Identification of novel variants in the COL4A4 gene in Korean patients with thin basement membrane nephropathy.
Method: Genomic DNA was obtained from the peripheral blood lymphocytes. For the analysis of the COL4A4 gene, all the exons including splicing sites were amplified by PCR and screened by direct sequencing analysis.
Results: Eight novel COL4A4 sequence variants were found in these patients. Two of these variants, G199R and G1606E, were possibly pathogenic variants affecting the phenotype. None of these variants were observed in 286 chromosomes from normal Korean control subjects. In addition, 39 polymorphisms including 7 novel SNPs were identified in this study.
Interpretation & conclusion: The frequency of COL4A4 mutations in Korean patients with TBMN is low and the other cases may have mutations in other genes like COL4A3. Screening of the COL4A3 gene and finding a novel causative gene for TBMN will help clarify the pathogenesis of this disorder and perhaps for distinguishing TBMN from Alport syndrome.
Key words COL4A4--glomerular basement membrane--Korean population--mutation--thin basement membrane nephropathy
Thin basement membrane nephrophathy [TBMN; Mendelian Inheritance in Man (MIM)#141200] is the most common cause of an inherited renal disease that occurs in at least 1 per cent of the world population (1,2). It is characterized by persistent glomerular haematuria, proteinuria and normal renal function, and is caused by abnormally thin glomerular basement membrane (GBM) (3,4).
Type 4 collagen A is a major structural component of GBM containing six different collagen chains, designated [alpha]1 through [alpha]6 which are encoded by the genes COL4A1 through COL4A6, respectively. Alpha chains 3, 4, and 5 are strongly expressed in GBM (5). In normal individuals, [alpha]3, [alpha]4, and [alpha]5 chains form triple-helical molecules which together constitute cross-linked protein meshwork (6). Because this network makes the structural skeleton of the postnatal GBM, it seems to be essential for the maintenance of strength of the GBM (7).
TBMN is inherited in an autosomal dominant fashion. Heterozygous mutations have been shown to occur in COL4A3 and COL4A4, which are important parts of the framework for the basement membrane (8-15). Some individuals with TBMN are thought to be carriers for genes that cause Alport syndrome (AS)8. Mutations are scattered throughout these genes without any "hot spots" and most mutations result in single nucleotide substitutions and lead to missense or nonsense mutations. In addition, six insertion or deletion mutations have been identified.
It is a major clinical challenge to differentiate between TBMN characterized by nonprogressive haematuria and Alport syndrome with progressive haematuria as the main symptom. Apparently, mutations in the COL4A3 or COL4A4 genes result in a clinical spectrum of disease, ranging from TBMN to autosomal dominant or recessive Alport syndrome depending on the nature of the mutation and gene dosage (9). However, detection of mutations is difficult because of the large size of the COL4A3 and COL4A4 genes, the need for direct sequencings of exons to maximize mutation detection, the need to detect splicing mutations, and the large number of polymorphisms that exist in these two genes.
A few studies have been done to find the cause of TBMN in the Caucasian populations (8-16). However, no information regarding frequency or mutation spectrum in both of the genes has been available for the Asian populations. Moreover, most of the studies have used single strand conformational polymorphisms (SSCP) analysis to detect the mutations. Because of the low detection rate of SSCP analysis, it may underestimate the frequency of COL4A3 and COL4A4 mutations in patients with TBMN.
In this study, genetic analysis of the COL4A4 gene was done in Korean patients to find the cause of TBMN by direct sequencing analysis and to correlate these mutations with clinical features.
