11/5/2013

OBJECTIVES

AGENDA

• Review current prenatal aneuploidy screening and testing options.

• History of prenatal genetic screening • Maternal serum screening • Diagnostic testing

• Summarize and compare new technologies in prenatal screening, specifically, non-invasive prenatal testing (NIPT).

The Prenatal Genetic Testing Odyssey:

• Cell free fetal DNA testing • Methodology • Sensitivity and specificity

Review Of Traditional And New Technologies • Describe new advances in carrier screening beyond ethnicitybased conditions.

JA N I N E ROS E N BE RG, MS, LCG C

• Carrier screening • Ethnicity-based • Expanded

HISTORY OF PRENATAL ANEUPLOIDY SERUM SCREENING

1984 Maternal serum alpha-fetoprotein (AFP) used in conjunction with maternal age

NUCHAL TRANSLUCENCY (NT) OBTAINED BETWEEN ~11+0 AND 13+6 GESTATIONAL WEEKS

http://www.fetalmedicine.com/

HISTORY OF PRENATAL ANEUPLOIDY SERUM SCREENING

1990s

2000s

2011- present

•

Triple screen is developed with additional analytes of human chorionic gonadotropin (hCG) and unconjugated estriol (uE3) • Down syndrome (DS) detection rate: 69%

Combination screenings utilize both first and second trimester modalities • Integrated • DS detection rate: 94-96%

Cell-free fetal DNA (cff-DNA) testing is clinically available • Sensitivity for DS: 98%

•

Quad screen is introduced with the addition of inhibin A (DIA) • DS detection rate: 81%

•

Serum integrated • DS detection rate: 85-88%

•

•

Nuchal translucency (NT) measurement • DS detection rate: 64-70%

Stepwise sequential • DS detection rate: 95%

•

Contingent sequential • DS detection rate: 88-94%

•

First trimester screening is developed combining NT with free β-hCG, and pregnancy associated plasma protein A (PAPP-A) measurements • DS detection rate: 82-87%

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DIAGNOSTIC TESTING Chorionic Villus Sampling

Amniocentesis

Gestational Age

10-12 weeks

15+ weeks

Type of Sample

Placental

Amniotic fluid

Risk of Miscarriage 1/200* Miscellaneous

DIAGNOSTIC TESTING CHORIONIC VILLUS SAMPLING (CVS) Transabdominal

DIAGNOSTIC TESTING AMNIOCENTESIS

Transcervical

10% http://www.panoramatest.com/

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CFF DNA AND ANEUPLOIDY

CFF DNA AND ANEUPLOIDY

• Analyzes cell-free DNA in maternal circulation to detect common fetal aneuploidies • • • • •

Euploid fetus

COUNTING METHOD The total number of cf-fetal fragments vs. cf-maternal fragments of any one chromosome is proportional to the size of the chromosome, and is consistent from sample to sample, and patient to patient.

 ~10% of the DNA fragments in a pregnant woman’s blood are from the fetus ( )  ~90% are from the mother ( )

Down syndrome Trisomy 18 Trisomy 13 Sex chromosome aneuploidies Other less common conditions that are only available through certain laboratories

CFF DNA AND ANEUPLOIDY:

Fetus with trisomy 21, 18 or 13

Sequencing tells you which chromosome the combined maternal and fetal fragments come from.

• Also can evaluate for fetal gender • Benefit: X-linked genetic conditions Chromosome 1 Provided by Sequenom CMM

