Clinical impact of additional findings detected by genome-wide non-invasive prenatal testing: Follow-up results of the TRIDENT-2 study.

In the TRIDENT-2 study, all pregnant women in the Netherlands are offered genome-wide non-invasive prenatal testing (GW-NIPT) with a choice of receiving either full screening or screening solely for common trisomies. Previous data showed that GW-NIPT can reliably detect common trisomies in the general obstetric population and that this test can also detect other chromosomal abnormalities (additional findings). However, evidence regarding the clinical impact of screening for additional findings is lacking. Therefore, we present follow-up results of the TRIDENT-2 study to determine this clinical impact based on the laboratory and perinatal outcomes of cases with additional findings. Between April 2017 and April 2019, additional findings were detected in 402/110,739 pregnancies (0.36%). For 358 cases, the origin was proven to be either fetal (n = 79; 22.1%), (assumed) confined placental mosaicism (CPM) (n = 189; 52.8%), or maternal (n = 90; 25.1%). For the remaining 44 (10.9%), the origin of the aberration could not be determined. Most fetal chromosomal aberrations were pathogenic and associated with severe clinical phenotypes (61/79; 77.2%). For CPM cases, occurrence of pre-eclampsia (8.5% [16/189] vs 0.5% [754/159,924]; RR 18.5), and birth weight <2.3rd percentile (13.6% [24/177] vs 2.5% [3,892/155,491]; RR 5.5) were significantly increased compared to the general obstetric population. Of the 90 maternal findings, 12 (13.3%) were malignancies and 32 (35.6%) (mosaic) pathogenic copy number variants, mostly associated with mild or no clinical phenotypes. Data from this large cohort study provide crucial information for deciding if and how to implement GW-NIPT in screening programs. Additionally, these data can inform the challenging interpretation, counseling, and follow-up of additional findings.

Further delineation of the rare GDACCF (global developmental delay, absent or hypoplastic corpus callosum, dysmorphic facies syndrome): genotype and phenotype of 22 patients with ZNF148 mutations.

BACKGROUND: Pathogenic variants in the zinc finger protein coding genes are rare causes of intellectual disability and congenital malformations. Mutations in the ZNF148 gene causing GDACCF syndrome (global developmental delay, absent or hypoplastic corpus callosum, dysmorphic facies; MIM #617260) have been reported in five individuals so far. METHODS: As a result of an international collaboration using GeneMatcher Phenome Central Repository and personal communications, here we describe the clinical and molecular genetic characteristics of 22 previously unreported individuals. RESULTS: The core clinical phenotype is characterised by developmental delay particularly in the domain of speech development, postnatal growth retardation, microcephaly and facial dysmorphism. Corpus callosum abnormalities appear less frequently than suggested by previous observations. The identified mutations concerned nonsense or frameshift variants that were mainly located in the last exon of the ZNF148 gene. Heterozygous deletion including the entire ZNF148 gene was found in only one case. Most mutations occurred de novo, but were inherited from an affected parent in two families. CONCLUSION: The GDACCF syndrome is clinically diverse, and a genotype-first approach, that is, exome sequencing is recommended for establishing a genetic diagnosis rather than a phenotype-first approach. However, the syndrome may be suspected based on some recurrent, recognisable features. Corpus callosum anomalies were not as constant as previously suggested, we therefore recommend to replace the term 'GDACCF syndrome' with 'ZNF148-related neurodevelopmental disorder'.

Truncating SRCAP variants outside the Floating-Harbor syndrome locus cause a distinct neurodevelopmental disorder with a specific DNA methylation signature.

