1. Medical Genetics & Genomics Guri Tzivion, PhD tzivion@windsor.edu Extension 506 BCHM 590: Summer 2015 Windsor University School of Medicine

2. BCHM 590 MD2 Genetics Class 28 Block 2 Review Part 1

3. Material for Block 2 exam  Transgenic animals & gene therapy: 2-3 questions  Cloning & the human genome project: 1-2 questions  Quantitative genetics and multifactorial traits: 2-3 questions  Population genetics: 1-2 questions  Inherited genetic diseases: 2-3 questions  Cancer and cancer heredity: 5-6 questions  Genetic counseling: 2-3 questions  Molecular Diagnostics: Linkage, PCR, RFLP & SNPs: 1-2 q

4. Gene Knockout

7. STEPS IN GENE THERAPY: 1.Identification of the defective gene. 2.Cloning of normal healthy gene. 3.Identification of target cell / tissue / organ. 4.Insertion of the normal functional gene into the host DNA. METHOD: Introduction of FUNCTIONAL GENES into appropriate cells Transferred gene (TRANSGENE) encodes & produces proteins The Proteins encoded by Transgene corrects the disorder

8. Gene Therapy: Approaches: Two ways to deliver genes: 1. Ex vivo approach 2. In vivo approach

9. 1. Ex vivo approach: Target cells are removed from the body and grown in vitro. Target cells are removed from the body and grown in vitro. The gene of interest is inserted into the cultured cells. The gene of interest is inserted into the cultured cells. The transgenic cells are re-introduced into the donor. The transgenic cells are re-introduced into the donor.

10. 2. In vivo approach: Direct Gene Transfer Cloned therapeutic gene is introduced directly into the affected tissue, without removing cells from the body. Specially designed vehicles are needed.

11. TYPES OF GENE THERAPY: 1. SOMATIC CELL THERAPY 2. GERM LINE THERAPY

12. 1. GENE AUGMENTATION THERAPY: If a disease is caused by a mutation causing loss of function, introduction of a FUNCTIONAL COPY OF THE GENE into the cell will restore the normal function of the gene. Examples: 1.Deficiency of ADA 2.Haemophilia

13. Therapeutic Cloning

15. Benefits Can be used to cure diseases If a patient receives stem cells cloned containing his own genetic material, then his/her immune system would not reject them as foreign material Research going on to find cure for Parkinson’s, paralysis, damaged heart muscles, arthritis and diabetes mellitus

16. Basic SCNT Methodology

17. Challenges to successful SCNT Reprogram a nucleus from a differentiated stage (somatic cell) to an embryonic stage Properly activate genes necessary for early embryonic development and suppress differentiation associated genes

18. Postnatal problems with SCNT Abnormally high birth weight Respiratory and metabolic abnormalities – underdeveloped lungs – Pulmonary hypertension “Adult clone sudden death syndrome” - cloned pigs died of heart failure at less than 6 months

19. Premature aging of cloned animals Telomeres are specialized structures at the ends of linear chromosomes that shorten with age Cloned cattle have widely varying telomere lengths Dolly’s telomeres – shorter than an age-matched control – consistent with a 6-year old mammary cell telomere

20. The Human Genome Project

22. Contributions of the human genome project Promoted the discovery of more than 1,800 disease genes. As a result of the Human Genome Project, today’s researchers can find a gene suspected of causing an inherited disease in a matter of days, rather than the years it took before the genome sequence was in hand. There are now more than 1,000 genetic tests for human disease conditions. These tests enable patients to learn their genetic risks for disease and also help healthcare professionals diagnose disease. At least 350 biotechnology-based products resulting from the Human Genome Project are currently in clinical trials.

23. DNA-Polymerase + Nucleotides Primers Denaturation 95°C Annealing 50-60°C Extension 68°C Denaturation, annealing Extension x30 Steps in PCR

24. Microarray Technology: Detection of differentially-expressed genes

25. Multifactorial Traits

26. Polygenic & Multifactorial Disorders Polygenic traits are controlled by two or more genes Multifactorial traits/disorders are polygenic traits with an environmental component

27. Pleiotropy Pleiotropy: when one gene affects more than one seemingly unrelated phenotypic character. Most genes are pleiotropic, for example, dwarfism, phenylketonuria and sickle cell anemia.

