Although chronic kidney disease (CKD) is recognized as an important risk factor for ischemic stroke, genetic factors underlying predisposition to ischemic stroke in individuals with or without CKD remain largely unknown. The aim of the present study was to identify genetic variants that confer susceptibility to ischemic stroke in individuals with or without CKD in order to allow prediction of genetic risk for such individuals separately. The study population comprised 974 individuals with CKD, including 227 subjects with ischemic stroke and 747 controls, and 3,470 individuals without CKD, including 612 subjects with ischemic stroke and 2,858 controls. The 150 polymorphisms examined in the present study were selected by genome-wide association studies of ischemic stroke and myocardial infarction with the use of the GeneChip Human Mapping 500K Array Set (Affymetrix). In individuals with CKD, an initial Chi-square test revealed that the A→G polymorphism (rs10992119) of the receptor tyrosine kinase-like orphan receptor 2 gene (ROR2) was significantly (false discovery rate for allele frequency, 0.0478) associated with ischemic stroke. Multivariable logistic regression analysis with adjustment for covariates revealed that the A→G polymorphism of ROR2 was significantly (P=0.0100) associated with ischemic stroke (recessive model; odds ratio 1.57; 95% CI 1.12–2.23), with the G allele representing a risk factor for this condition. A stepwise forward selection procedure demonstrated that this polymorphism was a significant (P=0.0095) and independent determinant of ischemic stroke. In individuals without CKD, no polymorphism was significantly related to ischemic stroke. Genotyping for ROR2 may prove informative for assessment of the genetic risk for ischemic stroke in Japanese individuals with CKD. Determination of the genotype for this polymorphism may prove informative for assessment of the genetic risk for ischemic stroke in such individuals.
Ischemic stroke is a multifactorial and polygenic disease, and is strongly influenced by a genetic component (1–3). Although genome-wide association studies have implicated various candidate genes underlying predisposition to ischemic stroke (4–6), the genes that confer susceptibility to this condition remain to be identified definitively.
It is increasingly recognized that chronic kidney disease (CKD) is an independent risk factor for atherosclerotic diseases including ischemic stroke. Individuals with CKD are at increased risk not only for end stage renal disease but also for a poor cardiovascular outcome and premature death (7–9). Furthermore, many uncertainties exist regarding genetic contribution to ischemic stroke in individuals with CKD.
We performed an association study for 150 polymorphisms of 144 candidate genes and ischemic stroke in 4,444 Japanese individuals with or without CKD. The aim of the present study was to identify genetic variants that confer susceptibility to ischemic stroke in individuals with or without CKD in order to allow prediction of genetic risk for such individuals separately.
Materials and methodsStudy population
The study population comprised 4,444 unrelated Japanese individuals (2,113 men, 2,331 women) who either visited outpatient clinics or were admitted to one of the five participating hospitals (Gifu Prefectural General Medical Center, Gifu Prefectural Tajimi Hospital in Gifu Prefecture, Japan; and Hirosaki University Hospital, Reimeikyo Rehabilitation Hospital, and Hirosaki Stroke Center in Aomori Prefecture, Japan) between October 2002 and March 2008, due to various symptoms or for an annual health checkup, or individuals who were recruited to a population-based prospective cohort study of aging and age-related diseases in Nakanojo, Gunma Prefecture, Japan. The study subjects comprised 974 people with CKD (227 subjects with ischemic stroke and 747 controls) and 3,470 people without CKD (612 subjects with ischemic stroke and 2,858 controls). Estimated glomerular filtration rate (eGFR) was calculated with the use of the simplified prediction equation proposed by the Japanese Society of Nephrology and based on that described in the Modification of Diet in Renal Disease study (10): eGFR (ml min−1 1.73 m−2) = 194 x [age (years)]−0.287 x [serum creatinine (mg/dl)]−1.094 (x 0.739 if female). The National Kidney Foundation-Kidney Disease Outcome Quality Initiative guidelines recommend a diagnosis of CKD when eGFR is <60 ml min−1 1.73 m−2 (11), on the basis of which 974 subjects in the present study were diagnosed with CKD. The diagnosis of ischemic stroke was based on the occurrence of a new and abrupt focal neurological deficit, with neurological symptoms and signs persisting for >24 h; it was confirmed by positive findings in computed tomography or magnetic resonance imaging (or both) of the head. The type of stroke was determined according to the Classification of Cerebrovascular Diseases III (12). Individuals with cardiogenic embolic infarction, lacunar infarction alone, transient ischemic attack, moyamoya disease, or cerebral venous sinus thrombosis were excluded from the study, as were those with atrial fibrillation in the absence or presence of valvular heart disease. The 3,605 control subjects were recruited from community-dwelling individuals or the patients who visited outpatient clinics regularly for treatment of various common diseases. They had no history of ischemic or hemorrhagic stroke or other cerebral diseases; of coronary heart disease, aortic aneurysm, or peripheral arterial occlusive disease; or of other atherosclerotic, thrombotic, embolic, or hemorrhagic disorders. The study protocol complied with the Declaration of Helsinki and was approved by the Committees on the Ethics of Human Research of Mie University Graduate School of Medicine, Hirosaki University Graduate School of Medicine, Gifu International Institute of Biotechnology, Tokyo Metropolitan Institute of Gerontology and the participating hospitals. Written informed consent was obtained from each participant.
