MICA associations with oral squamous cell carcinoma
M. Ivanova (Sofia, BG)
Major histocompatibility complex class I-related chain A (MICA) is a ligand of the Natural killer group 2, member D (NKG2D) receptor. Recent studies have shown that MICA is up regulated in tumors from epithelial origin, playing a key role in immunological surveillance and different alleles are associated with diseases related to NK activity. The aim of our study was to analyze associations of MICA polymorphism with oral squamous cell carcinoma (OSSC). Twenty seven patients with histologically proven OSSC were included in the study. The majority of patients had G2-G3 tumors according to Anneroth's classification. The control group included healthy subjects from the Bulgarian population. MICA genotyping was performed by a PCR-SSO kit (LABType SSO MICA, OneLambda) and PCR-SBT. Our results showed statistically significant protective association for MICA*12:01 allele (Pc<0.05, OR-0.07), encoding a full length protein. Interestingly this allele had a higher frequency in the healthy Bulgarian population compared to other European populations. With the highest frequency in patients with OSSC was observed MICA*08:01 allele, encoding truncated protein. However the difference with the control group was with a borderline significance (Pc=0.053). Although our data are preliminary considering the small number of patients analyzed, the associations observed support the model that alleles encoding truncated, ectopic and soluble MICA molecules play an important role in OSSC by down regulation of NKG2D on NK and CD8+ T cells leading to aberrant immunological surveillance.
CR1 polymorphisms, serum levels of CR1 protein and mRNA expression: an anti-inflammatory role in pemphigus foliaceus
D. Augusto (Curitiba, BR)
Pemphigus foliaceus (PF) is an autoimmune disease endemic in Brazil, characterized by epidermal blisters and auto-antibodies against desmoglein-1. The recognition of the auto-antigen activates the complement system, which collaborates with the acantholytic process. Complement receptor 1 (CR1) regulates the complement system by destabilizing C3 and C5 convertases and preventing tissue injury. In order to evaluate if there is an association between common CR1 single nucleotide polymorphisms (SNPs) and the susceptibility to PF, we developed multiplex sequence-specific PCR assays and simultaneously genotyped nine SNPs including rs2274567 (His1208Arg) in exon 22, rs3737002 (Thr1408Met) in exon 26, and rs17047660 (Lys1590Glu) in exon 29, in 282 PF patients and 404 controls. We also measured the expression of soluble CR1 (sCR1) in 27 controls and 53 PF patients, in addition to the mRNA levels in 63 controls. We identified 13 haplotypes. Genotype distribution was in Hardy and Weinberg (HW) equilibrium in both groups. The GTATCTACA and GCATCTACA haplotypes may have a protective effect against development of the disease (P=0.03, OR=0.62 and P=0.02, OR=0.23, respectively) while GCACCTACG haplotype may increase susceptibility (P=0.0001, OR=4.47). Among patients with active disease, those under treatment had higher sCR1 serum levels than those prior to treatment (P=0.02). Among patients under treatment, those with localized lesions had higher sCR1 levels than those having generalized disease (P=0.0004). Carriers of the GCATCTACA haplotype allele presented lower CR1 mRNA expression levels (p=0.02). The results lead us to suggest that CR1 haplotypes may modulate gene expression and susceptibility to PF. Furthermore, corticoid treatment seems to increase sCR1 serum level, and higher sCR1 levels may play a protective role in individuals with PF.