Truncating variants in exons 33 and 34 of the SNF2-related CREBBP activator protein (SRCAP) gene cause the neurodevelopmental disorder (NDD) Floating-Harbor syndrome (FLHS), characterized by short stature, speech delay, and facial dysmorphism. Here, we present a cohort of 33 individuals with clinical features distinct from FLHS and truncating (mostly de novo) SRCAP variants either proximal (n = 28) or distal (n = 5) to the FLHS locus. Detailed clinical characterization of the proximal SRCAP individuals identified shared characteristics: developmental delay with or without intellectual disability, behavioral and psychiatric problems, non-specific facial features, musculoskeletal issues, and hypotonia. Because FLHS is known to be associated with a unique set of DNA methylation (DNAm) changes in blood, a DNAm signature, we investigated whether there was a distinct signature associated with our affected individuals. A machine-learning model, based on the FLHS DNAm signature, negatively classified all our tested subjects. Comparing proximal variants with typically developing controls, we identified a DNAm signature distinct from the FLHS signature. Based on the DNAm and clinical data, we refer to the condition as "non-FLHS SRCAP-related NDD." All five distal variants classified negatively using the FLHS DNAm model while two classified positively using the proximal model. This suggests divergent pathogenicity of these variants, though clinically the distal group presented with NDD, similar to the proximal SRCAP group. In summary, for SRCAP, there is a clear relationship between variant location, DNAm profile, and clinical phenotype. These results highlight the power of combined epigenetic, molecular, and clinical studies to identify and characterize genotype-epigenotype-phenotype correlations.

Rapid exome sequencing as a first-tier test in neonates with suspected genetic disorder: results of a prospective multicenter clinical utility study in the Netherlands.

The introduction of rapid exome sequencing (rES) for critically ill neonates admitted to the neonatal intensive care unit has made it possible to impact clinical decision-making. Unbiased prospective studies to quantify the impact of rES over routine genetic testing are, however, scarce. We performed a clinical utility study to compare rES to conventional genetic diagnostic workup for critically ill neonates with suspected genetic disorders. In a multicenter prospective parallel cohort study involving five Dutch NICUs, we performed rES in parallel to routine genetic testing for 60 neonates with a suspected genetic disorder and monitored diagnostic yield and the time to diagnosis. To assess the economic impact of rES, healthcare resource use was collected for all neonates. rES detected more conclusive genetic diagnoses than routine genetic testing (20% vs. 10%, respectively), in a significantly shorter time to diagnosis (15 days (95% CI 10-20) vs. 59 days (95% CI 23-98, p < 0.001)). Moreover, rES reduced genetic diagnostic costs by 1.5% (€85 per neonate). CONCLUSION:  Our findings demonstrate the clinical utility of rES for critically ill neonates based on increased diagnostic yield, shorter time to diagnosis, and net healthcare savings. Our observations warrant the widespread implementation of rES as first-tier genetic test in critically ill neonates with disorders of suspected genetic origin. WHAT IS KNOWN: • Rapid exome sequencing (rES) enables diagnosing rare genetic disorders in a fast and reliable manner, but retrospective studies with neonates admitted to the neonatal intensive care unit (NICU) indicated that genetic disorders are likely underdiagnosed as rES is not routinely used. • Scenario modeling for implementation of rES for neonates with presumed genetic disorders indicated an expected increase in costs associated with genetic testing. WHAT IS NEW: • This unique prospective national clinical utility study of rES in a NICU setting shows that rES obtained more and faster diagnoses than conventional genetic tests. • Implementation of rES as replacement for all other genetic tests does not increase healthcare costs but in fact leads to a reduction in healthcare costs.

All-in-one whole exome sequencing strategy with simultaneous copy number variant, single nucleotide variant and absence-of-heterozygosity analysis in fetuses with structural ultrasound anomalies: A 1-year experience.

OBJECTIVE: We performed a 1-year evaluation of a novel strategy of simultaneously analyzing single nucleotide variants (SNVs), copy number variants (CNVs) and copy-number-neutral Absence-of-Heterozygosity from Whole Exome Sequencing (WES) data for prenatal diagnosis of fetuses with ultrasound (US) anomalies and a non-causative QF-PCR result. METHODS: After invasive diagnostics, whole exome parent-offspring trio-sequencing with exome-wide CNV analysis was performed in pregnancies with fetal US anomalies and a non-causative QF-PCR result (WES-CNV). On request, additional SNV-analysis, restricted to (the) requested gene panel(s) only (with the option of whole exome SNV-analysis afterward) was performed simultaneously (WES-CNV/SNV) or as rapid SNV-re-analysis, following a normal CNV analysis. RESULTS: In total, 415 prenatal samples were included. Following a non-causative QF-PCR result, WES-CNV analysis was initially requested for 74.3% of the chorionic villus (CV) samples and 45% of the amniotic fluid (AF) samples. In case WES-CNV analysis did not reveal a causative aberration, SNV-re-analysis was requested in 41.7% of the CV samples and 17.5% of the AF samples. All initial analyses could be finished within 2 weeks after sampling. For SNV-re-analysis during pregnancy, turn-around-times (TATs) varied between one and 8 days. CONCLUSION: We show a highly efficient all-in-one WES-based strategy, with short TATs, and the option of rapid SNV-re-analysis after a normal CNV result.

