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PURPOSE: ARF1 was previously implicated in periventricular nodular heterotopia (PVNH) in only five individuals and systematic clinical characterisation was not available. The aim of this study is to provide a comprehensive description of the phenotypic and genotypic spectrum of ARF1-related neurodevelopmental disorder. METHODS: We collected detailed phenotypes of an international cohort of individuals (n=17) with ARF1 variants assembled through the GeneMatcher platform. Missense variants were structurally modelled, and the impact of several were functionally validated. RESULTS: De novo variants (10 missense, 1 frameshift, 1 splice altering resulting in 9 residues insertion) in ARF1 were identified among 17 unrelated individuals. Detailed phenotypes included intellectual disability (ID), microcephaly, seizures and PVNH. No specific facial characteristics were consistent across all cases, however microretrognathia was common. Various hearing and visual defects were recurrent, and interestingly, some inflammatory features were reported. MRI of the brain frequently showed abnormalities consistent with a neuronal migration disorder. CONCLUSION: We confirm the role of ARF1 in an autosomal dominant syndrome with a phenotypic spectrum including severe ID, microcephaly, seizures and PVNH due to impaired neuronal migration.

Abstract Background We previously described the KINSSHIP syndrome, an autosomal dominant disorder associated with intellectual disability (ID), mesomelic dysplasia and horseshoe kidney, caused by de novo variants in the degron of AFF3. Mouse knock-ins and overexpression in zebrafish provided evidence for a dominant-negative mode of action, wherein an increased level of AFF3 resulted in pathological effects. Methods Evolutionary constraints suggest that other modes-of-inheritance could be at play. We challenged this hypothesis by screening ID cohorts for individuals with predicted-to-be damaging variants in AFF3. We used both animal and cellular models to assess the deleteriousness of the identified variants. Results We identified an individual with a KINSSHIP-like phenotype carrying a de novo partial duplication of AFF3 further strengthening the hypothesis that an increased level of AFF3 is pathological. We also detected seventeen individuals displaying a milder syndrome with either heterozygous Loss-of-Function (LoF) or biallelic missense variants in AFF3. Consistent with semi-dominance, we discovered three patients with homozygous LoF and one compound heterozygote for a LoF and a missense variant, who presented more severe phenotypes than their heterozygous parents. Matching zebrafish knockdowns exhibit neurological defects that could be rescued by expressing human AFF3 mRNA, confirming their association with the ablation of aff3. Conversely, some of the human AFF3 mRNAs carrying missense variants identified in affected individuals did not rescue these phenotypes. Overexpression of mutated AFF3 mRNAs in zebrafish embryos produced a significant increase of abnormal larvae compared to wild-type overexpression further demonstrating deleteriousness. To further assess the effect of AFF3 variation, we profiled the transcriptome of fibroblasts from affected individuals and engineered isogenic cells harboring + / + , KINSSHIP/KINSSHIP, LoF/ + , LoF/LoF or KINSSHIP/LoF AFF3 genotypes. The expression of more than a third of the AFF3 bound loci is modified in either the KINSSHIP/KINSSHIP or the LoF/LoF lines. While the same pathways are affected, only about one third of the differentially expressed genes are common to the homozygote datasets, indicating that AFF3 LoF and KINSSHIP variants largely modulate transcriptomes differently, e.g. the DNA repair pathway displayed opposite modulation. Conclusions Our results and the high pleiotropy shown by variation at this locus suggest that minute changes in AFF3 function are deleterious.

The endocannabinoid system is a highly conserved and ubiquitous signalling pathway with broad-ranging effects. Despite critical pathway functions, gene variants have not previously been conclusively linked to human disease. We identified nine children from eight families with heterozygous, de novo truncating variants in the last exon of DAGLA with a neuro-ocular phenotype characterized by developmental delay, ataxia and complex oculomotor abnormality. All children displayed paroxysms of nystagmus or eye deviation accompanied by compensatory head posture and worsened incoordination most frequently after waking. RNA sequencing showed clear expression of the truncated transcript and no differences were found between mutant and wild-type DAGLA activity. Immunofluorescence staining of patient-derived fibroblasts and HEK cells expressing the mutant protein showed distinct perinuclear aggregation not detected in control samples. This report establishes truncating variants in the last DAGLA exon as the cause of a unique paediatric syndrome. Because enzymatic activity was preserved, the observed mislocalization of the truncated protein may account for the observed phenotype. Potential mechanisms include DAGLA haploinsufficiency at the plasma membrane or dominant negative effect. To our knowledge, this is the first report directly linking an endocannabinoid system component with human genetic disease and sets the stage for potential future therapeutic avenues.