24 autosomal STR microsatellites provide new insights into the Mexican Mestizo population from Mexico City and the Highlands
H. Flores-Aguilar (México City, MX)
STRs are versatile and informative genetic markers that have facilitated a great understanding of the genetic diversity present in natural populations, being a common tool in forensics, paternity and anthropological studies. Data collection is conducted based on many populations and allele frequency (AF) must be established in making an identification or any study. Mexican Mestizos are of mixed descent. Their ancestry is the result of admixture between the indigenous people of Asian ancestry that inhabited the country, different Europeans, and to a lesser extent, Africans. The aim of the study was to describe the population genetics in Mexico City and Highlands, by using the PowerPlex21 & GenePrint 24(Promega) & Global filer & Identifiler (Thermo Fisher Sci.) systems. We investigated 24 autosomal STRs in genomic DNA extracted from blood samples of 721 healthy Mexican Mestizos (57.4% males; 42.6% females). The analysis was performed with a 3500 Genetic Analyzer (Thermo Fisher Sci.). The genotyping process was done with the Gene Mapper ID-X v1.4 software. We compared the frequency with other studies in Mexicans from different regions. Heterozygosity (HE), Linkage disequilibrium (LD); Hardy-Weinberg equilibrium (HWE), Power of Discrimination (PD), Match Probability (MP) and Power of Exclusion (PE) were estimated with the PowerStat software. A total of 339 alleles at the 24 STR loci were found with their corresponding AF, ranging 0.069-50.704. These STRs are highly polymorphic being the most diverse FGA, D18551, Penta-E, D21S11, D6S1043 and SE33 (30-18 alleles). Penta E showed the highest PD(0.985) and SE33,the highest PE (0.947). Concordantly, these STR loci showed the highest heterozygosity together with D6S1043; one allele was not in HWE at; vWA, D8S1179, Penta D, D12S391, D19S433, D16S539; two alleles at TH01, Penta E, D21S11; 3 at D18S51, FGA, CSF1P0, since they were rare alleles. LD displayed no association between paired loci. Our data are concordant with those shown in Monterrey (North Mexico) but show different AF from them & those found in other Mexico regions. The most frequent alleles >20% were at; D21S11; D6S1043; vWA;TH01;CSF1P0; D16S539; D8S1179; D3S1358; D19S433, D2S391; D7S820. STR loci provide highly informative polymorphic data for population genetics, paternity testing and forensic identification.
Preliminary study regarding the association between Tumor Necrosis Factor Alpha gene polymorphisms and Childhood Idiopathic Nephrotic Syndrome in Romanian pediatric patients
I. TIERANU (Bucharest, RO)
Childhood idiopathic nephrotic syndrome (INS) is one of the most common glomerular diseases, characterized by heavy proteinuria, hypoalbuminemia, dyslipidemia and generalized edema. Patients with resistance to steroid treatment have a high risk of progressing to chronic kidney disease, which is considered a public health problem worldwide. New evidence suggests that several cytokines, including tumor necrosis factor alpha (TNF-alpha) may play an important role in the pathogenesis of this disease. Our objective was to analyze two single nucleotide polymorphisms (SNPs) of TNF-alpha gene in 70 patients with INS and 159 healthy controls. The two SNPs (rs1799724/-857C/T and rs1800629/-308G/A) were genotyped by TaqMan Genotyping Assays C_11918223_10 and C_7514879_10 (Real-time PCR System, Applied Biosystems, USA). Association tests were performed with the software PLINK v 1.07 and p values <0.05 were considered significant. Controls and patients were in Hardy-Weinberg equilibrium for the investigated SNPs. Minor alleles frequencies were 11.6% in INS patients versus 13.2% in controls for 857*T allele and 14.3% in INS versus 18.3% in controls for 308*A allele. Although the minor alleles were more frequent in controls than in patients, the difference was not statistically significant (p=0.28, OR 0.74 and p=0.63, OR 0.86). An analysis regarding the distribution of the two TNF-alpha polymorphisms was conducted in steroid sensitive INS patients (80%) versus steroid resistant patients (20%). We found a low frequency of 857*T allele in steroid resistant patients (8.3%) compared to steroid sensitive patients (15.3%) and controls (13.2%), but not statistically significant (p>0.05).We conclude that neither -857C/T, nor -308G/A polymorphisms of TNF-alpha gene are associated with the susceptibility and the response to steroid treatment of INS in our population. Given the small sample size used, future studies are necessary to clarify the results observed in the present study.