28. Examples of polygenic and multifactorial disorders Autism & epilepsy Dibetes mellitus & Glaucoma Hypertension, Ischemic heart disease & Ischemic stroke Manic depression & Schizophernia Multiple sclerosis & Parkinson disease Crohn disease (inflammatory bowel disease and ulcerative colitis), Asthma, Psoriasis & Rheumatoid arthritis Congenital malformations, for example, neural tube defects, congenital dislocation of the hip (CDH), cleft lip and palate and congenital heart disease.

29. Multifactorial Disorders Other examples: Alzheimer's disease & mood disorders Breast, ovarian, colon and other cancers Hypothyroidism & asthma

30. How evidence is gathered for genetic factors in complex diseases: Familial risks: what is the incidence of a disorder in relatives compared with the incidence in the general population? Twin studies: what is the incidence in monozygotic compared with dizygotic twins? Adoption studies: what is the incidence in adopted children of the disorders which their parent had? Population and Migration studies: what is the incidence in people from a particular ancestry group when they move to a different geographical area?) Evidence from these types of studies can estimate the heritability of a condition - the proportion of the aetiology ascribed to genetic factors rather than environmental factors

31. Incomplete & co-dominance Heterozygotes show an intermediate phenotype RR = red flowers rr = white flowers Rr = pink flowers make 50% less color

32. AP Biology Incomplete dominance true-breeding red flowers true-breeding white flowers X 100% 100% pink flowers F 1 generation (hybrids) 25% white F 2 generation 25% red 1:2:1 P self-pollinate 50% pink

33. Co-dominance Alleles affect the phenotype in separate, distinguishable ways ABO blood groups: 3 alleles I A, I B, i both I A & I B are dominant to i allele I A & I B alleles are co-dominant to each other Determine the presences of oligosaccharides on the surface of red blood cells

34. Blood types genotypephenotype status I A I A I A itype A type A oligosaccharides on surface of RBC __ I B I B I B itype B type B oligosaccharides on surface of RBC __ I A I B type AB both type A & type B oligosaccharides on surface of RBC universal recipient i ii itype O no oligosaccharides on surface of RBC universal donor

35. Epistasis One gene masks another: Coat color in mice, pigment (C) or no pigment (c); More pigment (black=B) or less (brown=b, recessive) cc = albino, regardless of B/b allele 9:3:3:1 becomes 9:3:4

36. Polygenic inheritance Some phenotypes are determined by the additive effects of 2 or more genes on a single character Phenotypes on a continuum Examples of human traits: Skin color Height Weight Eye color Intelligence Some behaviors

37.  Monogenic traits, e.g., pituitary dwarfism Qualitative Traits (the phenotypes fall into different and distinct categories). 120 cm 165 cm aa AA or Aa aa AA or Aa Height of individuals with pituitary dwarfism & of normal population

38.  Monogenic traits, e.g., Phenylketonuria (PKU) 0~5% 45%~50% 100% aa Aa AA aa Aa AA PAH (phenylalanine hydroxylase) activity of PKU, carrier & normal individuals

39.  Polygenic traits Quantitative Traits 130 140 150 160 170 180 190 200 Height (cm) 8070605040302010Variability  Gaussian (normal) distribution  The bell-shaped curve

40. Threshold605040302010 Low High VariabilityThreshold Affected

41. Genotypes: 4 n Phenotypes: 1 + 2n

42. Calculating probability of recurrence (having another affected child to the same parents)

43. © 2009 NHS National Genetics Education and Development CentreSupporting Genetics Education for Health www.geneticseducation.nhs.uk ProbandSiblings of proband Families with one child with multifactoria l condition 50 brothers and sisters

44. © 2009 NHS National Genetics Education and Development CentreSupporting Genetics Education for Health www.geneticseducation.nhs.uk ProbandSiblings of proband 50 brothers and sisters 2 affected with multifactorial condition Families with one child with multifactoria l condition

45. © 2009 NHS National Genetics Education and Development CentreSupporting Genetics Education for Health www.geneticseducation.nhs.uk ProbandSiblings of proband Therefore probability of recurrence = 1/25 (4%) 2/50 affected with multifactorial condition Families with one child with multifactoria l condition

46. Autosomal Recessive Traits: Only expressed in individuals that have two copies of the relevant gene. More frequent with inbreeding, isolated groups. Autosomal Dominant Traits: Expressed even if only one copy of the gene is inherited. Effects sometimes show up later in life. Sex-linked Traits: Associated with genes on the X chromosome. Chromosomal Abnormalities: Deletions, Duplications, Inversions, Translocations Nondisjunction and Aneuploidy: Extra or missing chromosomes

47. Direct transmission from an affected parent to an affected child. Transmission can occur from affected father to affected son. 1:1 ratio of affected vs. unaffected progeny with one affected parent. AUTOSOMAL DOMINANT INHERITANCE (Affected children always have an affected parent.)

