Posts in Category: Genetics

Heart Failure in Pediatric Patients With Congenital Heart Disease

Heart Failure in Pediatric Patients With Congenital Heart Disease

Robert B. Hinton, Stephanie M. Ware

Genes causing congenital heart disease (CHD) and cardiomyopathy form a complex network. The network diagrams were generated using ToppCluster (www.toppcluster.cchmc.org) software. Gene lists were derived from clinically available next-generation sequencing panels for cardiomyopathy genes (n=50) and CHD genes (n=44). A, Abstracted cluster network showing a selected subset of features from the following categories: Gene ontology (GO): molecular function; GO: biological processes; GO: cellular component; human phenotype; mouse phenotype; pathway; disease (cardiomyopathy and CHD). The features are color coded by category and connected to cardiomyopathy (white circle) and CHD gene nodes. B, Gene level network with nodes (blue) selected from features in GO: biological processes category. The network illustrates that regulation of cellular component of movement, actin filament-based processes, and cardiac chamber morphogenesis are shared in common between cardiomyopathy and CHD genes, whereas regulation of force of heart contraction and tube development are unshared. [Powerpoint File]

Genetics and Genomics of Congenital Heart Disease

Genetics and Genomics of Congenital Heart Disease

Samir Zaidi, Martina Brueckner

NOTCH signaling in congenital heart disease (CHD) (A) the outline of NOTCH signaling pathway showing signal-sending cell in yellow and signal receiving cell in green. B, Syndromes and CHD associated with NOTCH pathway gene mutations. BAV indicates bicuspid aortic valve; CoA, coarctation of the aorta; HLHS, hypoplastic left heart syndrome; HTX, heterotaxy-associated defects; NA, not applicable; NICD, Notch intracellular domain; VSD, ventricular septal defect; TA, truncus arteriosus; and TOF, tetralogy of Fallot. [Powerpoint File]

Genetics and Genomics of Congenital Heart Disease

Genetics and Genomics of Congenital Heart Disease

Samir Zaidi, Martina Brueckner

A, Outline of human heart development. The x axis displays days of human and mouse gestation. B, The spectrum of congenital heart disease from mild to severe. The lesions indicated as “severe” are expected to require intervention in the first year of life. Classes of CHD based on proposed developmental-genetic mechanisms are indicated in parentheses. C, Genetic causes of CHD identified to date. ASD indicates atrial septal defect; CHD, congenital heart disease; CoA, coarctation of the aorta; CTD, conotruncal defect; HLHS, hypoplastic left heart syndrome; HTX, heterotaxy; LVO, left ventricular outflow obstruction; TGA, transposition of the great arteries; TOF, tetralogy of Fallot; and VSD, ventricular septal defect. [Powerpoint File]

Gene Editing With CRISPR/Cas9 RNA-Directed Nuclease

Gene Editing With CRISPR/Cas9 RNA-Directed Nuclease

Thomas Doetschman, Teodora Georgieva

Cas9 RNA-guided nuclease system. Schematic representation of the Streptococcus pyogenes Cas9 nuclease (green) targeted to genomic DNA by a single-guide RNA (sgRNA) consisting of an ≈20-nt guide sequence (blue) and a scaffold (red). The guide sequence is directly upstream of the protospacer adjacent motif (PAM), NGG (orange circles). Cas9 mediates a double-strand DNA break (DSB) ≈3 bp upstream of the PAM (red triangles). The break is repaired by 1 of 2 mechanisms: nonhomologous end joining (NHEJ) that creates random insertions or deletions at the target site or homology-directed repair (HDR). Two types of template can be used for HDR: small single-stranded DNA (ssDNA) oligonucleotide donor with short 60- 70-bp homology arms and a linear or circular dsDNA plasmid with long homology arms of 1 to 3 kb. [Powerpoint File]

Gene Editing With CRISPR/Cas9 RNA-Directed Nuclease

Gene Editing With CRISPR/Cas9 RNA-Directed Nuclease

Thomas Doetschman, Teodora Georgieva

Large deletions, marker insertions, and single-nucleotide polymorphism (SNP) insertions. A, Multiple exons can be deleted by nonhomologous end joining (NHEJ) by designing single-guide RNAs (sgRNAs) that flank the region to be deleted. In the presence of Cas9 double-strand DNA breaks (DSBs) will occur resulting in small indels at the site of end joining. The double bar at the sgRNA:gene hybridization site represents the DSB caused by Cas9. Cas9 can be introduced as either mRNA or recombinant protein. B, Marker gene insertion can be achieved through homology-directed repair (HDR) in which the homologous regions are introduced in a plasmid as would be done by gene targeting. C, A chromosomal region deletion has been done using HDR in which 2 sgRNAs were designed at each end of the region to be deleted. The homology template was an single-strand DNA (ssDNA) oligo with homology at each end of the region to be deleted. D, SNP insertion using HDR with ssDNA oligo as homology template. [Powerpoint File]

