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

2. Models of Disease Etiology Genetic (inherited) Environmental (somatic) Multifactorial (polygenic + somatic) 2Molecular Diagnostics

3. Family History of Phenotype is Illustrated on a Pedigree Diagram 3Molecular Diagnostics

4. Pedigree Diagrams Reveal Transmission Patterns 4Molecular Diagnostics

5. Pedigree Diagrams Reveal Transmission Patterns 5Molecular Diagnostics

6. Pedigree Diagrams Reveal Transmission Patterns 6Molecular Diagnostics

7. 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. 7Molecular Diagnostics

8. 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 (+/-) 8Molecular Diagnostics

9. 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. 9Molecular Diagnostics

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11. 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). 11Molecular Diagnostics

12. Hemachromatosis Type I 12Molecular Diagnostics

13. Exon 4 G->A Rsa1 sites 240 bp 140 bp 110 bp 30 bp MW +/+ +/+ m/m +/m +/+ +/+ PCR primer Mutation creates an Rsa1 site (Mut) (+) Agarose gel HFE C282Y Detection by PCR- RFLP 13Molecular Diagnostics

14. 153 bp 116 bp Exon 10 G->A 67 bp 37 bp +/+ +/m m/m MW MnlI sites PCR primer (+) (Mut) Mutation destroys an MnlI site Agarose gel Detection of Factor V Leiden (R506Q) Mutation by PCR-RFLP 14Molecular Diagnostics

15. 148 bp 123 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 15Molecular Diagnostics

16. A T Mut probe Flap A A Mutation present -> Cleavage FQ Complex formation Fluorescence in plate well indicates presence of mutation F Cleavage A C wt probe Flap Normal sample (no cleavage) Factor V Leiden (R506Q) Mutation Detection by INVADER TM Assay Molecular Diagnostics16

17. 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 17Molecular Diagnostics

18. 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 18Molecular Diagnostics

19. Mitochondrial inheritance Non-Mendelian Transmission Patterns Gonadal mosaicism Nucleotide repeat expansion 19Molecular Diagnostics

20. Nucleotide Repeat Expansion in Fragile X Mental Retardation Gene (FMR1) Molecular Diagnostics20

21. 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 restriction enzyme Full mutation Detection of Fragile X CGG Expansion Mutations by PCR and Southern Blot 21Molecular Diagnostics

22. 10–29 repeats (normal) > 40 repeats Huntington Disease Huntingtin 80–170 bp Labeled PCR primer Autoradiogram of polyacrylamide gel Detection of Huntingtin Gene CAG Expansion Mutations by PCR Molecular Diagnostics22

23. 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) 23Molecular Diagnostics

24. HV 1 HV 2 P L P H1 P H2 MELAS 3243A>G LHON 3460G>A MERRF 8344A>G NARP 8393T>G LHON 11778G>A LHON 14484T>C Areas deleted in KSS Mitochondrial Mutations Associated with Disease 24Molecular Diagnostics

25. 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 25Molecular Diagnostics

26. 551 bp 206 bp 345 bp MspI U C U C U C Agarose gel U = Uuncut, no MspI C = Cut, with MspI The presence of the mutation creates an MspI restriction enzyme site in the amplicon. Mutation present Detection of NARP Mitochondrial Point Mutation (ATPase VI 8993 T → C or G) by PCR-RFLP Molecular Diagnostics26

27. M M + + PvuII U C U C 16.6 kb (normal) Deletion mutant (Heteroplasmy) The restriction enzyme, PvuII cuts once in the circular mitochondrial DNA. M = Mutant + = Normal U = Uncut, No PvuII C = Cut with PvuII Autoradiogram Detection of KSS Mitochondrial Deletion Mutation by Southern Blot Molecular Diagnostics27

28. 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). 28Molecular Diagnostics

29. 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 29Molecular Diagnostics

30. DNA Methylation Detected by Methylation Specific PCR (MSP-PCR) Molecular Diagnostics30 Bisulfite treatment converts unmethylated C residues to U. PCR …GTC Me GATC Me GATC Me GTG… …GTCGATCGATCGTG… …GTC Me GATC Me GATC Me GTG… …GTUGATUGATUGTG…  G CTAG CTAG CAC CTAGCTAGCACG G Product No product PCR primer

31. 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 31Molecular Diagnostics

32. 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. 32Molecular Diagnostics

33. 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 33Molecular Diagnostics

34. 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 34Molecular Diagnostics