Selection and genotyping of polymorphisms
Our aim was to identify genetic variants associated with ischemic stroke in the Japanese population with or without CKD in a case-control association study by examining the relations of candidate gene polymorphisms to this condition. Polymorphisms examined in the present study (data not shown) were selected from genome-wide association studies of ischemic stroke and myocardial infarction (P-value for allele frequency <1.0×10−7) with the use of the GeneChip Human Mapping 500K Array Set (Affymetrix, Santa Clara, CA, USA) (13). We have not examined the relation of these polymorphisms to ischemic stroke in the absence or presence of CKD in our previous studies (14–17).
Venous blood (7 ml) was collected in tubes containing 50 mmol/l ethylenediaminetetraacetic acid (disodium salt), the peripheral blood leukocytes were isolated, and genomic DNA was extracted from these cells with a DNA extraction kit (Genomix; Talent, Trieste, Italy). Genotypes of the 150 polymorphisms were determined at G&G Science (Fukushima, Japan) by a method that combines the polymerase chain reaction and sequence-specific oligonucleotide probes with suspension array technology (Luminex, Austin, TX, USA). Primers, probes and other conditions for genotyping of polymorphisms related to ischemic stroke are shown in Table I. Detailed genotyping methodology has been described previously (18).
Statistical analysis
Quantitative data were compared between subjects with ischemic stroke and controls by the unpaired Student's t-test. Categorical data were compared by the Chi-square test. Allele frequencies were estimated by the gene counting method, and the Chi-square test was used to identify departure from Hardy-Weinberg equilibrium. In an initial screen, the genotype distributions (3×2) and allele frequencies (2×2) of each polymorphism were compared between subjects with ischemic stroke and controls by the Chi-square test. Given the multiple comparisons of genotypes, the false discovery rate (FDR) was calculated by the method of Benjamini and Hochberg (19) from the distribution of P-values for allele frequencies of the 150 polymorphisms. Polymorphisms with an FDR of <0.05 were further examined by multivariable logistic regression analysis with adjustment for covariates that differed significantly between subjects with ischemic stroke and controls. Multivariable logistic regression analysis was thus performed with ischemic stroke as a dependent variable and independent variables including age, gender (0, woman; 1, man), serum concentration of creatinine, and the prevalence of hypertension, diabetes mellitus and hypercholesterolemia (0, no history of these conditions; 1, positive history), the genotype of each polymorphism, and the P-value, odds ratio and 95% CI were calculated. Each genotype was assessed according to dominant, recessive and additive genetic models. Additive models included the additive 1 model (heterozygotes vs. wild-type homozygotes) and the additive 2 model (variant homozygotes vs. wild-type homozygotes), which were analyzed simultaneously with a single statistical model. We also performed a stepwise forward selection procedure to examine the effects of genotypes as well as other covariates on ischemic stroke. In the stepwise forward selection procedure, each genotype was examined according to a dominant or recessive model on the basis of statistical significance in the multivariable logistic regression analysis. With the exception of the initial screen by the Chi-square test (FDR <0.05), a P-value of <0.05 was considered statistically significant. Statistical significance was examined by two-sided tests performed with JMP version 5.1 software and JMP Genomics version 3.2 software (SAS Institute, Cary, NC, USA).
