1. Genetics in TSC and LAM April 16 th 2016 Kevin C. Ess, MD, PhD Chief, Pediatric Neurology Depts. of Pediatrics and Neurology Vanderbilt University Medical Center

2. Wake Forest Baptist Medical Center Thanks to Tamison Jewett, MD Section on Medical Genetics Department of Pediatrics Wake Forest Medical Center 2

3. Wake Forest Baptist Medical Center Outline Chromosomes and genes Autosomal dominant inheritance Mosaicism Types of gene mutations TSC1 and TSC2 genes – What is their role? Roles of geneticist and genetic counselor How to find labs that test for TSC gene mutations What does the future hold? How to find research studies

4. Wake Forest Baptist Medical Center Chromosomes

5. Wake Forest Baptist Medical Center Karyotype 5

8. Ideogram of the human chromosomes p-arm q-arm <= centromere autosomes Sex chromosomes acrocentric 

9. Wake Forest Baptist Medical Center Chromosomes versus genes

10. Wake Forest Baptist Medical Center Genes are made of “bases”

11. Wake Forest Baptist Medical Center Bases make up amino acids Methionine-Leucine-Glycine

12. Wake Forest Baptist Medical Center Amino acids make up proteins!

13. Wake Forest Baptist Medical Center The Human Genome Project, completed in 2003, told us that there are 20,000-25,000 structural genes in the human genome.

14. Wake Forest Baptist Medical Center For the purposes of this talk: Causative gene mutation=Pathogenic variant Other genetic changes considered variants, often of unknown clinical significance! 14

15. Wake Forest Baptist Medical Center Autosomal dominant inheritance

16. Wake Forest Baptist Medical Center Autosomal dominant pedigree

17. Wake Forest Baptist Medical Center Autosomal Dominant Inheritance Each affected individual has a similarly affected parent. Exceptions: New causative mutation, nonpenetrance Affected individuals have a 50% risk that each of their children will be affected. Females and males have an equal chance of being affected. Disease occurs in subsequent generations of a family. Examples: Tuberous Sclerosis Complex, Marfan syndrome, neurofibromatoses, achondroplasia

18. Wake Forest Baptist Medical Center Tenets of Autosomal Dominant Inheritance Father-to-son transmission is a hallmark (not REQUIRED, but when we see this, we know the condition is autosomal dominant) Reduced penetrance is a feature, with penetrance rates varying among conditions Variable expressivity is seen within and between families

19. Wake Forest Baptist Medical Center Penetrance vs. Variability An analogy: Think of a light with a dimmer switch: – “penetrance” refers to whether the light (trait) is on or off (can you see it at all?) – “variability” refers to how bright or dim the light (trait) is (mild, moderate, or severe)

20. Wake Forest Baptist Medical Center Mosaicism Somatic – Only SOME of the cells of the body have the causative gene mutation Can include all cell types Germline – Only SOME of the egg or sperm cells have the causative gene mutation Occurs ONLY in the egg or sperm cells

21. Wake Forest Baptist Medical Center Somatic mosaicism 21

22. Wake Forest Baptist Medical Center Germline mosaicism 22

23. Wake Forest Baptist Medical Center Parents of a child with TSC About 33% of individuals with TSC have a parent with TSC 2-10% of parents of children with TSC have mosaicism (either germline or somatic) About 66% of people with TSC have a de novo TSC1 or TSC2 pathogenic (disease- causing) variant, and neither parent has the same gene variant 23

24. Wake Forest Baptist Medical Center What are some reasons why we can’t detect TSC on examination of the parent of an affected child? Gonadal mosaicism in a parent Reduced penetrance in a parent Late onset of signs/symptoms in a parent Parent is unavailable for testing The pathogenic variant occurred for the first time in the affected child 24

25. Wake Forest Baptist Medical Center What is the recurrence risk for having a child with TSC? 50% when a parent has TSC 1-2% when the parents are clinically unaffected (due to possibility of gonadal mosaicism) 25

26. Wake Forest Baptist Medical Center Types of gene variants Point mutations – Silent – Missense – Nonsense Frameshift mutations – Insertions – Deletions

