Association of atherosclerotic cerebral infarction and human leukocyte antigen-DRB in a North Chinese Han population
Correspondence Address: Source of Support: Jilin Provincial Science & Technology Department (200505169), Conflict of Interest: None DOI: 10.4103/0028-3886.84330
Source of Support: Jilin Provincial Science & Technology Department (200505169), Conflict of Interest: None
Background and Objectives: Evidence from experimental and genetic studies suggest the existence of a potential link between the polymorphisms of human leukocyte antigen class II gene (HLA-DR) and ischemic stroke. This study addressed the association of HLA-DR gene with atherosclerotic cerebral infarction (ACI) in a North Chinese Han population. Material and Methods: The genotyping of HLA-DRB1 was determined by standard techniques based on polymerase chain reaction and sequence-specific oligonucleotides hybridization in a gene chip. Results: The relative risk (RR) of HLA-DRB1 * 04 and HLA-DRB1 * 03 (17) in patients with ACI and their first-degree relatives were significantly higher than those in the control group (RR=2.56 and 18.77, respectively; P <0.05). In contrast, the RR of HLA-DRB1 * 12 was dramatically reduced in patients with ACI in relation to healthy controls (RR=0.17; P <0.01). Conclusions: These data indicate that the polymorphisms in HLA-DRB1 may influence the risk of ACI in the North Han population of China. Further studies are necessary to validate the observation in larger samples.
Keywords: Atherosclerotic cerebral infarction, gene chip, Han nationality, human leukocyte antigen, north china
Stroke is the third leading cause of death in the United States, constituting approximately 700 000 cases each year.  The subtypes of stroke are transient ischemic attacks, cerebral infarction, and hemorrhage. Most strokes are cerebral ischemia and infarction.  The major risk factors for cerebral ischemia and infarction include family history, hypertension, diabetes mellitus, and other risk factors for the development of atherosclerosis such as hypercholesterolemia.  Notably, atherosclerosis is the leading etiologic factor of cerebral ischemia or infarction, especially among people aged 50 years or above.  Atherosclerotic disease accounts for approximately 25% of ischemic strokes.  Atherosclerotic plaque of the stenotic carotid artery is the underlying cause of the majority of ischemic strokes.  Thus, managing the underlying atherosclerosis is important for preventing recurrent stroke or cerebral ischemic events.
Epidemiologic studies suggest a polygenic basis for stroke, , and a large number of candidate gene association studies have attempted to identify the genes involved in stroke.  A meta-analysis of genetic studies in ischemic stroke in 2004 demonstrated that statistically significant associations with ischemic stroke were identified for factor V Leiden Arg506Gln, methylenetetrahydrofolate reductase C677T, prothrombin G20210A, and angiotensin-converting enzyme insertion/deletion.  A recent, multicentric case-control study revealed that the apolipoprotein E ε4 genotype did not predict the risk of ischemic stroke but was associated with severity of subclinical intracranial atherosclerosis in men.  Moreover, immunogenetic profile, such as the alleles of human leukocyte antigen (HLA) HLA-B51, -DRB1*0802, -DQA1*0401, and -DQB1*0402, were found to be associated with a predisposition to idiopathic childhood ischemic stroke.  This finding confirms the existence of a genetic cause for ischemic stroke.
The human major histocompatibility complex is called HLA. The major HLA antigens consist of HLA-A, -B, -C and -DR, -DP, -DQ. HLAs are essential elements for immune function. HLA molecules were identified by their prominent role in transplantation reactions and in the immune responses to various immunogens and infections.  An HLA-related genetic susceptibility has been found in diseases that clearly involve immune defense mechanisms such as allograft rejections, infections, and autoimmune disorders. , Moreover, there is evidence for an HLA association in cancer,  leukemia,  atherosclerosis,  and ischemic disease. ,, In addition, genes within the HLA complex are the proven genetic risk factors in idiopathic childhood ischemic stroke, ,, suggesting that HLA molecules are involved in ischemic stroke.
