugated goat antirabbit IgG (Invitrogen, Paisley, UK) as the second antibody.
Generation and purification of antipeptide antibodies
Polyclonal antisera were generated against the amino-acid sequence VFYTLPCICHHS, which was specific for the alternatively spliced variant of COL4A5 (Figure 2C). The peptide was synthesized, purified and coupled to keyhole limpet haemocyanin by QIAGEN (Tokyo, Japan). The conjugates were mixed 1:1 with Freund's complete adjuvant and subcutaneously injected into two rabbits. Animals were injected with the mixture four times at 2 week intervals, and serum was obtained simultaneously at the times of the third and fourth immunization and 2 weeks after the final injection. The specificity of the antisera was initially tested using standard immuno-dot blot analysis. Antipeptide antibodies were precipitated from sera at high titre, using 40% saturated ammonium sulfate solution. The precipitant was resuspended in phosphate-buffered saline (PBS) and then dialysed against it.
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PCR
RT–PCR was performed on hair roots and peripheral blood mononuclear cells (PBMCs) in four overlapping segments spanning the entire COL4A5 open reading frame. PBMCs were collected from heparinized blood by Ficoll-Paque Plus (Amersham Biosciences, Uppsala, Sweden) density-gradient centrifugation within 30 min of blood collection. Total RNA was extracted from hair roots and PBMCs of the patient and her mother, using a nucleic acid purification system (MagExtractor MFX-2000, Toyobo, Osaka, Japan) according to the manufacturer's instructions. Messenger RNA (mRNA) was converted into complementary DNA (cDNA) using a Ready-to-go T-primed First-Strand Kit (Amersham Biosciences, NJ, USA). PCR was performed using 35 cycles comprising 5 s denaturation (at 94°C), 1 s annealing (at 55.5°C) and 2 s extension (at 72°C). Primers for RT–PCR were synthesized according to King et al. [9].
Genomic DNA was obtained from the PBMCs using a DNeasy Tissue Kit (Qiagen, CA, USA) according to the manufacturer's instruction. Primers for amplification of the fragment including the entire length of intron 49 were designed based on the sequences of exons 49 and 50 (the primer sequences were 5'-AGATTCCCCATTGTCCTCAG-3' and 5'-CATAGTAGTTACAGGTACCC-3'). PCR was performed under the same conditions as those described for RT–PCR.
DNA sequence analysis
The RT–PCR and genomic-PCR product were subcloned into T-vector pCRII (Invitrogen, CA, USA) and sequenced. Sequence data were analysed by the programme DNASIS Ver. 1.00 (Hitachi Software, Tokyo, Japan), and subsequent homology searches were performed using BLAST (available on the Internet at http://www.ncbi.nlm.nih.gov/BLAST/).
X-chromosome inactivation analysis
The Assessment of X-chromosome inactivation patterns involved the digestion of DNA samples with methylation-sensitive restriction endonucleases HpaII and HhaI [10,11]. Purified genomic DNA (500 ng) was digested in a 20 µl volume of buffer solution with 5U HpaII and HhaI (Takara Biotechnology, Tokyo, Japan) and incubated overnight at 37°C. The digested DNA was purified and concentrated to a 10 µl solution by a standard procedure of phenol:chloroform extraction and ethanol precipitation. Two microlitres of (HpaII + HhaI)-digested or undigested DNA were used for PCR of the polymorphic locus ‘HUMARA’, using the forward primer 5'-TCCAGAATCTGTTCCAGAGCGTGC-3' and the reverse primer 5'-CTCTACGATGGGCTTGGGGAGAAC-3'. The PCR was performed for 40 cycles of amplification, each comprising 1 min at 95°C, 1 min at 62°C and 1 min at 72°C, after an initial 5 min denaturation step at 95°C, and with a final extension step of 10 min at 72°C. Ten microlitres of each PCR product were electrophoresed with an identical volume of loading buffer (Takara Biotechnology) in a 3% agarose gel. X-chromosome patterns were then visualized with ethidium bromide staining under an ultraviolet illuminator.
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Results |
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Morphological investigation
The light microscopic lesions showed moderate injury, and 7 out of 16 glomeruli showed global sclerosis. Four glomeruli showed segmental sclerosis and hyalinosis with mesangial hypercellularity (Figure 1A), and others showed mild hypertrophy. Double contours and irregularity of the GBM were seen, and tubular atrophy and interstitial fibrosis were seen in 60–70% of the samples. Foam cells were also found.
Immunofluorescence staining for the 5 chain of type IV collagen showed that it was well preserved in the glomeruli (Figure 1B), and the 2 chain also well stained (data not shown, technical positive control).
Ultrastructure analysis revealed minimal mesangial proliferation without apparent dense deposition, and the GBM showed focal thinning and occasional basket-woven features. Subepithelial or subendothelial deposition was not seen, but endothelial cells were mildly swollen and podocytes showed occasional foot process effacement (Figure 1C).
RT–PCR
RT–PCR products from three of the four segments of COL4A5 cDNA derived from our patient and her mother were of the same size as those from normal controls (data not shown). However, the PCR bands for the terminal region from exon 41 to the 3'-untranslated region, which contains a non-collagenous (NC) domain, suggested a product (1726 bp, Figure 2C) that was larger than the control, while the product from this region in DNA derived from hair roots showed the expected size of 1596 bp (Figure 2A).