Complement receptor 1 polymorphism is associated with leprosy in Brazil
D. Augusto (Curitiba, BR)
Leprosy is a chronic infectious disease caused by the obligate intracellular pathogens Mycobacterium leprae and M. lepromatosis, which invade macrophages and Schwann cells. Complement receptor 1 (CR1) binds to C3b/C4b fragments and to mannose binding lectin (MBL) deposited on opsonized bacteria, facilitating bacterial entrance into phagocytes. Here, we developed a multiplex PCR sequence-specific assay and genotyped nine single nucleotide polymorphisms (SNPs) in 213 leprosy patients and 297 controls: rs6656401 (intron 4), rs3849266 (intron 21), rs2274567 (exon 22), rs3737002 (exon 26), rs11118131 (intron 26), rs11118167 (intron 28), rs17047660 (exon 29), rs4844610 (intron 37) and rs12034383 (intron 37). We measured the mRNA and soluble CR1 levels in a subset of up to 80 samples. We identified 18 haplotypes, whose frequencies differed between ethnic groups (p<0.000001). Afro-Brazilians with A alleles from polymorphism rs6656401 and rs4844610 presented almost four times increased susceptibility to leprosy (OR=3.89, p=0.003). Euro-Brazilians with the intronic rs3849266T presented higher susceptibility to leprosy (OR=1.63, p=0.028). Carriers of the rs11118167C presented higher CR1 mRNA expression in comparison to T/T homozygotes (p=0.036). Euro-Brazilians with the variant rs12034383A exhibited higher sCR1 levels compared to G/G homozygotes (p=0.0175). A negative correlation between sCR1 and MBL levels was also observed (r= -0.52; p=0.007). The results lead us to suggest that CR1 polymorphisms modulate gene expression and sCR1 levels, as well as susceptibility to leprosy, with the different effects in distinct ethnic groups.
The genetic profile of KIR receptors in Serbian population
B. Jovanovic (Beograd, RS)
Killer cell immunoglobulin-like receptors (KIRs) are expressed on natural killer (NK) cells and on some types of T-cells. KIR genes encode inhibitory and activating receptors. Polymorphism of KIR genes is present in variation of alleles and haplotype variation in gene content. Distribution of KIR genes varies between individuals and across populations. The aim of this study was to analyze KIR gene and genotype frequencies in a sample of 97 randomly selected, healthy, unrelated individuals from different parts of Serbia. KIR genotyping was performed by PCR SSO (One Lambda) and PCR SSP (Innotrain, Olerup). The observed frequencies of KIR genes and genotypes were determined by direct counting. KIR genotypes were assigned according to the Allele Frequencies KIR database. Framework genes KIR3DL3, KIR3DP1, KIR2DL4, KIR3DL2 were found in all individuals of study sample and pseudogene KIR2DP1 almost in all individuals (95.8%). The frequency of inhibitory KIR genes was higher (77.7%) than frequency of activating (39.2%) except KIR2DS4 gene which was found with frequency of 94.8%. KIR2DS5 gene was present with smallest frequency of 27.8%. The frequency of other activating KIR genes are: KIR2DS2 (56%), KIR2DS3 and KIR2DS1 (37.1%), KIR3DS1(38.1%). Inhibitory KIR genes were present in almost all individuals, KIR2DL1 (96.9%), KIR3DL1(93.8%), KIR2DL3 (86.6%) and with lower frequency KIR2DL2 and KIR2DL5 (55.6%). 28 different genotypes were observed. Among AA genotypes (28.8%), the most frequent was genotype ID1 (27.8%) and other was genotype ID203 (1.03%). Among BX genotypes (71.1%), the most frequent was genotype ID5 (12.4%) followed by ID2 (8.2%), ID4 (6.2%), ID3 (5.2%), ID6 (4.1%). Sixteen KIR genotypes were observed only once each. This data about the distribution of KIR genes and genotypes will be useful as a reference for further analysis of KIR genes diversity and diseases association study in Serbian population.
NOD2/CARD15 polymorphisms may protect from acute myocardial infarction
M. Petrek (Olomouc, CZ)
Acute myocardial infarction (AMI) is a complex disease of polygenic and multifactorial nature. Inflammatory components play a role in its pathogenesis. Of many polymorphic genes, NOD2/CARD15 may be of relevance. In this study we analysed the association between AMI and NOD2/CARD15 gene variants (rs2066844, rs2066845, rs2066847). 250 AMI patients and 100 healthy control subjects, both of Czech origin, were enrolled. MassArray iPLEX methodology (Agena Bioscience, San Diego, CA) was used for SNP genotyping. Genotype and allele frequencies were determined; the distribution of genotypes complied to Hardy-Weinberg equilibrium. Significant difference in C allele frequencies was observed between the patient group (0.04) and the control subjects (0.08) for rs2066847; no difference was detected for the other two SNPs tested. This finding, along with predominance of carriers of NOD2/CARD15 rs2066847 variant among healthy controls, suggests its possible protective character against AMI (Odds Ratio, OR 0.40; p=0.008). The plausible protective effect of the C allele of rs2066847 must be replicated in other centres/other ethnicities, otherwise it is considered preliminary and pertinent for Czech population only.