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Autoimmune lymphoproliferative syndrome (ALPS) is characterized by chronic nonmalignant lymphadenopathy, splenomegaly, cytopenias, and other autoimmune manifestations. ALPS is caused by lymphocyte accumulation from defects in FAS-mediated apoptosis. Heterozygous germline or somatic pathogenic single nucleotide variants in FAS are the most common molecular etiology of ALPS. Through the Centralized Sequencing Program at the National Institute of Allergy and Infectious Diseases, we performed exome sequencing on subjects with a clinical diagnosis of ALPS, with a subset receiving copy number variant (CNV) analysis. In this cohort, we identified 3 subjects from unrelated families with CNVs at the FAS locus. One subject had a de novo ∼0.828 Mb copy number loss encompassing all of FAS. The second subject had a maternally inherited ∼1.004 Mb copy number loss encompassing all of FAS. The third subject had a paternally inherited ∼0.044 Mb copy number loss encompassing exons 7 through 9 of FAS. Subjects with deletions in FAS had clinical presentations and biomarker profiles similar to those with ALPS and with germline and somatic FAS variants. We demonstrate that CNV analysis should be pursued if there is clinical and biomarker evidence of ALPS because it can lead to a molecular diagnosis and appropriate treatment when FAS sequencing is inconclusive.

Sterile alpha motif (SAM) and Src homology-3 (SH3) domain-containing 3 (SASH3), also called SH3-containing lymphocyte protein (SLY1), is a putative adaptor protein that is postulated to play an important role in the organization of signaling complexes and propagation of signal transduction cascades in lymphocytes. The SASH3 gene is located on the X-chromosome. Here, we identified 3 novel SASH3 deleterious variants in 4 unrelated male patients with a history of combined immunodeficiency and immune dysregulation that manifested as recurrent sinopulmonary, cutaneous, and mucosal infections and refractory autoimmune cytopenias. Patients exhibited CD4+ T-cell lymphopenia, decreased T-cell proliferation, cell cycle progression, and increased T-cell apoptosis in response to mitogens. In vitro T-cell differentiation of CD34+ cells and molecular signatures of rearrangements at the T-cell receptor α (TRA) locus were indicative of impaired thymocyte survival. These patients also manifested neutropenia and B-cell and natural killer (NK)-cell lymphopenia. Lentivirus-mediated transfer of the SASH3 complementary DNA–corrected protein expression, in vitro proliferation, and signaling in SASH3-deficient Jurkat and patient-derived T cells. These findings define a new type of X-linked combined immunodeficiency in humans that recapitulates many of the abnormalities reported in mice with Sly1–/– and Sly1Δ/Δ mutations, highlighting an important role of SASH3 in human lymphocyte function and survival.

Disease-causing mutations in a linkage-specific deubiquitylase provide insights into chromatin remodeling during embryogenesis., Reversible modification of proteins with linkage-specific ubiquitin chains is critical for intracellular signaling. Information on physiological roles and underlying mechanisms of particular ubiquitin linkages during human development are limited. Here, relying on genomic constraint scores, we identify 10 patients with multiple congenital anomalies caused by hemizygous variants in OTUD5, encoding a K48/K63 linkage–specific deubiquitylase. By studying these mutations, we find that OTUD5 controls neuroectodermal differentiation through cleaving K48-linked ubiquitin chains to counteract degradation of select chromatin regulators (e.g., ARID1A/B, histone deacetylase 2, and HCF1), mutations of which underlie diseases that exhibit phenotypic overlap with OTUD5 patients. Loss of OTUD5 during differentiation leads to less accessible chromatin at neuroectodermal enhancers and aberrant gene expression. Our study describes a previously unidentified disorder we name LINKED (LINKage-specific deubiquitylation deficiency–induced Embryonic Defects) syndrome and reveals linkage-specific ubiquitin cleavage from chromatin remodelers as an essential signaling mode that coordinates chromatin remodeling during embryogenesis.

Inborn errors of immunity (IEI) are a genetically heterogeneous group of disorders with a broad clinical spectrum. Identification of molecular and functional bases of these disorders is important for diagnosis, treatment, and an understanding of the human immune response. We identified 6 unrelated males with neutropenia, infections, lymphoproliferation, humoral immune defects, and in some cases bone marrow failure associated with 3 different variants in the X-linked gene TLR8, encoding the endosomal Toll-like receptor 8 (TLR8). Interestingly, 5 patients had somatic variants in TLR8 with

