Chapter 13

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

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. + + + Normal phenotype + + + + + + Abnormal phenotype - + - 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 (+/-)

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 Cell membrane HFE Gene product 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 G->A 153 bp 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. Q F A 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 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 FMR-1 CGG(CGG) 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 Southern blot Full mutation PCR 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. Huntingtin Detection of Huntingtin Gene CAG Expansion Mutations by PCR Labeled PCR primer 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. HV 2 P H1 LHON P 14484T>C MELAS H2 P L 3243A>G HV 1 LHON Areas deleted in KSS 3460G>A LHON 11778G>A MERRF 8344A>G NARP 8393T>G Mitochondrial Mutations Associated with Disease

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.