Ninety patients (51 men and 39 women; mean age: 42±10.0 years) diagnosed with HFRS were enrolled in this study. Patients were recruited from 12 districts in Hubei Province, China and were unrelated. All patients were diagnosed during hospitalization from 1986 to 1995 according to standard clinical criteria (Xiong et al., 2011), which mainly include: age≥14 years, febrile time≤4 days, typical HFRS symptoms and signs (e.g., fever, proteinuria, hematuria), and potential exposure history to wild rodents or other HFRS patients. Patient sera were subsequently confirmed to be positive by indirect IgM/IgG enzyme-linked immunosorbent assays (ELISAs) performed in our laboratory, using mixed antigens of HTNV and SEOV. Other kidney disorders, diabetes, cardiovascular and hematologic disease, viral hepatitis, and autoimmune disease were excluded.
The control subjects included 101 healthy, unrelated blood donors (56 men and 45 women; mean age: 39±11.0 years) without a history of HFRS-like disease recruited from hospital. Both patients and controls were of Han Chinese descent. There were no significant differences in the distributions of ages and sexes between patients with HFRS and healthy controls. Written informed consent was obtained from all patients and controls in the study. The Research Ethics Committee of Wuhan University approved this project.
Patients with HFRS were classified into four clinical types (mild, medium, severe, and gravis), according to the diagnostic criteria outlined in Prevention and Treatment Strategy of HFRS (Ministry of Health, People's Republic of China, 1997; Liu et al., 2008b). Criteria for classification of types included severity of toxemic symptoms, blood pressure, extent of hemorrhage, severity of kidney damage (proteinuria occurrence and duration of oliguric period as well as levels of urea nitrogen), and the occurrence and severity of complications. Only 70 patients were included in this analysis, as clinical data for disease classification were not available for the remaining 20 patients.
Blood samples were collected in ethylene diamine tetra acetic acid (EDTA)-anticoagulant tubes and stored at -80 ℃ until use. Total genomic DNA was extracted from 500-1000 μL of blood by standard proteinase K/phenol methods (Ahmad et al., 1995). All DNA samples were quantified using a NANODROP 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and stored at -20 ℃ until use.
SNP genotype data for Han Chinese in Beijing, China (CHB) were obtained from the NCBI dbSNP database (http://www.ncbi.nlm.nih.gov/snp) and HAPMAP database (http://snp.cshl.org/cgi-perl/gbrowse/hapmap27_B36/). A total of five SNPs were selected for genotyping: two for the intron regions of ITGAV (which encodes an α chain integrin subunit, located at chromosome 2) and three for the exon regions of ITGB3 (which encodes a β chain integrin subunit, located at chromosome 17). The two intronic SNPs from the 5′ and 3′ ends of ITGAV were rs3768777 (A/G) and rs3738919 (A/C); and the three exonic SNPs with high allele frequencies from ITGB3 were rs5918 (C/T, exon 10), rs13306487 (A/G, exon 10), and rs5921 (A/G, exon 3). Detailed information of the selected SNPs in this study is given in Table 1.