The diagnosis of mitochondrial disorders remains a challenging and often unmet need. We sought to investigate a sibling pair with suspected mitochondrial disease and a clinical presentation notable for global developmental delay, poor growth, sensorineural hearing loss, and brain MRI abnormalities, both with early death. Following uninformative exome and genome sequencing of the family quartet, RNA sequencing was pursued as an orthogonal testing strategy. RNA sequencing of fibroblasts from the older sibling identified the presence of a cryptic exon in intron 1 of NDUFB10, that included an in-frame stop codon. NDUFB10 encodes a subunit of mitochondrial OXPHOS complex I. Differential expression analysis relative to control samples suggested significantly decreased expression. The cryptic exon was found to contain a rare intronic variant, NM_004548.3:c.131-442G>C, that was homozygous in both affected siblings and absent from population allele frequency databases. Immunoblot and quantitative proteomic analysis of fibroblasts from the older sibling revealed decreased abundance of complex I subunits associated with NDUFB10, providing evidence of isolated complex I deficiency. Biallelic variants in NDUFB10 have previously been reported in a single individual with infantile-onset mitochondrial disease. We present data implicating a deep intronic variant in NDUFB10 as the cause of mitochondrial disease in two further individuals. This variant results in loss of expression and overall destabilization of mitochondrial OXPHOS complex I and highlights the importance of RNA sequencing as a complementary diagnostic tool in patients undergoing genome-wide diagnostic evaluation.

Embryonic development occurs through commitment of pluripotent stem cells to differentiation programs that require highly coordinated changes in gene expression. Chromatin remodeling of gene regulatory elements is a critical component of how such changes are achieved. While many factors controlling chromatin dynamics are known, mechanisms of how different chromatin regulators are orchestrated during development are not well understood. Here, we describe LINKED (LINKage-specific-deubiquitylation-deficiency-induced Embryonic Defects) syndrome, a novel multiple congenital anomaly disorder caused by hypomorphic hemizygous missense variants in the deubiquitylase OTUD5/DUBA. Studying LINKED mutations in vitro, in mouse, and in models of neuroectodermal differentiation of human pluripotent stem cells, we uncover a novel regulatory circuit that coordinates chromatin remodeling pathways during early differentiation. We show that the K48-linkage-specific deubiquitylation activity of OTUD5 is essential for murine and human development and, if reduced, leads to aberrant cell-fate specification. OTUD5 controls differentiation through preventing the degradation of multiple chromatin regulators including ARID1A/B and HDAC2, mutation of which underlie developmental syndromes that exhibit phenotypic overlap with LINKED patients. Accordingly, loss of OTUD5 during early differentiation leads to less accessible chromatin at neural and neural crest enhancers and thus aberrant rewiring of gene expression networks. Our work identifies a novel mechanistic link between phenotypically related developmental disorders and an essential function for linkagespecific ubiquitin editing of substrate groups (i.e. chromatin remodeling complexes) during early cellfate decisions – a regulatory concept, we predict to be a general feature of embryonic development.

De novo variants in DDX3X account for 1-3% of unexplained intellectual disability (ID) cases and are amongst the most common causes of ID especially in females. Forty-seven patients (44 females, 3 males) have been described. We identified 31 additional individuals carrying 29 unique DDX3X variants, including 30 postnatal individuals with complex clinical presentations of developmental delay or ID, and one fetus with abnormal ultrasound findings. Rare or novel phenotypes observed include respiratory problems, congenital heart disease, skeletal muscle mitochondrial DNA depletion, and late-onset neurologic decline. Our findings expand the spectrum of DNA variants and phenotypes associated with DDX3X disorders.

We describe a case of acute liver failure and myopericarditis due to herpes simplex virus–1 (HSV-1) in an immunocompetent adult. We estimate that, at the height of viremia, the patient contained a quantity of HSV-1 virions approaching that of human cells. The patient recovered with acyclovir that was dose-adjusted for neurotoxicity and developed a vigorous anti-HSV-1 T-cell response.

ObjectiveTo expand the clinical spectrum of lysyl-tRNA synthetase (KARS) gene–related diseases, which so far includes Charcot-Marie-Tooth disease, congenital visual impairment and microcephaly, and nonsyndromic hearing impairment.MethodsWhole-exome sequencing was performed on index patients from 4 unrelated families with leukoencephalopathy. Candidate pathogenic variants and their cosegregation were confirmed by Sanger sequencing. Effects of mutations on KARS protein function were examined by aminoacylation assays and yeast complementation assays.ResultsCommon clinical features of the patients in this study included impaired cognitive ability, seizure, hypotonia, ataxia, and abnormal brain imaging, suggesting that the CNS involvement is the main clinical presentation. Six previously unreported and 1 known KARS mutations were identified and cosegregated in these families. Two patients are compound heterozygous for missense mutations, 1 patient is homozygous for a missense mutation, and 1 patient harbored an insertion mutation and a missense mutation. Functional and structural analyses revealed that these mutations impair aminoacylation activity of lysyl-tRNA synthetase, indicating that defective KARS function is responsible for the phenotypes in these individuals.ConclusionsOur results demonstrate that patients with loss-of-function KARS mutations can manifest CNS disorders, thus broadening the phenotypic spectrum associated with KARS-related disease.

