Peter Benn

Non‐invasive prenatal testing for aneuploidy: current status and future prospects

Non‐invasive prenatal testing (NIPT) for aneuploidy using cell‐free DNA in maternal plasma is revolutionizing prenatal screening and diagnosis. We review NIPT in the context of established screening and invasive technologies, the range of cytogenetic abnormalities detectable, cost, counseling and ethical issues. Current NIPT approaches involve whole‐genome sequencing, targeted sequencing and assessment of single nucleotide polymorphism (SNP) differences between mother and fetus. Clinical trials have demonstrated the efficacy of NIPT for Down and Edwards syndromes, and possibly Patau syndrome, in high‐risk women. Universal NIPT is not cost‐effective, but using NIPT contingently in women found at moderate or high risk by conventional screening is cost‐effective. Positive NIPT results must be confirmed using invasive techniques. Established screening, fetal ultrasound and invasive procedures with microarray testing allow the detection of a broad range of additional abnormalities not yet detectable by NIPT. NIPT approaches that take advantage of SNP information potentially allow the identification of parent of origin for imbalances, triploidy, uniparental disomy and consanguinity, and separate evaluation of dizygotic twins. Fetal fraction enrichment, improved sequencing and selected analysis of the most informative sequences should result in tests for additional chromosomal abnormalities. Providing adequate prenatal counseling poses a substantial challenge given the broad range of prenatal testing options now available. Copyright

Position statement from the Aneuploidy Screening Committee on behalf of the Board of the International Society for Prenatal Diagnosis

Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT, USA Prenatal Diagnosis Unit, Institute of Gynecology, Obstetrics and Neonatology, Hospital Clinic, Maternitat Campus, University of Barcelona Medical School, Catalonia, Spain Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong Department of Obstetrics and Gynecology, Columbia University Medical Center, New York, NY, USA Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, PA, USA Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands Department of Obstetrics and Gynecology, Albert Einstein College of Medicine, New York, NY, USA Department of Obstetrics and Gynecology, University of Calgary, Calgary, AB, Canada Department of Obstetrics and Gynecology, Assaf Harofe Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA, USA Department of Obstetrics and Gynecology, Washington University in St Louis, St Louis, MO, USA Laboratory for Infectious Diseases and Perinatal Screening, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands Prenatal Screening Unit, Clinical Biochemistry Department, Barking Havering & Redbridge University Hospitals, King George Hospital, Goodmayes, UK Genetics Program, North York General Hospital, Toronto, ON, Canada Department of Mathematics and Statistics, University of Plymouth, Plymouth, UK Prenatal Diagnosis Unit, Genetic Institute, Sourasky Medical Center, Tel Aviv, Israel *Correspondence to: Peter Benn. E-mail: benn@nso1.uchc.edu This Statement replaces the January 2011 Statement (Prenatal Diagnosis 2011;31:519–522) and the Rapid Response Statement (Prenatal Diagnosis 2012;32:1–2).

Prenatal Detection of Down Syndrome using Massively Parallel Sequencing (MPS): a rapid response statement from a committee on behalf of the Board of the International Society for Prenatal Diagnosis, 24 October 2011

Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT, USA Prenatal Diagnosis Unit, Institute of Gynecology, Obstetrics and Neonatology, Hospital Clinic, Maternitat Campus, University of Barcelona Medical School, Catalonia, Spain Department of Obstetrics and Gynecology, Columbia University Medical Center, New York, NY, USA Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, PA, USA Department of Obstetrics and Gynecology, Albert Einstein College of Medicine, New York, NY, USA Department of Obstetrics and Gynecology, University of Calgary, Calgary, AB, Canada Department of Obstetrics and Gynecology, Assaf Harofe Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Department of Obstetrics and Gynecology, Washington University in St Louis, St Louis, MO, USA Laboratory for Infectious Diseases and Perinatal Screening, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands Prenatal Screening Unit, Clinical Biochemistry Department, Barking Havering and Redbridge University Hospital, King George Hospital, Goodmayes, UK Department of Mathematics and Statistics, University of Plymouth, Plymouth, UK Prenatal Diagnosis Unit, Genetic Institute, Sourasky Medical Center, Tel Aviv, Israel *Correspondence to: Peter Benn. E-mail: benn@nso1.uchc.edu