PurposeDiphthamide is a post-translationally modified histidine essential for messenger RNA translation and ribosomal protein synthesis. We present evidence for DPH5 as a novel cause of embryonic lethality and profound neurodevelopmental delays (NDDs).MethodsMolecular testing was performed using exome or genome sequencing. A targeted Dph5 knockin mouse (C57BL/6Ncrl-Dph5em1Mbp/Mmucd) was created for a DPH5 p.His260Arg homozygous variant identified in 1 family. Adenosine diphosphate-ribosylation assays in DPH5-knockout human and yeast cells and in silico modeling were performed for the identified DPH5 potential pathogenic variants.ResultsDPH5 variants p.His260Arg (homozygous), p.Asn110Ser and p.Arg207Ter (heterozygous), and p.Asn174LysfsTer10 (homozygous) were identified in 3 unrelated families with distinct overlapping craniofacial features, profound NDDs, multisystem abnormalities, and miscarriages. Dph5 p.His260Arg homozygous knockin was embryonically lethal with only 1 subviable mouse exhibiting impaired growth, craniofacial dysmorphology, and multisystem dysfunction recapitulating the human phenotype. Adenosine diphosphate-ribosylation assays showed absent to decreased function in DPH5-knockout human and yeast cells. In silico modeling of the variants showed altered DPH5 structure and disruption of its interaction with eEF2.ConclusionWe provide strong clinical, biochemical, and functional evidence for DPH5 as a novel cause of embryonic lethality or profound NDDs with multisystem involvement and expand diphthamide-deficiency syndromes and ribosomopathies.

Telangiectases and arteriovenous malformations (AVMs) are the characteristic lesions of Hereditary Hemorrhagic Telangiectasia (HHT). Somatic second-hit loss-of-function variations in the HHT causative genes, ENG and ACVRL1, have been described in dermal telangiectasias. It is unclear if somatic second-hit mutations also cause the formation of AVMs and nasal telangiectasias in HHT. To investigate the genetic mechanism of AVM formation in HHT, we evaluated multiple affected tissues from fourteen individuals. DNA was extracted from fresh/frozen tissue of 15 nasal telangiectasia, 4 dermal telangiectasia, and 9 normal control tissue biopsies, from nine unrelated individuals with HHT. DNA from six formalin-fixed paraffin-embedded (FFPE) AVM tissues (brain, lung, liver, and gallbladder) from five individuals was evaluated. A 736 vascular malformation and cancer gene next-generation sequencing (NGS) panel was used to evaluate these tissues down to 1% somatic mosaicism. Somatic second-hit mutations were identified in three in four AVM biopsies (75%) or half of the FFPE (50%) samples, including the loss of heterozygosity in ENG in one brain AVM sample, in which the germline mutation occurred in a different allele than a nearby somatic mutation (both are loss-of-function mutations). Eight of nine (88.9%) patients in whom telangiectasia tissues were evaluated had a somatic mutation ranging from 0.68 to 1.96% in the same gene with the germline mutation. Six of fifteen (40%) nasal and two of four (50%) dermal telangiectasia had a detectable somatic second hit. Additional low-level somatic mutations in other genes were identified in several telangiectasias. This is the first report that nasal telangiectasias and solid organ AVMs in HHT are caused by very-low-level somatic biallelic second-hit mutations.

The Genome Empowerment Scale (GEmS), developed as a research tool, assesses perspectives of parents of children with undiagnosed disorders about to undergo exome or genome sequencing related to the process of empowerment. We defined genomic healthcare empowerment as follows: perceived ability to understand and seek new information related to the genomic sequencing, manage emotions related to the diagnostic process and outcomes, and utilize genomic sequencing information to the betterment of the individual/child and family. The GEmS consists of four scales, two are primarily emotion-focused (Meaning of a Diagnosis, and Emotional Management of the Process) and two are action-oriented (Seeking Information and Support, and Implications and Planning). The purpose of this research was to provide a strategy for interpreting results from the GEmS and present illustrative cases. These illustrations should serve to facilitate use of the GEmS in the clinical and research arena, particularly with respect to guiding genetic counseling processes for parents of children with undiagnosed conditions.

