Harald Surowy

Exploring the genetics of irritable bowel syndrome: a GWA study in the general population and replication in multinational case-control cohorts

Objective IBS shows genetic predisposition, but adequately powered gene-hunting efforts have been scarce so far. We sought to identify true IBS genetic risk factors by means of genome-wide association (GWA) and independent replication studies. Design We conducted a GWA study (GWAS) of IBS in a general population sample of 11 326 Swedish twins. IBS cases (N=534) and asymptomatic controls (N=4932) were identified based on questionnaire data. Suggestive association signals were followed-up in 3511 individuals from six case-control cohorts. We sought genotype-gene expression correlations through single nucleotide polymorphism (SNP)-expression quantitative trait loci interactions testing, and performed in silico prediction of gene function. We compared candidate gene expression by real-time qPCR in rectal mucosal biopsies of patients with IBS and controls. Results One locus at 7p22.1, which includes the genes KDELR2 (KDEL endoplasmic reticulum protein retention receptor 2) and GRID2IP (glutamate receptor, ionotropic, delta 2 (Grid2) interacting protein), showed consistent IBS risk effects in the index GWAS and all replication cohorts and reached p=9.31×10−6 in a meta-analysis of all datasets. Several SNPs in this region are associated with cis effects on KDELR2 expression, and a trend for increased mucosal KDLER2 mRNA expression was observed in IBS cases compared with controls. Conclusions Our results demonstrate that general population-based studies combined with analyses of patient cohorts provide good opportunities for gene discovery in IBS. The 7p22.1 and other risk signals detected in this study constitute a good starting platform for hypothesis testing in future functional investigations.

Age- and Tumor Subtype–Specific Breast Cancer Risk Estimates for CHEK2*1100delC Carriers

PURPOSE CHEK2*1100delC is a well-established breast cancer risk variant that is most prevalent in European populations; however, there are limited data on risk of breast cancer by age and tumor subtype, which limits its usefulness in breast cancer risk prediction. We aimed to generate tumor subtype- and age-specific risk estimates by using data from the Breast Cancer Association Consortium, including 44,777 patients with breast cancer and 42,997 controls from 33 studies genotyped for CHEK2*1100delC. PATIENTS AND METHODS CHEK2*1100delC genotyping was mostly done by a custom Taqman assay. Breast cancer odds ratios (ORs) for CHEK2*1100delC carriers versus noncarriers were estimated by using logistic regression and adjusted for study (categorical) and age. Main analyses included patients with invasive breast cancer from population- and hospital-based studies. RESULTS Proportions of heterozygous CHEK2*1100delC carriers in controls, in patients with breast cancer from population- and hospital-based studies, and in patients with breast cancer from familial- and clinical genetics center-based studies were 0.5%, 1.3%, and 3.0%, respectively. The estimated OR for invasive breast cancer was 2.26 (95%CI, 1.90 to 2.69; P = 2.3 × 10(-20)). The OR was higher for estrogen receptor (ER)-positive disease (2.55 [95%CI, 2.10 to 3.10; P = 4.9 × 10(-21)]) than it was for ER-negative disease (1.32 [95%CI, 0.93 to 1.88; P = .12]; P interaction = 9.9 × 10(-4)). The OR significantly declined with attained age for breast cancer overall (P = .001) and for ER-positive tumors (P = .001). Estimated cumulative risks for development of ER-positive and ER-negative tumors by age 80 in CHEK2*1100delC carriers were 20% and 3%, respectively, compared with 9% and 2%, respectively, in the general population of the United Kingdom. CONCLUSION These CHEK2*1100delC breast cancer risk estimates provide a basis for incorporating CHEK2*1100delC into breast cancer risk prediction models and into guidelines for intensified screening and follow-up.

