Detection of Multiple HPV Types in Liquid Biopsies of Cervical Neoplasia.

BACKGROUND: More than 95% of cervical cancers and their precancerous lesions are caused by human papillomavirus (HPV). Cell-free (cf) HPV DNA detection in blood samples may serve as a monitoring tool for cervical cancer. METHODS: In our methodological study, an HPV panel for simultaneous detection of 24 types using mass spectrometry-based analysis was developed for liquid biopsy approaches and tested on HPV positive cell lines, plasmid controls, and cervical high-grade squamous intraepithelial lesions (HSIL) in positive smear samples (n = 52). It was validated in cfDNA blood samples (n = 40) of cervical cancer patients. RESULTS: The HPV panel showed proficient results in cell lines and viral plasmids with a limit of detection of 1 IU (international units)/µL for HPV16/18 and 10GE/µL for HPV11/31/33/39/45/51/52/58/59 and a specificity of 100% for the tested HPV types. In cervical smear samples, HPV DNA was detected with a sensitivity of 98.14%. The overall agreement between the new HPV panel and clinical records was 97.2% (κ = 0.84). In cervical cancer cfDNA, 26/40 (65.0%) tested positive for any HPV type, with most infections due to hrHPV (24/26). HPV positive samples were found in all FIGO stages, with the highest positivity ratio in FIGO III and IV. Even the lowest stage, FIGO I, had 12/23 (52.2%) patients with a positive HPV plasma status. CONCLUSIONS: This proof-of-concept paper shows that the described assay produces reliable results for detecting HPV types in a multiplex mass spectrometry-based assay in cervical smear and cfDNA with high specificity and sensitivity in both cohorts. The assay shows potential for liquid biopsy-based applications in monitoring cervical cancer progression.

Low-Frequency Blood Group Antigens in Switzerland.

BACKGROUND: High-frequency blood group antigens (HFA) are present in >90% of the human population, according to some reports even in >99% of individuals. Therefore, patients lacking HFA may become challenging for transfusion support because compatible blood is hardly found, and if the patient carries alloantibodies, the cross-match will be positive with virtual every red cell unit tested. METHODS: In this study, we applied high-throughput blood group SNP genotyping on >37,000 Swiss blood donors, intending to identify homozygous carriers of low-frequency blood group antigens (LFA). RESULTS: 326 such individuals were identified and made available to transfusion specialists for future support of patients in need of rare blood products. CONCLUSION: Thorough comparison of minor allele frequencies using population genetics revealed heterogeneity of allele distributions among Swiss blood donors which may be explained by the topographical and cultural peculiarities of Switzerland. Moreover, geographically localized donor subpopulations are described which contain above-average numbers of individuals carrying rare blood group genotypes.

MNSs genotyping by MALDI-TOF MS shows high concordance with serology, allows gene copy number testing and reveals new St(a) alleles.

Results of genotyping with true high-throughput capability for MNSs antigens are underrepresented, probably because of technical issues, due to the high level of nucleotide sequence homology of the paralogous genes GYPA, GYPB and GYPE. Eight MNSs-specific single nucleotide polymorphisms (SNP) were detected using matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry (MALDI-TOF MS) in 5800 serologically M/N and S/s pre-typed Swiss blood donors and 50 individuals of known or presumptive black African ethnicity. Comparison of serotype with genotype delivered concordance rates of 99·70% and 99·90% and accuracy of genotyping alone of 99·88% and 99·95%, for M/N and S/s, respectively. The area under the curve of peak signals was measured in intron 1 of the two highly homologous genes GYPB and GYPE and allowed for gene copy number variation estimates in all individuals investigated. Elevated GYPB:GYPE ratios accumulated in several carriers of two newly observed GYP*401 variants, termed type G and H, both encoding for the low incidence antigen St(a). In black Africans, reduced GYPB gene contents were proven in pre-typed S-s-U- phenotypes and could be reproduced in unknown specimens. Quantitative gene copy number estimates represented a highly attractive supplement to conventional genotyping, solely based on MNSs SNPs.

High-throughput Kell, Kidd, and Duffy matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry-based blood group genotyping of 4000 donors shows close to full concordance with serotyping and detects new alleles.