Abstract Background Aortopathies are a group of disorders characterized by aneurysms, dilation, and tortuosity of the aorta. Because of the phenotypic overlap and genetic heterogeneity of diseases featuring aortopathy, molecular testing is often required for timely and correct diagnosis of affected individuals. In this setting next generation sequencing (NGS) offers several advantages over traditional molecular techniques. Methods The purpose of our study was to compare NGS enrichment methods for a clinical assay targeting the nine genes known to be associated with aortopathy. RainDance emulsion PCR and SureSelect RNA-bait hybridization capture enrichment methods were directly compared by enriching DNA from eight samples. Enriched samples were barcoded, pooled, and sequenced on the Illumina HiSeq2000 platform. Depth of coverage, consistency of coverage across samples, and the overlap of variants identified were assessed. This data was also compared to whole-exome sequencing data from ten individuals. Results Read depth was greater and less variable among samples that had been enriched using the RNA-bait hybridization capture enrichment method. In addition, samples enriched by hybridization capture had fewer exons with mean coverage less than 10, reducing the need for followup Sanger sequencing. Variants sets produced were 77% concordant, with both techniques yielding similar numbers of discordant variants. Conclusions When comparing the design flexibility, performance, and cost of the targeted enrichment methods to whole-exome sequencing, the RNA-bait hybridization capture enrichment gene panel offers the better solution for interrogating the aortopathy genes in a clinical laboratory setting.

Abstract Background Hereditary hemorrhagic telangiectasia (HHT) is a vascular disorder characterized by epistaxis, arteriovenous malformations, and telangiectases. The majority of the patients have a mutation in the coding region of the activin A receptor type II-like 1 (ACVRL1) or Endoglin (ENG) gene. However, in approximately 15% of cases, sequencing analysis and deletion/duplication testing fail to identify mutations in the coding regions of these genes. Knowing its vital role in transcription and translation control, we were prompted to investigate the 5'untranslated region (UTR) of ENG. Methods and Results We sequenced the 5'UTR of ENG for 154 HHT patients without mutations in ENG or ACVRL1 coding regions. We found a mutation (c.-127C > T), which is predicted to affect translation initiation and alter the reading frame of endoglin. This mutation was found in a family with linkage to the ENG, as well as in three other patients, one of which had an affected sibling with the same mutation. In vitro expression studies showed that a construct with the c.-127C > T mutation alters the translation and decreases the level of the endoglin protein. In addition, a c.-9G > A mutation was found in three patients, one of whom was homozygous for this mutation. Expression studies showed decreased protein levels suggesting that the c.-9G > A is a hypomorphic mutation. Conclusions Our results emphasize the need for the inclusion of the 5'UTR region of ENG in clinical testing for HHT.

Abstract Background Connective tissue diseases characterized by aortic aneurysm, such as Marfan syndrome, Loeys-Dietz syndrome and Ehlers Danlos syndrome type IV are heterogeneous and despite overlapping phenotypes, the natural history, clinical manifestations and interventional course for each diagnosis can be quite unique. The majority of mutations involved in the etiology of these disorders are missense and nonsense mutations. However, large deletions and duplications undetected by sequencing may be implicated in their pathogenesis, and may explain the apparent lack of genotype-phenotype correlation in a subset of patients. The objective of this study was to search for large pathogenic deletions and/or duplications in the FBN1, TGFβR1, and TGFβR2 genes using multiplex-ligation dependent probe amplification (MLPA) in patients with aortopathy, in whom no mutations in the FBN1, TGFβR1, and TGFβR2 genes were identified by sequencing. Methods The study included 14 patients from 11 unrelated families with aortic aneurysm. Of those, six patients (including 3 first-degree relatives), fulfilled the revised Ghent criteria for Marfan syndrome, and eight had predominantly aortic aneurysm/dilatation with variable skeletal and craniofacial involvement. MLPA for FBN1, TGFβR1, and TGFβR2 was carried out in all patients. A 385 K chromosome 15 specific array was used in two patients with a deletion of the entire FBN1 in order to define its size and boundaries. Results We identified two novel large deletions in the FBN1 gene in four patients of two unrelated families who met clinical diagnostic criteria for Marfan syndrome. One patient was found to have a FBN1 deletion encompassing exons 1-5. The other three patients had a 542 Kb deletion spanning the whole FBN1 gene and five additional genes (SLC24A5, MYEF2, CTXN2, SLC12A1, DUT) in the chromosome 15. Conclusions Our findings expand the number of large FBN1 deletions, and emphasize the importance of screening for large genomic deletions in connective tissue disorders featuring aortopathies, especially for those with classic Marfan phenotype.

