The 6%–9% risk of an untoward outcome previously established by Warburton for prenatally detected de novo balanced chromosomal rearrangements (BCRs) does not account for long-term morbidity. We performed long-term follow-up (mean 17 years) of a registry-based nationwide cohort of 41 individuals carrying a prenatally detected de novo BCR with normal first trimester screening/ultrasound scan. We observed a significantly higher frequency of neurodevelopmental and/or neuropsychiatric disorders than in a matched control group (19.5% versus 8.3%, p = 0.04), which was increased to 26.8% upon clinical follow-up. Chromosomal microarray of 32 carriers revealed no pathogenic imbalances, illustrating a low prognostic value when fetal ultrasound scan is normal. In contrast, mate-pair sequencing revealed disrupted genes (ARID1B, NPAS3, CELF4), regulatory domains of known developmental genes (ZEB2, HOXC), and complex BCRs associated with adverse outcomes. Seven unmappable autosomal-autosomal BCRs with breakpoints involving pericentromeric/heterochromatic regions may represent a low-risk group. We performed independent phenotype-aware and blinded interpretation, which accurately predicted benign outcomes (specificity = 100%) but demonstrated relatively low sensitivity for prediction of the clinical outcome in affected carriers (sensitivity = 45%–55%). This sensitivity emphasizes the challenges associated with prenatal risk prediction for long-term morbidity in the absence of phenotypic data given the still immature annotation of the morbidity genome and poorly understood long-range regulatory mechanisms. In conclusion, we upwardly revise the previous estimates of Warburton to a morbidity risk of 27% and recommend sequencing of the chromosomal breakpoints as the first-tier diagnostic test in pregnancies with a de novo BCR.

With the aim of assessing the long-term morbidity risk of BCRs, we used the Danish nationwide health registries to perform an epidemiological study of an unbiased cohort of all prenatally detected de novo BCRs found in Denmark in the period 1970–2008. We combined these results with a clinical and molecular reexamination of the available live-born BCR carriers to detail the clinical description and to evaluate the prognostic value of CMA and MPS. The consequence of the breakpoints in each BCR was evaluated by two independent groups, one of which was blinded to all clinical data. Based on the obtained results, we present a clinical workflow for pregnancies with a de novo BCR.

Many diagnostic laboratories have replaced prenatal karyotyping with CMA since the method can reveal both aneuploidies and submicroscopic imbalances within a few days.However, CMA will not detect chromosomal rearrangements unless they are associated with submicroscopic deletions and/or duplications. Such imbalances have been reported in up to 40% of carriers with a postnatally detected de novo BCR ascertained because of an abnormal phenotype,while a recent whole-genome sequencing study suggested that only 15.5% and 18.8% of individuals ascertained for a severe congenital anomaly and a postnatally detected de novo BCR without prior microarray screening harbored imbalances at the breakpoints greater than 1 Mb and 100 kb, respectively.However, the frequency of imbalances in unselected prenatally detected BCRs is unknown. If a BCR is truly balanced, it can nevertheless be disease causing if one of the involved chromosomal breakpoints disrupts an autosomal dosage-sensitive gene or an X-linked recessive gene,unmasks a recessive mutation in the normal allele,or is associated with long-range position effects (LRPEs).Molecular mapping of BCRs with NGS-based whole-genome paired-end or mate-pair sequencing (MPS)has greatly facilitated the detection of breakpoints that disrupt genes and topologically associated domains (TADs) that encompass candidate genes within which LRPEs might occur.Although MPS has been applied to a limited number of prenatally detected BCRs,the improved capability for prediction of pathogenicityover conventional cytogenetic approaches has not been evaluated in a diagnostic setting, where the clinical knowledge about the fetus is sparse or lacking. Furthermore, the almost total lack of NGS-mapped breakpoints from healthy BCR carriers might challenge our ability to prenatally discriminate likely pathogenic from likely benign BCRs.

Despite remarkable advances in molecular cytogenetics in terms of non-invasive prenatal testing (NIPT), chromosomal microarray (CMA), and next-generation sequencing (NGS) technologies, karyotyping of fetal cells remains the primary diagnostic method for prenatal detection of chromosomal abnormalities in most countries. Given the microscopic resolution of karyotyping (∼5–10 Mb) and thus the limited genomic context that can be inferred for abnormal findings, prediction of morbidity risk from prenatal detection of de novo apparently balanced chromosomal rearrangements (BCRs), including reciprocal translocations, inversions, and insertions, represents a profound diagnostic challenge. In the largest study to date based on 377,357 prenatal samples, the risk of severe congenital anomaly was found to be 6.1% for carriers of a prenatally detected de novo balanced reciprocal translocation (n = 163) and 9.4% for carriers of a de novo balanced inversion (n = 32) when excluding those ascertained due to abnormal ultrasound findings.However, clinical information was limited to the first 2 years or less after birth for the large majority of the cohort (85%), thus precluding accurate prediction of risk beyond early developmental anomalies (e.g., later-onset and diagnosed disorders). A more recent study of 16 prenatally detected de novo BCR carriers with a mean follow-up time of 6.7 years which reported a long-term morbidity similar to the expected in the normal population lacked a matched control group and was likely biased toward inclusion of healthy carriers.The genetic counseling implications for improved prediction of adverse outcomes are thus substantial; at present, the detection of a de novo BCR results in a parental decision to terminate 20%–31% of pregnancies.

