D. Van Opstal

Additional value of prenatal genomic array testing in fetuses with isolated structural ultrasound abnormalities and a normal karyotype: a systematic review of the literature

To establish the prevalence of submicroscopic genetic copy number variants (CNVs) in fetuses with a structural ultrasound anomaly (restricted to one anatomical system) and a normal karyotype. The aim was to determine the diagnostic and prognostic value of genomic array testing in these pregnancies.

Prenatal detection and outcome of congenital diaphragmatic hernia (CDH) associated with deletion of chromosome 15q26: two patients and review of the literature.

Congenital diaphragmatic hernia (CDH) is a severe birth defect characterized by a defect in the diaphragm with pulmonary hypoplasia and postnatal pulmonary hypertension. Approximately 50% of CDH cases are associated with other non‐pulmonary congenital anomalies (so called non‐isolated CDH) and in 5–10% of cases there is a chromosomal etiology. The majority of CDH cases are detected prenatally. In some cases prenatal chromosome analysis reveals a causative chromosomal anomaly, most often aneuploidy. Deletion of 15q26 is the most frequently described structural chromosomal aberration in patients with non‐isolated CDH. In this paper we report on two patients with a deletion of 15q26 and phenotypes similar to other patients with CDH caused by 15q26 deletions. This phenotype consists of intra‐uterine growth retardation, left‐sided CDH, cardiac anomalies and characteristic facial features, similar to those seen in Fryns syndrome. We propose that when this combination of birth defects is identified, either pre‐ or postnatally, further investigations to confirm or exclude a deletion of 15q26 are indicated, since the diagnosis of this deletion will have major consequences for the prognosis and, therefore, can affect decision making.

Abnormal non‐invasive prenatal test results concordant with karyotype of cytotrophoblast but not reflecting abnormal fetal karyotype

We present a unique case in which non‐invasive and invasive prenatal diagnoses showed abnormal, but discordant, results. A patient with abnormal non‐invasive prenatal test (NIPT) results, indicating a 99% risk for monosomy X, was referred to our center for genetic counseling and confirmatory studies. Cytogenetic analysis of uncultured mesenchymal core of chorionic villi (CV) revealed a mosaic male karyotype consisting of two abnormal cell lines: one with monosomy X and the other with an isodicentric chromosome Y. Array analysis of the trophoblast confirmed the NIPT results. Based on the CV results, the patient opted for termination of pregnancy. After extensive counseling by a clinical geneticist about the possible outcomes and by a gynecologist about the risk of a second‐trimester abortion procedure, the patient agreed to undergo early amniocentesis. Amniocentesis confirmed that the fetus had a male karyotype with an isodicentric chromosome Y, and the single nucleotide polymorphism (SNP) array profile suggested absence of the monosomy X cell line. The male infant was expected to be infertile. The patient finally decided to continue the pregnancy. Our case confirms that NIPT results are comparable with those of short‐term cultured CV investigating the cytotrophoblast. Our patient was not aware that the NIPT results reveal the placental karyotype, which sometimes may be different from the fetal karyotype. Pretest counseling and providing the risk figures for false‐positive and false‐negative NIPT results are of great importance in order to discourage women from terminating pregnancies based on NIPT results alone. Copyright

Recurrence of DiGeorge syndrome: prenatal detection by FISH of a molecular 22q11 deletion.

We report on a prenatal diagnosis by FISH of a familial 22q11 deletion associated with DiGeorge syndrome (DGS). The deletion was seen in the proband with symptoms of full DGS, in the physically normal father, and in a subsequent pregnancy. After birth this child showed hypocalcaemia, a T cell deficit, and a right sided aortic arch.

Whole-genome array as a first-line cytogenetic test in prenatal diagnosis

The main goal of cytogenetic prenatal diagnosis is to inform prospective parents about the chromosomal status of their fetus. A chromosomal aberration usually causes an abnormal phenotype in childhood. Prenatal whole-genome cytogenetic diagnosis was for a long time dependent on karyotyping, which requires time-consuming cell culturing, has a limited resolution (5–10 Mb) and is dependent on optimal harvesting and chromosome staining conditions. Nowadays, genomic microarray technology allows whole-genome testing at a higher resolution and it can be applied to uncultured fetal material, allowing shorter reporting times when compared with classical cytogenetic techniques. Genomic microarray testing has been recommended for routine postnatal cytogenetics in cases of intellectual disability and/or multiple congenital anomalies1, and for prenatal diagnosis in cases of fetal ultrasound anomalies2. However, its implementation for all indications in prenatal genetic diagnosis is still under discussion3–6. The main arguments against offering prenatal array testing for all indications are the possibility of detecting: (1) CNVs causing well-described clinically significant anomalies not related to the initial indication (unexpected diagnoses); (2) CNVs associated with a variable expressivity and heterogeneity of clinical features, with an as yet unquantifiable chance of an abnormal phenotype if found prenatally (so called susceptibility loci (SL) for neurodevelopmental disorders); and (3) variants of unknown clinical significance (VOUS). Such findings may complicate genetic counseling7–12 and these issues raise further questions, such as which outcomes of genomic microarrays should be reported to pregnant couples? Should they be offered a choice regarding about which possible array outcomes they wish to be informed? Is extensive genetic pretest counseling in every case necessary and feasible in clinical practice? Since this new technology is already present in prenatal clinics, rather than debating whether array testing should be performed for all referrals of invasive cytogenetic prenatal diagnosis, we should instead be discussing how to meet this challenge. In this Editorial, we review the current international status regarding the use of array technology for prenatal diagnosis. We discuss platforms and testing resolution, indications, counseling, (possible problematic) findings and the decision regarding what should be reported to the future parents.