TRIDENT-2: National Implementation of Genome-wide Non-invasive Prenatal Testing as a First-Tier Screening Test in the Netherlands.

The Netherlands launched a nationwide implementation study on non-invasive prenatal testing (NIPT) as a first-tier test offered to all pregnant women. This started on April 1, 2017 as the TRIDENT-2 study, licensed by the Dutch Ministry of Health. In the first year, NIPT was performed in 73,239 pregnancies (42% of all pregnancies), 7,239 (4%) chose first-trimester combined testing, and 54% did not participate. The number of trisomies 21 (239, 0.33%), 18 (49, 0.07%), and 13 (55, 0.08%) found in this study is comparable to earlier studies, but the Positive Predictive Values (PPV)-96% for trisomy 21, 98% for trisomy 18, and 53% for trisomy 13-were higher than expected. Findings other than trisomy 21, 18, or 13 were reported on request of the pregnant women; 78% of women chose to have these reported. The number of additional findings was 207 (0.36%); these included other trisomies (101, 0.18%, PPV 6%, many of the remaining 94% of cases are likely confined placental mosaics and possibly clinically significant), structural chromosomal aberrations (95, 0.16%, PPV 32%,) and complex abnormal profiles indicative of maternal malignancies (11, 0.02%, PPV 64%). The implementation of genome-wide NIPT is under debate because the benefits of detecting other fetal chromosomal aberrations must be balanced against the risks of discordant positives, parental anxiety, and a potential increase in (invasive) diagnostic procedures. Our first-year data, including clinical data and laboratory follow-up data, will fuel this debate. Furthermore, we describe how NIPT can successfully be embedded into a national screening program with a single chain for prenatal care including counseling, testing, and follow-up.

The broader phenotypic spectrum of congenital caudal abnormalities associated with mutations in the caudal type homeobox 2 gene.

The caudal type homeobox 2 (CDX2) gene encodes a developmental regulator involved in caudal body patterning. Only three pathogenic variants in human CDX2 have been described, in patients with persistent cloaca, sirenomelia and/or renal and anogenital malformations. We identified five patients with de novo or inherited pathogenic variants in CDX2 with clinical phenotypes that partially overlap with previous cases, that is, imperforate anus and renal, urogenital and limb abnormalities. However, additional clinical features were seen including vertebral agenesis and we describe considerable phenotypic variability, even in unrelated patients with the same recurrent p.(Arg237His) variant. We propose CDX2 variants as rare genetic cause for a multiple congenital anomaly syndrome that can include features of caudal regression syndrome and VACTERL. A causative role is further substantiated by the relationship between CDX2 and other proteins encoded by genes that were previously linked to caudal abnormalities in humans, for example, TBXT (sacral agenesis and other vertebral segmentation defects) and CDX1 (anorectal malformations). Our findings confirm the essential role of CDX2 in caudal morphogenesis and formation of cloacal derivatives in humans, which to date has only been well characterized in animals.

Embryo tracking system for high-throughput sequencing-based preimplantation genetic testing.

