Introduction
The incidence of type 2 diabetes mellitus (DM) is
rapidly increasing and has become one of the most common chronic
diseases worldwide (1). According
to recent estimates, the prevalence of type 2 DM will increase to
439 million adults (7.7%) by 2030 (2). Asia in particular is a major site of
emerging epidemics, mainly due to the transition in nutritional
habits and changes in lifestyle (2). Since type 2 DM is associated with an
increased incidence of cardiovascular disease and long-term
mortality, it imposes a significant economic burden on healthcare
wordwide (3,4). Furthermore, the contribution of
intensive lifestyle intervention or glycemic control to the
reduction of cardiovascular mortality remain controversial
(5,6). Therefore, aggressive disease
prevention and early detection should be a global strategy
priority.
Family history is one of the major risk factors for
type 2 DM (7), suggesting that
genetic factors significantly contribute to the development and
progression of type 2 DM (8). To
date, genome-wide association studies (GWASs) have identified
various loci and ~40 genes associated with predisposition to type 2
DM (9–13); however, the genes that contribute to
genetic susceptibility to type 2 DM in Japanese individuals have
yet to be definitively identified.
We previously demonstrated that the α-kinase 1 gene
(ALPK1) is a susceptibility locus for chronic kidney disease
in Japanese individuals with DM by a GWAS (14). Although genetic variants of
ALPK1 have been associated with chronic kidney disease in
individuals with DM, whether ALPK1 is a susceptibility locus
for DM has not been elucidated. Thus, we conducted an association
study for the rs2074388 (A→G, Asp565Gly) or rs2074379 (A→G,
Ile732Met) variants of ALPK1 and type 2 DM in
community-dwelling Japanese individuals, in order to provide a
basis for the personalized prevention of this disease.
Materials and methods
Study population
The study population comprised 5,959
community-dwelling individuals (495 subjects with type 2 DM and
5,464 controls) who were recruited to a population-based cohort
study in Inabe, Mie, Japan, between 2010 and 2012. DM was defined
as a fasting plasma glucose level of ≥6.93 mmol/l (126 mg/dl), a
blood glycosylated hemoglobin (hemoglobin A1c) content
of ≥6.9%, or administration of antidiabetic medication. Type 2 DM
was defined according to the criteria of the World Health
Organization previously described (15,16).
Individuals with type 1 DM, maturity-onset diabetes of the young,
DM associated with mitochondrial diseases or single gene disorders,
pancreatic diseases including severe pancreatitis and pancreatic
tumors, other metabolic or endocrinologic diseases, or severe liver
and renal dysfunction, were excluded from the study. Individuals on
medication that may cause secondary DM were also excluded. The
control individuals had a fasting plasma glucose level of <6.05
mmol/l (110 mg/dl) and a blood hemoglobin A1c content of
<6.2% and had no history of DM or of receiving antidiabetic
medication.
The study protocol complied with the Declaration of
Helsinki and was approved by the Human Research Ethics Committees
of Mie University Graduate School of Medicine and Inabe General
Hospital. Written informed consent was obtained from each
subject.
Genotyping of polymorphisms
Venous blood (5 ml) was collected into tubes
containing 50 mmol/l ethylenediaminetetraacetic acid (disodium
salt), peripheral blood leukocytes were isolated and genomic DNA
was extracted from these cells with the SMITEST EX-R&D DNA
extraction kit (Medical and Biological Laboratories, Co., Ltd,
Nagoya, Japan). The genotypes of rs2074388 and rs2074379 variants
of ALPK1 were determined at G&G Science Co., Ltd.
(Fukushima, Japan) by the multiplex bead-based Luminex assay, a
method that combines polymerase chain reaction (PCR) and
sequence-specific oligonucleotide probes with suspension array
technology (Luminex, Austin, TX, USA). Genotyping involved PCR
amplification, hybridization, streptavidin-phycoerythrin reaction
and measurement of fluorescence. The genotyping methodology was
previously described in detail (14,17).
