Molecular Detection of Inherited Diseases

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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