Common variant of ALPK1 is not associated with gout: a replication study

Gout is one of the most kinds of common inflammatory arthritis as a consequence of hyperuricemia. Alpha-protein kinase 1 (ALPK1) gene locates in a gout-susceptibility locus on chromosome 4q21–31, and encodes ALPK1 protein which plays a pivotal role in the phosphorylation of myosin 1. In the previous genetic study of Taiwanese populations, 3 single nucleotide polymorphisms (SNPs), rs11726117, rs231247 and rs231253, in ALPK1 gene were reported to have a significant association with gout. However, no replication study has been performed to confirm this association. Therefore, we first conducted a replication study with clinically defined gout patients in a different population. Linkage disequilibrium (LD) analyzes of the 3 SNPs in ALPK1 revealed that these SNPs are in strong LD in a Japanese population. Among the 3 SNPs of ALPK1, rs11726117 (M861T) is the only missense SNP. Therefore, rs11726117 was genotyped in a Japanese population of 903 clinically defined gout cases and 1,302 controls, and was evaluated for a possible association with gout. The minor allele frequencies of rs11726117 were 0.26 and 0.25 in the case and control groups, respectively. The association analysis has not detected a significant association between rs11726117 and gout susceptibility in a Japanese population (p = 0.44). Because ABCG2, a major causative gene for gout, also locates in the gout-susceptibility locus on chromosome 4q, these findings suggest that among genes in a gout-susceptibility locus, not ALPK1 but ABCG2 could be important as a gout-susceptible gene.

ALPK1 is thought to play a pivotal role in the phosphorylation of myosin 1 and the apical trafficking of raft-associated sucrose-isomaltase [9]. In the previous study of Taiwanese Han and Taiwan aborigines, Ko et al. [8] reported that 3 single nucleotide polymorphisms (SNPs), rs11726117, rs231247 and rs231253, in ALPK1 gene are associated with gout. However, no replication study has been performed to confirm the association between ALPK1 and gout.
In the present study, we therefore investigated the association between gout and ALPK1 with Japanese gout cases and controls.

Study participants
As cases, 903 male Japanese patients with primary gout were collected from the outpatients of Midorigaoka Hospital (Osaka, Japan), Kyoto Industrial Health Association (Kyoto, Japan) and Jikei University Hospital (Tokyo, Japan). Gout diagnoses were obtained according to the criteria established by the American College of Rheumatology [10]. For controls, 1,302 male Japanese individuals were collected from the Japan Multi-Institutional Collaborative Cohort Study (J-MICC Study) [11]. Exclusion criteria for the controls were high serum uric acid (SUA) levels ([7.0 mg/dl) and any gout history. The mean ages with standard deviation of case and control groups were 55.2 ± 12.9 and 52.7 ± 8.4 years old, respectively, and their respective mean body-mass index was 24.7 ± 3.3 and 23.2 ± 2.8 kg/m 2 . In this study, all subjects provided written informed consent. This study was approved by the institutional ethical committees, and all procedures involved in this study were performed in accordance with the Declaration of Helsinki.
The v 2 test was used for association analysis with SPSS v.22.0J (IBM Japan Inc., Tokyo, Japan).

Results
In the previous genetic analysis of the Taiwanese populations by Ko et al. [8], the genotype distributions are very similar among the 3 SNPs (rs11726117, rs231247 and rs231253) of ALPK1 (Table 1). Therefore, we hypothesized that these SNPs are in linkage disequilibrium. To confirm this hypothesis, the HapMap JPT data have been analyzed. According to the hypothesis, the 3 SNPs were in strong linkage disequilibrium (r 2 C 0.99; Table 1) among the Japanese population in HapMap data, indicating that minor alleles of the 3 SNPs exist in one haplotype. Therefore, in this study, rs11726117 was genotyped to reveal its association with gout, because only this SNP is the nonsynonymous mutation (M861T) among these SNPs; rs231247 is a synonymous mutation (R1084R) and rs231253 is in the 3 0 untranslated region (3 0 UTR). Table 2 shows the genotyping result of rs11726117 for 903 gout patients and 1,302 controls. The call rate was 97.4 %. The frequencies of genotypes were in Hardy-Weinberg equilibrium (p = 0.43).
As compared with the control group, the genotype distribution of rs11726117 (C/C, C/T or T/T) in the case group was not significantly different (p = 0.75; Table 2).
The minor allele (T) frequencies of the variant were 0.26 and 0.25 in case and control groups, respectively, indicating that rs11726117 is a common missense mutation. The association analysis has not detected a significant association between rs11726117 and gout susceptibility in the allele frequency mode (p = 0.44; Table 2).

Discussion
ALPK1 gene locates in a gout-susceptibility locus (between microsatellite markers 4DS3243 and 4DS1625) on chromosome 4q21-31 [16]. In the Taiwanese populations, ALPK1 was previously reported to be associated with gout susceptibility [8].
ALPK1 belongs to the alpha-kinase family and plays a role in the phosphorylation of myosin 1 [9]. A recent genome-wide association study (GWAS) revealed the possible relationship between ALPK1 SNPs and chronic kidney disease (CKD) [17]. As hyperuricemia is highly correlated with CKD risk [18,19], together with the renal expression of ALPK1 [17], ALPK1 could be a possible susceptible gene for gout/hyperuricemia. However, the present study detected no significant association between ALPK1 and gout. This may be partly due to the difference of the investigated population. In addition, we previously reported that ABCG2, which also locates in a gout-susceptibility locus on chromosome 4q21-31 ( Fig. 1), is strongly associated with gout [2,20]. Taken together, these findings suggest that among genes in a gout-susceptibility locus, not ALPK1 but ABCG2 is important as a susceptible gene for gout (Fig. 1). Although further studies of ALPK1 are necessary to reveal the relationship between ALPK1 SNPs and gout, our study at least revealed that rs11726117 of ALPK1 is not a strong genetic risk for gout.  Fig. 1 The locations of ALPK1 and ABCG2 in the gout-susceptibility locus. Gout-susceptibility locus was previously identified between D4S3243 and D4S1625 on chromosome 4q21-31. Both ALPK1 and ABCG2 locate in this locus Common variant of ALPK1 is not associated 3