27. Wake Forest Baptist Medical Center Not all gene mutations cause disease! Genes are constantly changing; GENE CHANGE IS THE KEY TO VARIATION BETWEEN INDIVIDUALS – Some variants do not result in any detectable abnormality – Some variants are “pathogenic” (disease- causing) – Some variants are of uncertain significance (VOUS= variant of uncertain significance) 27

28. Wake Forest Baptist Medical Center Point mutations-milder outcome

29. Wake Forest Baptist Medical Center Frameshift mutations-more severe outcome

30. Wake Forest Baptist Medical Center DNA mutation analogy Normal sequence – THE RED CAT ATE THE RAT Point mutation – THC RED CAT ATE THE RAT Insertion – THE CRE DCA TAT ETH ERA T Deletion – THE (R)EDC ATA TET HER AT 30

31. Wake Forest Baptist Medical Center There are two known TSC genes TSC1 – Located on chromosome 9q – Encodes a protein called “hamartin” – Mutations in this gene are found in 24% of people with TSC TSC2 – Located on chromosome 16p – Encodes a protein called “tuberin” – Mutations in this gene are found in 66% of people with TSC

32. Wake Forest Baptist Medical Center TSC pathogenic variants TSC1 variants (24%)- almost all cause truncation of the hamartin protein (incomplete formation) – 53% are small deletions and insertions – 23% are nonsense (premature truncation) – 15% are splice-site (affects protein formation) – 5% are large deletions/rearrangements – 4% are missense (abnormal protein is made) 32

33. Wake Forest Baptist Medical Center TSC pathogenic variants (cont.) TSC2 variants (66%) – 35% are small deletions and insertions – 29% are missense – 15% are nonsense – 15% are splice site – 6% are large deletions and rearrangements 33

34. Wake Forest Baptist Medical Center Hamartin and Tuberin Hamartin (TSC1) and tuberin (TSC2) functions are known to: – Work together to regulate cell proliferation – Have tumor suppressor function

35. Wake Forest Baptist Medical Center TSC1 TSC2 and the mTOR pathway -an intracellular signaling pathway important in regulating the cell cycle -mTOR is important in cell growth and proliferation -TSC1 and 2 keep mTOR in check -when TSC1 or 2 cannot form the complex that inhibits mTOR, there is unchecked cell growth and proliferation

36. Wake Forest Baptist Medical Center TSC2 Loss TSC1 and TSC2 are Tumor Suppressor Genes But sporadic LAM has never been transmitted mother to daughter……

37. ‘Sporadic’ LAM is caused by somatic mutations in tuberous sclerosis (TSC2) genes Carsillo PNAS 2000

38. TSC Mutations in TSC-one germ line, one somatic TSC Mutations in sporadic LAM-two somatic “Sporadic LAM is tuberous sclerosis in two organs”

39. Wake Forest Baptist Medical Center Clue #2-LAM is metastatic! AJRCCM Karbowniczek 2003 The cells that comprise the LAM lesion that recurs in transplanted lungs are derived from the recipient.

40. Wake Forest Baptist Medical Center Rapamycin—how does it work?

41. Wake Forest Baptist Medical Center Clinical Geneticists Reviews prenatal, past medical, developmental, and family histories Examines the individual (and parents, if possible) carefully Discusses his/her findings with care givers Works with care givers to make a plan for further testing/management Follows the individual over time as indicated 41

42. Wake Forest Baptist Medical Center Genetic counseling Is a process of communication aimed at defining and addressing the complex psychosocial issues associated with the diagnosis of a genetic disorder

43. Wake Forest Baptist Medical Center Genetic counseling includes: Obtaining a history that includes family and ethnic information Advising patients of risk to themselves and to other family members Offering genetic testing/prenatal diagnosis Outlining management options Providing access to resources/support

44. Wake Forest Baptist Medical Center Genetic counseling must include awareness of: Insurance issues Legal issues Cultural beliefs Religious beliefs Educational level

45. Wake Forest Baptist Medical Center Genetic counseling (cont.) Standard of practice is to provide NONDIRECTIVE counseling by informing and educating patients, thereby empowering them to make decisions best suited to their needs/views.

46. Wake Forest Baptist Medical Center For a list of laboratories offering clinical genetic testing for TSC, go to www.genetests.org 46

47. Wake Forest Baptist Medical Center 47