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Project-based learning, with its emphasis on 'learning by doing', is the dominant teaching method in industrial design. Learners are supposed to be motivated to tackle complex problems such as those in the dynamic field of sustainability. However, it is still unclear how the process of increasing motivation within projects can be systematically targeted for specific sustainability challenges and directed towards potential later pro-environmental behavior. The project-based learning method presented in this paper, framed by a normative decision-making model, aims to intrinsically motivate industrial design students to engage in the exemplary circular economy field of metal recycling and at the same time promote necessary professional competencies on the metal, alloy, product and system level. It is demonstrated which specific intervention measures can be suitable to achieve this goal and how they can be methodically employed. Preliminary quantitative evaluation results indicate that the project-based learning method can indeed strongly motivate the target group.•Connection to a normative decision-making model from social psychology for the domain of environmental protection as a conceptual framework•Consideration of intervention measures at various levels (metal, alloy, product, system)•Use of generative toolkits to translate the acquired knowledge into practical application during design tasks., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors. Published by Elsevier B.V.)

Patients with undiagnosed and/or rare disorders frequently manifest dysmorphic and neurological features. There is a lack of information on the effectiveness of telehealth in the evaluation of these disorders. We thus compared an unassisted virtual physical examination (PE) with an in-person PE in undiagnosed individuals and also assessed participant telehealth satisfaction. Twenty-six individuals enrolled in the Undiagnosed Diseases Network study underwent an in-home synchronous virtual PE, and a subsequent in-person PE, by the same clinician. The participants completed surveys on telehealth usability and provider empathy. On PE, general appearance and craniofacial features showed near perfect agreement (κ = 0.81-1.00) between the telehealth and in-person evaluations. Specific components of the neurological examination demonstrated substantial agreement (speech, gait, coordination; κ > 0.61), whereas others had moderate agreement (muscle tone, strength; κ = 0.41-0.60), and a few had none to slight agreement (skin; κ < 0.20). Some systems could not be examined in the virtual PE. Importantly, features relevant to diagnosis or management were missed on the virtual PE in only 2/26 individuals. The participants were satisfied with the quality of the telehealth interaction, as well as empathy demonstrated by providers in the virtual interface. Telehealth is effective for PEs in undiagnosed diseases and is acceptable to affected individuals., (© 2024 Wiley Periodicals LLC.)

Although next-generation sequencing has enabled diagnoses for many patients with Mendelian disorders, the majority remain undiagnosed. Here, we present a sibling pair who were clinically diagnosed with Escobar syndrome, however targeted gene testing was negative. Exome sequencing (ES), and later genome sequencing (GS), revealed compound heterozygous TTN variants in both siblings, a maternally inherited frameshift variant [(NM&#95;133378.4):c.36812del; p.(Asp12271Valfs*10)], and a paternally inherited missense variant [(NM&#95;133378.4):c.12322G > A; p.(Asp4108Asn)]. This result was considered nondiagnostic due to poor clinical fit and limited pathogenicity evidence for the missense variant of uncertain significance (VUS). Following initial nondiagnostic RNA sequencing (RNAseq) on muscle and further pursuit of other variants detected on the ES/GS, a reanalysis of noncanonical splice sites in the muscle transcriptome identified an out-of-frame exon retraction in TTN, near the known VUS. Interim literature included reports of patients with similar TTN variants who had phenotypic concordance with the siblings, and a diagnosis of a congenital titinopathy was given 4 years after the TTN variants had been initially reported. This report highlights the value of reanalysis of RNAseq with a different approach, expands the phenotypic spectrum of congenital titinopathy and also illustrates how a perceived phenotypic mismatch, and failure to consider known variants, can result in a prolongation of the diagnostic journey., (© 2024 Wiley Periodicals LLC.)

The maintenance of lipid asymmetry on the plasma membrane is regulated by flippases, such as ATP8A2, ATP11A, and ATP11C, which translocate phosphatidylserine and phosphatidylethanolamine from the outer leaflet to the inner leaflet. We previously identified a patient-derived point mutation (Q84E) in ATP11A at the phospholipid entry site, which acquired the ability to flip phosphatidylcholine (PtdCho). This mutation led to elevated levels of sphingomyelin (SM) in the outer leaflet of the plasma membrane. We herein present two de novo ATP11A dominant mutations (E114G and S399L) in heterozygous patients exhibiting neurological and developmental disorders. These mutations, situated near the predicted phospholipid exit site, similarly confer the ability for ATP11A to recognize PtdCho as a substrate, resulting in its internalization into cells. Cells expressing these mutants had increased SM levels on their surface, attributed to the up-regulated expression of the sphingomyelin synthase-1 gene, rendering them more susceptible to SM phosphodiesterase-mediated cell lysis. Corresponding mutations in ATP11C and ATP8A2, paralogs of ATP11A, exerted similar effects on PtdCho-flipping activity and increased SM levels on the cell surface. Molecular dynamics simulations, based on the ATP11C structure, suggest that the E114G and S399L mutations enhance ATP11C's affinity toward PtdCho. These findings underscore the importance of the well-conserved exit and entry sites in determining phospholipid substrate specificity and indicate that aberrant flipping of PtdCho contributes to neurological disorders., Competing Interests: Competing interests statement:The authors declare no competing interest.

Members of the leucine rich repeat (LRR) and PDZ domain (LAP) protein family are essential for animal development and histogenesis. Densin-180, encoded by LRRC7, is the only LAP protein selectively expressed in neurons. Densin-180 is a postsynaptic scaffold at glutamatergic synapses, linking cytoskeletal elements with signalling proteins such as the α-subunit of Ca 2+ /calmodulin-dependent protein kinase II. We have previously observed an association between high impact variants in LRRC7 and Intellectual Disability; also three individual cases with variants in LRRC7 had been described. We identify here 33 individuals (one of them previously described) with a dominant neurodevelopmental disorder due to heterozygous missense or loss-of-function variants in LRRC7. The clinical spectrum involves intellectual disability, autism, ADHD, aggression and, in several cases, hyperphagia-associated obesity. A PDZ domain variant interferes with synaptic targeting of Densin-180 in primary cultured neurons. Using in vitro systems (two hybrid, BioID, coimmunoprecipitation of tagged proteins from 293T cells) we identified new candidate interaction partners for the LRR domain, including protein phosphatase 1 (PP1), and observed that variants in the LRR reduced binding to these proteins. We conclude that LRRC7 encodes a major determinant of intellectual development and behaviour., (© 2024. The Author(s).)

Biallelic pathogenic variants in CDC45 are associated with Meier-Gorlin syndrome with craniosynostosis (MGORS type 7), which also includes short stature and absent/hypoplastic patellae. Identified variants act through a hypomorphic loss of function mechanism, to reduce CDC45 activity and impact DNA replication initiation. In addition to missense and premature termination variants, several pathogenic synonymous variants have been identified, most of which cause increased exon skipping of exon 4, which encodes an essential part of the RecJ-orthologue's DHH domain. Here we have identified a second cohort of families segregating CDC45 variants, where patients have craniosynostosis and a reduction in height, alongside common facial dysmorphisms, including thin eyebrows, consistent with MGORS7. Skipping of exon 15 is a consequence of two different variants, including a shared synonymous variant that is enriched in individuals of East Asian ancestry, while other variants in trans are predicted to alter key intramolecular interactions in α/β domain II, or cause retention of an intron within the 3'UTR. Our cohort and functional data confirm exon skipping is a relatively common pathogenic mechanism in CDC45, and highlights the need for alternative splicing events, such as exon skipping, to be especially considered for variants initially predicted to be less likely to cause the phenotype, particularly synonymous variants., (© 2024. The Author(s).)