CFF DNA AND ANEUPLOIDY: COUNTING METHOD TCCGCCCAGGCCATGAGGGACCTGGAAATGGCTGAT GGCCCTGGGGACAGTCTCCAATCCACTGAGTCATCT GACACGGTGGAGCTCGGCCACACCAGGCCCAGCTGG GGCCCTGGGGACAGTCTCCAATCCACTGAGTCATCT ACAGTGGTGGGGCCCATCCCTGGGTGAGGCTCAGTT GGCCCTGGGGACAGTCTCCAATCCACTGAGTCATCT GGCCCTGGGGACAGTCTCCAATCCACTGAGTCATCT GGCCCTGGGGACAGTCTCCAATCCACTGAGTCATCT TCCGCCCAGGCCATGAGGGACCTGGAAATGGCTGAT GACACGGTGGAGCTCGGCCACACCAGGCCCAGCTGG GGCCCTGGGGACAGTCTCCAATCCACTGAGTCATCT ACAGTGGTGGGGCCCATCCCTGGGTGAGGCTCAGTT GGCCCTGGGGACAGTCTCCAATCCACTGAGTCATCT GGCCCTGGGGACAGTCTCCAATCCACTGAGTCATCT GACACGGTGGAGCTCGGCCACACCAGGCCCAGCTGG GGCCCTGGGGACAGTCTCCAATCCACTGAGTCATCT

Sequencing tells you which chromosome the DNA fragment comes from.

1

2

3

4

5

6

7

8

9

10

11

12

13 14

15

16

17

18 19

20

chr21 chr10 chr14 chr10 chr21 chr10 chr10 chr10 chr21 chr14 chr10 chr21 chr10 chr10 chr14 chr10

21 22

X

Chromosome 21 Provided by Sequenom CMM

CFF DNA AND ANEUPLOIDY:

CFF DNA AND ANEUPLOIDY:

COUNTING METHOD

SINGLE NUCLEOTIDE POLYMORPHISM (SNP) METHOD

DNA MPS (massively parallel sequencing) does not differentiate which DNA fragments come from the mother and which from the fetus.

• What is a SNP? • A variation in the DNA sequence where one base pair (or single nucelotide) differs between the chromosomes in a pair

• There are over 10-30 million SNPs in the human genome The over-representation of chromosome 21– specific fragments/sequences underlying a pregnancy affected with fetal trisomy 21 can be measured

Y

Provided by Sequenom CMM

Unaffected Fetus

Fetus with Trisomy 21

Provided by Sequenom CMM

http://www.ibbl.lu

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CFF DNA AND ANEUPLOIDY:

SENSITIVITY AND SPECIFICITY

SINGLE NUCLEOTIDE POLYMORPHISM (SNP) METHOD

SENSITIVITY AND SPECIFICITY Counting Method

• Sensitivity/ true positive rate • The proportion of actual positives which are correctly identified as such (e.g. the percentage of fetuses with Down syndrome who are correctly detected as being affected)

• Specificity/ true negative rate • The proportion of negatives which are correctly identified as such (e.g. the percentage of fetuses who are correctly identified as not having Down syndrome)

Sequenom (MaterniT21+)

Ariosa

SNP Method Verinata

(Harmony)

(Verifi)

Natera (Panorama)

Sensitivity

Specificity

Sensitivity

Specificity

Sensitivity

Specificity

Sensitivity

Specificity

Trisomy 21

99.1%

99.9%

99%

99.97%

>99.9%

99.8%

>99%

>99%

Trisomy 18

>99.9%

99.6%

98%

99.93%

97.4%

99.6%

>99%

>99%

Trisomy 13

91.7%

99.7%

XX/XY

Accuracy of Y chromosome: 99.4%

8/10

99.9%

87.5%

>99.9%

>99%

>99%

XX: >99% XY: >99%

>99% >99%

XX: 97.6% XY: 99.1%

99.2% 98.9%

XX: >99% XY: >99%

>99% >99%

99.5% 99.5% 100% 100%

XO: 95.0% Limited data for remainder

99.0% Limited data for remainder

XO: 91.7% Limited data for remainder

>99% Limited data for remainder

N/A

N/A

N/A

>99%

>99%

Sex Chromosome Aneuploidy

96.2%*

99.7%*

XO: 96.3% XXX: 1/1 XXY: 6/6 XYY: 2/3

Triploidy/ Vanishing Twin

N/A

N/A

N/A

*Overall sex chromosome aneuploidy statistics

RECOMMENDATIONS FROM PROFESSIONAL SOCIETIES • American College of Obstetrics & Gynecology (ACOG) • “Patients at increased risk of aneuploidy can be offered testing with cell free fetal DNA.” • ACOG’s definition of “increased risk of aneuploidy” • • • • •