HLA loci are some of the most genetically variable coding loci in mammals.  Over 100 alleles of HLA loci have been detected in the human population; of these, the most variable are HLA-B and HLA-DRB1.  Moreover, it has been speculated that the HLA-DR and HLA-DP peptide complex delivers the strongest avidity between T-cell receptor, leading to reactive T-cell-mediated immune responses.  Therefore, in this study, we tested whether the specific alleles in the HLA-DR region were associated with atherosclerotic cerebral infarction (ACI) in the Han population in North China by using a case-control study of 143 patients and 131 first-degree relatives from 71 families and 150 ethnicity- and gender-matched healthy controls. The results provide insight into the polymorphisms of HLA-DRB1 in Han population as well as into the role of genetic factors in the development of ACI.
This was a case-controlled study. All subjects were of Han Chinese origin from North China. The 143 patients included in the present study were from 71 families with no consanguinity. All patients were diagnosed to be ACI according to the standard criteria of cerebral vascular disease. Patients with ACI included 79 males and 55 females. The mean age at onset was 51.32 ± 12.51 years. All the 131 first-degree relatives included 59 males and 72 females (15 brothers, 34 sisters, and 82 sons and daughters) and the mean age was 30.12 ± 8.21 years and none of them had history of ACI. The control group included 150 unrelated, North Chinese, healthy Han subjects (85 males and 65 females and mean age, 50.54 ± 13.01 years). Exclusion criteria for control subjects included central nervous system-related diseases, a self-reported history of hypertension, hyperlipidemia, diabetes mellitus, atherosclerotic peripheral arterial disease, and brain tumor. Written informed consent was obtained from all the subjects, and this study was approved by the institution's research ethical committee for Human Subject Research.
DNA extraction and HLA-DRB1 detection by a gene chip assay
A sample of 2.7 ml of whole blood was collected in heparinized tubes. Genomic DNA was extracted from white blood cell fractions using the Qiagen Blood Kit (Qiagen, Chatsworth, CA, USA). The genotyping of HLA-DRB1 was then determined by standard techniques based on polymerase chain reaction (PCR) and sequence-specific oligonucleotides hybridization. Briefly, the PCR amplifiers were hybridized to a gene chip, which detect 19 alleles of HLA-DRB1. The allele of designation of HLA-DRB1 was from the WHO HLA Nomenclature Committee. All the primers for the alleles of HLA-DRB1 were designed and synthesized by Shanghai Gene Research Institutes.
The PCR was carried out in a final volume of 25 μl, which contained 100 μM each dNTPs, 1 mM MgCl 2 , 2.5 units of Taq polymerase (TeleChem, Sunnyvale, CA, USA), 2.5 μl of reaction buffer (100 mM Tris-HCl at pH 9.0, containing 500 mM KCl and 15 mM MgCl 2 ), 100 nM each primers, and 100 ng isolated DNA. The reaction was performed for a total of 35 cycles with the use of a PCR cycler (Telechem). PCR conditions were as follows: preheating for 5 minutes at 95°C, followed by 35 cycles of 30 seconds at 95°C, 30 seconds at 58°C, and 2 minutes at 72°C, and a final extension of 5 minutes at 72°C.
The resultant amplifiers were then hybridized with a gene chip (Shanghai Boxin Genchip Inc., Shanghai, China). In brief, the gene chip membranes were equilibrated with ×2 sodium chloride and sodium citrate at room temperature for 10 minutes. Membrane was prehybridized using prehybridization buffer and by shaking at 35°C for 30 minutes. Membranes were then hybridized with 500 ml of hybridization buffer containing denatured amplimers (15 ml) and by shaking at 48°C for at least 1 hour. The chips were washed two times in washing buffer for 5 minutes at room temperature and at 35°C, respectively. Following this stringent washing, the chips were equilibrated with ×1 phosphate-buffered saline (pH 7.4) containing 0.05% Tween-20 and 0.1% SDS and by shaking at room temperature for 5 minutes twice. The chips were then washed with ddH 2 O and dried at room temperature. The gene chips were scanned by ScanArray4000 (Perkin Elmer, USA). [Figure 1] and [Figuer 2] show the microarray scanning data from two cases with HLA-DRB alleles, respectively.
