1. Molecular Detection of Inherited Diseases
Chapter 13
Molecular Detection of Inherited Diseases

2. 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.

3. Models of Disease Etiology
Genetic (inherited)
Environmental (somatic)
Multifactorial (polygenic + somatic)

4. Family History of Phenotype is Illustrated on a Pedigree Diagram
male affected male deceased male
female affected female deceased female

5. Pedigree Diagrams Reveal Transmission Patterns
Autosomal dominant (AD)
Autosomal recessive (AR)
Sex-linked (X-linked recessive)

6. 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.

7. 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 (+/-)

8. 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.

9. 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.

10. 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).

11. Hemachromatosis Type I
H63D and
S65C
mutations S
NH2 S S
C282Y
mutation
b2 Microglobulin S S S
HFE
Gene product
Cell membrane
Cytoplasm
COOH

12. HFE C282Y Detection by PCR-RFLP
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

13. Detection of Factor V Leiden (R506Q) Mutation by PCR-RFLP
PCR primer
Exon 10
PCR primer
MnlI sites
(+)
(Mut)
+/+ +/m m/m MW
153 bp
G->A
116 bp
Mutation destroys an MnlI site
67 bp
37 bp
Agarose gel

14. Detection of Factor V Leiden (R506Q) Mutation by SSP-PCR
PCR primer
Exon 10
Sequence-specific PCR primers
Longer primer ends on mutated base A and makes a larger amplicon
G->A
148 bp
(Mut)
(+)
123 bp
Agaros gel

15. 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 F
Fluorescence in plate well indicates presence of mutation
Cleavage

16. 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

17. 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

18. Non-Mendelian Transmission Patterns
Gonadal mosaicism
Nucleotide repeat expansion
Mitochondrial inheritance

19. Nucleotide Repeat Expansion in Fragile X Mental Retardation Gene (FMR1)
Normal
CGG(CGG)
FMR-1
5–55
Amplification
Premutation (Carrier)
FMR-1
CGGCGGCGG(CGG)
56–200
Amplification and methylation
Full mutation (affected)
FMR-1
CGGCGGCGGCGGCGGCGG(CGG)
200–2000+

20. Detection of Fragile X CGG Expansion Mutations by PCR and Southern Blot
Full mutation
50–90
(premutation)
Inactive X in
females cleaved by methylation-
specific restriction
enzyme
20–40
(normal)
Due to their large size, Southern blot is required to detect full mutations.
Premutations can
be detected by PCR.

21. Detection of Huntingtin Gene CAG Expansion Mutations by PCR
Labeled PCR primer
Huntingtin
80–170 bp
>
40 repeats
Huntington
Disease
10–29 repeats
(normal)
Autoradiogram of polyacrylamide gel

22. 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)

23. Mitochondrial Mutations Associated with Disease
HV 1
HV 2 P L H1 H2
MELAS
3243A>G
LHON
3460G>A
MERRF
8344A>G
NARP
8393T>G
11778G>A
14484T>C
Areas
deleted in
KSS

24. 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

25. Detection of NARP Mitochondrial Point Mutation (ATPase VI 8993 T→C or G) by PCR-RFLP
The presence of
the mutation
creates an MspI
restriction
enzyme site in the
amplicon.
U = Uuncut, no MspI
C = Cut, with MspI
MspI U C U C U C
551 bp
345 bp
Mutation
present
206 bp
Agarose gel

26. 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.
M = Mutant
+ = Normal
U = Uncut, No PvuII
C = Cut with PvuII
16.6 kb (normal)
Deletion mutant
(Heteroplasmy)
Autoradiogram

27. 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).

28. 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

29. DNA Methylation Detected by Methylation Specific PCR (MSP-PCR)
…GTCMeGATCMeGATCMeGTG… …GTCGATCGATCGTG…
Bisulfite treatment converts unmethylated C residues to U.
PCR
…GTCMeGATCMeGATCMeGTG… …GTUGATUGATUGTG…
G CTAG CTAG CAC CTAGCTAGCACG G
PCR primer
PCR primer
Product No product

30. 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

31. 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.

32. 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

33. 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

34. Summary
Mendelian (AR, AD, and sex-linked) and non-Mendelian 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.

35. 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.