Disclosures Consultant, Teva Pharmaceuticals Inc. Distributor of Verifi® Consultant, FugeneGenetics. Distributor of Harmony® Member, Clinical Expert panel Illumina Reproductive & Genomic HealthManufacturer of Sequencing Products, Verifi®

1. NIPT for Microdeletions
& Single Gene Disorders
Yuval Yaron, MD
Tel Aviv Sourasky Medical Center

2. Disclosures
• Consultant, Teva Pharmaceuticals Inc.
Distributor of Verifi®
• Consultant, FugeneGenetics.
Distributor of Harmony®
• Member, Clinical Expert panel
Illumina Reproductive & Genomic Health
Manufacturer of Sequencing Products, Verifi®

3. Human Genome = Encyclopedia

4. Chromosome = Volume

5. Gene = Page

6. Genome = 3,000,000,000
Exome = 2% of the Genome

7. Genetic Aberrations
• Chromosomal
o Numerical
o Structural
• Submicroscopic
o Deletions
o Duplications
• Sequence variants
o Polymorphisms
o Mutations

8. Genetic Aberrations
• Chromosomal
• Numerical
• Structural
• Submicroscopic
• Deletions
• Duplications
• Sequence variants
• Polymorphisms
• Mutations

9. Basic NIPT Detects ~83% of
Chromosomal Anomalies
Modified from Wellesley et al. European Journal of Human Genetics 2012;20:521–526

10. Basic NIPT would Miss 17% of Anomalies
Modified from Wellesley et al. European Journal of Human Genetics 2012;20:521–526

11. NIPT for Rare Autosomal Trisomies
• Some laboratories include NIPT screening for:
trisomy 9, trisomy 16 and trisomy 22
• However most of such conceptuses
spontaneously abort in the 1st trimester
(unless mosaic).
• With each added condition there is the
potential for an additional 0.1% to the false
positive rates
• Questionable benefit

12. Genetic Aberrations
• Chromosomal
• Numerical
• Structural
• Submicroscopic
o Deletions
o Duplications
• Sequence variants
• Polymorphisms
• Mutations

13. Microdeletions

14. Common Microdeletions
Prader Willi
4 Mb del 15q11-13
Williams-Beuren
1.5 Mb del 7q11.23
DiGeorge / VCF
3.5 Mb del 22q11

15. Known Microdeletions

16. Chromosomal Microarray (CMA)

17. Comparative Genomic Hybridization

18. Detects Deletions & Duplications

19. Risk of Significant Deletion/Duplication
• Autism or MR + dysmorphism 10%-20%
• Fetus with ultrasound anomaly 5%-7%
• No Risk, normal karyotype 0.5%

21. CNVs Following Normal Karyotype
Total
CNV
with Clinical
Significance
(%)
Pathogenic
CNV
(%)
CNV of Uncertain Clinical
Significance
Benign CNV
(%)
Indication for
Prenatal
Diagnosis
Potential
Clinical
Significance
(%)
Likely Benign
CNV
(%)
6.02.83.22.532.7US Anomaly
1.70.51.31.931.9Maternal Age
1.60.41.21.833.9Screen Positive
1.30.50.80.830.1Other *
2.50.91.61.832.3All

22. Genetic Counseling for
Variants of Unclear Significance (VOUSs)

23. NIPT for Microdeletions

24. Srinavasan A, et al. Noninvasive detection of fetal subchromosome abnormalities via deep
sequencing of maternal plasma. Am J Human Genet 2013;92:1-10
Fetal Microdeletions Detected by NIPT

25. Commercial Microdeletion Panels
Syndrome Locus Estimated
prevalence
Deletion size
(Mb)
≥3 Mb
DiGeorge/
velocardiofacial syndrome
22q11.2 1:2000 - 1:4000 3 85%
Monosomy 1p36 1p36 1:5000 - 1:10,000 1.5 - 10.5 85%
Angelman syndrome or
Prader-Willi syndrome
15q11.2-q13 1:20,000 5 – 6 70%
5p- Cri-du-Chat syndrome 5p15 1:50,000 10 - 30 99%
4p- Wolf-Hirschhorn syndrome 4p16.3 1:50,000 1.9 – 30 99%
Yaron et al. 2015, Obstet Gynecol, in Press

26. 22q11.2 Deletion
● Overlaps Velocardiofacial/DiGeorge Syndrome
● De novo (~90%) or inherited (~10%)
● Variable phenotype
● Congenital heart defect (74%)
● Palate abnormalities (69%)
● Learning difficulties (70–90%)
● Characteristic facial features
● Thymic and parathyroid abnormalities

27. 1p36 Deletion Syndrome
• Significant developmental delay
• Behavioral problems
• Hypotonia (95%)
• Brain abnormalities (90%)
• Congenital heart defects (70%)
• Eye problems (50%%)
• Distinct facial features
• Growth abnormalities