Results
The characteristics of the study subjects are documented in Table II. In individuals with CKD, age, the frequency of males, prevalence of hypertension, hypercholesterolemia and diabetes mellitus, as well as systolic and diastolic blood pressure, serum concentrations of total cholesterol, low-density lipoprotein (LDL)-cholesterol and creatinine, fasting plasma glucose level and blood glycosylated hemoglobin content were greater, whereas serum concentration of high-density lipoprotein (HDL)-cholesterol and eGFR were lower in the subjects with ischemic stroke than in the controls. In individuals without CKD, age, the frequency of males, the prevalence of hypertension and diabetes mellitus, as well as systolic and diastolic blood pressure, serum concentrations of LDL-cholesterol and creatinine, fasting plasma glucose level and blood glycosylated hemoglobin content were greater, whereas the serum concentration of HDL-cholesterol was lower in subjects with ischemic stroke than in the controls.
Evaluation of allele frequencies by the Chi-square test revealed that eleven polymorphisms were related (P-value for allele frequency of <0.05) to the prevalence of ischemic stroke in individuals with CKD. Among these polymorphisms, the A→G polymorphism (rs10992119) of the receptor tyrosine kinase-like orphan receptor 2 gene (ROR2) was significantly (FDR for allele frequency of <0.05) associated with the prevalence of ischemic stroke (Table III). The genotype distributions for 11 polymorphisms related to ischemic stroke in individuals with CKD are also documented in Table III. In individuals without CKD, another set of 11 polymorphisms was related (P-value for allele frequency of <0.05) to the prevalence of ischemic stroke, but no polymorphism was significantly (FDR for allele frequency of <0.05) related to ischemic stroke (Table IV). The genotype distributions for 11 polymorphisms related to ischemic stroke in individuals without CKD are also shown in Table IV. In control subjects, the genotype distributions of these polymorphisms with the exception of those of POLR1D and RUVBL2 were in Hardy-Weinberg equilibrium (data not shown).
Multivariable logistic regression analysis with adjustment for age, gender, serum concentration of creatinine, and the prevalence of hypertension, diabetes mellitus and hypercholesterolemia revealed that the A→G polymorphism of ROR2 (recessive and additive 2 models) was significantly (P<0.05) associated with ischemic stroke in individuals with CKD, with the G allele representing a risk factor for this condition (Table V). A stepwise forward selection procedure was performed to examine the effects of the ROR2 genotype as well as of age, gender, serum concentration of creatinine, and the prevalence of hypertension, diabetes mellitus and hypercholesterolemia on ischemic stroke (Table VI). Hypertension, diabetes mellitus, gender, age, ROR2 genotype (recessive model) and hypercholesterolemia, in descending order of statistical significance, were significant (P<0.05) and independent determinants of ischemic stroke in individuals with CKD.
Finally, we examined the relation of the A→G polymorphism of ROR2 to intermediate phenotypes, including systolic and diastolic blood pressure, fasting plasma glucose level, blood glycosylated hemoglobin content, serum concentrations of total cholesterol, triglycerides and HDL-cholesterol, and Body Mass Index (BMI). The Chi-square test did not detect any relation between ROR2 genotype and either of the intermediate phenotypes (data not shown).
Discussion
The main cause of ischemic stroke is atherothrombosis, with the principal and treatable risk factors including hypertension, diabetes mellitus, dyslipidemia and smoking (20). In addition to these conventional risk factors, genetic variants are important in the etiology of ischemic stroke (21). Prediction of the risk for ischemic stroke on the basis of genetic information would be useful for deciding how aggressively to target the clinical risk factors that are currently amenable to treatment. In this study we examined the possible relations of 150 polymorphisms to the prevalence of ischemic stroke in 4,444 Japanese individuals and showed that the A→G polymorphism (rs10992119) in intron 1 of ROR2 was significantly associated with ischemic stroke in individuals with CKD.
