Genetics and Reproductive Options for SMA Families Annual SMA Conference Dallas, Texas Friday, June 15, 2017

Transcription

1 Genetics and Reproductive Options for SMA Families 2018 Annual SMA Conference Dallas, Texas Friday, June 15, 2017

2 Part 1: SMA and Genetics Louise R Simard, PhD Part 2: SMA Carrier Screening Melissa Gibbons, MS CGC Part 3: Reproductive Options for SMA Families Harvey J Stern, MD, PhD 2

3 Part 1: SMA and Genetics Louise R. Simard, PhD University of Manitoba, Faculty of Medicine Dept. Biochemistry & Medical Genetics Winnipeg, MB, Canada R3E 0J Ph.D. U Toronto Medical Genetics Ste. Justine Hospital Research Centre, U Montreal Independent research program in SMA DNA diagnostics Consultant, incl. SMA testing 2006-present Professor, Department Head, U Manitoba Questions are welcome! 3

4 Part 1: SMA and Genetics SMA lower Motor Neuron Disease with a wide range of clinical presentation (severity) Outline SMA Gene SMN1 and SMN2 SMN1 Mutations SMA severity SMA Genetics Chromosomes as vehicles of the DNA genomic blueprint Autosomal Recessive Inheritance SMA Molecular Tests Diagnosis Carrier 4

5 Part 1: SMA and Genetics SMA is a lower motor neuron disease that recurs in families (inherited). 0 1 Disease Severity Wide Clinical Presentation! Type 2 Intermediate SMA < 18 months Never stand unaided Decreased life expectancy Type 0 Prenatal SMA In utero Respiratory support Death before 1 month Type 3 Kugelberg-Welander 18 months Stand alone but loss of mobility Normal life expectancy Type 1 Werdnig-Hoffman < 6 months Never sit unaided Death before 2 years Type 4 Adult SMA 21 years of age Progressive muscle weakness Normal life expectancy Image adapted from from Principles of Neural Science, ER Kandel, JH Schwartz, TM Jessell, eds., 4 th Ed. McGraw Hill. Loss of MN cells leads to Muscle Atrophy 5

6 Part 1: SMA and Genetics Identifying the SMA gene 1 gene 5 SMA types Nature (1990) 344: Lancet (1990) 336: Nature (1990) 345: Nature (1990) Lefebvre et al., (Melki laboratory, INSERM Institut Necker) 6

7 Part 1: SMA and Genetics Genome Decoration Page/NCBI Genetic Code = Blueprint of life ~20,000 genes in the human genome G C T A T A A T CG CG AT T A GC C G DNA = four nucleic acids (C, G, T, A) Different sequence of A-C-T-G = gene C (cytosine) pairs with G (guanine) T (thymine) pairs with A (adenine) 7

8 SMN1 vs. SMN2 gene Chr 5q13 Mutations in SMN1 Cause 5q-SMA DNA (a) SMN2 T 7 > 99 % identical SMN1 C 7 STOP STOP RNA (b) Promoter T Promoter C STOP STOP science-environment mrna (c) Start Start protein unfunctional protein functional protein ~10% Full-length transcripts ~90% Nonfunctional SMN protein 100% Full-length transcripts Functional Wild Type SMN protein 8

9 Part 1: SMA and Genetics % patients Deletions (missing DNA) is a common mutation in SMA SMN Very large deletions are often associated with type I and type II SMA. Type I SMAs most often cannot make ANY SMN In Type II & III SMA, the mutation is often confined to SMN1 exon type I type II type III SMN null SMN E7 Simard et al., (1997) Am J Med Genet 72:

10 Part 1: SMA and Genetics SMA locus is very variable On average: SMN2 SMN2 SMN1 SMN1 But also find: SMN1 SMN2 SMN1 SMN1 SMN2 SMN1 No apparent Clinical effect SMN2 SMN2 SMN1 SMN2 SMA causing Chromosomes 10

11 % patients Part 1: SMA and Genetics SMN2 is a major modifier gene SMN2 copy number decreases with increasing severity Taken from Feldkotter et al., (2002) Am J Hum Genet 70: # SMN2 copies type I type II type III n=188 n=110 n=77 11

12 Part 1: SMA and Genetics SMA is inherited as an Autosomal Recessive Disorder The fundamental unit of inheritance is DNA. Each cell has over 5 feet of DNA! It must be compacted to fit inside a cell in the form of chromosomes. We have 23 pairs of chromosomes; each set is inherited from our biological parents MTIwNjA4NjMzNTA5MDg2NzMy/ gregor-mendel jpg SMA autosomal recessive mode of inheritance ******************************************** There must be a mutation (error) in both copies of a gene (maternal and paternal) for the genetic disorder to be passed onto a child. 12

