Report on the Molecular Approaches to Osteoarthritis Symposium,Imperial College London,UK, 18–20 April 2004

Estrogen and estrogen receptors (ERs) are known to play important roles in the pathophysiology of osteoarthritis (OA). To investigate ER-α gene polymorphisms for its associations with primary knee OA, we conducted a case–control association study in patients with primary knee OA (n = 151) and healthy individuals (n = 397) in the Korean population. Haplotyping analysis was used to determine the relationship between three polymorphisms in the ER-α gene (intron 1 T/C, intron 1 A/G and exon 8 G/A) and primary knee OA. Genotypes of the ER-α gene polymorphism were determined by PCR followed by restriction enzyme digestion (PvuII for intron 1 T/C, XbaI for intron 1 A/G, and BtgI for exon 8 G/A polymorphism). There was no significant difference between primary knee OA patients and healthy control individuals in the distribution of any of the genotypes evaluated. However, we found that the allele frequency for the exon 8 G/A BtgI polymorphism (codon 594) was significantly different between primary knee OA patients and control individuals (odds ratio = 1.38, 95% confidence interval = 1.01–1.88; P = 0.044). In haplotype frequency estimation analysis, there was a significant difference between primary knee OA patients and control individuals (degrees of freedom = 7, χ² = 21.48; P = 0.003). Although the number OA patients studied is small, the present study shows that ER-α gene haplotype may be associated with primary knee OA, and genetic variations in the ER-α gene may be involved in OA.     

The association between estrogen receptor alpha polymorphisms and the risk of prostate cancer in Slovak population

The aim of our study was to evaluate the effect of two polymorphisms in the estrogen receptor alpha, PvuII and XbaI, on the development of prostate cancer within Slovak population, as well as their correlation with selected clinical characteristics. The study was performed using 311 prostate cancer patients and 256 healthy male controls. Both polymorphisms were significantly associated with higher risk of prostate cancer development. At the same time, the CC genotype of PvuII polymorphism (OR = 1.98; 95 % CI 0.94–4.21; p = 0.05) and the AG genotype of XbaI polymorphism (OR = 1.74; 95 % CI 1.0–3.02; p = 0.04) significantly contributed to the development of low-grade carcinoma, while the AG and GG genotypes of the XbaI polymorphism contributed mainly to the development of high-grade prostate cancer (OR = 1.83; 95 % CI 1.12–3.01; p = 0.01 and OR = 2.13; 95 % CI 1.06–4.19; p = 0.03, respectively). Similarly, the AG and GG genotypes of XbaI polymorphism showed significant association with prostate cancer in patients with serum PSA level ≥10 ng/ml. Both polymorphisms were found at the same time to be more frequent in patients diagnosed before the age of 60. We conclude on the basis of these results that PvuII and XbaI polymorphisms of estrogen receptor alpha might be associated with prostate cancer risk within Slovak population. Although this is a pilot study and, as such, more detailed investigations are needed to confirm the role of these polymorphisms in prostate cancer development and progression within said Slovak population, our results might still provide a valuable basis for further research with larger patient groups.     

A PvuII restriction fragment length polymorphism of the glucose-6-phosphate dehydrogenase gene is an African-specific marker

Summary The site of a PvuII restriction fragment length polymorphism (RFLP) of the human glucose-6-phosphate dehydrogenase (G6PD) gene has been located in intron V, 60 bp upstream of G6PD exon VI. A population survey shows this RFLP to be specific for African populations, with frequencies of the rarer allele (PvuII type 2 site present) of 0.32–0.40 in Kenyans, Nigerians, Zambians, and West Indians. This allele has not been found in the European, Asian and Middle Eastern populations studied. Such population-specific markers may be useful in the study of population affinities and may provide insight into prehistoric migrations of peoples.     

