Lack of functional KL-VS polymorphism of the KLOTHO gene in the Korean population

Abstract

The functional variant of the Klotho "KL-VS" stretch, which includes six polymorphisms in linkage disequilibrium, is reportedly associated with healthy aging and longevity in European and American populations. Among Asian populations, this variant has been observed in the Indian population but not in the Iranian population. An association between KL-VS polymorphism and aging has not been reported in Koreans. To investigate whether the KL-VS polymorphism could be associated with healthy aging and longevity in a Korean population, we analyzed genotype and allele frequencies of the KL-VS variant in a large Korean population sample. The KL-VS variant was not found in 874 Korean individuals. Thus, it is not possible to test its association to aging in the East Asian populations.

Keywords
Aging; KLOTHO gene; KL-VS polymorphism; population genetics

Klotho is a member of the glycosidase family 1 and a single-pass type-I transmembrane protein. It contains a signal sequence at the N-terminus and an extracellular domain, which is composed of two internal repeats, KL1 and KL2. These repeats exhibit 20-40% sequence homology to β-glycosidases (Kuro-o et al., 1997). The protein translated from the Klotho gene exists in both secreted and membrane-bound forms (Matsumura et al., 1998). The Klotho gene is expressed primarily in the prostate, placenta, and kidney (Shiraki-Iida et al., 1998) and may play an important role in the regulation of calcium homeostasis (Nabeshima 2002), suppression of insulin and Wnt signaling (Liu et al., 2007), amelioration of vascular endothelial dysfunction, increase of nitric oxide production, and reduction of elevated blood pressure (Saito et al., 2000).

Klotho is an age-regulating protein. KLOTHO-deficient mice exhibit phenotypes resembling premature human aging (Kuro-o et al., 1997). Deletion of the KLOTHO gene in mice leads to premature aging phenotypes including arteriosclerosis, osteopenia, and shortened life span. On the other hand, over-expression of this gene extends the lifespan of transgenic mice by 20-30% (Kurosu et al., 2005).

The human KLOTHO gene is located on chromosome 13q12 and contains five exons. The expression "KL-VS polymorphism or variant" is used to describe a specific haplotype in a block of six SNPs, in perfect linkage disequilibrium. Of these six SNPs, rs9536314 (F352V) and rs9527025 (C370S) result in amino acid substitutions (Arking et al., 2002). KL-VS refers to the V352 and S370 alleles of these SNPs and corresponds to a variant that shows reduced activity. The KL-VS polymorphism may alter the level of secreted Klotho form and the catalytic activities of Klotho protein (Arking et al., 2002; Dubal et al., 2014). The KL-VS variant spans exon 2 and its flanking sequence, and is common in Caucasians (Low et al., 2005; Freathy et al., 2006; Riancho et al., 2007; Novelli et al., 2008; Tsezou et al., 2008; Invidia et al., 2010; Nzietchueng et al., 2011; Tavakkoly-Bazzaz et al., 2011). KL-VS can influence KLOTHO gene expression in vitro, and it has been inconsistently associated with human longevity in European and American populations (Majumdar et al., 2010). An association between KL-VS polymorphism and human longevity in Asian populations is unexplored and unknown. Here, we analyzed the genomic DNA of 874 healthy Koreans to investigate the frequency of the KL-VS variant of the KLOTHO gene and its possible association with human longevity in Koreans.

Participants were 874 healthy Korean controls (418 males and 456 females) consisting of 101 individuals ≤ 40 years of age, 671 individuals 41-79 years of age, and 102 individuals ≥ 80 years of age. The subjects were recruited during routine checkups at the Chuncheon Sacred Heart Hospital. Informed consent was obtained from all individuals. The study was approved by the Ethical Committee of Chonbuk National University. Genomic DNA was extracted from 200 μL whole blood using QIAamp^® DNA blood Mini Kit (QIAGEN, Valencia, CA, USA). Polymerase chain reaction (PCR) was performed with sense primer (5'-AGGCTCATGCCAAAGTCTGG-3') and antisense primer (5'-GTTTCCATGATGAACTTTTTGAGG-3'). After purification by using QIAquick^® Gel Extraction Kit (QIAGEN), the PCR products were directly sequenced with a model 3730 capillary electrophoresis sequencer (Applied Biosystems, Foster City, CA, USA). Statistical analyses were carried out using Statistical Analysis Software (SAS) version 9.3 (SAS Institute, Cary, NC, USA). The genotypes and allele frequencies of the KL-VS polymorphism were compared using the chi-square or Fisher's exact test.