Genomic medicine has been transformed by next-generation sequencing (NGS), inclusive of exome sequencing (ES) and genome sequencing (GS). Currently, ES is offered widely in clinical settings, with a less prevalent alternative model consisting of hybrid programs that incorporate research ES along with clinical patient workflows. We were among the earliest to implement a hybrid ES clinic, have provided diagnoses to 45% of probands, and have identified several novel candidate genes. Our program is enabled by a cost-effective investment by the health system and is unique in encompassing all the processes that have been variably included in other hybrid/clinical programs. These include careful patient selection, utilization of a phenotype-agnostic bioinformatics pipeline followed by manual curation of variants and phenotype integration by clinicians, close collaborations between the clinicians and the bioinformatician, pursuit of interesting variants, communication of results to patients in categories that are predicated upon the certainty of a diagnosis, and tracking changes in results over time and the underlying mechanisms for such changes. Due to its effectiveness, scalability to GS and its resource efficiency, specific elements of our paradigm can be incorporated into existing clinical settings, or the entire hybrid model can be implemented within health systems that have genomic medicine programs, to provide NGS in a scientifically rigorous, yet pragmatic setting., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Although genomic research offering next-generation sequencing (NGS) has increased the diagnoses of rare/ultra-rare disorders, populations experiencing health disparities infrequently participate in these studies. The factors underlying non-participation would most reliably be ascertained from individuals who have had the opportunity to participate, but decline. We thus enrolled parents of children and adult probands with undiagnosed disorders who had declined genomic research offering NGS with return of results with undiagnosed disorders (Decliners, n = 21) and compared their data to those who participated (Participants, n = 31). We assessed: (1) practical barriers and facilitators, (2) sociocultural factors-genomic knowledge and distrust, and (3) the value placed upon a diagnosis by those who declined participation. The primary findings were that residence in rural and medically underserved areas (MUA) and higher number of barriers were significantly associated with declining participation in the study. Exploratory analyses revealed multiple co-occurring practical barriers, greater emotional exhaustion and research hesitancy in the parents in the Decliner group compared to the Participants, with both groups identifying a similar number of facilitators. The parents in the Decliner group also had lower genomic knowledge, but distrust of clinical research was not different between the groups. Importantly, despite their non-participation, those in the Decliner group indicated an interest in obtaining a diagnosis and expressed confidence in being able to emotionally manage the ensuing results. Study findings support the concept that some families who decline participation in diagnostic genomic research may be experiencing pile-up with exhaustion of family resources - making participation in the genomic research difficult. This study highlights the complexity of the factors that underlie non-participation in clinically relevant NGS research. Thus, approaches to mitigating barriers to NGS research participation by populations experiencing health disparities need to be multi-pronged and tailored so that they can benefit from state-of -the art genomic technologies., (© 2023 National Society of Genetic Counselors.)

Background: A recurrent de novo variant (c.892C>T) in NACC1 causes a neurodevelopmental disorder with epilepsy, cataracts, feeding difficulties, and delayed brain myelination (NECFM). An unusual and consistently reported feature is episodic extreme irritability and inconsolability. We now characterize these episodes, their impact on the family, and ascertain treatments that may be effective. Parents of 14 affected individuals provided narratives describing the irritability episodes, including triggers, behavioral and physiological changes, and treatments. Simultaneously, parents of 15 children completed the Non-communicating Children's Pain Checklist-Revised (NCCPC-R), a measure to assess pain in non-verbal children., Results: The episodes of extreme irritability include a prodromal, peak, and resolving phase, with normal periods in between. The children were rated to have extreme pain-related behaviors on the NCCPC-R scale, although it is unknown whether the physiologic changes described by parents are caused by pain. Attempted treatments included various classes of medications, with psychotropic and sedative medications being most effective (7/15). Nearly all families (13/14) describe how the episodes have a profound impact on their lives., Conclusions: NECFM caused by the recurrent variant c.892C>T is associated with a universal feature of incapacitating episodic irritability of unclear etiology. Further understanding of the pathophysiology can lead to more effective therapeutic strategies., (© 2023. Institut National de la Santé et de la Recherche Médicale (INSERM).)

Purpose: Mendelian etiologies for acute encephalopathies in previously healthy children are poorly understood, with the exception of RAN binding protein 2 (RANBP2)-associated acute necrotizing encephalopathy subtype 1 (ANE1). We provide clinical, genetic, and neuroradiological evidence that biallelic variants in ribonuclease inhibitor (RNH1) confer susceptibility to a distinctive ANE subtype., Methods: This study aimed to evaluate clinical data, neuroradiological studies, genomic sequencing, and protein immunoblotting results in 8 children from 4 families who experienced acute febrile encephalopathy., Results: All 8 healthy children became acutely encephalopathic during a viral/febrile illness and received a variety of immune modulation treatments. Long-term outcomes varied from death to severe neurologic deficits to normal outcomes. The neuroradiological findings overlapped with ANE but had distinguishing features. All affected children had biallelic predicted damaging variants in RNH1: a subset that was studied had undetectable RNH1 protein. Incomplete penetrance of the RNH1 variants was evident in 1 family., Conclusion: Biallelic variants in RNH1 confer susceptibility to a subtype of ANE (ANE2) in previously healthy children. Intensive immunological treatments may alter outcomes. Genomic sequencing in children with unexplained acute febrile encephalopathy can detect underlying genetic etiologies, such as RNH1, and improve outcomes in the probands and at-risk siblings., Competing Interests: Conflict of Interest Rebecca Ganetzky is a paid consultant for Minovia Therapeutics and Nurture Genomics. Neal Sondheimer is employed by Synlogic, Inc. All other authors declare no conflicts of interest., (Copyright © 2023 American College of Medical Genetics and Genomics. Published by Elsevier Inc. All rights reserved.)

Congenital heart disease (CHD) affecting the conotruncal region of the heart, occurs in 40-50% of patients with 22q11.2 deletion syndrome (22q11.2DS). This syndrome is a rare disorder with relative genetic homogeneity that can facilitate identification of genetic modifiers. Haploinsufficiency of TBX1, encoding a T-box transcription factor, is one of the main genes responsible for the etiology of the syndrome. We suggest that genetic modifiers of conotruncal defects in patients with 22q11.2DS may be in the TBX1 gene network. To identify genetic modifiers, we analyzed rare, predicted damaging variants in whole genome sequence of 456 cases with conotruncal defects and 537 controls, with 22q11.2DS. We then performed gene set approaches and identified chromatin regulatory genes as modifiers. Chromatin genes with recurrent damaging variants include EP400, KAT6A, KMT2C, KMT2D, NSD1, CHD7 and PHF21A. In total, we identified 37 chromatin regulatory genes, that may increase risk for conotruncal heart defects in 8.5% of 22q11.2DS cases. Many of these genes were identified as risk factors for sporadic CHD in the general population. These genes are co-expressed in cardiac progenitor cells with TBX1, suggesting that they may be in the same genetic network. The genes KAT6A, KMT2C, CHD7 and EZH2, have been previously shown to genetically interact with TBX1 in mouse models. Our findings indicate that disturbance of chromatin regulatory genes impact the TBX1 gene network serving as genetic modifiers of 22q11.2DS and sporadic CHD, suggesting that there are some shared mechanisms involving the TBX1 gene network in the etiology of CHD., (© 2023. The Author(s).)