The duplicated gene copy of the ovine growth hormone gene contains a PvuII polymorphism in the second intron

PvuII restriction fragment length polymorphism (RFLP) was found at the growth hormone locus in sheep carrying the GH2 allele where the gene is duplicated. By restriction analysis and using the polymerase chain reactibn we demonstrated that this RFLP is due to a mutation at the Pvu II site located in the second intron of the 3′ copy of the GH2 allele.     

DNA multiallelic systems reveal gene/longevity associations not detected by diallelic systems. The APOB locus

To identify possible genetic factors affecting human longevity we compared allele pools at two candidate loci for longevity between a sample of 143 centenarians (S) and a control sample of 158 individuals (C). The candidate loci were APOB and TPO, which code for apolipoprotein B and thyroid peroxidase, respectively. Both restriction fragment length (RFL) (XbaI₂₄₈₈ and EcoRI₄₁₅₄) and variable number of tandem repeat (VNTR) (3′APOB-VNTR) polymorphisms were analysed at the APOB locus; the TPO-VNTR polymorphism (intron 10) was analysed at the TPO locus. The main result of the investigation was that there is an association between the APOB locus and longevity that is revealed only when multiallelic polymorphisms are considered. In particular: (i) the frequency of 3′APOB-VNTR alleles with fewer than 35 repeats is significantly lower in cases than in controls; (ii) the linkage disequilibrium between the XbaI-RFLP and the EcoRI-RFLP is significantly different from 0 in cases but not in controls; (iii) the EcoRI-RFLP and XbaI-RFLP allele frequencies do not discriminate between cases and controls. The differences observed between case and control allele pools are specific to the APOB locus, since no significant difference was observed at the TPO locus. Received: 27 November 1995 / Revised: 24 July 1996     

Polymorphisms of estrogen receptors alpha and beta in idiopathic, infertile Brazilian men: a case-control study

Estrogen plays an important role in the human reproductive system and its action is mediated mainly by two specific receptors: α (ERα) and β (ERβ). There are polymorphic variants in both ER genes, and studies showed their association with reproductive outcomes. We aimed to determine the distribution of ERα and ERβ gene polymorphisms in idiopathic, infertile Brazilian patients in a case–control study comprising 187 idiopathic, infertile Brazilian men with nonobstructive azoospermia (NOA, n = 78) or severe oligozoospermia (SO, n = 109) and 216 fertile men. Detection of ERα (PvuII and XbaI) and ERβ (AluI and RsaI) gene polymorphisms were performed using TaqMan PCR. The results were analyzed statically, and a P‐value < 0.05 was considered significant. Single‐marker analysis revealed that neither PvuII nor XbaI polymorphisms of the ERα gene were associated either with NOA group (P = 0.662 and P = 0.527, respectively) or SO group (P = 0.777 and P = 1.0, respectively). Regarding ERβ polymorphisms, no statistical difference was observed between the AluI polymorphism and NOA group compared to controls (P = 1.0) or between SO group and controls (P = 0.423). We found similar results with the RsaI polymorphism. Statistical analysis did not reveal a difference between NOA (P = 0.740) and SO (P = 0.920) groups compared to controls. Combined genotypes of ERα and ERβ polymorphisms did not identify a haplotype associated with idiopathic infertility. Thus, in the Brazilian population, genetic variations in both estrogen receptors alpha (PvuII and XbaI) and beta (AluI and RsaI) were not relevant to idiopathic infertility. Mol. Reprod. Dev. 78:665–672, 2011. © 2011 Wiley‐Liss, Inc.     

