Association of NFKB1, NKX2-5, GATA4 and RANKL Gene Polymorphisms with Sporadic Congenital Heart Disease in Greek Patients.

Congenital heart disease (CHD) is a group of structural defects of the heart and the great vessels, and one of the leading causes of death among infants and young adults. Several gene variants are involved in diverse mechanisms of cardiac and vessel development and could thus be considered candidate mutated genes for a congenital heart defect or a specific variant could predispose a person to CHD. In the present study, variants in four such genes are investigated for the first time in a group of young Greek CHD patients: the NFKB1 gene polymorphism (-94ins/ delATTG), rs28362491, NKX2-5 gene polymorphism rs2277923, GATA4 gene polymorphism rs11785481 and RANKL gene polymorphism rs4531631. A total of 43 CHD patients and 100 healthy adults were included in the study. The polymerase chain reaction-restriction fragment length polymorphism (PRC-RFLP) method was used to genotype the aforementioned polymorphisms of NFKB1, NKX2-5, GATA4 and RANKL. The association analysis identified that there was a protective association between CHD and the A allele of rs2277923 polymorphism (p = 0.004). The D allele of the rs28362491 polymorphism is also a likely risk factor for causing CHD (p = 0.006). The differences of the rs4531631 and rs11785481 variant contribution had no statistical significance between the groups (p >0.05). In conclusion, our results revealed that the rs28362491 and rs2277923 gene polymorphisms, but not the rs4531631 and rs11785481 polymorphisms, may contribute to CHD risk in a cohort of Greek CHD patients.

Genetic analysis of the human Insulin-like 3 gene: absence of mutations in a Greek paediatric cohort with testicular maldescent.

This study investigated the hypothesis that genetic alterations of the human insulin-like 3 (INSL3) gene are associated with testicular maldescent (TMD). Genomic DNA was extracted and amplified from peripheral blood samples of 170 unrelated children with all possible phenotypical expressions of TMD and 50 volunteers with normal external genitalia from the general paediatric population (controls). PCR-single strand conformation polymorphism analysis was used to screen INSL3 gene for genetic variants. For rapid screening of a detected nonsilent genetic alteration, restriction assay using endonuclease Eag I was further employed. Products were analysed on 2% agarose gel and restriction patterns were visualised by ethidium bromide. Differences in genotype and allelic distributions of nonsilent genetic alterations were evaluated between (i) patients-controls, (ii) familial-sporadic, (iii) bilateral-unilateral and (iv) intra-abdominal-inguinal cases of TMD. No mutations were detected. Three common INSL3 gene polymorphisms (27G>A, 126G>A, 178G>A) unrelated to any particular phenotype of TMD were detected both in patients and controls. These results indicate that INSL3 gene mutations are not a common cause of TMD in the human.

The (TAAAA)n polymorphism of sex hormone-binding globulin gene is not associated with testicular maldescent.

The aim of this family-based study was to investigate the potential association/genetic linkage of the (TAAAA)n polymorphism of sex hormone-binding globulin gene proximal promoter with testicular maldescent (TMD). Genomic DNA was extracted from the peripheral blood of 487 subjects (174 index families): (i) 180 children with all phenotypes of TMD, (ii) 307 parents (156 mothers and 151 fathers). Conventional polymerase chain reaction amplification products were electrophoresed on 10% nondenaturating polyacrylamide gel and visualised by silver staining. After excluding ambiguous parental-child trios and most cases of index families with missing parental genotypes, 429 individuals were left for analysis: 138 completely typed nuclear families (five included a second affected child) and five child-parent couples (one parent missing). Eight fathers presented history of TMD, that is, a total of 156 cases with TMD were analysed. Alleles were analysed with the affected family-based control method and logistic regression-based extension of the transmission disequilibrium test for multiallelic loci. (ΤΑΑΑΑ)n polymorphism analysis revealed six alleles based on repeat numbers (n=5-10). No association/genetic linkage between the (TAAAA)n polymorphism and TMD was detected. Other factors should be investigated to potentially explain the genetic predisposition that seems to exist in at least a subgroup of these patients.

Altered sequence of the ETS1 transcription factor may predispose to rheumatoid arthritis susceptibility.

OBJECTIVE: ETS1 belongs to the ETS family of transcription factors that regulate the expression of various immune-related genes. The aim of this study was to identify whether the ETS1 single nucleotide polymorphism (SNP) rs11221332, described in Caucasian subjects, plays a role in rheumatoid arthritis (RA) susceptibility. METHODS: We genotyped this polymorphism in 136 unrelated patients with RA and 147 healthy individuals with no history of autoimmune disease. Genotyping was performed with a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay and the data were analysed using SPSS statistical software. RESULTS: A statistically significant difference was observed in the distribution of the rs11221332 genotypes between RA patients and controls (p = 0.041). Comparing the distribution of rs11221332 alleles between the groups studied, a greater difference was found [odds ratio (OR) 1.504, 95% confidence interval (CI) 1.036-2.183; p = 0.039]. CONCLUSIONS: The present study revealed, for first time, the positive association of a polymorphism in the sequence of the ETS1 transcription factor with RA susceptibility. Further studies in other ethnic groups of patients are needed to confirm the results of the present genetic association study related to ETS1, a widely used transcription factor in the regulation of the expression of various genes.