Material & Methods
Patients: Forty five unrelated patients with TBMN (29 males and 16 females) with a mean age of 30 yr (range 17 to 62 yr) were recruited consecutively from January 2002 to January 2007 from the Department of Nephrology, Kyungpook National University Hospital, Daegu in Korea. All patients had more than 3 red blood cells per high-power field on phase-contrast microscopy of a midstream urine specimen on at least two separate visits. Patients who underwent renal biopsy and were diagnosed as a thin basement membrane disease without any other significant histologic evidence of giomerulopathy were included in this analysis. The patients who had additional glomerulopathies as well as thin basement membrane disease and refused to participate in genetic analysis were excluded. Patients with clinical or histologic evidence of systemic lupus erythematosus, vasculitis, collagen diseases, and drug abuse were also excluded.
The diagnosis of TBMN was confirmed by diffuse GBM thinning on electron microscopy (Hitachi H-7000 transmission electron microscope Hitachi, Tokyo, Japan) of the biopsied renal tissue in all the patients. One hundred forty three unrelated Koreans who were diagnosed their normal renal function from Kyungpook National University Hospital were used as controls. All subjects provided written informed consent for participation according to the protocol approved by the Ethics Committee of Kyungpook National University Hospital prior to the study.
Amplification of exons of the COL4A4 gene: Genomic DNA was obtained from the peripheral blood lymphocytes using FlexiGene DNA kit (QIAGEN Hilden, Germany). The final concentration of each DNA pool was adjusted to 2.5 ng/[micro]l.
For the analysis of the COL4A4 gene, all the exons were amplified by polymerase chain reaction (PCR) using the appropriate intronic primer sets which were designed by using Primer 3 software (http://www-genome, wi.mit.edu/cgi-bin/primer/primer3_www.cgi). PCR reaction was performed in a total of 25 [micro]l reaction, containing 0.2 mM of each deoxynucleotide, 15 pmol of each forward and reverse primers (Genomine, Korea), 1.0-1.5 mM [MgCl.sub.2] (SolGent Co., Ltd., Korea), 10 mM Tris-HC1 (pH 8.3), 50 mM KC1, 0.75 U of Taq DNApolymerase (SolGent Co., Ltd., Korea), and 25 ng of genomic DNA by use of the DNA Engine[R] Thermal cycler (BIO-RAD, USA). PCR conditions were as follows: 35 cycles of denaturation at 95[degrees]C for 30 sec; annealing at 55[degrees]C or 57[degrees]C, depending on the primers for 30 sec; and extension at 72[degrees]C for 1 min. The first denaturation step and the last extension step were at 95[degrees]C for 2 min and 72[degrees]C for 10 min, respectively.
Five microliters of the PCR products were separated and visualized on a 2 per cent agarose gel. Fifteen microliters of this PCR product were then treated with 0.3 U of shrimp alkaline phosphatase and 3 U of exonuclease I (USB Corporation, USA) at 37[degrees]C for 1 h, followed by incubation at 80[degrees]C for 15 min. This was diluted with an equal volume of d[H.sub.2]O, and 6 [micro]l was used for the final sequencing reaction.
Sequencing analysis of the COL4A4 gene: Sequencing reactions were performed in both directions on the PCR products in reactions containing 5 pmol of primer, 0.25 [micro]l of ABI Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA) and 1 [micro]l of 5X dilution buffer (400 mM Tris-HCl, pH 9 and 10 mM [MgCl.sub.2]). Cycling conditions were 95[degrees]C for 2 min followed by 35 cycles of 94[degrees]C for 20 sec, 55[degrees]C for 20 sec, and 60[degrees]C for 4 min. Sequencing reaction products were ethanol precipitated, and the pellets were resuspended in 10 [micro]l of formamide loading dye. An ABI 3130XL DNA sequencer was used to resolve the products, and data were analyzed by using ABI sequencing Analysis v.5.0 (Applied Biosystems, USA) and LASERGENE-SeqMan software (DNASTAR, Madison, WI, USA).
Statistical analysis: Allele frequencies (p) and heterozygosities (H=1-[p.sup.2]-[q.sup.2]=2p, q=1-p) of all polymorphisms were calculated. Chi square test was used to estimate whether individual polymorphisms were in Hardy-Weinberg equilibrium. D'-based linkage disequilibrium patterns and haplotypes were calculated and represented using the Haploview algorithm that searches for a spine of strong |D '| running from one marker to another (http://www.broad.mit.edu/mpg/ haploview/).
