Novel single nucleotide polymorphisms of the cytokeratin 19 pseudogene are associated with primary biliary cirrhosis

Hepatology Research 25 (2003) 281
/286 www.elsevier.com/locate/ihepcom

Novel single nucleotide polymorphisms of the cytokeratin 19 pseudogene are associated with primary biliary cirrhosis Yukiko Daimon a, Kiyofumi Yamanishi b, Yoshiki Murakami a, Toshihiko Kirishima a, Yoshito Ito a, Masahito Minami a, Takeshi Okanoue a,* a

Third Department of Internal Medicine, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 6028566, Japan b Department of Dermatology, Kyoto Prefectural University of Medicine, Kyoto, Japan Received 27 February 2002; received in revised form 22 August 2002; accepted 8 October 2002

Abstract Primary biliary cirrhosis (PBC) is characterized by chronic inflammation and destruction of intrahepatic bile ducts. However, the pathogenesis of PBC has not been fully delineated. We examined whether patients with PBC harbor genomic mutations of the cytokeratin 19 (CK19 ) gene since that gene is specifically expressed in biliary epithelial cells. Thirty-six patients with PBC, 26 patients with other liver diseases, and 36 healthy volunteers were enrolled in this study, but there were no significant differences in the genomic sequence of the CK19 gene between those groups. On the other hand, novel single nucleotide polymorphisms (SNPs) of the CK19 pseudogene, C341T, T524G and A754G, were frequently detected in PBC patients. These results suggest that those novel SNPs of the CK19 pseudogene may be associated with PBC and may prove useful for predicting susceptibility to PBC. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Cytokeratin 19; CK19 pseudogene; Primary biliary cirrhosis; Single nucleotide polymorphism

1. Introduction Primary biliary cirrhosis (PBC) is characterized by the destruction of intra-hepatic bile ducts that ultimately leads to liver cirrhosis and death. However, the molecular mechanism(s) of PBC

Abbreviations: PBC, primary biliary cirrhosis; CK, cytokeratin; SNP, single nucleotide polymorphism. * Corresponding author. Tel.: /81-75-251-5519; fax: /8175-251-0710. E-mail address: [email protected] (T. Okanoue).

remains unknown, although autoimmune processes may play an important role in its pathogenesis [1,2]. The familial clustering of PBC suggests that genetic backgrounds are involved in susceptibility to PBC [3
/5]. Indeed, the major histocompatibility complex regions C4B*2, HLA DRB1*0801 (DRB1*0803 in Japan), and DPB1*0301 (DPB1*0501 in Japan) have been identified as susceptibility alleles for PBC [6
/12]. Cytokeratins (CKs) polymerize to form 10-nm intermediate filaments which function as a cellular cytoskeleton in epithelial cells. CKs are classified into more than 20 types by virtue of their distinct

1386-6346/03/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. doi:10.1016/S1386-6346(02)00280-2

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isoelectric points and molecular weights [13]. Mutations in 13 of those CK types have been identified in epidermal, oral, and ocular diseases [14
/16]. CK8 and CK18 are expressed in normal liver parenchymal cells, whereas cytokeratin 19 (CK19 ) is expressed in biliary epithelial cells [13,17]. Transgenic mice that over-express a mutant CK18 gene show mild chronic hepatitis, fragile hepatocytes, and marked susceptibility to drug-induced liver injury [14,18
/20]. Ku et al. have reported that mutations in the CK8 and CK18 genes are associated with cryptogenic liver diseases [14,21]. However, the association of CK19 with diseases of the biliary duct system has not been studied. In this study, we analyzed the CK19 gene and its pseudogene in PBC patients and found that three novel single nucleotide polymorphisms (SNPs) of the CK19 pseudogene are associated with PBC.

2. Materials and methods 2.1. Patients Thirty-six patients with PBC, 26 patients with other liver diseases, and 36 healthy volunteers were enrolled in this study. Those patients who had liver diseases other than PBC were as follows: 16, chronic hepatitis type C; 2, chronic hepatitis type B; 4, fatty liver; 1, idiopathic portal hypertension; 1, TTV-virus infection; 1, primary sclerosing cholangitis; 1, autoimmune hepatitis. Informed consent was obtained from all subjects in this study. All patients were Japanese and there were no significant differences between the groups with respect to age (59.59/12.7 vs. 53.89/15.0 years old) or gender (Table 1). All PBC patients were positive