Predisposition for TMPRSS2-ERG Fusion in Prostate Cancer by Variants in DNA Repair Genes

The somatic fusion of TMPRSS2 to ETS oncogenes is a common event in prostate cancer (PCa). We hypothesized that defects in DNA repair may lead to an increase of chromosomal rearrangements and thus to the occurrence of ETS oncogene fusion. We have previously conducted a genome-wide linkage analysis in TMPRSS2-ERG fusion-positive PCa families, revealing potential susceptibility loci on chromosomes 5q14, 9q21, 10q26, 11q24, 12q15, 13q12, 18q, and Xq27. In the present study, nine candidate genes from these regions were selected from the context of DNA repair and screened for mutations in TMPRSS2-ERG fusion-positive families. Thirteen nonsynonymous variants, 5 of which had a minor allele frequency of <0.05, were genotyped in 210 familial cases, 47 of which with a known TMPRSS2-ERG status, 329 sporadic cases, and 512 controls. Significant association of TMPRSS2-ERG fusion-positive PCa was found with rare variants in the genes for POLI [variant F532S: P = 0.0011; odds ratios (OR), 4.62; 95% confidence interval (95% CI), 1.84-11.56] and ESCO1 (variant N191S: P = 0.0034; OR, 4.27; 95% CI, 1.62-11.28). Additional findings, regardless of TMPRSS2-ERG status, were the overrepresentation of a rare BRCA2 variant (V2728I: P = 0.03; OR, 6.16; 95% CI, 1.19-32.00) in familial PCa and of a common allele of RMI1 (variant N455S: P = 0.02; OR, 1.33; 95% CI, 1.04-1.70) in unselected PCa cases. The DNA repair genes POLI and ESCO1 are proposed as susceptibility genes for TMPRSS2-ERG fusion-positive PCa that warrant further investigation. (Cancer Epidemiol Biomarkers Prev 2009;18(11):3030–5)

FANCM c.5791C>T nonsense mutation (rs144567652) induces exon skipping, affects DNA repair activity and is a familial breast cancer risk factor

Numerous genetic factors that influence breast cancer risk are known. However, approximately two-thirds of the overall familial risk remain unexplained. To determine whether some of the missing heritability is due to rare variants conferring high to moderate risk, we tested for an association between the c.5791C>T nonsense mutation (p.Arg1931*; rs144567652) in exon 22 of FANCM gene and breast cancer. An analysis of genotyping data from 8635 familial breast cancer cases and 6625 controls from different countries yielded an association between the c.5791C>T mutation and breast cancer risk [odds ratio (OR) = 3.93 (95% confidence interval (CI) = 1.28-12.11; P = 0.017)]. Moreover, we performed two meta-analyses of studies from countries with carriers in both cases and controls and of all available data. These analyses showed breast cancer associations with OR = 3.67 (95% CI = 1.04-12.87; P = 0.043) and OR = 3.33 (95% CI = 1.09-13.62; P = 0.032), respectively. Based on information theory-based prediction, we established that the mutation caused an out-of-frame deletion of exon 22, due to the creation of a binding site for the pre-mRNA processing protein hnRNP A1. Furthermore, genetic complementation analyses showed that the mutation influenced the DNA repair activity of the FANCM protein. In summary, we provide evidence for the first time showing that the common p.Arg1931* loss-of-function variant in FANCM is a risk factor for familial breast cancer.

Heritability of baseline and induced micronucleus frequencies

The scoring of micronuclei (MN) is widely used in biomonitoring and mutagenicity testing as a surrogate marker of chromosomal damage inflicted by clastogenic agents or by aneugens. Individual differences in the response to a mutagenic challenge are known from studies on cancer patients and carriers of mutations in DNA repair genes. However, it has not been studied to which extent genetic factors contribute to the observed variability of individual MN frequencies. Our aim was to quantify this heritable genetic component of both baseline and radiation-induced MN frequencies. We performed a twin study comprising 39 monozygotic (MZ) and 10 dizygotic (DZ) twin pairs. Due to the small number of DZ pairs, we had to recruit controls from which 38 age- and gender-matched random control pairs (CPs) were generated. For heritability estimates, we used biometrical modelling of additive genetic, common environmental, and unique environmental components (ACE model) of variance and direct comparison of variance between the sample groups. While heritability estimates from MZ to DZ comparisons produced inconclusive results, both estimation methods revealed a high degree of heritability (h(2)) for baseline MN frequency (h(2) = 0.68 and h(2) = 0.72) as well as for the induced frequency (h(2) = 0.68 and h(2) = 0.57) when MZ were compared to CP. The result was supported by the different intraclass correlation coefficients of MZ, DZ and CP for baseline (r = 0.63, r = 0.31 and r = 0.0, respectively) as well as for induced MN frequencies (r = 0.79, r = 0.74 and r = 0.0, respectively). This study clearly demonstrates that MN frequencies are determined by genetic factors to a major part. The strong reflection of the genetic background supports the idea that MN frequencies represent an intermediate phenotype between molecular DNA repair mechanisms and the cancer phenotype and affirms the approaches that are made to utilise them as predictors of, for example, cancer risk.