13 Part 1: SMA and Genetics SMA is inherited as an Autosomal Recessive Disorder Affected individuals have mutations in both copies of a recessive gene. Carriers have a mutation in only one copy of a recessive gene and is asymptomatic. Males and females can be carriers. 1 in 4 Chance 25% Taken from ghr.nlm.nih.gov 1 in 2 1 in 4 Chance Chance (50%) 25% Having an affected child is a surprise because carrier parents are asymptomatic. From this point onwards genetic counseling becomes possible. Males and females can be affected. If both parents are carriers, the risk of having a child with the disease is 25%. 13

14 Part 1: SMA and Genetics >95% of patients have deleterious mutations in the SMN1 gene (deletions, gene conversions, point mutations) deletion T 7 SMN2 C X SMN1 7 gene conversion T 7 SMN2 T SMN1SMN2 point mutation 14

15 Part 1: SMA and Genetics Molecular Diagnostic Tests SMN2 T 7 SMN1 C 7 Detection of homozygous mutations presence/absence of SMN1 Ex7 = diagnosis Detection of heterozygotes Number of copies of exon 7 = carrier testing Analysis of point mutations = diagnosis Not yet routine, specialized labs only 15

16 Part 1: SMA and Genetics Diagnostic Tests: SMN1 mutations Diagnosis of SMA patients All or None test! Van der Steege et al. (1996) Am J Hum Genet 58: SMN1 SMN2 (187 pb) (164 pb) Compared to EMG & muscle biopsy, the DNA test is: non-invasive sensitive, detects >95% of all SMAs accurate, all lacking SMN1 exon 7 have SMA ideal for newborn screening Limitation: Some SMA individuals have a point mutation in the SMN1 gene. Such mutations are not detected by the deletion or quantitative PCR assay. Taken from Passon et al. (2010) Mol Cell Probes 24:310 16

17 Part 1: SMA and Genetics Diagnostic Tests: Carrier Detection 2 vs. 1 copy of SMN1 exon 7 test (dosage) Detection of heterozygotes SMN quantitative test 0:4 0:4 0:3 1:2 1:3 SMN1:SMN2 copies 2:1 2:2 McAndrew et al. (1997) Am J Hum Genet 60: Rochette et al., (1997) Neurogenetics 1: SMN1 SMN2 Taken from Passon et al. (2010) Mol Cell Probes 24:310 95% of SMA patients are lacking SMN1 exon 7 Because SMA is an autosomal recessive disorder, most SMA carriers should have only 1 copy of SMN1 exon 7 17

18 Part 1: SMA and Genetics Diagnostic Challenges Rarely able to give a 100% Yes or No result. These tests do not detect small mutations in the SMN1 gene. Taken from Alías et al. (2009) Human Genetics 125:29 On average: SMN2 SMN1 But. SMN2 SMN2 SMN1 SMN2 SMN1 SMN1 2 to 5% of individuals have both SMN1 genes on the same chromosome (2+0 genotype) - so are carriers 18

19 A Genetic Counselor is a valuable resource to help you understand the genetics of SMA, opportunities for testing (diagnosis, carrier status, prenatal) and how to interpret test results. 19

20 Part 2: SMA Carrier Screening Melissa Gibbons, MS CGC University Colorado Health Sciences Ctr. Children s Hospital Colorado Genetic Counselor 20

21 Part 3: Reproductive Options for SMA Families Harvey J. Stern MD, PhD Director, Reproductive Genetics Genetics & IVF Institute 21

22 Workshop Goals What are the genetic aspects of SMA especially as they relate to reproduction? What are the reproductive choices for families at-risk for SMA? 22

23 RECESSIVE DISEASE

24 Reproductive Options for SMA Families Very difficult decisions about family building 24

25 Reproductive Options for SMA Families Decisions regarding childbearing are personal and reflect our own ethical, moral and religious views. This is not a one size fits all type of issue. Couples who are at-risk for a genetic disorder should be allowed to make up their own minds after considering all their reproductive options. 25

26 Reproductive Options for SMA Families Decisions are influenced by the couple s experience with SMA. Those who have had a child or sib affected will react differently from a couple identified to be at-risk by genetic screening. Decisions are also influenced by couple s attitudes towards prenatal diagnosis and assisted reproduction ( playing God ) 26

27 Reproductive Options for SMA Families Most families do not wish to bring another child with SMA into the world. 27

28 Reproductive Options for SMA Families Have no or no more children. (Most Common Choice) Adoption. Use a gamete donor. (pre-tested for SMA) 28