The KL-VS polymorphism was not found in the Korean population sample (Table 1). The genotype and allele frequencies of the KL-VS polymorphism in the Korean population were significantly different from those previously reported in European and American populations. The present data are similar to data from Iranians but significantly different from data from Indian subjects (Table 1).

Table 1
Genotype and allele frequencies of the Klotho KL-VS variant sequence in various populations.

Population	Age, yr	Total	Genotype, n (%)			P-value	Allele, %		P-value	Reference
			WT/WT	WT/ VS	VS/VS		WT	VS
UK Caucasian	29-35	1619	1158 (71.5)	409 (25.3)	52 (3.2)	-	84.2	15.8	-	Freathy et al., 2006
Bohemian Czech	New Born	390	307 (78.7)	73 (18.7)	10 (2.6)	0.016	88.1	11.9	0.006	Arking et al., 2003
	Elderly ≥ 75	415	308 (74.2)	103 (24.8)	4 (0.1)	0.040	86.6	13.4	0.078
Baltimore Caucasian	New Born	420	309 (73.6)	100 (23.8)	11 (2.6)	0.652	85.5	14.5	0.347
	Elderly ≥ 65	723	530 (73.3)	185 (25.6)	8 (1.1)	0.012	86.1	13.9	0.088
Baltimore African American	New Born	226	156 (69.0)	58 (25.7)	12 (5.3)	0.259	81.9	18.1	0.213
	Elderly ≥ 65	242	169 (69.8)	68 (28.1)	5 (2.1)	0.439	83.9	16.1	0.878
American	Mean 54.3	143	95 (66.4)	44 (30.8)	4 (2.8)	0.350	81.8	18.2	0.302	Low et al., 2005
US Caucasian	<35	332	241 (72.6)	85 (25.6)	6 (1.8)	0.390	85.4	14.6	0.425	Novelli et al., 2008
	93-105	708	517 (73.0)	170 (24.0)	21 (3.0)	0.757	85.0	15.0	0.451
Spanish	18-86 (mean 48.0)	438	330 (75.3)	104 (23.8)	4 (0.9)	0.021	87.2	12.8	0.025	Riancho et al., 2007
Greek	Men (mean 61.5), Women (mean 65.8)	383	309 (80.7)	71 (18.4)	3 (0.8)	<0.001	90.0	10.0	<0.001	Tsezou et al., 2008
Italian	Men <66, Women <73	463	348 (75.2)	103 (22.2)	12 (2.6)	0.300	86.3	13.7	0.113	Invidia et al., 2010
	Men 66-88, Women 73-91	300	203 (67.7)	94 (31.3)	3 (1.0)	0.140	83.3	16.7	0.613
	Men >88, Women >91	326	236 (72.4)	80 (24.5)	10 (3.1)	0.950	84.7	15.3	0.746
French	25-88 (mean 57.5)	629	436 (69.3)	172 (27.4)	21 (3.3)	0.579	83.0	17.0	0.340	Nzietchueng et al., 2011
Iranian	Mean 55	53	53 (100)	0 (0)	0 (0)	<0.001	100	0	<0.001	Tavakkoly-Bazzaz et al., 2011
Indian	≤ 40	375	270 (72.0)	99 (26.4)	6 (1.6)	0.237	85.2	14.8	0.479	Majumdar et al., 2010
	>40	199	140 (70.4)	53 (26.6)	6 (3.0)	0.911	83.7	16.3	0.801
Korean	≤ 40 (mean 27.0)	101	101 (100)	0 (0)	0 (0)	<0.001	100	0	<0.001	This study
	40-79 (mean 62.9)	671	671 (100)	0 (0)	0 (0)	<0.001	100	0	<0.001
	≥ 80 (mean 85.8)	102	102 (100)	0 (0)	0 (0)	<0.001	100	0	<0.001

The KL-VS polymorphism was absent in a large Korean population sample. The KL-VS polymorphism is present in Caucasians, Americans, and Indians, but apparently not in Iranian, Korean, and Japanese populations (Majumdar et al., 2010). The differences in the distribution of genotype and allele frequencies of this polymorphism suggest the possibility that the evolutionary distances are closer between Europeans and Americans than between Europeans and East Asians. The results obtained from SNP markers in human populations showed that European populations were closer to the Amerian populations than East Asians (Shriver et al., 2004; Fazeli and Vallian, 2012). In our previous studies, we reported that the genotype frequencies of polymorphisms of certain genes are remarkably different between Koreans and Europeans (Jeong et al., 2011, 2014).