Purpose: Next-generation sequencing (NGS) has revolutionized the diagnostic process for rare/ultrarare conditions. However, diagnosis rates differ between analytical pipelines. In the National Institutes of Health-Undiagnosed Diseases Network (UDN) study, each individual's NGS data are concurrently analyzed by the UDN sequencing core laboratory and the clinical sites. We examined the outcomes of this practice., Methods: A retrospective review was performed at 2 UDN clinical sites to compare the variants and diagnoses/candidate genes identified with the dual analyses of the NGS data., Results: In total, 95 individuals had 100 diagnoses/candidate genes. There was 59% concordance between the UDN sequencing core laboratories and the clinical sites in identifying diagnoses/candidate genes. The core laboratory provided more diagnoses, whereas the clinical sites prioritized more research variants/candidate genes (P < .001). The clinical sites solely identified 15% of the diagnoses/candidate genes. The differences between the 2 pipelines were more often because of variant prioritization disparities than variant detection., Conclusion: The unique dual analysis of NGS data in the UDN synergistically enhances outcomes. The core laboratory provided a clinical analysis with more diagnoses and the clinical sites prioritized more research variants/candidate genes. Implementing such concurrent dual analyses in other genomic research studies and clinical settings can improve both variant detection and prioritization., Competing Interests: Conflict of Interest The authors declare no conflicts of interest., (Copyright © 2022 American College of Medical Genetics and Genomics. Published by Elsevier Inc. All rights reserved.)

Exome sequencing (ES) and genome sequencing (GS) have radically transformed the diagnostic approach to undiagnosed rare/ultrarare Mendelian diseases. Next-generation sequencing (NGS), the technology integral for ES, GS, and most large (100+) gene panels, has enabled previously unimaginable diagnoses, changes in medical management, new treatments, and accurate reproductive risk assessments for patients, as well as new disease gene discoveries. Yet, challenges remain, as most individuals remain undiagnosed with current NGS. Improved NGS technology has resulted in long-read sequencing, which may resolve diagnoses in some patients who do not obtain a diagnosis with current short-read ES and GS, but its effectiveness is unclear, and it is expensive. Other challenges that persist include the resolution of variants of uncertain significance, the urgent need for patients with ultrarare disorders to have access to therapeutics, the need for equity in patient access to NGS-based testing, and the study of ethical concerns. However, the outlook for undiagnosed disease resolution is bright, due to continual advancements in the field.

Advanced bioinformatics algorithms allow detection of multiple-exon copy-number variations (CNVs) from exome sequencing (ES) data, while detection of single-exon CNVs remains challenging. A retrospective review of Baylor Genetics' clinical ES patient cohort identified four individuals with homozygous single-exon deletions of TBCK (exon 23, NM&#95;001163435.2), a gene associated with an autosomal recessive neurodevelopmental phenotype. To evaluate the prevalence of this deletion and its contribution to disease, we retrospectively analyzed single nucleotide polymorphism (SNP) array data for 8194 individuals undergoing ES, followed by PCR confirmation and RT-PCR on individuals carrying homozygous or heterozygous exon 23 TBCK deletions. A fifth individual was diagnosed with the TBCK-related disorder due to a heterozygous exon 23 deletion in trans with a c.1860+1G>A (NM&#95;001163435.2) pathogenic variant, and three additional heterozygous carriers were identified. Affected individuals and carriers were from diverse ethnicities including European Caucasian, South Asian, Middle Eastern, Hispanic American and African American, with only one family reporting consanguinity. RT-PCR revealed two out-of-frame transcripts related to the exon 23 deletion. Our results highlight the importance of identifying single-exon deletions in clinical ES, especially for genes carrying recurrent deletions. For patients with early-onset hypotonia and psychomotor delay, this single-exon TBCK deletion might be under-recognized due to technical limitations of ES., (© 2022 Wiley Periodicals LLC.)

DROSHA encodes a ribonuclease that is a subunit of the Microprocessor complex and is involved in the first step of microRNA (miRNA) biogenesis. To date, DROSHA has not yet been associated with a Mendelian disease. Here, we describe two individuals with profound intellectual disability, epilepsy, white matter atrophy, microcephaly and dysmorphic features, who carry damaging de novo heterozygous variants in DROSHA. DROSHA is constrained for missense variants and moderately intolerant to loss-of-function (o/e = 0.24). The loss of the fruit fly ortholog drosha causes developmental arrest and death in third instar larvae, a severe reduction in brain size and loss of imaginal discs in the larva. Loss of drosha in eye clones causes small and rough eyes in adult flies. One of the identified DROSHA variants (p.Asp1219Gly) behaves as a strong loss-of-function allele in flies, while another variant (p.Arg1342Trp) is less damaging in our assays. In worms, a knock-in that mimics the p.Asp1219Gly variant at a worm equivalent residue causes loss of miRNA expression and heterochronicity, a phenotype characteristic of the loss of miRNA. Together, our data show that the DROSHA variants found in the individuals presented here are damaging based on functional studies in model organisms and likely underlie the severe phenotype involving the nervous system., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Nuclear deubiquitinase BAP1 (BRCA1-associated protein 1) is a core component of multiprotein complexes that promote transcription by reversing the ubiquitination of histone 2A (H2A). BAP1 is a tumor suppressor whose germline loss-of-function variants predispose to cancer. To our knowledge, there are very rare examples of different germline variants in the same gene causing either a neurodevelopmental disorder (NDD) or a tumor predisposition syndrome. Here, we report a series of 11 de novo germline heterozygous missense BAP1 variants associated with a rare syndromic NDD. Functional analysis showed that most of the variants cannot rescue the consequences of BAP1 inactivation, suggesting a loss-of-function mechanism. In T cells isolated from two affected children, H2A deubiquitination was impaired. In matching peripheral blood mononuclear cells, histone H3 K27 acetylation ChIP-seq indicated that these BAP1 variants induced genome-wide chromatin state alterations, with enrichment for regulatory regions surrounding genes of the ubiquitin-proteasome system (UPS). Altogether, these results define a clinical syndrome caused by rare germline missense BAP1 variants that alter chromatin remodeling through abnormal histone ubiquitination and lead to transcriptional dysregulation of developmental genes., Competing Interests: Declaration of interests The Department of Molecular and Human Genetics at Baylor College of Medicine receives revenue from clinical genetic testing completed at Baylor Genetics Laboratory. K.Mc., R.E.S., and I.M.W. are employees of GeneDx, Inc., (Copyright © 2021 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

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., (© 2021 National Society of Genetic Counselors.)

Schizophrenia occurs in about one in four individuals with 22q11.2 deletion syndrome (22q11.2DS). The aim of this International Brain and Behavior 22q11.2DS Consortium (IBBC) study was to identify genetic factors that contribute to schizophrenia, in addition to the ~20-fold increased risk conveyed by the 22q11.2 deletion. Using whole-genome sequencing data from 519 unrelated individuals with 22q11.2DS, we conducted genome-wide comparisons of common and rare variants between those with schizophrenia and those with no psychotic disorder at age ≥25 years. Available microarray data enabled direct comparison of polygenic risk for schizophrenia between 22q11.2DS and independent population samples with no 22q11.2 deletion, with and without schizophrenia (total n = 35,182). Polygenic risk for schizophrenia within 22q11.2DS was significantly greater for those with schizophrenia (p adj  = 6.73 × 10 -6 ). Novel reciprocal case-control comparisons between the 22q11.2DS and population-based cohorts showed that polygenic risk score was significantly greater in individuals with psychotic illness, regardless of the presence of the 22q11.2 deletion. Within the 22q11.2DS cohort, results of gene-set analyses showed some support for rare variants affecting synaptic genes. No common or rare variants within the 22q11.2 deletion region were significantly associated with schizophrenia. These findings suggest that in addition to the deletion conferring a greatly increased risk to schizophrenia, the risk is higher when the 22q11.2 deletion and common polygenic risk factors that contribute to schizophrenia in the general population are both present., (© 2020. The Author(s), under exclusive licence to Springer Nature Limited.)