29 Reproductive Options for SMA Families Natural conception with 1 st or 2 nd trimester prenatal testing. Preimplantation Genetic Diagnosis 29

30 What is PGD? Involves the use of assisted reproduction technologies (IVF) to provide a method of prenatal diagnosis. Offers an alternative to traditional methods of prenatal diagnosis including chorionic villous sampling and amniocentesis, with the option of termination of affected pregnancies. 30

31 Development of PGD This year represents the 28th anniversary of the first application of PGD in humans. (Handyside et al. Nature ) Since 1990, approximately 400,000 PGD cycles have been performed worldwide. This has resulted in over 100,000 PGD babies born after the procedure. 31

32 PGD for Genetic Disease affected affected affected Transfer unaffected embryos to the patient 32

33 In-Vitro Fertilization (IVF) 33

34 Questions About PGD Why do I need to do IVF, I have no trouble getting pregnant? 34

35 Questions About PGD IVF is used to increase the odds of producing a healthy child. There is no change in the genetic material (egg or sperm). By creating multiple embryos, one increases the chance that good quality embryos which are not affected with SMA will be present and available to be returned to the uterus. 35

36 IVF Procedure

37 IVF/PGD Procedure Components of IVF/PGD Cycle: 1. Down-regulation (preparation) 2. Ovarian Stimulation - FSH 3. Egg Retrieval 4. Fertilization (ICSI) 5. Embryo biopsy and testing 6. Embryo transfer to uterus 37

38 Parts Of An IVF Cycle 38

39 Normal Ovulation 39

40 Embryo Development Day 5 Day 4 Day 3 Day 2 4 cell 2 cell 8 cell Morula Blastocyst Fertilization Adapted from Moore and Persaud, Saunders,

41

42 IVF: Monitoring Visit Follicle measurement Estradiol levels Days of FSH Stimulation 42

43 Stimulated Ovary 43

44 Egg Retrieval 44

45 Egg Aspiration 45

46 Egg Ready For Fertilization 46

47 Fertilization by Intracytoplasmic Sperm Injection ICSI ICSI 47

48 Embryo Development 2 cell 4 cell 3 cell 8 cell From: Veeck LL, An Atlas of Human Gametes and Conceptuses, 48 Parthenon Publishing, 1999

49 Embryo Development Morula stage embryos Blastocyst stage embryos 49 From: Veeck LL, An Atlas of Human Gametes and Conceptuses, Parthenon Publishing, 1999

50 Embryo Transfer 50

51 PGD for Single Gene Disorders Analysis involves whole genome amplification with multiplex PCR amplification of the mutation along with 3-4 linked polymorphic markers which generate a chromosomal haplotype. At the same time, the embryos are also tested by a chromosomal microarray for abnormalities (ie. Down syndrome) which can lead to failed implantation or pregnancy loss. 51

52 PGD for SMA SMN 1 & 2 = unaffected SMN 2 only = affected Embryo Biopsy 52 SMN gene

53 SMN1 SMN 1 & 2 Present NO SMN1 Affected

54

55

56

57 24 Chromosome Analysis by Microarray Performed concurrently with SMA testing to identify the embryos with the best potential to make a baby as well as being free of SMA. 57

58 Accuracy of PGD Using the combination of mutational analysis and linked markers the accuracy of the test 98-99%. Contamination with external DNA is a major concern. Analyses done in surgical clothing in biohazard hood. 58

59 Specific Issues with IVF/PGD Patient Discomfort: Medications are given by injection (subq). Abdominal distention and discomfort and some nausea are common. Egg retrieval done under anesthesia. Ovarian hyperstimulation syndrome 1-2%. 59

60 Questions about IVF/PGD Is IVF Safe? What will I feel? Does it increase cancer risk? Are the babies born normal? What are the costs? What about insurance? 60

61 Questions About IVF/PGD What is the success rate of IVF/PGD? Varies, particularly with maternal age, but in large series 50-60% of patients became pregnant per IVF/PGD cycle. Improved with concurrent testing for chromosome abnormalities. 61

62 Thank you!!! 62

63 Part 3: Reproductive Options for SMA Families Harvey J. Stern MD, PhD Director, Reproductive Genetics Genetics & IVF Institute 1

64 Workshop Goals What are the genetic aspects of SMA especially as they relate to reproduction? What are the reproductive choices for families at-risk for SMA? 2

65 Reproductive Options for SMA Families Very difficult decisions about family building 3

66 Reproductive Options for SMA Families Decisions regarding childbearing are personal and reflect our own ethical, moral and religious views. This is not a one size fits all type of issue. Couples who are at-risk for a genetic disorder should be allowed to make up their own minds after considering all their reproductive options. 4