Next‐generation sequencing (NGS) has been instrumental in solving the genetic basis of rare inherited diseases, especially neurodevelopmental syndromes. However, functional workup is essential for precise phenotype definition and to understand the underlying disease mechanisms. Using whole exome (WES) and whole genome sequencing (WGS) in four independent families with hypotonia, neurodevelopmental delay, facial dysmorphism, loss of white matter, and thinning of the corpus callosum, we identified four previously unreported homozygous truncating PPP1R21 alleles: c.347delT p.(Ile116Lysfs*25), c.2170_2171insGGTA p.(Ile724Argfs*8), c.1607dupT p.(Leu536Phefs*7), c.2063delA p.(Lys688Serfs*26) and found that PPP1R21 was absent in fibroblasts of an affected individual, supporting the allele's loss of function effect. PPP1R21 function had not been studied except that a large scale affinity proteomics approach suggested an interaction with PIBF1 defective in Joubert syndrome. Our co‐immunoprecipitation studies did not confirm this but in contrast defined the localization of PPP1R21 to the early endosome. Consistent with the subcellular expression pattern and the clinical phenotype exhibiting features of storage diseases, we found patient fibroblasts exhibited a delay in clearance of transferrin‐488 while uptake was normal. In summary, we delineate a novel neurodevelopmental syndrome caused by biallelic PPP1R21 loss of function variants, and suggest a role of PPP1R21 within the endosomal sorting process or endosome maturation pathway.

Histones mediate dynamic packaging of nuclear DNA in chromatin, a process that is precisely controlled to guarantee efficient compaction of the genome and proper chromosomal segregation during cell division and to accomplish DNA replication, transcription, and repair. Due to the important structural and regulatory roles played by histones, it is not surprising that histone functional dysregulation or aberrant levels of histones can have severe consequences for multiple cellular processes and ultimately might affect development or contribute to cell transformation. Recently, germline frameshift mutations involving the C-terminal tail of HIST1H1E, which is a widely expressed member of the linker histone family and facilitates higher-order chromatin folding, have been causally linked to an as-yet poorly defined syndrome that includes intellectual disability. We report that these mutations result in stable proteins that reside in the nucleus, bind to chromatin, disrupt proper compaction of DNA, and are associated with a specific methylation pattern. Cells expressing these mutant proteins have a dramatically reduced proliferation rate and competence, hardly enter into the S phase, and undergo accelerated senescence. Remarkably, clinical assessment of a relatively large cohort of subjects sharing these mutations revealed a premature aging phenotype as a previously unrecognized feature of the disorder. Our findings identify a direct link between aberrant chromatin remodeling, cellular senescence, and accelerated aging.

SON is a key component of the spliceosomal complex and a critical mediator of constitutive and alternative splicing. Additionally, SON has been shown to influence cell-cycle progression, genomic integrity, and maintenance of pluripotency in stem cell populations. The clear functional relevance of SON in coordinating essential cellular processes and its presence in diverse human tissues suggests that intact SON might be crucial for normal growth and development. However, the phenotypic effects of deleterious germline variants in SON have not been clearly defined. Herein, we describe seven unrelated individuals with de novo variants in SON and propose that deleterious variants in SON are associated with a severe multisystem disorder characterized by developmental delay, persistent feeding difficulties, and congenital malformations, including brain anomalies.

Each year in the United States, thousands of cases of sudden and unexpected deaths of infants, children, and young adults are assigned an undetermined cause of death after postmortem investigation and autopsy. Heritable genetic variants have been suggested as the cause of up to a third of sudden death (SD) cases. Elucidation of the genetic variants involved in SD cases is important to not only help establish cause and manner of death of these individuals, but to also aid in determining whether familial genetic testing should be considered. Previously, these types of postmortem screenings have not been a feasible option for most county medical examiners’ and coroners’ offices. We sequenced full exons of 64 genes associated with SD in the largest known cohort (351) of infant and young SD decedents using massively parallel sequencing at 1 yr of age), were found to have a reportable genetic variant contributing to SD. These percentages represent an estimate lower than those previously reported. Overall yields and results likely vary between studies due to differences in evaluation techniques and reporting. Additionally, we recommend ongoing assessment of data, including nonreported novel variants, as technology and literature continually advance. This study demonstrates a strategy to implement molecular autopsies in medicolegal investigations of young SD decedents.