Biallelic loss-of-function variants in the thrombospondin-type laminin G domain and epilepsy-associated repeats (TSPEAR) gene have recently been associated with ectodermal dysplasia and hearing loss. The first reports describing a TSPEAR disease association identified this gene is a cause of nonsyndromic hearing loss, but subsequent reports involving additional affected families have questioned this evidence and suggested a stronger association with ectodermal dysplasia. To clarify genotype-phenotype associations for TSPEAR variants, we characterized 13 individuals with biallelic TSPEAR variants. Individuals underwent either exome sequencing or panel-based genetic testing. Nearly all of these newly reported individuals (11/13) have phenotypes that include tooth agenesis or ectodermal dysplasia, while three newly reported individuals have hearing loss. Of the individuals displaying hearing loss, all have additional variants in other hearing-loss-associated genes, specifically TMPRSS3, GJB2, and GJB6, that present competing candidates for their hearing loss phenotype. When presented alongside previous reports, the overall evidence supports the association of TSPEAR variants with ectodermal dysplasia and tooth agenesis features but creates significant doubt as to whether TSPEAR variants are a monogenic cause of hearing loss. Further functional evidence is needed to evaluate this phenotypic association., (© 2021 The Authors. American Journal of Medical Genetics Part A published by Wiley Periodicals LLC.)

The Polycomb group (PcG) gene RNF2 (RING2) encodes a catalytic subunit of the Polycomb repressive complex 1 (PRC1), an evolutionarily conserved machinery that post-translationally modifies chromatin to maintain epigenetic transcriptional repressive states of target genes including Hox genes. Here, we describe two individuals, each with rare de novo missense variants in RNF2. Their phenotypes include intrauterine growth retardation, severe intellectual disabilities, behavioral problems, seizures, feeding difficulties and dysmorphic features. Population genomics data suggest that RNF2 is highly constrained for loss-of-function (LoF) and missense variants, and both p.R70H and p.S82R variants have not been reported to date. Structural analyses of the two alleles indicate that these changes likely impact the interaction between RNF2 and BMI1, another PRC1 subunit or its substrate Histone H2A, respectively. Finally, we provide functional data in Drosophila that these two missense variants behave as LoF alleles in vivo. The evidence provide support for deleterious alleles in RNF2 being associated with a new and recognizable genetic disorder. This tentative gene-disease association in addition to the 12 previously identified disorders caused by PcG genes attests to the importance of these chromatin regulators in Mendelian disorders., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Purpose: The NIH Undiagnosed Diseases Network (UDN) evaluates participants with disorders that have defied diagnosis, applying personalized clinical and genomic evaluations and innovative research. The clinical sites of the UDN are essential to advancing the UDN mission; this study assesses their contributions relative to standard clinical practices., Methods: We analyzed retrospective data from four UDN clinical sites, from July 2015 to September 2019, for diagnoses, new disease gene discoveries and the underlying investigative methods., Results: Of 791 evaluated individuals, 231 received 240 diagnoses and 17 new disease-gene associations were recognized. Straightforward diagnoses on UDN exome and genome sequencing occurred in 35% (84/240). We considered these tractable in standard clinical practice, although genome sequencing is not yet widely available clinically. The majority (156/240, 65%) required additional UDN-driven investigations, including 90 diagnoses that occurred after prior nondiagnostic exome sequencing and 45 diagnoses (19%) that were nongenetic. The UDN-driven investigations included complementary/supplementary phenotyping, innovative analyses of genomic variants, and collaborative science for functional assays and animal modeling., Conclusion: Investigations driven by the clinical sites identified diagnostic and research paradigms that surpass standard diagnostic processes. The new diagnoses, disease gene discoveries, and delineation of novel disorders represent a model for genomic medicine and science.

Trafficking protein particle (TRAPP) complexes, which include the TRAPPC4 protein, regulate membrane trafficking between lipid organelles in a process termed vesicular tethering. TRAPPC4 was recently implicated in a recessive neurodevelopmental condition in four unrelated families due to a shared c.454+3A>G splice variant. Here, we report 23 patients from 17 independent families with an early-infantile-onset neurodegenerative presentation, where we also identified the homozygous variant hg38:11:119020256 A>G (NM&#95;016146.5:c.454+3A>G) in TRAPPC4 through exome or genome sequencing. No other clinically relevant TRAPPC4 variants were identified among any of over 10,000 patients with neurodevelopmental conditions. We found the carrier frequency of TRAPPC4 c.454+3A>G was 2.4-5.4 per 10,000 healthy individuals. Affected individuals with the homozygous TRAPPC4 c.454+3A>G variant showed profound psychomotor delay, developmental regression, early-onset epilepsy, microcephaly and progressive spastic tetraplegia. Based upon RNA sequencing, the variant resulted in partial exon 3 skipping and generation of an aberrant transcript owing to use of a downstream cryptic splice donor site, predicting a premature stop codon and nonsense mediated decay. These data confirm the pathogenicity of the TRAPPC4 c.454+3A>G variant, and refine the clinical presentation of TRAPPC4-related encephalopathy.

Background: Resources within the Undiagnosed Diseases Network (UDN), such as genome sequencing (GS) and model organisms aid in diagnosis and identification of new disease genes, but are currently difficult to access by clinical providers. While these resources do contribute to diagnoses in many cases, they are not always necessary to reach diagnostic resolution. The UDN experience has been that participants can also receive diagnoses through the thoughtful and customized application of approaches and resources that are readily available in clinical settings., Methods: The UDN Genetic Counseling and Testing Working Group collected case vignettes that illustrated how clinically available methods resulted in diagnoses. The case vignettes were classified into three themes; phenotypic considerations, selection of genetic testing, and evaluating exome/GS variants and data., Results: We present 12 participants that illustrate how clinical practices such as phenotype-driven genomic investigations, consideration of variable expressivity, selecting the relevant tissue of interest for testing, utilizing updated testing platforms, and recognition of alternate transcript nomenclature resulted in diagnoses., Conclusion: These examples demonstrate that when a diagnosis is elusive, an iterative patient-specific approach utilizing assessment options available to clinical providers may solve a portion of cases. However, this does require increased provider time commitment, a particular challenge in the current practice of genomics., (© 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Purpose: Guidelines by professional organizations for assessing variant pathogenicity include the recommendation to utilize biologically relevant transcripts; however, there is variability in transcript selection by laboratories., Methods: We describe three patients whose genomic results were incorrect, because alternative transcripts and tissue expression patterns were not considered by the commercial laboratories., Results: In individual 1, a pathogenic coding variant in a brain-expressed isoform of CKDL5 was missed twice on sequencing, because the variant was intronic in the transcripts considered in analysis. In individual 2, a microdeletion affecting KMT2C was not reported on microarray, since deletions of proximal exons in this gene are seen in healthy individuals; however, this individual had a more distal deletion involving the brain-expressed KMT2C isoform, giving her a diagnosis of Kleefstra syndrome. Individual 3 was reported to have a pathogenic variant in exon 10 of OFD1 on exome, but had no typical features of the OFD1-related disorders. Since exon 10 is spliced from the more biologically relevant transcripts of OFD1, it was determined that he did not have an OFD1 disorder., Conclusion: These examples illustrate the importance of considering alternative transcripts as a potential confounder when genetic results are negative or discordant with the phenotype.