Gene Editing With CRISPR/Cas9 RNA-Directed Nuclease

Gene Editing With CRISPR/Cas9 RNA-Directed Nuclease

Thomas Doetschman, Teodora Georgieva

High-throughput screens. A, High-throughput gene knockout screens in which a lentiviral single-guide RNA (sgRNA) library for multiple sites in the early coding region of thousands of genes results in cell clones in which each single gene is knocked out by nonhomologous end joining repair in which indels occur. Several clones will represent each gene. A screen for a specific phenotypic outcome, such as drug resistance or altered growth, will identify genes involved in those processes. B, High-throughput screen for long noncoding RNAs (lncRNAs). A lentiviral library of paired sgRNAs, each pair of which flanks an individual lncRNA, is transduced into cells along with Cas9. Several sgRNA pairs (color matched) are designed for each lncRNA. Phenotyping clones with similar characteristics will identify lncRNAs associated with that phenotype. C, High-throughput regulatory region screen. A lentiviral library consisting of sgRNAs for tiling at ≈10-bp intervals in the regulatory region of multiple genes can be used to identify regulatory regions for each gene. A similar approach has been used to make a library of cells in which a guide RNA (gRNA or CRISPR RNA) is inserted into a modified Rosa locus in which the hairpin RNA (or tracrRNA) is adjacent to the gRNA insertion site thereby generating a sgRNA for each cell. ssDNA indicates single-strand DNA. [Powerpoint File]

Gene Editing With CRISPR/Cas9 RNA-Directed Nuclease

Gene Editing With CRISPR/Cas9 RNA-Directed Nuclease

Thomas Doetschman, Teodora Georgieva

CRISPR-mediated modulation of gene expression. A, CRISPR interference system with dCas9 (dead, nuclease-deficient Cas9 colored in red) fused to the negative transcriptional effector KRAB (Krüppel-associated box of the Kox1 gene, colored red). A lentiviral library of single-guide RNAs for tiling at gene regulatory regions can identify enhancer elements and their location flanking the transcriptional start site (TSS) that affect gene expression. B, CRISPR activation system with dCas9 fused to a tethered string of an antibody epitope. Cotransduction of an epitope-specific antibody fused to multiple Herpes virus transcriptional activation genes VP16 will bind to the string of epitopes bringing multiple VP16s to the transcriptional machinery, thereby activating the gene. VP64 is a complex of 4 VP16 molecules. Figure derived from Figure 5A from Tanenbaum et al. [Powerpoint File]

Surprises From Genetic Analyses of Lipid Risk Factors for Atherosclerosis

Surprises From Genetic Analyses of Lipid Risk Factors for Atherosclerosis

Kiran Musunuru, Sekar Kathiresan

The cumulative effects of genetic variants that raise plasma low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglyceride (TG) levels on the risk of myocardial infarction (MI). SD indicates standard deviation. [Powerpoint File]

RNA Splicing: Regulation and Dysregulation in the Heart

RNA Splicing: Regulation and Dysregulation in the Heart

Maarten M.G. van den Hoogenhof, Yigal M. Pinto, Esther E. Creemers

Two-step splicing reaction. Splicing occurs by a 2-step trans-esterification reaction to remove introns and to join exons together. In the first step, U1 small nuclear ribonucleoprotein (snRNP) assembles at the 5′ splice site of an exon and U2 snRNP at the branch point sequence (BPS), just upstream of the 3′splice site of the adjacent/downstream exon. This configuration is known as the prespliceosome. Hereafter, U1 and U2 are joined by the snRNPs U5 and U4–U6 complexes to form the precatalytic spliceosome. Next, U4–U6 complexes unwind, releasing U4 and U1 from the prespliceosomal complex. This allows U6 to base pair with the 5′ splice site and the BPS. The 5′ splice site gets cleaved, which leads to a free 3′ OH-group at the upstream exon, and a branched intronic region at the downstream exon, called the intron lariat. During the second step, U5 pairs with sequences in both the 5′ and 3′ splice site, positioning the 2 ends together. The 3′ OH-group of the upstream (5′) exon fuses with the 3′ intron–exon junction, thereby conjoining the 2 exons and excising the intron in the form of a lasso-shaped intron lariat. Finally, the spliceosome disassembles, and all components are recycled for future splicing reactions. [Powerpoint File]