Based on the findings, we classified each breakpoint to be either pathogenic, likely pathogenic, VUS, or benign and made a combined classification for each BCR. This classification was compared with an independent, blinded classification carried out according to a published interpretation strategy.When the blinded analyses predicted “pathogenic” or “likely pathogenic” for a carrier with a NPD, it was considered in accordance with the clinical outcome and thereby a successful prediction. When the BCR of a healthy carrier was classified as “benign” or “VUS,” it was also considered a successful prediction.

When a breakpoint was found to disrupt a protein-coding or non-coding gene, the gene was evaluated using PubMed, Online Mendelian Inheritance in Man (OMIM), haploinsufficiency-index (HI),and the probability of loss-of-function intolerance (pLI)extracted from ExAC.We also overlapped the MPS-defined breakpoints with a gene panel comprising genes with de novo loss-of-function mutations (dnLoF) reported in amalgamated large-scale genomic studieswith entries from ClinVar and the Deciphering Developmental Disorders Study.In addition, we searched for overlapping deletions and duplications in DECIPHER, Clinical Genome Resource, and Database of Genomic Variants, and for overlapping CNV gains and losses from individuals with neurodevelopmental and/or neuropsychiatric disorders (NPDs) versus control subjects.Potential LRPEs for each breakpoint were studied by using previously annotated TADs and topological boundary regionsfor human embryonic stem cells and IMR90 fibroblasts. All known disease-associated genes and genes with a pLI > 0.9 within each disrupted TAD were considered. As we primarily observed brain-related phenotypes among the carriers, we used the 3D Genome Browser to extract Hi-C and Virtual 4C data from neurogenic precursor cells for the listed genes and genomic positions. The breakpoints were also overlapped with 128 schizophrenia-associated regions, representing 108 loci identified by genome-wide association studiesand with mouse phenotypes obtained from Mouse Genome Informatics. In addition, we compared the disrupted genes with published protein-protein interactions networks implicated in NPDs,fragile X mental retardation protein interaction partners,and the postsynaptic density proteome.

Metaphase chromosomes from cultured peripheral blood lymphocytes were prepared according to standard protocols, G-banded,and karyotyped using a Leica CW4000 karyotyping system. FISH was performed as explained in detail elsewhere.The signals were analyzed using a Leica DMRXA microscope equipped with a Leica DFC 340 FX camera and CW4000 CytoFISH software. Painting probes and centromere-specific probes were purchased from Cambio Ltd. and ONCOR, Amersham Buchler, respectively. The BAC clone RP11-586I18 was kindly provided by the Wellcome Trust Sanger Institute. The FISH results were illustrated using Cytogenetic Data Analysis System (CyDAS Package)and karyotypes were written in accordance with ISCN 2016.

Mate-pair libraries were prepared using the Mate Pair Library v2 kit (Illumina), and paired-end sequenced (2×36 bases) on a Genome Analyzer IIx (Illumina). Paired reads were aligned to the hg19 reference genome, and those that aligned to different chromosomes or with unexpected strand orientations were extracted and analyzed to identify potential translocation and inversion breakpoints as previously decribed.For all carriers with sufficient DNA (n = 23), the breakpoints were confirmed by Sanger sequencing and the breakpoint junctions were analyzed as described previously.MPS was repeated for two of the unmappable BCRs (P64 and P87) on an Illumina Next 500 platform using 2×150 bp.

Genomic DNA was extracted from whole blood or saliva from the BCR carriers and their parents. The CMA analyses were performed with Affymetrix Genome-Wide Human SNP Array 6.0 (Affymetrix) using standard protocols as a service at AROS Applied Biotechnology. CEL files were analyzed with the Genotyping Console software (Affymetrix) according to the manufacturer’s recommendations using “copy number analysis single sample mode” and regional GC correction identifying copy-number variations (CNVs) encompassing >8 markers. We selected the size threshold of >100 kb for the initial analyses as these CNVs would be detected in most diagnostic laboratories using either SNP array or array CGH. We analyzed and classified the CNVs of the BCR carriers and their parents using the recommendations of the American College of Medical Genetics.Pathogenic and likely pathogenic CNVs were included in the final interpretation while benign findings and variants of unknown significance (VUS) without strong arguments for pathogenicity were disregarded as described by Vanakker et al.

The study was approved by DCCR, the Danish Departments of Clinical Genetics, the National Scientific Ethics Committee (H-KF-2006-5901 and H-C-2008-070), and the Danish Data Protection Agency (2009-41-3683 and 2009-41-3108). Informed consent was obtained from carriers participating in the clinical investigation when older than 15 years and from their parents.

The parents of the live-born BCR carriers were invited by letter in 2008 to participate in a clinical and molecular examination. Informed consent was obtained from 32/41 group A carriers (78%) and 7/9 group B carriers (78%). They were all seen by the same medical doctor (author C. Halgren) who performed a semi-structured clinical examination of the proband and a medical interview with the parents (see Supplemental Note ). In addition, clinical photographs were taken and blood and/or saliva samples for DNA analysis were collected. The clinical photos were evaluated by a panel of medical doctors with clinical genetics expertise (authors S.K., K.B.-N., N.T., and I.B.). Dysmorphic features, if any, were described according to Elements of Morphology.In the subsequent years, additional health-related information about the carriers was obtained both by sporadic contact with the families and systematically in 2016 when a second questionnaire was sent to all the participating carriers and/or their parents.

