Justo Lorenzo Bermejo

Atlas of the clinical genetics of human dilated cardiomyopathy

AIM Numerous genes are known to cause dilated cardiomyopathy (DCM). However, until now technological limitations have hindered elucidation of the contribution of all clinically relevant disease genes to DCM phenotypes in larger cohorts. We now utilized next-generation sequencing to overcome these limitations and screened all DCM disease genes in a large cohort. METHODS AND RESULTS In this multi-centre, multi-national study, we have enrolled 639 patients with sporadic or familial DCM. To all samples, we applied a standardized protocol for ultra-high coverage next-generation sequencing of 84 genes, leading to 99.1% coverage of the target region with at least 50-fold and a mean read depth of 2415. In this well characterized cohort, we find the highest number of known cardiomyopathy mutations in plakophilin-2, myosin-binding protein C-3, and desmoplakin. When we include yet unknown but predicted disease variants, we find titin, plakophilin-2, myosin-binding protein-C 3, desmoplakin, ryanodine receptor 2, desmocollin-2, desmoglein-2, and SCN5A variants among the most commonly mutated genes. The overlap between DCM, hypertrophic cardiomyopathy (HCM), and channelopathy causing mutations is considerably high. Of note, we find that >38% of patients have compound or combined mutations and 12.8% have three or even more mutations. When comparing patients recruited in the eight participating European countries we find remarkably little differences in mutation frequencies and affected genes. CONCLUSION This is to our knowledge, the first study that comprehensively investigated the genetics of DCM in a large-scale cohort and across a broad gene panel of the known DCM genes. Our results underline the high analytical quality and feasibility of Next-Generation Sequencing in clinical genetic diagnostics and provide a sound database of the genetic causes of DCM.

The balance between heritable and environmental aetiology of human disease.

The Human Genome Project and the ensuing International HapMap Project were largely motivated by human health issues. But the distance from a DNA sequence variation to a novel disease gene is considerable; for complex diseases, closing this gap hinges on the premise that they arise mainly from heritable causes. Using cancer as an example of complex disease, we examine the scientific evidence for the hypothesis that human diseases result from interactions between genetic variants and the environment.

Methylome analysis and integrative profiling of human HCCs identify novel protumorigenic factors

To identify new tumor‐suppressor gene candidates relevant for human hepatocarcinogenesis, we performed genome‐wide methylation profiling and vertical integration with array‐based comparative genomic hybridization (aCGH), as well as expression data from a cohort of well‐characterized human hepatocellular carcinomas (HCCs). Bisulfite‐converted DNAs from 63 HCCs and 10 healthy control livers were analyzed for the methylation status of more than 14,000 genes. After defining the differentially methylated genes in HCCs, we integrated their DNA copy‐number alterations as determined by aCGH data and correlated them with gene expression to identify genes potentially silenced by promoter hypermethylation. Aberrant methylation of candidates was further confirmed by pyrosequencing, and methylation dependency of silencing was determined by 5‐aza‐2′‐deoxycytidine (5‐aza‐dC) treatment. Methylation profiling revealed 2,226 CpG sites that showed methylation differences between healthy control livers and HCCs. Of these, 537 CpG sites were hypermethylated in the tumor DNA, whereas 1,689 sites showed promoter hypomethylation. The hypermethylated set was enriched for genes known to be inactivated by the polycomb repressive complex 2, whereas the group of hypomethylated genes was enriched for imprinted genes. We identified three genes matching all of our selection criteria for a tumor‐suppressor gene (period homolog 3 [PER3], insulin‐like growth‐factor–binding protein, acid labile subunit [IGFALS], and protein Z). PER3 was down‐regulated in human HCCs, compared to peritumorous and healthy liver tissues. 5‐aza‐dC treatment restored PER3 expression in HCC cell lines, indicating that promoter hypermethylation was indeed responsible for gene silencing. Additionally, functional analysis supported a tumor‐suppressive function for PER3 and IGFALS in vitro. Conclusion: The present study illustrates that vertical integration of methylation data with high‐resolution genomic and transcriptomic data facilitates the identification of new tumor‐suppressor gene candidates in human HCC. (HEPATOLOGY 2012;56:1817–1827)

A genome-wide association study identifies 6p21 as novel risk locus for dilated cardiomyopathy

AIMS Dilated cardiomyopathy (DCM) is one of the leading causes for cardiac transplantations and accounts for up to one-third of all heart failure cases. Since extrinsic and monogenic causes explain only a fraction of all cases, common genetic variants are suspected to contribute to the pathogenesis of DCM, its age of onset, and clinical progression. By a large-scale case-control genome-wide association study we aimed here to identify novel genetic risk loci for DCM. METHODS AND RESULTS Applying a three-staged study design, we analysed more than 4100 DCM cases and 7600 controls. We identified and successfully replicated multiple single nucleotide polymorphism on chromosome 6p21. In the combined analysis, the most significant association signal was obtained for rs9262636 (P = 4.90 × 10(-9)) located in HCG22, which could again be replicated in an independent cohort. Taking advantage of expression quantitative trait loci (eQTL) as molecular phenotypes, we identified rs9262636 as an eQTL for several closely located genes encoding class I and class II major histocompatibility complex heavy chain receptors. CONCLUSION The present study reveals a novel genetic susceptibility locus that clearly underlines the role of genetically driven, inflammatory processes in the pathogenesis of idiopathic DCM.