All statistical analyses were performed with the computer software SPSS12.0 (SPSS Inc., Chicago, IL, USA). The strength of the statistical association between the disease and HLA-DRB alleles was expressed by relative risk (RR) and 95% confident intervals. Statistical significance was examined by the χ2 test. When the P value was less than 0.05, the association with each individual allele was considered to be significant.
The HLA-DRB alleles in atherosclerotic cerebral infarction patients and healthy controls
The frequencies of the HLA-DRB alleles in patients with ACI and control population are shown in [Table 1]. The HLA-DRB1*04 allele showed a significant positive association with the disease (RR=2.56; χ2 =11.847; P=0.0006). Notably, the frequency of HLA-DRB1*03(17) was dramatically higher in patients with ACI than in controls (RR=18.772; χ2 =14.830; P=0.0001) and showed highest risk for ACI when compared with other HLA-DRB alleles. On the other hand, the frequency of HLA-DRB1*12 (RR=0.168; χ2 =44.55; P<0.0001) was significantly lower in patients with ACI than in controls. Thus, the haplotypes HLA-DRB1*04 and HLA-DRB1*03 (17) are positively related to ACI, and haplotype HLA- DRB1*12 is a protective HLA-DRB1 allele for ACI in North Han population of China.
The HLA-DRB alleles in atherosclerotic cerebral infarction patients and their first-degree relatives
Frequencies of the HLA-DRB1 alleles in patients with ACI and their first-degree relatives are shown in [Table 2]. There were no significant differences in the studied HLA-DRB1 alleles between patients with ACI and their first-degree relatives (P>0.05), suggesting that ACI probands and their first-degree relatives display the same susceptibility of HLA-DRB1 alleles.
The HLA-DRB alleles in the first-degree relatives of atherosclerotic cerebral infarction patients and healthy controls
Frequencies of HLA-DRB1 alleles in the first-degree relatives of patients with ACI and healthy controls are shown in [Table 3]. Frequencies of the HLA-DRB1*04 and -DRB1*03 (17) alleles were significantly higher in the first-degree relatives of patients with ACI than in healthy controls (RR=2.496, χ2 =10.725, P=0.001 and RR=26.865, χ2 =21.558, P<0.0001, respectively). Similarly, to patients with ACI, the frequency of HLA-DRB1*12 (RR=0.23; χ2 =25.758; P<0.0001) in the first-degree relatives of patients with ACI was significantly lower than that in controls.
Stroke is a multifactorial disorder in which environmental and genetic factors are implicated in disease susceptibility and severity. ,,, A family history of stroke is considered an important risk factor for stroke.  Studies with twins, siblings, and relatives have provided substantial evidence for heritability of stroke.  Moreover, the incidence of ischemic stroke differs among ethnic groups, which may be attributed to differences in both the distribution and frequency of genetic variants, as well as in environmental factors such as diet, exercise, and other lifestyle aspects. ,,, Therefore, it is important to examine the relationship between gene polymorphisms and stroke in some ethnic groups with high or low incidence of stroke. In the present study, 143 patients and 131 first-degree relatives of ischemic stroke victims from 71 families and 150 ethnicity- and gender-matched healthy controls in the North Chinese Han population were recruited to determine the HLA-DRB1 alleles.
Stroke can be ischemic and hemorrhagic, ischemic stroke being more common. The main cause of ischemic stroke is atherothrombosis, with major risk factors including hypertension, hypercholesterolemia, and diabetes mellitus.  Given that vascular inflammation has been recognized as an important player of atherosclerotic disease, inflammatory genes may play a pivotal role in the pathogenesis of ischemic stroke. Indeed, many genetic epidemiological studies have identified many genes that are related to the prevalence of ischemic stroke, including various proinflammatory genes such as lymphotoxin-alpha (LTA), interleukin 6 (IL6), and arachidonate 5-lipoxygenase-activating protein gene (ALOX5AP). , These genes have recently been identified as a susceptibility locus for coronary heart disease. , Identification of these susceptibility genes will likely contribute to early diagnosis and treatment of ischemic stroke.
