10 Clinical DNA Diagnosis

  1. Single Base Substitutions
    Missense mutations: replace one amino acid with another in the gene product

    Nonsense mutations: replace an amino acid with a stop codon

    Splice site mutations: create or destroy signals for exon/intron splicing

    Silent Mutation: base pair substitution results in no change to amino acid sequence

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  2. Deletions
    • the absence of a portion of the DNA, usually in the coding or regulatory sequence
    • might be less damaging if in multiples of 3 bps
  3. Microdeletions
    • *deletion not visible in the Karyotype
    • a constellation of findings due to a specific large deletion including several genes
    • eg. Williams syndrome
  4. Insertions
    • mutations that result in extra DNA within the coding sequence of a gene
    • eg. some forms of Crohn’s disease
  5. Duplications
    • type of insertion that are due to repeated regions of DNA, often including whole genes
    • regions can be duplicated next to one another (end to end, in “tandem”) or elsewhere on the genome
    • eg. Charcot-Marie-Tooth disease
  6. Frameshift Mutations
    • produced by deletions, insertions or splicing errors resulting in the shift of the reading frame
    • they often result in a stop codon that produces a truncated polypeptide
  7. Promoter Mutations
    • affect the binding of RNA polymerase to the promoter site
    • can result in the reduced production of mRNA & therefore decreased production of a protein
    • eg. Dyskeratosis congenita (premature aging + bone marrow disease)
  8. Tandem Repeat Expansions
    • when repeats increase from small to large number genetic diseases occur
    • expansion of the repeat sequences leads to more severe disease in the patient’s offspring (anticipation)
    • eg. Fragile X, Huntington’s - only expands in male meiosis, Duchenne muscular dystrophy, myotonic dystrophy (triplet repeat disorders)
  9. Loss of Function Mutation (3)
    • mutations result in gene products that have reduced function, no function, &/or may interfere with the function of the normal product
    • are generally inherited in an autosomal recessive disease
    • eg. cystic fibrosis, phenylketonuria
  10. Null Mutation (Null LoF Mutation)
    • often inherited in an AR manner
    • mutation carriers are healthy because 50% function is sufficient
    • if 2 mutated copies are passed on → child will be diseased
  11. Haploinsufficiency
    • occurs when a diploid organism has only a single functional copy of a gene
    • the other copy inactivated by mutation
    • the single functional copy does not produce enough of a gene product (typically a protein) to bring about a wild-type condition
    • this leads to an abnormal or diseased state
  12. Incomplete Dominance (Dosage Effect)
    • depending on whether a mutation is inherited heterozygously or homozygously, the disease can be autosomal dominant or autosomal recessive
    • autosomal dominant when half of the normal protein product is insufficient for normal function
    • disease is likely mild with 1 mutation (Aa)
    • autosomal recessive disease is severe (but not lethal) with 2 mutations (aa)
    • eg. Familial Hypercholesterolemia
  13. Familial Hypercholesterolemia
    • 1 LDLR mutation (Aa): somewhat elevated cholesterol levels (350-550 mg/dL compared to normal 200) with increased risk for heart disease (AD)
    • 2 LDLR mutations (aa): VERY elevated cholesterol levels (650-1,000 mg/dL) + early arteriosclerosis with visible deposits of cholesterol in the skin, eyelids, & cornea
    • treatment: statin, remove cholesterol from blood like dialysis, or liver transplant (so cholesterol can be processed)
    • (fun fact: cholesterol deposits in achilles tendon is so rare outside of FH that it’s considered diagnostic)
  14. Dominant Negative
    • 1 mutation causes a mutant product that’s “useless & gets in the way” - mutant protein interferes with the function of the normal protein
    • disease is inherited in an AD manner (Aa)
    • eg. osteogenesis imperfecta (OI) - has 8 different subtypes & 4 result from mutations in the pro-collagen genes
  15. Gain of Function Mutations
    • requires a very specific mutation - relatively rare
    • cause the protein product to actively function in a new, abnormal way
    • disease inherited in an AD manner (can be inherited or de novo)
    • eg. Achondroplasia caused by a mutation (only 2 have been identified) in the FGFR3 gene - causes receptor to be constitutively on resulting in poor bone growth (prevents chondrocyte proliferation → short bones)