BACKGROUND: After the ABO (ABO) and Rh (RHD and RHCE) blood group systems, Kell (KEL), Kidd (SLC14A1), and Duffy (DARC) represent the second most important clinically relevant antigens. STUDY DESIGN AND METHODS: Samples from 4000 Swiss blood donors, with serologic prevalues for K/k, Kp(a/b), Jk(a/b), and Fy(a/b), and 48 additional samples of presumptive black African origin were genotyped using high-throughput matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry, applying one single-multiplex polymerase chain reaction/primer-extension reaction simultaneously detecting 15 single-nucleotide polymorphisms. RESULTS: Genotype/phenotype concordance for K/k, Kp(a/b), Jk(a/b), and all Fy(a/b) specificities were 100, 99.98, 99.93, and 99.20%, respectively. Discrepancies were caused by erroneous serologic profiles (n = 33), mainly attributed to weakly expressed Fy(x) (n = 28). Only three discrepancies had a genetic basis. They could all be explained by newly observed silenced alleles: one KEL*02N.34 and one FY*02N.03 with predicted R700Q and G261R amino acid exchanges, respectively, and one JK*B, with an as-yet-unidentified silencing cause. According to NCBI SNP database entry for rs8176034, another new allele, KEL*02.38, had been expected, and we formally demonstrated its presence. We furthermore identified individuals with rare phenotypes, such as Js(a/b) heterozygotes among Caucasians, rare alleles, the "Swiss" JK*01N.03, and rare genotypes, such as one Fy(x) homozygote. CONCLUSION: Genotyping proved its practicability in the daily routine setting and qualitatively outperformed serology. Technology is ideal for time-insensitive donor genotyping and allows for a broad range of throughput needs. Consequently, from a technologic point of view, serotyping should be replaced by genotyping for donors' blood groups encoded by KEL, SLC14A1, and DARC.

Matrix-assisted laser desorption/ionisation, time-of-flight mass spectrometry-based blood group genotyping--the alternative approach.

Although matrix-assisted laser desorption/ionisation, time-of-flight mass spectrometry (MALDI-TOF MS) has previously been reported for high throughput blood group genotyping, those reports are limited to only a few blood group systems. This review describes the development of a large cooperative Swiss-German project, aiming to employ MALDI-TOF MS for the molecular detection of the blood groups Rh, Kell, Kidd, Duffy, MNSs, a comprehensive collection of low incidence antigens, as well as the platelet and granulocyte antigens HPA and HNA, representing a total of 101 blood group antigens, encoded by 170 alleles, respectively. Recent reports describe MALDI-TOF MS as a technology with short time-to-resolution, ability for high throughput, and cost-efficiency when used in genetic analysis, including forensics, pharmacogenetics, oncology and hematology. Furthermore, Kell and RhD genotyping have been performed on fetal DNA from maternal plasma with excellent results. In summary, this article introduces a new technological approach for high throughput blood group genotyping by means of MALDI-TOF MS. Although all data presented are preliminary, the observed success rates, data quality and concordance with known blood group types are highly impressive, underlining the accuracy and reliability of this cost-efficient high throughput method.

N-acetyltransferase 2, exposure to aromatic and heterocyclic amines, and receptor-defined breast cancer.

The role of N-acetyltransferase 2 (NAT2) polymorphism in breast cancer is still unclear. We explored the associations between potential sources of exposure to aromatic and heterocyclic amines (AHA), acetylation status and receptor-defined breast cancer in 1020 incident cases and 1047 population controls of the German GENICA study. Acetylation status was assessed as slow or fast. Therefore, NAT2 haplotypes were estimated using genotype information from six NAT2 polymorphisms. Most probable haplotypes served as alleles for the deduction of NAT2 acetylation status. The risks of developing estrogen receptor alpha (ER) and progesterone receptor (PR)-positive or negative tumors were estimated for tobacco smoking, consumption of red meat, grilled food, coffee, and tea, as well as expert-rated occupational exposure to AHA with logistic regression conditional on age and adjusted for potential confounders. Joint effects of these factors and NAT2 acetylation status were investigated. Frequent consumption of grilled food and coffee showed higher risks in slow acetylators for receptor-negative tumors [grilled food: ER-: odds ratio (OR) 2.57, 95% confidence interval (CI) 1.07-6.14 for regular vs. rare; coffee: ER-: OR 2.55, 95% CI 1.22-5.33 for >or=4 vs. 0 cups/day]. We observed slightly higher risks for never smokers that are fast acetylators for receptor-positive tumors compared with slow acetylators (ER-: OR 1.32, 95% CI 1.00-1.73). Our results support differing risk patterns for receptor-defined breast cancer. However, the modifying role of NAT2 for receptor-defined breast cancer is difficult to interpret in the light of complex mixtures of exposure to AHA.

