Lecture 11 Flashcards
Models of Disease Etiology
Genetic
Environmental
Multifactorial
Genetic- inherited- cystic fibrous
Environmental - somatic - change in DNA to cells that isnt germ or egg cell- skin cancer
Multifactorial - polygenic ± somatic -
higher risk of heart disease with certain genes
Pedigree
-Family history of phenotype is illustrated on a pedigree diagram
-reveal transmission patterns
autosomal dominate - men and women 50% for children to express
autosomal recessive -
Transmission Patterns
Gain-of-function
Loss-of-function
Dominant negative
-autosomal dominant, autosomal recessive and sex linked recessive -observed in single-gene disorders by one genetic mutation
-prediction of a pattern is called Mendelian inheritance
-observable traits
what is a Autosomal recessive transmission
-most observed
-mutant phenotype is not observed in the heterozygous (normal/mutant) state -must be homozygous (mutant/mutant) to show the abnormal phenotype
Gain-of-function -mutations usually display a dominant phenotype
Loss-of-function -mutations usually display a recessive phenotype
Dominant negative -observed with loss of function in multimeric proteins expressed on both chromosomes
Loss of Heterozygosity
- it is the Loss of the normal allele, revealing the mutant allele caused by somatic mutations or deletions (environmental and not inherited)
-abnormal phenotype a hemizygous (mutant/deletion) state caused by Autosomal recessive mutations
Hemochromatosis Type I
molecular detection of single gene
disorders:
-* Overabsorption of iron from food caused by mutations in the gene for a membrane iron transporter protein
-directs iron stores to overload them
-autosomal recessive
-HFE C282Y Detection -detected by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP)
Thrombophilic state
molecular detection of single gene
disorders:
- Caused by the Leiden mutation in the gene for coagulation factor 5 (F5) and mutations in the gene for coagulation factor 2 (F2)
-detection by PCR-RFLP,(SSP-PCR) - 2 primers
Few diseases have simple transmission patterns due to
Variable expressivity
Genetic heterogeneity
Incomplete penetrance
Variable expressivity—range of phenotypes from the same genetic mutation
Genetic heterogeneity—different mutations causing the same phenotype observed in diseases with multiple genetic components. Alzheimer’s
Incomplete penetrance—presence of mutation but no abnormal phenotype
Non-Mendelian Transmission Patterns
Gonadal mosaicism:
Genomic imprinting:
Nucleotide-repeat expansion:
Mitochondrial inheritance:
Single-gene disorders or disorders with multiple genetic components with non-classical patterns of transmission:
Gonadal mosaicism: somatic mutation in germline cells (gonads) egg or sperm only offspring has the phenotype and the 1st gen does not
Genomic imprinting: nucleotide or histone modifications that do not change the DNA sequence
Nucleotide-repeat expansion: increased allele sizes disrupt gene function.
Fragile X -(FMR1) - CGG mutations detected by PCR and Southern Blot . Seen on the telomere on the x chromosome . Repeats larger than 200 get methylated on the 5’ end and turns off the gene causing fragile x
- methylation creates the bands at the bottom on the gel
- Huntingtin Gene CAG Expansion Mutations by PCR - presents at 30-40. repeats predict the disease severity
Mitochondrial inheritance: maternal inheritance of mitochondrial genes
Human Disorders Due to Mitochondrial Mutations
- Kearns–Sayre syndrome (KSS) (mitochondrial deletion) detected by southern blot
- Pigmentary retinopathy, chronic progressive external ophthalmoplegia (CPEO)
- Leber hereditary optic neuropathy (LHON)
- Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS)
- Myoclonic epilepsy with ragged red fibers (MERFF)
- Deafness
- Neuropathy, ataxia, retinitis pigmentosa (NARP) detected by PCR RFLP
- Subacute necrotizing encephalomyelopathy with neurogenic muscle weakness, ataxia, retinitis pigmentosa (Leigh with NARP)
usually SNP
Mitochondrial Mutations
Homoplasmy
Heteroplasmy
Homoplasmy—all mitochondria in a cell are the same
Heteroplasmy—some mitochondria are normal, and others have mutations
* The severity of the disease phenotype depends on the amount of mutant and normal mitochondria present
* the earlier it occurs the more severe
NARP - PCR RFLP
mitochondrial DNA is digested by restriction enzymes. if the patient has a mutation creating a binding site for the RE creating two fragments. In affected Mito DNA will appear as one large product
KSS - southern blot
in normal patient when DNA is digested only one band of 16.6 is created if there is a mutation there is 5 base deletion - 2 bands seen
Next-Generation Sequencing & Genetic
Testing
Germline mutations
Targeted sequencing panels
Exome sequencing
Genome sequencing
Germline mutations—50% to 100% allele frequency. Sprem or egg - future generations not you
* Mutations discovered by NGS are confirmed by Sanger sequencing
Targeted sequencing panels cover multiple known disease-causing gene mutations
Exome sequencing covers 85% of disease-causing mutations - protein coating of DNA which is where most of the disease is found
* Target/exome enrichment is performed by probe hybridization
* Sensitivity may be lower than targeted gene panel
Genome sequencing covers coding and noncoding regions
Genetic Testing Limitations
- Therapeutic targets (except for gene therapy) are phenotypic
- Non-symptomatic diagnosis where disease phenotype is not (yet) expressed may raise ethical concerns
- Most disease and normal traits are multicomponent systems
Advantages of Molecular Genetic Testing
- Rapid definitive
- DNA analysis has predictive value for disease risk, diagnosis and therapeutic intervention
- Gene-variant databases for referral
- Gene discovery for inherited diseases has moved from research to clinical applications
Target Microorganisms for Molecular-based
Testing
Those that are difficult or time-consuming to isolate safer and shorter
* Mycobacteria
Hazardous organisms
* Histoplasma, Coccidioides
Those without reliable testing methods
* HIV, hepatitis C virus
High-volume tests- urine is less invasive and pcr is faster
* N. gonorrhoeae, C. trachomatis
Applications of Molecular-Based Testing
- Rapid or high-throughput identification of microorganisms
- Detection and analysis of resistance genes
- Genotyping
- Classification
- Discovery of new microorganisms