Results & Discussion
All 47 COL4A4 coding exons covering the splicing sites were screened for the mutations by direct sequencing analysis. A total of 47 sequence variants were identified in 45 TBMN patients. Table I presents known and novel polymorphisms found in the COL4A4 gene. Seven novel polymorphisms were found for the first time in this study; IVS8-83T>C, IVS 10+47 G>A, IVS10+67 T>C, IVS11-72 G>T, D682G, IVS28-154 T>C and IVS32-165 T>C. In addition, the existence of 32 database single nucleotide polymorphisms (SNPs) (dbSNPs) were confirmed in the Korean population. The allele frequencies of these known SNPs in the Koreans were similar to those of other populations (11,12). Interestingly, five variants changing amino acids were identified in the exons. Four of them were already reported as a polymorphism in another study (11,12), but it was unclear whether a substitution glycine for aspartic acid in exon 26 (D682G) was mutation or polymorphism. This variant was found in 143 normal controls, so it ruled out the possibility that D682G was pathogenic.
After filtering all SNPs with a minor allele frequency (MAF) less than 0.2 (n=7) or length polymorphisms (n=2) or SNPs with significant deviation from the Hardy-Weinberg equilibrium (P<0.05) (n=3) or SNPs showing less than 75 per cent success rate of genotyping (n=3), the linkage disequilibrium (LD) pattern of the 25 SNPs within the gene was constructed (Fig. a). It showed that COL4A4 could be parsed into four haplotype blocks with each block having strong LD. All the blocks except block 2 have three common haplotypes with frequencies of at least 10 per cent (Fig. b). This result can be useful for the application of association studies with tagging SNP or haplotype analysis.
Eight novel COL4A4 variants were identified from the TBMN patients in this study (Table II). There were two heterozygous glycine missense variants that produced arginine or glutamic acid substitutions (G199R and G1606E). An arginine for glycine substitution in exon 10 (G199R) was present in only one of the 45 patients with TBMN and in none of the 143 normal subjects. It affected a glycine within a highly-conserved region of the collagenous domain gly-X-Y repeats, segregated with haematuria in the family in which it occurred, and was considered pathogenic. Another glycine substitution which is a glycine to glutamic acid change at position 1606 in exon 48 was found in an individual with TBMN. This substitution occurred in the noncollagenous domain (NC1) of [alpha]4 chain. This variant was not found in the 143 healthy individuals, and glycine substitution in the type IV collagen chains is often pathogenic, so it was considered 'possibly pathogenic'. In addition, two synonymous variants and four intronic variants were found (Table II). Although these splicing variants were only found in the patients with TBMN and were not detected in the normal subjects, further studies such as functional analysis will need to be done to consider these as pathogenic.
Clinically, 10 patients (22.2%) had gross haematuria, 11 patients (24.4%) had proteinuria more than 150 mg/day and 5 patients (11.1%) had proteinuria more than 500 mg/day. Also, two patients (4.4%) had hypertension and 8 patients (17.8%) had duplication of glomerular basement membrane on electron microscopy. There were no significant differences in clinical features, such as gross haematuria, proteinuria, hypertension and the frequency of duplication of glomerular basement membrane, between the variants and non variants group in patients with TBMN (Table III). Based on these results, suggested novel mutation of COL4A4 in patients with TBMN did not cause worse clinical manifestations in our patients. Thus, the degree of haematuria or proteinuria and the frequency of duplication of glomerular basement membrane did not seem to depend on these COL4A4 mutations.