The registry-based case-control study involved the 41 unbiased carriers in group A and a matched control group comprising 205 live-born individuals (5 control subjects per case subject) with a normal prenatal chromosome analysis obtained from the DCCR. The carriers and control subjects were strictly matched according to prenatal indications and with a decreasing priority according to gender, maternal age (±2.5 years), examination year, sampling procedure, and cytogenetic laboratory. By registry linkage, all available medical data were obtained from the Danish National Patient Register,the Danish Psychiatric Central Research Register,the Danish Register of Causes of Death,the Danish Medical Birth Registry,and the Danish Cancer Registry,using the personal identification number as a key.These nationwide medical registries hold information regarding hospital contacts (see Supplemental Note ) whereas contacts with general or specialized practitioners are not included. Medical diagnoses were confirmed by scrutiny of the original medical records. The morbidity prevalence among the carriers was compared to that of the control group using Fisher’s exact test (two-sided). Registry data were also obtained for group B carriers, but were not included in the case-control study to minimize ascertainment bias.

The Danish Cytogenetic Central Register (DCCR) holds information on pre- and postnatal chromosome examinations performed in Denmark (population ∼5.7 million people), including 191,977 prenatal samples registered in the period 1970–2008. In the entire database, we searched for prenatally detected de novo BCRs defined as apparently balanced reciprocal translocations, inversions, insertions, and complex rearrangements. We excluded Robertsonian translocations and common inversion variants as defined by Gardner et al.In total, 122 prenatal samples with a reported de novo BCR were found in DCCR ( Table S1 ). Evaluation of the prenatal and parental karyotypes revealed four BCRs which were inherited from a healthy parent and one unbalanced BCR, resulting in 50 live-born prenatally detected de novo BCR carriers, 65 terminated pregnancies, and 2 spontaneous abortions. The karyotypes were also evaluated in relation to type of rearrangement and the cytogenetically determined location of the breakpoints ( Tables S1 and S2 ). The carriers were grouped according to ascertainment ( Table S3 ) into an unbiased group A of 41 live-born carriers ( Figure 1 ) and a potential biased group B of 9 live-born carriers referred due to abnormal ultrasound, elevated biochemical pregnancy markers, or abnormal results in the combined first trimester screening.

The nationwide cohort of prenatally detected carriers of a de novo balanced chromosomal rearrangement (BCR) was reexamined both by a registry and a clinical study. To reduce the risk of ascertainment bias in the case-control study, the carriers were grouped into an unbiased group A (with invasive prenatal test because of e.g., maternal age or anxiety) and a potentially biased group B (with invasive prenatal test because of abnormal fetal ultrasound scan and/or maternal serum markers). Chromosomal microarray and mate-pair sequencing were performed for all carriers with available DNA.

The two independent research groups classified the BCRs concordantly in 26 of 27 (96.3%) carriers ( Table 1 ). By the blinded evaluation of 11 NPD-affected BCR carriers, 5 were classified as pathogenic or likely pathogenic in accordance with a sensitivity of 45%. Two of these five carriers had a complex BCR (P76 and P83) and two others (P71 and P62) had disruption of a known or suggested NPD gene: ARID1B and NPAS3. The fifth affected carrier (P119) with a blinded classification of likely pathogenic had disruption of solute carrier family 8 member A1 (SLC8A1 [MIM: 182305 ]) with high pLI. The unblinded research group (with knowledge about the phenotype) suggested LRPE based on the other breakpoint disrupting a TAD containing the Mowat-Wilson syndrome (MWS [MIM: 235730 ]) gene zinc finger E-box binding homeobox 2 (ZEB2 [MIM: 605802 ]) since the individual had ID and severe constipation that potentially could be related to MWS ( Figure 4 ). LRPE was also suggested by the unblinded research group in P124, with disruption of the evolutionarily conserved HOXC cluster ( Figure 5 ). All the BCRs of the healthy carriers (16/16) were classified as benign or VUS by the blinded evaluation, concordant with the clinical outcome (specificity = 100%). Among the 22 BCRs that were not classified as pathogenic or likely pathogenic by the blinded evaluation, six carriers (27.3%; 5/17 unbiased group A carriers and 1/5 potentially biased group B carriers) had a NPD diagnosis.

The HOXC cluster is located within a ∼140 kb region that is almost devoid of transposons,highlighting its evolutionary importance. The HiC-heatmap and the Virtual 4C originate from a neuronal progenitor cell line (H1-NPC). The interaction of SNP rs71227279 within exon 1 of HOTAIR, fits with the topologically associated domains (TAD), and with chromatin loops.

The HiC-heatmap and the Virtual 4C originates from a neuronal progenitor cell line (H1-NPC). The ZEB2-interactions in H1-NPC fits exactly with a ∼3.5 Mb large TAD in both hESC and IMR90 cells.The Virtual 4C data show that ZEB2 has interactions distal to the breakpoint. Moreover, a forebrain and limb-specific enhancer (hs609)has been removed by the inv(2) breakpoint. SCZ indicates a schizophrenia GWAS locus.