Background: The X-chromosome gene USP9X encodes a deubiquitylating enzyme that has been associated with neurodevelopmental disorders primarily in female subjects. USP9X escapes X inactivation, and in female subjects de novo heterozygous copy number loss or truncating mutations cause haploinsufficiency culminating in a recognizable syndrome with intellectual disability and signature brain and congenital abnormalities. In contrast, the involvement of USP9X in male neurodevelopmental disorders remains tentative., Methods: We used clinically recommended guidelines to collect and interrogate the pathogenicity of 44 USP9X variants associated with neurodevelopmental disorders in males. Functional studies in patient-derived cell lines and mice were used to determine mechanisms of pathology., Results: Twelve missense variants showed strong evidence of pathogenicity. We define a characteristic phenotype of the central nervous system (white matter disturbances, thin corpus callosum, and widened ventricles); global delay with significant alteration of speech, language, and behavior; hypotonia; joint hypermobility; visual system defects; and other common congenital and dysmorphic features. Comparison of in silico and phenotypical features align additional variants of unknown significance with likely pathogenicity. In support of partial loss-of-function mechanisms, using patient-derived cell lines, we show loss of only specific USP9X substrates that regulate neurodevelopmental signaling pathways and a united defect in transforming growth factor β signaling. In addition, we find correlates of the male phenotype in Usp9x brain-specific knockout mice, and further resolve loss of hippocampal-dependent learning and memory., Conclusions: Our data demonstrate the involvement of USP9X variants in a distinctive neurodevelopmental and behavioral syndrome in male subjects and identify plausible mechanisms of pathogenesis centered on disrupted transforming growth factor β signaling and hippocampal function., (Copyright © 2019 Society of Biological Psychiatry. All rights reserved.)

The 22q11.2 deletion syndrome (22q11.2DS) results from non-allelic homologous recombination between low-copy repeats termed LCR22. About 60%-70% of individuals with the typical 3 megabase (Mb) deletion from LCR22A-D have congenital heart disease, mostly of the conotruncal type (CTD), whereas others have normal cardiac anatomy. In this study, we tested whether variants in the hemizygous LCR22A-D region are associated with risk for CTDs on the basis of the sequence of the 22q11.2 region from 1,053 22q11.2DS individuals. We found a significant association (FDR p < 0.05) of the CTD subset with 62 common variants in a single linkage disequilibrium (LD) block in a 350 kb interval harboring CRKL. A total of 45 of the 62 variants were associated with increased risk for CTDs (odds ratio [OR) ranges: 1.64-4.75). Associations of four variants were replicated in a meta-analysis of three genome-wide association studies of CTDs in affected individuals without 22q11.2DS. One of the replicated variants, rs178252, is located in an open chromatin region and resides in the double-elite enhancer, GH22J020947, that is predicted to regulate CRKL (CRK-like proto-oncogene, cytoplasmic adaptor) expression. Approximately 23% of patients with nested LCR22C-D deletions have CTDs, and inactivation of Crkl in mice causes CTDs, thus implicating this gene as a modifier. Rs178252 and rs6004160 are expression quantitative trait loci (eQTLs) of CRKL. Furthermore, set-based tests identified an enhancer that is predicted to target CRKL and is significantly associated with CTD risk (GH22J020946, sequence kernal association test (SKAT) p = 7.21 × 10 -5 ) in the 22q11.2DS cohort. These findings suggest that variance in CTD penetrance in the 22q11.2DS population can be explained in part by variants affecting CRKL expression., (Copyright © 2019 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

While genomic sequencing (ES/GS) has the potential to diagnose children with difficult to diagnose phenotypes, the goal should be not only a diagnosis, but also to empower parents to seek next steps for their children and to emotionally manage the outcome, whether or not a diagnosis is secured. To help achieve this goal, objective measures are needed to assess the process of parental empowerment related to genome sequencing. We present the validity and reliability of the Genome Empowerment Scale (GEmS), developed using a healthcare empowerment theoretical model. To evaluate its psychometric properties, 158 parents of 117 children with an undiagnosed condition undergoing genomic sequencing completed the GEmS, measures for criterion validity and for depression and anxiety. Factor analysis resulted in a four factor solution: (a) meaning of a diagnosis; (b) emotional management of the process; (c) seeking information and support and (d) implications and planning. Reliability and validity analyses show that the GEmS has good psychometric properties. The inter-relationships among the factors revealed a profile that may identify parents at risk for a poorer outcome who may benefit from targeted genetic counseling. The GEmS, an objective measure of parental genomic empowerment, can be utilized for future research and translational applications., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Despite growing evidence of diagnostic yield and clinical utility of whole exome sequencing (WES) in patients with undiagnosed diseases, there remain significant cost and reimbursement barriers limiting access to such testing. The diagnostic yield and resulting clinical actions of WES for patients who previously faced insurance coverage barriers have not yet been explored., Methods: We performed a retrospective descriptive analysis of clinical WES outcomes for patients facing insurance coverage barriers prior to clinical WES and who subsequently enrolled in the Undiagnosed Diseases Network (UDN). Clinical WES was completed as a result of participation in the UDN. Payer type, molecular diagnostic yield, and resulting clinical actions were evaluated., Results: Sixty-six patients in the UDN faced insurance coverage barriers to WES at the time of enrollment (67% public payer, 26% private payer). Forty-two of 66 (64%) received insurance denial for clinician-ordered WES, 19/66 (29%) had health insurance through a payer known not to cover WES, and 5/66 (8%) had previous payer denial of other genetic tests. Clinical WES results yielded a molecular diagnosis in 23 of 66 patients (35% [78% pediatric, 65% neurologic indication]). Molecular diagnosis resulted in clinical actions in 14 of 23 patients (61%)., Conclusions: These data demonstrate that a substantial proportion of patients who encountered insurance coverage barriers to WES had a clinically actionable molecular diagnosis, supporting the notion that WES has value as a covered benefit for patients who remain undiagnosed despite objective clinical findings., (© 2019 National Society of Genetic Counselors.)

Encoding the slow skeletal muscle isoform of myosin binding protein-C, MYBPC1 is associated with autosomal dominant and recessive forms of arthrogryposis. The authors describe a novel association for MYBPC1 in four patients from three independent families with skeletal muscle weakness, myogenic tremors, and hypotonia with gradual clinical improvement. The patients carried one of two de novo heterozygous variants in MYBPC1, with the p.Leu263Arg variant seen in three individuals and the p.Leu259Pro variant in one individual. Both variants are absent from controls, well conserved across vertebrate species, predicted to be damaging, and located in the M-motif. Protein modeling studies suggested that the p.Leu263Arg variant affects the stability of the M-motif, whereas the p.Leu259Pro variant alters its structure. In vitro biochemical and kinetic studies demonstrated that the p.Leu263Arg variant results in decreased binding of the M-motif to myosin, which likely impairs the formation of actomyosin cross-bridges during muscle contraction. Collectively, our data substantiate that damaging variants in MYBPC1 are associated with a new form of an early-onset myopathy with tremor, which is a defining and consistent characteristic in all affected individuals, with no contractures. Recognition of this expanded myopathic phenotype can enable identification of individuals with MYBPC1 variants without arthrogryposis., (© 2019 Wiley Periodicals, Inc.)