Genetic variation in Fc gamma receptor IIa and risk of coronary heart disease: negative results from two large independent populations.

BACKGROUND: The role of the Fc gamma receptor IIa (Fc gamma RIIa), a receptor for C-reactive protein (CRP), the classical acute phase protein, in atherosclerosis is not yet clear. We sought to investigate the association of Fc gamma RIIa genotype with risk of coronary heart disease (CHD) in two large population-based samples. METHODS: Fc gamma RIIa-R/H131 polymorphisms were determined in a population of 527 patients with a history of myocardial infarction and 527 age and gender matched controls drawn from a population-based MONICA- Augsburg survey. In the LURIC population, 2227 patients with angiographically proven CHD, defined as having at least one stenosis >or= 50%, were compared with 1032 individuals with stenosis <50%. RESULTS: In both populations genotype frequencies of the Fc gamma RIIa gene did not show a significant departure from the Hardy-Weinberg equilibrium. Fc gamma RIIa R(-131) --> H genotype was not independently associated with lower risk of CHD after multivariable adjustments, neither in the MONICA population (odds ratio (OR) 1.08; 95% confidence interval (CI) 0.81 to 1.44), nor in LURIC (OR 0.96; 95% CI 0.81 to 1.14). CONCLUSION: Our results do not confirm an independent relationship between Fc gamma RIIa genotypes and risk of CHD in these populations.

Polymorphic loci of E2F2, CCND1 and CCND3 are associated with HER2 status of breast tumors.

Overexpression of the human epidermal growth factor receptor 2 (HER2) in breast tumors is associated with bad prognosis. Therefore, it is highly relevant to further improve understanding of the regulatory mechanisms of HER2 expression. In addition to gene amplification, transcriptional regulation plays a crucial role in HER2 overexpression. In this study, we analyzed 3 polymorphisms E2F2_-5368_A>G, CCND1_870_A>G and CCND3_-677_C>T located in genes involved in cell cycle regulation in the GENICA population-based and age-matched breast cancer case-control study from Germany. We genotyped 1,021 cases and 1,015 controls by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Statistical analyses were performed by conditional logistic regression. We observed no differences in genotype frequencies between breast cancer cases and controls. Subgroup analysis showed associations between carriers of the E2F2_-5368_G allele (OR: 0.60, 95% CI: 0.42-0.85), carriers of the CCND1_870_G allele (OR: 0.66, 95% CI: 0.45-0.96) and carriers of the CCND3_-677_T allele (OR: 1.72, 95% CI: 1.20-2.49) and HER2 expression in breast tumors. This finding points to an association of an increased expression of these cell cycle regulators with lower expression of HER2. An explanation for this observation might be that low expression of E2F2, CCND1 and CCND3 decrease levels of factors down-regulating HER2. We conclude that the analyzed polymorphisms located in E2F2, CCND1 and CCND3 are potential markers for HER2 status of breast tumors.

Joint analysis of individual participants' data from 17 studies on the association of the IL6 variant -174G>C with circulating glucose levels, interleukin-6 levels, and body mass index.

BACKGROUND: Several studies have investigated associations between the -174G>C single nucleotide polymorphism (rs1800795) of the IL6 gene and phenotypes related to type 2 diabetes mellitus (T2DM) but presented inconsistent results. AIMS: This joint analysis aimed to clarify whether IL6 -174G>C was associated with glucose and circulating interleukin-6 concentrations as well as body mass index (BMI). METHODS: Individual-level data from all studies of the IL6-T2DM consortium on Caucasian subjects with available BMI were collected. As study-specific estimates did not show heterogeneity (P>0.1), they were combined by using the inverse-variance fixed-effect model. RESULTS: The main analysis included 9440, 7398, 24,117, or 5659 non-diabetic and manifest T2DM subjects for fasting glucose, 2-hour glucose, BMI, or circulating interleukin-6 levels, respectively. IL6 -174 C-allele carriers had significantly lower fasting glucose (-0.091 mmol/L, P=0.014). There was no evidence for association between IL6 -174G>C and BMI or interleukin-6 levels, except in some subgroups. CONCLUSIONS: Our data suggest that C-allele carriers of the IL6 -174G>C polymorphism have lower fasting glucose levels on average, which substantiates previous findings of decreased T2DM risk of these subjects.