Gene ITGAV ITGB3 SNP rs3768777 rs3738919 rs5918 rs5921 rs13306487 Location in gene Intron Intron Exon Exon Exon Position 37665539 37730678 4014008 4022879 4023066 Location in mRNA - - 196 1377 1564 Nucleotide base A-G A-G C-T A-G A-G Amino acid mutation - - Pro-Leu Ile-Val Gln-Arg MAF in CHB 0.232 0.058 0.007 0.004 0.050
Table 1. Background information of selected SNPs in this study
TaqMan SNP genotyping assays and Bi-PASA test were employed to amplify the target regions. Genotyping of rs5918 was conducted on DNA specimens according to the TaqMan Genotyping Assay protocol, using 5′ nuclease assays with allele-specific TaqMan probes (Applied Biosystems, Foster City, CA, USA) on a Bio-Rad CFX96 instrument (Bio-Rad, Hercules, CA, USA). The reaction protocol was as follows: 95 ℃ for 10 min; 40 cycles of 92 ℃ for 15 s and 60 ℃ for 1 min; and 72 ℃ for 5 min. Genotyping of rs3768777, rs3738919, rs13306487, and rs5921 was conducted by Bi-PASA test, since the SNP sites were located in single nucleotide mutation-rich regions, which are not easily analyzed by TaqMan SNP genotyping assays. Bi-PASA test was also performed because it involves fewer steps, costs less, and is easier to interpret (Liu et al., 1997). The amplification was carried out according to the following protocol: initial denaturation at 95 ℃ for 3 min; 95 ℃ for 30 s, 30 s touchdown from 68 ℃ to 60 ℃ in 0.8 ℃ steps, followed by 26 cycles at 60 ℃ for 30 s and at 72 ℃ for 30 s; and a final extension at 72 ℃ for 5 min. PCR primers used for rs3768777, rs3738919, rs13306487, and rs5921 SNPs amplification are listed in Table 2.
SNP Primers rs3768777 rs3768777-p 5′-GTTGCTAATGTTCCGCGTTGCA-3′ rs3768777-q 5′-GTAGTAGAAGATGGTCCTATCCACG-3′ rs3768777-a 5′-GGGGGGGGGCTCATCACCCCACCCCCA-3′ rs3768777-b 5′-GGGGGGGGCGTGCTCCTAACGCTAACAT-3′ rs3738919 rs3738919-p 5′-ATTTCCAGGTGGAACTTCTTTTGGA-3′ rs3738919-q 5′-TCACAATTCAGATTTTTGCCACTGG-3′ rs3738919-a 5′-GGGGGGGGGCGACACAAAGGAAATTTAGA-3′ rs3738919-b 5′-GGGGGGGGGCGGTGTGACACTTTACAAAG-3′ rs13306487 rs13306487-p 5′-AAGGCTGAGGAACTCCAGATTG-3′ rs13306487-q 5′-TGTTTCCAGTGGTTGCAGGTAT-3′ rs13306487-a 5′-GGGGGGGGGCCGAATGCAGCCCCCA-3′ rs13306487-b 5′-GGGGGGGGGCACGGGCTGACCCTCCC-3′ rs5921 rs5921-p 5′-AAGGCTGAGGAACTCCAGATTG-3′ rs5921-q 5′-TGTTTCCAGTGGTTGCAGGTAT-3′ rs5921-a 5′-GGGGGGGGGCTCAAGGACAGCCTGATCA-3′ rs5921-b 5′-GGGGGGGGGCATCAAAGGTGACCTGGAC-3′
Table 2. PCR primers used for SNPs amplification
The amplified fragments were readily distinguishable by electrophoresis through a 2% agarose gel. The results obtained for the genotypes were further validated by sequencing analysis of 10 randomly selected samples (data not shown).
Statistical analysis was performed using SPSS 17.0 software. The genotypic frequencies of all SNPs in the different groups were calculated, and their distributions were analyzed by Hardy-Weinberg equilibrium statistics. Genotypic and allelic frequencies between the HFRS and control groups were assessed by χ2 analysis. Genotype and allele distributions between clinical types were analyzed by Mann-Whitney U tests. Differences with P values of less than 0.05 were considered statistically significant.
Blood collection and DNA isolation
SNP selection and primer design
Genotyping of candidate SNPs of integrin αvβ3
All the genotype frequencies of the studied SNPs were subjected to Hardy-Weinberg Equilibrium (HWE) statistics. The observed values of candidate SNP genotype frequencies in each group demonstrated no significant differences compared with the corresponding expected values in each group (P > 0.05). Genotype distributions in each group did not deviate from HWE and were in accordance with genetic equilibrium.