Background Exome sequencing is now being incorporated into clinical care for pediatric and adult populations, but its integration into prenatal diagnosis has been more limited. One reason for this is the paucity of information about the clinical utility of exome sequencing in the prenatal setting. Methods We retrospectively reviewed indications, results, time to results (turnaround time, TAT), and impact of exome results for 146 consecutive “fetal exomes” performed in a clinical diagnostic laboratory between March 2012 and November 2017. We define a fetal exome as one performed on a sample obtained from a fetus or a product of conception with at least one structural anomaly detected by prenatal imaging or autopsy. Statistical comparisons were performed using Fisher’s exact test. Results Prenatal exome yielded an overall molecular diagnostic rate of 32% (n = 46/146). Of the 46 molecular diagnoses, 50% were autosomal dominant disorders (n = 23/46), 41% were autosomal recessive disorders (n = 19/46), and 9% were X-linked disorders (n = 4/46). The molecular diagnostic rate was highest for fetuses with anomalies affecting multiple organ systems and for fetuses with craniofacial anomalies. Out of 146 cases, a prenatal trio exome option designed for ongoing pregnancies was performed on 62 fetal specimens, resulting in a diagnostic yield of 35% with an average TAT of 14 days for initial reporting (excluding tissue culture time). The molecular diagnoses led to refined recurrence risk estimates, altered medical management, and informed reproductive planning for families. Conclusion Exome sequencing is a useful diagnostic tool when fetal structural anomalies suggest a genetic etiology, but other standard prenatal genetic tests did not provide a diagnosis. Electronic supplementary material The online version of this article (10.1186/s13073-018-0582-x) contains supplementary material, which is available to authorized users.

De novo variants in DDX3X account for 1-3% of unexplained intellectual disability (ID), one of the most common causes of ID, in females. Forty-seven patients (44 females, 3 males) have been described. We identified 29 additional individuals carrying 27 unique DDX3X variants in the setting of complex clinical presentations including developmental delay or ID. In addition to previously reported manifestations, rare or novel phenotypes were identified including respiratory problems, congenital heart disease, skeletal muscle mitochondrial DNA depletion, and late-onset neurologic decline. Our findings expand the spectrum of DNA variants and phenotypes associated with DDX3X disorders.

Item does not contain fulltext N-alpha-acetylation is a common co-translational protein modification that is essential for normal cell function in humans. We previously identified the genetic basis of an X-linked infantile lethal Mendelian disorder involving a c.109T>C (p.Ser37Pro) missense variant in NAA10, which encodes the catalytic subunit of the N-terminal acetyltransferase A (NatA) complex. The auxiliary subunit of the NatA complex, NAA15, is the dimeric binding partner for NAA10. Through a genotype-first approach with whole-exome or genome sequencing (WES/WGS) and targeted sequencing analysis, we identified and phenotypically characterized 38 individuals from 33 unrelated families with 25 different de novo or inherited, dominantly acting likely gene disrupting (LGD) variants in NAA15. Clinical features of affected individuals with LGD variants in NAA15 include variable levels of intellectual disability, delayed speech and motor milestones, and autism spectrum disorder. Additionally, mild craniofacial dysmorphology, congenital cardiac anomalies, and seizures are present in some subjects. RNA analysis in cell lines from two individuals showed degradation of the transcripts with LGD variants, probably as a result of nonsense-mediated decay. Functional assays in yeast confirmed a deleterious effect for two of the LGD variants in NAA15. Further supporting a mechanism of haploinsufficiency, individuals with copy-number variant (CNV) deletions involving NAA15 and surrounding genes can present with mild intellectual disability, mild dysmorphic features, motor delays, and decreased growth. We propose that defects in NatA-mediated N-terminal acetylation (NTA) lead to variable levels of neurodevelopmental disorders in humans, supporting the importance of the NatA complex in normal human development.

Deformed epidermal autoregulatory factor-1 (DEAF1), a transcription factor essential for central nervous system and early embryonic development, has recently been implicated in a series of intellectual disability-related neurodevelopmental anomalies termed, in this study, as DEAF1-associated neurodevelopmental disorder (DAND). We identified six potentially deleterious DEAF1 variants in a cohort of individuals with DAND via clinical exome sequencing (CES) and in silico analysis, including two novel de novo variants: missense variant c.634G > A p.Gly212Ser in the SAND domain and deletion variant c.913_915del p.Lys305del in the NLS domain, as well as c.676C > T p.Arg226Trp, c.700T > A p.Trp234Arg, c.737G > C p.Arg246Thr, and c.791A > C p.Gln264Pro. Luciferase reporter, immunofluorescence staining, and electrophoretic mobility shift assays revealed that these variants had decreased transcriptional repression activity at the DEAF1 promoter and reduced affinity to consensus DEAF1 DNA binding sequences. In addition, c.913_915del p.K305del localized primarily to the cytoplasm and interacted with wild-type DEAF1. Our results demonstrate that variants located within the SAND or NLS domains significantly reduce DEAF1 transcriptional regulatory activities and are thus, likely to contribute to the underlying clinical concerns in DAND patients. These findings illustrate the importance of experimental characterization of variants with uncertain significance identified by CES to assess their potential clinical significance and possible use in diagnosis.

