Indian J Med Res 129, May 2009, pp 525-533

Identification of novel variants in the COL4A4 gene in Korean patients with thin basement membrane nephropathy Jeong-In Baek, 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

Received January 4, 2008 Background & objectives: The α4 chain of the type 4 collagen family is an important component of the glomerular basement membrane (GBM) in the kidney. It is encoded by the COL4A4 gene, and mutations of this gene are known to be associated with thin basement membrane nephropathy (TBMN). To better understand the contribution of variants in the COL4A4 gene to TBMN, we investigated the sequence of the complete COL4A4 gene in 45 Korean patients with TBMN. 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 population1,2. 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 α1 through α6 which are encoded by the genes COL4A1 through COL4A6, respectively. Alpha chains 3, 4, and 5 are strongly expressed in GBM5. In normal individuals, α3, α4, and α5 chains form triple-helical molecules which together constitute 525

526

INDIAN J MED RES, MAY 2009

cross-linked protein meshwork6. Because this network makes the structural skeleton of the postnatal GBM, it seems to be essential for the maintenance of strength of the GBM7. 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 membrane8-15. 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 dosage9. 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 populations8-16. 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 glomerulopathy 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/µl. 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://wwwgenome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). PCR reaction was performed in a total of 25 µl reaction, containing 0.2 mM of each deoxynucleotide, 15 pmol of each forward and reverse primers (Genomine, Korea), 1.0-1.5 mM MgCl2 (SolGent Co., Ltd., Korea), 10 mM Tris-HCl ((pH 8.3), 50 mM KCl, 0.75 U of Taq DNA polymerase (SolGent Co., Ltd., Korea), and 25 ng of genomic DNA by use of the DNA Engine® Thermal cycler (BIO-RAD, USA). PCR conditions were as follows: 35 cycles of denaturation at 95oC for 30 sec; annealing at 55oC or 57oC, depending on the primers for 30 sec; and extension at 72oC for 1 min. The first denaturation step and the last extension step were at 95oC for 2 min and 72oC for 10 min, respectively. Five microliters of the PCR products were separated and visualized on a 2 per cent agarose gel.

BAEK et al: IDENTIFICATION OF NOVEL VARIANTS IN THE COL4A4 GENE IN KOREANS

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 37oC for 1 h, followed by incubation at 80oC for 15 min. This was diluted with an equal volume of dH2O, and 6 µl 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 µl of ABI Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA) and 1 µl of 5X dilution buffer (400 mM Tris-HCl, pH 9 and 10 mM MgCl2). Cycling conditions were 95oC for 2 min followed by 35 cycles of 94oC for 20 sec, 55oC for 20 sec, and 60oC for 4 min. Sequencing reaction products were ethanol precipitated, and the pellets were resuspended in 10 µl 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 LASERGENESeqMan software (DNASTAR, Madison, WI, USA). Statistical analysis: Allele frequencies (p) and heterozygosities (H=1-p²-q²=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/ 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, IVS10+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 populations11,12. Interestingly, five variants changing amino acids were identified in the exons. Four of them were already reported as a polymorphism in another study11,12, but it was unclear whether a substitution glycine for

527

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 ( C

Synonymous Glu 34

Nucleotide Effect on change coding sequence

IVS7-121 T>G IVS8-83 T>C IVS9+162 G>T IVS9-111 G>A IVS10+47 G>A IVS10+67 T>C IVS10-39 A>G IVS11-72 G>T IVS12+104 G>A IVS14-38 insCAATATTTC IVS15+175 C>G IVS17-149 A>G P482S 2096 G>A Pro482→Ser in the CD IVS22-150 G>C D682G 2699 A>G Asp682→Gly in the CD IVS27+25 ins polyA IVS28-154 T>C IVS28-138 T>C IVS28-5 C>T IVS32-165 T>C IVS32-50 T>C L1004P 3665 A>G Leu1004→Pro in the CD IVS33+92 G>T IVS33-128 G>T IVS34+129 G>C IVS34-66 A>G G1198G 4248 C>T Synonymous Gly 1198

Q34Q

SNP ID Variant

Exon 3

Location

3

2

2

1

3

position

Codon

0.51 ( T ) 0.62 ( T ) 0.49 ( C ) 0.50 0.42 ( T )

0.44 ( C ) 0.08 ( C ) 0.08 ( C ) 0.49 ( A )

0.33 ( G ) -

Unknown Unknown 0.41 ( A ) 0.36 ( A ) 0.05 ( A ) Unknown 0.37 ( C ) 0.03 ( A ) 0.49 ( G )

0.00 ( C )

CEU

0.63 ( T ) 0.37 ( G ) 0.36 ( G ) 0.38 ( A ) 0.39 ( T )

0.44 ( T ) 0.10 ( C ) 0.09 ( C ) 0.38 ( A )

0.20 ( G ) -

Unknown Unknown 0.43 ( A ) 0.38 ( A ) 0.33 ( A ) Unknown 0.37 ( C ) 0.23 ( A ) 0.34 ( A )

0.04 ( C )