STUDY QUESTION: Can the embryo tracking system (ETS) increase safety, efficacy and scalability of massively parallel sequencing-based preimplantation genetic testing (PGT)? SUMMARY ANSWER: Applying ETS-PGT, the chance of sample switching is decreased, while scalability and efficacy could easily be increased substantially. WHAT IS KNOWN ALREADY: Although state-of-the-art sequencing-based PGT methods made a paradigm shift in PGT, they still require labor intensive library preparation steps that makes PGT cost prohibitive and poses risks of human errors. To increase the quality assurance, efficiency, robustness and throughput of the sequencing-based assays, barcoded DNA fragments have been used in several aspects of next-generation sequencing (NGS) approach. STUDY DESIGN, SIZE, DURATION: We developed an ETS that substantially alleviates the complexity of the current sequencing-based PGT. With (n = 693) and without (n = 192) ETS, the downstream PGT procedure was performed on both bulk DNA samples (n = 563) and whole-genome amplified (WGAed) few-cell DNA samples (n = 322). Subsequently, we compared full genome haplotype landscapes of both WGAed and bulk DNA samples containing ETS or no ETS. PARTICIPANTS/MATERIALS, SETTING, METHODS: We have devised an ETS to track embryos right after whole-genome amplification (WGA) to full genome haplotype profiles. In this study, we recruited 322 WGAed DNA samples derived from IVF embryos as well as 563 bulk DNA isolated from peripheral blood of prospective parents. To determine possible interference of the ETS in the NGS-based PGT workflow, barcoded DNA fragments were added to DNA samples prior to library preparation and compared to samples without ETS. Coverages and variants were determined. MAIN RESULTS AND THE ROLE OF CHANCE: Current PGT protocols are quality sensitive and prone to sample switching. To avoid sample switching and increase throughput of PGT by sequencing-based haplotyping, six control steps should be carried out manually and checked by a second person in a clinical setting. Here, we developed an ETS approach in which one step only in the entire PGT procedure needs the four-eyes principal. We demonstrate that ETS not only precludes error-prone manual checks but also has no effect on the genomic landscape of preimplantation embryos. Importantly, our approach increases efficacy and throughput of the state-of-the-art PGT methods. LIMITATIONS, REASONS FOR CAUTION: Even though the ETS simplified sequencing-based PGT by avoiding potential errors in six steps in the protocol, if the initial assignment is not performed correctly, it could lead to cross-contamination. However, this can be detected in silico following downstream ETS analysis. Although we demonstrated an approach to evaluate purity of the ETS fragment, it is recommended to perform a pre-PGT quality control assay of the ETS amplicons with non-human DNA, such that the purity of each ETS molecule can be determined prior to ETS-PGT. WIDER IMPLICATIONS OF THE FINDINGS: The ETS-PGT approach notably increases efficacy and scalability of PGT. ETS-PGT has broad applicative value, as it can be tailored to any single- and few-cell sequencing approach where the starting specimen is scarce, as opposed to other methods that require a large number of cells as the input. Moreover, ETS-PGT could easily be adapted to any sequencing-based diagnostic method, including PGT for structural rearrangements and aneuploidies by low-pass sequencing as well as non-invasive prenatal testing. STUDY FUNDING/COMPETING INTEREST(S): M.Z.E. is supported by the EVA (Erfelijkheid Voortplanting & Aanleg) specialty program (grant no. KP111513) of Maastricht University Medical Centre (MUMC+), and the Horizon 2020 innovation (ERIN) (grant no. EU952516) of the European Commission. TRIAL REGISTRATION NUMBER: N/A.

Liquid biopsy: state of reproductive medicine and beyond.

Liquid biopsy is the process of sampling and analyzing body fluids, which enables non-invasive monitoring of complex biological systems in vivo. Liquid biopsy has myriad applications in health and disease as a wide variety of components, ranging from circulating cells to cell-free nucleic acid molecules, can be analyzed. Here, we review different components of liquid biopsy, survey state-of-the-art, non-invasive methods for detecting those components, demonstrate their clinical applications and discuss ethical considerations. Furthermore, we emphasize the importance of artificial intelligence in analyzing liquid biopsy data with the aim of developing ethically-responsible non-invasive technologies that can enhance individualized healthcare. While previous reviews have mainly focused on cancer, this review primarily highlights applications of liquid biopsy in reproductive medicine.

Mutations in RPSA and NKX2-3 link development of the spleen and intestinal vasculature.