Evaluation of <Emphasis Type="Italic">ERα</Emphasis> and <Emphasis Type="Italic">VDR</Emphasis> gene polymorphisms in relation to bone mineral density in Turkish postmenopausal women

It has been suggested that the estrogen receptor alpha (ERα) and vitamin D receptor (VDR) genes as possibly implicated in reduced bone mineral density (BMD) in osteoporosis. The present study investigated the relation of ERα PvuII/XbaI polymorphisms and VDR FokI/TaqI polymorphisms with BMD in Turkish postmenopausal women. Eighty-one osteoporotic and 122 osteopenic postmenopausal women were recruited. For detection of the polymorphisms, polymerase chain reaction-restriction fragment lenght polymorphism techniques have been used. BMD was measured at the lumbar spine and hip by dual-energy X-ray absorptiometry. Distributions of ERα (PvuII dbSNP: rs2234693, XbaI dbSNP: rs9340799) and VDR genotypes (FokI dbSNP rs10735810, TaqI dbSNP: rs731236) were similar in study population. Although overall prevalence of osteoporosis had no association with these genotypes, the prevalence of decreased femoral neck BMD values were higher in the subjects with ERα PvuII “PP” and ERα XbaI “XX” genotypes than in those with “Pp/pp” genotypes and “xx” genotype, respectively (P < 0.05). Furthermore, subjects with VDR FokI “FF” genotype had lower BMD values of femoral neck and total hip compared to those with “Ff” genotype (P < 0.05). In the logistic regression analysis, we confirmed the presence of relationships between the VDR FokI “FF” genotypes, BMI ≤ 27.5, age ≥ 55 and the increased risk of femoral neck BMD below 0.8 value in postmenopausal women. The present data suggests that the ERα PvuII/XbaI and VDR FokI polymorphisms may contribute to the determination of bone mineral density in Turkish postmenopausal women.     

Association of Dental Fluorosis with Polymorphisms of Estrogen Receptor Gene in Chinese Children

Dental fluorosis (DF) is one of the important performances of endemic fluorosis. Some studies indicated that estrogen receptor (ESR) gene polymorphisms were associated with bone metabolism-related diseases. Therefore, it is possible that the variation in ESR genotypes will be associated with DF status. A case?Ccontrol study was conducted among children aged 8?C12 years with (n?=?75) or without (n?=?165) DF in China to investigate the relationship between ESR gene polymorphisms and DF. Gene polymorphisms were genotyped using the PCR-RFLP procedure. Children carrying R allele of ER RsaI had significantly increased risk of DF (Odds ratio (OR)?=?1.821; 95% confidence interval (CI), 1.013?C3.274) compared to children carrying r allele of ER RsaI in endemic fluorosis villages. For children with high-loaded fluoride status, carrying X allele of ESR?? XbaI had a significantly decreased risk of DF (OR?=?0.542; 95% CI, 0.314?C0.936) compared to carrying x allele. This study provides the first evidence of an association between polymorphisms in the ESR gene with DF in high-fluoride-exposed populations. Further studies are needed to confirm the association.     

Apolipoprotein B-100 XbaI gene polymorphism in gallbladder cancer

Genetic polymorphisms in the apolipoprotein B (apoB) gene have been reported to be associated with altered serum lipids and susceptibility to cholesterol gallstones (GS). Gallstones are among the well-known risk factors for carcinoma of the gallbladder (GBC). In the present study, the association between the XbaI polymorphism of the apo B gene was examined in patients with GBC and GS and in normal controls in a north Indian population. DNA samples from patients with GBC (n=153), GS (n=117) and healthy subjects (n=137) were analysed for the apoB-XbaI polymorphism by polymerase chain reaction followed by restriction fragment length polymorphism. The genotype X+/– was less frequent in patients with GBC (39.2%) than in those with GS (68.3%) and in normal subjects (66.4%; P<0.00001). In contrast, there was an increase in the homozygous X–/– genotype in patients with GBC (54.9%) as compared with those with GS (23.9%) and normal subjects (25.5%; P<0.00001). The frequency of the X– allele was found to be significantly increased in GBC patients with or without GS (odds ratio=2.3 and 1.7, respectively). We suggest that the apoB-XbaI gene polymorphism confers susceptibility to carcinoma of the gallbladder under specific environmental conditions.     