Among the 27 BCRs mapped by MPS and with normal CMA, 11 carriers (40.7%) had an NPD diagnosis without congenital malformations (mean follow-up time: 22.2 years), 1 had a mild congenital malformation (hemivertebra) without NPD and otherwise healthy (follow-up time 23 years), whereas 15 (55.6%) were healthy with normal development (mean follow-up time: 23.9 years) ( Table 1 ). The complex BCRs were found only among the affected carriers (2 of 11 balanced BCRs with an NPD diagnosis). Among the simple BCRs, non-genic breakpoints were found in five healthy (P73, P75, P90, P91, P110) and one affected (P59) carrier. Disruption of a gene with either HI < 10% or pLI > 0.9 was found both among the affected (7/11; 63.6%) and unaffected (7/16; 43.8%) carriers ( Tables 1 and S7 ), whereas disruption of a known or suggested autosomal-dominant NPD-associated gene was found exclusively in affected carriers: in AT-rich interaction domain 1B (ARID1B [MIM: 614556 ]) in P71; in neuronal PAS domain protein 3 (NPAS3 [MIM: 609430 ]) in P62; and in CUGBP Elav-like family member 4 (CELF4 [MIM: 612679 ]) in P83. When using the dnLoF gene list,we found three BCR carriers with truncation of a dnLoF gene that has been reported in affected individuals previously: one affected carrier (P71) with disruption of ARID1B and two unaffected carriers (P96 and P157) with disruption of phosphodiesterase 4D interacting protein (PDE4DIP [MIM: 608117 ]) and adhesion G protein-coupled receptor V1 (ADGRV1 [MIM: 602851 ]). In addition, when comparing CNV gains and losses, protein-protein interactions networks, and fragile X mental retardation protein interaction partners, we found no difference between affected and unaffected, and only one of the disrupted genes (leucine rich repeat transmembrane neuronal 4, LRRTM4 [MIM: 610870 ] in P151 with NPD) overlapped with the postsynaptic density proteome ( Table S7 ). Three breakpoints overlapping with or near schizophrenia GWAS loci were all from affected carriers.

Among the balanced BCRs, two demonstrated complexity that was cryptic to karyotyping (2/27; 7.4%): P76 with chromotripsis and a total of seven breakpoints involving chromosome 2 and 11 ( Figure 3 ) and P83 with a complex translocation/inversion with four breakpoints, that all turned out to be associated with deletions below our threshold of 100 kb in size (range 45–58 kb; a detailed description of the molecular and phenotypic findings has been published previously). In addition, the BCR that was found to be unbalanced by CMA (P94) had one breakpoint on chromosome 1 and five breakpoints on chromosomes 2 and 21 each, illustrating that the 2.8 Mb deletion of chromosome 21 was not an independent event, but in fact was associated with the structural rearrangement detected by karyotyping. The remaining 25 carriers had simple balanced BCRs involving two breakpoints only.

Partial karyogram of an assumed simple two-way translocation t(2;11)(p23;q23)dn where mate-pair sequencing (MPS) and Sanger sequencing revealed chromothripsis (P76). The brackets indicate regions on the normal chromosomes 2 and 11 that were shown by MPS to involve a total of 7 breakpoints. The chromosomal segments involved and their arrangement on the derivative chromosomes are shown on the Circos plot as well as on the colored diagrams below.

The breakpoints were determined in 28/36 carriers with available DNA (77.8%), including 27 balanced BCRs and the single unbalanced BCR (P94). Enough DNA was available for confirmation with Sanger sequencing in 23 carriers, hence 47 breakpoint-junction sequences were characterized ( Table S6 and Supplemental Note ). Short deletions (1–25 base pairs [bp]) and duplications (1–17 bp) were found in 17 and 18 breakpoints, respectively, while larger deletions (3,144–7,600 bp) were found in 6 breakpoints. Short stretches of microhomology (2–8 bp) were detected in half of the breakpoint-junctions (23/46; 50.0%), while the remaining breakpoint-junctions showed no microhomology (0–1 bp), and this distribution was similar for mapped breakpoints that derived from NPD-affected and healthy carriers (10/19; 52.6% and 13/27; 48.1%, respectively). These findings are in accordance with non-homologous end joining or microhomology-mediated end joining as the breakage-repair mechanisms in the large majority of the BCRs in this study (22/23; 95.7%). We observed Alu- and L1-mediated deletions (5,924 bp and 3,925 bp, respectively) associated with the breakpoints of one BCR (P75), indicating that the translocation had likely occurred as a secondary event after the free ends were generated due to the small deletions on chromosomes 1 and 14.

DNA was available for MPS from 36 BCR carriers (group A: n = 29 and group B: n = 7) and failed to reveal the breakpoints for 8 BCRs (8/36; 22.2%, 7 autosomal-autosomal and 1 X-autosomal translocation, all 8 from the unbiased ascertained group A). The mean physical coverage of the MPS analyses was 24.9 (range 16.1–37.1) in carriers in which we detected clusters of breakpoint-spanning reads, compared to 23.9 (range 20.9–29.6) in carriers in which we could not detect the breakpoints, indicating that the lack of detection is not related to lower coverage. Postnatal chromosome analysis of the eight unmapped BCRs revealed that in each BCR at least one breakpoint was localized to an acrocentric p-arm or a pericentromeric region, thus suggesting these BCRs were not detectable using standard alignment of mate-pair reads given the highly repetitive regions. We confirmed this interpretation by FISH mapping for seven of the eight BCRs ( Table 2 Figure 2 , and Supplemental Note ). None of the seven carriers of unmappable autosomal-autosomal BCRs had NPD, suggesting that these individuals might represent a low-risk group. However, this was not statistically different from the proportion among carriers with mappable autosomal-autosomal BCRs, among whom 12 had NPD out of a total of 28 (p = 0.070, Fisher’s exact test, two-sided). In contrast, the single female carrier of an X-autosomal translocation had NPD.

