By Susan Klugman, Siobhan Dolan

from GeneWatch 28-1 | Jan-May 2015

Advances in genetic technology are allowing a more rapid and cost-efficient capacity to perform clinical genome-scale sequencing via whole exome and whole genome sequencing. At the same time, non invasive prenatal screening using cell free fetal DNA is providing access to fetal DNA and the potential to perform genetic testing prenatally without incurring the risk of miscarriage. The excitement about these two developments coinciding has prompted calls for whole genome sequencing on cell free fetal DNA. This certainly is an exciting prospect, but on the front line in the clinical prenatal reproductive genetics setting, there are many challenges to consider before this becomes a reality.

Clinical Genome-scale Sequencing

Whole genome sequencing (WGS) involves sequencing an individual's entire genome, whereas whole exome sequencing (WES) involves sequencing the exome, the protein coding regions, which constitute approximately 1 percent of the genome. WES can be used to target particular genes of clinical interest and can have a lower cost but may miss mutations due to insufficient capture of some exons.

Clinical genome-scale sequencing is used currently in the pediatric or adult setting when a patient presents with clinical findings that have a high suspicion for a genetic etiology and traditional genetic testing has been negative or inconclusive. Traditional genetic testing includes karyotype analysis, to determine the chromosomal complement of an individual, and targeted testing based on clinical features that can diagnose syndromes such as Williams-Beuren syndrome or Familial Hypertrophic Cardiomyopathy. Commonly, evaluation of intellectual disability, developmental delay or multiple congenital anomalies in a child will prompt consideration of genome-scale sequencing when karyotype, targeted testing and possibly chromosomal microarray analysis are negative.

Candidates for genome-scale sequencing are often already undertaking a plan of clinical care and genetic testing results may or may not alter that care. One of the most important outcomes of genetic testing can be that test results offer insight into etiology, which helps parents answer the lingering question as to why their child has a particular clinical phenotype. Such information might be useful for parents who are planning future pregnancies or for family members who share the same genetic variants. In cases where well-defined syndromes are diagnosed, genetic information can allow for anticipatory guidance for care for a range of clinical outcomes that the patient might experience.

However, genome-scale sequencing might provide results of interpretive uncertainty. Benign familiar variants are commonly found and variants of uncertain significance can occur as well. Also, secondary findings may be revealed, defined as genetic findings that are not relevant to the reason the test was performed. An example might be the identification of a BRCA1 mutation (an indicator of increased risk for breast and ovarian cancer) in a 2-year old child being evaluated for developmental delay. Possible results need to be explained to the patient or parents (in the case of a child) so that testing is performed only after obtaining informed consent indicating that parents understand the risk of interpretative uncertainty, incidental findings and variants of uncertain significance. Many parents are likely able to tolerate results of interpretive uncertainty because their child's care is ongoing and genetic information is seen as "bonus" or extra information that might help explain the "why" but does not dramatically change the plan of care. In the example of a 2-year old child being evaluated for developmental delay, the child will continue to receive developmental assessment, physical therapy, occupational therapy, early intervention or head start, and routine pediatric care, regardless of whether the genetic testing results identify a mutation. 

Non-invasive Prenatal Screening (NIPS)

To date, cell free fetal DNA is being used clinically to screen for trisomies 21, 13 and 18, sex chromosome aneuploidies (Turner syndrome, Klinefelter syndrome) as well as a few microdeletions (22q11 deletion, Angelman, Cri-du-chat, 1p36 deletion, and Prader-Willi syndrome), depending on the laboratory. Led by several major companies that are developing this technology, non invasive prenatal screening (NIPS) is a highly sensitive screening test for a group of significant genetic conditions that offers results early in pregnancy, thereby better quantifying patients' risk. However, NIPS is not a diagnostic test and results indicating increased risk need to be confirmed by a diagnostic test, either chorionic villus sampling (CVS) in the first trimester or amniocentesis in the second trimester.