Association between paraoxonase-1 gene Q192R and L55M polymorphisms and risk of gastric cancer: A case-control study from Iran

The aim of the present study was to examine the relation between two paraoxonase1 (PON1) polymorphisms, Q192R and L55M and susceptibility to gastric cancer in an Iranian population. In this case-control study the PON1 polymorphisms were assessed in 90 gastric cancer patients and 90 healthy controls by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method. Regarding PON1 Q192R polymorphism, a significant increase in the R allele in the patient group compared with the controls (p value?=?0.0006) While the Q allele was more frequent in the control group. No significant difference was found in the genotype or allele frequency of the L55M polymorphism between healthy individuals and patients with gastric cancer. Our results demonstrated the protective effect of Q allele against gastric cancer.     

Association of polymorphisms at restriction enzyme recognition sites of apolipoprotein B and E gene with dyslipidemia in children undergoing primary nephrotic syndrome

Background Dyslipidemia, a common complication, is very prevalent in children with primary nephrotic syndrome (PNS). Recent studies have shown that genetic basis may be involved in the onset of HLP secondary to PNS. ApoB and E have been identified as the important candidate genes for lipid abnormalities. Objective: To investigate the association of apolipoprotein B (apoB) and E (apoE) genetic polymorphisms (Xba I, EcoR I, Msp I, and Hha I) with parameters describing the serum lipid profiles in children undergoing PNS. Methods: Genomic DNA was extracted from 250 children diagnosed with PNS and 200 healthy controls with neither allergic nor renal disease. ApoB (Xba I, EcoR I, and Msp I) and apoE (Hha I) genotypes were determined by PCR-restriction fragment length polymorphism (RFLP) analysis. The fasting serum lipoprotein (a) [Lp(a)], total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), apolipoprotein A1 (apoA1), apoB, and total protein from a 24-h urine sample were measured. Results: No significant differences in genotypes and alleles frequencies were observed for the apoB Xba I, EcoR I, Msp I and the apoE Hha I restriction sites in PNS patients as compared to controls (P > 0.05). Patients and controls with X + allele exhibited significantly higher serum levels of Lp(a), TC, nonHDL-C, LDL-C, LDL-C/HDL-C ratio, and apoB than that with X− allele (P < 0.05), whereas for apoA1/B ratio the opposite was found (P < 0.01). E−/E− carriers had significantly higher Lp(a), TC, HDL-C, and apoA1 concentrations than did E+/E− or E+/E+ carriers in control group (P < 0.05). Healthy children carrying the rare EcoR I allele had higher mean Lp(a), TC, and HDL-C levels than homozygotes for E+ (P < 0.05). Higher Lp(a) serum concentrations were observed in patients with E− allele (P < 0.05). No significant differences in lipid parameters were determined for the apoB Msp I and apoE Hha I the polymorphisms study (P > 0.05). When genetic variations were compared with urinary protein excretion, the Xba I X− allele was more frequent in patients with elevated proteinuria (P < 0.01). Conclusion: Presence of Xba I X+ allele and/or EcoR I E− at the apoB gene may be risk factors for lipid abnormalities secondary to childhood PNS.     

p53 protein expression and oestrogen and progesterone receptor status in invasive ductal breast carcinomas

p53 protein expression and oestrogen and progesterone receptor status in invasive ductal breast carcinomas The p53 protein expression and oestrogen and progesterone receptors status was investigated in correlation to the grade of malignancy of primary breast carcinomas. Our material constituted imprints from surgical biopsies of 75 invasive ductal breast cancer cases. The p53 protein expression was investigated immunocytologically using the monoclonal antibody p53 DO-7 (DAKO). A biochemical DCC method was applied for the detection of oestrogen and progesterone receptors for all tumours. Fifty-one percent of breast cancer cases were p53 protein positive. A statistically significant association of p53 protein expression and high tumour grade was found (chi2=23.72, d.f.=2, P < 0.001). A statistically significant association was also found between oestrogen and progesterone receptor positive cases and the grade of malignancy (P < 0.001). A negative association between p53 protein expression and oestrogen (ER) and progesterone receptors (PgR) positivity was found. From our results it appears that it is possible to distinguish from grade II tumours two subgroups of cases, one with low malignancy potential and p53 (-), ER (+), PgR (+), and another subgroup with high malignancy potential and phenotype p53 (+), ER (-), PgR (-). The last subset of patients could actually benefit from adjuvant therapy.     

