Cardiac Transcription Factors and Regulatory Networks.

Cardiac development is a fine-tuned process governed by complex transcriptional networks, in which transcription factors (TFs) interact with other regulatory layers. In this chapter, we introduce the core cardiac TFs including Gata, Hand, Nkx2, Mef2, Srf, and Tbx. These factors regulate each other's expression and can also act in a combinatorial manner on their downstream targets. Their disruption leads to various cardiac phenotypes in mice, and mutations in humans have been associated with congenital heart defects. In the second part of the chapter, we discuss different levels of regulation including cis-regulatory elements, chromatin structure, and microRNAs, which can interact with transcription factors, modulate their function, or are downstream targets. Finally, examples of disturbances of the cardiac regulatory network leading to congenital heart diseases in human are provided.

Technologies to Study Genetics and Molecular Pathways.

Over the last few decades, the study of congenital heart disease (CHD) has benefited from various model systems and the development of molecular biological techniques enabling the analysis of single gene as well as global effects. In this chapter, we first describe different models including CHD patients and their families, animal models ranging from invertebrates to mammals, and various cell culture systems. Moreover, techniques to experimentally manipulate these models are discussed. Second, we introduce cardiac phenotyping technologies comprising the analysis of mouse and cell culture models, live imaging of cardiogenesis, and histological methods for fixed hearts. Finally, the most important and latest molecular biotechniques are described. These include genotyping technologies, different applications of next-generation sequencing, and the analysis of transcriptome, epigenome, proteome, and metabolome. In summary, the models and technologies presented in this chapter are essential to study the function and development of the heart and to understand the molecular pathways underlying CHD.

Human Genetics of Tetralogy of Fallot and Double-Outlet Right Ventricle.

Tetralogy of Fallot (TOF) and double-outlet right ventricle (DORV) are conotruncal defects resulting from disturbances of the second heart field and the neural crest, which can occur as isolated malformations or as part of multiorgan syndromes. Their etiology is multifactorial and characterized by overlapping genetic causes. In this chapter, we present the different genetic alterations underlying the two diseases, which range from chromosomal abnormalities like aneuploidies and structural mutations to rare single nucleotide variations affecting distinct genes. For example, mutations in the cardiac transcription factors NKX2-5, GATA4, and HAND2 have been identified in isolated TOF cases, while mutations of TBX5 and 22q11 deletion, leading to haploinsufficiency of TBX1, cause Holt-Oram and DiGeorge syndrome, respectively. Moreover, genes involved in signaling pathways, laterality determination, and epigenetic mechanisms have also been found mutated in TOF and/or DORV patients. Finally, genome-wide association studies identified common single nucleotide polymorphisms associated with the risk for TOF.

Intensified lipid-lowering treatment with alirocumab in patients with coronary heart disease.

BACKGROUND: Atherosclerotic cardiovascular disease is the leading cause of death and disability in the Western world. OBJECTIVE: To characterise adults with confirmed coronary heart disease (CHD) and primary heterozygous familial or non-familial hypercholesterolaemia or mixed dyslipidaemia who received alirocumab in a real-world setting. METHODS: This open, prospective, multicentre, non-interventional study, conducted in Germany, enroled patients with confirmed CHD who were treated with alirocumab according to its summary of product characteristics. Prescription was at the physician's discretion and independent of study participation. Patients were followed for 12 weeks after alirocumab initiation. RESULTS: In total, 245 patients (mean age 62.2 years; 34.0% female) were documented at 90 sites. Overall, 47.7% had familial hypercholesterolaemia, 48.9% non-familial hypercholesterolaemia and 43.8% mixed dyslipidaemia; 74.6% had hypertension and 29.2% diabetes mellitus. The most common lipid-lowering therapy in the 12 months preceding alirocumab was a statin, often in combination with ezetimibe (73.5%). Statin contraindications were documented for 46.2% patients and statin intolerance for 63.8%. The mean low-density lipoprotein cholesterol (LDL-C)-level prior to alirocumab was 150.5±51.6 mg/dL. Alirocumab prescription was in compliance with German national recommendations and/or European guidelines. The most common starting dose was 75 mg every other week. Overall, 57% patients reached target LDL-C levels (<70 mg/dL) after 12 weeks of treatment. Alirocumab was generally well tolerated. CONCLUSION: In a real-world setting in Germany, alirocumab was prescribed for patients with atherosclerotic cardiovascular disease who had high baseline LDL-C levels with or without statin intolerance. Efficacy and safety were consistent with findings observed in the ODYSSEY Phase III programme.

