1. 35th Annual Perinatal Medicine & Women’s Health Care Symposium
“Genetic Testing and Genetic Screening in the Preconception and Prenatal Setting”
35th Annual Perinatal Medicine & Women’s Health Care Symposium
September 17, 2015
Myra J. Wick

2. Disclosure Summary
Listed below are individuals with control of the content of this program who have disclosed…
Relevant financial relationship(s) with industry:
None
No relevant financial relationship(s) with industry:
Speaker: Myra Wick
References to off-label usage(s) of pharmaceuticals or instruments in their presentation:

3. Objectives
The learner will understand the basics of Expanded Carrier Screening (ECS)
The learner will be familiar with the concepts of preimplantation genetic screening (PGS) and preimplantation genetic testing (PGD)
The learner will understand the benefits and limitations of cell free DNA (cfDNA) screening.

4. What is Genetic Screening?
Allows early detection of, predisposition to or exclusion of a genetic disease.
May involve general population or specific subpopulations (not necessarily based on current health status).
Unique- results may imply risk to family members; family members may/may not wish to be included.
Results may be used for health-related reproductive or lifestyle choices.
European Journal of Human Genetics (2003) 11, Suppl 2, S5–S7

5. Carrier Screening
“…testing with the aim of identifying heterozygote carriers of mutations that put two carriers at risk of having a child with a disorder causing serious disability or early death.”- A. Beaudet

6. History of Carrier Screening “Programs”
1970’s - testing for Tay-Sachs in the Ashkenazi Jewish population
1980’s- thalassemia screening programs in the Mediterranean region
“Universal carrier screening”
1993- for cystic fibrosis (CF)
2010- More than 100 disorders- Counsyl
2015- Next generation sequencing (NGS)

7. Ethnic Based Screening
Screening for genetic conditions known to occur with an increased frequency within a certain ethnic group
Autosomal recessive conditions
Purpose: To detect couples at risk for prenatally diagnosable genetic diseases
Multi-ethnic society
What is your ethnicity?
Ashkenazy Jewish? African American? French Canadian? Mediterranean?

8. Carrier Frequencies based on Ethnic Origin
Population
Condition
Carrier Frequency
African-American
Sickle Cell
Cystic Fibrosis
Beta-Thalassemia
1 in 10
1 in 65
1 in 75
Ashkenazi Jewish
Gaucher disease
Tay-Sachs disease
Dysautonomia
Canavan disease
1 in 15
1 in 25
1 in 30
1 in 32
1 in 40
Asian
Alpha-Thalassemia
1 in 20
1 in 50
European American
French Canadian, Cajun
Tay Sachs disease
Hispanic
1 in 46
1 in in 50
Mediterranean
1 in 29

9. Cystic Fibrosis
Most common life threatening AR condition in the non-Hispanic white population.
Median survival=37 years
CFTR (CF transmembrane regulator) gene
Chromosome 7
Incidence 1:2500
Prenatal and preconception screening introduced in 2001

10. CF Screening
1,000+ mutations have been identified within the CFTR gene
The ∆F508 mutation is the most common
66% of all CF causing mutations worldwide
The remaining mutations vary greatly in their frequency/distribution
Majority are either private or limited to a small number of individuals
ACMG and ACOG recommend a core mutation panel of 23 mutations; most laboratories test for >100 mutations

11. Residual Risk? Important Concept in Genetic Screening

12. Ethnic Based Screening
Screening for genetic conditions known to occur with an increased frequency within a certain ethnic group
Autosomal recessive conditions
Purpose: To detect couples at risk for prenatally diagnosable genetic diseases
Multi-ethnic society
What is your ethnicity?
Ashkenazi Jewish? African American? French Canadian? Mediterranean?