Estimating the single nucleotide polymorphism genotype misclassification from routine double measurements in a large epidemiologic sample.

Previously, estimation of genotype misclassification of single nucleotide polymorphisms (SNPs) as encountered in epidemiologic practice and involving thousands of subjects was lacking. The authors collected representative data on approximately 14,000 subjects from 8 studies and 646,558 genotypes assessed in 2005 by means of matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Overall discordance among 57,805 double genotypes from routine quality control was 0.36%. Fitting different misclassification models by maximum likelihood assuming identical misclassification for all SNPs, the estimated misclassification probabilities ranged from 0.0000 to 0.0035. When applying the misclassification simulation and extrapolation (MC-SIMEX) method for the first time to genetic data to account for the misclassification in a reanalysis of adiponectin-encoding (APM1) gene SNP associations with plasma adiponectin in 1,770 subjects, the authors found no impact of this small error on association estimates but increased estimates for a more substantial error. This study is the first to provide large-scale epidemiologic data on SNP genotype misclassification. The estimated misclassification in this example was small and negligible for association estimates, which is reassuring and essential for detecting SNP associations. In situations with more substantial error, the presented approach using duplicate genotyping and the MC-SIMEX method is practical and helpful for quantifying the genotyping error and its impact.

Evidence for a pathogenic role of different mutations at codon 188 of PRNP.

Clinical and pathological changes in familial Creutzfeldt-Jakob disease (CJD) cases may be similar or indistinguishable from sporadic CJD. Therefore determination of novel mutations in PRNP remains of major importance. We identified two different rare mutations in codon 188 of the prion protein gene (PRNP) in four patients suffering from a disease clinically very similar to the major subtype of sporadic CJD. Both mutations result in an exchange of the amino acid residue threonine for a highly basic residue, either arginine (T188R) or lysine (T188K). The T188R mutation was found in one patient and the T188K mutation in three patients. The prevalence of mutations at codon 188 of PRNP was tested in 593 sporadic CJD cases and 735 healthy individuals. Neither mutation was found. The data presented here argue in favor of T188K being a pathogenic mutation causing genetic CJD. Since one individual with this mutation, who is the father of a clinically affected patient with T188K mutation, is now 79 years old and shows no signs of disease, this mutation is likely associated with a penetrance under 100%. Further observations will have to show whether T188R is a pathogenic mutation.

On the replication of genetic associations: timing can be everything!

The failure of researchers to replicate genetic-association findings is most commonly attributed to insufficient statistical power, population stratification, or various forms of between-study heterogeneity or environmental influences.(1) Here, we illustrate another potential cause for nonreplications that has so far not received much attention in the literature. We illustrate that the strength of a genetic effect can vary by age, causing "age-varying associations." If not taken into account during the design and the analysis of a study, age-varying genetic associations can cause nonreplication. By using the 100K SNP scan of the Framingham Heart Study, we identified an age-varying association between a SNP in ROBO1 and obesity and hypothesized an age-gene interaction. This finding was followed up in eight independent samples comprising 13,584 individuals. The association was replicated in five of the eight studies, showing an age-dependent relationship (one-sided combined p = 3.92 x 10(-9), combined p value from pediatric cohorts = 2.21 x 10(-8), combined p value from adult cohorts = 0.00422). Furthermore, this study illustrates that it is difficult for cross-sectional study designs to detect age-varying associations. If the specifics of age- or time-varying genetic effects are not considered in the selection of both the follow-up samples and in the statistical analysis, important genetic associations may be missed.

Quantifying the contribution of genetic variants for survival phenotypes.