48. Affected individuals can be either male or female. However, affected children not necessarily have affected parents. Affected parents can have affected offspring. AUTOSOMAL RECESSIVE INHERITANCE

49. SEX-LINKED RECESSIVE TRAITS More affected males than females. Affected grand- father to grand- son thru carrier female. Females do not usually manifest the disorder.

50. Genes on sex chromosomes Y chromosome SRY: sex-determining region Master regulator for maleness Turns on genes for production of male hormones (pleiotropy) X chromosome Other traits beyond sex determination Hemophilia Duchenne muscular dystrophy Color-blindness

51. Genetics of Populations Population a localized group of individuals belonging to same species Gene pool = The total genes in a population Evolution on the smallest scale occurs when the relative frequency of alleles in a population changes over a succession of generations = microevolution

52. Genetics of a Non-evolving Population The gene pool is in stasis This is described by Hardy-Weinberg principal The frequencies of alleles in a population’s gene pool remain constant over the generations unless acted on by agents other than sexual recombination i.e. shuffling the deck has no effect on the overall genetic make-up of the population

53. The Hardy-Weinberg Principle The frequency of an allele in a population will remain constant over time, provided that the following conditions are met: The population is large and randomly breeding There are no conditions acting on the population to change the allele frequency

54. The Hardy-Weinberg Equation This example is the simplest case: 2 alleles, one is dominant For this case: if p = frequency of one allele q = the frequency of the other Then: p + q = 1 probability of AA (AA genotype) = p 2 probability of aa (aa genotype = a phenotype) = q 2 probability of Aa (Aa genotype) = 2pq Therefore: p 2 + 2pq + q 2 = 1

55. Uses of Hardy-Weinberg You can calculate the frequency of a gene in a population if you know the frequency of the phenotypes Important in genetic disease counseling

56. Relevance to Evolution A population at genetic equilibrium does not evolve Hardy-Weinberg tells us what to expect in non- evolving populations Therefore it is a baseline for comparing actual populations where gene pools may be changing. Can determine if the population is evolving

57. Genetic Equilibrium Hardy-Weinberg equilibrium is maintained only if the population meets all 5 of the following criteria: Very large population size Isolation from other populations migration can affect the gene pool No net mutations Random mating No natural selection no difference in reproductive success Describes an ideal that rarely exists in nature

58. Altering Genetic Equilibrium For evolution to take place something must upset the genetic equilibrium of the population: Factors that change genetic equilibrium are: Genetic drift Migration (Gene flow) Non-random mating (Isolation) Mutation Natural selection

59. Sample Questions 3. Which of the following is NOT a condition of the Hardy- Weinberg law? A.Mating within the population occurs at random with respect to the loci under consideration. B.The population is infinitely large or, in practical terms, is large enough that sampling errors and random effects are negligible. C.No more than one other factor—mutation, migration, or random genetic drift—can be acting at one time during the period under study. D.There is no selective advantage for any of the genotypes under consideration; all genotypes are equally viable and fertile. E.All of the above are conditions of the Hardy-Weinberg law.

60. Sample Questions 3. Which of the following is NOT a condition of the Hardy- Weinberg law? A.Mating within the population occurs at random with respect to the loci under consideration. B.The population is infinitely large or, in practical terms, is large enough that sampling errors and random effects are negligible. C.No more than one other factor—mutation, migration, or random genetic drift—can be acting at one time during the period under study. D.There is no selective advantage for any of the genotypes under consideration; all genotypes are equally viable and fertile. E.All of the above are conditions of the Hardy-Weinberg law.

61. For tomorrow, make sure to go through the material and identify specific questions. We will practice tomorrow also some example questions. Friday will be a special class for students who need extra help or have specific questions or gaps. We can also go over more sample questions. Email me if you are interested !!!

62. BCHM 590 MD2 Genetics Class 29 Block 2 Review Part 2