Restriction fragment length polymorphisms at the heat shock protein HSP70 gene(s) in pigs*

Pigs from a population consisting of eight US breeds or strains and three Chinese breeds were examined by restriction fragment length polymorphism (RFLP) analysis of the heat shock protein HSP70 gene(s). Limited polymorphisms with PstI and PvuII restriction enzymes were observed, but there were no polymorphisms with BomIII and BglI.     

Polymorphisms at the GLUT1 (HepG2) and GLUT4 (muscle/adipocyte) glucose transporter genes and non-insulin-dependent diabetes mellitus (NIDDM)

Summary In order to determine the possible contribution of the GLUT1 (HepG2) glucose transporter gene to the inheritance of non-insulin-dependent diabetes mellitus (NIDDM), two restriction fragment length polymorphisms (RFLPs) and the related haplotypes at this locus were studied in 48 Italian diabetic patients and 58 normal subjects. Genotype frequencies for the XbaI polymorphism were significantly different between patients and controls (XbaI: ² = 9.80, df= 2, P < 0.0079). A significant difference was also found in the allele frequencies between NIDDM patients and controls (² =9.39, df = 1, P < 0.0022), whereas no differences were found for the StuI RFLP. No linkage disequilibrium was detected between the XbaI and StuI RFLPs in this sample. The analysis of the four haplotype frequencies (X1S1, X1S2, X2S1, X2S2) revealed a significant difference between diabetic patients and controls (² = 14.26, df =3, P < 0.002). By comparing single haplotype frequencies, a significant difference between the two groups was found for the X1S1 and X2S2 haplotypes. A two-allele RFLP at the GLUT4 (muscle/adipocyte) glucose transporter gene, detected with the restriction enzyme KpnI, was also examined; no differences were found between patients and controls for this RFLP. The finding of an association between polymorphic markers at the GLUT1 transporter and NIDDM suggests that this locus may contribute to the inherited susceptibility to the disease in this Italian population.     

Study on the Association Between Estrogen Receptor Gene (ESR) and Reproduction Traits in Landrace Pigs

To study the potential use of estrogen receptor gene (ESR) as a genetic marker to improve the reproductive traits of pigs, the genotypes of the ESR PCR product digested by Pvu II were determined in 2 239 litters from 612 Landrace sows. The data of the first, second, and later parities were separately evaluated. Although the frequency of the B allele was much lower than that of the A allele, likelihood ratio test showed that the gene frequencies were in Hardy-Weinberg equilibrium. The effects of ESR of different parities were not equal. In summary, the sows with the BB genotype showed better performance for total number of piglets born (TNB) and number of piglets born alive (NBA), but had a lower average piglet weight at birth (AWB). It was concluded that ESR could be used as a marker for the selection of litter size in the Landrace population.     

Meta-Analysis of Associations of IL1 Receptor Antagonist and Estrogen Receptor Gene Polymorphisms with Systemic Lupus Erythematosus Susceptibility