Frequency of submicroscopic chromosomal aberrations in pregnancies without increased risk for structural chromosomal aberrations: systematic review and meta‐analysis

To establish, based on a systematic literature review, the frequency of pathogenic submicroscopic chromosomal aberrations in fetuses that are not at increased risk for unbalanced structural chromosomal aberrations, with the aim of determining whether high‐resolution testing for submicroscopic aberrations is beneficial in a general pregnant population.

SNP array detects chromosome aberrations that we thought do not exist: the first case of an isochromosome Xp (i(X)(p10))

It is known that half of the patientswith Turner syndrome showa mosaic karyotype. In most of the mosaic cases, the karyotype consists of cell lines with different numbers of chromosome X, and an isochromosome Xq is also often detected. The occurrence of an isochromosome Xp is rare, however, reported only in a dicentric form containing a part of euchromatic material of the long Xq arm between the centromeres. It has been hypothesized that isochromosome Xpmight be viable only if the inactivation center XIST locus (q13) is retained in the remainingmaterial of the long arm. IsochromosomeXpwithout XIST was reported in cancer cells, but as far as we know, a case of constitutional i(X)(p10) has never been described. Single nucleotide polymorphism (SNP) array has been implemented in routine prenatal diagnosis in our laboratory, and according to our local protocol, SNP array testing is performed, in all cases of sex chromosomal aneuploidy in order to exclude structural abnormalities of the X chromosome and any additional (sub)microscopic abnormalities that might influence pregnancy management and the patient’s decision making. A 31-year-old woman, gravida 2, para 0, pregnant after ovulation stimulation with gonadotrophins, was referred for advanced fetal ultrasound because of suspected cystic hygroma at routine first trimester ultrasound examination at 13weeks of gestation. The ultrasound examination confirmed the presence of cystic hygroma, and consequently, transabdominal chorionic villus sampling was performed. Rapid aneuploidy testing with QF-PCR (quantitative fluorescent polymerase chain reaction Devyser Complete Kit v2 on DNA from the uncultured mesenchymal core of chorionic villi) showed a monosomy X. The pregnancy was terminated because of the ultrasound abnormalities and the abnormal QF-PCR results. HumanCytoSNP-12 array (Illumina) was performed as described previously, and an abnormal chromosome X plot was seen (Figure 1). B-allele frequency was suggestive for a whole-arm aberration of the short arm of chromosome X. In this case, karyotyping and fluorescence in situ hybridization (FISH) were performed to visualize the aberration and to investigate the percentage of the different cell lines. Karyotyping of 17 metaphases in long-term cultured (LTC)-villi revealed only a cell line with monosomy X, which indicates that if a routine karyotyping was performed, the isochromosome Xp would not have been detected. Subsequent FISH analysis of LTC-villi revealed an i(X)(p10) in 11 out of 107 of analyzed metaphases (Figure 2). Array results on DNA isolated from cultured villi also revealed low-level mosaicism of an i(X)(p10) of about 10% fitting the combined results of FISH and karyotyping of LTC-villi (11/124 metaphases= 8.8%). Uncultured material, however, showed a higher percentage (~60%) of cells with i(X)(p10) (Figure 1) suggesting strong growth selection in favor of 45,X cells. This phenomenon could explain why isochromosome Xp was never detected by karyotyping. Detection of low-level mosaicism requires many metaphases and therefore possibly longer cell culturing, which can result in loss of the i(Xp). Until now, an isochromosome for the short arm of chromosome X lacking XIST was always considered to be lethal because there would be a functional Xp trisomy. Unfortunately, fetal tissue was not available for confirmation of the presence of i(X)(p10) in the fetus itself after the pregnancy was terminated. Therefore, it is possible that the cell line with the isochromosome was confined to the placenta only and that the fetus itself consisted of cells with monosomy X, although low-level or tissue-specific generalized mosaicism cannot be excluded. Our case supports the statement of Prakash et al. who showed the high utility of SNP array genotyping when addressed to clinical and research questions in Turner syndrome. This case also proves that an SNP array may even detect chromosome abnormalities that we thought cannot exist because of the genetic imbalance present. It is well-known that the array technique detects abnormalities that are not detectable with