In summary, we identified eight novel variants which were only detected in the TBMN patients along with numerous SNPs in this study. Although all six amino acid substitutions and intronic variants assumed to be pathogenic, the overall detection rate of COL4A4 mutations in the patients with TBMN was low. The low mutation yield can be attributed to mutations in the COL4A3 gene and possibly other genes. Therefore, further studies such as screening of the COL4A3 gene and finding a novel causative gene for TBMN will be of interest for clarifying the pathogenesis of this disorder and perhaps for distinguishing TBMN from AS.
To date, many studies for TBMN and Alport syndrome have been performed. But, genetic studies for the aetiological factor of TBMN are insufficient compared with Alport syndrome. Especially according to the Human Mutation Database, only three pathogenic mutations associated with TBMN in COL4A4 gene have been identified (www.hgmd.cfac.uk) (13,17). Though further functional studies that detect aetiological effect of these mutations were not done, the detection of novel mutations in COL4A4 gene will provide useful information on the genetic cause of TBMN and their molecular mechanisms.
The authors acknowledge the patients and their physicians for their cooperation. This study was supported by a grant of the Korea Healthcare technology R&D project, Ministry for Health, Welfare and Family Affairs, Republic of Korea (A080560).
Received January 4, 2008
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(17.) Frasca GM, Onetti-Muda A, Marl F, Longo I, Scala E, Pescucci C, et al. Thin glomerular basement membrane disease: clinical significance of a morphological diagnosis--a collaborative study of the Italian Renal Immunopathology Group. Nephrol Dial Transplant 2005; 20 : 545-51.
Reprint requests: Dr Un-Kyung Kim, Department of Biology, Kyungpook National University, 1370 Sankyuk-dong, Buk-gu Daegu 702-701, South Korea e-mail: email@example.com
Jeong-In Back, Su-Jin Choi, Sun-Hee Park *, Ji-Young Choi *, Chan-Duck Kim *, Yong-Lim Kim * & Un-Kyung Kim
Department of Biology, College of Natural Sciences, Kyungpook National University &
* Department of Nephrology, Kyungpook National University Hospital, Daegu, Korea
Table I. Identification of known and novel single nucleotide polymorphisms (SNPs) of the COL4A4 gene Location SNP ID Variant Nucleotide change Exon 3 rs3817617 Q34Q 756 T>C Intron 7 rsl2465531 IVS7-121 T>G -- lntron 8 novel IVS8-83 T>C -- Intron 9 rs4306711 IVS9+162 G>T -- Intron 9 rs6436654 IVS9-111 G>A -- Intron 10 novel IVS10+47 G>A -- Intron 10 novel IVS10+67 T>C -- Intron 10 rs12475686 IVS10-39 A>G -- Intron l1 novel IVS11-72 G>T -- lntron 12 rs 10174459 * IVS12+104 G>A -- Intron 14 rs11274738 IVS14-38 insCAATATTTC -- Intron 15 rs1574391 IVS15+175 C>G -- Intron 17 rs2894632 IVS17-149 A>G -- Exon 21 rs3736633 P482S 2096 G>A Intron 22 rs10187611 IVS22-150 G>C -- Exon 26 novel D682G 2699 A>G Intron 27 rs10653430 IVS27+25 ins polyA -- Intron 28 novel IVS28-154 T>C -- Intron 28 rs2141829 * IVS28-138 T>C -- Intron 28 rs3769641 * IVS28-5 C>T -- Intron 32 novel IVS32-165 T>C -- Intron 32 rs2272205 IVS32-50 T>C -- Exon 33 rs4675141 L1004P 3665 A>G Intron 33 rs2272204 IVS33+92 G>T -- Intron 33 rs2251223 IVS33-128 G>T -- Intron 34 rs2272203 IVS34+129 G>C -- Intron 34 rs2272200 IVS34-66 A>G -- Exon 39 rs10203363 G1198G 4248 C>T Exon 39 rs2229812 K1228K 4338 G>A Intron 40 rsl3423714 IVS40+9 G>C -- Intron 41 rs 1917127 IVS41+34 C>T -- Exon 42 rs2272199 M1327V 4633 A>G(T>C) Exon 42 rs3817490 P1360P 4734 A>G(T>C) Intron 43 rs3752896 IVS43-36 C>T -- Exon 44 rs3752895 P1403S 4861 G>A(C>T) Intron 44 rs10188770 IVS44-24 A>G -- Intron 46 rsl3419076 IVS46-8 G>A -- Exon 47 rs 10187726 V1516V 5202 T>C Exon 48 rs2228557 F1644F 5586 C>T Location Effect on Codon coding sequence position Exon 3 Synonymous 3 Glu 34 Intron 7 -- -- lntron 8 -- -- Intron 9 -- -- Intron 9 -- -- Intron 10 -- -- Intron 10 -- -- Intron 10 -- -- Intron l1 -- -- lntron 12 -- -- Intron 14 -- -- Intron 15 -- -- Intron 17 -- -- Exon 21 Pro482 [right arrow] Ser in 1 the CD Intron 22 -- -- Exon 26 Asp682 [right arrow] Gly in 2 the CD Intron 27 -- -- Intron 28 -- -- Intron 28 -- -- Intron 28 -- -- Intron 32 -- -- Intron 32 -- -- Exon 33 Leu 1004 [right arrow] Pro in 2 the CD Intron 33 -- -- Intron 33 -- -- Intron 34 -- -- Intron 34 -- -- Exon 39 Synonymous Gly 3 1198 Exon 39 Synonymous Lys 3 1228 Intron 40 -- -- Intron 41 -- -- Exon 42 Metl327 [right arrow] Val in 1 the CD Exon 42 Synonymous Pro 3 1360 Intron 43 -- -- Exon 44 Pro 143 [right arrow] Ser in 1 the CD Intron 44 -- -- Intron 46 -- -- Exon 47 Synonymous Val 3 1516 Exon 48 Synonymous Phe 3 1644 Allele frequency(minor) Location Heterozygosity CEU HCB KOR Exon 3 0.00(C) 0.04(C) 0.06(C) 0.11 Intron 7 Unknown Unknown 0.13(T) 0.23 lntron 8 -- -- 0.10(C) 0.18 Intron 9 Unknown Unknown 0.38(T) 0.47 Intron 9 0.41(A) 0.43(A) 0.45(A) 0.49 Intron 10 -- -- 0.36(G) 0.46 Intron 10 -- -- 0.36(C) 0.46 Intron 10 0.36(A) 0.38(A) 0.47(A) 0.50 Intron l1 -- -- 0.15(T) 0.26 lntron 12 0.05(A) 0.33(A) 0.33(A) 0.44 Intron 14 Unknown Unknown 0.32 0.44 Intron 15 0.37(C) 0.37(C) 0.34(C) 0.45 Intron 17 0.03(A) 0.23(A) 0.30(A) 0.42 Exon 21 0.49(G) 0.34(A) 0.48(G) 0.50 Intron 22 0.33(G) 0.20(G) 0.24(G) 0.37 Exon 26 -- -- 0.11(G) 0.19 Intron 27 -- -- -- -- Intron 28 -- -- 0.24(C) 0.38 Intron 28 0.44(C) 0.44(T) 0.24(T) 0.37 Intron 28 0.08(C) 0,10(c) 0.03(C) 0.06 Intron 32 -- -- 0.43(C) 0.49 Intron 32 0.08(C) 0.09(c) 0.10(c) 0.17 Exon 33 0.49(A) 0.38(A) 0.43(A) 0.49 Intron 33 0.51(T) 0.63(T) 0.43(T) 0.49 Intron 33 0.62(T) 0.37(G) 0.47(G) 0.50 Intron 34 0.49(C) 0.36(G) 0.32(G) 0.44 Intron 34 0.50 0.38(A) 0.44(A) 0.49 Exon 39 