Complex Diseases

• Human diseases can be mimicked with the help of in vitro and in vivo models
• Animal models


  • Generated by gene transfer and tools for gene targeting
• Genomic engineering


  Uses programmable nuclease and homologous recombination
• CRISPR/Cas


  A highly flexible system that can be adapted for various applications in DNA and RNA engineering
• Describe technologies to analyse whole human genomes in a single experiment
• Describe applications of personalised medicine using pharmacogenomics
• Understand major differences between monogenic and complex diseases and identify strategies to analyse complex diseases
• High-throughput sequencing is realised by immobilising DNA target fragments
• DNA is then amplified by adaptor-primed PCR
• Sequencing-by-synthesis


  A method based on the reversible incorporation of dye terminators
• Illumina second-generation sequencing approach uses reversible terminator sequencing
• Illumina reversible terminator sequencing


  1. Genome fragmentation

  2. Genomic selection & enrichment

  3. DNA capture and amplification

  4. DNA library and sequencing
• Single-molecule sequencing


  An optical system (zero-mode waveguide, ZMV) detects the incorporated base in real time
• Single-molecule real-time (SMRT) sequencing


  • No PCR
  • No termination
  • Still sequencing by synthesis
• Nanopore sequencing


  A very long, single DNA molecule passes through a "nanopore" in a membrane positioned in an electrical field
• Nanopore sequencing involves real-time sequencing with no PCR and no sequencing by synthesis
• High-throughput sequence analysis of an individual's DNA identifies thousands of variants compared to reference genomes
• Pathogenic variants


  Can cause monogenic diseases
• Variants


  Can contribute to the susceptibility to a polygenic or multifactorial disease
• Variants


  Can have an effect on how drugs act or which side effects drugs exert
• Individual variation of drug responses


  Can be pharmacokinetic (variable regarding the drug uptake, distribution and metabolism) or pharmacodynamic (variable regarding the responsiveness of the drug target)
• All these can be influenced by genetic factors, drug interactions, age & disease
• Some ethical issues that might be raised by high-throughput DNA sequencing include incidental findings, confidentiality and privacy, neonatal screening, genetic discrimination, and genetic manipulation
• Drug disposition


  Can be divided into four stages: Absorption, Distribution, Metabolism, Excretion (ADME)
• Type A adverse drug reactions

  An exaggerated response to a standard dose; variants make an individual particularly sensitive to the drug
• Type B adverse drug reactions


  An unexpected response unrelated to the normal action of the drug; variants facilitate particular interactions that would not normally occur
• 6.5% of hospital admissions were related to adverse drug reactions, with a fatality rate of 0.15%
• Phase 1 drug metabolism


  Mainly involves monooxygenation, with a variety of P450 cytochromes contributing
• Phase 2 drug metabolism


  Can involve acetylation, glucuronidation, sulfation, methylation
• Glutathione S-transferases (GSTs)

  Are generally involved in detoxification of xenobiotics and certain carcinogens
• Deletions of GST genes are common because of unequal crossover in gene clusters