2. An introduction to genetics for cat breeders Kathryn Robson MRC Weatherall Institute of Molecular Medicine University of Oxford

3. Genetics Just over 150 years old as a science Gregor Mendel (1822-1884) Coined the terms “recessive” and “dominant” in reference to certain traits Demonstrated that these traits were heritable Proposed the existence of genes which he called factors

4. Genes Most genes code for proteins Humans have over 20,000 genes Each chromosome contains many thousands of genes Chromosomes are made up of DNA and protein

5. Structure of a chromosome There are almost 3 meters of DNA in a cell

6. Structure of a chromosome Workshop 1 3m piece of string and cells

7. How many chromosomes? The diploid genome refers to the total number of chromosomes in a cell Chromosomes come in pairs apart from the sex chromosomes One of each pair is inherited from each parent AnimalDiploid no. chromosomes Human46 Cat38 Dog78 Sheep54 Cow60

8. Individual chromosome paints identify specific chromosomes

9. Genes provide the instructions The DNA code comprises 4 building blocks These are abbreviated to A, G, T and C A codon is comprised of three of these building blocks e.g. GAT or GGG Each codon codes for an amino acid which is the building block of protein

10. Proteins These can be structural e.g. the proteins that make up skin These can be catalysts that speed up the chemical reactions in the body These can be the factory workers and part of a production line These can be the defense system e.g. antibodies These can be part of the management team e.g. insulin

11. Congenital vs inherited disorders Congenital disorders – Quite often are developmental – Usually a one off – Manifest at birth or soon after Inherited disorders – Due to errors in the DNA, generally inherited from one or both parents – May manifest at birth or much later in life

12. Mutations These are alterations in the DNA code The come in different types – Deletions – Insertions – Missense – Nonsense In some instances they change the instructions

13. Deletions and insertions The cat has a black mate. The cat has a black mat. The cart has a black mate.

14. Missense and nonsense mutations The cat has a black mate. The rat has a black mate. The hat has a black mate. The cat has a bl.ck mate.

15. Mutations in genes give rise to inherited diseases What do these genes have in common? Myosin heavy chainCaveolin 3Calreticulin 3 Myosin light chain kinase 2Troponin T type 2 Actin  cardiac muscle 1 Cardiac myosin binding protein 3 Tropomyosin 15’ AMP activated protein kinase subunit  2 Myosin light chain 3Troponin I type 3Cardiac LIM protein Myosin light chain 2TitinVinculin Myosin heavy chain 6Troponin C type 1Junctophilin 2 MyozeninNexilinPhospholambin Myopallidin

16. Mutations in genes give rise to inherited diseases What do these gene have in common? Mutations in the genes listed give rise to different forms of cardiomyopathy in humans Many of them are muscle proteins uniquely found in heart muscle

17. The complexity of heart muscle

18. The order of the instructions is important 1.ONLY I hit him in the eye yesterday. (No one else did.) 2.I ONLY hit him in the eye yesterday. (Did not slap him.) 3.I hit ONLY him in the eye yesterday. (I did not hit others.) 4.I hit him ONLY in the eye yesterday. (I did not hit outside the eye.) 5.I hit him in ONLY the eye yesterday. (Not other organs.) 6.I hit him in the ONLY eye yesterday. (He doesn't have another eye.) 7.I hit him in the eye ONLY yesterday. (Not today.) 8.I hit him in the eye yesterday ONLY. (Did not wait for today.)

19. Mitosis This is the term for cell division With the exception of the cells that go on to form the egg and sperms cells, all cells in the body undergo mitosis In this way they form identical copies of themselves This is for example a natural part of repair and growth

20. Mitosis

21. Meiosis This is the term for cell division that ends in the production of eggs and sperm In this form of division the number of chromosomes in each cell must be reduced from two copies of each chromosome down to one When the egg and sperm fuse the number of chromosomes goes back to two pairs of each, one from each parent

22. Cell division Workshop 2 Shuffling cards

23. Gametes These are the sperm and the egg cells Each gamete has only one copy of each chromosome When the egg is fertilised by the sperm the chromosomes exist as pairs, one of each pair coming from each parent

24. Dominant genes The black/seal gene is a dominant gene The tabby gene is a dominant gene You only need one copy of either of these genes to see that these genes are present If you have one copy of a gene you are a heterozygote These genes are found on the autosomes

25. Recessive genes The chocolate gene is a recessive gene The dilute gene is a recessive gene You need both copies of either of these genes for their expression to be visible You are therefore a homozygote for these genes These genes are found on the autosomes

26. A genetic cross Both parents are homozygous for different alleles at the same locus SS ss Ss The sire is homozygous for S and the dam homozygous for s Each of their gametes only contain S or s respectively Each kitten has inherited S from the sire and s from the dam All kittens regardless of gender are Ss, they are heterozygotes F1 generation

27. A genetic cross Both parents are heterozygotes for the two alleles at the same locus Ss SS s S s The gametes from the sire are either S or s The gametes from the dam are either S or s Four possible combinations

28. A genetic cross Imagine two parents heterozygous for the black gene and the dilute gene B b BbBb BbBb BB BbBb BbBb bBbB bBbB bb Three kittens statistically should be seal and one blue Two out of the three kittens are heterozygotes and carry the dilute gene