ROR2 encodes a receptor tyrosine kinase and type 1 transmembrane protein that exists on the cell surface (22,23). Receptor tyrosine kinases are glycoproteins that play important roles in regulating intracellular signaling pathways that control cell proliferation, differentiation, migration, metabolism and apoptosis (24,25). Human ROR2 is expressed in many tissues during development and plays an important role in developmental morphogenesis, including skeletal morphogenesis and bone and cartilage formation (26). Mutations in ROR2 have been reported to cause either of two genetic skeletal disorders: loss-of-function mutations cause Robinow syndrome whereas gain-of-function mutations cause Brachydactyly type B (27,28). Recently, ROR2 was demonstrated to function as an oncogene and was found to be expressed in gastric cancer with signet ring cell features (29). An increase in ROR2 mRNA expression was observed in the majority of renal cell carcinomas, and protein expression was confirmed at the tumor cell level (30). However, the association between ROR2 and atherosclerotic disease has not been reported. In the present study, we showed that the A→G polymorphism of ROR2 (rs10992119) was significantly associated with the prevalence of ischemic stroke in individuals with CKD, with the G allele representing a risk factor for this condition. Given that the ROR genotype did not relate to intermediate phenotypes and that rs10992119 is located in intron 1, the underlying mechanism remains unclear.
There were several limitations in our study. i) We used eGFR instead of a directly measured GFR to define CKD. We did not have information regarding the underlying renal disease for each subject; ii) it is possible that the polymorphism associated with ischemic stroke in the present study is in linkage disequilibrium with other polymorphisms in the same gene or in other nearby genes that are actually responsible for the development of this condition; iii) given that the association of the ROR2 polymorphism with ischemic stroke in the present study was not replicated in independent subject panels, our study was considered to be hypothesis generating; iv) given that the study population comprised only Japanese individuals, validation of our findings is required in other ethnic groups; and v) the functional relevance of the identified polymorphism of ROR2 with the pathogenesis of ischemic stroke remains to be determined.
In conclusion, our present results suggest that ROR2 may be a susceptibility locus for ischemic stroke in Japanese individuals with CKD, although the functional relevance to this condition was not determined. Determination of the genotype for this polymorphism may prove informative for the assessment of the genetic risk for ischemic stroke in such individuals. Validation of our findings will require their replication with independent subject panels of various ethnic groups.
This work was supported in part by the Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (nos. 18209023, 18018021 and 19659149 to Y.Y.).