for anti-mitochondrial antibody (AMA) or were diagnosed based on histology and on biochemical and clinical data. 2.2. DNA extraction from peripheral blood mononuclear cells To 5 ml heparinized whole blood, 45 ml of 1% NH4Cl was added and incubated at 37 8C for 1 h with shaking. The hemolysed solution was centrifuged 3000 /g for 15 min. The precipitated peripheral blood mononuclear cells were subjected to DNA extraction using a ‘G NOMETM kit’ (BIO 101, Joshua Way, CA) according to the manufacturer’s instructions. 2.3. PCR amplification and direct DNA sequencing The CK19 gene and the CK19 pseudogene were amplified by PCR using the primer pairs listed in Table 2. Those primers were designed based on the sequence of Genbank accession No. NM2276 [22] for the CK19 gene, and on M33101 and NT_007158.5 [23] for the CK19 pseudogene. The primer pairs of S5-AS7, S1-AS6, and S6-AS8 were used for PCR of the CK19 pseudogene; the S2, S3, AS1, AS2, AS3, AS4, and AS5 primers were used to amplify the CK19 gene. PCR was performed using a GeneAmp PCR system 9600-R (PerkinElmer Cetus, Norwalk, CT). The PCR conditions are summarized in Table 2. PCR products were purified using a QIAquick Gel Extraction Kit (QIAGEN Inc., Chatsworth, CA), according to the manufacturer’s instructions. BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (Applied Biosystems Inc., Foster City, CA) and a PRIZM 377XL DNA sequencer (Applied Biosys-

Table 1 Baseline of patients and controls in this study

Male: female Age (total) Age (female)

PBC patients (n /36)

Other liver disease patients (n/26)

Healthy controls (n/36)

5:31 59.59/12.7a (34
/84 y.o.) 58.99/13.1

8:18 57.29/10.7a (32
/83 y.o.) 57.69/12.2

5:31 51.29/15.0a (29
/79 y.o.) 54.29/14.0

No significant differences were noted in their ages among three groups. a The numbers shown are mean9/S.D.

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Table 2 The list of all primers used and PCR amplification conditions Sense primer

S1 S2

Antisense primer

S4

GGG GGT TGC TCC GTC CGT GC GTG CTC CGC CTC GCC ATG ACT TCC TAC AGC TGA GAC GGA ACA GGC TCT GCG CAT GAG CGT ACC TGG AGA TGC AGA TCG AA

S5 S6

CAG GGT CGC TCC ATC CGT CG GCA GAT GAC TTC CGA ACC AA

S3

Primer pair S1-AS1 Sl-AS2 S2-AS3 S2-AS4 S3-AS5 S4-AS6 S5-AS7 S1-AS6 S6-AS8

PCR condition (1) (1) (2) (2) (2) (3) (3) (1) (1)

AS1 AS2 AS3 AS4 AS5 AS6 AS7 AS8

GTC GTT GAG GTT CTG CAT GG CTG CAT CTC CAG GTC GGT CC TGT CCC GCA GGT CCT GGA TGG TCG TGT AGT CAG GTA GGC CAG CTC TTC CTT CAG GCC TTC CAC TCA GGA TCT TGG CGA GAT CGG TGC CCG TCC CTT CCT TCC CAT CCC TC CTG AGC CGA ATC CAC CTC CC TTC ATG CTC AGC CGT GAC TG

PCR product 2
/299 2
/641 18
/450 18
/674 539
/783 628
/1306 89
/830 89
/1381 593
/1005

CK19 CK19 CK19 CK19 CK19 CK19 CK19 pseudogene CK19 pseudogene CK19 pseudogene

PCR condition: (1) 94 8C 2 min, (94 8C l min, 55 8C 1 min, 72 8C 3 min) 35 cycle, 72 8C 10 min; (2) 94 8C 2 min, (94 8C 1 min, 65 8C 1 min, 72 8C 3 min) 35 cycle, 72 8C 10 min; (3) 94 8C 2 min, (94 8C 1 min, 57 8C 1 min, 72 8C 3 min) 35 cycle, 72 8C 10 min.

tems Inc.) were used for direct sequencing of the PCR products. 2.4. Statistical analysis Data were analyzed using Wilcoxon’s twosample test, x2-test, t -test, and Fisher’s test.

3. Results To elucidate whether the genotypes of the CK19 gene and the CK19 pseudogene are associated with PBC, we analyzed the genomic sequence of those genes in 36 Japanese patients with PBC and in 62 Japanese individuals without PBC. We examined the genomic sequence of the CK19 gene in those samples, but no mutations or SNPs were found. Because the CK19 pseudogene is highly homologous to the CK19 gene, we designed PCR primers suitable to amplify the CK19 pseudogene

specifically. In the CK19 pseudogene, variations of C341T, T524G and A754G have been found. In the prevalence of 341TT, there was no significant difference between PBC patients and non-PBC subjects but there was a 3.1-fold increase in the frequency of 341 TT in PBC as compared to non-PBC subjects (13.9 vs. 4.8%). In the prevalence of 524GG, there was no significant difference between PBC patients and non-PBC subjects, but there was a 4.3-fold increase in the frequency of 524GG in PBC as compared to non-PBC subjects (13.9 vs. 3.2%). The frequency of 524TT was significantly lower in PBC patients as compared to non-PBC subjects (P /0.0376). The frequency of 754GG was significantly higher in PBC patients (P /0.0163) and the frequency of 754AA was significantly lower in PBC patients as compared to non-PBC subjects (P /0.0376) (Table 3). The frequency of the allelic C341T transition was 14% in PBC patients, whereas it was 5% in subjects without PBC (P/0.0320 in two-tailed

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Fisher’s exact test). Similarly, the frequency of the allelic T524G transversion was 28% in PBC patients, whereas it was 12% in subjects without PBC (P /0.0071). The frequency of the allelic A754G transition was 26% in PBC patients, whereas it was 10% in subjects without PBC (P /0.0043) (Table 4). The relative risk of carriers with an allelic variation of either A754G or T524G to develop PBC was estimated to be 1.99 and the relative risk of carriers with an allelic variation of either C341T to develop PBC was estimated to be 2.87 (Table 5).