Maternal age ≥35 years at time of delivery History of a prior pregnancy with a trisomy- not a family history of a trisomy Ultrasound abnormality indicating elevated risk Positive serum screening Parental balanced Robertsonian translocation with increased risk for trisomies 21 or 13

• “Cell free fetal DNA testing should not be part of routine prenatal laboratory assessment, but should be an informed patient choice after pretest counseling.” • “Cell free fetal DNA testing should not be offered to low-risk women or women with multiple gestations because it has not been sufficiently evaluated in these groups.”

CELL FREE FETAL DNA TESTING • Benefits • • • • • •

No increased risk for miscarriage Highest detection rates across all non-invasive testing options Ability to screen for aneuploidies beyond traditional maternal serum screening Able to be completed very early in pregnancy Ultrasound not required Capability to expand

• Limitations • • • •

CARRIER SCREENING

Not diagnostic Does not screen for all aneuploidies or genetic conditions New technology with limited long term data Insurance coverage is a work in progress • Expensive! (~2700)

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CARRIER SCREENING • Testing used to identify usually asymptomatic individuals who have a gene mutation for an autosomal recessive or X-linked recessive disorder • Traditionally, offerings vary based on ethnicity

CARRIER FREQUENCIES FOR COMMON CONDITIONS Population

Condition

Carrier Frequency

African American

Sickle cell disease Cystic fibrosis Beta-thalassemia

1/12 1/61 1/75

Ashkenazi Jewish

Gaucher disease Cystic fibrosis Tay-Sachs disease Familial dysautonomia Canavan disease

1/15 1/24 1/30 1/32 1/40

Asian

Alpha-thalassemia Beta-thalassemia Cystic fibrosis

1/20 1/50 1/94

Caucasian (non-Hispanic )

Cystic fibrosis

1/25

Caucasian (Hispanic)

Cystic fibrosis

1/58

French Canadian

Tay-Sachs disease

1/30

Mediterranean

Beta-thalassemia Sickle cell disease

1/25 1/40

RECOMMENDATIONS FROM PROFESSIONAL SOCIETIES

RECOMMENDATIONS FROM PROFESSIONAL SOCIETIES

REGARDING HEMOGLOBINOPATHY SCREENING

REGARDING INDIVIDUALS OF ASHKENAZI JEWISH DESCENT

• American College of Obstetrics & Gynecology (ACOG) • “Individuals of African, Southeast Asian, and Mediterranean descent are at increased risk for being carriers of hemoglobinopathies and should be offered carrier screening and, if both parents are determined to be carriers, genetic counseling.” • “A complete blood count and hemoglobin electrophoresis are appropriate laboratory tests for screening for hemoglobinopathies. Solubility tests alone are inadequate for

screening because they fail to identify important transmissible hemoglobin gene abnormalities affecting fetal outcome.”

RECOMMENDATIONS FROM PROFESSIONAL SOCIETIES REGARDING CYSTIC FIBROSIS (CF) CARRIER SCREENING

• National Institutes of Health (NIH) • “Genetic testing for CF should be offered to adults with a positive family history of CF, to partners of people with CF, to couples currently planning a pregnancy, and to couples seeking prenatal testing. The panel does not recommend offering CF genetic testing to the general population or newborn infants.”

• American College of Obstetrics & Gynecology (ACOG) • “It is important that CF screening continues to be offered to women of reproductive age. It is becoming increasingly difficult to assign a single ethnicity to individuals. It is reasonable, therefore, to offer CF carrier screening to all patients. Screening is most efficacious in the non-Hispanic white and Ashkenazi Jewish populations.” • CF screening should consist of direct DNA analysis of the most common 23 CFTR mutations; complete analysis of the CFTR gene by DNA sequencing is not recommended for routine carrier screening.