Idiopathic intestinal varicosis is a developmental disorder defined by dilated and convoluted submucosal veins in the colon or small bowel. A limited number of families with idiopathic intestinal varices has been reported, but the genetic cause has not yet been identified. We performed whole-exome and targeted Sanger sequencing of candidate genes in five intestinal varicosis families. In four families, mutations in the RPSA gene were found, a gene previously linked to congenital asplenia. Individuals in these pedigrees had intestinal varicose veins and angiodysplasia, often in combination with asplenia. In a further four-generation pedigree that only showed intestinal varicosities, the RPSA gene was normal. Instead, a nonsense mutation in the homeobox gene NKX2-3 was detected which cosegregated with the disease in this large family with a LOD (logarithm of the odds) score of 3.3. NKX2-3 is a component of a molecular pathway underlying spleen and gut vasculature development in mice. Our results provide a molecular basis for familial idiopathic intestinal varices. We provide evidence for a relationship between the molecular pathways underlying the development of the spleen and intestinal mucosal vasculature that is conserved between humans and mice. We propose that clinical management of intestinal varices, should include assessment of a functional spleen.

Rapid whole exome sequencing in pregnancies to identify the underlying genetic cause in fetuses with congenital anomalies detected by ultrasound imaging.

OBJECTIVE: The purpose of this study was to explore the diagnostic yield and clinical utility of trio-based rapid whole exome sequencing (rWES) in pregnancies of fetuses with a wide range of congenital anomalies detected by ultrasound imaging. METHODS: In this observational study, we analyzed the first 54 cases referred to our laboratory for prenatal rWES to support clinical decision making, after the sonographic detection of fetal congenital anomalies. The most common identified congenital anomalies were skeletal dysplasia (n = 20), multiple major fetal congenital anomalies (n = 17) and intracerebral structural anomalies (n = 7). RESULTS: A conclusive diagnosis was identified in 18 of the 54 cases (33%). Pathogenic variants were detected most often in fetuses with skeletal dysplasia (n = 11) followed by fetuses with multiple major fetal congenital anomalies (n = 4) and intracerebral structural anomalies (n = 3). A survey, completed by the physicians for 37 of 54 cases, indicated that the rWES results impacted clinical decision making in 68% of cases. CONCLUSIONS: These results suggest that rWES improves prenatal diagnosis of fetuses with congenital anomalies, and has an important impact on prenatal and peripartum parental and clinical decision making.

Deleterious de novo variants of X-linked ZC4H2 in females cause a variable phenotype with neurogenic arthrogryposis multiplex congenita.

Pathogenic variants in the X-linked gene ZC4H2, which encodes a zinc-finger protein, cause an infrequently described syndromic form of arthrogryposis multiplex congenita (AMC) with central and peripheral nervous system involvement. We present genetic and detailed phenotypic information on 23 newly identified families and simplex cases that include 19 affected females from 18 families and 14 affected males from nine families. Of note, the 15 females with deleterious de novo ZC4H2 variants presented with phenotypes ranging from mild to severe, and their clinical features overlapped with those seen in affected males. By contrast, of the nine carrier females with inherited ZC4H2 missense variants that were deleterious in affected male relatives, four were symptomatic. We also compared clinical phenotypes with previously published cases of both sexes and provide an overview on 48 males and 57 females from 42 families. The spectrum of ZC4H2 defects comprises novel and recurrent mostly inherited missense variants in affected males, and de novo splicing, frameshift, nonsense, and partial ZC4H2 deletions in affected females. Pathogenicity of two newly identified missense variants was further supported by studies in zebrafish. We propose ZC4H2 as a good candidate for early genetic testing of males and females with a clinical suspicion of fetal hypo-/akinesia and/or (neurogenic) AMC.

Disruption of POGZ Is Associated with Intellectual Disability and Autism Spectrum Disorders.

