Introduction: the new genetics-Jacques S. Abramowicz

Ultrasound is dead. NIPT can do it all. Nathan Fox

You still need ultrasound in 2016. Steve Warsof

Discussion and Q&A

1.Good luck2.Maternal age. Pray and Good luck!3.Amniocentesis. Pregnancy loss!4.AFP5.Genetic sonogram6.Triple/quadruple screen7.First screen 8.cffDNA

Maternal age LB 2nd trim.

25 years 1/1250 1/887

30 years 1/965 1/685

35 years 1/386 1/274

38 years 1/182 1/129

40 years 1/100 1/78

45 years 1/31 1/22

35 years 1/386 1/274

Screening Options:

First Trimester Screening

Quadruple Marker Screen

Integrated, Sequential, or Contingency Screens

Anatomy Scan

Benefit(s)

Non-invasive = no risk

Identifies women from low risk pool who are at increased risk

Disadvantage(s)

Risk calculation only

False positive/negative

Limited to trisomy 18, 13, 21

Timing, insurance coverage

Patient anxiety

Invasive Prenatal Diagnosis testing Options:

Chorionic Villus Sampling (CVS)

Amniocentesis

Benefit(s)

Diagnostic information on all aneuploidies

Additional testing available such as microarray, PCR

Disadvantage(s)

Invasive, risk of pregnancy loss (1/300-1/500)

German pathologist, identified fetal trophoblasts in maternal lungs of 14 women who died from eclampsia (1883)

Schmorl G., Pathologisch-anatomische Untersuchungen über Puerperal-Eklampsie. Verlag FCW Vogel, Leipzig; 1883

Christian Georg Schmorl (1861-1932)

1893: Schmorl

1980’s: Development of cell sorting: separation of single cells Development of PCR and FISH: analysis of single cells

1990’s: analysis of whole fetal cells in maternal plasma

1997: Lo and others report on cell free fetal DNA

2002: National Institute of Child Health and Human Development Fetal Cell IsolationStudy (NIFTY) trial

2011: Introduction of noninvasive prenatal diagnostic tests

Non-Invasive DNA Screening

NIDS

cffDNA represents ~10% of total DNA in maternal plasma (Lo 1997, Chiu 2011)Much higher percentage than intact fetal cellscffDNA made up of short (<200 bp) DNA fragments (Chan 2004)Reliably detected after 7 wks gestation (Birch 2005)Higher concentrations late in gestationShort half life (16 min), undetectable by 2 hrs postpartum (Lo 1999)Quantitative differences in chromosome fragments can identify fetuses with Down syndrome, trisomy 18, trisomy 13, and sex chromosome abnormalies

Background

Early applications of NIPT included:determination of Rhesus D blood-group status

fetal sex

diagnosis of autosomal dominant disorders of paternal inheritance.

Available clinically since November 2011 in the United States Analyzes cell-free fetal DNA circulating in maternal blood: (cffDNA) Placental and fetal-derived cells, possibly through the breakdown of

fetal cells in circulation ~10-15% of cell-free DNA circulating in maternal blood is from the

fetus Quantitative differences in chromosome fragments can identify

fetuses with trisomy 21, 18, 13, and sex chromosome abnormalies

By far most accurate performance for T21/18

Benn et al, Ultrasound Obstet Gynecol 2013, 42: 15-33

Often referred to as “non-invasive prenatal testing” or NIPT

Considered screening - sensitivity, specificity are not 100%

Can be performed after 10 weeks gestation

Results in approximately 7-10 days

ACOG has endorsed the use of this technology in patients who have a risk factor, given that validation studies were performed in high risk population:

AMA

Abnormal maternal serum screen

Ultrasound abnormality

Previous pregnancy with/family history of aneuploidy

Massively Parallel Shotgun Sequencing (MPSS)Utilized by most NIDS labs

Next-generation Aneuploidy Testing Using Single Nucleotide Polymorphisms (NATUS)