Various polymorphisms of the KLOTHO gene including the KL-VS polymorphism and G-395A, G1110C, C1818T, and C2298T SNPs have been reported (Kawano et al., 2002). Genetic association studies of the KLOTHO gene have been reported in sickle cell anemia, coronary artery disease (CAD), ischemic stroke, type 2 diabetes, hypertension, and hemodialysis (Arking et al., 2003; Friedman et al., 2009; Wang et al., 2010). Among these KLOTHO polymorphisms, genetic association studies have been carried out to demonstrate an association between functional polymorphism of KLOTHO KL-VS and human aging and cognition, because the functional polymorphism of KLOTHO KL-VS was associated with modulation of its activity and trafficking of the protein (Dubal et al., 2014). Some studies reported a positive correlation with longevity, but other studies did not report such an association (Di Bona et al., 2014).

In conclusion, the KL-VS polymorphism of the KLOTHO gene was not found in our large Korean population sample, and hence it does not appear to be an effector of aging and human longevity in Koreans.

Acknowledgement

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2014R1A1A2057943) and the Basic Science Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012R1A1A2003686) and research funds of Chonbuk National University in 2013.

References

• Arking DE, Becker DM, Yanek LR, Fallin D, Judge DP, Moy TF, Becker LC and Dietz HC (2003) KLOTHO allele status and the risk of early-onset occult coronary artery disease. Am J Hum Genet 72:1154-1161.
• Arking DE, Krebsova A, Macek Sr M, Macek Jr M, Arking A, Mian IS, Fried L, Hamosh A, Dey S, McIntosh I, et al. (2002) Association of human aging with a functional variant of klotho. Proc Natl Acad Sci USA 99:856-861.
• Di Bona D, Accardi G, Virruso C, Candore G and Caruso C (2014) Association of Klotho polymorphisms with healthy aging: A systematic review and meta-analysis. Rejuvenation Res 17:212-216.
• Dubal DB, Yokoyama JS, Zhu L, Broestl L, Worden K, Wang D, Sturm VE, Kim D, Klein E, Yu GQ, et al. (2014) Life extension factor klotho enhances cognition. Cell Rep 7:1065-1076.
• Fazeli Z and Vallian S (2012) Phylogenetic relationship analysis of Iranians and other world populations using allele frequencies at 12 polymorphic markers. Mol Biol Rep 39:11187-11199.
• Freathy RM, Weedon MN, Melzer D, Shields B, Hitman GA, Walker M, McCarthy MI, Hattersley AT and Frayling TM (2006) The functional "KL-VS" variant of KLOTHO is not associated with type 2 diabetes in 5028 UK Caucasians. BMC Med Genet 7:e51.
• Friedman DJ, Afkarian M, Tamez H, Bhan I, Isakova T, Wolf M, Ankers E, Ye J, Tonelli M, Zoccali C, et al. (2009) Klotho variants and chronic hemodialysis mortality. J Bone Miner Res 24:1847-1855.
• Invidia L, Salvioli S, Altilia S, Pierini M, Panourgia MP, Monti D, De Rango F, Passarino G and Franceschi C (2010) The frequency of Klotho KL-VS polymorphism in a large Italian population, from young subjects to centenarians, suggests the presence of specific time windows for its effect. Biogerontology 11:67-73.
• Jeong BH, Lee KH, Lee YJ, Yun J, Park YJ, Cho HJ, Kim YH, Cho YS, Choi EK, Carp RI, et al. (2011) Absence of association between two HECTD2 polymorphisms and sporadic Creutzfeldt-Jakob disease. Dement Geriatr Cogn Disord 31:146-151.
• Jeong BH, Kim HJ, Lee KH, Carp RI and Kim YS (2014) RARB and STMN2 polymorphisms are not associated with sporadic Creutzfeldt-Jakob disease (CJD) in the Korean population. Mol Biol Rep 41:2389-2395.
• Kawano K, Ogata N, Chiano M, Molloy H, Kleyn P, Spector TD, Uchida M, Hosoi T, Suzuki T, Orimo H, et al. (2002) Klotho gene polymorphisms associated with bone density of aged postmenopausal women. J Bone Miner Res 17:1744-1751.