Purpose: Diagnosing monogenic diseases facilitates optimal care, but can involve the manual evaluation of hundreds of genetic variants per case. Computational tools like Phrank expedite this process by ranking all candidate genes by their ability to explain the patient's phenotypes. To use these tools, busy clinicians must manually encode patient phenotypes from lengthy clinical notes. With 100 million human genomes estimated to be sequenced by 2025, a fast alternative to manual phenotype extraction from clinical notes will become necessary., Methods: We introduce ClinPhen, a fast, high-accuracy tool that automatically converts clinical notes into a prioritized list of patient phenotypes using Human Phenotype Ontology (HPO) terms., Results: ClinPhen shows superior accuracy and 20× speedup over existing phenotype extractors, and its novel phenotype prioritization scheme improves the performance of gene-ranking tools., Conclusion: While a dedicated clinician can process 200 patient records in a 40-hour workweek, ClinPhen does the same in 10 minutes. Compared with manual phenotype extraction, ClinPhen saves an additional 3-5 hours per Mendelian disease diagnosis. Providers can now add ClinPhen's output to each summary note attached to a filled testing laboratory request form. ClinPhen makes a substantial contribution to improvements in efficiency critically needed to meet the surging demand for clinical diagnostic sequencing.

The "diagnostic odyssey" is well known and described in genetic counseling literature. Studies addressing the psychological, emotional, and financial costs of not having a diagnosis have shown how it permeates the lives of patients and families. The Undiagnosed Diseases Network aims to end this odyssey by providing diagnoses to individuals with undiagnosed conditions through multidisciplinary evaluations, whole exome and genome sequencing, and basic science research. It also provides an opportunity to learn from patients and families and to better understand their journeys and the impact of receiving a diagnosis. Seven cases are presented that outline challenges that come from working with chronically undiagnosed and newly diagnosed patients in a time when sequencing for clinical diagnosis is rapidly increasing. They illuminate the emotional journey of patients and families searching for a diagnosis and the mental health problems, financial distress, and chaos that can accompany not having answers. They also illustrate the surprising reactions patients and families can have to receiving a diagnosis, including anger, grief, and disappointment. While the lessons learned from these families are not novel, new strategies are presented for handling these challenges in undiagnosed and ultra-rare populations, groups that will increase with the rise of clinical sequencing., (© 2019 National Society of Genetic Counselors.)

Purpose: Sixty to seventy-five percent of individuals with rare and undiagnosed phenotypes remain undiagnosed after exome sequencing (ES). With standard ES reanalysis resolving 10-15% of the ES negatives, further approaches are necessary to maximize diagnoses in these individuals., Methods: In 38 ES negative patients an individualized genomic-phenotypic approach was employed utilizing (1) phenotyping; (2) reanalyses of FASTQ files, with innovative bioinformatics; (3) targeted molecular testing; (4) genome sequencing (GS); and (5) conferring of clinical diagnoses when pathognomonic clinical findings occurred., Results: Certain and highly likely diagnoses were made in 18/38 (47%) individuals, including identifying two new developmental disorders. The majority of diagnoses (>70%) were due to our bioinformatics, phenotyping, and targeted testing identifying variants that were undetected or not prioritized on prior ES. GS diagnosed 3/18 individuals with structural variants not amenable to ES. Additionally, tentative diagnoses were made in 3 (8%), and in 5 individuals (13%) candidate genes were identified. Overall, diagnoses/potential leads were identified in 26/38 (68%)., Conclusions: Our comprehensive approach to ES negatives maximizes the ES and clinical data for both diagnoses and candidate gene identification, without GS in the majority. This iterative approach is cost-effective and is pertinent to the current conundrum of ES negatives.

A set of glutamylases and deglutamylases controls levels of tubulin polyglutamylation, a prominent post-translational modification of neuronal microtubules. Defective tubulin polyglutamylation was first linked to neurodegeneration in the Purkinje cell degeneration ( pcd ) mouse, which lacks deglutamylase CCP1, displays massive cerebellar atrophy, and accumulates abnormally glutamylated tubulin in degenerating neurons. We found biallelic rare and damaging variants in the gene encoding CCP1 in 13 individuals with infantile-onset neurodegeneration and confirmed the absence of functional CCP1 along with dysregulated tubulin polyglutamylation. The human disease mainly affected the cerebellum, spinal motor neurons, and peripheral nerves. We also demonstrate previously unrecognized peripheral nerve and spinal motor neuron degeneration in pcd mice, which thus recapitulated key features of the human disease. Our findings link human neurodegeneration to tubulin polyglutamylation, entailing this post-translational modification as a potential target for drug development for neurodegenerative disorders., (© 2018 The Authors.)

The burden of living with an undiagnosed condition is high and includes physical and emotional suffering, frustrations, and uncertainty. For patients and families experiencing these stressors, higher levels of empowerment may be associated with better outcomes. Thus, it is important to understand the experiences of patients with undiagnosed conditions and their families affected by undiagnosed conditions in order to identify strategies for fostering empowerment. In this study, we used the Genetic Counseling Outcome Scale (GCOS-24) to assess levels of empowerment and support group participation in 35 adult participants and 67 parents of child participants in the Undiagnosed Diseases Network (UDN) prior to their UDN in-person evaluation. Our results revealed significantly lower empowerment scores on the GCOS-24 in adult participants compared to parents of child participants [t(100) = - 3.01, p = 0.003, average difference = - 11.12, 95% CI (- 3.78, - 18.46)] and no significant association between support group participation and empowerment scores. The majority of participants (84.3%, 86/102) are not currently participating in any support groups, and participation rates were not significantly different for adult participants and parents of child participants (11.4 vs. 19.7%, respectively, FE p = 0.40). Open-ended responses provided additional insight into support group participation, the challenges of living with undiagnosed conditions, and positive coping strategies. Future research will evaluate the extent to which empowerment scores change as participation in the UDN unfolds.

Background: The majority of undiagnosed diseases manifest with objective findings that warrant further investigation. The Undiagnosed Diseases Network (UDN) receives applications from patients whose symptoms and signs have been intractable to diagnosis; however, many UDN applicants are affected primarily by subjective symptoms such as pain and fatigue. We sought to characterize presenting symptoms, referral sources, and demographic factors of applicants to the UDN to identify factors that may determine application outcome and potentially differentiate between those with undiagnosed diseases (with more objective findings) and those who are less likely to have an undiagnosed disease (more subjective symptoms)., Methods: We used a systematic retrospective review of 151 consecutive Not Accepted and 50 randomly selected Accepted UDN applications. The primary outcome was whether an applicant was Accepted, or Not Accepted, and, if accepted, whether or not a diagnosis was made. Objective and subjective symptoms and information on prior specialty consultations were collected from provider referral letters. Demographic data and decision data on network acceptance were gathered from the UDN online portal., Results: Fewer objective findings and more subjective symptoms were found in the Not Accepted applications. Not Accepted referrals also were from older individuals, reported a shorter period of illness, and were referred to the UDN by their primary care physicians. All of these differences reached statistical significance in comparison with Accepted applications. The frequency of subspecialty consults for diagnostic purposes prior to UDN application was similar in both groups., Conclusions: The preponderance of subjective and lack of objective findings in the Not Accepted applications distinguish these from applicants that are accepted for evaluation and diagnostic efforts through the UDN. Not Accepted applicants are referred primarily by their primary care providers after multiple specialist consultations fail to yield answers. Distinguishing between patients with undiagnosed diseases with objective findings and those with primarily subjective findings can delineate patients who would benefit from further diagnostic processes from those who may have functional disorders and need alternative pathways for management of their symptoms., Trial Registration: clinicaltrials.gov NCT02450851 , posted May 21st 2015.