The (TTTA)n polymorphism of aromatase (CYP19) gene is associated with age at menarche.

BACKGROUND: Twin studies have shown that age at menarche may be subject to hereditary influences but the specific determinants are unknown. Estrogens are known to have an important role in menarche. Since the enzyme aromatase is responsible for the conversion of androgens to estrogens, the aromatase (CYP19) gene could be a candidate gene for the regulation of menarche. The aim of this study was to investigate the possible association of the CYP19(TTTA)(n) polymorphism with age at menarche. METHODS: We studied 130 healthy adolescent females from a closed community in North-Western Greece. Information on menarche was obtained through interviews. The BMI was recorded. The CYP19(TTTA)(n) polymorphism was genotyped. RESULTS: The mean age at menarche was 12.9 ± 1.2 years and the BMI = 19.8 ± 2.3 kg/m(2). Genotype analysis revealed 5 CYP19(TTTA)(n) alleles containing 7-11 TTTA repeats. Girls homozygous for the allele with 7 TTTA repeats had earlier menarche (12.45 ± 0.9 years) than girls carrying other genotypes (13.0 ± 1.2 years, P = 0.025), whereas the BMI was not different between these two subgroups. Carriers of the allele with 11 TTTA repeats had later menarche compared with non-carriers (14.1 ± 0.75 versus 12.8 ± 1.2 years, P< 0.001), whereas no difference was found in BMI values. Comparing girls with early menarche (<12 years, 25th percentile) with girls with late menarche (>13.75 years, 75th percentile), we found that 31% of the girls with early menarche were homozygous for the (TTTA)(7) allele compared with 6.9% among girls with late menarche (P = 0.018). In addition, none of the girls carrying the (TTTA)(11) allele was found among the subgroup with early menarche, whereas 24.1% of girls with late menarche had the (TTTA)(11) allele (P = 0.001). No association between other alleles and age at menarche was found. CONCLUSIONS: There is evidence for a genetic contribution of the CYP19 gene to the age at menarche.

The role of microRNA-146a (miR-146a) and its target IL-1R-associated kinase (IRAK1) in psoriatic arthritis susceptibility.

MicroRNAs have shown different expression patterns in immune diseases. The present study explores the association of miRNA-146a variant rs2910164 and of two IRAK1 (target of miR-146a) polymorphisms rs3027898 and rs1059703 with psoriasic arthritis (PsA). Twenty-nine PsA and 66 controls were enrolled in the study. To study if the statistical significant differences between patients with PsA and controls are independent to psoriasis, we expanded the study in 49 patients with ankylosing spondylitis (AS). Strong statistical significant difference was observed in IRAK1 rs3027898 polymorphism distribution between patients with PsA and controls (P = 0.003), as between patients with AS and controls (P < 0.001). Marginally significant difference was observed in distribution of IRAK1 rs1059703 genotypes between patients with PsA and controls (P = 0.058), but no difference was observed in miRNA-146a rs2910164 distribution (P = 0.394). This is the first study that addresses IRAK1 rs3027898 polymorphism association with PsA susceptibility, but further studies could help to understand the extent of the proposed association.

The role of tumor necrosis factor (TNF)-alpha and TNF receptor polymorphisms in susceptibility to ankylosing spondylitis.

OBJECTIVE: Ankylosing spondylitis (AS) is a chronic inflammatory disease affecting the sacroiliac joint and vertebral column. Tumor necrosis factor alpha (TNF-alpha), a cytokine that acts via two tumor necrosis factor receptors (TNFR1 and TNFR2), may be implicated in the pathogenesis of AS. The aim of the present study was to examine the role of the polymorphisms 36A>G (TNFR1), 676T>G (TNFR2), -857C>T (TNF-alpha), -308G>A (TNF-alpha), and -238G>A (TNF-alpha) in AS susceptibility. METHODS: Forty-nine AS patients and 68 randomly chosen healthy volunteers were enrolled in the study. Polymerase chain reaction coupled with a restriction fragment length polymorphism assay was performed in the genotype analysis of each variant. RESULTS: The polymorphisms 36A>G (TNFR1) and -238G>A (TNF-alpha were not found to be in Hardy-Weinberg equilibrium in the patient group and therefore were excluded from the statistical analysis. A marginally statistically significant difference was observed in the distribution of 676T>G (TNFR2) genotypes between AS patients and controls (p=0.054) and was revealed to be more significant in the 676T>G allele distribution between the two groups (p=0.031). The complex genotype TNFR2 676TG/ TNF-alpha -857CC (p=0.041) was also differently distri-buted between AS patients and controls. CONCLUSIONS: The TNFR2 676T allele is reported here for first time to be differently distributed between AS patients and controls. The higher frequency of the wild type TNFR2 676T allele in AS patients suggests the functional ability of TNFR2 to support increased TNF-alpha mediated immunoactivity.

