molecular detection of inherited diseases
DESCRIPTION
Molecular Detection of Inherited Diseases. Chapter 13. Models of Disease Etiology. Genetic (inherited) Environmental (somatic) Multifactorial (polygenic + somatic). Family History of Phenotype is Illustrated on a Pedigree Diagram. Pedigree Diagrams Reveal Transmission Patterns. - PowerPoint PPT PresentationTRANSCRIPT
Molecular Detection of Inherited Diseases
Chapter 13
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Models of Disease Etiology Genetic (inherited) Environmental (somatic) Multifactorial (polygenic + somatic)
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Family History of Phenotype is Illustrated on a Pedigree Diagram
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Pedigree Diagrams Reveal Transmission Patterns
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Pedigree Diagrams Reveal Transmission Patterns
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Pedigree Diagrams Reveal Transmission Patterns
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Transmission Patterns AR, AD, or sex-linked patterns are observed in
single-gene disorders (diseases caused by one genetic mutation).
Prediction of a transmission pattern assumes Mendelian inheritance of the mutant allele.
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Transmission Patterns Gain of function mutations usually display a dominant
phenotype. Loss of function mutations usually display a recessive
phenotype. Dominant negative patterns are observed with loss of function in
multimeric proteins.
++ ++
+- ++
Normal phenotype
Abnormal phenotype
+
+
+
-
Homozygous (+/+)
Heterozygous (+/-)
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Autosomal Recessive (AR) Transmission
AR is the most frequently observed transmission pattern. The mutant phenotype is not observed in the heterozygous
(normal/mutant) state. A mutation must be homozygous (mutant/mutant) to show
the abnormal phenotype. AR mutations also result in an abnormal phenotype in a
hemizygous (mutant/deletion) state. Loss of the normal allele, revealing the mutant allele, is
called loss of heterozygosity, or LOH. LOH results from somatic (environmental, not inherited) mutations or
deletions of the normal allele.
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Examples of Molecular Detection of Single Gene Disorders
Hemachromatosis I: overabsorption of iron from food caused by mutations in the gene for a membrane iron transporter (hemachromatosis type I gene: HFE).
Thrombophilic state caused by the Leiden mutation in the gene for coagulation factor V (F5) and/or specific mutations in the gene for coagulation factor II (F2).
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Hemachromatosis Type I1212 Molecular DiagnosticsMolecular Diagnostics
Exon 4
G->ARsa1 sites
240 bp
140 bp110 bp 30 bp
MW +/+ +/+ m/m +/m +/+ +/+
PCR primer
PCR primer
Mutation creates an Rsa1 site
(Mut) (+)
Agarose gel
HFE C282Y Detection by PCR-RFLP
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153 bp116 bp
Exon 10
G->A
67 bp 37 bp
+/+ +/m m/m MW
MnlI sites
PCR primer
PCR primer
(+)(Mut)
Mutation destroys an MnlI site
Agarose gel
Detection of Factor V Leiden (R506Q) Mutation by PCR-RFLP
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148 bp123 bp
Exon 10
G->A
PCR primer
Sequence-specific PCR primers
Longer primer ends on mutated base A and makes a larger amplicon
(Mut) (+)
Agaros gel
Detection of Factor V Leiden (R506Q) Mutation by SSP-PCR
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A
T
Mut probeFlap
A
A
Mutation present -> Cleavage
F Q
Complex formation
Fluorescence in plate well indicates presence of mutation
FCleavage
A
C
wt probeFlap
Normal sample(no cleavage)
Factor V Leiden (R506Q) Mutation Detection by INVADERTM Assay
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Few Diseases Have Simple Transmission Patterns Due To: Variable expressivity: range of phenotypes from the
same genetic mutation Genetic heterogeneity: different mutations cause
the same phenotype Often observed in diseases with multiple genetic
components Incomplete penetrance: presence of mutation but no
abnormal phenotype
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Non-Mendelian Transmission Patterns
Single-gene disorders or disorders with multiple genetic components with nonclassical patterns of transmission: Gonadal mosaicism: somatic mutation in germ-line cells
(gonads) Genomic imprinting: nucleotide or histone modifications
that do not change the DNA sequence Nucleotide repeat expansion: increased allele sizes disrupt
gene function Mitochondrial inheritance: maternal inheritance of
mitochondrial genes
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Mitochondrial inheritance
Non-Mendelian Transmission Patterns
Gonadal mosaicism
Nucleotide repeat expansion
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Nucleotide Repeat Expansion in Fragile X Mental Retardation Gene (FMR1)
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PCR Southern blot
Premutations can be detected by PCR.