Background Exon-targeted microarrays can detect small (

This corrects the article DOI: 10.1038/gim.2016.18.

Purpose: Truncating mutations in the maternally imprinted, paternally expressed gene MAGEL2, which is located in the Prader-Willi critical region 15q11-13, have recently been reported to cause Schaaf -Yang syndrome, a Prader-Willi-like disease that manifests as developmental delay/intellectual disability, hypotonia, feeding difficulties, and autism spectrum disorder. The causality of the reported variants in the context of the patients' phenotypes was questioned, as MAGEL2 whole -gene deletions seem to cause little or no clinical phenotype.Methods: Here we report a total of 18 newly identified individuals with Schaaf-Yang syndrome from 14 families, including 1 family with 3 individuals found to be affected with a truncating variant of MAGEL2, 11 individuals who are clinically affected but were not tested molecularly, and a presymptomatic fetal sibling carrying the pathogenic MAGEL2 variant.Results: All cases harbor truncating mutations of MAGEL2, and nucleotides c.1990-1996 arise as a mutational hotspot, with 10 individuals and 1 fetus harboring a c.1996dupC (p.Q666fs) mutation and 2 fetuses harboring a c.1996deIC (p.Q666fs) mutation. The phenotypic spectrum of Schaaf-Yang syndrome ranges from fetal akinesia to neurobehavioral disease and contractures of the small finger joints.Conclusion: This study provides strong evidence for the pathogenicity of truncating mutations of the paternal allele of MAGEL2, refines the associated clinical phenotypes, and highlights implications for genetic counseling for affected families.

Here we describe a patient who presented with a history of congenital diaphragmatic hernia, inguinal hernia, and recurrent umbilical hernia. She also has joint laxity, hypotonia, and dysmorphic features. A unifying diagnosis was not identified based on her clinical phenotype. As part of her evaluation through the Undiagnosed Diseases Network, trio whole-exome sequencing was performed. Pathogenic variants in FBN1 and TRPS1 were identified as causing two distinct autosomal dominant conditions, each with de novo inheritance. Fibrillin 1 (FBN1) mutations are associated with Marfan syndrome and a spectrum of similar phenotypes. TRPS1 mutations are associated with trichorhinophalangeal syndrome types I and III. Features of both conditions are evident in the patient reported here. Discrepant features of the conditions (e.g., stature) and the young age of the patient may have made a clinical diagnosis more difficult in the absence of exome-wide genetic testing.

WOS: 000393996800025, PubMed: 27577878, Background: Primary immunodeficiency diseases (PIDDs) are clinically and genetically heterogeneous disorders thus far associated with mutations in more than 300 genes. The clinical phenotypes derived from distinct genotypes can overlap. Genetic etiology can be a prognostic indicator of disease severity and can influence treatment decisions. Objective: We sought to investigate the ability of whole-exome screening methods to detect disease-causing variants in patients with PIDDs. Methods: Patients with PIDDs from 278 families from 22 countries were investigated by using whole-exome sequencing. Computational copy number variant (CNV) prediction pipelines and an exome-tiling chromosomal microarray were also applied to identify intragenic CNVs. Analytic approaches initially focused on 475 known or candidate PIDD genes but were nonexclusive and further tailored based on clinical data, family history, and immunophenotyping. Results: A likely molecular diagnosis was achieved in 110 (40%) unrelated probands. Clinical diagnosis was revised in about half (60/ 110) and management was directly altered in nearly a quarter (26/ 110) of families based on molecular findings. Twelve PIDD-causing CNVs were detected, including 7 smaller than 30 Kb that would not have been detected with conventional diagnostic CNV arrays. Conclusion: This high-throughput genomic approach enabled detection of disease-related variants in unexpected genes; permitted detection of low-grade constitutional, somatic, and revertant mosaicism; and provided evidence of a mutational burden in mixed PIDD immunophenotypes., South-Eastern Norway Health Authority; American Women's club of Oslo; National Human Genome Research InstituteUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Human Genome Research Institute (NHGRI); National Heart, Lung, and BloodUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Heart Lung & Blood Institute (NHLBI) [U54HG006542]; Jeffrey Modell Foundation; NIHUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [AI-120989], Funding for the work performed in Oslo was provided by the South-Eastern Norway Health Authority, and A. S.-P. received research scholarship from the American Women's club of Oslo. The BHCMG is supported by the National Human Genome Research Institute and the National Heart, Lung, and Blood (U54HG006542). Funding was also provided by the Jeffrey Modell Foundation and NIH AI-120989 (to J.S.O.).