Intellectual disability (ID) and autism spectrum disorders (ASD) are genetically heterogeneous, and a significant number of genes have been associated with both conditions. A few mutations in POGZ have been reported in recent exome studies; however, these studies do not provide detailed clinical information. We collected the clinical and molecular data of 25 individuals with disruptive mutations in POGZ by diagnostic whole-exome, whole-genome, or targeted sequencing of 5,223 individuals with neurodevelopmental disorders (ID primarily) or by targeted resequencing of this locus in 12,041 individuals with ASD and/or ID. The rarity of disruptive mutations among unaffected individuals (2/49,401) highlights the significance (p = 4.19 × 10(-13); odds ratio = 35.8) and penetrance (65.9%) of this genetic subtype with respect to ASD and ID. By studying the entire cohort, we defined common phenotypic features of POGZ individuals, including variable levels of developmental delay (DD) and more severe speech and language delay in comparison to the severity of motor delay and coordination issues. We also identified significant associations with vision problems, microcephaly, hyperactivity, a tendency to obesity, and feeding difficulties. Some features might be explained by the high expression of POGZ, particularly in the cerebellum and pituitary, early in fetal brain development. We conducted parallel studies in Drosophila by inducing conditional knockdown of the POGZ ortholog row, further confirming that dosage of POGZ, specifically in neurons, is essential for normal learning in a habituation paradigm. Combined, the data underscore the pathogenicity of loss-of-function mutations in POGZ and define a POGZ-related phenotype enriched in specific features.

Haploinsufficiency of CUX1 Causes Nonsyndromic Global Developmental Delay With Possible Catch-up Development.

OBJECTIVE: Developmental delay (DD) with favorable intellectual outcome and mild intellectual disability (ID) are mostly considered to be of complex genetic and environmental origin, but, in fact, often remain unclear. We aimed at proving our assumption that also mild cases of DD and ID may be of monogenic etiology. METHODS: We clinically evaluated 8 individuals and performed exome sequencing or array copy number analysis and identified variants in CUX1 as the likely cause. In addition, we included a case from the public database, DECIPHER. RESULTS: All 9 individuals harbored heterozygous null-allele variants in CUX1, encoding the Cut-homeobox 1 transcription factor that is involved in regulation of dendritogenesis and cortical synapse formation in layer II to IV cortical neurons. Six variants arose de novo, while in one family the variant segregated with ID. Of the 9 included individuals, 2 were diagnosed with moderate ID, 3 with mild ID, and 3 showed a normal age-related intelligence at ages 4, 6, and 8 years after a previous history of significant DD. INTERPRETATION: Our results suggest that null-allele variants, and thus haploinsufficiency of CUX1, cause an isolated phenotype of DD or ID with possible catch-up development. This illustrates that such a developmental course is not necessarily genetic complex, but may also be attributed to a monogenic cause. Ann Neurol 2018;84:200-207.

NBEA: Developmental disease gene with early generalized epilepsy phenotypes.

NBEA is a candidate gene for autism, and de novo variants have been reported in neurodevelopmental disease (NDD) cohorts. However, NBEA has not been rigorously evaluated as a disease gene, and associated phenotypes have not been delineated. We identified 24 de novo NBEA variants in patients with NDD, establishing NBEA as an NDD gene. Most patients had epilepsy with onset in the first few years of life, often characterized by generalized seizure types, including myoclonic and atonic seizures. Our data show a broader phenotypic spectrum than previously described, including a myoclonic-astatic epilepsy-like phenotype in a subset of patients. Ann Neurol 2018;84:796-803.

PURA syndrome: clinical delineation and genotype-phenotype study in 32 individuals with review of published literature.

BACKGROUND: De novo mutations in PURA have recently been described to cause PURA syndrome, a neurodevelopmental disorder characterised by severe intellectual disability (ID), epilepsy, feeding difficulties and neonatal hypotonia. OBJECTIVES: To delineate the clinical spectrum of PURA syndrome and study genotype-phenotype correlations. METHODS: Diagnostic or research-based exome or Sanger sequencing was performed in individuals with ID. We systematically collected clinical and mutation data on newly ascertained PURA syndrome individuals, evaluated data of previously reported individuals and performed a computational analysis of photographs. We classified mutations based on predicted effect using 3D in silico models of crystal structures of Drosophila-derived Pur-alpha homologues. Finally, we explored genotype-phenotype correlations by analysis of both recurrent mutations as well as mutation classes. RESULTS: We report mutations in PURA (purine-rich element binding protein A) in 32 individuals, the largest cohort described so far. Evaluation of clinical data, including 22 previously published cases, revealed that all have moderate to severe ID and neonatal-onset symptoms, including hypotonia (96%), respiratory problems (57%), feeding difficulties (77%), exaggerated startle response (44%), hypersomnolence (66%) and hypothermia (35%). Epilepsy (54%) and gastrointestinal (69%), ophthalmological (51%) and endocrine problems (42%) were observed frequently. Computational analysis of facial photographs showed subtle facial dysmorphism. No strong genotype-phenotype correlation was identified by subgrouping mutations into functional classes. CONCLUSION: We delineate the clinical spectrum of PURA syndrome with the identification of 32 additional individuals. The identification of one individual through targeted Sanger sequencing points towards the clinical recognisability of the syndrome. Genotype-phenotype analysis showed no significant correlation between mutation classes and disease severity.