ReferencesBakSGaistDSindrupSHSkyttheAChristensenKGenetic liability in stroke: a long-term follow-up study of Danish twinsStroke337697742002Tournier-LasserveENew players in the genetics of strokeN Engl J Med347171117122002TouzéERothwellPMHeritability of ischaemic stroke in women compared with men: a genetic epidemiological studyLancet Neurol61251332007MatarínMBrownWMScholzSA genome-wide genotyping study in patients with ischaemic stroke: initial analysis and data releaseLancet Neurol64144202007KuboMHataJNinomiyaTA nonsynonymous SNP in PRKCH (protein kinase C eta) increases the risk of cerebral infarctionNat Genet392122172007IkramMASeshadriSBisJCGenomewide association studies of strokeN Engl J Med360171817282009IrieFIsoHSairenchiTThe relationships of proteinuria, serum creatinine, glomerular filtration rate with cardiovascular disease mortality in Japanese general populationKidney Int69126412712006YahalomGSchwartzRSchwammenthalYChronic kidney disease and clinical outcome in patients with acute strokeStroke40129613032009MuntnerPHeJHammLLoriaCWheltonPKRenal insufficiency and subsequent death resulting from cardiovascular disease in the United StatesJ Am Soc Nephrol137457532002MatsuoSImaiEHorioMRevised equations for estimated GFR from serum creatinine in JapanAm J Kidney Dis539829922009LeveyASEckardtKUTsukamotoYDefinition and classification of chronic kidney disease: a position statement from Kidney Disease: Improving Global Outcomes (KDIGO)Kidney Int67208921002005A committee established by the Director of the National Institute of Neurological Disorders and Stroke, National Institutes of HealthSpecial Report from the National Institute of Neurological Disorders and Stroke. Classification of Cerebrovascular Diseases IIIStroke216376761990YamadaYFukuNTanakaMIdentification of CELSR1 as a susceptibility gene for ischemic stroke in Japanese individuals by a genome-wide association studyAtherosclerosis2071441492009OguriMKatoKYokoiKAssociation of a polymorphism of BCHE with ischemic stroke in Japanese individuals with chronic kidney diseaseMol Med Rep27797852009YamadaYKatoKOguriMAssociation of genetic variants with atherothrombotic cerebral infarction among Japanese individuals with metabolic syndromeInt J Mol Med218018082008YamadaYMetokiNYoshidaHGenetic factors for ischemic and hemorrhagic stroke in Japanese individualsStroke39221122182008YamadaYMetokiNYoshidaHGenetic risk for ischemic and hemorrhagic strokeArterioscler Thromb Vasc Biol26192019252006ItohYMizukiNShimadaTHigh-throughput DNA typing of HLA-A, -B, -C and -DRB1 loci by a PCR-SSOP-Luminex method in the Japanese populationImmunogenetics577177292005BenjaminiYHochbergYControlling the false discovery rate: a practical and powerful approach to multiple testingJ Royal Stat Soc Ser B572893001995GoldsteinLBAdamsRBeckerKPrimary prevention of ischemic stroke: a statement for healthcare professionals from the Stroke Council of the American Heart AssociationStroke322802992001HumphriesSEMorganLGenetic risk factors for stroke and carotid atherosclerosis: insights into pathophysiology from candidate gene approachesLancet Neurol32272352004MasiakowskiPCarrollRDA novel family of cell surface receptors with tyrosine kinase-like domainJ Biol Chem26726181261901992ForresterWCThe Ror receptor tyrosine kinase familyCell Mol Life Sci5983962002StommelJMKimmelmanACYingHCoactivation of receptor tyrosine kinases affects the response of tumor cells to targeted therapiesScience3182872902007SchlessingerJCell signaling by receptor tyrosine kinasesCell1032112252000YodaAOishiIMinamiYExpression and function of the Ror-family receptor tyrosine kinases during development: lessons from genetic analyses of nematodes, mice and humansJ Recept Signal Transduct Res231152003AfzalARRajabAFenskeCDRecessive Robinow syndrome, allelic to dominant brachydactyly type B, is caused by mutation of ROR2Nat Genet254194222000OldridgeMFortunaAMMaringaMDominant mutations in ROR2, encoding an orphan receptor tyrosine kinase, cause brachydactyly type BNat Genet242752782000KatohMKatohMComparative genomics on ROR1 and ROR2 orthologsOncol Rep14138113842005WrightTMBrannonARGordanJDRor2, a developmentally regulated kinase, promotes tumor growth potential in renal cell carcinomaOncogene28251325232009Tables