4. Discussion CK19 is expressed predominantly in epithelial cells, but not in hematopoietic or lymphatic cells. In the liver, CK19 is expressed only in biliary epithelial cells [13,14]. Since PBC is characterized by the damage of intrahepatic bile ducts, we assumed that mutations in the CK19 gene might be a predisposition to PBC. However, neither polymorphisms nor mutations of the CK19 gene were found in PBC patients. On the other hand, we identified several novel SNPs in the CK19 pseudogene. Moreover, the allelic substitution of C341T, T524G or A754G of the CK19 pseudogene is significantly frequent in PBC patients. These Table 3 The association between the polymorphism of CK19 pseudogene and PBC No. of patients

P

PBC patients (n/36)

Subjects without PBC (n/62)

341 CC 341 CT 341 TT

31 (86%) 0 (0%) 5 (14%)

59 (95%) 0 (0%) 3 (5%)

0.1390
/ 0.1390

524 TT 524 TG 524 GG

21 (58%) 10 (28%) 5 (14%)

49 (79%) 11 (18%) 2 (3%)

0.0376 0.3085 0.0946

754 AA 754 AG 754 GG

21 (58%) 11 (31%) 4 (11%)

49 (79%) 13 (21%) 0 (0%)

0.0376 0.3338 0.0163

results suggest that those polymorphisms of the CK19 pseudogene might be associated with PBC. Previous studies indicated the association of DRB1*0803 with PBC. The gene frequency of DRB1*0803 in PBC vs. controls was 16.7 vs. 9.3% in Fukuoka 24.8 vs. 8.6% in Kanazawa [25] and 35.5 vs. 7.4% in Kouchi [7]. The frequency of those allelic substitutions in the CK19 pseudogene is almost the same as those of the HLA DR8 gene in PBC [8,9]. Both of CK19 pseudogene and HLA DR gene are located on chromosome 6. But the former located on long arm and the latter located on short arm, so SNPs of CK19 pseudogene was considered to be independent factor from HLA DR in pathogenesis of PBC. PBC has a preponderance of middle-aged female, but there are no significant differences in age and gender between subjects with SNPs and subjects without SNPs. There were no significant differences in staging of PBC or positive ratio of AMA between subjects with SNPs and subjects without SNPs. Pseudogenes are believed to arise from the integration of reverse transcripts of RNAs into the genome at an early stage of evolution [23,26]. Those pseudogenes are homologous to their source genes, but contain nucleotide changes that prevent the production of functional gene products [29]. As a result, pseudogenes have no coding potential. Indeed, we detected no CK19 pseudogene transcripts in liver tissues even using reverse transcription-PCR. Recently, the identification of SNPs throughout the human genome has focused not only on coding regions (cSNPs) [28,29], but also on non-coding regions [27,30,32]. Some groups have shown that polymorphisms in non-coding regions, including pseudogenes and introns, are associated with some diseases, such as lung cancer, prostate cancer, multiple myeloma, epidermal diseases, and dementia [32
/37]. It is unlikely that the CK19 pseudogene is directly involved in the pathogenesis of PBC, because no transcripts of the pseudogene were identified in liver tissues. Instead, polymorphisms of the CK19 pseudogene C341T, T524G and A754G might be linked to the genetic disequilibrium of an adjacent possible PBC sensi-

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Table 4 The association between the polymorphism of CK19 pseudogene and PBC No. of alleles

P

PBC patients (n/36; 72 alleles)

Subjects without PBC (n/62; 124 alleles)

341 C 341 T

62 (86%) 10 (14%)

118 (95%) 6 (5%)

0.03020

524 T 524 G

52 (72%) 20 (28%)

109 (88%) 15 (12%)

0.0071

754 A 754 G

53 (74%) 19 (26%)

111 (90%) 13 (10%)

0.0047

Table 5 Relative risk of CK19 pseudogene in comparison with controls and PBC patients Marker

Relative risk

‘T’ at position 341 ‘G’ at position 524 ‘G’ at position 754

2.87 1.99 1.99

[8]

[9]

[10]

tivity gene. Such variations in the CK19 pseudogene may be useful to predict PBC susceptibility in individuals and may lead to the identification of possible PBC sensitivity gene(s).

[11]

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