  16. Benefits of DNA-based Tests
    • 1. Definitive Diagnosis: Clinical diagnosis of a genetic disease can be confirmed by molecular analysis
    • 2. Presymptomatic Diagnosis: Individuals at risk for disease can be identified before the disease has become clinically evident allowing use of prophylactic measures for screening & treatment (eg. colorectal cancer)
    • 3. Preimplantation/Prenatal Diagnosis: Allows for diagnosis of genetic conditions with known mutations in a blastocyst or fetus
    • 4. Genotype-Phenotype Correlations: Identification of certain mutant alleles may help predict specific prognosis, clinical phenotype, or therapeutic response
  17. Challenges of DNA-based Tests
    • 1. Heterogeneity: Mutations in several different genes may produce the same clinical syndrome (eg. “kidney disease” is caused by multiple genes)
    • 2. Allelic disorders: Different mutations in the same gene may produce different clinical syndromes
    • 3. Variable expression: Genetic diseases may be variable within families & between families (knowing the mutation does not necessarily predict phenotype)
    • 4. Non-paternity can be inadvertently discovered
    • 5. Genetic discrimination concerns may arise
  18. Karyotype Analysis
    • can detect visible chromosomal abnormalities (deletions, duplications, translocations, inversions, insertions) but it LACKS sensitivity
    • the changes that can be seen via Karyotype analysis are 3 megabases of DNA
  19. FISH (Fluorescent in situ hybridization)
    • detects known submicroscopic (micro)deletion & duplication syndromes
    • can detect subtelomeric deletions & duplications
    • FISH probes are ~150 kb of DNA - looking for + or -: a deletion/duplication is there or it isn’t
  20. Methylation Specific RT-PCR
    • treatment of DNA with bisulfite converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected
    • can tell what parts of genome are or aren't silenced (methylated)
  21. Allele Specific Oligonucleotide Testing (ASO)
    • a panel of common mutations in a given gene or several genes is premade
    • exons from the gene of interest are amplified using PCR, labeled, & hybridized to said filter with allele specific oligonucleotides representing possible mutations
    • an ASO is a short piece of synthetic DNA complementary to the sequence of a target DNA; it acts as a probe for the presence of the target in a Southern blot assay
  22. Short Tandem Repeat (STR) Analysis
    • compares specific loci on DNA from 2 or more samples
    • measures the exact number of repeating units
    • probes are attached to desired regions on the DNA & a polymerase chain reaction (PCR) is employed to discover the lengths of the short tandem repeats
    • in lecture, she uses this to describe how you’d check paternity
    • can detect variations in the number of repetitive sequences scattered throughout the genome
  23. Multiple Ligation-dependent Probe Amplification (MLPA)
    • multiple exons can be amplified at once (using PCR) & run out on a gel to determine presence of region
    • can be used to determine if there’s a large deletion, duplication, or rearrangement in a gene
  24. Population Screening
    • the process of looking for mutations in someone who is at higher than usual risk for a disease or a disease predisposition, but is not yet affected
    • provides presymptomatic detection
    • aids with reproductive decision making
    • the benefits should outweigh the costs
    • optimize sensitivity & specificity
    • provides “screening” not “testing” - screening gives you the odds of having a disease, testing proves whether you DO or DON’T have it
  25. Testing
    the process of targeted disease-specific laboratory procedures to show that someone is definitively affected or not affected with a disease
  26. What’s the best example of presymptomatic testing?
    the Newborn Screen
  27. Population Predispositions
    Caucasians: cystic fibrosis, spinal muscular atrophy

    Ashkenazi Jews: Tay-Sachs, Gaucher, Canavan disease, Niemann Pick type A, mucolipidosis IV, familial dysautonomia, Bloom syndrome, Fanconi anemia type C, cystic fibrosis, glycogen storage disease IA, maple syrup urine disease, Usher syndromes IF and III, nemaline myopathy, hyperinsulinemia, dehydrolipoamide dehydrogenase deficiency, Joubert, Walker-Warburg

    African Americans: thalassemia, sickle cell anemia, hemoglobin C

    Known familial single gene disorders: eg. Fragile X

    Known familial translocations
Card Set
10 Clinical DNA Diagnosis
Genetics Exam 2