Partial karyogram showing the normal and the derivative chromosomes 1 and 22 and the fluorescent in situ hybridization (FISH) results from a phenotypically normal t(1;22) translocation carrier (P160). The two-color experiment was performed with a centromere 1 probe (D1Z5, red signals, red arrows) and a 1qh probe (D1Z1, green signals, green arrows) showing that the centromere 1 probe hybridized to the normal chromosome 1 and the derivative 1 (der(1)) and not to the derivative 22 (der(22)) in accordance with an intact centromere on chromosome 1. The 1qh probe hybridized to the normal chromosome 1 and both of the derivative chromosomes, demonstrating that the chromosomal breakpoint is within the heterochromatic region on chromosome 1q12. The centromere 22 probe gave signals on chromosome 22 and der(22) in addition to a small signal on der(1) in some of the cells (not shown) resulting in the revised ISCN karyotype: 46,XX,t(1;22)(q12;p11.1).ish t(1;22)(D1Z5+,D1Z1+,DZ22Z1?;D1Z1+,D1Z5-,D22Z1+).

Only the informative probes are included in the karyotype presented in this table. See Supplemental Note for further details regarding the FISH probes and results.

Karyotypes and Fluorescent In Situ Hybridization Results from Carriers where the Balanced Chromosomal Rearrangement Could Not be Mapped by Mate-Pair Sequencing

Table 2 Karyotypes and Fluorescent In Situ Hybridization Results from Carriers where the Balanced Chromosomal Rearrangement Could Not be Mapped by Mate-Pair Sequencing

Among the 32 group A carriers who were available for Affymetrix SNP 6.0 screening, we searched for deletions and duplications > 100 kb and found no pathogenic or likely pathogenic CNVs. In the potentially biased group B, we identified a pathogenic CNV in one of the seven carriers: a 2.8 Mb de novo deletion arr[GRCh37] 21q22.12q22.13(36028883_38854467)x1 encompassing 16 protein-coding genes including dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A [MIM: 600855 ]) in a t(1;2) translocation carrier ascertained because of prenatally detection of holoprosencephaly (P94).

The mean follow-up time of the clinical examination was 21.9 years (range 9–38 years, Table S5 ) resulting in confirmation and further extension of the diagnoses detected by the registry-based study, including description of potential dysmorphic facial features ( Table S4 and Supplemental Note ). Furthermore, the clinical examination detected three additional group A carriers (P59, P76, P87) and one additional group B carrier (P151) with NPD ( Table S4 ). These diagnoses were not found in the national registries due to later hospital admission or because the carriers were diagnosed without hospitalization (e.g., by a private psychiatrist). Combining both the registry and the clinical examination, we found 26.8% (11/41) with NPD and 7.3% (3/41) with milder congenital malformations among prenatally detected de novo BCR carriers with normal first trimester screening/ultrasound scan (group A). Among the potentially biased carriers (group B), 33.3% (3/9) had NPD with or without congenital malformations.

The registry-based epidemiology study was carried out for the 41 live-born BCR carriers with an unbiased ascertainment (group A) and they had a mean follow-up time of 17 years (range 3–34 years). We revealed disease diagnoses in 12 BCR carriers (29.2%) including 3 (7.3%) with minor congenital malformations and normal cognitive development ( Table S4 ). Only one of these malformations (congenital hip luxation) was apparent at birth. Eight carriers (19.5%) without congenital malformations were diagnosed with NPDs, including intellectual disability (ID), learning disabilities, attention and/or behavioral disorders, autism spectrum disorders (ASD), and mood disorders with a median age of onset of 7.5 years (range 2–17 years; Tables 1 and S4 ), emphasizing the importance of long-term follow-up data in such carriers. Furthermore, one carrier was hospitalized several times, most likely suffering from a dysfunction of the autonomic nervous system. Among the 205 control subjects, 8 (3.9%) had congenital malformations and normal cognitive development: 5 presented with isolated congenital heart malformations; 1 presented with cochlear aplasia, vaginal atresia, and a single kidney; 1 with urethral valve and congenital hip luxation; and 1 with congenital arthrogryposis multiplex with bilateral knee luxation. An additional 17 of the control subjects (8.3%) had NPDs, some with associated congenital malformations. The congenital malformations reported for the BCR carriers were milder and the frequency was not significantly higher compared to the control subjects (7.3% versus 3.9%, p = 0.40, Fisher’s exact test, two-sided), whereas the frequency of NPDs was significantly higher among BCR carriers than control subjects (19.5% versus 8.3%, p = 0.04, Fisher’s exact test, two-sided). Registry data were also retrieved for the group B carriers but without calculating risk estimates because this group was likely biased because of their ascertainment. Indeed, among the nine carriers, we found two carriers with brain malformations, both ascertained by abnormal prenatal ultrasound ( Table S4 ).

Analyst 1 had full knowledge about the phenotypes whereas analyst 2 performed the classification blinded. Abbreviations: ACC, agenesis of corpus callosum; ADD, attention deficit disorder; ASD, autism spectrum disorder; BCR, balanced chromosomal rearrangements; HI, haploinsufficiency index; ID, intellectual disability; NPD, neurodevelopmental and/or neuropsychiatric disorders; TAD, topological associated domain; VUS, variant of unknown significance; pLI, probability of loss-of-function intolerance. Low HI is used for scores < 10% and high pLI for scores > 0.9.

Defined as carriers with a NPD diagnosis having a BCR with the classification pathogenic or likely pathogenic by the blinded analyst or healthy carriers having a BCR with the classification benign or VUS by the blinded analyst.

a Defined as carriers with a NPD diagnosis having a BCR with the classification pathogenic or likely pathogenic by the blinded analyst or healthy carriers having a BCR with the classification benign or VUS by the blinded analyst.

