Single Gene Mutations and Inheritance I April 3, 2008

1. Single Gene Mutations and Inheritance IApril 3, 2008 Lisa Schimmenti, M.D.

2. Objectives • Understand the basic types of single gene mutations • Understand how mutations lead to abnormal protein expression • Understand how a dominant mutation is passed from parent to child (inherited) • Know retinoblastoma genetics: dominant inheritance, but recessive on the cellular level.

3. Mutations are a change in DNA sequence that leads to a change in protein expression

4. U A A G A A C U G Met Ala The genetic code A codon is made of 3 base pairs 64 codons total 1 codon (AUG) encodes methionine and starts translation of all proteins 60 codons encode 20 amino acids (redundant code) 3 codons stop protein translation ASCO

5. Genetics Vocabulary • Allele: refers to different forms of the same gene • Example: • wildtype allele • mutant allele • null allele

6. More new words • Phenotype: the physical appearance of a trait • Genotype: the allele associated with a trait

7. Disease-Associated Mutations A mutation is a change in the normal base pair sequence that has been proven to associate with a disease state Commonly used to define DNA sequence changes that alter protein function ASCO

8. Disease-Associated Mutations Alter Protein Function Functional protein Nonfunctional or missing protein ASCO

9. Classes of Mutations

10. Type of mutations • Missense: nucleotide change leads to amino acid change • Nonsense: change leads to stop codon • Insertion: addition of nucleotides that lead to frameshift • Deletions: deletion of nucleotides • Splice site: changes RNA splicing • Expansion of repeat units

11. Nucleotide substitutionscan occur anywhere in the genome A C Pyr -> Pyr Pur -> Pur Transitions G T Pyr -> Pur Pur -> Pyr Transversions

12. Normal sequence ATG CCG TTT Met Pro Phe Mutant Sequence ATG CTG TTT Met Leu Phe Missense mutation in coding sequence

13. Nucleotide changes favor certain spots • CpG dinucleotide • C frequently methylated • Spontaneous deamidation to T • 8.5 x more likely to change than any other dinucleotide pair

14. Insertions/Deletions • Insertions (1 bp or >1 bp) • Deletions (1 bp or >1 bp)

15. ≤ 3 bp change (coding) • ins or del • 3 bp - ins or del one AA (most common mutation in CF delF508) • 2 bp ins or del - frame shift • 1 bp ins or del - frame shift • 1 bp substitution • Silent - “wobble” • Nonsense - AA to “stop” • Missense • Conservative - changes AA to same or similar AA • Non-conservative - changes AA by function or charge • May not change a basepair but may change the RNA splice recognition site

16. Changes in > 3 bp • Whole gene deletions or duplications • Often due to repeated sequences and mispairing in replications • Portion of gene deleted or fragment inserted • Often due to aberrant splicing

17. Normal Missense Nonsense Frameshift (1 bp deletion) Frameshift (1 bp insertion) Frameshift (3bp deletion) Triplet repeat expansion What does it look like? THE BIG RED DOG RAN OUT. THE BIG RAD DOG RAN OUT. THE BIG RED. THE BGR EDD OGR ANO…. THE BIG RED DOO GRA NOU THE BIG DOG RAN OUT. THE BIG BIG BIG BIG BIG RED DOG RAN OUT. Modifed from ASCO

18. Non-coding mutations that affect gene function • Promoter/enhancer element Trinucleotide repeats in 5 and 3’UTRs • Splice sites

19. Factors that influence how mutations alter gene function • Increase or decrease expression of gene product • Is one altered allele sufficient to cause disease? • Modifiers • Proportion of cells affected • Parent of origin

20. Effects of mutation on gene product • Null allele (loss of function) - no gene product • Hypomorph - decreased amt/activity • Gain of function - increased amt/activity • Dominant negative - antagonizes normal product • Neomorph - novel activity of product

21. Predicting that a gene product won’t do the job • Deletion, nonsense, frameshift of sequence is deleterious • Mutation in splice site usually bad • Missense mutations • Depends on location in protein • Is it non-conservative? • Is the AA conserved in evolution?

22. Which ones will cause disease? • All but silent or conservative missense sequence changes are likely to significantly alter product function • Among frameshifts, location of mutation alters likelihood of severity • Mutations in coding sequence are identified most frequently…but this may change

23. You only see the ones that hurt... • Sequence changes or variants can be • Silent (no change in product): Polymorphism • We have thousands of these • Manifesting (changing RNA or protein product): Mutation We all carry 7-10 gene mutations AHHHH!