Blood-based DNA methylation as biomarker for breast cancer: a systematic review

Multiple studies have investigated global DNA methylation profiles and gene-specific DNA methylation in blood-based DNA to develop powerful screening markers for cancer. This systematic review summarizes the current evidence on methylation studies that investigated methylation level of blood-derived DNA of breast cancer (BC) patients in comparison to healthy controls by conducting a systematic literature review in PubMed and Web of Science. Essential results, such as methylation levels of BC cases and healthy controls, p values, and odds ratios, were extracted from these studies by two investigators independently. Overall, 45 publications met the inclusion criteria for this review. DNA from whole blood, as well as cell-free DNA (cfDNA) from serum or plasma, was used in these studies. The most common method used for measuring global DNA methylation was the investigation of repetitive elements as surrogates and the application of array-based genome-wide methylation analysis. For measuring gene-specific methylation level, methylation-specific PCR and pyrosequencing were the most frequently used methods. Epigenome-wide blood DNA hypomethylation in BC patients were reported in several studies; however, the evidence is still not conclusive. The most frequently investigated gene in whole blood was BRCA1, which was found more frequently methylated in patients compared to controls. RASSF1A was the most widely investigated gene in cfDNA of serum or plasma, which was also found more frequently methylated in patients compared to controls. Several of the eligible studies reported the associations of global hypomethylation and increased BC risk. Studies investigated associations between gene-specific methylation and BC risk, while got heterogeneous results. But two studies reported that hypermethylation of ATM gene was associated with increased BC risk, which suggest the potential use of this gene for BC risk stratification. Overall, our review suggests the possibility of using blood-based DNA methylation marker as promising marker for BC risk stratification, as several studies found associations between certain methylation level in blood and BC risk. However, so far, the evidence is still quite limited. Optimal markers are yet to be developed and promising results needed to be validated in prospective study cohorts and tested in large screening populations.

Capture and Amplification by Tailing and Switching (CATS): An ultrasensitive ligation-independent method for generation of DNA libraries for deep sequencing from picogram amounts of DNA and RNA

Massive parallel sequencing (MPS) technologies have paved the way into new areas of research including individualized medicine. However, sequencing of trace amounts of DNA or RNA still remains a major challenge, especially for degraded nucleic acids like circulating DNA. This together with high cost and time requirements impedes many important applications of MPS in medicine and fundamental science. We have established a fast, cheap and highly efficient protocol called ‘Capture and Amplification by Tailing and Switching’ (CATS) to directly generate ready-to-sequence libraries for MPS from nanogram and picogram quantities of both DNA and RNA. Furthermore, those DNA libraries are strand-specific, can be prepared within 2–3 h and do not require preliminary sample amplification steps. To exemplify the capacity of the technique, we have generated and sequenced DNA libraries from hundred-picogram amounts of circulating nucleic acids isolated from human blood plasma, one nanogram of mRNA-enriched total RNA from cultured cells and few nanograms of bisulfite-converted DNA. The approach for DNA library preparation from minimal and fragmented input described here will find broad application in diverse research areas such as translational medicine including therapy monitoring, prediction, prognosis and early detection of various human disorders and will permit high-throughput DNA sequencing from previously inaccessible material such as minute forensic and archeological samples.