0.42(T) 0.39(T) 0.48(C) 0.50 Exon 39 0.43(A) 0.46(A) 0.46(G) 0.50 Intron 40 0.42(G) 0.44(G) 0.49(G) 0.50 Intron 41 0.42(C) 0.38(C) 0.47(T) 0.50 Exon 42 0.43(A) 0.44(A) 0.47(G) 0.50 Exon 42 0.43(A) 0.44(A) 0.49(G) 0.50 Intron 43 0.44(T) 0.37(T) 0.49(C) 0.50 Exon 44 0.43(G) 0.44(G) 0.49(G) 0.50 Intron 44 0.43(A) 0.44(A) 0.50 0.50 Intron 46 0.44(G) 0.46(G) 0.46(A) 0.50 Exon 47 0.50 0.43(T) 0.38(T) 0.47 Exon 48 0.42(T) 0.44(T) 0.46(C) 0.50 Location Reference /database Exon 3 NCBI database Intron 7 NCBI database lntron 8 Present study Intron 9 NCBI database Intron 9 NCBI database Intron 10 Present study Intron 10 Present study Intron 10 NCBI database Intron l1 Present study lntron 12 NCBI database Intron 14 NCBI database Intron 15 NCBI database Intron 17 NCBI database Exon 21 Longo et al, 2002 (12) Intron 22 NCBI database Exon 26 Present study Intron 27 NCBI database Intron 28 Present study Intron 28 NCBI database Intron 28 Badenas et al, 2002 (11) Intron 32 Present study Intron 32 NCBI database Exon 33 Longo et al, 2002 (12) Intron 33 NCBI database Intron 33 NCBI database Intron 34 NCBI database Intron 34 NCBI database Exon 39 Badenas et al, 2002 (11) Exon 39 NCBI database Intron 40 NCBI database Intron 41 NCBI database Exon 42 NCBI database Exon 42 Longo et al, 2002 (12) Intron 43 NCBI database Exon 44 Badenas et al, 2002 (11) Intron 44 Badenas et al, 2002 (11) Intron 46 NCBI database Exon 47 Longo et al, 2002 (12) Exon 48 Badenas et al, 2002 (11) The information of dbSNP ID is based on NCBI database (http://www.ncbi.nlm.nih.gov/). CEU; Utah residents with ancestry from northern and western Europe; HCB, Han Chinese in Beijing; * P values of deviation from Hardy-Weinberg Equilibrium Table II. Characteristics of novel COL4A4 variants identified from TBMN patients in this study Allele frequency Location Mutation Nucleotide Codon change position Major Minor Intron 3 IVS3-62 C>T -- -- 0.99 0.01 Intron 7 IVS7+6 A>G -- -- 0.99 0.01 Exon 10 G 199R 1249 G>A 1 0.99 0.01 Exon 32 P959P 3531 C>T 3 0.99 0.01 Exon 36 S1 102S 3960 C>T 3 0.99 0.01 Intron 44 IVS44-12 T>C -- -- 0.99 0.01 Intron 45 IVS45+3 C>T -- -- 0.99 0.01 Exon 48 G1606E 5471 G>A 2 0.99 0.01 Table III. Comparison of clinical features between variants and non-variants group Variants group Non variants group (n=8) (n=37) Gross haematuria 2 (25) 8 (21.6) Proteinuria 1 (12.5) 10 (27) (> 150 mg/day) Proteinuria 0 (0) 5 (13.5) (> 500 mg/day) Hypertension 0 (0) 2 (5.4) Duplication of GBM 1 (12.5) 7 (18.9) GBM, glomerular basement membrane Values in parentheses represent percentages
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|Author:||Baek, Jeong-In; Choi, Su-Jin; Park, Sun-Hee; Choi, Ji-Young; Kim, Chan-Duck; Kim, Yong-Lim; Kim, Un-|
|Publication:||Indian Journal of Medical Research|
|Date:||May 1, 2009|
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