Ubiquitination is a posttranslational modification that regulates many cellular processes including protein degradation, intracellular trafficking, cell signaling, and protein-protein interactions. Deubiquitinating enzymes (DUBs), which reverse the process of ubiquitination, are important regulators of the ubiquitin system. OTUD6B encodes a member of the ovarian tumor domain(OTU)-containing subfamily of deubiquitinating enzymes. Herein, we report biallelic pathogenic variants in OTUD6B in 12 individuals from 6 independent families with an intellectual disability syndrome associated with seizures and dysmorphic features. In subjects with predicted loss-of-function alleles, additional features include global developmental delay, microcephaly, absent speech, hypotonia, growth retardation with prenatal onset, feeding difficulties, structural brain abnormalities, congenital malformations including congenital heart disease, and musculoskeletal features. Homozygous Otud6b knockout mice were subviable, smaller in size, and had congenital heart defects, consistent with the severity of loss-of-function variants in humans. Analysis of peripheral blood mononuclear cells from an affected subject showed reduced incorporation of 19S subunits into 26S proteasomes, decreased chymotrypsin-like activity, and accumulation of ubiquitin-protein conjugates. Our findings suggest a role for OTUD6B in proteasome function, establish that defective OTUD6B function underlies a multisystemic human disorder, and provide additional evidence for the emerging relationship between the ubiquitin system and human disease.

Objective To describe a series of patients with pathogenic variants in FLNA and progressive lung disease necessitating lung transplantation. Study Design We conducted a retrospective chart review of 6 female infants with heterozygous presumed loss-of-function pathogenic variants in FLNA whose initial presentation was early and progressive respiratory failure. Results Each patient received lung transplantation at an average age of 11 months (range, 5-15 months). All patients had pulmonary arterial hypertension and chronic respiratory failure requiring tracheostomy and escalating levels of ventilator support before transplantation. All 6 patients survived initial lung transplantation; however, 1 patient died after a subsequent heart-lung transplant. The remaining 5 patients are living unrestricted lives on chronic immunosuppression at most recent follow-up (range, 19 months to 11.3 years post-transplantation). However, in all patients, severe ascending aortic dilation has been observed with aortic regurgitation. Conclusions Respiratory failure secondary to progressive obstructive lung disease during infancy may be the presenting phenotype of FLNA -associated periventricular nodular heterotopia. We describe a cohort of patients with progressive respiratory failure related to a pathogenic variant in FLNA and present lung transplantation as a viable therapeutic option for this group of patients.

ABL1 is a proto-oncogene well known as part of the fusion gene BCR-ABL1 in the Philadelphia chromosome of leukemia cancer cells. Inherited germline ABL1 changes have not been associated with genetic disorders. Here we report ABL1 germline variants cosegregating with an autosomal dominant disorder characterized by congenital heart disease, skeletal abnormalities, and failure to thrive. The variant c.734A>G (p.Tyr245Cys) was found to occur de novo or cosegregate with disease in five individuals (families 1-3). Additionally, a de novo c.1066G>A (p.Ala356Thr) variant was identified in a sixth individual (family 4). We overexpressed the mutant constructs in HEK 293T cells and observed increased tyrosine phosphorylation, suggesting increased ABL1 kinase activities associated with both the p.Tyr245Cys and p.Ala356Thr substitutions. Our clinical and experimental findings, together with previously reported teratogenic effects of selective BCR-ABL inhibitors in humans and developmental defects in Abl1 knockout mice, suggest that ABL1 has an important role during organismal development.

ATPase family AAA-domain containing protein 3A (ATAD3A) is a nuclear-encoded mitochondrial membrane protein implicated in mitochondrial dynamics, nucleoid organization, protein translation, cell growth, and cholesterol metabolism. We identified a recurrent de novo ATAD3A c.1582C>T (p.Arg528Trp) variant by whole-exome sequencing (WES) in five unrelated individuals with a core phenotype of global developmental delay, hypotonia, optic atrophy, axonal neuropathy, and hypertrophic cardiomyopathy. We also describe two families with biallelic variants in ATAD3A, including a homozygous variant in two siblings, and biallelic ATAD3A deletions mediated by nonallelic homologous recombination (NAHR) between ATAD3A and gene family members ATAD3B and ATAD3C. Tissue-specific overexpression of borR534W, the Drosophila mutation homologous to the human c.1582C>T (p.Arg528Trp) variant, resulted in a dramatic decrease in mitochondrial content, aberrant mitochondrial morphology, and increased autophagy. Homozygous null bor larvae showed a significant decrease of mitochondria, while overexpression of borWT resulted in larger, elongated mitochondria. Finally, fibroblasts of an affected individual exhibited increased mitophagy. We conclude that the p.Arg528Trp variant functions through a dominant-negative mechanism that results in small mitochondria that trigger mitophagy, resulting in a reduction in mitochondrial content. ATAD3A variation represents an additional link between mitochondrial dynamics and recognizable neurological syndromes, as seen with MFN2, OPA1, DNM1L, and STAT2 mutations.