Particularly in studies based on population representative samples, it is of major interest what impact a genetic variant has on the phenotype of interest, which cannot be answered by mere association estimates alone. One possible measure for quantifying the phenotype's variance explained by the genetic variant is R(2). However, for survival outcomes, no clear definition of R(2) is available in the presence of censored observations. We selected three criteria proposed for this purpose in the literature and compared their performance for single nucleotide polymorphism (SNP) data through simulation studies and for mortality data with candidate SNPs in the general population-based KORA cohort. The evaluated criteria were based on: (1) the difference of deviance residuals, (2) the variation of individual survival curves, and (3) the variation of Schoenfeld residuals. Our simulation studies included various censoring and genetic scenarios. The simulation studies revealed that the deviance residuals' criterion had a high dependence on the censoring percentage, was generally not limited to the range [0; 1] and therefore lacked interpretation as a percentage of explained variation. The second criterion (variation of survival curves) hardly reached values above 60%. Our requirements were best fulfilled by the criterion based on Schoenfeld residuals. Our mortality data analysis also supported the findings in simulation studies. With the criterion based on Schoenfeld residuals, we recommend a powerful and flexible tool for genetic epidemiological studies to refine genetic association studies by judging the contribution of genetic variants to survival phenotype.

Association of the MC4R V103I polymorphism with the metabolic syndrome: the KORA Study.

OBJECTIVE: Epidemiological studies showing an association between the melanocortin-4-receptor (MC4R) 103I variant (rs2229616) and decreased BMI are complemented by functional studies; these suggest a mechanism for appetite regulation and a linkage signal for physical activity and dietary intake for the region encompassing the MC4R. This study aims to provide epidemiological evidence for showing the association of this polymorphism with features of the metabolic syndrome and with parameters related to energy expenditure and dietary habits as potential mediators. METHODS AND PROCEDURES: We analyzed this polymorphism in 7,888 adults of a population-based cross-sectional study applying regression-based statistical models. RESULTS: Carriers of the MC4R 103I (3.7%) exhibited a significantly decreased waist circumference (-1.46 cm, P = 0.020), decreased glycosylated hemoglobin (HbA(1c)) (-0.09%, P = 0.040), and increased HDL-cholesterol (HDL-C) (+1.76 mg/dl, P = 0.056), but no change in blood pressure. The odds of having three or more components of the metabolic syndrome were substantially reduced among carriers of MC4R 103I (odds ratio (OR) = 0.46, P = 0.003). Controlling for BMI reduced the HbA(1c) and HDL-C association. Mediator analyses revealed a borderline association of MC4R 103I with carbohydrate intake (OR = 1.26, P = 0.059) possibly mediating association with leanness. DISCUSSION: Our representative study of well-phenotyped Europeans is the first to describe the association of the MC4R V103I with the metabolic syndrome and with a nutrient-related phenotype. Our data support the idea that this polymorphism plays a role in appetite regulation that not only affects BMI, but also other features of the metabolic syndrome. It further establishes that the association of the MC4R V103I with obesity and related phenotypes is genuine.

Breast cancer: a candidate gene approach across the estrogen metabolic pathway.

Polymorphisms within the estrogen metabolic pathway are prime candidates for a possible association with breast cancer risk. We investigated 11 genes encoding key proteins of this pathway for their potential contribution to breast cancer risk. Of these CYP17A1, CYP19A1, EPHX1, HSD17B1, SRD5A2, and PPARG2 participate in biosynthesis, CYP1A1, CYP1B1, COMT, GSTP1, and SOD2 in catabolism and detoxification. We performed a population-based case-control study with 688 incident breast cancer cases and 724 controls from Germany and genotyped 18 polymorphisms by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), PCR based RFLP (restriction fragment length polymorphism), and TaqMan allelic discrimination. Genotype frequencies were compared between cases and controls and odds ratios were calculated by conditional logistic regression. Further statistical analyses were based on cluster analysis, multifactor dimensionality reduction, logic regression, and global testing. Single factor analyses pointed to CYP1B1_1294_GG as a possible breast cancer risk modulator (OR = 2.57; 95% CI: 1.34-4.93) and two way stratification suggested associations between BMI > or = 30 kg/m(2) and COMT_472_GG (P = 0.0076 and P = 0.0026), BMI < 20 kg/m(2) and HSD17B1_937_GG (P = 0.0082) as well as CYP17A1_-34_CC and HRT use > or =10 years (P = 0.0063). Following correction for multiple testing none of these associations remained significant. No significant association between breast cancer risk and genetic polymorphisms was observed in multifactor analyses. The tested polymorphisms of the estrogen metabolic pathway may not play a direct role in breast cancer risk. Therefore, future association studies should be extended to other polymorphisms and other regulatory pathways.