De novo mutations in the SET nuclear proto-oncogene, encoding a component of the inhibitor of histone acetyltransferases (INHAT) complex in patients with nonsyndromic intellectual disability.

The role of disturbed chromatin remodeling in the pathogenesis of intellectual disability (ID) is well established and illustrated by de novo mutations found in a plethora of genes encoding for proteins of the epigenetic regulatory machinery. We describe mutations in the "SET nuclear proto-oncogene" (SET), encoding a component of the "inhibitor of histone acetyltransferases" (INHAT) complex, involved in transcriptional silencing. Using whole exome sequencing, four patients were identified with de novo mutations in the SET gene. Additionally, an affected mother and child were detected who carried a frameshift variant in SET. Four patients were found in literature. The de novo mutations in patients affected all four known SET mRNA transcripts. LoF mutations in SET are exceedingly rare in the normal population and, if present, affect only one transcript. The pivotal role of SET in neurogenesis is evident from in vitro and animal models. SET interacts with numerous proteins involved in histone modification, including proteins encoded by known autosomal dominant ID genes, that is, EP300, CREBBP, SETBP1, KMT2A, RAC1, and CTCF. Our study identifies SET as a new component of epigenetic regulatory modules underlying human cognitive disorders, and as a first member of the Nucleosome Assembly Protein (NAP) family implicated in ID.

Heterozygous variants in ACTL6A, encoding a component of the BAF complex, are associated with intellectual disability.

Pathogenic variants in genes encoding components of the BRG1-associated factor (BAF) chromatin remodeling complex have been associated with intellectual disability syndromes. We identified heterozygous, novel variants in ACTL6A, a gene encoding a component of the BAF complex, in three subjects with varying degrees of intellectual disability. Two subjects have missense variants affecting highly conserved amino acid residues within the actin-like domain. Missense mutations in the homologous region in yeast actin were previously reported to be dominant lethal and were associated with impaired binding of the human ACTL6A to ß-actin and BRG1. A third subject has a splicing variant that creates an in-frame deletion. Our findings suggest that the variants identified in our subjects may have a deleterious effect on the function of the protein by disturbing the integrity of the BAF complex. Thus, ACTL6A gene mutation analysis should be considered in patients with intellectual disability, learning disabilities, or developmental language disorder.

Detection of clinically relevant copy-number variants by exome sequencing in a large cohort of genetic disorders.

PURPOSE: Copy-number variation is a common source of genomic variation and an important genetic cause of disease. Microarray-based analysis of copy-number variants (CNVs) has become a first-tier diagnostic test for patients with neurodevelopmental disorders, with a diagnostic yield of 10-20%. However, for most other genetic disorders, the role of CNVs is less clear and most diagnostic genetic studies are generally limited to the study of single-nucleotide variants (SNVs) and other small variants. With the introduction of exome and genome sequencing, it is now possible to detect both SNVs and CNVs using an exome- or genome-wide approach with a single test. METHODS: We performed exome-based read-depth CNV screening on data from 2,603 patients affected by a range of genetic disorders for which exome sequencing was performed in a diagnostic setting. RESULTS: In total, 123 clinically relevant CNVs ranging in size from 727 bp to 15.3 Mb were detected, which resulted in 51 conclusive diagnoses and an overall increase in diagnostic yield of ~2% (ranging from 0 to -5.8% per disorder). CONCLUSIONS: This study shows that CNVs play an important role in a broad range of genetic disorders and that detection via exome-based CNV profiling results in an increase in the diagnostic yield without additional testing, bringing us closer to single-test genomics.Genet Med advance online publication 27 October 2016.