HCB

0.43 ( T ) 0.47 ( G ) 0.32 ( G ) 0.44 ( A ) 0.48 ( C )

0.24 ( C ) 0.24 ( T ) 0.03 ( C ) 0.43 ( C ) 0.10 ( C ) 0.43 ( A )

0.24 ( G ) 0.11 ( G )

0.13 ( T ) 0.10 ( C ) 0.38 ( T ) 0.45 ( A ) 0.36 ( G ) 0.36 ( C ) 0.47 ( A ) 0.15 ( T ) 0.33 ( A ) 0.32 0.34 ( C ) 0.30 ( A ) 0.48 ( G )

0.06 ( C )

KOR

Allele frequency (minor)

0.49 0.50 0.44 0.49 0.50

0.38 0.37 0.06 0.49 0.17 0.49

0.37 0.19

0.23 0.18 0.47 0.49 0.46 0.46 0.50 0.26 0.44 0.44 0.45 0.42 0.50

0.11

Contd....

NCBI database NCBI database NCBI database NCBI database Badenas et al, 200211

NCBI database Present study NCBI database Badenas et al, 200211 Present study NCBI database Longo et al, 200212

NCBI database Present study

NCBI database Present study NCBI database NCBI database Present study Present study NCBI database Present study NCBI database NCBI database NCBI database NCBI database Longo et al, 200212

NCBI database

Heterozygosity Reference / database

Table I. Identification of known and novel single nucleotide polymorphisms (SNPs) of the COL4A4 gene

528 INDIAN J MED RES, MAY 2009

P1360P

IVS44-24 A>G IVS46-8 G>A V1516V

F1644F

Intron 44 rs10188770 Intron 46 rs13419076 Exon 47 rs10187726

Exon 48

4338 G>A Synonymous Lys 1228 4633 Met1327→Val in A>G(T>C) the CD 4734 Synonymous Pro A>G(T>C) 1360 4861 Pro1430→Ser in G>A(C>T) the CD 5202 T>C Synonymous Val 1516 5586 C>T Synonymous Phe 1644

Nucleotide Effect on chage coding sequence

3

3

1

3

1

3

position

Codon

0.42 ( T )

0.43 ( A ) 0.44 ( G ) 0.50

0.44 ( T ) 0.43 ( G )

0.43 ( A )

0.42 ( G ) 0.42 ( C ) 0.43 ( A )

0.43 ( A )

CEU

0.44 ( T )

0.44 ( A ) 0.46 ( G ) 0.43 ( T )

0.37 ( T ) 0.44 ( G )

0.44 ( A )

0.44 ( G ) 0.38 ( C ) 0.44 ( A )

0.46 ( A )

HCB

0.46 ( C )

0.50 0.46 ( A ) 0.38 ( T )

0.49 ( C ) 0.49 ( G )

0.49 ( G )

0.49 ( G ) 0.47 ( T ) 0.47 ( G )

0.46 ( G )

KOR

Allele frequency (minor)

0.50

0.50 0.50 0.47

0.50 0.50

0.50

0.50 0.50 0.50

0.50

Badenas et al, 200211

Badenas et al, 200211 NCBI database Longo et al, 200212

NCBI database Badenas et al, 200211

Longo et al, 200212

NCBI database NCBI database NCBI database

NCBI database

Heterozygosity Reference / database

The information of dbSNP ID is based on NCBI database (http://www.ncbi.nlm.nih.gov/). http://www.ncbi.nlm.nih.gov/). CEU; Utah residents with ancestry from northern and western Europe; HCB, Han http://www.ncbi.nlm.nih.gov/ Chinese in Beijing; *P values of deviation from Hardy-Weinberg Equilibrium

rs2228557

IVS43-36 C>T P1403S

Intron 43 rs3752896 Exon 44 rs3752895

rs3817490

Exon 42

K1228K

IVS40+9 G>C IVS41+34 C>T M1327V

rs2229812

SNP ID Variant

Intron 40 rs13423714 Intron 41 rs1917127 Exon 42 rs2272199

Exon 39

Location

BAEK et al: IDENTIFICATION OF NOVEL VARIANTS IN THE COL4A4 GENE IN KOREANS 529

530

INDIAN J MED RES, MAY 2009

Fig. LD coefficients and haplotypes of 25 dbSNPs in the COL4A4 gene. a. Linkage disequilibrium coefficient (D’) and LD blocks among 25 dbSNPs in the COL4A4 gene. The triangles indicate BLOCKs 1, 2, 3 and 4. b. Haplotypes constructed from 18 SNPs in 4 blocks and their frequencies in the Korean population. Only SNPs with frequencies over 0.02 are shown.