The rs5918 SNP was detected by TaqMan SNP genotyping assays, as described above, and the C/T or T/T genotype was represented in different amplification curves by real-time quantitative PCR (data not shown). As shown in Figure 1, Bi-PASA test produced two fragments for the homozygote and three fragments for heterozygote.
The distributions of genotypes and allelic frequencies of four candidate SNPs of integrin αvβ3 (rs5918, rs3768777, rs3738919, and rs13306487) were comparable between patients with HFRS and controls (all P > 0.05; Table 3). In addition, there was only one genotype of the rs5921 locus (G/G) detected both in healthy individuals and patients with HFRS (Figure 1 and Table 3). There were three genotype variants of rs3768777 (A/G, G/G, and A/A) in the HFRS and control populations; however, the allelic frequencies between groups were equivalent (P > 0.05). For rs5918, rs3738919, and rs13306487, two types of mutant alleles were observed. The majority were homozygous (T/T, C/C, and G/G genotypes, respectively) with similar distributions in patient and control cohorts (all P > 0.05).
SNP Group Genotypes Allele frequency (%) aa ab bb χ2 P a b χ2 P rs5918 Controls 0 1 100 0.467 0.494 1(0.5) 201(99.5) 0.464 0.496 Patients 0 2 88 2(1.1) 178(98.9) rs3768777 Controls 6 35 60 0.354 0.838 47(23.3) 155(76.7) 0.000 0.988 Patients 4 34 52 42(23.3) 138(76.7) rs3738919 Controls 0 14 87 0.328 0.567 14(6.9) 188(93.1) 0.306 0.580 Patients 0 10 80 10(5.6) 170(94.4) rs13306487 Controls 0 6 95 0.721 0.396 6(3.0) 196(97.0) 0.073 0.402 Patients 0 3 87 3(1.7) 177(98.3) rs5921 Controls 0 0 101 - - - - - - Patients 0 0 90
Table 3. Comparison of genotypes and allele frequencies of candidate SNPs between patients with HFRS and controls
We further analyzed the associations between two more highly detectable SNP sites and disease severity of HFRS. Table 4 illustrated the demographic and clinical characteristics of 70 of the patients included in this analysis. The numbers of patients with mild, moderate, severe, and gravis types were 9, 49, 11, and 1, respectively. As shown in Table 5, the distributions of genotypes at rs3768777 and rs3738919 sites among patients with difference clinical types of HFRS did not differ significantly (all P > 0.05), nor did the allele frequencies of a and b.
Clinical features and laboratory data Mild type (n=9) Medium type (n=49) Severe type (n=11) Gravis type (n=1) Age (years)a 25.6±6 34.2±12.9 36.6±18.3 17 Sex (man/women) 7/2 38/11 7/4 1/0 WBC count at admission (×109/L) < 10 4 14 2 ≥10 5 35 9 1 Platelet count at admission (×109/L) ≥50 4 12 1 < 50 5 37 10 1 Maximum serum creatinine at oliguric phase (μmol/L)a 221.6±83.1 434.6±243.0 698.6±308.6 760.4 Patient hospital duration (days)a 10.7±2.2 12.0±4.6 15.6±12.3 2.0 Note: aData are presented as means±SDs
Table 4. Demographic, clinical, and hematochemical characteristics in patients with different clinical types of HFRS
SNP Clinical type Genotypes Allele frequency (%) aa ab bb a b rs3768777 Mild 1 1 7 3(8.6) 15(14.3) Medium 3 19 27 25(71.5) 73(69.5) Severe 0 6 5 6(17.1) 16(15.2) Gravis 0 1 0 1(2.8) 1(1.0) rs3738919 Mild 0 2 7 2(25.0) 16(12.1) Medium 0 4 45 4(50.0) 94(71.2) Severe 0 2 9 2(25.0) 20(15.2) Gravis 0 0 1 0(0.0) 2(1.5)
Table 5. Genotypes and allelic frequencies of rs3768777 and rs3738919 in the integrin αvβ3 gene in patients with different clinical types of HFRS