Chapter 13Chapter 13

Molecular Detection of Inherited Diseases

ObjectivesObjectives

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 EtiologyModels of Disease Etiology

Genetic (inherited) Environmental (somatic) Multifactorial (polygenic + somatic)

Family History of Family History of PhenotypePhenotype is is Illustrated on a Illustrated on a PedigreePedigree Diagram Diagram

male affected male deceased male

female affected female deceased female

Pedigree Diagrams Reveal Pedigree Diagrams Reveal Transmission PatternsTransmission Patterns

Sex-linked (X-linked recessive)

Autosomal recessive (AR)Autosomal dominant (AD)

Transmission PatternsTransmission 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 PatternsTransmission 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 (+/-)

Autosomal Recessive (AR) Autosomal Recessive (AR) TransmissionTransmission

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 HeterozygosityLoss 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 Examples of Molecular Detection of Single Gene Disorders 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).

S

S

S

S

S

S

COOH

NH2

C282Ymutation

H63D andS65Cmutations

Cell membrane

Cytoplasm

2 Microglobulin

HFEGene product

Hemachromatosis Type I

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

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

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

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

Few Diseases Have Simple Few Diseases Have Simple Transmission Patterns Due To: 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 Non-Mendelian Transmission PatternsPatterns

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

Mitochondrial inheritance

Non-Mendelian Transmission Non-Mendelian Transmission PatternsPatterns

Gonadal mosaicism Nucleotide repeat expansion

CGG(CGG)5–55

CGGCGGCGG(CGG)56–200

CGGCGGCGGCGGCGGCGG(CGG)200–2000+

Normal

Premutation (Carrier)

Full mutation (affected)

Amplification

Amplification and methylation

FMR-1

FMR-1

FMR-1

Nucleotide Repeat Expansion in Fragile Nucleotide Repeat Expansion in Fragile X Mental Retardation Gene (X Mental Retardation Gene (FMRFMR1)1)

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 infemales cleaved by methylation-specific restrictionenzyme

Full mutation

Detection of Fragile X CGG Expansion Detection of Fragile X CGG Expansion Mutations by PCR and Southern BlotMutations by PCR and Southern Blot

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

Human Disorders Due to Human Disorders Due to Mitochondrial MutationsMitochondrial 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)

HV 1 HV 2

PL

PH1PH2MELAS

3243A>G

LHON3460G>A

MERRF8344A>G NARP

8393T>G

LHON11778G>A

LHON14484T>C

Areasdeleted in KSS

Mitochondrial Mutations Mitochondrial Mutations Associated with DiseaseAssociated with Disease

Mitochondrial MutationsMitochondrial 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

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

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

Genomic ImprintingGenomic 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 Example of Diseases Affected by Genomic ImprintingGenomic 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 DNA Methylation Detected by Methylation Specific PCR (MSP-PCR)Methylation Specific PCR (MSP-PCR)

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 Other Methods for Detection of DNA MethylationDNA Methylation

Methylation-sensitive single-nucleotide primer extension

PCR-RFLP with methylation sensitive restriction enzymes

Southern blot with methylation-sensitive restriction enzymes

Genetic Testing LimitationsGenetic 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 InheritanceMultifactorial Inheritance(Complex Traits)(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 ComplexitiesGenetic 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

SummarySummary

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.

SummarySummary

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.