Cadherins constitute a family of transmembrane proteins that mediate calcium-dependent cell-cell adhesion. The extracellular domain of cadherins consists of extracellular cadherin (EC) domains, separated by calcium binding sites. The EC interacts with other cadherin molecules in cis and in trans to mechanically hold apposing cell surfaces together. CDH2 encodes N-cadherin, whose essential roles in neural development include neuronal migration and axon pathfinding. However, CDH2 has not yet been linked to a Mendelian neurodevelopmental disorder. Here, we report de novo heterozygous pathogenic variants (seven missense, two frameshift) in CDH2 in nine individuals with a syndromic neurodevelopmental disorder characterized by global developmental delay and/or intellectual disability, variable axon pathfinding defects (corpus callosum agenesis or hypoplasia, mirror movements, Duane anomaly), and ocular, cardiac, and genital anomalies. All seven missense variants (c.1057G>A [p.Asp353Asn]; c.1789G>A [p.Asp597Asn]; c.1789G>T [p.Asp597Tyr]; c.1802A>C [p.Asn601Thr]; c.1839C>G [p.Cys613Trp]; c.1880A>G [p.Asp627Gly]; c.2027A>G [p.Tyr676Cys]) result in substitution of highly conserved residues, and six of seven cluster within EC domains 4 and 5. Four of the substitutions affect the calcium-binding site in the EC4-EC5 interdomain. We show that cells expressing these variants in the EC4-EC5 domains have a defect in cell-cell adhesion; this defect includes impaired binding in trans with N-cadherin-WT expressed on apposing cells. The two frameshift variants (c.2563_2564delCT [p.Leu855Valfs∗4]; c.2564_2567dupTGTT [p.Leu856Phefs∗5]) are predicted to lead to a truncated cytoplasmic domain. Our study demonstrates that de novo heterozygous variants in CDH2 impair the adhesive activity of N-cadherin, resulting in a multisystemic developmental disorder, that could be named ACOG syndrome (agenesis of corpus callosum, axon pathfinding, cardiac, ocular, and genital defects).

Abstract Background Despite numerous molecular and computational advances, roughly half of patients with a rare disease remain undiagnosed after exome or genome sequencing. A particularly challenging barrier to diagnosis is identifying variants that cause deleterious alternative splicing at intronic or exonic loci outside of canonical donor or acceptor splice sites. Results Several existing tools predict the likelihood that a genetic variant causes alternative splicing. We sought to extend such methods by developing a new metric that aids in discerning whether a genetic variant leads to deleterious alternative splicing. Our metric combines genetic variation in the Genome Aggregate Database with alternative splicing predictions from SpliceAI to compare observed and expected levels of splice-altering genetic variation. We infer genic regions with significantly less splice-altering variation than expected to be constrained. The resulting model of regional splicing constraint captures differential splicing constraint across gene and exon categories, and the most constrained genic regions are enriched for pathogenic splice-altering variants. Building from this model, we developed ConSpliceML. This ensemble machine learning approach combines regional splicing constraint with multiple per-nucleotide alternative splicing scores to guide the prediction of deleterious splicing variants in protein-coding genes. ConSpliceML more accurately distinguishes deleterious and benign splicing variants than state-of-the-art splicing prediction methods, especially in “cryptic” splicing regions beyond canonical donor or acceptor splice sites. Conclusion Integrating a model of genetic constraint with annotations from existing alternative splicing tools allows ConSpliceML to prioritize potentially deleterious splice-altering variants in studies of rare human diseases.

The transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) family of cytokines critically regulates vascular morphogenesis and homeostasis. Impairment of TGF-β or BMP signaling leads to heritable vascular disorders, including hereditary hemorrhagic telangiectasia (HHT). Drosha, a key enzyme for microRNA (miRNA) biogenesis, also regulates the TGF-β and BMP pathway through interaction with Smads and their joint control of gene expression through miRNAs. We report that mice lacking Drosha in the vascular endothelium developed a vascular phenotype resembling HHT that included dilated and disorganized vasculature, arteriovenous fistulae, and hemorrhages. Exome sequencing of HHT patients who lacked known pathogenic mutations revealed an overrepresentation of rare nonsynonymous variants of DROSHA Two of these DROSHA variants (P100L and R279L) did not interact with Smads and were partially catalytically active. In zebrafish, expression of these mutants or morpholino-directed knockdown of Drosha resulted in angiogenesis defects and abnormal vascular permeability. Together, our studies point to an essential role of Drosha in vascular development and the maintenance of vascular integrity, and reveal a previously unappreciated link between Drosha dysfunction and HHT.

The RASopathies are a group of disorders due to variations of genes associated with the Ras/MAPK pathway. Some of the RASopathies include neurofibromatosis type 1 (NF1), Noonan syndrome, Noonan syndrome with multiple lentigines, cardiofaciocutaneous (CFC) syndrome, Costello syndrome, Legius syndrome, and capillary malformation-arteriovenous malformation (CM-AVM) syndrome. In combination, the RASopathies are a frequent group of genetic disorders. This report summarizes the proceedings of the 4th International Symposium on Genetic Disorders of the Ras/MAPK pathway and highlights gaps in the field. © 2016 Wiley Periodicals, Inc.