Little is known about the psychosocial profiles of parents who have a child with an undiagnosed chronic illness. The National Institutes of Health Undiagnosed Diseases Network (UDN) evaluates individuals with intractable medical findings, with the objective of discovering the underlying diagnosis. We report on the psychosocial profiles of 50 parents whose children were accepted to one of the network's clinical sites. Parents completed questionnaires assessing anxiety, depression, coping self-efficacy, and health care empowerment at the beginning of their child's UDN clinical evaluation. Parents of undiagnosed children had high rates of anxiety and depression (~ 40%), which were significantly inversely correlated with coping self-efficacy, but not with health care empowerment. Coping self-efficacy, depressive, and anxiety symptoms were better in parents with older children and with longer duration of illness. Gender differences were identified, with mothers reporting greater health care engagement than fathers. Overall, our findings suggest that parents of children with undiagnosed diseases maintain positive coping self-efficacy and remain actively engaged in health care and to a lesser degree tolerance for uncertainty, but these come with a high emotional cost to the parents. As the parents' psychological needs may not be obvious, these should be ascertained and the requisite support provided.

The 17 genes of the T-box family are transcriptional regulators that are involved in all stages of embryonic development, including craniofacial, brain, heart, skeleton and immune system. Malformation syndromes have been linked to many of the T-box genes. For example, haploinsufficiency of TBX1 is responsible for many structural malformations in DiGeorge syndrome caused by a chromosome 22q11.2 deletion. We report four individuals with an overlapping spectrum of craniofacial dysmorphisms, cardiac anomalies, skeletal malformations, immune deficiency, endocrine abnormalities and developmental impairments, reminiscent of DiGeorge syndrome, who are heterozygotes for TBX2 variants. The p.R20Q variant is shared by three affected family members in an autosomal dominant manner; the fourth unrelated individual has a de novo p.R305H mutation. Bioinformatics analyses indicate that these variants are rare and predict them to be damaging. In vitro transcriptional assays in cultured cells show that both variants result in reduced transcriptional repressor activity of TBX2. We also show that the variants result in reduced protein levels of TBX2. Heterologous over-expression studies in Drosophila demonstrate that both p.R20Q and p.R305H function as partial loss-of-function alleles. Hence, these and other data suggest that TBX2 is a novel candidate gene for a new multisystem malformation disorder.

PurposeTo describe examples of missed pathogenic variants on whole-exome sequencing (WES) and the importance of deep phenotyping for further diagnostic testing.MethodsGuided by phenotypic information, three children with negative WES underwent targeted single-gene testing.ResultsIndividual 1 had a clinical diagnosis consistent with infantile systemic hyalinosis, although WES and a next-generation sequencing (NGS)-based ANTXR2 test were negative. Sanger sequencing of ANTXR2 revealed a homozygous single base pair insertion, previously missed by the WES variant caller software. Individual 2 had neurodevelopmental regression and cerebellar atrophy, with no diagnosis on WES. New clinical findings prompted Sanger sequencing and copy number testing of PLA2G6. A novel homozygous deletion of the noncoding exon 1 (not included in the WES capture kit) was detected, with extension into the promoter, confirming the clinical suspicion of infantile neuroaxonal dystrophy. Individual 3 had progressive ataxia, spasticity, and magnetic resonance image changes of vanishing white matter leukoencephalopathy. An NGS leukodystrophy gene panel and WES showed a heterozygous pathogenic variant in EIF2B5; no deletions/duplications were detected. Sanger sequencing of EIF2B5 showed a frameshift indel, probably missed owing to failure of alignment.ConclusionThese cases illustrate potential pitfalls of WES/NGS testing and the importance of phenotype-guided molecular testing in yielding diagnoses.

Identifying the underlying causes of disease requires accurate interpretation of genetic variants. Current methods ineffectively capture pathogenic non-coding variants in genic regions, resulting in overlooking synonymous and intronic variants when searching for disease risk. Here we present the Transcript-inferred Pathogenicity (TraP) score, which uses sequence context alterations to reliably identify non-coding variation that causes disease. High TraP scores single out extremely rare variants with lower minor allele frequencies than missense variants. TraP accurately distinguishes known pathogenic and benign variants in synonymous (AUC = 0.88) and intronic (AUC = 0.83) public datasets, dismissing benign variants with exceptionally high specificity. TraP analysis of 843 exomes from epilepsy family trios identifies synonymous variants in known epilepsy genes, thus pinpointing risk factors of disease from non-coding sequence data. TraP outperforms leading methods in identifying non-coding variants that are pathogenic and is therefore a valuable tool for use in gene discovery and the interpretation of personal genomes.While non-coding synonymous and intronic variants are often not under strong selective constraint, they can be pathogenic through affecting splicing or transcription. Here, the authors develop a score that uses sequence context alterations to predict pathogenicity of synonymous and non-coding genetic variants, and provide a web server of pre-computed scores.

Background: Patients' stories of their illnesses help bridge the divide between patients and providers, facilitating more humane medical care. Illness narratives have been classified into three types: restitution (expectation of recovery), chaos (suffering and loss), and quest (unexpected positive effect from illness). Undiagnosed patients have unique illness experiences and obtaining their narratives would provide insights into the medical and emotional impact of living with an undiagnosed illness. Adults and children with undiagnosed diseases apply to be evaluated by the Undiagnosed Diseases Network (UDN). Written illness narratives from 40 UDN applicants, including 20 adult probands who applied for themselves and 20 parents who applied for their children, were analyzed for: 1) narrative content and 2) narrative type., Results: Narrative content: could be grouped into three themes: 1) Expectations of the UDN: the majority felt they had no further healthcare options and hoped the UDN would provide them with a diagnosis, with the adults expecting to return to their previously healthy life and the parents wanting information to manage their child's healthcare. 2) Personal medical information: the narratives reported worsening of symptoms and some offered opinions regarding the cause of their illness. The proband narratives had few objective findings, while parental narratives had detailed objective information. 3) Experiences related to living with their undiagnosed illness: frustration at being undiagnosed was expressed. The adults felt they had to provide validation of their symptoms to providers, given the lack of objective findings. The parents worried that something relevant to their child's management was being overlooked. Narrative type: All the narratives were of the chaos type, but for different reasons, with the probands describing loss and suffering and the parents expressing fear for their child's future. The parental narratives also had elements of restitution and quest, with acceptance of "a new normal", and an emphasis on the positive aspects of their child's illness which was absent from the probands., Conclusions: These narratives illustrate the chaos that coexists with being undiagnosed. The differences between the proband and parental narratives suggest that these two groups have different needs that need to be considered during their evaluation and management.

Whole-exome sequencing (WES) has increasingly enabled new pathogenic gene variant identification for undiagnosed neurodevelopmental disorders and provided insights into both gene function and disease biology. Here, we describe seven children with a neurodevelopmental disorder characterized by microcephaly, profound developmental delays and/or intellectual disability, cataracts, severe epilepsy including infantile spasms, irritability, failure to thrive, and stereotypic hand movements. Brain imaging in these individuals reveals delay in myelination and cerebral atrophy. We observe an identical recurrent de novo heterozygous c.892C>T (p.Arg298Trp) variant in the nucleus accumbens associated 1 (NACC1) gene in seven affected individuals. One of the seven individuals is mosaic for this variant. NACC1 encodes a transcriptional repressor implicated in gene expression and has not previously been associated with germline disorders. The probability of finding the same missense NACC1 variant by chance in 7 out of 17,228 individuals who underwent WES for diagnoses of neurodevelopmental phenotypes is extremely small and achieves genome-wide significance (p = 1.25 × 10 -14 ). Selective constraint against missense variants in NACC1 makes this excess of an identical missense variant in all seven individuals more remarkable. Our findings are consistent with a germline recurrent mutational hotspot associated with an allele-specific neurodevelopmental phenotype in NACC1., (Copyright © 2017 American Society of Human Genetics. All rights reserved.)