Systemic lupus erythematosus (SLE) is an autoimmune disease that affects a number of different organs and tissues. Interleukin-1 (IL1) and estrogen are considered potential elements in the pathology of SLE. Recently, the variable number of tandem repeats (VNTR) polymorphism in the IL1 receptor antagonist gene (IL1-RN) and PvuII (rs2234693) and XbaI (rs9340799) polymorphisms in the estrogen receptor 1 gene (ESR1) have been associated with a predisposition to SLE. However, the evidence for these associations is inconclusive. We therefore conducted a meta-analysis to validate the roles of these polymorphisms in SLE susceptibility. We searched four databases and identified a total of 17 eligible articles comprising 24 studies. The Newcastle-Ottawa quality assessment scale was used to assess the qualities of the selected studies. We assessed the strengths of the associations using odds ratios (ORs) with 95% confidence intervals (95% CIs). Regarding the IL-1RN VNTR, the 2 allele significantly increased SLE susceptibility (2 vs. L: OR = 1.34, 95% CI = 1.03–1.73, P = 0.03). The ESR1 PvuII CC/CT genotype was also associated with SLE susceptibility (CC/CT vs. TT: OR = 1.25, 95% CI = 1.06–1.47, P = 0.01), and the difference was especially pronounced among Asians (CC/CT vs. TT: OR = 1.33, 95% CI = 1.04–1.69, P = 0.02). No significant association between the ESR1 XbaI polymorphism and SLE susceptibility was observed in the overall analysis. However, a marginally significant association between the GG/GA genotype was found in individuals of Asian descent (GG/GA vs. AA: OR = 1.30, 95% CI = 1.01–1.67, P = 0.04). These results indicate that the IL1-RN VNTR 2 allele, ESR1 PvuII CC/CT genotype and ESR1 XbaI GG/GA genotype may increase SLE susceptibility, especially in Asian individuals.     

Genetic variants of fibroblast growth factor receptor 2 (<Emphasis Type="Italic">FGFR2</Emphasis>) are associated with breast cancer risk in Chinese women of the Han nationality

Fibroblast growth factor receptor 2 (FGFR2), a recently described risk factor for breast cancer, plays important roles in cell growth, invasiveness, motility, and angiogenesis. In attempt to investigate whether FGFR2 polymorphisms are associated with a risk of breast cancer in Chinese women of the Han nationality, we genotyped single-nucleotide polymorphisms (SNPs) of seven FGFR2 sites (rs2981582, rs17102287, rs17542768, rs10510097, rs11200012, rs3750817, rs2981578) in 816 women including 388 breast cancer patients and 428 healthy controls via the polymerase chain reaction single-strand conformation polymorphism procedure as well as sequence detection. Our results suggest that the A allele and AA genotype of SNP rs2981578 appear to be protective factors associated with breast cancer, while the CT genotype of SNP rs3750817 is a putative risk factor.     

A PvuII polymorphism in the 5′ flanking region of the apolipoprotein AIV gene: its use to study genetic variation determining serum lipid and apolipoprotein concentration

Summary We have used a 1.05-kb unique genomic fragment from the 5 end of the apolipoprotein (apo) CIII gene to identify a restriction fragment length polymorphism (RFLP) detected with the restriction enzyme PvuII, in the apoCIII-apoAIV intergenic region. In a sample of 220 normolipidaemic individuals from the UK population, the frequency of the rare allele, VB2 is 0.054. The PvuII polymorphism is in apparent linkage equilibrium with three other RFLPs of this gene cluster, detected with the restriction enzymes XmnI, PstI and SstI, but in linkage disequilibrium with an RFLP in the apoCIII gene also detected with PvuII. Taken together, these five RFLPs have a PIC (polymorphism information content) value of 0.8, and therefore are informative for genetic studies. Individuals with the genotype VB1VB2 had lower mean concentrations of apoAI, and HDL-cholesterol than individuals with the genotype VB1VB1. However these differences were not statistically significant.     

An unreported RFLP for probe 218 EP6 that is useful in linkage analysis of adult polycystic kidney disease

Probe 218 EP6 is known to recognise two restriction fragment length polymorphisms (RFLPs) after digestion of genomic DNA withPvuII. We report a rare allele that segregates in Mendelian fashion, in a family where adult polycystic kidney disease was being tracked using linked polymorphisms.