Utilized by Natera laboratory

Can detect triploidy

Has higher rate of un-reportable results

Massively parallel shotgun sequencing (MPSS) and counting: high-throughput technique that uses miniaturized platforms for sequencing large numbers of small DNA sequences called reads from the entire genome. There is no way to distinguish the origin of the individual reads; the only difference is in the total number of reads that are detected

Targeted Sequencing (t-MPS): instead of sequencing random genomic fragments from all chromosomes, targeted sequencing selectively amplifies specific genomic regions of interest (such as nonpolymorphic loci on chromosomes 21 and 18, or specific polymorphic loci of interest) and then reads and counts only those specific sequences. This significantly reduces total number of reads analyzed, with concomitant improvement in efficiency and 10-fold reduction in overall costs

Determines the quantity of “circulating cell free fetal DNA” in the maternal serum

Although the term ccffDNA is used, it is important to recognize that DNA is trophoblastic in origin

Measures amount of ccffDNA from chromosomes 21, 18, 13, X and Y as compared to a control

Commercial labs continuing to add more chromosomes (16, 22) and microdeletions (1p36, 22q11.2, 15q11-13, 5p, 4p, etc.)

Requires special blood tubes (black and tan) and significant quantify of serum 12-20mL in order to obtain sufficient DNA from the pregnancy

Patients with high BMI have increased rate of quantify not sufficient (un-reportable results)

Can be performed in twin or triplet gestations (at select laboratories)

Massively Parallel Shotgun

Sequencing

Targeted Sequencing

Sequenom Illumina Ariosa

Targeted Sequencing

Natera

COUNTING SNPs

Panorama®HarmonyTM

NxGen MDx

Various (Progenity,

Counsyl, LabCorp)

MaterniT21TM

Quest

Common autosomal aneuploidies compatible with live-birth– Down syndrome (trisomy 21), trisomy 18, trisomy 13

Autosomal aneuploidies commonly seen in early SAB– Trisomy 16, trisomy 22, trisomy 9

Sex aneuploidies, clinically mild compared to autosomal aneuploidies– Monosomy X (Turner), XXX, XXY (Klinefelter), XYY

Triploidy (69 chromosomes)– SNP-based only

Select microdeletion syndromes– 22q11.2 (VCFS/DiGeorge syndrome), 1p36 , 15q11.2 (Prader

Willi/Angelman), 5p (Cri du Chat), 4p16.3(Wolf-Hirschhorn syndrome) & 8q24 (Langer-Giedion syndrome, or trichorhinophalangeal syndrome type 2)

Chromosomal microarray analysis is a method of measuring gains and losses of DNA throughout the human genome.

CMA platforms use thousands of DNA probes spread across the genome to detect gains and losses of genetic material.

Extracted DNA from the patient (fetus) is compared with a reference (normal) genome.

Allows identification of abnormal copy number changes (gains and losses).

CMA looks for duplicated (extra) or deleted (missing) chomosomal segments, sometimes called copy number variants (CNVs).

1MB (megabase) = 1 million base pairs

Microdeletions are 100kb to several MB

Karyotype (and some NIDS) can usually only detect >7-10 MB

Outcome will depend on the size & the genes involved

http://www.nchpeg.org/microarray/

deletions and duplications : copy number variations (CNVs)

letters

words

sentences

page

book

Maternal Age

Example of a5-condition

Microdeletions Panel2

Down Syndrome1

1Snijders, et al. Ultrasound Obstet Gynecol 1999;13:167–170. 2Combined prevalence using higher end of published ranges from Gross et al. Prenatal Diagnosis 2011; 39, 259-266; and www.genetests.org. Total prevalence may range from 1/1071 - 1/2206.

1/2000

1/1000

1/500

1/250

20 22 24 26 28 30 32 34

These include:Microdeletions and microduplications of chromosome segments, which

are too small to see under a microscope, but may contain multiple genesMost abnormalities of chromosome number (trisomy, monosomy, etc.), including Down syndromeMost unbalanced rearrangements of chromosome structure (translocations, etc.)