13. Expanded Carrier Screening (ESC) What is it?
Panels which simultaneously screen for multiple genetic conditions (>100)
Most are autosomal recessive
Most are rare
Some are X-linked or autosomal dominant
Same panel offered for all individuals
Multiethnic society
More efficient, less expensive
For reproductive age couples, preconception testing is ideal

14. ECS: When/Who When Preconception (ideally) Prenatally Who
Women of reproductive age; sequential testing of partner if positive
Concurrent testing of patient and partner
Time to consider reproductive options
Egg or Sperm Donor (many banks require some screening for all donors)

15. Interpretation and Counseling
Negative
Residual risk
Negative test does not eliminate risk to offspring
CF residual risk: 1/270x1/25x1/4=1/27,000
Paternity
Patient positive
Partner testing
Significance for patient
Fx and premature ovarian failure/FX Tremor Ataxia Syndrome
Significance for family members
Patient and partner positive
Pregnancy risk
Characteristics of disease, variability of symptoms

16. What is offered (Counsyl)? “It’s like an ultrasound for your DNA”

17. “It’s like an ultrasound for your DNA”

18. Other Laboratories: Emory Genetics Laboratory
Targeted mutation analysis for 725 mutations in 145 genes (does not include MTHFR, Prothrombin)
Sequencing analysis
Pricing similar to Counsyl
“Custom” panels available
Program for Jewish Health
36 disease AJ panel + SMA and Fx
Natera
Horizon Multi-disease
Genetic Carrier Screening- 39 disorders including SMA and FX

19. Sequenom HerediT…

20. Mayo Experience- 106 Patients
Patients with positive carrier status for one disorder (total number of carriers identified):
-Bardet Biedl- 2
-Alpha-1 antitrypsin deficiency- 2
-Medium-chain acyl-CoA dehydrogenease deficiency (MCAD)
-Familial Mediterranean fever- 3
- Biotinidase deficiency- 1
- Factor XI deficiency -1
- Hereditary fructose intolerance-1 patient with 2 mutations, affected
- Congenital disorders of glycosylation 1a- 1
- Congenital deafness due to GJB2- 3
- Glycogen storage disease type V- 2
- Fragile X- 2
- CF -3
- SCAD -1
- Phenylalanine hydroxylase (PAH)- 1
- Sickle trait -1
-Spinal muscular atrophy- 3
- Smith Lemli Opitz -1

21. Mayo Experience- 106 patients
Positive Carrier Status for More Than One Mutation:
-Gracile, Pompe and SMA
-Pseudocholinesterase and SMA
-Fragile X and SMA
-Congenital disorders of glycosylation 1A and CF
-Biotinidase, CF and galactosemia
-CF and Gaucher
-Niemann Pick C and primary hyperoxaluria
-Niemann Pick C, congenital deafness due to GJB2 and biotinidase
-Factor XI and MCAD
-Congenital deafness due to GJB2 and SMA
-Factor XI, hereditary fructose intolerance and Pendred syndrome

22. Carrier Screening and Societal Guidelines
ACOG
Hemoglobinopathies: African, Mediterranean and SE Asian ancestry
AJ: Tay-Sachs, Cystic fibrosis (CF), Canavan, Familial Dysautonomia
Population: (CF)
Family history based: Spinal muscular atrophy (SMA), Fragile X (Fx)
Expanded carrier screening: Obstet Gyn 2015; 3:653.
National society of genetic counselors:
Population screening: CF
Family history based: Fx
ACMG:
AJ: Tay-Sachs, Cystic fibrosis (CF), Canavan, Familial Dysautonomia, Niemann-Pick, Bloom syndrome, Fanconi Anemia Group C, Mucolipidosis IV, Gaucher
Population: SMA and CF
ECS: Guidelines published in 2013 (Grody et al, 2013;Genet Med 15:482), Obstet Gyn 2015; 3:653.

23. Expanded Carrier Screening ACMGG, ACOG, NSGC, PQF, SMFM Obstet Gyn 2015;3:653
All individuals are offered screening for same set of conditions
May include more than 100 genetic conditions
Pretest education (verbal, videos, online, pamphlets)
Less well defined phenotypes
Rare conditions and frequency, detection rate, residual risk
Interpretation of negative results
Panels may change over time
Most disorders are AR, some X-lined or AD
Molecular methods may not be the method of choice
Consent
Post-Test counseling

24. ACMG recommendations for Screening Panels (Grody et al, Genet Med 2013;15:482)
The disorders should be of such a nature that most at risk individuals would consider prenatal diagnosis to facilitate reproductive decision making.
Patients must provide consent for screening of adult onset disorders, and be aware of implications to self and family members.
For each condition, causative gene(s), mutations and mutation frequencies are known, such that residual risks can be assessed.
Validated clinical association between mutations detected and severity of the disorder.
Compliance with ACMG Standards and Guidelines for Clinical Laboratories.