28. Prader-Willi Syndrome
• Infantile hypotonia
• Hypothalamic hypogonadism
• Mild to moderate DD
• Initial FTT
• Later Obesity
• Short stature

29. Prader Willi Syndrome
• Paternal copy expressed
• Maternal copy silenced

30. Angelman Syndrome
• Severe DD
• Movement disorder-ataxia
• Acquired microcephaly
• Seizures
• Unique behavior
(Happy Puppet)

31. Angelman Syndrome
● Maternal copy expressed
● Paternal copy silenced

32. 5p Deletion /Cri du Chat Syndrome
• Cat Like Cry
• Severe DD / Behavioral problems
• Microcephaly
• Dysmorphic Features
• Deletions 3 - 30 Mb
• 90% de novo
• 10% parental balanced translocation

33. 4p- Deletion/ Wolf-Hirschhorn Syndrome
• Syndrome
• Microcephaly
• Micrognathia,
• Short philtrum,
• Prominent glabella
• Ocular hypertelorism,
• Dysplastic ears and periauricular tags
• Growth and mental retardation

34. Microdeletion Panel - NATERA

35. Microdeletion Panel - ILLUMINA

36. Microdeletion Panel - SEQUENOM

37. MaterniT® GENOME - SEQUENOM

38. NIPT Microdeletion Panels - Pros
• Microdeletions are relatively common
• Some more severe than Down syndrome
• Are not part of routine serum screening
• Incidence is independent of age or ethnicity
• Many have no ultrasound findings
• Can occur spontaneously without family history

39. NIPT Microdeletion Panels - Cons
• Not validated in clinical studies
• Most studies used spiked serum samples
• Not all del22q cases detected
(only 85% 3Mb deletions)
• Not all Prader Willi/Angelman cases detected
(only ~70% due to deletions)
• Microdeletion burden not significantly altered

40. NIPT Microdeletion Panels - Cons
• In high-risk patients (i.e. with U/S anomalies)
o Increased risk of missed diagnoses
• In low-risk patients
o Low PPV and increased false positive rates

41. Predictive Values (PPV & NPV)
• Positive Predictive Value
o Given an abnormal result -
what is the chance of truly having the Condition ?
o “How worried should I be ?”
• Negative Predictive Value
o Given an normal result -
what is the chance of truly NOT having the Condition ?
o “How reassured Can I be ?”

42. PPV Influenced by Prevalence

43. Positive Predictive Values T21
52.5%
91.8%
99.2%
0% 20% 40% 60% 80% 100%
1:1000
1:100
1:10
Prevalence
Based on Gil MM et al. Ultrasound Obstet Gynecol 2015; 45: 249–266

44. Positive Predictive Values
In a 35 year old patient assuming 99.9% sensitivity at 0.1% FPR
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
T21 T18 T13 22q11 1p36 15q 5p-
Yaron et al. Obstet Gynecol, in Press

45. Genetic Aberrations
• Chromosomal
o Numeric
o Structural
• Submicroscopic Copy Number Variants (CNVs)
o Deletions
o Duplications
• Sequence variants
o Single nucleotide polymorphisms (SNPs)
o Pathogenic mutations

46. Paternally-Inherited &
de novo Dominant Mutations

47. Paternally-Inherited Mutations
Lo and Chiu Nature Reviews Genetics 2007;8:71–77

48. Paternally inherited or de novo
autosomal dominant disorders
Monogenic disorder Approach to NIPD
Achondroplasia • Detection or exclusion of paternal or
de novo allele reported as
diagnostic.
• Technologies used include a variety
of PCR approaches, dPCR and NGS
Apert syndrome
Early onset primary dystonia I
Thanatophoric dysplasia
Huntington's disease
• Not fully accurate. Invasive testing
recommended for diagnosis

49. Paternally Inherited Mutations
Autosomal dominant disease

50. Autosomal recessive conditions where
parents carry different altered alleles
Monogenic disorder Approach to NIPD
a & β-thalassaemia • Exclusion of paternal allele reduces
risk to zero.
• Detection of the paternal allele
indicates an increased risk (to 50%)
and invasive testing is required for
definitive diagnosis.
• Technologies used include PCR,
dPCR and NGS have been reported
CAH
Cystic fibrosis
Leber congenital amaurosis
Propionic acidemia
Frasers syndrome
AR polycystic kidney disease

51. Paternally Inherited Mutations
Autosomal recessive disease

52. Autosomal recessive conditions where
parents carry the same altered allele
Monogenic disorder Approach to NIPD
Sickle cell anaemia
• Estimation of allelic ratios
required, so accurate estimation
of fetal fraction needed.
• Currently can only be done using
dPCR or NGS.
• Limited application in female
fetuses.
Y-chromosome sequences can be
used in male-bearing pregnancies
β-thalassaemia

53. Autosomal Recessive Conditions

54. Autosomal Recessive Conditions

55. Depth of Sequencing

59. Is This the Future of Amniocentesis ?