RARE TRISOMY MOSAICISM DIAGNOSED IN AMNIOCYTES, INVOLVING AN AUTOSOME OTHER THAN CHROMOSOMES 13, 18, 20, AND 21: KARYOTYPE/PHENOTYPE CORRELATIONS

In order to determine the significance of trisomy mosaicism of an autosome other than chromosomes 13, 18, 20, and 21, 151 such cases diagnosed prenatally through amniocentesis were reviewed. These rare trisomy mosaicism cases include 54 from 17 cytogenetic laboratories, 34 from a previous North American mosaicism survey, and 63 from published reports. All were cases of true mosaicism with information available on pregnancy outcome, and with no evidence of biased ascertainment. There were 11 cases of 46/47,+2; 2 of 46/47,+3; 2 of 46/47,+4; 5 of 46/47,+5; 3 of 46/47,+6; 8 of 46/47,+7; 14 of 46/47,+8; 25 of 46/47,+9; 2 of 46/47,+11; 23 of 46/47,+12; 5 of 46/47,+14; 11 of 46/47,+15; 21 of 46/47,+16; 7 of 46/47,+17; 1 of 46/47,+19; and 11 of 46/47,+22. As to the risk of an abnormal outcome, the data showed a very high risk (>60 per cent) for 46/47,+2, 46/47,+16, and 46/47,+22; a high risk (40–59 per cent) for 46/47,+5, 46/47,+9, 46/47,+14, and 46/47,+15; a moderately high risk (20–39 per cent) for 46/47,+12; a moderate risk (up to 19 per cent) for 46/47,+7 and 46/47,+8; a low risk for 46/47,+17; and an undetermined risk, due to lack of cases, for the remaining autosomal trisomy mosaics. Most cases were evaluated at birth or at termination, so subtle abnormalities may have escaped detection and developmental retardation was not evaluated at all. Comparison of the phenotypes of prenatally diagnosed abnormal cases and postnatally diagnosed cases with the same diagnosis showed considerable concordance. Since the majority of anomalies noted are prenatally detectable with ultrasound, an ultrasound examination should be performed in all prenatally diagnosed cases. In cytogenetic confirmation studies, the data showed much higher confirmation rates in cases with abnormal outcomes than in cases with normal outcomes [81 per cent vs. 55 per cent for fibroblasts (from skin, fetal tissue, and/or cord); 88 per cent vs. 46 per cent for placental cells; 22 per cent vs. 10 per cent for blood cells]. The confirmation rate reached 85 per cent when both fibroblasts and placental tissues were studied in the same case (with trisomic cells found in one or the other, or both). Therefore, one must emphasize that both fibroblasts and placental tissues should be studied. Except for 46/47,+8 and 46/47,+9, PUBS is of limited value for prenatal diagnosis of rare trisomy mosaicism. DNA studies for UPD are suggested for certain chromosomes with established imprinting effects, such as chromosomes 7, 11, 14, and 15, and perhaps for chromosomes 2 and 16, where imprinting effects are likely.