HLA complex are the strongest predisposing genetic factors.  HLA is located on the surface of B lymphocytes, macrophages, dendritic cells, and activated T-cells. The HLA complex genes that are primarily involved are most often those encoding the peptide-presenting HLA class I or class II molecules. The class II region of the HLA system contains three distinct gene loci: HLA-DP, HLA-DQ, and HLA-DR.  All loci are presumed to be involved in antigen presentation to CD4 + T lymphocytes, which results in antigen-specific immune responses and thus controls the predisposition for, or protection from, certain immune-associated diseases.  CD4 + T lymphocytes recognize combinations of polymorphic regions that present on DR molecules of distinct specificity.  Moreover, certain specific HLA-DR/DQ alleles have been found to be involved in the pathogenesis of particular autoimmune diseases such as rheumatoid arthritis, type 1 diabetes mellitus, and multiple sclerosis. ,,, It is also remarkable that the same autoimmune disease can be associated in different populations with distinct and partially overlapping sets of HLA-DQ alleles,  suggesting that the variability of HLA-DQ alleles is likely to contribute to the development of some disease conditions in different ethnic populations.
Some case-controlled studies have demonstrated that genes linked to class II alleles, such as HLA-DR, may contribute to the etiologic process of cardiac and cerebral damage. ,, In particular, a gene in HLA-D region has been shown to be comprised of risk genes in ischemic stroke. , Haplotype HLA-DRB1*0802, -DRA1*0401, -DQB1*0402 has especially been identified as a high-risk haplotype for idiopathic childhood ischemic stroke.  We also previously reported that HLA-DR B1*0301 gene may be susceptible for promoting ACI in the population of Northeastern China.  In the present study, a gene-dosing effect of HLA-DRB1*04 and HLA-DRB1*03 (17) is suggested by its high frequency in patients with ACI. In contrast, the low frequency of HLA-DRB1*12 in ACI patients and their first-degree relatives indicates a protective HLD-DRB1 allele for ACI.
Development of atherosclerotic plaques represents a complex interplay of environmental risk factor exposure and a multigenic profile with intermediate phenotypes. Inherited determinants may increase the risk of ACI; however, they are insufficient to induce the disease by themselves. Environmental factors, such as infections, probably contribute to the development of disease. Emerging literature points to several immunologic and genetic susceptibility factors that may provide insight into the atherosclerotic plaque progression in the development of stroke.  In brief, exposure of memory T-lymphocytes to infectious or endogenous antigens may result in rapid clonal expansion of T-cell variable b chain subtypes and stimulate macrophages to release intracellular enzymes, causing localized vascular injury, and initiating atherosclerotic plaque formation. This represents an acquired susceptibility based on previous infectious burden exposure. The effects of infectious agents can further be influenced by an individual's HLA class II molecule profiles, which can affect susceptibility to specific organisms. Moreover, functional polymorphisms of HLA class II genes can predispose patients to a more robust inflammatory expression after risk factor exposure. Taken together, the immunologic and genetic susceptibility factors may interact to initiate and promote the development of atherosclerotic ischemic stroke. However, the nature of the association between HLA-DR and ischemic stroke remains to be elucidated.
In summary, the present immunogenetic data indicate that HLA-DRB1*04 and -DRB1*03 (17) may be genetic susceptibility factors for cerebral ischemic stroke in the North Chinese Han population. Identification of HLA-DRB1 susceptibility genes will thus likely contribute to the prevention, early diagnosis, and treatment of ischemic stroke. However, further analyses are necessary to validate the observation in larger samples.
This work was supported by a grant from Jilin Provincial Science & Technology Department (200505169). The authors have no financial disclosures to declare and no conflicts of interest to report.
[Figure 1], [Figuer 2]
[Table 1], [Table 2], [Table 3]