Primers, probes and conditions for genotyping of polymorphisms related (P-value for allele frequency of <0.05) to ischemic stroke by the Chi-square test.
Gene
SNP
Sense primer (5′→3′)
Antisense primer (5′→3′)
Probe 1 (5′→3′)
Probe 2 (5′→3′)
Annealing (°C)
Cycles
Individuals with CKD
ROR2
A→G (rs10992119)
TGCAATGCACCGAGGAGCAAGT
CTTGCCCTGGGGCATCTGAAG
CAGCGACTAAATCAGCCACGC
GTGGCGTGGCCGATTTAGTC
60
50
SORCS2
C→T (rs17828052)
AGGCTCTCCTGTATCTACAGATG
CACCAGGCAAGCAAAGAGAGGTA
TGACTTACTTTCTTCGAGCATTT
AAACACTTAAATGCTCAAAGAAAGT
60
50
LLGL2
A→G (rs1671021)
GCTCCTGGCCTCACCTTGCG
GCTGCTCTACAAACTCAGCACTG
CTGGGCACTGAAGTTCTCGTT
CCAACGAGAACCTCAGTGCC
60
50
CELSR1
A→C (rs9615362)
TGGAAACCTAGTTTGGTTAAGTGTT
CACCAGGGAGCCACACATGTC
AGGCAGGTGGTGTTATCCCA
CATTGTCTGGGATACCACCAC
60
50
CDH4
A→G (rs11698886)
AGTGTGTCAATGTCCCATCCTTG
TCCCCAGAGCCTGAGCTCAGC
GGAAGCCTCAACATTCTCTCTG
GGCAGAGAGAACGTTGAGGC
60
50
RAB6C
C→G (rs6719989)
GCTGGGGAAGCCGAAGCGC
GACGGAGGGTGGCGGAGCC
CGCAGATGTATCCGGGATCT
GCAGATGTATCCCGGATCTCT
60
50
PPP1R12B
G→T (rs930734)
ACTCCCCATGGGGTGGCTCG
CTTGGTCTTAGAGGCGTCCAGT
CTCCTACAGATGCGCCTTTGA
AGGGTCAAAGGCTCATCTGTA
60
50
CCDC86
G→T (rs480081)
GGGCTGCTTCTTGGAGAATGAC
CCTTTGCTGGGACGTACTCAGT
CTTGCAGATTTGAACTCCATGT
CCGGGGACATGGATTTCAAAT
60
50
AKAP12
A→G (rs756009)
CGAGGATGACCCAGGCCTCAA
CCACACTTTGCCGGCCTCCAG
AGTGAGGGCAACTTTGCTAA
GAGGGCAACTTCGCTAAATCC
60
50
ZC3H3
A→G (rs7464822)
CCGTGCTCTCTGTTGTTCCAC
CTGCGTCTCCAGCTGACAGTG
TGTTGTCATTAAACATCAGGTAG
TGTTGTCATTAAACACCAGGTAG
60
50
RUVBL2
C→T (rs1062708)
GGACGTGATCACCATCGACAAG
GAGCCCATAGCGTCGTAGTCG
GCAAGATCTCCAAGTTGGGC
GAGCGGCCCAACTTGGAGA
60
50
Individuals without CKD
CELSR1
C→T (rs4044210)
CCATGGCCCTGAGGTCCACG
GCTGGCTGCCCCCAGAGCTC
CCACGAAGGAGATGGTGGTA
TTAACTGTACCACCATCTCCTT
60
50
CELSR1
C→T (rs6007897)
GGAGACGGAGGACTCCAGCTC
CTTGCTGTCGACATCTTTGACAAG
TCTTCATGGATGGCGTCGAAT
CCGCGATTCGACGCCATCC
60
50
PTPRN2
C→T (rs1638021)
CAGCCCTTCCCACCTACCAG
CCCAGGTCTCCCAGCCTCAG
AGCGAACCTTTGAGCTTTGC
CCAGCGGCAAAGTTCAAAGG
60
50
SEMA3F
A→G (rs12632110)
GGTGCTGCACCGTGGATGTGA
CTCCAGATCACTCCTCTACTACA
TGTGAGTCCTTGCACAGTGGT
CATTTCACCACTGCGCAAGGA
60
50
ZNF607
G→T (rs17306508)
GCAGAGGCTCCCGCAGTGAC
TGGACCCCTGGCTCCGGTTC
CCTTTTGGTTCCACCTGAAGA
CCGCCTTCTTCATGTGGAAC
60
50
ITPK1
C→T (rs2295394)
GCCCTGCGGCAGGCACTGG
GGCGTGCTGCCCTGTCTGGT
ATGATGTCGATGCCGAAGAGT
TGATGTCGATGCCAAAGAGTG
60
50
POLR1D
C→T (rs14105)
GGCGGAACCTCGAGCGCGGA
AAAAGAGGGCGATAAGGAACCAG
GAAAGAAGAAAACCCGAAGAAAC
AAAGAAGAAAACCCAAAGAAACAC
60
50
NDST1
C→G (rs2545342)
CCTTTCATGAGCTCTTTCTTAGCT
CATCAGAAACCTCTTTCAAGAATGC
CAGAGGATCAAGCAATAATCAG
GACAGAGGATCAAGGAATAATCA
60
50
RUVBL2
C→T (rs753307)
CAGATGGCGGCAGTGAGTGAC
GGATGAAAATTCCCTGCGTCTGA
ATTCTAGGATGAAATCGGAACC
CTAGGATGAAATCAGAACCCTG
60
50
CARD14
C→T (rs8068452)
ATGGTGCCGTGAAACCTCGAAG
CAGGTTCCAGAACCTTCCGCTA
AGTAGAGTCTGCCTCCATATCA
TCGATGATATGGAAGCAGACTC
60
50
RABGAP1L
C→G (rs12078839)
CTTGCTATTTCAGCCATTGCTGAA
TATAGTGGTGGAGCTGGAAATAGA
GGAAGGAACCAACTGGAGAGT
TCTGACTCTCCACTTGGTTCC
60
50
Characteristics of the study subjects.
