Diagnostic

• DNA basics

  • Chromosomes in the cell (chromatin)
  • Histon-bound -> nucleosome
  • Chromatid diploid
  • 22 pairs -> 44 autosomes +XX/XY (gonosomes)
• Difficult classification (mutation or polymorphism) is standardized according to certain characteristics
• Human genome

  • 3.1 billion nucleotides
  • 98% not protein-coding (~40,000)
  • ~19,600 protein-coding genes
  • SINEs/LINEs: short/long interspersed nuclear elements
  • Variant = neutral term for genetic aberrations (mutation=possibly pathogenic, polymorphism=considered benign)
  • SNV = single nucleotide variant
  • VUS = variant of unknown clinical significance
• DNA polymorphisms

  • missense: false AA, possibly pathogenic depending on 3° structure
  • splicing: at start/end of an intron, can cause wrong splicing
  • stop/nonsense: mostly pathogenic, more severe with earlier transcription termination
  • indels: single nucleotide insertion or deletion
  • frameshift (1-2 bases) vs. in-frame (3 bases)
• Chromosomes

  • Giemsa staining for visualization and determination -> AT vs GC rich region
  • p- = short arm; q- = long arm
  • karyogram: sorted after size (1>22 +X/Y) and band pattern (giemsa)
  • centromere locations: metacentric (X), submetacentric (k), acrocentric (v) chr. 13, 14, 15, 21, 22
• PCR steps

  1. Denaturation + primer hybridization

  2. DNA synthesis/primer elongation

  3. N-times repetition (25-35x) -> fragments (2n)

  4. Product verification via gelelctrophoresis (by size/negative charge - -> +)
• Nomenclature of polymorphisms

  • genomic (c.) and protein (p.) level
  • position + type of change
  • exchange: c.4G>C / p.(Ala2Pro)
  • deletion: c.7_9delCTG / p.(Val3del)
  • insertion with direct termination: c.15_16insT / p.(Lys6*)
  • insertion with termination after 3 AA: c.9_10insT / p.(Gln4Ser*3)
• Chromosomal polymorphisms

  • numeric: e.g. trisomy 13, 18, 21, Turner (X0) and Klinefelter syndrome (XXY)
  • structural: translocation, deletion, inversion, insertion, duplication
• PCR reagents

  • Nucleotides = building blocks (deoxynucleotide triphosphate, dNTP)
  • DNA-Polymerase = catalytic enzyme -> DNA synthesis/elongation
  • Primer = oligonucleotides (specific to amplified region, Amplicon)
  • Buffer = reaction conditions, ions, enzyme-cofactors
• PCR applications

  • Crucial for any DNA sequencing
  • Basis for molecular genetic analysis/diagnosis
  • qPCR: information on DNA/RNA content (in cycle 5-15)
• Sanger sequencing

  1. template strand + 1 primer

  2. mixture of deoxy- and marked dideoxy-nucleotides (4 dNTPs + 1 specific ddXTP)

  3. incorporation of ddXTP -> chain termination/stop of synthesis + colored light signal

  4. capillary electrophoresis with fluorescence excitation
• NGS steps


  • Random fragmentation
  • Adapter aligation (library prep)
  • Clonal Amplification -> Clusters on flow cell
  • Cyclic sequencing (paired end) -> all nucleotides = marked reversible chain terminators
• NGS (Next Generation Sequencing)


  • high-throughput + massive-parallel sequencing
  • Sequencing by synthesis, sbs (in real time)
  • Short read length (150 bases per sequencing)
  • WES = whole exome sequencing
  • WGS = whole genome sequencing
  • Panel sequencing = sub selection of genes/target regions
• Sanger steps

  1. DNA preparation

  2. PCR amplification

  3. PCR-product purification

  4. Sequencing reaction

  5. Sequence purification

  6. Capillary gelelectrophoresis

  7. Sequence analysis
• EPCAM: deletion in EPCAM gene cause inactivation of neighboring MSH2 gene
• Genetic predisposition to malignant tumor growth in specific regions
• Endometrium cancer (20-60%)
• HNPCC/ Lynch syndrome = hereditary non-polyose colon carcinoma
• Prevalence of HNPCC/Lynch syndrome is ~1:500
• Colon cancer (60-80%) in 75% due to spontaneous mutation
• Mutations in DNA-mismatch repair genes

  • MLH1
  • MSH2
  • MSH6
  • PMS2
• Penetrance of HNPCC/Lynch syndrome: 60-80% (of people with mutation develop the disease), dependent on other mutations and immune system
• CF is autosomal recessive (AR)
• CF results in Cl-ion channel defect leading to thickening of mucous especially in the lungs (chronic bronchitis) and other organs
• Colon carcinomas in younger age (~44 yrs)
• CF incidence is 1:2000-3000
• CF is caused by a mutation of the CFTR gene (phosphorylation-activated anion channel cystic fibrosis transmembrane conductance regulator)
• Heterozygotes for CF may have a possible selection advantage (against tuberculosis and typhus)
• CF has different grades and symptoms due to different mutations (R117H vs F508del)
• CBAV: congenital bilateral absence of vas deferens
• Homozygotes for CF may face a deadly outcome without treatment
• Drug therapy for CF is dependent on the underlying mutation and can be very expensive
• Genotype does not always influence phenotype in CF
• Penetrance in CF: proportion of people with the genetic variant that exhibit symptoms of the genetic disorder
• Intron splicing marker: T-allele variant (5, 7 or 9) influences splicing, TG repeats following T-allele (10-13 repeats), polymorphic thymidine tract (IVS8-5T, -7T, -9T) best: IVS8-9T following 10 TG repeats
• R117H: CFTR mutation p.Arg117His, c.350G>A in exon 4, Class IV, protein partially functional, rather mild mutation but clinically variable outcome (often no CF but infertility)
• F508del: Foundermutation, 70% of CFTR mutations, often severe in homozygotes
• Rare diseases have an incidence of <1:2000 and are often due to genetic predisposition
• Described genes in OMIM Catalog: 16824, Protein-coding genes in humans: ~19,600
• Hereditary tumorsyndromes often involve genetic predisposition and follow the 2-hit model for tumor suppressor genes