PEARL: A Non-interventional Study of Real-World Alirocumab Use in German Clinical Practice.

BACKGROUND: Several lipid guidelines recommend that proprotein convertase subtilisin/kexin type 9 inhibitors should be considered for patients with atherosclerotic cardiovascular disease who are inadequately treated with maximally tolerated lipid-lowering treatment. OBJECTIVES: The PEARL study assessed the efficacy and safety of the proprotein convertase subtilisin/kexin type 9 inhibitor alirocumab in patients with hypercholesterolemia in a real-world setting. METHODS: PEARL was an open, prospective, multicenter, non-interventional study conducted in Germany. Patients (n = 619) for whom treating physicians decided to use alirocumab 75 or 150 mg every 2 weeks according to German guidelines (low-density lipoprotein cholesterol > 1.8/2.6 mmol/L [> 70/100 mg/dL], depending on cardiovascular risk, despite maximally tolerated statin therapy with/without other non-alirocumab lipid-lowering therapy) were enrolled and followed for 24 weeks. Physicians could adjust the alirocumab dose based on their clinical judgment. The primary efficacy endpoint was low-density lipoprotein cholesterol reduction from baseline (prior to alirocumab therapy) to week 24. RESULTS: Overall, 72.8% of patients reported complete or partial statin intolerance. Mean low-density lipoprotein cholesterol was 4.7 mmol/L (180.5 mg/dL) and 2.3 mmol/L (89.8 mg/dL) at baseline and week 24, respectively. Least-squares mean percentage change from baseline to week 24 in low-density lipoprotein cholesterol was - 48.6%. Initial alirocumab dose was 75 mg in 72.9% of patients and 150 mg in 24.5% of patients; 19.6% of patients received an alirocumab dose increase (75 to 150 mg) and 1.6% of patients received a dose decrease. Adverse events were reported in 10.3% of patients, with myalgia being the most common. CONCLUSIONS: In a real-world setting in Germany, alirocumab was used in patients who had high baseline low-density lipoprotein cholesterol levels with/without statin intolerance. Efficacy and safety were consistent with findings observed in the ODYSSEY Phase III program.

Muscle-relevant genes marked by stable H3K4me2/3 profiles and enriched MyoD binding during myogenic differentiation.

Post-translational modifications of histones play a key role in the regulation of gene expression during development and differentiation. Numerous studies have shown the dynamics of combinatorial regulation by transcription factors and histone modifications, in the sense that different combinations lead to distinct expression outcomes. Here, we investigated gene regulation by stable enrichment patterns of histone marks H3K4me2 and H3K4me3 in combination with the chromatin binding of the muscle tissue-specific transcription factor MyoD during myogenic differentiation of C2C12 cells. Using k-means clustering, we found that specific combinations of H3K4me2/3 profiles over and towards the gene body impact on gene expression and marks a subset of genes important for muscle development and differentiation. By further analysis, we found that the muscle key regulator MyoD was significantly enriched on this subset of genes and played a repressive role during myogenic differentiation. Among these genes, we identified the pluripotency gene Patz1, which is repressed during myogenic differentiation through direct binding of MyoD to promoter elements. These results point to the importance of integrating histone modifications and MyoD chromatin binding for coordinated gene activation and repression during myogenic differentiation.

Comparative DNA methylation and gene expression analysis identifies novel genes for structural congenital heart diseases.