Genetic variation in adipokine genes and risk of colorectal cancer

OBJECTIVE Obesity has been related to an increased risk of colorectal cancer (CRC). Adipokines produced by the adipose tissue are directly linked to obesity and may thus contribute to the pathogenesis of CRC. We hypothesized that potentially functional polymorphisms in the adipokine genes leptin (LEP), leptin receptor (LEPR), resistin (RETN), and adiponectin (ADIPOQ) may be associated with CRC. DESIGN AND METHODS We studied the association of four putatively functional single nucleotide polymorphisms (SNPs) with CRC risk using a hospital-based study design with 702 cases and 752 controls from the Czech Republic. We used likelihood ratio tests to select the best model to represent the relationship between genotypes and risk of CRC. Age-adjusted odds ratios (ORs) under the best model were calculated for each SNP. Previous genotyping data on insulin (INS)-related genes were used to explore interactions between genes in obesity- and diabetes-related pathways by using two independent methods, logistic regression, and multifactor-dimensionality reduction. RESULTS A trend to associate between the RETN SNP rs1862513 (C-420G) and CRC risk was observed (per allele OR 1.18, 95% confidence interval (0.99-1.40). Statistically, significant interactions were observed between the INS SNP rs3842754 (+1127INSPstI) genotypes and both the LEPR SNP rs1137101 (Q223R) and the ADIPOQ SNP rs266729 (C-11374G) genotypes. CONCLUSIONS Our results suggest that variants in the adipokine genes may affect CRC risk in combination with variants in diabetes-related genes.

LGR5 is a marker of poor prognosis in glioblastoma and is required for survival of brain cancer stem-like cells

In various types of cancers including glioblastoma, accumulating evidence show the existence of cancer stem‐like cells (CSCs), characterized by stem cell marker expression, capability of differentiation and self‐renewal, and high potential for tumor propagation in vivo. LGR5, whose expression is positively regulated by the Wnt signaling pathway, is a stem cell marker in intestinal mucosa and hair follicle in the skin. As Wnt signaling is also involved in brain development, the function of LGR5 in the maintenance of brain CSCs is to be assessed. Our study showed that the LGR5 transcript level was increased in CSCs. Co‐immunofluorescence staining demonstrated the co‐localization of CD133‐ and LGR5‐positive cells in glioblastoma tissue sections. Functionally, silencing of LGR5 by lentiviral shRNA‐mediated knockdown induced apoptosis in brain CSCs. Moreover, LGR5 depletion led to a downregulation of L1 cell adhesion molecule expression. In line with an important function in glioma tumorigenesis, LGR5 expression increased with glioma progression and correlated with an adverse outcome. Our findings suggest that LGR5 plays a role in maintenance and/or survival of brain CSCs.

Genome-wide methylation screen in low-grade breast cancer identifies novel epigenetically altered genes as potential biomarkers for tumor diagnosis

Aberrant DNA methylation constitutes a well‐established epigenetic marker for breast cancer. Changes in methylation early in cancer development may be clinically relevant for cancer detection and prognosis‐based therapeutic decisions. In the present study, a combination of methyl‐CpG immunoprecipitation (MCIp) and human CpG island (CGI) arrays was applied to compare genome‐wide DNA methylation profiles in 10 low‐grade in situ and invasive breast cancers against 10 normal breast samples. In total, 214 CGIs were found to be hypermethylated in ≥6 of 10 tumors. Functional term enrichment analyses revealed an overrepresentation of homeobox genes and genes involved in transcription and regulation of transcription. Significant hypermethylation of 11 selected genes in tumor vs. normal tissue was validated in two independent sample sets (45 tumors and 11 controls, 43 tumors and 8 controls) using quantitative EpiTyper technology. In tumors, median methylation levels of BCAN, HOXD1, KCTD8, KLF11, NXPH1, POU4F1, SIM1, and TCF7L1 were ≥30% higher than in normal samples, representing potential biomarkers for tumor diagnosis. Using the 90th percentile of methylation levels in normal tissue as cutoff value, 62–92% of in situ samples (n=13), 72–97% of invasive samples from the first validation set (n=32), and 86–100% of invasive samples from the second validation set (n=43) were classified as hypermethylated. Hypermethylation of KLF11 and SIM1 might also be associated with increased risk of developing metastases. In summary, early methylation changes are frequent in the low‐grade pathway of breast cancer and may be useful in the development of differential diagnostic and possibly also prognostic markers.—Faryna, M., Konermann, C., Aulmann, S., Bermejo, J. L., Brugger, M., Diederichs, S., Rom, J., Weichenhan, D., Claus, R., Rehli, M., Schirmacher, P., Sinn, H.‐P., Plass, C., Gerhauser, C. Genome‐wide methylation screen in low‐grade breast cancer identifies novel epigenetically altered genes as potential biomarkers for tumor diagnosis. FASEB J. 26, 4937–4950 (2012). www.fasebj.org

Melanocortin receptor 1 variants and melanoma risk: a study of 2 European populations.