Due to their large size, Southern blot is required to detect full mutations.
20–40(normal)
50–90(premutation) Inactive X in
females cleaved by methylation-specific restrictionenzyme
Full mutation
Detection of Fragile X CGG Expansion Mutations by PCR and Southern Blot
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10–29 repeats(normal)
>40 repeatsHuntingtonDisease
Huntingtin
80–170 bp
Labeled PCR primer
Autoradiogram of polyacrylamide gel
Detection of Huntingtin Gene CAG Expansion Mutations by PCR
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Human Disorders Due to Mitochondrial Mutations
Kearnes Sayre syndrome (KSS) 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 (MERRF) Deafness Neuropathy, ataxia, retinitis pigmentosa (NARP) Subacute necrotizing encephalomyelopathy with
neurogenic muscle weakness, ataxia, retinitis pigmentosa (Leigh with NARP)
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HV 1 HV 2
PL
PH1PH2MELAS
3243A>G
LHON3460G>A
MERRF8344A>G NARP
8393T>G
LHON11778G>A
LHON14484T>C
Areasdeleted in KSS
Mitochondrial Mutations Associated with Disease
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Mitochondrial Mutations Homoplasmy: all mitochondria in a cell are the
same Heteroplasmy: some mitochrondria are normal and
others have mutations The severity of the disease phenotype depends on
the amount of mutant and normal mitochondria present
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551 bp206 bp345 bp
MspI U C U C U C
Agarose gel
U = Uuncut, no MspIC = Cut, with MspI
The presence of the mutationcreates an MspIrestrictionenzyme site in the amplicon.
Mutationpresent
Detection of NARP Mitochondrial Point Mutation (ATPase VI 8993 T→C or G) by PCR-RFLP
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M M + +PvuII U C U C
16.6 kb (normal)
Deletion mutant
(Heteroplasmy)
The restriction enzyme,PvuII cuts once in the circularmitochondrial DNA.
M = Mutant+ = NormalU = Uncut, No PvuIIC = Cut with PvuII
Autoradiogram
Detection of KSS Mitochondrial Deletion Mutation by Southern Blot
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Genomic Imprinting Gene silencing due to methylation of C residues
and other modifications. Genomic imprinting occurs during production of egg
and sperm. The phenotypic effects of imprinting are revealed in
diseases in which the maternal or paternal allele is lost (uniparental disomy/deletion).
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Example of Diseases Affected by Genomic Imprinting
Prader-Willi Syndrome: caused by regional deletion or mutation in the paternally inherited chromosome 15
Angelman Syndrome: a different disease phenotype caused by regional deletion or mutation in the maternally inherited chromosome 15
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DNA Methylation Detected by Methylation Specific PCR (MSP-PCR)
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Bisulfite treatment converts unmethylated C residues to U.
PCR
…GTCMeGATCMeGATCMeGTG… …GTCGATCGATCGTG…
…GTCMeGATCMeGATCMeGTG… …GTUGATUGATUGTG… G CTAG CTAG CAC CTAGCTAGCACG G
Product No product
PCR primer PCR primer
Other Methods for Detection of DNA Methylation
Methylation-sensitive single-nucleotide primer extension
PCR-RFLP with methylation sensitive restriction enzymes
Southern blot with methylation-sensitive restriction enzymes
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Genetic Testing Limitations Intergenic mutations in splice sites or regulatory
regions may be missed by analysis of gene coding regions.
Therapeutic targets (except for gene therapy) are phenotypic.
Nonsymptomatic diagnosis where disease phenotype is not (yet) expressed may raise ethical concerns.
Most disease and normal traits are multicomponent systems.
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Multifactorial Inheritance(Complex Traits)
Complex traits have no distinct inheritance pattern. Complex traits include normal traits affected by
multiple loci and/or environmental factors (height, blood pressure).
Quantitative traits are complex traits with phenotypes defined by thresholds. Obesity, BMI 27 kg/m Diabetes, fasting glucose 126 mg
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Genetic Testing Complexities Variable expressivity: a single genetic mutation
results in a range of phenotypes Genetic heterogeneity: the same phenotype results
from mutations in different genes (includes diseases with multiple genetic components)
Penetrance: presence of mutation without the predicted phenotype
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