EP06.03: Be aware of a diagnostic delay: Rotterdam's experience with NIPT in 1071 high-risk pregnancies

Objectives: Congenital heart defect (CHD) is the one of the major causes for neonatal lethality. And ultrasound screening enables the early diagnosis of CHD in prenatal stage. Emerging studies by using chromosomal microarray analysis of prenatal samples show that pathogenic copy number variants (CNVs) are highly associated with CHD. Methods: Here, we collected DNA samples from 145 unrelated fetuses with CHD diagnosed by ultrasound screening and subjected them for CNV analysis by low-pass whole-genome sequencing. CNV classification was performed according to the guideline from the American College of Medical Genetics. Maternal ages are 27.6±3.73, while the gestational weeks are 24.4±5.2. Sixty-one samples out of them are intra-cardiac defects (42.1%) and the remaining samples are extra-cardiac abnormalities (57.9%).Subtype classification of two groups is different, in intracardiac defect, the top were TOF (21.3%), TGA(14.8%) and AVSD (11.8%), while, the other were VSD (21.4%), AVSD (16.7%) and PS (13.1%). Results: Aneuploidies were identified in 29 (20%) fetuses, 27 out of them were with extra-cardiac abnormalities. Pathogenic CNVs were detected in seven cases, including a heterozygous loss at 22q13 harbouring FBLN1 and a gain at 10q23.31q26.3 with SHOC2 and ANKRD1.There were 344 unknown clinical significance micro-duplication or micro-deletion found in 91 samples, the number of CNV per sample was more in extra-cardiac abnormalities group when compared to intra-cardiac defect (p-value = 0.1514). 7.53% of which were predicted to be pathogenic by DECIPHER and GeneReviews. Conclusions: Our study demonstrates the potential clinical diagnostic utility of genomic imbalance profiling in CHD patients.

P16.05: The influence of chromosomal microarray and NIPT on the diagnostic yield in 6,811 high-risk pregnancies without ultrasound anomalies

Methods: On 3rd June 2016 Embase and PubMed databases were systematically searched for all relevant articles on prevalence of pathogenic submicroscopic CNVs in fetuses tested due to advanced maternal age or parental anxiety. Relevant full text articles were analysed and based on the extracted data the prevalence of submicroscopic CNVs was calculated. Results: Meta-analysis was conducted in a pooled cohort of 10,614 fetuses based on the 10 largest studies (N > 300) of a total of 19 relevant studies. In 0.84%, 95%CI [0.55%, 1.30%] of fetuses a submicroscopic pathogenic aberration was detected prenatally. The onset/penetrance of the submicroscopic findings was studied in 10,314 fetuses out of 8 papers that presented aberrant cases with all necessary details. The prevalence of early onset syndromic disorders due to a submicroscopic aberration was calculated to be 1:270, based on 0.37%, 95%CI [0.27%, 0.52%] cases where aberrations were specified. Conclusions: This systematic review shows that a significant proportion of fetuses in a general pregnant population carry a submicroscopic pathogenic CNV. Based on these figures all women should be informed on their individual risk for all pathogenic chromosome aberrations and not only for common trisomies.

P16.04: Prevalence of submicroscopic chromosome aberrations in pregnancies without increased risk for structural chromosome aberrations: a systematic review of the literature

33 (8.2%). Among fetuses with structural ultrasound abnormalities and normal karyotype(n=369) that underwent CMA, the detection rate of copy number variants (CNV) was 22.2% (82/369), including pathogenic CNV in 11.4% (42/369) and variants of uncertain clinical significance (VOUS) in 10.8% (40/369). In the groups of cardiac, central nervous, musculoskeletal, facial and urogenital malformations, the rate of the pathogenic CNV were 16.6% (38/229), 21.9% (14/64), 15.5% (15/97), 13.5% (7/52) and 18.8% (6/32) respectively. Ultrasound anomaly restricted to one anatomical system (n=238) had a 10.5% (n=25) chance of carrying a pathogenic CNV, while this chance increased to 13.0% (n=17) for multiple ultrasound anomalies (n=131). Conclusions: In the assessment of genetic abnormalities in pregnancies with fetal anomalies, the diagnostic yield may be increased by 22% if CMA is used as a complementary tool to conventional cytogenetics. Our results suggest that CMA is valuable in prenatal genetic diagnosis of fetuses with fetal anomalies.