Statistical analysis
Quantitative data were compared between subjects
with type 2 DM and controls using the unpaired Student’s t-test and
categorical data were compared using the Chi-square test. The
allele frequencies were estimated with the gene-counting method and
the Chi-square test was used to identify departures from
Hardy-Weinberg equilibrium. Genotype distributions and allele
frequencies of the rs2074388 and rs2074379 variants of ALPK1
were compared between subjects with type 2 DM and controls using
the Chi-square test. A multivariable logistic regression analysis
was performed with type 2 DM as a dependent variable and
independent variables including age, gender (0, female; 1, male),
body mass index, smoking status (0, non-smoker; 1, current or
former smoker) and ALPK1 genotype; the P-values, odds ratios
and 95% confidence intervals were calculated. The ALPK1
genotype was assessed according to dominant (0, wild-type
homozygote; 1, heterozygote and variant homozygote) and recessive
(0, wild-type homozygote and heterozygote; 1, variant homozygote)
genetic models. We investigated the linkage disequilibrium between
the rs2074388 and rs2074379 variants of ALPK1 and the
association of the haplotypes of these polymorphisms to type 2 DM.
P<0.05 was considered to indicate statistically significant
differences. Statistical significance was assessed using two-sided
tests performed with JMP Genomics software, version 6.0 (SAS
Institute, Cary, NC, USA). Linkage disequilibrium and haplotype
analysis of these polymorphisms were performed with SNPAlyze
software, version 6 (Dynacom Co., Ltd., Yokohama, Japan).
Results
Subject characteristics
The baseline characteristics of the subjects are
presented in Table I. Age, the
frequency of male gender, body mass index and the prevalence of
smoking, hypertension, dyslipidemia and chronic kidney disease were
higher among subjects with type 2 DM compared to controls.
 | Table ICharacteristics of subjects with type
2 diabetes mellitus (DM) and controls. |
Table I
Characteristics of subjects with type
2 diabetes mellitus (DM) and controls.
| Characteristics | DM | Controls | P-value |
|---|
| No. of subjects | 495 | 5,464 | |
| Age (years) | 60.2±9.6 | 51.9±12.8 | <0.0001 |
| Gender (male/female,
%) | 74.9/25.1 | 53.6/46.4 | <0.0001 |
| Body mass index
(kg/m2) | 24.8±3.8 | 22.8±3.3 | <0.0001 |
| Current or former
smoker (%) | 48.7 | 38.5 | <0.0001 |
| Hypertension (%) | 58.2 | 25.3 | <0.0001 |
| Systolic blood
pressure (mmHg) | 128±17 | 119±16 | <0.0001 |
| Diastolic blood
pressure (mmHg) | 78±12 | 73±12 | <0.0001 |
| Dyslipidemia (%) | 69.3 | 47.0 | <0.0001 |
| Serum total
cholesterol (mmol/l) | 5.14±0.94 | 5.18±0.84 | 0.2324 |
| Serum triglycerides
(mmol/l) | 1.52±1.09 | 1.23±0.84 | <0.0001 |
| Serum HDL-cholesterol
(mmol/l) | 1.49±0.39 | 1.67±0.44 | <0.0001 |
| Serum LDL-cholesterol
(mmol/l) | 3.25±0.89 | 3.23±0.81 | 0.9922 |
| Chronic kidney
disease (%) | 14.8 | 7.1 | <0.0001 |
| Fasting plasma
glucose (mmol/l) | 7.85±2.43 | 5.34±0.48 | <0.0001 |
| Blood hemoglobin
A1c (%) | 6.6±1.3 | 5.2±0.3 | <0.0001 |
| Serum creatinine
(μmol/l) | 59.6±14.8 | 56.0±13.4 | <0.0001 |
| eGFR (ml
min−1 1.73 m−2) | 77.5±17.8 | 80.0±15.1 | 0.0002 |
Comparison of genotype distributions and
allele frequencies
The comparison of genotype distributions or allele
frequencies using the Chi-square test revealed that the rs2074388
and rs2074379 variants of ALPK1 were significantly
associated with type 2 DM (P<0.05) (Table II). The genotype distributions of
the two polymorphisms were in Hardy-Weinberg equilibrium among
subjects with type 2 DM and controls.
