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Molecular Diagnostics
Molecular Detection of Inherited
Diseases
Chapter 13
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Molecular Diagnostics
Models of Disease Etiology
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Genetic (inherited)
Environmental (somatic)
Multifactorial (polygenic + somatic)
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Molecular Diagnostics
Family History of Phenotype is
Illustrated on a Pedigree Diagram
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Molecular Diagnostics
Pedigree Diagrams Reveal
Transmission Patterns
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Molecular Diagnostics
Pedigree Diagrams Reveal
Transmission Patterns
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Molecular Diagnostics
Pedigree Diagrams Reveal
Transmission Patterns
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Molecular Diagnostics
Transmission Patterns
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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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Molecular Diagnostics
Transmission Patterns
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Gain of function mutations usually display a dominant
phenotype.
Loss of function mutations usually display a recessive
phenotype.
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Dominant negative patterns are observed with loss of function in
multimeric proteins.
Homozygous (+/+)
+
+
+
Heterozygous (+/-)
-
+
+
+
+
+
+
-
+
Normal phenotype
Abnormal phenotype
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Molecular Diagnostics
Autosomal Recessive (AR)
Transmission
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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.
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LOH results from somatic (environmental, not inherited) mutations or
deletions of the normal allele.
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Molecular Diagnostics
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Molecular Diagnostics
Examples of Molecular Detection of
Single Gene Disorders
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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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Molecular Diagnostics
Hemachromatosis Type I
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Molecular Diagnostics
HFE C282Y Detection by PCRRFLP
PCR primer
Exon 4
PCR primer
Mutation creates an Rsa1 site
G->A
Rsa1 sites
(Mut)
(+)
MW +/+ +/+ m/m +/m +/+ +/+
240 bp
140 bp
110 bp
30 bp
Agarose gel
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Molecular Diagnostics
Detection of Factor V Leiden
(R506Q) Mutation by PCR-RFLP
PCR primer
Exon 10
PCR primer
MnlI sites
(+)
(Mut)
+/+ +/m m/m MW
G->A
153 bp
116 bp
67 bp
Mutation destroys an MnlI site
37 bp
Agarose gel
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Molecular Diagnostics
Detection of Factor V Leiden
(R506Q) Mutation by SSP-PCR
PCR primer
Exon 10
Sequence-specific PCR primers
G->A
Longer primer ends on mutated
base A and makes a larger
amplicon
148 bp
123 bp
Agaros gel
(Mut)
(+)
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Molecular Diagnostics
Factor V Leiden (R506Q) Mutation
Detection by INVADERTM Assay
Flap
Mut probe
Flap
A
wt probe
A
T
Mutation present -> Cleavage
C
Normal sample
(no cleavage)
A
Complex formation
F
Q
A
Cleavage
F
Fluorescence in plate well
indicates presence of mutation
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Molecular Diagnostics
Few Diseases Have Simple
Transmission Patterns Due To:
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Variable expressivity: range of phenotypes from the
same genetic mutation
Genetic heterogeneity: different mutations cause
the same phenotype
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Often observed in diseases with multiple genetic
components
Incomplete penetrance: presence of mutation but no
abnormal phenotype
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Molecular Diagnostics
Non-Mendelian Transmission
Patterns
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Single-gene disorders or disorders with multiple
genetic components with nonclassical patterns of
transmission:
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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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Molecular Diagnostics
Non-Mendelian Transmission
Patterns
Gonadal mosaicism
Nucleotide repeat expansion
Mitochondrial inheritance
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Molecular Diagnostics
Nucleotide Repeat Expansion in Fragile
X Mental Retardation Gene (FMR1)
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Molecular Diagnostics
Detection of Fragile X CGG Expansion
Mutations by PCR and Southern Blot
Southern blot
PCR
50–90
(premutation)
20–40
(normal)
Premutations can
be detected by PCR.
Full mutation
Inactive X in
females
cleaved by
methylationspecific
restriction
enzyme
Due to their large size, Southern blot
is required to detect full mutations.
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Molecular Diagnostics
Detection of Huntingtin Gene CAG
Expansion Mutations by PCR
Labeled PCR
primer
Huntingtin
80–170 bp
10–29 repeats
(normal)
Autoradiogram of polyacrylamide gel
>40 repeats
Huntington
Disease
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Molecular Diagnostics
Human Disorders Due to
Mitochondrial Mutations
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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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Molecular Diagnostics
Mitochondrial Mutations
Associated with Disease
HV 1
P
H1
MELAS
3243A>G
P
H2
LHON
3460G>A
MERRF
8344A>G
HV 2
LHON
14484T>C
P
L
Areas
deleted in
KSS
LHON
11778G>A
NARP
8393T>G
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Molecular Diagnostics
Mitochondrial Mutations
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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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Molecular Diagnostics
Detection of NARP Mitochondrial
Point Mutation (ATPase VI 8993 T→C
or G) by PCR-RFLP
U = Uuncut, no MspI
C = Cut, with MspI
MspI U C U C U C
551 bp
The presence of
the mutation
creates an MspI
restriction
enzyme site in the
amplicon.
345 bp
206 bp
Agarose gel
Mutation
present
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Molecular Diagnostics
Detection of KSS Mitochondrial
Deletion Mutation by Southern
Blot
M M + +
PvuII U C U C
The restriction enzyme,
PvuII cuts once in the circular
mitochondrial DNA.
16.6 kb (normal)
M = Mutant
(Heteroplasmy)
+ = Normal
U = Uncut, No PvuII
C = Cut with PvuII
Autoradiogram
Deletion mutant
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Molecular Diagnostics
Genomic Imprinting
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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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Molecular Diagnostics
Example of Diseases Affected by
Genomic Imprinting
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Prader-Willi Syndrome: caused by regional deletion
or mutation in the paternally inherited chromosome
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Angelman Syndrome: a different disease phenotype
caused by regional deletion or mutation in the
maternally inherited chromosome 15
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Molecular Diagnostics
DNA Methylation Detected by
Methylation Specific PCR (MSP-PCR)
…GTCMeGATCMeGATCMeGTG…
…GTCGATCGATCGTG…
Bisulfite treatment
converts
unmethylated C
residues to U.
…GTCMeGATCMeGATCMeGTG…
G CTAG CTAG CAC
PCR primer
Product
PCR
…GTUGATUGATUGTG…
CTAGCTAGCACG
G
PCR primer
No product
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Molecular Diagnostics
Other Methods for Detection of DNA
Methylation
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Methylation-sensitive single-nucleotide primer
extension
PCR-RFLP with methylation sensitive restriction
enzymes
Southern blot with methylation-sensitive restriction
enzymes
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Molecular Diagnostics
Genetic Testing Limitations
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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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Molecular Diagnostics
Multifactorial Inheritance
(Complex Traits)
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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.
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Obesity, BMI 27 kg/m
Diabetes, fasting glucose 126 mg
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Molecular Diagnostics
Genetic Testing Complexities
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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