Depending on the platform, CMA may also detect:Excessive homozygosity, suggestive of risk for recessive disease or imprinting disordersTriploidy and other duplications of the entire chromosome set (tetraploidy, etc.)

As with traditional karyotype, mosaicism of greater than 20-25% can be detected by CMA testing. Detection rates vary with the specific testing platform

http://www.nchpeg.org/microarray/

Cannot detect balanced chromosome rearrangements (identifies dosage differences, not positional differences)

Cannot identify triploidy (in general)

Possible Pitfalls: Identification of a copy variant of unknown significance (~1.5%)Requires parental bloods for comparisonPossible out of pocket expense to the patient

• Fetal anomalies on ultrasound and invasive diagnostic testing declined

• A chromosome rearrangement in patient or partner• A previous child or pregnancy with chromosome anomaly• Abnormal nuchal translucency results• Advanced maternal age• Traditional serum screen positive• Other failed noninvasive prenatal test results• Concerns about deletions or duplications genome-wide• Desire for the most comprehensive fetal chromosome

testing without the risks associated with an invasive procedure

• Most genetic mutations identified by CMA analysis are not associated with increasing maternal age; therefore, use of this test for prenatal diagnosis should not be restricted to women aged 35 years and older.

• In cases of IUFD or stillbirth when further cytogenetic analysis is desired, CMA on fetal tissue (amniotic fluid, placenta, or products of conception) is recommended because of increased likelihood of obtaining results and improved detection of causative abnormalities.

• Limited data are available on the clinical utility of CMA analysis to evaluate 1st and 2nd trimesters pregnancy losses; therefore, not recommended at this time.

Maternal blood cfDNA (noninvasive) testing

Identify > 95% of genome-wide deletions or duplications ≥ 7 Mb.

Discordant results: placental, maternal, or fetal mosaicism or neoplasm; vanishing twin; prior maternal organ transplant; or other causes.

Positive MaterniT GENOME test result: genetic counseling; invasive prenatal diagnosis for confirmation of test results.

Negative MaterniT GENOME test result: does not ensure unaffected pregnancy.

May get uninformative result (insufficient sequencing coverage, noise or artifacts in the region, amplification or sequencing bias, or insufficient fetal fraction)

Will not identify pregnancies at risk for neural tube defects or ventral wall defects.

cfDNA testing for whole chromosome abnormalities (including sex chromosomes) and for subchromosomal abnormalities can lead to the potential discovery of both fetal and maternal genomic abnormalities that could have minor, or no, clinical significance.

Evaluating the significance of a positive or a non-reportable test result may involve both invasive prenatal testing and additional studies on the mother. Such investigations may lead to detection of maternal chromosomal or subchromosomalabnormalities, which on occasion may be associated with benign or malignant maternal neoplasms.

cfDNA testing may not accurately identify fetal triploidy, balanced rearrangements, or the precise location of subchromosomal duplications or deletions (detected by prenatal diagnosis with CVS or amniocentesis.

The ability to report results may be impacted by maternal body mass index (BMI), maternal weight, and/or maternal systemic lupus erythematosus (SLE).

The results, including benefits and limitations, should be discussed with a qualified health care provider.

Pregnancy management decisions, including termination of the pregnancy, should not be based on the results of this test alone.

Fetal anomalies on ultrasound in patient who declines diagnostic testing

Chromosome rearrangement in patient or partner

Previous child or pregnancy with chromosome anomaly

Abnormal nuchal translucency results

Advanced maternal age

Traditional serum screen positive

Other failed noninvasive prenatal test results

Concerns about deletions or duplications genome-wide

From Sequenom website

1.Good luck2.Maternal age. Pray and Good luck!3.Amniocentesis. Pregnancy loss!4.AFP5.Genetic sonogram6.Triple/quadruple screen7.First screen 8.cffDNA9. Is ultrasound dead????