Chapter 13
Molecular Detection of Inherited Diseases
Objectives
 Describe Mendelian patterns of inheritance as
exhibited by pedigree diagrams.
 Give examples of laboratory methods designed
to detect single-gene disorders.
 Discuss non-Mendelian inheritance and give
examples of these types of inheritance, such as
mitochondrial disorders and trinucleotide repeat
expansion diseases.
 Show how genomic imprinting (epigenetics) can
affect disease phenotype.
Models of Disease Etiology
 Genetic (inherited)
 Environmental (somatic)
 Multifactorial (polygenic + somatic)
Family History of Phenotype is
Illustrated on a Pedigree Diagram
male
affected male
deceased male
female
affected female
deceased female
Pedigree Diagrams Reveal
Transmission Patterns
Autosomal dominant (AD)
Autosomal recessive (AR)
Sex-linked (X-linked recessive)
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.
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.
Homozygous (+/+)
+
+
+
Heterozygous (+/-)
-
+
+
+
+
+
+
-
+
Normal phenotype
Abnormal phenotype
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.
Loss of Heterozygosity
 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.
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
(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).
Hemachromatosis Type I
H63D and
S65C
mutations
S
C282Y
mutation
S
S
S
S
NH2
b2 Microglobulin
S
Cytoplasm
Cell membrane
HFE
Gene product
COOH
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
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
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)
(+)
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
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
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
Non-Mendelian Transmission
Patterns
Gonadal mosaicism
Nucleotide repeat expansion
Mitochondrial inheritance
Nucleotide Repeat Expansion in Fragile
X Mental Retardation Gene (FMR1)
Normal
CGG(CGG)
5–55
FMR-1
Amplification
Premutation (Carrier)
CGGCGGCGG(CGG)
56–200
Amplification and methylation
Full mutation (affected)
CGGCGGCGGCGGCGGCGG(CGG)
200–2000+
FMR-1
FMR-1
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.
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
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)
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
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
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
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
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).
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
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
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
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.
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
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
Summary
 Mendelian (AR, AD, and sex-linked) and nonMendelian patterns of inheritance are exhibited by
pedigree diagrams.
 Frequently occurring point mutations are easily
detected by a variety of molecular methods
including PCR, PCR-RFLP, SSP-PCR, and
Southern blot.
 Non-Mendelian patterns of inheritance are exhibited
by nucleotide repeat expansions, mitochondrial
mutations, gonadal mosaicism, and genomic
imprinting.
Summary
 Gene silencing on methylation of C residues
affects phenotype without changing the DNA
sequence.
 Although molecular methods are ideal for
detection of DNA lesions, molecular analysis
may not always be the optimal strategy for
laboratory testing.