Glucocorticoid receptor variants may predispose to rheumatoid arthritis susceptibility.

OBJECTIVES: Deregulation of glucocorticoid (GC) secretion could be associated with rheumatoid arthritis (RA). The GC receptor (GR) has two isoforms. In the present study, we explored the role of GR-alpha polymorphisms rs33388, rs33389, and Bcl I, and the GR-beta variant rs6198 in RA susceptibility. METHODS: One hundred and thirty-six RA patients and 148 ethnic matching controls were studied. Polymorphisms rs33388 and Bcl I were genotyped by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP), and variants rs33389 and rs6198 by polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) coupled with sequencing. Arlequin and SPSS softwares were used in the statistical analysis. RESULTS: The polymorphisms studied were in Hardy-Weinberg equilibrium in both groups. A marginally statistical significant difference was observed in the distribution of rs33388 genotypes between RA patients and controls (p = 0.053). When the A and T alleles were compared, the statistical significance was p = 0.025. Specific complex genotypes were also differentially distributed: the GR-alpha complex genotypes (a) [homozygote (homo) wild-type (wt) rs33388-homo wt rs33389] (11% RA vs. 21% controls; p = 0.023), (b) [homo wt rs33388-homo wt rs33389-homo non-wt Bcl I] (0.7% RA vs. 4.7% controls; p = 0.042), and (c) the GR-beta complex genotype [homo wt rs33388-homo wt rs33389-homo non-wt Bcl I-homo wt rs6198] (0.7% RA vs. 4.7% controls; p = 0.042). CONCLUSIONS: GR-alpha and GR-beta polymorphisms are potentially associated with RA susceptibility. However, additional studies in larger and other ethnic groups of patients are needed to confirm the results of the present study.

Absence of SLC22A12 gene mutations in Greek Caucasian patients with primary renal hypouricaemia.

OBJECTIVE: Primary renal hypouricaemia is a hereditary clinical disorder characterized by increased renal urate clearance due to isolated renal tubular defect of uric acid transport. There have been only a few studies on primary renal hypouricaemia in Caucasian populations. Defects in the SLC22A12 gene, which encodes the renal urate transporter URAT1, have been reported to be related to the disease pathogenesis. This study was undertaken to elucidate whether SLC22A12 gene mutations are responsible for low serum uric acid levels in Greek people. MATERIAL AND METHODS: Nine Greek Caucasian subjects with primary renal hypouricaemia were included in the study. All had serum uric acid less than 2.5 mg dL(-1) (0.14 mmol L(-1)), fractional excretion of uric acid more than 10% and no other known causes of hypouricaemia. Mutation analysis of the SLC22A12 gene was performed. RESULTS: No mutation was found--only the previously reported silent polymorphism 1246T > C (His 42His) in exon 2 of the SLC22A12 gene. CONCLUSIONS: No previously reported mutation of URAT1 was associated with primary renal hypouricaemia in Greek subjects.

Combined estrogen receptor alpha and estrogen receptor beta genotypes influence the age of menarche.

BACKGROUND: Age at menarche has a strong genetic influence. We reported recently an association between the XbaI (351A-->C)and PvuII (397T-->C) polymorphisms of the estrogen receptor (ER)alpha gene with the age of menarche in Greek adolescents. In the present study, we examined whether ERbeta genotypes alone, or in combination with ERalpha genotypes, may also influence onset of menarche. METHODS: We performed genotyping for the single nucleotide polymorphisms 1730A-->G and 1082G-->A of the ERbeta gene and examined their association with the age of menarche in the same cohort of 145 Greek girls. We then looked for a possible effect of combined ERalpha and beta genotypes on the age of menarche. RESULTS: Menarche occurred 7 months later in girls with the AA genotype of the 1730A-->G polymorphism than in girls with the AG genotype (mean +/- SD: 13.23 +/- 1.24 versus 12.66 +/- 1.26 years, respectively; P = 0.005). The 1082G-->A polymorphism was not detected in any of the girls examined. A significant effect of combined ERalpha and beta genotypes was also apparent. Menarche occurred 11 months later in girls bearing the AA/TT,AA (ERalpha, ERbeta) genotypes compared with girls with the CC/CC,AG genotype (13.30 +/- 1.27 nersus 12.41 +/- 1.28 years; P = 0.042). The difference remained significant after adjusting for body mass index (P = 0.034). CONCLUSION: Combined ERalpha and ERbeta polymorphisms may influence the age of menarche.

Y-chromosomal STR haplotypes in a population sample from continental Greece, and the islands of Crete and Chios.

Eight Y-chromosomal short tandem repeats (STRs)--DYS19, DYS389-I, DYS389-II, DYS390, DYS391, DYS392, DYS393, and DYS385--were typed in a population sample (n = 113) of unrelated males from seven different regions of Greece (Macedonia, Thessaly, Epirus, Central Greece, Peloponnese, Crete Island, and Chios Island).