2. Single Gene Mutations and Inheritance I April 3, 2008 Lisa Schimmenti, M.D.

3. 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.

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

5. ASCO 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 3 codons stop protein translation 60 codons encode 20 amino acids (redundant code) UA A A UG Met G C A Ala

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

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

8. ASCO 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

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

10. Classes of Mutations

11. 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

12. Nucleotide substitutions can occur anywhere in the genome A TG C Transversions Pyr -> Pur Pur -> Pyr Transitions Pyr -> Pyr Pur -> Pur

13. Missense mutation in coding sequence Normal sequence ATG CCG TTT MetProPhe Mutant Sequence ATG CTG TTT MetLeuPhe

14. 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

15. Insertions/Deletions Insertions (1 bp or >1 bp) Deletions (1 bp or >1 bp)

16. ≤ 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

17. 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

18. 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. Normal Missense Nonsense Frameshift (1 bp deletion) Frameshift (1 bp insertion) Frameshift (3bp deletion) Triplet repeat expansion Modifed from ASCO

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

20. 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

21. 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

22. 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?

23. 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

24. 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!

25. 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)

26. Autosomal Dominant

27. 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

28. 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

29. Autosomal Dominant probabilities D d d d DdDd dd DdDd Daughters 50% normal 50% affected Sons 50% normal 50% affected Mother Father

30. 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

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

32. 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: I a I b I null

33. ABO blood typing: co-dominant alleles A/-B/- BB B/- ABAB A/--/- ABAB BB A/-

34. 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

35. Autosomal dominant: new mutation dd Dd

36. 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

37. 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

38. 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)

39. Effects of homozygosity dd Dd dd Dd DD

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

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

42. 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 /

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

44. 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

45. Tumor Suppressor Genes Clinical 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

46. Variation: reduced penetrance ddDd dd Dd dd Dd dd

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

48. 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

49. Autosomal Dominant: sex limited trait Dddd Dd dd Dd

50. 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

51. Autosomal Dominant: variation in age of onset Dddd Dd dd Dd dd Dd

52. Variation: Altered age of onset Huntington’s Chorea – Progressive dementia – Choreiform movements Autosomal Dominant pattern Age of onset usually after 30 Triplet repeat expansion in the Huntington gene that can expand

53. What to expect for the next lecture: Recessive inheritance X-linked inheritance –Dominant –Recessive Population genetics for docs

54. Example Question Find the one true statement about dominant inheritance : –A. Male are more commonly affected than females. –B. Two copies of the gene carry mutations to have the phenotype –C. Only gain of function mutations are dominant. –D. Haploinsufficiency mutations can only cause dominant disorders. –E. Mutations can be passed from fathers to sons.