Position statement from the Chromosome Abnormality Screening Committee on behalf of the Board of the International Society for Prenatal Diagnosis

President President-Elect Past President Secretary Treasurer Lucas Otano MD, PhD (Argentina) Ignatia B. Van den Veyver MD (USA) Jan M.M. van Lith MD, PhD (Netherlands) Louise Wilkins-Haug MD (USA) Antoni Borrell MD, PhD (Spain) Directors Peter Benn PhD, DSc (USA) Lyn Chitty PhD (UK) Rossa Chiu (Hong Kong) Roland Devlieger MD, PhD (Belgium) Sylvie Langlois MD, CCMG (Canada) Anthony O. Odibo MD, MSCE (USA) R. Doug Wilson MD, Msc, FRCSC (Canada) Yuval Yaron MD (Israel) Diana W. Bianchi MD, ex officio (USA) Position Statement from the Chromosome Abnormality Screening Committee on Behalf of the Board of the International Society for Prenatal Diagnosis

Maternal cfDNA screening for Down syndrome – a cost sensitivity analysis

This study aimed to determine the principal factors contributing to the cost of avoiding a birth with Down syndrome by using cell‐free DNA (cfDNA) to replace conventional screening.

Changes in the utilization of prenatal diagnosis.

OBJECTIVE: The impact of prenatal screening for Down syndrome has largely been assessed under the assumption that screening protocols and policies are fully used. To measure the overall effectiveness in actual clinical practice, we analyzed the tests performed by a single cytogenetics laboratory. METHODS: We reviewed all amniotic fluid and chorionic villus samples (CVS) processed by the University of Connecticut Health Centers cytogenetics laboratory for the years 1991 to 2002. We evaluated trends in the use of prenatal testing, referral indications, and the numbers of cytogenetic abnormalities identified. RESULTS: The number of women receiving amniocentesis or CVS declined more than 50% from 1,988 in 1991 to 933 in 2002 (P < .001), despite an increase in the number of women of advanced maternal age in the population served. There was a 68% decline in the number of women who underwent invasive prenatal testing solely on the basis of their age (1,314 in 1991 to 423 in 2002, P < .001). The number of Down syndrome fetuses detected prenatally increased from 20 to 31 (P = .08), representing approximately one half of the affected pregnancies present in the population served. Between 1991 and 2002, the proportion of antenatal cytogenetic tests with a significant chromosomal abnormality increased from 1 in 43 (2.3%) to 1 in 14 (7.0%; P < .001). CONCLUSION: Advances in maternal serum screening and second-trimester ultrasonography have resulted in more judicious use of amniocentesis and chorionic villus sampling. LEVEL OF EVIDENCE: II-2

Cytogenetic studies of bone marrow fibroblasts cultured from patients with myelofibrosis and myeloid metaplasia

Summary. Cytogenetic studies of bone marrow fibroblasts and blood cells from peripheral blood or bone marrow were performed in 19 patients with myelofibrosis with myeloid metaplasia (group 1), nine patients with other myeloproliferative syndromes without myelofibrosis (group 2), and 12 patients with anaemia secondary to iron deficiency or chronic inflammatory disease (group 3). Clonal cell populations with abnormal karyotypes were seen in the bone marrow or blood in five of 14 (36%) group 1 patients, one of nine (11%) group 2 patients and none (0%) of the group 3 patients. Abnormal karyotypes of bone marrow fibroblasts were found in three of 16 (19%) of patients of group 1, and in two of nine (22%) and two of 12 (17%) patients each of groups 2 and 3, respectively. Since abnormal karyotypes can be found in bone marrow fibroblasts cultured from normal subjects, and since the abnormalities seen in the bone marrow fibroblasts differed from those found in bone marrow or blood cells, it is suggested that abnormal karyotypes found in bone marrow fibroblasts cultured from patients with primary myelofibrosis do not necessarily reflect neoplasia. The results of this study are compatible with the widely accepted hypothesis that in patients presenting with ‘primary’ myelofibrosis, the fibrosis is a secondary reactive process.

Advances in prenatal screening for Down syndrome: I. general principles and second trimester testing.