AIMS: For the majority of congenital heart diseases (CHDs), the full complexity of the causative molecular network, which is driven by genetic, epigenetic, and environmental factors, is yet to be elucidated. Epigenetic alterations are suggested to play a pivotal role in modulating the phenotypic expression of CHDs and their clinical course during life. Candidate approaches implied that DNA methylation might have a developmental role in CHD and contributes to the long-term progress of non-structural cardiac diseases. The aim of the present study is to define the postnatal epigenome of two common cardiac malformations, representing epigenetic memory, and adaption to hemodynamic alterations, which are jointly relevant for the disease course. METHODS AND RESULTS: We present the first analysis of genome-wide DNA methylation data obtained from myocardial biopsies of Tetralogy of Fallot (TOF) and ventricular septal defect patients. We defined stringent sets of differentially methylated regions between patients and controls, which are significantly enriched for genomic features like promoters, exons, and cardiac enhancers. For TOF, we linked DNA methylation with genome-wide expression data and found a significant overlap for hypermethylated promoters and down-regulated genes, and vice versa. We validated and replicated the methylation of selected CpGs and performed functional assays. We identified a hypermethylated novel developmental CpG island in the promoter of SCO2 and demonstrate its functional impact. Moreover, we discovered methylation changes co-localized with novel, differential splicing events among sarcomeric genes as well as transcription factor binding sites. Finally, we demonstrated the interaction of differentially methylated and expressed genes in TOF with mutated CHD genes in a molecular network. CONCLUSION: By interrogating DNA methylation and gene expression data, we identify two novel mechanism contributing to the phenotypic expression of CHDs: aberrant methylation of promoter CpG islands and methylation alterations leading to differential splicing.

Outlier-based identification of copy number variations using targeted resequencing in a small cohort of patients with Tetralogy of Fallot.

Copy number variations (CNVs) are one of the main sources of variability in the human genome. Many CNVs are associated with various diseases including cardiovascular disease. In addition to hybridization-based methods, next-generation sequencing (NGS) technologies are increasingly used for CNV discovery. However, respective computational methods applicable to NGS data are still limited. We developed a novel CNV calling method based on outlier detection applicable to small cohorts, which is of particular interest for the discovery of individual CNVs within families, de novo CNVs in trios and/or small cohorts of specific phenotypes like rare diseases. Approximately 7,000 rare diseases are currently known, which collectively affect ∼6% of the population. For our method, we applied the Dixon's Q test to detect outliers and used a Hidden Markov Model for their assessment. The method can be used for data obtained by exome and targeted resequencing. We evaluated our outlier-based method in comparison to the CNV calling tool CoNIFER using eight HapMap exome samples and subsequently applied both methods to targeted resequencing data of patients with Tetralogy of Fallot (TOF), the most common cyanotic congenital heart disease. In both the HapMap samples and the TOF cases, our method is superior to CoNIFER, such that it identifies more true positive CNVs. Called CNVs in TOF cases were validated by qPCR and HapMap CNVs were confirmed with available array-CGH data. In the TOF patients, we found four copy number gains affecting three genes, of which two are important regulators of heart development (NOTCH1, ISL1) and one is located in a region associated with cardiac malformations (PRODH at 22q11). In summary, we present a novel CNV calling method based on outlier detection, which will be of particular interest for the analysis of de novo or individual CNVs in trios or cohorts up to 30 individuals, respectively.

Rare and private variations in neural crest, apoptosis and sarcomere genes define the polygenic background of isolated Tetralogy of Fallot.

Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart disease. Its genetic basis is demonstrated by an increased recurrence risk in siblings and familial cases. However, the majority of TOF are sporadic, isolated cases of undefined origin and it had been postulated that rare and private autosomal variations in concert define its genetic basis. To elucidate this hypothesis, we performed a multilevel study using targeted re-sequencing and whole-transcriptome profiling. We developed a novel concept based on a gene's mutation frequency to unravel the polygenic origin of TOF. We show that isolated TOF is caused by a combination of deleterious private and rare mutations in genes essential for apoptosis and cell growth, the assembly of the sarcomere as well as for the neural crest and secondary heart field, the cellular basis of the right ventricle and its outflow tract. Affected genes coincide in an interaction network with significant disturbances in expression shared by cases with a mutually affected TOF gene. The majority of genes show continuous expression during adulthood, which opens a new route to understand the diversity in the long-term clinical outcome of TOF cases. Our findings demonstrate that TOF has a polygenic origin and that understanding the genetic basis can lead to novel diagnostic and therapeutic routes. Moreover, the novel concept of the gene mutation frequency is a versatile measure and can be applied to other open genetic disorders.