Sequence analysis
Sequencing data for the RT–PCR product of the COL4A5 region containing the NC-domain from hair roots of the patient and her mother showed that this product had no mutations and was similar to the normal control sequence. However, the longer transcripts of COL4A5 derived from PBMCs of the patient revealed a novel alternatively spliced transcript of the COL4A5 gene, which contained the entire length of intron 49 without a splice-site mutation. Sequencing of both the RT–PCR product and genomic-PCR product revealed that there were two point mutations in intron 49 (c.4821 + 121, T>C; c.4822-151_150, ins T), and the longer RT–PCR band seen in the PBMCs of the patient and her mother had an additional 130 bp, giving a product of 1726 bp (Figure 2C). These mutations were not seen in our normal controls and have not been previously reported, based on an examination of the Ensembl single nucleotide polymorphism database (http://www.ensembl.org). This alternative splicing induced an in-frame stop codon that was 13 codons from the 3' end of the normal exon 49. Sequence data for the patient's mother were identical to those for the patient herself.
Detection of an alternatively spliced variant of COL4A5 in renal specimens
Immunofluorescence using the polyclonal antibody against the peptide specifically present in the alternatively spliced form of COL4A5 revealed linear staining of the GBM in renal specimens from the patient, while only weak non-specific signals were obtained for normal control specimens (Figure 2D).
X-chromosome inactivation analysis
To study the X-chromosome inactivation pattern of the patients, CAG repeat polymorphism of the ‘HUMARA’ gene was analysed by PCR combined with methylation-sensitive endonuclease (HpaII and HhaI) digestion. The PCR products using the undigested male control genome as a template were of a single size, while no amplification was observed with the digested genome. In the normal female control, two bands were observed with or without digestion, indicating random X-chromosome inactivation. When the genomic template of the patient and her mother were used without digestion, double bands occurred in the gel, while with digestion the number of common bands between the patient and her mother diminished. This indicated that the X-chromosome carrying the normal allele had been selectively inactivated (Figure 3).
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Discussion |
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Alternative splicing of mRNAs greatly expands the number of gene products that may be produced from a single coding sequence. Recently, expressed sequence tag analysis has indicated a much higher rate of alternative splicing in human genes than was previously believed to occur [12], and it is now thought that generation of an increased diversity of proteins from a single gene through alternative splicing gives great advantages for adaptation to different environments [13]. RT–PCR analysis for COL4A5 revealed that PBMCs from our patient and her mother selectively expressed mutant transcripts, whereas normal COL4A5 transcripts were detected in hair roots. Similar mutant transcripts were reported in a Japanese patient in 1993, but these were due to a point mutation at the 3' end of exon 49 [14], whereas the isoform identified in our patient did not have a point mutation at the splice site. It has recently been suggested that intronic sequences may work as splicing enhancers or silencers [15,16], and it is possible that the mutations in intron 49 or those in other introns caused the alternative splicing. Preferential activation of the donor site in exon 50 in total RNA extracted from PBMCs of the patients was also consistent with an analysis using the Splice Site Predictor Program from the Berkeley Drosophila Genome Project (http://www.fruitfly.org/seq_tools/splice.html). This programme scores 5' splice-donor sites and 3' splice-acceptor sites between 0 and 1.0, with scores approaching 1.0 indicating a closer match to the consensus sequence. The donor site in exon 50 yielded a score of 0.92, whereas that in exon 49 scored below 0.4. Moreover, the uncommon X-chromosome between the patient and her mother, which carried a normal allele, was predominantly inactivated in the PBMCs of the patient, leading to a preferential transcription of the truncated form of COL4A5 in these cells. Guo et al. [17] have described an Alport female with a severe phenotype in whom 90% of the COL4A5 mRNA in white blood cells and kidney was derived from a mutant alleleError, and Vetrie and coworkers [18], in a study of X-chromosome inactivation patterns in white blood cells of female patients, found a random inactivation pattern in most subjects and occasional subjects with extreme skewing of inactivation. Taken together with our data, these studies suggest that tissue-specific distribution of an alternatively spliced COL4A5 isoform reflects tissue-specific functional alteration, and that additional non-random inactivation of the X-chromosome with a normal allele influences the phenotype of heterozygous X-linked Alport syndrome.
Nakanishi et al. [19] showed an inverse correlation between expression of the 5 chain of type IV collagen in the epidermal basement membrane and urinary protein excretion in heterozygous females, and this study supported the notion that the presence in heterozygous females of a normal COL4A5 allele, and consequently some level of 5 chain expression in basement membranes, is indeed protective of renal function. In our patient, clinical data and the light- and electron-microscopy findings in renal biopsy specimens were characteristic of Alport syndrome, whereas immunofluorescence staining for the 5 chain of type IV collagen in glomeruli indicated that it was well preserved. However, the anti-5 chain of type IV collagen monoclonal antibody that we used recognizes the amino-acid sequence CQISEQKRPIDVEFQK [20], and the truncated isoform of COL4A5 also contains this sequence. Immunofluorescence data obtained with an antibody against the amino-acid sequence VFYTLPCICHHS, which is specifically present in the truncated COL4A5 isoform, showed fine staining along the GBM in the patient's kidney, but not in the normal control. These results show the importance of specific antibody–epitope recognition in immunostaining analysis.
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Conclusion |
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Tissue-specific distribution of a splicing isoform of COL4A5 with skewed inactivation of the X-chromosome carrying a normal allele causes moderately severe renal insufficiency in female Alport syndrome patients, and we suggest that these mechanisms induce phenotypic variation in heterozygous X-linked Alport syndrome.
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Acknowledgments |
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This study was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The HeLa cells used for conditioning of PCR and electrophoresis were supplied by RIKEN BRC Cell Bank, Tsukuba, Ibaraki, Japan. The authors are grateful to the patient and her mother for consenting to the genetic analysis. The authors also thank the student at the University of Tsukuba and the nurse and patient at our university hospital for providing samples for collection of normal control data.
Conflict of interest statement. None declared.
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[Abstract/Free Full Text]
Accepted in revised form: 27. 1.06
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