Among the 191,977 prenatal samples in DCCR, a de novo BCR was detected in 117 pregnancies (0.06%), including 105 reciprocal translocations (∼1:2,000), 9 inversions (∼1:20,000), 2 complex rearrangements (∼1:95,000), and 1 insertional translocation (∼1:190,000). 65 samples (55.6%) were terminated electively, 2 samples (1.7%) were lost due to spontaneous abortion, and 50 samples (42.7%) defined a live-born BCR cohort of 48 carriers of an apparently balanced simple reciprocal translocation and 2 carriers of an apparently balanced inversion ( Table S2 ). We found no significant difference between the live-born and the terminated de novo BCR pregnancies with respect to ascertainment (advanced maternal age versus other indications, Table S3 ) or involvement of the X chromosome or a heterochromatic region ( Table S2 ). However, all pregnancies with a cytogenetically determined insertional translocation (1/1; 100%) or complex rearrangement (2/2; 100%) and most pregnancies with an inversion (7/9; 78%) were terminated ( Table S2 ).

Discussion

This study combined registry data with clinical and molecular re-examination of prenatally detected de novo BCR carriers to perform an unbiased evaluation of long-term morbidity risk. These analyses revealed a 2- to 3-fold increased risk of NPD compared to the matched control group and a limited prognostic value of CMA in the presence of a cytogenetically balanced rearrangement. The prognostic value of MPS was more promising in the context of BCRs. The most distinguishing factor was that blinded interpretation of MPS was able to accurately predict a benign outcome for all healthy carriers, while also predicting an untoward outcome due to disruption of known disease-associated genes/regulatory domains in almost half of the affected carriers.

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Kirk E.P. Long-term health and development of children diagnosed prenatally with a de novo apparently balanced chromosomal rearrangement. 1 Warburton D. De novo balanced chromosome rearrangements and extra marker chromosomes identified at prenatal diagnosis: clinical significance and distribution of breakpoints. We found a 20% risk of NPD among the prenatally detected unbiased de novo BCR carriers, which is higher than the previously reported risk of an untoward outcome (6%–9%).While striking, this morbidity estimate is also likely to be an underestimate since we encountered three additional unbiased carriers with NPD during the subsequent clinical studies, resulting in a total morbidity risk of 27%. The population cytogenetic study of Warburtondid not capture later-onset disorders like NPD because the majority of carriers were followed for only 2 years or less. In the study by Sinnerbrink et al.,the carriers were followed for a longer period (mean 6.7 years), but they lacked a matched control group and only 16 of the 58 live-born carriers could be included in the study. We encountered no carriers with severe congenital malformations in contrast to Warburton,but that study preceded the availability of CMA data and it is thus unknown whether a higher frequency of chromosomal imbalances in her cohort could explain the difference. Another explanation could be a potential difference in who were offered invasive prenatal testing due to differences in the national prenatal screening programs between the two studies, which might have resulted in fewer carriers with severe congenital malformations in our cohort.

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et al. The genomic landscape of balanced cytogenetic abnormalities associated with human congenital anomalies. 61 Moysés-Oliveira M.

Guilherme R.S.

Meloni V.A.

Di Battista A.

de Mello C.B.

Bragagnolo S.

Moretti-Ferreira D.

Kosyakova N.

Liehr T.

Carvalheira G.M.

et al. X-linked intellectual disability related genes disrupted by balanced X-autosome translocations. Am. J. Med. Genet. B Neuropsychiatr. The prognostic value of CMA in pregnancies with normal first trimester screening and ultrasound scan in the presence of a de novo BCR seems to be low as we found no carriers with a pathogenic deletion or duplication (>100 kb) in the unbiased group A. In the potentially biased group B, only one carrier had a pathogenic CNV, and this was associated with a severe brain malformation that was also detected by prenatal ultrasound. The lack of associated imbalances in the large majority of carriers with an NPD phenotype points toward a pathogenic effect of the breakpoints themselves. We were able to map 77% of the BCRs by MPS, which is a lower rate than a recent study of affected BCR carriers.The technique relies on the ability to sequence and align both ends of millions of individual DNA fragments, where the ends of the breakpoint spanning fragments are discordant with regard to distance (deletion, duplication) or orientation (inversions) or align to different chromosomes (translocation) in the annotated human genome. A significant fraction of the generated mate-pair reads is discarded because one or both ends cannot be realigned to the genome. Reasons for this could be that they involve repetitive sequences and/or are located in the remaining unannotated part (gaps) of the genome. Indeed, we confirmed by FISH that at least one breakpoint of each of seven unmappable BCRs were within chromosomal regions with repetitive sequences. All seven autosomal-autosomal unmappable BCRs were found in healthy carriers, potentially reflecting lower risk due to the location of breakpoint(s) within non-genic regions. In contrast, the only (female) carrier of a X-autosomal translocation (P87) had ID and dysmorphic features, which may be related to the X chromosome rearrangement and the increased morbidity risk of X chromosome breakpoints.It is also noteworthy that if the mapped BCRs were separated from the unmapped case subjects, which could be predicted with some certainty by karyotyping alone and confirmed by MPS/FISH, 40% of the remaining mappable BCR carriers had an NPD diagnosis.

9 Redin C.

Brand H.

Collins R.L.

Kammin T.

Mitchell E.

Hodge J.C.

Hanscom C.

Pillalamarri V.

Seabra C.M.

Abbott M.A.

et al. The genomic landscape of balanced cytogenetic abnormalities associated with human congenital anomalies. , 62 Chiang C.