25. Implications of ECS??

26. Preimplantation Genetic Testing: PGS and PGD
Preimplantation genetic screening (PGS)
Screening for aneuploidy
Detect abnormal chromosome complement arising from parental balanced translocation
Preimplantation genetic diagnosis (PGD)
Screening for single gene disorders

27. Preimplantation genetic testing

28. When to biopsy? Blastocyst-stage (day 5-6) 5-10 trophectoderm cells
Lower percentage of cell loss
More starting DNA
Lower rates of mosaicism
Fewer embryos tested
May increase live birth rate
No impact on blastocyst reproductive potential when compared with cleavage-stage biopsy (day 3)

29. Biopsy Video

30. PGD Parental mutations must be known
Collection of DNA samples from family members
Design period
Reports of 500 diseases currently diagnosed
Adult onset disorders
Ethics
HLA matching
Technical challenges
Potential for false negative results
ESHRE-PGD consortium data
clinical pregnancy rate of 29% per embryo transfer (Human Reproduction Update 2012;18:234)

31. Preimplantation Genetic Diagnosis

32. Why PGS?

33. Maternal Age Versus Day3 and Day5 Aneuploidy Rates
Percentage of embryos
Maternal age
Data from Genesis Genetics

34. “Clinical Indications” for PGS (J Obset Gynaecol Can 2015;37(5):451 and BMJ 2014;349:g7611))
Recurrent unexplained pregnancy loss or recurrent miscarriage with aneuploidy
Repeated failure of implantation
Severe male factor infertility
Coupled with PGS
IVF with single embryo transfer
Highest implantation rate associated with elective single embryo transfer of euploid embryo (eSET)

35. PGS Methodology- “Historical”
Technical difficulties
Signal overlaps
Probe failure
Limit to total number of probes
FISH with day 3 embryo biopsy associated with decreased live birth rates
Not recommended (J Obset Gynaecol Can 2015;37(5):451)

36. “New” PGS Methodology “Comprehensive chromosome screening”
Chromosome microarray
qPCR
Next Generation Sequencing (NGS)
Chromosome microarray

37. Next generation sequencing and chromosome array results
Yang et al. BMC Medical Genomics (2015) 8:30
DOI /s

38. Results

39. Current Recommendations J Obset Gynaecol Can 2015;37(5):451
1. Pretest counseling
2. Reduce risk of child with know genetic disorder
Invasive pre or postnatal testing to confirm PGD/PGS results
Trophectoderm biopsy safe
Lab related:
PGD with multiplex PCR on trophectoderm biopsy
Chromosome screening on trophectoderm biopsy for translocation carriers
FISH with Day 3 biopsy should not recommended
Education and counseling by genetics professional:
Discuss limitations and small risk for error
Studies ongoing as to whether PGS improves live birth rates
PCS with comprehensive chromosome screening increases implantation rates in patients with good prognosis

40. Prenatal Testing/Screening Cell free DNA (cfDNA, NIPT, NIPS)

41. Reasons for Prenatal Screening and Prenatal Testing
Screen for chromosomal aneuploidies
Major cause of intellectual disability, long-term morbidity, and fetal/childhood mortality
Down syndrome (trisomy 21)
most common live-born aneuploidy
ACOG guidelines

42. Maternal Age Related Risk

43. Prenatal Screening: Down Syndrome and Trisomy 18
Traditional testing
First trimester screening
Nuchal translucency ultrasound + 2 proteins in mom’s blood
Second Trimester screening
Four proteins in maternal blood
~85% detection rate, 5% false positive
Ultrasound (US) Evaluation at weeks
New and evolving- Free fetal DNA testing (Noninvasive prenatal screening).