BAEK et al: IDENTIFICATION OF NOVEL VARIANTS IN THE COL4A4 GENE IN KOREANS

531

532

INDIAN J MED RES, MAY 2009

Table II. Characteristics of novel COL4A4 variants identified from TBMN patients in this study Location

Mutation

Nucleotide Codon Allele frequency change position Major Minor

Intron 3 IVS3-62 C>T Intron 7 IVS7+6 A>G Exon 10 G199R 1249 G>A Exon 32 P959P 3531 C>T Exon 36 S1102S 3960 C>T Intron 44 IVS44-12 T>C Intron 45 IVS45+3 C>T -

1 3 3 -

0.99 0.99 0.99 0.99 0.99 0.99 0.99

0.01 0.01 0.01 0.01 0.01 0.01 0.01

Exon 48

2

0.99

0.01

G1606E

5471 G>A

Table III. Comparison of clinical features between variants and non-variants group

Gross haematuria Proteinuria (> 150 mg/day) Proteinuria (> 500 mg/day) Hypertension Duplication of GBM

Variants group (n=8)

Non variants group (n=37)

2 (25) 1 (12.5)

8 (21.6) 10 (27)

0 (0)

5 (13.5)

0 (0) 1 (12.5)

2 (5.4) 7 (18.9)

GBM, glomerular basement membrane Values in parentheses represent percentages

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.cf.ac.uk) www.hgmd.cf.ac.uk 13, 17. Though www.hgmd.cf.ac.uk) 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. Acknowledgment 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).

References 1.

Kincaid-Smith P. Thin basement membrane disease. In: Massry SG, Glassock RJ, editors. Textbook of nephrology, Part 13. Baltimore: Williams and Wilkins; 1995. p. 760-4.

2.

Savige J, Rana K, Tonna S, Buzza M, Dagher H, Wang YY. Thin basement membrane nephropathy. Kidney Int 2003; 64 : 1169-78.

3.

Thorner PS. Alport syndrome and thin basement membrane nephropathy. Nephron Clin Pract 2007; 106 : c82-8.

4.

Gregory MC. The clinical features of thin besement membrane nephropathy. Semin Nephrol 2005; 25 : 140-5.

5.

Peissel B, Geng L, Kalluri R, Kashtan C, Rennke HG, Gallo GR, et al. Comparative distribution of the alpha 1(IV), alpha 5(IV), and alpha 6(IV) collagen chains in normal human adult and fetal tissues and in kidneys from X-linked Alport syndrome patients.. J Clin Invest 1995; 96 : 1948-57.

6.

Gunwar S, Ballester F, Noelken ME, Sado Y, Ninomiya Y, Hudson BG. Glomerular basement membrane. Identification of a novel disulfide-cross-linked network of alpha3, alpha4, and alpha5 chains of type IV collagen and its implications for the pathogenesis of Alport syndrome. J Biol Chem 1998; 273 : 8767-75.

7.

Tryggvason K, Patrakka J. Thin basement membrane nephropathy. J Am Soc Nephrol 2006; 17 : 813-22.

8.

Buzza M, Wang YY, Dagher H, Babon J, Cotton RG, Powell H, et al. COL4A4 mutation in thin basement membrane disease previously described in Alport syndrome. Kidney Int 2001; 60 : 480-3.

9.

Mochizuki T, Lemmink HH, Mariyama M, Antignac C, Gubler MC, Pirson Y, et al. Identification of mutations in the alpha 3(IV) and alpha 4(IV) collagen genes in autosomal recessive Alport syndrome. Nat Genet 1994; 8 : 77-81.

10. Ozen S, Ertoy D, Heidet L, Cohen-Solal L, Ozen H, Besbas N, et al. Benign familial hematuria associated with a novel COL4A4 mutation. Pediatr Nephrol 2001; 16 : 874-7.

BAEK et al: IDENTIFICATION OF NOVEL VARIANTS IN THE COL4A4 GENE IN KOREANS 11. Badenas C, Praga M, Tazón B, Heidet L, Arrondel C, Armengol A, et al. Mutations in the COL4A4 and COL4A3 genes cause familial benign hematuria. J Am Soc Nephrol 2002; 13 : 1248-54. 12. Longo I, Porcedda P, Mari F, Giachino D, Meloni I, Deplano C, et al. COL4A3/COL4A4 mutations: from familial hematuria to autosomal-dominant or recessive Alport syndrome. Kidney Int 2002; 61 : 1947-56. 13. Buzza M, Dagher H, Wang YY, Wilson D, Babon JJ, Cotton RG, et al. Mutations in the COL4A4 gene in thin basement membrane disease. Kidney Int 2003; 63 : 447-53. 14. Gross O, Netzer KO, Lambrecht R, Seibold S, Weber M. Novel COL4A4 splice defect and in-frame deletion in a large consanguine family as a genetic link between benign familial

533

haematuria and autosomal Alport syndrome. Nephrol Dial Transplant 2003; 18 : 1122-7. 15. Tazón Vega B, Badenas C, Ars E, Lens X, Milá M, Darnell A, et al. Autosomal recessive Alport's syndrome and benign familial hematuria are collagen type IV diseases. Am J Kidney Dis 2003; 42 : 952-9. 16. Rana K, Tonna S, Wang YY, Sin L, Lin T, Shaw E, et al. Nine novel COL4A3 and COL4A4 mutations and polymorphisms identified in inherited membrane diseases. Pediatr Nephrol 2007; 22 : 652-7. 17. Frascá GM, Onetti-Muda A, Mari 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 protected]