The Role of DNA Methylation in Cardiovascular Risk and Disease: Methodological Aspects, Study Design, and Data Analysis for Epidemiological Studies

The Role of DNA Methylation in Cardiovascular Risk and Disease: Methodological Aspects, Study Design, and Data Analysis for Epidemiological Studies

Jia Zhong*, Golareh Agha*, Andrea A. Baccarelli

DNA methylation is usually associated with inactive transcription and suppressed gene expression. The figure depicts the molecular mechanism linking DNA methylation and inactive transcription. Cytosine is methylated to 5-methylcytosine by DNA methyltransferase (DNMT). Binding of a methyl CpG-binding protein to methylated sequences prevents access to this sequence by transcription factors (TFs), thereby suppressing transcription. MBP indicates methyl CpG-binding protein; SAM, S-adenosylmethionine; and SAH, S-adenosylhomocysteine. [Powerpoint File]

Lncing Epigenetic Control of Transcription to Cardiovascular Development and Disease

Lncing Epigenetic Control of Transcription to Cardiovascular Development and Disease

Gizem Rizki, Laurie A. Boyer

Future directions for the study of long noncoding RNAs (lncRNAs) in cardiovascular biology. There still remain many unanswered questions about the exact functions and molecular mechanisms of lncRNAs that govern diverse aspects of cardiac physiology. Some of the outstanding questions and promising areas of research are outlined in the schematic. CM indicates cardiomyocyte (illustration credit: Ben Smith). [Powerpoint File]

Lncing Epigenetic Control of Transcription to Cardiovascular Development and Disease

Lncing Epigenetic Control of Transcription to Cardiovascular Development and Disease

Gizem Rizki, Laurie A. Boyer

Epigenetic regulation by cardiovascular long noncoding RNAs (lncRNAs). Mechanisms of action of lncRNAs, such as Bvht, Fendrr, and Mhrt, that regulate cardiac gene expression by modulating chromatin modifying protein complexes (A), and Kcnq-overlapping lncRNA 1 (Kcnq1ot1), which regulates chromatin organization and structure (B) are depicted. BAF indicates Brg1/Brm-associated factor; CM, cardiomyocyte; CP, cardiac progenitor cells; ESCs, embryonic stem cells; MES, mesodermal cells; PRC, polycomb repressive complex; and TrxG/MLL, trithorax/mixed lineage leukemia (illustration credit: Ben Smith). [Powerpoint File]

Lncing Epigenetic Control of Transcription to Cardiovascular Development and Disease

Lncing Epigenetic Control of Transcription to Cardiovascular Development and Disease

Gizem Rizki, Laurie A. Boyer

Long noncoding RNAs in the transcriptional circuitry of cardiovascular development. A, Many long noncoding RNAs (lncRNAs) are important for the development of different cell types in the cardiovascular system. Specific examples important for developmental transitions, such as Braveheart and Fendrr, as well as formation of cardiovascular cell types, such as Sencr and Malat1 are depicted. B, Transcriptional profiling studies have identified many lncRNAs that are differentially expressed during cardiac development. Many of these transcripts still await validation and further characterization (illustration credit: Ben Smith). [Powerpoint File]

Genetics of Sudden Cardiac Death

Genetics of Sudden Cardiac Death

Connie R. Bezzina, Najim Lahrouchi, Silvia G. Priori

Schematic representation of a cardiomyocyte displaying the proteins involved in the pathogenesis of the primary electric disorders. A, potassium (IKr; B), calcium (ICaL), and (C) sodium (INa) channel structures and subunits are shown. CASQ2 indicates calsequentrin-2; PLN, cardiac phospholamban; RyR2, ryanodine receptor 2; SERCA2a, sarcoplasmic/endoplasmic reticulum calcium ATPase 2a; and SR, sarcoplasmic reticulum (Illustration credit: Ben Smith). [Powerpoint File]

Genetics of Sudden Cardiac Death

Genetics of Sudden Cardiac Death

Connie R. Bezzina, Najim Lahrouchi, Silvia G. Priori

Schematic representation of a cardiomyocyte exhibiting proteins involved in the pathogenesis of the inherited cardiomyopathies, including sarcomeric, cytoskeletal, desmosomal proteins, and nuclear envelope proteins. MLP indicates cysteine and glycine-rich protein 3 (also known as muscle LIM [Lin-11, Islet-1, Mec-3] protein) (Illustration credit: Ben Smith). [Powerpoint File]