44. Nuchal Translucency

45. Sensitivity and Specificity of Traditional Screening
Screening Test
Detection Rate* at False Positive Rate of:
1% %
2nd trim. QUAD
60%
81%
1st trim. combined
72%
85%
Serum integrated
70%
86%
Full integrated
87%
95%
*from: Malone FD et al. First-trimester or second-trimester screening, or both, for Down's syndrome.
(FASTER Research Consortium). N Engl J Med. 2005; 353:

46. Traditional Prenatal Screening
↑ Maternal age → ↑ detection rate, ↑FPR

47. Gold Standard Invasive Testing
CVS
Performed at weeks
Risk of loss ~1% (likely lower)
Amnio
Generally >15 weeks
Risk of loss ~0.5% (likely lower)

48. Holy Grail of Prenatal Screening?
Goals
Reduce false positive and false negative results
Reduce risk/possible exposure associated with amniocentesis & CVS
Decrease maternal anxiety
Holy Grail

49. Cell Free Fetal DNA and Non-Invasive Prenatal Screening
1997: Lo et al report fetal DNA in maternal serum
Clinical testing for +21 offered in 2011
Trisomy 13, 18, 21, Fetal sex determination followed shortly
Competing commercial laboratories
Patents, IP and legal battles

50. What is “Cell Free DNA”? Released through cellular death (apoptosis)
DNA cleaved into small fragments ( bp)
Released into blood stream as cfDNA
During pregnancy maternal blood contains both fetal and maternal cfDNA
Most of cell free fetal DNA Is placental in origin.
Rapidly cleared after delivery

51. Mass Parallel Shotgun Sequencing (MPSS)
1. DNA is fragmented and analyzed in 36-bp lengths (reads)
3. The sequences derived from each chromosome is plotted against what should normally be present, indicated by the dashed line slightly above 1.0
2. The reads are aligned against the human genome sequence and counted
4. The amount of DNA in the chromosomes of interest is normalized against the DNA from other chromosomes to determine the relative number of reads present in a given sample

52. NIPT Aneuploidy Testing with Massively Parallel Genomic Sequencing
Massively Parallel Shotgun Sequencing (MPSS)
©2008 by National Academy of Sciences
Chiu R W K et al. PNAS 2008;105:

53. Detection of Fetal Aneuploidy
MPS Enables Precise Molecular Counting
Fetal
cfDNA
= DNA counts
Fetus with trisomy contributes ~50% more cfDNA from the trisomic chromosome
Millions of “counts” allows detection of small relative cfDNA increase
Do NOT distinguish or separate fetal from maternal cfDNA
Maternal
cfDNA
Diploid 21 Fetus
Trisomy 21 Fetus

54. cfDNA Screening Bioinformatic capabilities – molecular counting
cell free DNA from blood sample
Precise, automated sequencing methods - “next generation sequencing”

55. Initial Studies in High Risk Patients

56. 2015 ACMG Genetics and Genomics Review Course

57. Other Developments in cfDNA?
-Microdeletions
- Low risk pregnancy

58. Microdeletion Syndromes
What are Microdeletions?
“Complex clinical phenotype due to dosage imbalance of unrelated genes which happen to be contiguous on a chromosome”

60. NIPS and Microdeletions
Rare syndromes
Validation issues
No societal guidelines
? PPV

61. Performance in Low Risk Population?
NEJM 2015;125:375

62. Limitations “False Positive” results Mosaicism Vanishing twin
“No call”
Maternal obesity
Aneuploidy
Maternal malignancy
Twin gestation
Will not detect:
Single gene disorders, structural chromosome abnormalities, translocations, congenital malformations

63. Pretest counseling
Conventional screening for low risk
Patient may choose cfDNA regardless of risk status
Screen for common trisomies and sex chromosome composition, if requested
Recommend diagnostic test for positive cfDNA results
Management options not based on cfDNA results
Counseling for “no call” results
Not for microdeletion screening
Offer invasive testing for fetal anomalies on US
Negative result ≠ unaffected pregnancy
Offer MSAFP with cfDNA for detection of abdominal wall and NTD
Patients may decline all testing/screening

64. August 20, 2015….

65. The Future… cfDNA genome test Whole genome/whole exome in the NICU
Whole exome/whole genome via CVS/amnio

66. Questions?