Increased Radiosensitivity as an Indicator of Genes Conferring Breast Cancer Susceptibility

Purpose:This paper briefly summarizes the research on increased radiosensitivity in breast cancer patients measured by the micronucleus test (MNT) and its association to genetic variants in DNA repair genes. More preliminary data are presented on the distribution of chromosomes and chromosome fragments in micronuclei (MN) in order to gain more information on clastogenic and aneugenic effects and better understand the phenotype of increased radiosensitivity.Material and Methods:Reports of relevant studies obtained from a search of PubMed and studies referenced in those reports were reviewed. In four patients with high MN frequency (three cancer patients, one control) and four probands with low MN frequency, the presence of chromosome fragments or whole chromosomes in MN was determined by fluorescence in situ hybridization analysis for chromosomes 1, 7, and 17.Results:An increased MN frequency in breast cancer patients compared to controls has consistently been reported with high significance. Higher MN frequencies were observed in 20–50% of breast cancer patients. Chromosomal fragments of chromosome 17, but not of chromosomes 1 and 7 were more frequent in the probands with high MN frequency than in those with low frequency (p = 0.045).Conclusion:The MNT detects a cellular phenotype common to a portion of sporadic breast cancer patients. This phenotype is very likely to be genetically determined. For the genetic dissection of breast cancer susceptibility this phenotype may turn out to be more efficient than breast cancer itself. Additional parameters which can be measured simultaneously with the MN frequency may be able to further enhance its usefulness.Ziel:Diese Arbeit fasst die bisherige Forschung zur erhöhten Radiosensitivität bei Brustkrebspatientinnen zusammen, wie sie mit Hilfe des Mikronukleustests (MNT) bestimmt werden kann. Des Weiteren zeigen vorläufige Fluoreszenz-in-situ-Hybridisierungs-(FISH-)Analysen die Verteilung einzelner Chromosomen oder Chromosomenbruchstücke in Mikrokernen (MN), um aneugene oder klastogene Effekte zu detektieren und mehr Informationen über den Phänotyp der erhöhten Radiosensitivität zu erhalten.Material und Methodik:Als Grundlage für diese Übersicht dienten in PubMed veröffentlichte Untersuchungen sowie darin zitierte Studien. Des Weiteren wurden bei vier Probandinnen mit hohen MN-Raten (drei Krebspatientinnen, eine Kontrolle) und vier Probandinnen mit niedrigen MN-Raten MN mittels FISH auf die Frage hin untersucht, ob sie Fragmente oder die ganzen Chromosomen 1, 7 und 17 enthalten.Ergebnisse:Brustkrebspatientinnen und Kontrollen zeigen in der Literatur statistisch signifikante Unterschiede bezüglich der MN-Rate. Höhere MN-Raten wurden bei 20–50% der Brustkrebspatientinnen beobachtet. Die Chromosomenfragmente von Chromosom 17 kamen häufiger bei Probandinnen mit hoher MN-Rate als bei Probandinnen mit niedriger MN-Rate vor (p = 0,045).Schlussfolgerung:Der MNT detektiert einen Phänotyp, welcher einen Anteil der sporadischen Brustkrebspatientinnen erfasst. Dieser Phänotyp ist wahrscheinlich genetisch determiniert. Für genetische Untersuchungen zur Brustkrebssuszeptibilität könnte sich dieser intermediäre Phänotyp als geeigneter erweisen als das Auftreten der Erkrankung Brustkrebs selbst. Zusätzliche Parameter, die zusammen mit der MN-Rate bestimmt werden können, könnten seine Anwendbarkeit verbessern.