Characteristic
Subjects with CKD
Subjects without CKD
Ischemic stroke
Controls
P-value
Ischemic stroke
Controls
P-value
No. of subjects
227
747
612
2858
Age (years)
72.5±7.52
70.5±9.37
0.0010
68.5±10.4
64.6±11.3
<0.0001
Gender (males/females, %)
68.7/31.3
48.5/51.5
<0.0001
55.9/44.1
43.8/56.2
<0.0001
Body mass index (kg/m2)
23.3±3.3
23.6±3.5
0.3000
23.7±3.4
23.3±3.3
0.0899
Current or former smoker (%)
17.2
21.3
0.1784
15.9
18.6
0.1114
Hypertension (%)
85.0
49.4
<0.0001
65.0
37.1
<0.0001
Systolic blood pressure (mmHg)
154±28
138±23
<0.0001
150±27
137±20
<0.0001
Diastolic blood pressure (mmHg)
83±16
79±14
0.0008
85±16
73±12
<0.0001
Hypercholesterolemia (%)
37.0
29.7
0.0384
25.7
23.5
0.2518
Serum total cholesterol (mmol/l)
5.36±1.12
5.16±0.98
0.0251
5.09±1.03
5.12±0.88
0.6227
Serum triglyceride (mmol/l)
1.74±1.14
1.62±1.00
0.1922
1.53±0.82
1.49±1.02
0.3513
Serum HDL-cholesterol (mmol/l)
1.30±0.39
1.45±0.40
<0.0001
1.25±0.37
1.50±0.39
<0.0001
Serum LDL-cholesterol (mmol/l)
3.29±0.93
2.97±0.87
<0.0001
3.16±0.91
2.94±0.80
0.0002
Diabetes mellitus (%)
48.5
20.1
<0.0001
34.0
12.3
<0.0001
Fasting plasma glucose (mmol/l)
7.21±2.82
6.57±2.91
0.0058
7.26±2.90
6.39±2.59
<0.0001
Glycosylated hemoglobin (%)
6.08±1.40
5.55±1.23
0.0006
6.03±1.49
5.55±1.30
<0.0001
Serum creatinine (μmol/l)
96.9±89.0
81.5±60.4
0.0153
54.3±10.8
52.0±10.3
0.0003
eGFR (ml min−1 1.73 m−2)
47.5±10.8
50.8±8.9
<0.0001
77.8±15.1
78.5±15.9
0.4580
Quantitative data are the mean ± SD. Hypertension: systolic blood pressure of ≥140 mmHg, diastolic blood pressure of ≥90 mmHg, or taking antihypertensive medication. Hypercholesterolemia: serum total cholesterol of ≥5.72 mmol/l or taking lipid-lowering medication. Diabetes mellitus: fasting blood glucose of ≥6.93 mmol/l, glycosylated hemoglobin (hemoglobin A1c) content of ≥6.5%, or taking antidiabetes medication. HDL, high-density lipoprotein; LDL, low-density lipoprotein; eGFR, estimated glomerular filtration rate.
Polymorphisms related (P-value for allele frequency of <0.05) to ischemic stroke in subjects with CKD as determined by the Chi-square test.
Gene Symbol
Polymorphism
dbSNP
Ischemic stroke (%)
Controls (%)
P-value (genotype)
P-value (allele)
FDR (allele)
ROR2
A→G
rs10992119
0.0027
0.0006
0.0478
AA
6 (2.7)
52 (7.0)
AG
70 (31.1)
284 (38.3)
GG
149 (66.2)
406 (54.7)
SORCS2
C→T
rs17828052
0.0010
0.0056
0.2255
CC
179 (79.2)
625 (84.9)
CT
40 (17.7)
108 (14.7)
TT
7 (3.1)
3 (0.4)
LLGL2
A→G
rs1671021
0.0345
0.0121
0.2694
AA
181 (79.7)
529 (71.0)
AG
42 (18.5)
197 (26.4)
GG
4 (1.8)
19 (2.6)
CELSR1
A→C
rs9615362
0.0128
0.0135
0.2694
AA
0
0
AC
12 (5.3)
16 (2.2)
CC
213 (94.7)
726 (97.8)
CDH4
A→G
rs11698886
0.0634
0.0264
0.3277
AA
24 (10.7)
123 (16.6)
AG
108 (48.0)
355 (47.8)
GG
93 (41.3)
264 (35.6)
RAB6C
C→G
rs6719989
0.0714
0.0268
0.3277
CC
166 (73.8)
594 (80.1)
CG
54 (24.0)
141 (19.0)
GG
5 (2.2)
7 (0.9)
PPP1R12B
G→T
rs930734
0.0612
0.0300
0.3277
GG
130 (57.8)
362 (48.8)
GT
80 (35.5)
319 (43.0)
TT
15 (6.7)
61 (8.2)
CCDC86
G→T
rs480081
0.0864
0.0372
0.3277
GG
28 (12.5)
60 (8.1)
GT
95 (42.2)
302 (40.7)
TT
102 (45.3)
380 (51.2)
AKAP12
A→G
rs756009
0.0379
0.0386
0.3277
AA
0
0
AG
0
14 (1.9)
GG
225 (100)
728 (98.1)
ZC3H3
A→G
rs7464822
0.1331
0.0416
0.3277
AA
2 (0.9)
4 (0.5)
AG
29 (12.8)
63 (8.6)
GG
195 (86.3)
669 (90.9)
RUVBL2
C→T
rs1062708
0.0847
0.0474
0.3277
CC
73 (32.3)
210 (28.6)
CT
116 (51.3)
355 (48.2)
TT
37 (16.4)
171 (23.2)
Polymorphisms related (P-value for allele frequency of <0.05) to ischemic stroke in subjects without CKD as determined by the Chi-square test.