Application of high-throughput sequencing for studying genomic variations in congenital heart disease.

Congenital heart diseases (CHD) represent the most common birth defect in human. The majority of cases are caused by a combination of complex genetic alterations and environmental influences. In the past, many disease-causing mutations have been identified; however, there is still a large proportion of cardiac malformations with unknown precise origin. High-throughput sequencing technologies established during the last years offer novel opportunities to further study the genetic background underlying the disease. In this review, we provide a roadmap for designing and analyzing high-throughput sequencing studies focused on CHD, but also with general applicability to other complex diseases. The three main next-generation sequencing (NGS) platforms including their particular advantages and disadvantages are presented. To identify potentially disease-related genomic variations and genes, different filtering steps and gene prioritization strategies are discussed. In addition, available control datasets based on NGS are summarized. Finally, we provide an overview of current studies already using NGS technologies and showing that these techniques will help to further unravel the complex genetics underlying CHD.

Chemoresistance in non-small-cell lung cancer: can multidrug resistance markers predict the response of xenograft lung cancer models to chemotherapy?

OBJECTIVE: In chemotherapy for non-small-cell lung cancer (NSCLC), some patients seem to exhibit an intrinsic resistance or develop an acquired resistance under treatment. Results on resistance markers for possible treatment failure as shown in studies on selected lung cancer cell lines could not be completely confirmed in clinical trials. As these conflicting data require further research, we created a model between cell culture and the clinical need to study this problem. METHODS: Our study was based on patient-derived NSCLC xenografts in a mouse model, which revealed a high coincidence with the original tumour. Protein and messenger RNA (mRNA) expression of known resistance markers (breast cancer resistance protein (BCRP), multidrug resistance P-glycoprotein (MDR), lung cancer-related protein (LRP) and multidrug resistance protein 1 (MRP1)) were analysed by real-time polymerase chain reaction (PCR) and immunoblotting in 24 xenografts. Chemosensitivity to etoposide, carboplatin, gemcitabine, paclitaxel, cetuximab and erlotinib was determined in in vivo xenograft experiments and compared with the protein and mRNA expression of the multidrug resistance markers. RESULTS: With the exception of a single correlation between chemosensitivity and mRNA expression of etoposide and bcrp (mRNA expression of BCRP), we found no significant correlation between the response rates and protein- and mRNA expression levels in our 24 xenografts. The present results indicate that in vivo expression levels of multidrug resistance proteins and their mRNAs may not play a comparable role in chemoresistance of NSCLC, as pointed out in selected tumour cell lines. CONCLUSIONS: Patient-derived xenografts allow detailed investigation of therapy-related markers and their dynamic regulation in a well-standardised and clinically related way. As a consequence of our investigations, we regard multidrug resistance to be a multifactorial phenomenon, in which more factors than the markers analysed by the present study may be involved.

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Tumor cells that are nonsensitive to anticancer drugs frequently have a multidrug resistant (MDR) phenotype. Many studies with cell lines and patient material have been done to investigate the impact of different resistance markers at protein and mRNA level in drug resistance but with contradictory outcome. In the present study, 26 well-characterised patient-derived non-small cell lung cancer xenografts were used. The known chemosensitivity to etoposide, carboplatin, gemcitabine, paclitaxel and erlotinib was compared to the protein and mRNA expression of BCRP, LRP, MDR1, and MRP1. Further, four of these xenografts were short-term treated to analyse possible regulation mechanisms after therapeutic interventions. We found a borderline correlation between the bcrp mRNA expression and the response of xenografts to etoposide. All other constitutive mRNA and protein expression levels were not correlated to any drug response and were not significantly influenced by a short term treatment. The present results indicate that the expression levels of MDR proteins and mRNA investigated do not play an important role in the chemoresistance of NSCLC in the in vivo situation.