24. Polymorphism • A change in DNA sequence that is not disease causing • Occurs usually in greater than 1% of population • Usually does not change an amino acid sequence or produces a significant change (ie: valine for isoleucine)

25. Autosomal Dominant

26. Autosomal Dominant Inheritance • males and females equally affected • 1 in 2 chance of affected offspring from an affected parent • male to male transmission • structural genes, transcription factors • only one abnormal copy of the gene (allele) to have the phenotype

27. Mechanisms yielding dominant alleles • Haploinsufficiency: if one copy isn’t enough for function • Dominant negative: when a mutant product dimerizes or oligomerizes • Gain of Function: FGFR mutations causing activation of the receptor without a ligand present

28. Autosomal Dominant probabilities Mother D d Daughters 50% normal 50% affected Dd d dd Father Sons 50% normal 50% affected d dd Dd

29. Variations in the AD “rules” • Co-dominant expression • New mutation • Homozygosity for an AD trait • May be a more severe phenotype • Variable expression • Penetrance • Sex limited • Variation in age of onset

30. Variations:Co-dominant expression Definition: alleles that are both expressed when they occur in the heterozygous state. Example: ABO blood group antigens

31. ABO Blood Groups Defined by the presence or absence of two antigens on the surface of the red cell A and B • if you have A, you have anti-B antibodies • if you have B you have anti-A antibodies • if you have neither (O) you have both anti- A and -B • Absence of A or B = O • Three allele states: Ia Ib Inull

32. ABO blood typing: co-dominant alleles B/- BB A/- B/- B/- BB AB -/- A/- BB AB A/-

33. Variations: New Mutations • Occur at a rate of 10-4 to 10-7 per locus per cell division • “Hot spots” occur: CG pairs (8.5 x higher rate) • More easily seen in a very large gene • Paternal age effects

34. Autosomal dominant: new mutation dd dd dd dd dd dd dd dd dd dd dd Dd

35. Achondroplasia • Autosomal dominant • 100% penetrance • 1/26000: most common genetic dwarfism • 80% new mutations • Features • rhizomelic dwarfism (upper arms and thighs are short) • lumbar lordosis (lower spine curves out) • large head • small foramen magnum • intact intellect • Mutation in FGFR3: activating mutation

36. Fibroblast Growth Factor Receptors • Receptor tyrosine kinases • Four distinct genes: 1 through 4 • Critical in coordination of proportionate bone growth and development • Signaling regulates cellular processes

37. Achondroplasia and FGFR • Mutations in FGFR3 cause achondroplasia • Despite frequent new mutations almost all are caused by the same mutation (G -> A at nt 1138, causes gly 380 arg)

38. Effects of homozygosity dd dd dd dd Dd dd Dd dd Dd dd DD

39. Homozygosity for some dominant traits is lethal http://www.echt.chm.msu.edu/courseware/blockII/Pathology/382131.jpeg Homozygosity for achondroplasia causes very severe phenotype

40. AD and variable expression • Pleiotropy: one gene, many effects on different systems • Random chance • Modifier genes

41. Marfan Syndrome: variable expression • Skeletal findings- disproportionate long bone growth • Tall Stature • Eyes- dislocated lenses • Heart-dilation of aortic arch http://medgen.genetics.utah.edu/

42. AD traits are often variable in expression dd dd Dd dd Tall Dislocated lenses Dilated Ao Tall Dilated Ao dd dd Dd Dd dd Dislocated lenses Dilated Ao Normal Dd dd Dd

43. Variations: reduced penetrance • An individual may carry an altered allele and never manifest phenotypic evidence of this. • Penetrance: an “all or nothing” phenomenon in expression of a trait in a population • phenotype, NOT genotype

44. Tumor Suppressor GenesClinical Effects of Mutations • Cancers due to mutations are early in onset • Cancers due to mutations are often bilateral • Phenotype is inherited as a dominant trait BUT at the cellular level these are manifest as recessive: two abnormal alleles are required for manifestation • Genotype is that of one germline abnormal allele: malignancy occurs with environmental damage of second normal allele (Knudsen two hit hypothesis) • Penetrance reduced: no 2nd hit, no malignancy occurs

45. Variation: reduced penetrance dd dd dd Dd dd dd Dd dd Dd dd dd

46. Familial Retinoblastoma: a recessive state in the cell inherited as AD trait http://www.people.virginia.edu/~rjh9u/retblst.html http://www.retinoblastoma.com/Images/CT%20Scan.jpg

47. Familial Retinoblastoma Case history: 6 month old child found to have leucocoria (white pupil) On exam there is a large mass in the eye. The other eye has a small lesion Father gives the history that his mother had only one eye because the other was removed when she was a baby. He and his 2 sibs were healthy and have both eyes Explanation: REDUCED PENETRANCE of this trait

48. Autosomal Dominant: sex limited trait dd dd Dd dd dd dd Dd Dd dd dd dd dd Dd Dd

49. A sex-limited AD trait • Familial male precocious puberty • Pubertal onset in males by age 4. • Due to gain of function mutation that keeps the leutinizing hormone (LH) receptor in an “on” state

50. Autosomal Dominant: variation in age of onset dd dd Dd dd dd dd dd Dd dd Dd Dd dd Dd dd Dd dd