29. Seals and blues

30. Sex-linked genes genes red father The red gene is a sex-linked gene This gene is present on the X-chromosome Manifests itself in the F1 or first generation as a tortoiseshell female if the X chromosome comes from the father The males in the F1 generation only inherit their X chromosome from their mother as they have inherited their Y chromosome from the father and therefore do not display or express the red gene

31. A genetic cross Imagine two parents where the sire is red and the dam is black BB X Y BB X Y BB XX BB XX B B X X All male kittens are black because they inherit their mother’s X chromosome and their father’s Y chromosome All female kittens inherit an X chromosome from each parent, the one from their father carries the red gene They are therefore all tortoiseshell B X Y B X Y B X Y B X Y B B X X

32. Sex-linked genes genes 2 red mother The red gene is a sex-linked gene This gene is present on the X-chromosome Manifests itself in the F1 or first generation as a tortoiseshell female or red male if the X chromosome comes from a red mother The males in the F1 generation inherit their X chromosome from their mother as they have inherited their Y chromosome from the father and therefore are all red

33. A genetic cross Imagine two parents where the dam is red and the sire is black BB X Y BB X Y BB XX BB XX B B X X All male kittens are red because they inherit their mother’s red X chromosome and their father’s Y chromosome All female kittens inherit an X chromosome from each parent, the one from their mother carries the red gene They are therefore all tortoiseshell B X Y B X Y B X Y B X Y B B X X

34. Sex-linked genes genes 3 tortoiseshell mother The red gene is a sex-linked gene This gene is present on the X-chromosome Manifests itself in the F1 or first generation as a tortoiseshell female or red male if the X chromosome comes from their tortoiseshell mother 50% of the kittens will not inherit the red X chromosome and so will not be red or tortoiseshell

35. A genetic cross Imagine two parents where the dam is tortoieshell and the sire is black BB X Y BB X Y BB X BB X B B X X Half the male kittens are red because they inherit their mother’s red X chromosome and their father’s Y chromosome Half the female kittens inherit carry the red gene from their mother, half do not Those with the red gene are tortioiseshell Those kittens that do not inherit the X chromosome with the red gene are black (seal) B X Y B X Y B X Y B X Y B B X X

36. Genes work together Lilacs and creams To be lilac you need both two dilute genes and two chocolate genes To be cream you need two red genes if female and two dilute genes If male you need one red gene and two dilute genes

37. Chocolates and lilacs

38. X inactivation Males have one X and one Y chromosome Females have two X chromosomes One one X chromosome can be active in a cell The partner X chromosome is inactive, it has been silenced X inactivation is random Every tortoiseshell is unique

39. All four different colours of torties Note the different colour distribution on their heads

40. Reds and creams

41. A genetic cross Both parents are homozygous for different alleles at the same locus and one parent is a dominant tabby BB AA BB AA bb aa bb aa BA ba BA ba BA ba BA ba BA ba BA ba BA ba BA ba Sire is a dominant black (B) (seal) and dominant agouti (A) (tabby) Dam is homozygous for b and therefore dilute so is blue All 4 kittens are heterozygous for the agouti gene (tabby) All 4 kittens are black and carry the dilute gene (heterozygotes) F1 generation

42. A genetic cross Imagine two parents heterozygous for the black gene and the dilute gene and one parent is tabby Bb Aa Bb aa Bb aa BaBa Ba ba bA Ba ba Ba The eight different permutations are illustrated above 4/8 kittens have inherited A and are so are tabby 2/8 kittens have inherited bb and so are blue One of these kittens has inherited the Agouti gene and so is a blue tabby 1/8 kittens is a dominant black 1/8 kittens is a dominant black and is tabby BA Ba BA ba baba bA ba

43. A genetic cross Imagine two parents heterozygous for the black gene and the dilute gene and one parent is tabby Bb Aa Bb aa Bb aa BaBa Ba ba bA Ba ba Ba BA Ba BA ba baba bA ba Dominant Black tabby Heterozygote Black tabby Dominant Black Black heterozygote Heterozygote Black tabby Blue tabby Black heterozygote Blue

44. A genetic cross Imagine two parents heterozygous for the black gene and the dilute gene and one parent is tabby Bb Aa Bb aa Bb aa BaBa Ba ba bA Ba ba Ba BA Ba BA ba baba bA ba Dominant Black tabby Heterozygote Black tabby Dominant Black Black heterozygote Heterozygote Black tabby Blue tabby Black heterozygote Blue

45. Tabby points Note that unlike the torties their masks are all very similar

46. Tortie tabbies

47. Workshop 3 Using the 2 gametes-red and blue pieces of paper work out the colour of the kitten you have

48. Next generation sequencing Permits parallel sequencing Can sequence 16 genes at the same time e.g. CM genes Reduces time Reduces costs Once set up cost can be as little as£500 per patient/sample per panel run

49. Mutations in genes give rise to inherited diseases What do these genes have in common? Myosin heavy chainCaveolin 3Calreticulin 3 Myosin light chain kinase 2Troponin T type 2 Actin  cardiac muscle 1 Cardiac myosin binding protein 3 Tropomyosin 15’ AMP activated protein kinase subunit  2 Myosin light chain 3Troponin I type 3Cardiac LIM protein Myosin light chain 2TitinVinculin Myosin heavy chain 6Troponin C type 1Junctophilin 2 MyozeninNexilinPhospholambin Myopallidin

50. Acknowledgements Photographs – Barrie Tackley – Robert Fox – Sue Allen – Veronica Buckle

51. Thank you for your attention Questions? Kathryn.robson@imm.ox.ac.uk