The CYP1B1_1358_GG genotype is associated with estrogen receptor-negative breast cancer.

Cytochrome P450 1B1 (CYP1B1) is a major enzyme in the initial catabolic step of estradiol (E2) metabolism and belongs to the multitude of genes regulated by the estrogen receptor alpha (ERalpha). The common non-synonymous polymorphisms CYP1B1_1358_A>G and CYP1B1_1294_C>G increase CYP1B1 enzymatic activity. Given a relationship between CYP1B1 and breast tumor E2 level as well as E2 level and breast tumor ERalpha expression it is of interest to know whether CYP1B1 polymorphisms have an impact on the ERalpha status of breast cancer. We genotyped the GENICA population-based breast cancer case-control collection (1,021 cases, 1,015 controls) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and investigated in cases the association between genotypes and tumor ERalpha status (739 ERalpha positive cases; 212 ERalpha negative cases) by logistic regression. We observed a significant association between the homozygous variant CYP1B1_1358_GG genotype and negative ERalpha status (P = 0.005; OR 2.82, 95% CI: 1.37-5.82) with a highly significant Ptrend for CYP1B1_1358_A>G and negative ERalpha status (P = 0.003). We also observed an association of CYP1B1_1358_GG and negative PR status (P = 0.015; OR 2.36, 95% CI: 1.18-4.70) and a Ptrend of 0.111 for CYP1B1_1358_A>G and negative progesterone receptor (PR) status. We conclude that the CYP1B1_1358_A>G polymorphism has an impact on ERalpha status in breast cancer in that the CYP1B1_1358_GG genotype known to encode higher CYP1B1 activity is associated with ERalpha negativity.

A novel polymorphism in the promoter region of ERBB4 is associated with breast and colorectal cancer risk.

PURPOSE: The receptor tyrosine kinase ERBB4/HER4 plays a role in cell division, migration, differentiation, as well as apoptosis, and is frequently overexpressed in breast and colorectal tumors. To understand the role of genetic variations in the regulation of ERBB4 expression, we identified new polymorphisms and investigated their functional implication and risk association with breast and colorectal cancer. EXPERIMENTAL DESIGN: We screened colorectal tumors from 92 patients for genetic variants at the ERBB4 ATG -1000 bp 5'-regulatory region by denaturing high-performance liquid chromatography and sequencing. Variants were subjected to DNA-protein interaction analyses (electrophoretic mobility shift assay), reporter gene assays in breast cancer cell lines MDA134 and MDA157, and immunohistochemical analyses of breast tumors. We established genotype frequencies within a breast cancer case-control collection (1,021 cases, 1,015 population-based controls) and a colorectal cancer case-control collection (459 cases, 569 blood donors) using matrix-assisted laser desorption ionization/time of flight mass spectrometry. Adjusted odds ratios (OR) and 95% confidence intervals (CI) were assessed by multivariate logistic regression. RESULTS: We identified five new germ line variants -815 A>T, -782 G>T, -638 insTC, -267 C>G, and -219 del10bp. Two variants showed in vitro functional effects. The -782T allele showed lower protein binding affinity and lower promoter activity compared with the -782G allele, however, the -815T allele showed higher protein binding affinity and higher promoter activity. The -782T variant was identified as a risk allele for breast and colorectal cancer (OR, 1.59; 95% CI, 1.06-2.34 and OR, 2.21; 95% CI, 1.22-3.99, respectively). CONCLUSION: The ERBB4 -782 G>T polymorphism, by virtue of its in vitro functional implication and incidence, is a risk factor for breast and colorectal cancer.

Polymorphism of the DNA repair enzyme XRCC1 is associated with treatment prediction in anthracycline and cyclophosphamide/methotrexate/5-fluorouracil-based chemotherapy of patients with primary invasive breast cancer.