Gene Symbol
Polymorphism
dbSNP
Ischemic stroke (%)
Controls (%)
P-value (genotype)
P-value (allele)
FDR (allele)
CELSR1
C→T
rs4044210
0.0113
0.0121
0.5894
CC
0
0
CT
34 (5.6)
96 (3.4)
TT
577 (94.4)
2723 (96.6)
CELSR1
C→T
rs6007897
0.0115
0.0123
0.5894
CC
0
0
CT
32 (5.2)
89 (3.2)
TT
579 (94.8)
2730 (96.8)
PTPRN2
C→T
rs1638021
0.0463
0.0129
0.5894
CC
289 (47.3)
1479 (52.5)
CT
262 (42.9)
1117 (39.6)
TT
60 (9.8)
223 (7.9)
SEMA3F
A→G
rs12632110
0.0614
0.0192
0.5894
AA
144 (23.5)
571 (20.3)
AG
313 (51.2)
1417 (50.3)
GG
155 (25.3)
829 (29.4)
ZNF607
G→T
rs17306508
0.0832
0.0266
0.5894
GG
517 (84.9)
2498 (88.2)
GT
88 (14.5)
321 (11.3)
TT
4 (0.6)
14 (0.5)
ITPK1
C→T
rs2295394
0.0885
0.0291
0.5894
CC
364 (59.6)
1547 (54.9)
CT
215 (35.2)
1088 (38.6)
TT
32 (5.2)
184 (6.5)
POLR1D
C→T
rs14105
0.0908
0.0323
0.5894
CC
248 (40.7)
1059 (37.4)
CT
280 (46.0)
1305 (46.1)
TT
81 (13.3)
469 (16.5)
NDST1
C→G
rs2545342
0.1085
0.0362
0.5894
CC
5 (0.8)
37 (1.3)
CG
110 (18.0)
597 (21.2)
GG
497 (81.2)
2182 (77.5)
RUVBL2
C→T
rs753307
0.0111
0.0377
0.5894
CC
304 (49.9)
1358 (47.9)
CT
262 (43.0)
1159 (40.9)
TT
43 (7.1)
316 (11.2)
CARD14
C→T
rs8068452
0.0374
0.0447
0.5894
CC
36 (5.9)
257 (9.1)
CT
248 (40.7)
1137 (40.1)
TT
325 (53.4)
1439 (50.8)
RABGAP1L
C→G
rs12078839
0.0144
0.0451
0.5894
CC
480 (78.8)
2308 (81.5)
CG
118 (19.4)
506 (17.8)
GG
11 (1.8)
19 (0.7)
Multivariable logistic regression analysis of a polymorphism associated with ischemic stroke by the Chi-square test in individuals with CKD.
Gene
Polymorphism
Dominant
Recessive
Additive 1
Additive 2
P-value
OR (95% CI)
P-value
OR (95% CI)
P-value
OR (95% CI)
P-value
OR (95% CI)
ROR2
A→G (rs10992119)
0.0546
0.0100
1.57 (1.12–2.23)
0.1791
0.0280
2.76 (1.20–7.53)
OR, odds ratio; CI, confidence interval. Multivariable logistic regression analysis was performed with adjustment for age, gender, serum concentration of creatinine and the prevalence of hypertension, diabetes mellitus and hypercholesterolemia.
Effects of genotypes and other characteristics on ischemic stroke among individuals with CKD as determined by a stepwise forward selection procedure (P<0.05).