• American College of Obstetrics & Gynecology (ACOG) • “Carrier screening for Tay-Sachs disease, Canavan disease, cystic fibrosis, and familial dysautonomia should be offered to Ashkenazi Jewish individuals before conception or during early pregnancy.” • Carrier screening for other conditions should be made available if an individual or couple is interested.

• American College of Medical Genetics (ACMG)

EXPANDED CARRIER SCREENING

• “In addition [to the above], we recommend that carrier screening be offered for Fanconi anemia (Group C), Niemann-Pick (Type A), Bloom syndrome, mucolipidosis IV, and Gaucher disease.”

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SPINAL MUSCULAR ATROPHY (SMA)

CRITERIA FOR POPULATION SCREENING • In order for a genetic screening program to be successful, the following criteria should be met: • • • • •

Disorder is severe Frequency of carriers is high Testing is reliable in regards to having a high specificity and sensitivity Prenatal diagnosis is available Genetic counseling is available as needed

• Loss of motor neurons  weakness and atrophy of muscles • Severity and age of onset will vary based on type Type

Onset

Features

I

Birth or within first few months

Developmental delay, inability to support head or sit unassisted, difficulty with breathing and swallowing

II

Six to twelve months

Inability to stand or walking independently

III

Early childhood or adolescence

Able to stand and walk without assistance, but many require wheelchair assistance later in life

IV

After age of 30

Mild to moderate muscle weakness, tremor, twitching, or mild respiratory difficulties

GENETICS OF SMA • Autosomal recessive condition • Caused by mutations in the Survival Motor Neuron 1 (SMN1) gene • Modified by the number of Survival Motor Neuron 2 (SMN2) gene copies

• American College of Medical Genetics recommends that all couples be offered screening for SMA, as it is present in all populations neuromuscular.wustl.edu

CRITERIA FOR POPULATION SCREENING • In order for a genetic screening program to be successful, the following criteria should be met: • Disorder is severe • Frequency of carriers is high

DELVING EVEN FURTHER… • New genetic testing panels are available which offer carrier screening for 50+ conditions

• Patient is adopted

• ACOG and ACMG recommended conditions are included

• Patient and their partner are consanguineous

• They also include many conditions that haven’t been formally recommended for regular carrier screening

• Patient has a family history of a specific genetic condition and/or known genetic mutations for a specific condition

• SMA has a carrier frequency of 1/40-1/60

• Testing is reliable in regards to having a high specificity and sensitivity • Screening has an approximate 90% detection rate

• Prenatal diagnosis is available • Genetic counseling is available as needed

REASONS TO CONSIDER EXPANDED CARRIER SCREENING

• Some screen for severe conditions that onset at birth or during childhood • Others include conditions that are mild, may never lead to symptoms, and develop in adulthood • Some of the diseases screened for are common, especially in certain ethnic groups, while others are very rare

• May not know their ethnic background • May not have information regarding their family history

• Mutations that are present in the family may or may not be detected on the screening panel

• Patient is an information seeker and wants to screening for everything that is available

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RESULTS REPORT

RESULTS REPORT

RESULTS REPORT

• Reasons • Sometimes a negative result will not drastically reduce your risk

Counsyl test report

Counsyl test report

• Condition is extremely rare to begin with • Low test sensitivity

Counsyl test report

RECOMMENDATIONS FROM PROFESSIONAL SOCIETIES REGARDING EXPANDED CARRIER SCREENING

• American College of Medical Genetics (ACMG) • General population-based carrier screening should fit certain criteria, rather than simply including as many disorders as possible • Disorder is severe enough that at-risk couples would consider undergoing prenatal diagnosis • Informed consent is obtained for adult onset disorders, especially when results may impact the patient themselves

QUESTIONS? T H A N K YO U !

• Residual risk calculations must accurately take into account the patient’s ethnic background

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