Variation within the melanocortin receptor 1 (MC1R) gene, that influences phenotypic traits and susceptibility to melanoma, is abundant across the populations. We assessed and compared the risk of melanoma in 2 European populations, German and Spanish, by genotyping MC1R variants through direct DNA sequencing from 1,185 melanoma cases and 1,582 controls. The presence of any variant in both populations was associated with a significantly increased risk of melanoma (odds ratio OR = 1.67, 95% confidence interval CI 1.40–1.99). The population attributable fractions (PAF) associated with the MC1R variants in both populations was over 25%. However, the results showed a statistically significant (p < 0.0001) higher frequency of MC1R variants in the German (70%) than in the Spanish population (60%). The red‐hair colour (RHC) variants, though associated with increased risk in both populations, were more common in the German than in the Spanish population (p < 0.0001). Interestingly, non‐RHC variants increased the disease risk in the Spanish (OR = 1.60, 95% CI 1.20–2.14) but not in the German population (OR = 1.07, 95% CI 0.80–1.44). Although RHC variants explained a major proportion of the observed PAF in the German population, in the Spanish population the major contributor to the PAF was the non‐RHC V60L variant. We also observed reduced historic linkage disequilibrium between the variants V92M and T314T in the gene in German melanoma cases. In conclusion, our data underscored the unambiguous importance of the MC1R variants towards the population burden of melanoma. However, the variants that are associated with the disease differ between the investigated populations.

Folate metabolic gene polymorphisms and childhood acute lymphoblastic leukemia: a case–control study

We genotyped six folate metabolic pathway genes for 11 polymorphisms in 460 cases of childhood acute lymphoblastic leukemia (ALL) and 552 ethnically matched controls. None of the polymorphisms except the 66A>G (I22M) in the 5-methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR) gene showed any effect on disease risk. The carriers of the G-allele were associated with a marginal decreased risk of ALL (gender-adjusted global P=0.03; multiple-testing corrected P=0.25). Analysis of four polymorphisms in the MTRR gene showed statistically significant differences in haplotype distribution between cases and controls (global P<0.0001). The haplotypes GCAC (odds ratio (OR) 0.5, 95% confidence interval (CI) 0.4–0.6) and ATAC (OR 0.5, 95% CI 0.3–0.6) were associated with a reduced risk and the haplotypes ACAC (OR 2.3, 95% CI 1.8–2.9) and GTAC (OR 1.8, 95% CI 1.4–2.3) with an increased risk. The genotype-combination analyses indicated that the best model stratifies cases and controls based on the 66A>G and the 524C>T polymorphisms in the MTRR gene (global P=0.03). Our results suggest that, besides a weak association of childhood ALL with the 66A>G polymorphism, haplotypes within the MTRR gene may, in part, account for population-based differences in risk.

Tumor location and patient characteristics of colon and rectal adenocarcinomas in relation to survival and TNM classes

BackgroundOld age at diagnosis is associated with poor survival in colorectal cancer (CRC) for unknown reasons. Recent data show that colonoscopy is efficient in preventing left-sided cancers only. We examine the association of Tumor Node Metastasis (TNM) classes with diagnostic age and patient characteristics.MethodsThe Swedish Family-Cancer Database has data on TNM classes on 6,105 CRC adenocarcinoma patients. Ordinal logistic regression analysis was performed to model tumor characteristics according to age at diagnosis, tumor localization, gender, socioeconomic status, medical region and family history. The results were compared to results from survival analysis.ResultsThe only parameters systematically associated with TNM classes were age and tumor localization. Young age at diagnosis was a risk factor for aggressive CRC, according to stage, N and M with odds ratios (ORs) ranging from 1.80 to 1.93 for diagnosis before age 50 years compared to diagnosis at 80+ years. All tumor characteristics, particularly T, were worse for colon compared to rectal tumors. Right-sided tumors showed worse characteristics for all classifiers but M. The survival analysis on patients diagnosed since 2000 showed a hazard ratio of 0.55 for diagnosis before age 50 years compared to diagnosis at over 80 years and a modestly better prognosis for left-sided compared to right-sided tumors.ConclusionsThe results showed systematically more aggressive tumors in young compared to old patients. The poorer survival of old patients in colon cancer was not related to the available tumor characteristics. However, these partially agreed with the limited colonoscopic success with right-sided tumors.