OBJECTIVES: Outcome and survival in anthracycline-based and cyclophosphamide/methotrexate/5-fluorouracil-based chemotherapy of invasive breast cancer are unpredictable. Insights into treatment prediction are expected from studies searching for an association between genetic polymorphisms and treatment outcome effects. A common feature of treatment with chemoreagents is therapeutically induced DNA damage. Therefore, we tested the hypothesis of a relationship between event-free survival and genotype distributions of seven polymorphic DNA repair enzymes and four cell cycle regulators. BASIC METHODS: This case-case comparison included 180 patients with primary invasive breast cancer diagnosed between 1986 and 2000 and subjected to adjuvant chemotherapy (anthracycline/cyclophosphamide or cyclophosphamide/methotrexate/5-fluorouracil). Ninety-two patients were reported without recurrence and 88 were reported with recurrences or dead. Median clinical follow-up was 61.7 months. Constitutional DNA isolated from archived tissues was genotyped at 19 loci by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Statistical analyses included adjusted risk estimates, Kaplan-Meier analyses, Cox proportional hazard model, and permutation testing. MAIN RESULTS: Carriers of the XRCC1_1196_AA genotype had a reduced risk for recurrence/death (odds ratio adjusted 0.19; 95% confidence interval: 0.06-0.61), which was observed in survival analyses of all patients (P=0.003) and patients treated with chemotherapy but not radiotherapy (P=0.006). Multivariate analysis confirmed XRCC1 as a potential treatment predictor (hazard ratio 0.62; 95% confidence interval: 0.43-0.89). The result was stable upon permutation testing. No other significant associations were observed. CONCLUSION: The DNA repair enzyme XRCC1 is a potential treatment predictor for the outcome and survival of anthracycline and cyclophosphamide/methotrexate/5-fluorouracil-based chemotherapy of invasive breast cancer.

The association of a SNP upstream of INSIG2 with body mass index is reproduced in several but not all cohorts.

A SNP upstream of the INSIG2 gene, rs7566605, was recently found to be associated with obesity as measured by body mass index (BMI) by Herbert and colleagues. The association between increased BMI and homozygosity for the minor allele was first observed in data from a genome-wide association scan of 86,604 SNPs in 923 related individuals from the Framingham Heart Study offspring cohort. The association was reproduced in four additional cohorts, but was not seen in a fifth cohort. To further assess the general reproducibility of this association, we genotyped rs7566605 in nine large cohorts from eight populations across multiple ethnicities (total n = 16,969). We tested this variant for association with BMI in each sample under a recessive model using family-based, population-based, and case-control designs. We observed a significant (p < 0.05) association in five cohorts but saw no association in three other cohorts. There was variability in the strength of association evidence across examination cycles in longitudinal data from unrelated individuals in the Framingham Heart Study Offspring cohort. A combined analysis revealed significant independent validation of this association in both unrelated (p = 0.046) and family-based (p = 0.004) samples. The estimated risk conferred by this allele is small, and could easily be masked by small sample size, population stratification, or other confounders. These validation studies suggest that the original association is less likely to be spurious, but the failure to observe an association in every data set suggests that the effect of SNP rs7566605 on BMI may be heterogeneous across population samples.

Calpain-10 variants and haplotypes are associated with polycystic ovary syndrome in Caucasians.

PCOS is known to be associated with an increased risk of T2DM and has been proposed to share a common genetic background with T2DM. Recent studies suggest that the Calpain-10 gene (CAPN10) is an interesting candidate gene for PCOS susceptibility. However, contradictory results were reported concerning the contribution of certain CAPN10 variants, especially of UCSNP-44, to genetic predisposition to T2DM, hirsutism, and PCOS. By means of MALDI-TOF MS technique, we genotyped an expanded single nucleotide polymorphism panel, including the CAPN10 UCSNP-44, -43, -56, ins/del-19, -110, -58, -63, and -22 in a sample of 146 German PCOS women and 606 population-based controls. Statistical analysis revealed an association between UCSNP-56 and susceptibility to PCOS with an odds ratio (OR) of 2.91 (95% CI=1.51-5.61) for women carrying an AA genotype compared with GG. As expected, the 22-genotype of the ins/del-19 variant, which is in high linkage disequilibrium (r2=0.98) with UCSNP-56, was also significantly associated (OR=2.98, 95% CI=1.55-5.73). None of the additionally tested variants alone showed any significant association with PCOS. A meta-analysis including our study (altogether 623 PCOS cases and 1,224 controls) also showed significant association only with ins/del-19. The most common haplotype TGG3AGCA was significantly associated with a lower risk for PCOS (OR=0.487, P=0.0057). In contrast, the TGA2AGCA haplotype was associated with an increased risk for PCOS (OR=3.557, P=0.0011). By investigating a broad panel of CAPN10 variants, our results pointed to an allele dose-dependent association of UCSNP-56 and ins/del-19 with PCOS.