67 Reproductive Options for SMA Families Decisions are influenced by the couple s experience with SMA. Those who have had a child or sib affected will react differently from a couple identified to be at-risk by genetic screening. Decisions are also influenced by couple s attitudes towards prenatal diagnosis and assisted reproduction ( playing God ) 5

68 Reproductive Options for SMA Families Have no or no more children. (Most Common Choice) Adoption. Use a gamete donor. (pre-tested for SMA) Natural conception with 1 st or 2 nd trimester prenatal testing. Preimplantation Genetic Diagnosis 6

69 What is PGD? Involves the use of assisted reproduction technologies (IVF) to provide a method of prenatal diagnosis. Offers an alternative to traditional methods of prenatal diagnosis including chorionic villous sampling and amniocentesis, with the option of termination of affected pregnancies. 7

70 Development of PGD This year represents the 28th anniversary of the first application of PGD in humans. (Handyside et al. Nature ) Since 1990, approximately 500,000 PGD cycles have been performed worldwide. This has resulted in over 150,000 PGD babies born after the procedure. 8

71 PGD for Genetic Disease affected affected affected Transfer unaffected embryos to the patient 9

72 In-Vitro Fertilization (IVF) 10

73 Questions About PGD Why do I need to do IVF, I have no trouble getting pregnant? 11

74 Questions About PGD IVF is used to increase the odds of producing a healthy child. There is no change in the genetic material (egg or sperm). No genetic engineering. By creating multiple embryos, one increases the chance that good quality embryos which are not affected with SMA will be present and available to be returned to the uterus. 12

75 IVF Procedure

76 IVF/PGD Procedure Components of IVF/PGD Cycle: 1. Down-regulation (preparation) 2. Ovarian Stimulation - FSH 3. Egg Retrieval 4. Fertilization (ICSI) 5. Embryo biopsy and testing 6. Embryo transfer to uterus 14

77 Parts Of An IVF Cycle 15

78 Normal Ovulation 16

79 Day 5 Embryo Development Day 4 Day 3 Day 2 4 cell 2 cell 8 cell Morula Blastocyst Fertilization Adapted from 17 Moore and Persaud, Saunders, 1993

80

81 IVF: Monitoring Visit Follicle measurement Estradiol levels Days of FSH Stimulation 19

82 Stimulated Ovary 20

83 Egg Retrieval 21

84 Egg Aspiration 22

85 Egg Ready For Fertilization 23

86 Fertilization by Intracytoplasmic ICSI Sperm Injection ICSI 24

87 Embryo Development 2 cell 4 cell 3 cell 8 cell From: Veeck LL, An Atlas of Human Gametes and Conceptuses, 25 Parthenon Publishing, 1999

88 Embryo Development Morula stage embryos Blastocyst stage embryos 26 From: Veeck LL, An Atlas of Human Gametes and Conceptuses, Parthenon Publishing, 1999

89 Trophectoderm Embryo Biopsy 27

90 Embryo Transfer 28

91 PGD for Single Gene Disorders Analysis involves whole genome amplification with multiplex PCR amplification of the mutation along with 3-4 linked polymorphic markers which generate a chromosomal haplotype. At the same time, the embryos are also tested by a chromosomal microarray for abnormalities (ie. Down syndrome) which can lead to failed implantation or pregnancy loss. 29

92 PGD for SMA SMN 1 & 2 = unaffected SMN 2 only = affected Embryo Biopsy 30 SMN gene

93 SMN1 SMN 1 & 2 Present NO SMN1 Affected

94

95

96 24 Chromosome Analysis by Next Generation Sequencing Performed concurrently with SMA testing to identify the embryos with the best potential to make a baby as well as being free of SMA. 34

97 Accuracy of PGD Using the combination of mutational analysis and linked markers the accuracy of the test 98-99%. Contamination with external DNA is a major concern. Analyses done in surgical clothing in biohazard hood. 35

98 Specific Issues with IVF/PGD Patient Discomfort: Medications are given by injection (subq). Abdominal distention and discomfort and some nausea are common. Egg retrieval done under anesthesia. Ovarian hyperstimulation syndrome 1-2%. 36

99 Questions about IVF/PGD Is IVF Safe? What will I feel? Does it increase cancer risk? Are the babies born normal? What are the costs? What about insurance? 37

100 Questions About IVF/PGD What is the success rate of IVF/PGD? Varies, particularly with maternal age, but in large series 50-60% of patients became pregnant per IVF/PGD cycle. Improved with concurrent testing for chromosome abnormalities. 38

101 Thank you!!! 39