While NIPS is an excellent screening test, there are some challenges with implementation. An adequate fraction of cell free fetal DNA must be obtained and obese women are at increased risk for having an inadequate fetal fraction, leading to a failed test. In addition, confined placental mosaicism and vanishing twin as well as maternal cancers can influence cell free fetal DNA results, leading to an incorrect assessment of fetal status.

Furthermore, although NIPS is not a diagnostic test, some providers and patients misunderstand the distinction between screening and diagnostic testing for aneuploidy. NIPS can allow some to learn about certain aneuploidy risks while avoiding the risk of miscarriage. However, women with NIPS results showing increased risk are strongly advised to undertake a CVS or amniocentesis to confirm a definitive diagnosis. This misconception can lead to two potentially problematic outcomes. One is the termination of a pregnancy based on screening test results only. The second is a pregnant patient who receives an NIPS result showing increased risk but declines diagnostic testing and thus spends the duration of the pregnancy with uncertainty about the clinical diagnosis. Continued patient and provider education is needed, as well as access to genetic counseling, so that reliance on NIPS instead of invasive testing does not lead to missed diagnoses of aneuploidies as well as pregnancy termination decisions based only on screening tests.

Whole Genome Sequencing on Cell Free Fetal DNA

When these two technologies collide, there is great promise that risk-free diagnoses can be made early in pregnancy. In families with known genetic conditions, such as couples who are both identified as carriers of Tay-Sachs disease or Sickle cell anemia, identification of mutations in a fetus at 11 weeks gestation without the risk of miscarriage would be a wonderful clinical offering. In fact, one of the most successful uses of cell free fetal DNA testing to date has been screening fetuses at risk for Rh sensitization.

However, when WGS on cell free fetal DNA is proposed as an option for all pregnant patients, it raises concerns about the clinical utility of such testing in routine prenatal care. Information revealing variants of uncertain significance can be particularly problematic in the prenatal period, causing anxiety and stress in families who must make decisions about the pregnancy. What is relevant to consider is that the data obtained on a child is very different from the genetic information obtained on a fetus. We assume there is a bias of ascertainment in that all mutations and/or variants of uncertain significance obtained prenatally might not have the same implications if they are obtained in a child with an anomaly or disability. In order to be able to provide accurate predictions to parents, population-based datasets are needed regarding genetic variants in ALL children rather than simply datasets developed from affected children.

Parents' interest in the broadest possible number of genetic tests is often greatest when structural anomalies are noted on a prenatal ultrasound. Prenatal chromosomal microarray testing has been in clinical use for many years and the yield of an abnormal result after a normal karyotype is highest, approximately 6%, when structural anomalies are noted on the ultrasound. Thus the fetus with severe or multiple congenital anomalies is an area where WES and WGS are currently under investigation and may prove to have significant clinical utility. But, in the absence of a known fetal abnormality, genetic test results of interpretive uncertainty or uncertain significance are very difficult for both providers and patients to process. 

Uncertain genetic information prenatally has limited clinical utility and yet introduces much stress and anxiety, and there are arguably more risks than benefits of introducing such information into the prenatal period. Anecdotally, our pediatric colleagues have suggested that that anxiety of uncertain genetic test results identified prenatally can linger into the newborn period and negatively affect attachment and parenting.  Thus, we caution that pre-test counseling and a complete assessment of risks and benefits must be provided to parents so that appropriate candidates can be identified for WGS on cell free fetal DNA.

The jump to WGS on cell free fetal DNA might be in our near future but there are clinical, counseling and technical challenges to overcome first. The genomic era is an exciting time and maternal child health will certainly benefit from breakthroughs in genetics and genomics. Preterm birth, birth defects and infant mortality are all areas where genomic testing will hopefully contribute to a better understanding of causation and allow for the development of preventive strategies. However, continued research, data and education are all needed before we can translate these exciting advances into routine prenatal care in a way that consistently helps women and families achieve optimal healthy birth outcomes.

Susan Klugman, MD and Siobhan M. Dolan, MD, MPH, are both  Professors in the Department of Obstetrics & Gynecology and Women's Health at the Albert Einstein College of Medicine and an attending physicians in the Division of Reproductive Genetics at Montefiore Medical Center, the University Hospital for Einstein, in New York City.


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