Item does not contain fulltext 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.

Background Large-scale cohort-based whole exome sequencing of individuals with neurodevelopmental disorders (NDDs) has identified numerous novel candidate disease genes; however, detailed phenotypic information is often lacking in such studies. De novo mutations in pogo transposable element with zinc finger domain (POGZ) have been identified in six independent and diverse cohorts of individuals with NDDs ranging from autism spectrum disorder to developmental delay. Methods Whole exome sequencing was performed on five unrelated individuals. Sanger sequencing was used to validate variants and segregate mutations with the phenotype in available family members. Results We identified heterozygous truncating mutations in POGZ in five unrelated individuals, which were confirmed to be de novo or not present in available parental samples. Careful review of the phenotypes revealed shared features that included developmental delay, intellectual disability, hypotonia, behavioral abnormalities, and similar facial characteristics. Variable features included short stature, microcephaly, strabismus and hearing loss. Conclusions While POGZ has been associated with neurodevelopmental disorders in large cohort studies, our data suggest that loss of function variants in POGZ lead to an identifiable syndrome of NDD with specific phenotypic traits. This study exemplifies the era of human reverse clinical genomics ushered in by large disease-directed cohort studies; first defining a new syndrome molecularly and, only subsequently, phenotypically.

Item does not contain fulltext PURPOSE: Bosch-Boonstra-Schaaf optic atrophy syndrome (BBSOAS) is an autosomal-dominant disorder characterized by optic atrophy and intellectual disability caused by loss-of-function mutations in NR2F1. We report 20 new individuals with BBSOAS, exploring the spectrum of clinical phenotypes and assessing potential genotype-phenotype correlations. METHODS: Clinical features of individuals with pathogenic NR2F1 variants were evaluated by review of medical records. The functional relevance of coding nonsynonymous NR2F1 variants was assessed with a luciferase assay measuring the impact on transcriptional activity. The effects of two start codon variants on protein expression were evaluated by western blot analysis. RESULTS: We recruited 20 individuals with novel pathogenic NR2F1 variants (seven missense variants, five translation initiation variants, two frameshifting insertions/deletions, one nonframeshifting insertion/deletion, and five whole-gene deletions). All the missense variants were found to impair transcriptional activity. In addition to visual and cognitive deficits, individuals with BBSOAS manifested hypotonia (75%), seizures (40%), autism spectrum disorder (35%), oromotor dysfunction (60%), thinning of the corpus callosum (53%), and hearing defects (20%). CONCLUSION: BBSOAS encompasses a broad range of clinical phenotypes. Functional studies help determine the severity of novel NR2F1 variants. Some genotype-phenotype correlations seem to exist, with missense mutations in the DNA-binding domain causing the most severe phenotypes.Genet Med 18 11, 1143-1150.

Importance While congenital malformations and genetic diseases are a leading cause of early infant death, to our knowledge, the contribution of single-gene disorders in this group is undetermined. Objective To determine the diagnostic yield and use of clinical exome sequencing in critically ill infants. Design, Setting, and Participants Clinical exome sequencing was performed for 278 unrelated infants within the first 100 days of life who were admitted to Texas Children’s Hospital in Houston, Texas, during a 5-year period between December 2011 and January 2017. Exome sequencing types included proband exome, trio exome, and critical trio exome, a rapid genomic assay for seriously ill infants. Main Outcomes and Measures Indications for testing, diagnostic yield of clinical exome sequencing, turnaround time, molecular findings, patient age at diagnosis, and effect on medical management among a group of critically ill infants who were suspected to have genetic disorders. Results The mean (SEM) age for infants participating in the study was 28.5 (1.7) days; of these, the mean (SEM) age was 29.0 (2.2) days for infants undergoing proband exome sequencing, 31.5 (3.9) days for trio exome, and 22.7 (3.9) days for critical trio exome. Clinical indications for exome sequencing included a range of medical concerns. Overall, a molecular diagnosis was achieved in 102 infants (36.7%) by clinical exome sequencing, with relatively low yield for cardiovascular abnormalities. The diagnosis affected medical management for 53 infants (52.0%) and had a substantial effect on informed redirection of care, initiation of new subspecialist care, medication/dietary modifications, and furthering life-saving procedures in select patients. Critical trio exome sequencing revealed a molecular diagnosis in 32 of 63 infants (50.8%) at a mean (SEM) of 33.1 (5.6) days of life with a mean (SEM) turnaround time of 13.0 (0.4) days. Clinical care was altered by the diagnosis in 23 of 32 patients (71.9%). The diagnostic yield, patient age at diagnosis, and medical effect in the group that underwent critical trio exome sequencing were significantly different compared with the group who underwent regular exome testing. For deceased infants (n = 81), genetic disorders were molecularly diagnosed in 39 (48.1%) by exome sequencing, with implications for recurrence risk counseling. Conclusions and Relevance Exome sequencing is a powerful tool for the diagnostic evaluation of critically ill infants with suspected monogenic disorders in the neonatal and pediatric intensive care units and its use has a notable effect on clinical decision making.