• Kuro-o M, Matsumura Y, Aizawa H, Kawaguchi H, Suga T, Utsugi T, Ohyama Y, Kurabayashi M, Kaname T, Kume E, et al. (1997) Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature 390:45-51.
• Kurosu H, Yamamoto M, Clark JD, Pastor JV, Nandi A, Gurnani P, McGuinness OP, Chikuda H, Yamaguchi M, Kawaguchi H, et al. (2005) Suppression of aging in mice by the hormone Klotho. Science 309:1829-1833.
• Liu H, Fergusson MM, Castilho RM, Liu J, Cao L, Chen J, Malide D, Rovira II, Schimel D, Kuo CJ, et al. (2007) Augmented Wnt signaling in a mammalian model of accelerated aging. Science 317:803-806.
• Low AF, O'Donnell CJ, Kathiresan S, Everett B, Chae CU, Shaw SY, Ellinor PT and MacRae CA (2005) Aging syndrome genes and premature coronary artery disease. BMC Med Genet 6:e38.
• Majumdar V, Nagaraja D and Christopher R (2010) Association of the functional KL-VS variant of Klotho gene with early-onset ischemic stroke. Biochem Biophys Res Commun 403:412-416.
• Matsumura Y, Aizawa H, Shiraki-Iida T, Nagai R, Kuro-o M and Nabeshima Y (1998) Identification of the human klotho gene and its two transcripts encoding membrane and secreted klotho protein. Biochem Biophys Res Commun 242:626-630.
• Nabeshima Y (2002) Klotho: A fundamental regulator of aging. Ageing Res Rev 1:627-638.
• Novelli V, Viviani Anselmi C, Roncarati R, Guffanti G, Malovini A, Piluso G and Puca AA (2008) Lack of replication of genetic associations with human longevity. Biogerontology 9:85-92.
• Nzietchueng RE, Shamieh S, Benachour H, Labat C, Herbeth B, Ndiaye NC, Masson C, Visvikis-Siest S and Benetos A (2011) Klotho KL-VS genotype is involved in blood pressure regulation. Clin Chim Acta 412:1773-1777.
• Riancho JA, Valero C, Hernández JL, Ortiz F, Zarrabeitia A, Alonso MA, Peña N, Pascual MA, González-Macías J and Zarrabeitia MT (2007) Association of the F352V variant of the Klotho gene with bone mineral density. Biogerontology 8:121-127.
• Saito Y, Nakamura T, Ohyama Y, Suzuki T, Iida A, Shiraki-Iida T, Kuro-o M, Nabeshima Y, Kurabayashi M and Nagai R (2000) In vivo klotho gene delivery protects against endothelial dysfunction in multiple risk factor syndrome. Biochem Biophys Res Commun 276:767-772.
• Shriver MD, Kennedy GC, Parra EJ, Lawson HA, Sonpar V, Huang J, Akey JM and Jones KW (2004) The genomic distribution of population substructure in four populations using 8,525 autosomal SNPs. Hum Genomics 1:274-286.
• Shiraki-Iida T, Aizawa H, Matsumura Y, Sekine S, Iida A, Anazawa H, Nagai R, Kuro-o M and Nabeshima Y (1998) Structure of the mouse klotho gene and its two transcripts encoding membrane and secreted protein. FEBS Lett 424:6-10.
• Tavakkoly-Bazzaz J, Tabatabaei-Malazy O, Tajmir-Riahi M, Javidi D, Izadi M, Shahrabi-Farahani M, Amiri P and Amoli MM (2011) Absence of kl-vs variant of klotho gene in Iranian cardiac patients (comparison to the world populations). Dis Markers 31:211-214.
• Tsezou A, Furuichi T, Satra M, Makrythanasis P, Ikegawa S and Malizos KN (2008) Association of KLOTHO gene polymorphisms with knee osteoarthritis in Greek population. J Orthop Res 26:1466-1670.
• Wang HL, Xu Q, Wang Z, Zhang YH, Si LY, Li XJ, Yang QH and Xiao H (2010) A potential regulatory single nucleotide polymorphism in the promoter of the Klotho gene may be associated with essential hypertension in the Chinese Han population. Clin Chim Acta 411:386-390.

• Associate Editor: Mara H. Hutz

Publication Dates

• Publication in this collection
  16 June 2016
• Date of issue
  Jul-Sep 2016

History

• Received
  07 July 2015
• Accepted
  04 Dec 2015