DNA testing for disease

• Advantages of DNA testing:

  Can test for specific alleles (predict severity)

  Can test any cell at any stage of development (prenatal or embryonic screening)

  Can test for carrier status

  High sensitivity of PCR
• Neonatal screening:

  Early detection and treatment can lead to improved prognosis e.g. Phenylketonuria, CF, sickle-cell anaemia.
• Carrier screening:

  for autosomal recessive disorders e.g. alpha and beta thalassemia, sickle-cell anaemia, CF – risk of having an affected child
• Predictive screening:

  for presymptomatic diagnosis of late onset autosomal dominant disorders e.g. Huntington’s disease
• Embryo pre-implantation screening: (IVF)

  if high risk of affected child
• Invasive foetal screening:

  (chorionic villus sampling, amniocentesis) if high risk of affected child
• Non-inasive prenatal diagnosis (NIPD):

  uses blood sample from mother if high risk of affected child
• Methods of genetic testing:

  • PCR amplification of mutant/normal alleles (size differences)(allele-specific amplification)
  • Fluorescent in situ hybridisation (FISH)
  • Single Nucleotide Polymorphism (SNP) profiling
  • Whole genome sequencing
• Diagnosing repeat expansion diseases by PCR:

  • Huntington disease is caused by increased number of CAG repeats (>35) in the HTT gene
  • Extract DNA, perform PCR using primers flanking repeat sequence, separate products by gel electrophoresis
• Cystic fibrosis - Size differences in CFTR gene:

  • Common mutant allele (DF508 – deletion of codon for phenylalanine) found in > 75% CF patients in Northern Europe
  • Allele detected by analysis of PCR products using primers that flank the site of deletion
  • uses southern blot
• Allele-specific PCR:

  • test for it affectors
  • Design primer to not end when affected
  • if mismatched primer will not anneal so no extension
  • test for a base change
  • Two primers one that binds to normal allele and one binds to mutated allele
• Testing for multi-gene conditions:

  Diseases caused by variations in several different genes (e.g. multiple sclerosis, cancers)

  Certain combinations will give higher/lower susceptibility

  Need to identify high susceptibility alleles
• Single nucleotide polymorphisms (SNPS):

  Small changes (single base change) is associated with gene of interest

  These single base change is inherrited
• Genome-wide association studies (GWAS)

  • Genomes of many people with a certain genetic condition tested for millions of SNPs (on a microarray)
  • Compared SNPs with unaffected individuals
  • Statistically significant higher frequency amongst affected individuals carrying a certain SNPs = association
  • Design tests to look for high risk alleles
• SNPs detection:

  Microarray technology - design probe to stop just before SNPs

  polymerases adds complementary base which is labelled with light colour changes with base
• 23andMe issues:

  used faulty assumptions of DNA eg 12% norweigen

  can sell DNA to companies
• problems with SNP testing:

  Weak association with risk (exceptions are BRCA1/BRCA2)

  Poor predictive value

  Environmental factors very important

  Risk depends on combination of alleles from several-many genes
• Chromosomal disorder detection:

  FISH
• FISH:

  metaphase condensed chromatin or Interphase (can be used in Down's)

  fixed + stained

  washed with different probes - if binds DNA is present

  used for deletion detection
• Problems with current genetic testing:

  Restricted to one, or a few, disease alleles - testing for a few SNPs

  Need some prior knowledge of alleles/region to be tested
• Screening by whole genome sequencing:

  Use next generation, high-throughput sequencing technologies

  Can detect all possible alleles in all ~20,000 genes

  Currently too expensive

  Can be used to detect chromosome abnormalities
• Genome sequencing in the NHS:

  Aim was to sequence 100,000 NHS patients’ genomes by the end of 2017 (completed 5/12/18)

  rare congenital conditions (identify mutations)

  cancer (cancer vs. normal genome)

  infectious diseases (pathogen genomes and patient genomes)
• Non-invasive prenatal diagnosis by sequencing: alternative to aminocentesis

  Relies on free fetal DNA or RNA in maternal serum (3-6%)

  Uses ultra-high throughput sequencing to count the number of copies whole chromosomes (or alleles)

  theoretically diagnose all genetic diseases from one blood sample

  • many used for diagnosing down's before birth
• How to conduct NIPD

  Find a sequence that is repeated in a given chromosome
• Moral and ethical consequences of NIPD

  • increased number of abortions
  • Prevention of disabled births
  • Who chooses?
  • Eugenics
  • sex selection
  • desirable traits