The ASXL genes (ASXL1, ASXL2, and ASXL3) participate in body patterning during embryogenesis and encode proteins involved in epigenetic regulation and assembly of transcription factors to specific genomic loci. Germline de novo truncating variants in ASXL1 and ASXL3 have been respectively implicated in causing Bohring-Opitz and Bainbridge-Ropers syndromes, which result in overlapping features of severe intellectual disability and dysmorphic features. ASXL2 has not yet been associated with a human Mendelian disorder. In this study, we performed whole-exome sequencing in six unrelated probands with developmental delay, macrocephaly, and dysmorphic features. All six had de novo truncating variants in ASXL2. A careful review enabled the recognition of a specific phenotype consisting of macrocephaly, prominent eyes, arched eyebrows, hypertelorism, a glabellar nevus flammeus, neonatal feeding difficulties, hypotonia, and developmental disabilities. Although overlapping features with Bohring-Opitz and Bainbridge-Ropers syndromes exist, features that distinguish the ASXL2-associated condition from ASXL1- and ASXL3-related disorders are macrocephaly, absence of growth retardation, and more variability in the degree of intellectual disabilities. We were also able to demonstrate with mRNA studies that these variants are likely to exert a dominant-negative effect, given that both alleles are expressed in blood and the mutated ASXL2 transcripts escape nonsense-mediated decay. In conclusion, de novo truncating variants in ASXL2 underlie a neurodevelopmental syndrome with a clinically recognizable phenotype. This report expands the germline disorders that are linked to the ASXL genes., (Copyright © 2016. Published by Elsevier Inc.)

Due to the lack of empirical information on parental perceptions of primary results of whole exome sequencing (WES), we conducted a retrospective semi-structured interview with 19 parents of children who had undergone WES. Perceptions explored during the interview included factors that would contribute to parental empowerment such as: parental expectations, understanding of the WES and results, utilization of the WES information, and communication of findings to health/educational professionals and family members. Results of the WES had previously been communicated to families within a novel framework of clinical diagnostic categories: 5/19 had Definite diagnoses, 6/19 had Likely diagnoses, 3/19 had Possible diagnosis and 5/19 had No diagnosis. All parents interviewed expressed a sense of duty to pursue the WES in search of a diagnosis; however, their expectations were tempered by previous experiences with negative genetic testing results. Approximately half the parents worried that a primary diagnosis that would be lethal might be identified; however, the hope of a diagnosis outweighed this concern. Parents were accurately able to summarize their child's WES findings, understood the implications for recurrence risks, and were able to communicate these findings to family and medical/educational providers. The majority of those with a Definite/Likely diagnosis felt that their child's medical care was more focused, or there was a reduction in worry, despite the lack of a specific treatment. Irrespective of diagnostic outcome, parents recommended that follow-up visits be built into the process. Several parents expressed a desire to have all variants of unknown significance (VUS) reported to them so that they could investigate these themselves. Finally, for some families whose children had a Definite/Likely diagnosis, there was remaining frustration and a sense of isolation, due to the limited information that was available about the diagnosed rare disorders and the inability to connect to other families, suggesting that for families with rare genetic disorders, the diagnostic odyssey does not necessarily end with a diagnosis. Qualitative interviewing served a meaningful role in eliciting new information about parental motivations, expectations, and knowledge of WES. Our findings highlight a need for continued communication with families as we navigate the new landscape of genomic sequencing.

The overall understanding of the molecular etiologies of intellectual disability (ID) and developmental delay (DD) is increasing as next-generation sequencing technologies identify genetic variants in individuals with such disorders. However, detailed analyses conclusively confirming these variants, as well as the underlying molecular mechanisms explaining the diseases, are often lacking. Here, we report on an ID syndrome caused by de novo heterozygous loss-of-function (LoF) mutations in SON. The syndrome is characterized by ID and/or DD, malformations of the cerebral cortex, epilepsy, vision problems, musculoskeletal abnormalities, and congenital malformations. Knockdown of son in zebrafish resulted in severe malformation of the spine, brain, and eyes. Importantly, analyses of RNA from affected individuals revealed that genes critical for neuronal migration and cortex organization (TUBG1, FLNA, PNKP, WDR62, PSMD3, and HDAC6) and metabolism (PCK2, PFKL, IDH2, ACY1, and ADA) are significantly downregulated because of the accumulation of mis-spliced transcripts resulting from erroneous SON-mediated RNA splicing. Our data highlight SON as a master regulator governing neurodevelopment and demonstrate the importance of SON-mediated RNA splicing in human development., (Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

KCNH1 mutations have been identified in patients with Zimmermann-Laband syndrome and Temple-Baraitser syndrome, as well as patients with uncharacterized syndromes with intellectual disability and overlapping features. These syndromes include dysmorphic facial features, nail hypo/aplasia, thumb and skeletal anomalies, intellectual disability, and seizures. We report the epilepsy phenotype in patients with KCNH1 mutations. Demographic data, electroclinical features, response to antiepileptic drugs, and results of significant diagnostic investigations of nine patients carrying mutations in KCNH1 were obtained from referring centres. Epilepsy was present in 7/9 patients. Both generalized and focal tonic-clonic seizures were observed. Complete seizure control was achieved with pharmacological treatment in 2/7 patients; polytherapy was required in 4/7 patients. Status epilepticus occurred in 4/7 patients. EEG showed a diffusely slow background in 7/7 patients with epilepsy, with variable epileptiform abnormalities. Cerebral folate deficiency and an increase in urinary hypoxanthine and uridine were observed in one patient. Epilepsy is a key phenotypic feature in most individuals with KCNH1-related syndromes, suggesting a direct role of KCNH1 in epileptogenesis, although the underlying mechanism is not understood.

Despite the exciting advent of whole-exome sequencing (WES) in medical genetics practices, the optimal interpretation of results requires further actions such as reconsidering clinical information and obtaining further laboratory testing. There are no published data to guide clinicians in this process. In a retrospective study on 93 patients who underwent clinical WES, we set out to assess and resolve these practical challenges. With the laboratories reporting a molecular diagnostic rate of 25.8%, the medical geneticists and the laboratories were 90% concordant in their interpretation of the WES results. Divergence occurred when the medical geneticist reconsidered clinical information and/or additional information regarding pathogenicity of a variant. Variants of uncertain significance were reported in 86% of patients, with 53.7% needing follow-up, such as additional laboratory tests and genotyping of family members. By layering clinical data (e.g. mode of inheritance and phenotypic fit) on to the laboratory results, we developed clinical categories for the WES results. These categories of definite diagnosis (14/93), likely diagnosis (8/93), possible diagnosis (13/93) and no diagnosis (58/93) could be used to convey results to patients uniformly. Our framework for a clinically informed interpretation of the results enhances the utility of WES within medical genetics practices., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Individuals with 22q11.2 deletion syndrome (22q11.2DS) have an increased chance of developing a psychiatric disorder. While parents of children affected by 22q11.2DS typically receive counseling about risk for non-psychiatric health concerns, genetic counselors may be reluctant to discuss psychiatric risk. Further education of genetic counselors may be necessary to encourage discussion of psychiatric risk with these families. The goal of this project was to develop recommendations for genetic counselors to provide psychiatric risk information to families affected by 22q11.2DS. The recommendations were developed by synthesizing resources in the literature about risk communication. These recommendations were refined following an online focus group meeting with five health care professionals who were recruited for participation from 22q11.2DS clinics across the U.S.A. The focus group data revealed three themes related to discussion of psychiatric risk: 1) Stepwise approach, 2) Discussing treatment options and reducing risks, and 3) Addressing stigma. These recommendations may be used as a foundation for a future clinical protocol to encourage discussion about the risk for psychiatric illness at an earlier point in the diagnostic process for 22q11.2DS and to provide improved information, support and resources to affected families.