BACKGROUND Down syndrome is one of the most important causes of mental retardation in the population. In the absence of prenatal screening and diagnosis, prevalence at birth in the United States would currently exceed 1:600. The purpose of prenatal screening is to identify those women at the increased risk for an affected pregnancy and to maximize the options available to these women. TESTS AVAILABLE Second trimester serum screening involves combining the maternal age-specific risk for an affected pregnancy with the risks associated with the concentrations of maternal serum alpha-fetoprotein (MSAFP), unconjugated estriol (uE3), and human chorionic gonadotropin (hCG) (triple testing). A forth analyte, inhibin-A (INH-A), is increasingly being utilized (quadruple testing). Optimal second trimester screening requires the integration of a number of clinical variables, the most important of which is an accurate assessment of gestational age. In addition to Down syndrome, the triple and quadruple tests preferentially identify fetal trisomy 18, Turner syndrome, triploidy, trisomy 16 mosaicism, fetal death, Smith-Lemli-Opitz syndrome, and steroid sulfatase deficiency. Some programs modify the Down syndrome risks generated through maternal serum screening tests with fetal biometric data obtained by ultrasound. Other second trimester tests have shown promise, including the analysis of maternal urine and fetal cells in the maternal circulation, but none are in routine clinical use. CONCLUSION The second trimester triple and quadruple tests provide benchmarks for evaluating new screening protocols. The combination of fetal biometry, new test development as well as clarification of the role of co-factors that affect the concentrations of analytes in existing tests should lead to greater efficacy in second trimester screening for Down syndrome.

Practical and ethical considerations of noninvasive prenatal diagnosis.

CHROMOSOME ABNORMALITIES SUCH AS TRISOMY 21 (Down syndrome), other autosomal trisomies, sex chromosome abnormalities, and balanced and unbalanced translocations can be prenatally diagnosed. For many individuals, the availability of prenatal diagnosis removes the fear of having a child with a severe physical or mental disability. For chromosome abnormalities that are associated with significant morbidity and mortality, the public’s acceptance of prenatal diagnosis has been well established during the past 4 decades during which testing has been available. Current standards of care involve screening women to identify those at highest risk of having a fetus with a chromosome abnormality and then offering definitive diagnosis through the analysis of a chorionic villus sample or amniotic fluid cells. The American College of Obstetricians and Gynecologists recommends that all women be offered aneuploidy screening through various combinations of maternal serum tests and sonographic measurements. However, these screening tests are associated with relatively high false-positive rates and often involve multistep protocols that may take weeks to complete. Obtaining chorionic villus or amniocentesis specimens is expensive and requires an invasive procedure that carries a small risk to the fetus and the possibility of adverse maternal effects. Moreover, these invasive tests are usually not provided until late in the first trimester or second trimester. Amniocentesis and chorionic villus sampling have therefore imposed a rate-limiting step in the provision of prenatal diagnosis; in general only those patients at the highest risk of having a fetus with Down syndrome or another disorder of major clinical significance have been tested. For more than 30 years, these problems have spurred attempts to develop noninvasive prenatal diagnosis. The discovery of substantial amounts of conceptus-derived nucleic acids (DNA and RNA mostly from trophoblasts) in maternal blood has resulted in proof of principle and also in the development of noninvasive tests. Currently, it is possible to determine fetal sex, establish Rh genotype of the fetus, and diagnose genetic disorders or carrier status for paternally inherited mutations. A number of strategies have been proposed for noninvasive prenatal diagnosis for aneuploidy, including an RNA-based method for Down syndrome detection, which is expected to be commercially available within a year. The benefits of a noninvasive prenatal diagnosis are clear. It eliminates the dangers associated with invasive testing, and it allows for definitive diagnoses earlier in pregnancy, thus resulting in safer terminations of affected pregnancies, reduction of parental anxiety, and decreased medical costs. As a result, uptake of the testing is likely to be higher, which can translate into additional health and economic benefits for both individuals and, indirectly, society at large. The introduction of noninvasive prenatal diagnosis would require the ill-prepared medical system to change how patients are counseled and how cases are managed. Additionally, significant ethical issues are associated with noninvasive prenatal diagnosis. In this Commentary, we discuss these effects with a focus on the forthcoming introduction of noninvasive prenatal diagnosis for fetal Down syndrome.