Jacobsen J.C.

Ernst C.

Hanscom C.

Heilbut A.

Blumenthal I.

Mills R.E.

Kirby A.

Lindgren A.M.

Rudiger S.R.

et al. Complex reorganization and predominant non-homologous repair following chromosomal breakage in karyotypically balanced germline rearrangements and transgenic integration. 9 Redin C.

Brand H.

Collins R.L.

Kammin T.

Mitchell E.

Hodge J.C.

Hanscom C.

Pillalamarri V.

Seabra C.M.

Abbott M.A.

et al. The genomic landscape of balanced cytogenetic abnormalities associated with human congenital anomalies. 63 Collins R.L.

Brand H.

Redin C.E.

Hanscom C.

Antolik C.

Stone M.R.

Glessner J.T.

Mason T.

Pregno G.

Dorrani N.

et al. Defining the diverse spectrum of inversions, complex structural variation, and chromothripsis in the morbid human genome. MPS revealed short stretches of microhomology in half of the breakpoint junctions in this study both among healthy and affected carriers. Previous studies of larger cohorts of BCR carriers have found microhomology in 29% and 30.5%, respectively.By MPS we found complex rearrangements in seemingly simple BCRs in two balanced (7.4%) and one unbalanced (in total 10.7%) BCR, all in affected carriers, compatible with the notion that a complex rearrangement is a risk factor in itself. Support for this is also the much higher frequency of complex rearrangements (21%) found postnatally in affected BCR carriers.In our live-born cohort, no balanced insertional translocations were found but as shown by Collins et al.,this type of BCR might be associated with a higher risk of being complex and therefore are especially important to map when found prenatally.

9 Redin C.

Brand H.

Collins R.L.

Kammin T.

Mitchell E.

Hodge J.C.

Hanscom C.

Pillalamarri V.

Seabra C.M.

Abbott M.A.

et al. The genomic landscape of balanced cytogenetic abnormalities associated with human congenital anomalies. It is reassuring that, despite some differences, the evaluation and classification of the sequencing results from the 27 balanced mappable BCRs were largely concordant between two independent research groups. The estimated sensitivity of 45%–55% and specificity of 100% is encouraging, particularly given that one group was blinded to all phenotypic information. The interpretation used in the blinded analyses was also systematically applied to a cohort of 273 BCR carriers with known congenital anomalies and similarly found that the phenotypes could be directly attributed to the BCR breakpoints in less than 50% of probands based on current genome annotations,suggesting that the concordance reported here may be extensible to other cohorts. The high specificity in this small unbiased study is also reassuring as it shows that we rarely predict a negative clinical outcome in a healthy fetus.

11 Hearn T.

Renforth G.L.

Spalluto C.

Hanley N.A.

Piper K.

Brickwood S.

White C.

Connolly V.

Taylor J.F.N.

Russell-Eggitt I.

et al. Mutation of ALMS1, a large gene with a tandem repeat encoding 47 amino acids, causes Alström syndrome. 64 Santen G.W.E.

Aten E.

Sun Y.

Almomani R.

Gilissen C.

Nielsen M.

Kant S.G.

Snoeck I.N.

Peeters E.A.J.

Hilhorst-Hofstee Y.

et al. Mutations in SWI/SNF chromatin remodeling complex gene ARID1B cause Coffin-Siris syndrome. , 65 Tsurusaki Y.

Okamoto N.

Ohashi H.

Kosho T.

Imai Y.

Hibi-Ko Y.

Kaname T.

Naritomi K.

Kawame H.

Wakui K.

et al. Mutations affecting components of the SWI/SNF complex cause Coffin-Siris syndrome. 54 Pickard B.S.

Malloy M.P.

Porteous D.J.

Blackwood D.H.R.

Muir W.J. Disruption of a brain transcription factor, NPAS3, is associated with schizophrenia and learning disability. , 55 Kamnasaran D.

Muir W.J.

Ferguson-Smith M.A.

Cox D.W. Disruption of the neuronal PAS3 gene in a family affected with schizophrenia. 56 Visser R.

Gijsbers A.

Ruivenkamp C.

Karperien M.

Reeser H.M.

Breuning M.H.

Kant S.G.

Wit J.M. Genome-wide SNP array analysis in patients with features of sotos syndrome. 13 Halgren C.

Bache I.

Bak M.

Myatt M.W.

Anderson C.M.

Brøndum-Nielsen K.

Tommerup N. Haploinsufficiency of CELF4 at 18q12.2 is associated with developmental and behavioral disorders, seizures, eye manifestations, and obesity. The prediction was successful when a known or suggested disease-associated gene was disrupted, e.g., ARID1B was found to be disrupted in a carrier with agenesis of corpus callosum, ID, speech impairment, and ASD (P71). This individual was published online in 2011 together with seven unrelated individuals with various sized deletions encompassing ARID1B.14 The causal relation between mutations in ARID1B and NPD including Coffin-Siris syndrome (MIM: 135900 ) was confirmed shortly after.Similarly, NPAS3 was disrupted in a carrier with learning disabilities and attention disorder (P62), and this gene was originally linked to schizophrenia and IDby a t(9;14) translocation that disrupted the same intron as in P62. A third person with borderline IQ and need for special education has been reported with a deletion involving the first exon of NPAS3.In addition, a complex BCR with disruption of CELF4 was detected in a carrier with borderline IQ, developmental and behavioral disorders, myopia, obesity, and febrile seizures (P83).