| Table IIComparison of genotype distributions
or allele frequencies of the rs2074388 and rs2074379 variants of
ALPK1 by the Chi-square test between subjects with type 2
diabetes mellitus (DM) and controls. |
Table II
Comparison of genotype distributions
or allele frequencies of the rs2074388 and rs2074379 variants of
ALPK1 by the Chi-square test between subjects with type 2
diabetes mellitus (DM) and controls.
| Genotypes | DMa | Controlsa | P-value
(genotype) | P-value (allele) |
|---|
| rs2074388 | | | 0.0244 | 0.0095 |
| AA | 197 (39.8) | 2517 (46.1) | | |
| AG | 239 (48.3) | 2393 (43.8) | | |
| GG | 59 (11.9) | 554 (10.1) | | |
| Hardy-Weinberg
P-value | 0.2953 | 0.6723 | | |
| rs2074379 | | | 0.0304 | 0.0111 |
| AA | 198 (40.0) | 2516 (46.0) | | |
| AG | 238 (48.1) | 2397 (43.9) | | |
| GG | 59 (11.9) | 551 (10.1) | | |
| Hardy-Weinberg
P-value | 0.3283 | 0.5689 | | |
Multivariable logistic regression
analysis
A multivariable logistic regression analysis with
adjustment for age, gender, body mass index and smoking status
revealed that rs2074388 (dominant model) and rs2074379 (dominant
model) were significantly associated with type 2 DM, with the
G alleles of the two polymorphisms representing risk factors
for this disease (Table III).
| Table IIIMultivariable logistic regression
analysis of the rs2074388 and rs2074379 variants of ALPK1
and type 2 diabetes mellitus with adjustment for age, gender, body
mass index and smoking status. |
Table III
Multivariable logistic regression
analysis of the rs2074388 and rs2074379 variants of ALPK1
and type 2 diabetes mellitus with adjustment for age, gender, body
mass index and smoking status.
| Dominant | Recessive |
|---|
|
|
|
|---|
| Genotypes | P-value | OR (95% CI) | P-value | OR (95% CI) |
|---|
| rs2074388
(A→G) | 0.0051 | 1.32
(1.09–1.61) | 0.1693 | - |
| rs2074379
(A→G) | 0.0058 | 1.32
(1.08–1.60) | 0.1259 | - |
Haplotype analysis
As the rs2074388 and rs2074379 variants of
ALPK1 were in linkage disequilibrium [standard linkage
disequilibrium coefficient (r2)=0.9908, P<0.0001], we
performed a haplotype analysis for these polymorphisms. That
analysis revealed that the frequency of the major haplotype, A
(rs2074388)-A (rs2074379), was significantly lower (P<0.05),
whereas that of the minor haplotype G-G was significantly higher in
subjects with type 2 DM compared to controls (Table IV).
| Table IVAssociation of ALPK1
haplotypes with type 2 diabetes mellitus (DM). |
Table IV
Association of ALPK1
haplotypes with type 2 diabetes mellitus (DM).
| Haplotype | Overall
frequency | Frequency | Chi-square
P-value | Permutation
P-value |
|---|
|
|---|
| DM | Controls |
|---|
| A-A | 0.6754 | 0.6394 | 0.6787 | 0.0115 | 0.015 |
| G-G | 0.3226 | 0.3596 | 0.3193 | 0.0094 | 0.010 |
| G-A | 0.0011 | 0.0010 | 0.0011 | 0.9353 | 0.652 |
| A-G | 0.0008 |
1.6×10−12 | 0.0009 | 0.3407 | 0.358 |
Association of ALPK1 polymorphisms with
plasma glucose and hemoglobin A1c.