RAD51B in Familial Breast Cancer

Common variation on 14q24.1, close to RAD51B, has been associated with breast cancer: rs999737 and rs2588809 with the risk of female breast cancer and rs1314913 with the risk of male breast cancer. The aim of this study was to investigate the role of RAD51B variants in breast cancer predisposition, particularly in the context of familial breast cancer in Finland. We sequenced the coding region of RAD51B in 168 Finnish breast cancer patients from the Helsinki region for identification of possible recurrent founder mutations. In addition, we studied the known rs999737, rs2588809, and rs1314913 SNPs and RAD51B haplotypes in 44,791 breast cancer cases and 43,583 controls from 40 studies participating in the Breast Cancer Association Consortium (BCAC) that were genotyped on a custom chip (iCOGS). We identified one putatively pathogenic missense mutation c.541C>T among the Finnish cancer patients and subsequently genotyped the mutation in additional breast cancer cases (n = 5259) and population controls (n = 3586) from Finland and Belarus. No significant association with breast cancer risk was seen in the meta-analysis of the Finnish datasets or in the large BCAC dataset. The association with previously identified risk variants rs999737, rs2588809, and rs1314913 was replicated among all breast cancer cases and also among familial cases in the BCAC dataset. The most significant association was observed for the haplotype carrying the risk-alleles of all the three SNPs both among all cases (odds ratio (OR): 1.15, 95% confidence interval (CI): 1.11–1.19, P = 8.88 x 10−16) and among familial cases (OR: 1.24, 95% CI: 1.16–1.32, P = 6.19 x 10−11), compared to the haplotype with the respective protective alleles. Our results suggest that loss-of-function mutations in RAD51B are rare, but common variation at the RAD51B region is significantly associated with familial breast cancer risk.

Cell-free circulating DNA integrity is an independent predictor of impending breast cancer recurrence

Non-invasive blood-based molecule markers are evaluated as promising biomarkers these days. Here we investigated the potential of cell-free circulating DNA Integrity (cfDI) as blood-based marker for the prediction of recurrence during the follow-up of breast cancer patients within a prospective study cohort. cfDI was determined in plasma of 212 individuals, by measuring ALU and LINE1 repetitive DNA elements using quantitative PCR. A significant decrease of cfDI in recurrent breast cancer patients was observed. The group of patients who had impending recurrence during the follow-up had significant lower cfDI compared to the group of non-recurrent patients (P < 0.001 for ALU and LINE1 cfDI). cfDI could differentiate recurrent breast cancer patients from non-recurrent breast cancer subjects (area under the curve, AUC = 0.710 for ALU and 0.704 for LINE1). Univariate and multivariate analysis confirmed a significant association of recurrence and cfDI. Breast cancer patients with a lower cfDI had a much higher risk to develop recurrence than the patients with a higher cfDI (P = 0.020 for ALU cfDI and P = 0.019 for LINE1 cfDI, respectively). Further we show that cfDI is an independent predictor of breast cancer recurrence. In combination with other molecular markers, cfDI might be a useful biomarker for the prediction for breast cancer recurrence in clinic utility. We propose that cfDI might also be useful for the prediction of recurrence during the follow-up of other cancers.Non-invasive blood-based molecule markers are evaluated as promising biomarkers these days. Here we investigated the potential of cell-free circulating DNA Integrity (cfDI) as blood-based marker for the prediction of recurrence during the follow-up of breast cancer patients within a prospective study cohort. cfDI was determined in plasma of 212 individuals, by measuring ALU and LINE1 repetitive DNA elements using quantitative PCR. A significant decrease of cfDI in recurrent breast cancer patients was observed. The group of patients who had impending recurrence during the follow-up had significant lower cfDI compared to the group of non-recurrent patients (P < 0.001 for ALU and LINE1 cfDI). cfDI could differentiate recurrent breast cancer patients from non-recurrent breast cancer subjects (area under the curve, AUC = 0.710 for ALU and 0.704 for LINE1). Univariate and multivariate analysis confirmed a significant association of recurrence and cfDI. Breast cancer patients with a lower cfDI had a much higher risk to develop recurrence than the patients with a higher cfDI (P = 0.020 for ALU cfDI and P = 0.019 for LINE1 cfDI, respectively). Further we show that cfDI is an independent predictor of breast cancer recurrence. In combination with other molecular markers, cfDI might be a useful biomarker for the prediction for breast cancer recurrence in clinic utility. We propose that cfDI might also be useful for the prediction of recurrence during the follow-up of other cancers.