12 Kleinjan D.A.

van Heyningen V. Long-range control of gene expression: emerging mechanisms and disruption in disease. 19 Zepeda-Mendoza C.J.

Ibn-Salem J.

Kammin T.

Harris D.J.

Rita D.

Gripp K.W.

MacKenzie J.J.

Gropman A.

Graham B.

Shaheen R.

et al. Computational prediction of position effects of apparently balanced human chromosomal rearrangements. Despite the accurate prediction of a pathogenic variant for the above carriers, it is notable that 45%–55% of the BCRs of affected carriers were not predicted to be pathogenic or likely pathogenic. Some of these carriers may suffer from NPD because of another etiology (as in the control group), or the BCR may represent a pathogenic variant resulting from a yet unknown mechanism such as disruption of an unannotated disease locus or a position effect. Indeed, LRPE might be involved due to disruption of regulatory landscapes near developmental genes.Computational prediction of LRPE has recently been attempted by integration of high-resolution three-dimensional chromosomal structural data with transcriptional regulatory information and available phenotype data, though such predictions remain challenging.Our study is illustrative of these challenges: of the two potential LRPEs described herein, the blinded and unblinded groups were in agreement regarding the classification of one carrier and discordant for another. For the concordant carrier, the blinded group mentioned the disruption of SLC8A1 with high pLI, while the unblinded group suggested LRPE of the MWS-associated gene ZEB2 compatible with the clinical features of the case subject (ID, constipation). In the discordant carrier, the unblinded group predicted an LRPE due to disruption of the evolutionary conserved HOXC-cluster in a carrier with NPD, suggesting that phenotypic knowledge is a critical factor for such predictions—a knowledge that often is lacking prenatally. Indeed, we found no difference in the rate of BCR disruption of TADs harboring a known disease-associated gene among affected and unaffected carriers, emphasizing the lack of prognostic value of blinded prediction of LRPEs.

45 Lek M.

Karczewski K.J.

Minikel E.V.

Samocha K.E.

Banks E.

Fennell T.

O’Donnell-Luria A.H.

Ware J.S.

Hill A.J.

Cummings B.B.

et al. Exome Aggregation Consortium

Analysis of protein-coding genetic variation in 60,706 humans. , 66 Ropers H.H.

Wienker T. Penetrance of pathogenic mutations in haploinsufficient genes for intellectual disability and related disorders. It is also noteworthy that in this study, 7 of the 16 healthy carriers harbored a BCR that disrupted a gene with a high pLI and/or low HI, features that are used among other risk parameters for prediction of deleterious mutations. These analyses further emphasize that, while many loci in the genome are depleted for the expected number of loss-of-function mutations, the annotation of a gene as evolutionarily constrained, or intolerant to such variation, does not preclude the presence of such mutations in the general population, as demonstrated in multiple studies.

5 Wapner R.J.

Martin C.L.

Levy B.

Ballif B.C.

Eng C.M.

Zachary J.M.

Savage M.

Platt L.D.

Saltzman D.

Grobman W.A.

et al. Chromosomal microarray versus karyotyping for prenatal diagnosis. 22 Ordulu Z.

Kammin T.

Brand H.

Pillalamarri V.

Redin C.E.

Collins R.L.

Blumenthal I.

Hanscom C.

Pereira S.

Bradley I.

et al. Structural chromosomal rearrangements require nucleotide-level resolution: lessons from next-generation sequencing in prenatal diagnosis. 63 Collins R.L.

Brand H.

Redin C.E.

Hanscom C.

Antolik C.

Stone M.R.

Glessner J.T.

Mason T.

Pregno G.

Dorrani N.

et al. Defining the diverse spectrum of inversions, complex structural variation, and chromothripsis in the morbid human genome. Figure 6 A Clinical Workflow for Pregnancies with a De Novo Balanced Chromosomal Rearrangement Show full caption Prognostic factors associated with high and low risk for neurodevelopmental and/or neuropsychiatric disorders (NPD) are listed. Abbreviations are as follows: HI, haploinsufficiency index; LRPE, long-range position effects; pLI, the probability of loss-of-function intolerance; TADs, topologically associated domains. Based on the combined registry, clinical, and molecular study of an unbiased nationwide cohort, we present a clinical workflow for pregnancies with a de novo BCR ( Figure 6 ). CMA has a high diagnostic yield in pregnancies with a structural anomaly detected with ultrasoundand should obviously remain a first-tier screen. However, when a de novo BCR is detected, most of the affected carriers had truly balanced rearrangements and CMA had no prognostic value. Thus, we recommend whole-genome sequencing as the first-tier test in the presence of a de novo BCR. Rapid advances in NGS technology suggest that it is possible to detect the breakpoints of known BCRs within a few days, illustrating that it can be used in a prenatal diagnostic setting.Moreover, recent studies have shown that appropriate computational analyses of MPS data can readily detect genome-wide imbalances at higher resolution than CMA.We show that we can classify breakpoints associated with high-risk and low-risk genes/domains in a substantial fraction of de novo BCR pregnancies, and MPS data would thus improve the information content for genetic counseling. However, our study also highlights that the interpretation of sequencing results originating from a prenatally detected de novo BCR is not only time consuming but can also be very challenging due to the lack of phenotypic data related to many genes and genomic domains and a general lack of knowledge about BCRs in phenotypically normal carriers. Nevertheless, we expect that the rapidly growing knowledge about the human genome and about NPD in particular will allow us to predict the clinical outcome of an increasing fraction of de novo BCRs in the near future.