Finally, we assessed the association of rs2074388 or
rs2074379 to fasting plasma glucose level or blood hemoglobin
A1c content among all individuals, or individuals not on
antidiabetic medication. There were no significant differences in
the fasting plasma glucose level or blood hemoglobin A1c
content among the of rs2074388 or rs2074379 genotypes (data not
shown).
Discussion
We previously demonstrated that ALPK1 is a
susceptibility locus for chronic kidney disease in individuals with
DM by a GWAS (14). In this study,
we demonstrated that the rs2074388 and rs2074379 variants of
ALPK1 were significantly associated with the prevalence of
type 2 DM in community-dwelling Japanese individuals, with the
G alleles of the two polymorphisms representing risk factors
for this disease.
ALPK1 is a member of a newly discovered protein
kinase family that exhibits no sequence homology to the
conventional protein kinases (18).
It functions in apical transport by phosphorylating myosin-1a and
is a putative candidate for the regulation of intracellular
trafficking processes by phosphorylation (19). ALPK1 may act synergistically with
monosodium urate monohydrate crystals in promoting the production
of proinflammatory cytokines through the activation of nuclear
factor-κB and mitogen-activated protein kinase (ERK1/2 and p38)
signaling in cultured HEK293 cells, suggesting that ALPK1 may
contribute to the inflammatory process associated with the
development of gout (20).
Impaired insulin secretion and increased insulin
resistance are key components of type 2 DM (21). Although the contributions of these
factors to the onset and progression of type 2 DM may differ
between Caucasian and Asian populations, both factors are essential
for the diagnostic and therapeutic strategies of type 2 DM
(22). Recent studies demonstrated
that proinflammatory cytokines (interleukin-1β and tumor necrosis
factor) exert deleterious effects on insulin secretion and insulin
resistance (23,24). In addition, signal pathways
activated by proinflammatory cytokines, such as nuclear factor-κB
signaling, were shown to be responsible for these conditions
(25). As chronic inflammation may
be an important factor in the development of type 2 DM, the effects
of the rs2074388 or rs2074379 variants of ALPK1 on the
acceleration of the inflammatory process may account for their
association with type 2 DM.
Although the rs2074388 and rs2074379 variants of
ALPK1 were significantly associated with the prevalence of
type 2 DM, either polymorphism was not associated with fasting
plasma glucose level or blood hemoglobin A1c content
among all individuals or individuals not on antidiabetic
medication. Although the reason for this discrepancy has not been
fully elucidated, there are possible explanations: i) the number of
subjects with type 2 DM not on antidiabetic medication was limited;
ii) information regarding drug compliance obtained by a
questionnaire was incomplete; and iii) either polymorphism was not
associated with fasting plasma glucose level among control
individuals.
Our study had certain limitations: i) as the results
of the present study were not replicated, validation of our
findings may require their replication with other independent
subject panels or ethnic groups; ii) it is possible that rs2074388
or rs2074379 are in linkage disequilibrium with other polymorphisms
in the same gene or in other nearby genes that are actually
responsible for the development of type 2 DM; iii) although the
rs2074388 and rs2074379 variants of ALPK1 were significantly
associated with the prevalence of type 2 DM, either polymorphism
was not associated with fasting plasma glucose level or blood
hemoglobin A1c content; and iv) the functional relevance
of the rs2074388 or rs2074379 variants of ALPK1 to the
pathogenesis of type 2 DM has not been determined.
In conclusion, our results suggest that ALPK1
is a susceptibility gene for type 2 DM in community-dwelling
Japanese individuals. Determination of the genotypes for the
polymorphisms of ALPK1 may prove informative for the
assessment of the genetic risk for type 2 DM in the Japanese
population.
Acknowledgements
This study was supported by a Grant-in-Aid for
Scientific Research from the Ministry of Education, Culture,
Sports, Science and Technology of Japan (no. 24590746 to Y.Y.) and
by Research Grants from the Japan Health Foundation and Okasan Kato
Culture Promotion Foundation (to Y.Y.).
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