NGS In healthcare

• Main use of NGS in healthcare

  • Identify the genes that cause disease in polygenic diseases
• Common variations


  Present in at least 1% of the population
• Whether a particular SNP is pathogenic is usually unknown
• If the SNP occurs more frequently within a population that have a disease, it may be regarded as a risk allele for the disease
• Aim of GWAS


  Identify which common variants are often found in patients
• Microarray-based SNP typing


  1. Labelled patient or non-patient DNA allowed to hybridize to microarray

  2. Probes complimentary to all known common SNP loci in human genome (or the target gene) is spotted onto 1 microarray chip.

  3. Upon hybridization, fluorescence given off and detected by a laser

  
• Generally only identified SNPs that made a small total contribution to disease risk
• Missing heritability


  The genetic contribution that we know must exist but was not identified in the common-variant GWAS
• Rare variants: less than 1% frequency
• Rare variants cannot be studied using microarrays because they greatly outnumber common variants and individuals will have variants that are unknown

  
• NGS Rare-Variant-Association-Studies

  Study a family with multiple affected members
• Personalised medicine

  A more personalised approach to treatment based on genotype-phenotype correlations
• Genomics England: UK scientists completed a £300m government-backed project to sequence the genomes of 100,000 patients with cancer and very rare genetic diseases
• Illumina x NHS: Genomes were sequenced on the Illumina platform. NHS patient health records are key to the long-term project goals and will enable the genotype-phenotype correlations to be made
• Nanopore sequencing


  • Highly portable, can be used in the field
  • Can sequence viral genomes during viral outbreaks to characterise evolution and transmission paths
  • Example: used in the Ebola outbreak
• Genome editing


  Manipulating DNA at specific locations using the CRISPR-Cas9 system
• How to use CRISPR

  1. Identify the sequence of the genome causing the issue

  2. Create a specific guide RNA to recognise the particular stretch of DNA

  3. Guide RNA is attached to Cas9

  4. The complex is introduced into target cells

  5. Locates the target sequence and cuts the DNA

  6. Existing genome can be edited by modification, deletion or insertion (NHEJ)
• How Cas9-complex cuts DNA

  1. Locates and binds to a protospacer adjacent motif (PAM)

  2. Cas-9 unwinds DNA, allowing the sgRNA (guide RNA) to hybridise to the exposed DNA

  3. Nucleotides bind to the complimentary RNA

  4. Nuclease domains cut the DNA, producing a double stranded break
• Other uses of CRISPR-Cas9

  • De-activating nuclease domains and fusing other enzymes
  • Attach fluorescent molecules to visualise DNA sequence locations
  • Can be applied to multiple genes simultaneously
• CRISPR-Cas9 is potentially used to treat genetic diseases by reversing disease-causing mutations
• How CRISPR-Cas9 appears in nature

  1. Viral DNA is cut by bacteria and inserted in between CRISPR repeats in the bacteria genome

  2. This forms CRISPR-RNA. An enzyme cleaves them into 'guide RNAs'

  3. Guide RNA monomers interact with Cas9 enzymes

  4. The complex scans cytoplasm for sequence complementarity to the viral sequence

  5. Upon binding, Cas9 breaks the DNA
• How CRISPR is used to treat genetic disease

  1. Guide RNA can be designed to be complementary to a chromosome mutation and the flanking DNA

  2. Transfect cells with a vector that contains the correct sequence of the mutated gene

  3. Vector is added in high concentration so it's more likely to be incorporated than a homologous chromosome
• iPS stem cells


  Induced pluripotent stem cells that can differentiate into many types of adult cells
• How iPS stem cells are sourced

  1. Skin cells are expanded in culture

  2. Genetic reprogramming converts to iPS

  3. Culture conditions are manipulated to then convert the iPS into any adult cell, relevant for the disease in focus

  
• Editing the germ line


  Genome edits of germ line cells can be used to prevent genetic disease

  
• Pre-implantation genetic diagnosis (PGD) is offered by the NHS as an alternative to germ line editing
• Gene drive


  A genome edit could be driven through a wild population by making edits in the germ line in a way that they're always passed on to offspring
• How gene drive is done

  1. Immediately adjacent to the edited gene, driver elements are placed (genes encoding Guide RNA and Cas9)

  2. Guide RNA are designed to have sequence complementarity to a non-edited section of the edited gene

  3. Guide RNA has sequence complementarity in the same gene and recruits Cas9 to cut the homologous chromosome at the same location

  4. DNA is repaired and the second chromosome copies the edited gene – induced homozygosity

  
• If sperm is edited with drive elements, upon fertilisation, the drive element will enter the zygote. 100% chance for offspring to have the drive element
• Practical applications of gene drive


  • Currently being investigated as a countermeasure against mosquito-borne diseases, e.g. malaria
  • Aim: edit a gene in the mosquito genome that will inhibit its ability to transmit the infectious agent to humans

  
• The edited gene would spread through the mosquito population via gene drive due to their short life-cycle

  
• Ethical implications of gene drive


  • Ecosystem impacts
  • Public engagements
  • International guidelines – can cross borders
  • Safety? Reversal drives available in case something goes wrong

  
• Double stranded break repair model


  1. Resection: The break is processed by resection and leaves two non-overlapping 3' overhangs

  2. Invasion: The 3' end of the ssDNA invades a homologous DNA sequence, forming a D-loop. DNA polymerase extends the 3' overhang, using the homologous DNA as a template

  3. Second end capture: D-loop is captured and used as a template for synthesis of DNA in the upper 3' strand. Result: two Holliday junctions that need to be resolved

  4. Resolution: Holliday junction resolution results in either repair without crossing over or with crossing over