Variants in RNF213 lead to susceptibility to moyamoya disease, a rare cerebral angiopathy characterized by bilateral stenosis of the internal carotid arteries and development of a compensatory collateral network. We describe a 3-month-old female with seizures, arterial narrowing involving the internal carotid and intracranial arteries and inferior abdominal aorta, and persistently elevated transaminases. Whole exome sequencing demonstrated a novel de novo variant in RNF213, securing a molecular diagnosis and directing appropriate intervention. This report underscores the role of whole exome sequencing in cases for which a complex and atypical presentation may mask diagnosis. Furthermore, the early and severe presentation in our patient, in conjunction with a novel de novo RNF213 variant, suggests that specific variants in RNF213 may lead to a Mendelian form of disease rather than simply conferring susceptibility to multifactorial disease.

Whole-exome sequencing (WES) is increasingly used as a diagnostic tool in medicine, but prior reports focus on predominantly pediatric cohorts with neurologic or developmental disorders. We describe the diagnostic yield and characteristics of WES in adults. We performed a retrospective analysis of consecutive WES reports for adults from a diagnostic laboratory. Phenotype composition was determined using Human Phenotype Ontology terms. Molecular diagnoses were reported for 17.5% (85/486) of adults, which is lower than that for a primarily pediatric population (25.2%; P = 0.0003); the diagnostic rate was higher (23.9%) for those 18–30 years of age compared to patients older than 30 years (10.4%; P = 0.0001). Dual Mendelian diagnoses contributed to 7% of diagnoses, revealing blended phenotypes. Diagnoses were more frequent among individuals with abnormalities of the nervous system, skeletal system, head/neck, and growth. Diagnostic rate was independent of family history information, and de novo mutations contributed to 61.4% of autosomal dominant diagnoses. Early WES experience in adults demonstrates molecular diagnoses in a substantial proportion of patients, informing clinical management, recurrence risk, and recommendations for relatives. A positive family history was not predictive, consistent with molecular diagnoses often revealed by de novo events, informing the Mendelian basis of genetic disease in adults. Genet Med 18 7, 678–685.

Importance Clinical whole-exome sequencing is increasingly used for diagnostic evaluation of patients with suspected genetic disorders. Objective To perform clinical whole-exome sequencing and report (1) the rate of molecular diagnosis among phenotypic groups, (2) the spectrum of genetic alterations contributing to disease, and (3) the prevalence of medically actionable incidental findings such as FBN1 mutations causing Marfan syndrome. Design, Setting, and Patients Observational study of 2000 consecutive patients with clinical whole-exome sequencing analyzed between June 2012 and August 2014. Whole-exome sequencing tests were performed at a clinical genetics laboratory in the United States. Results were reported by clinical molecular geneticists certified by the American Board of Medical Genetics and Genomics. Tests were ordered by the patient’s physician. The patients were primarily pediatric (1756 [88%]; mean age, 6 years; 888 females [44%], 1101 males [55%], and 11 fetuses [1% gender unknown]), demonstrating diverse clinical manifestations most often including nervous system dysfunction such as developmental delay. Main Outcomes and Measures Whole-exome sequencing diagnosis rate overall and by phenotypic category, mode of inheritance, spectrum of genetic events, and reporting of incidental findings. Results A molecular diagnosis was reported for 504 patients (25.2%) with 58% of the diagnostic mutations not previously reported. Molecular diagnosis rates for each phenotypic category were 143/526 (27.2%; 95% CI, 23.5%-31.2%) for the neurological group, 282/1147 (24.6%; 95% CI, 22.1%-27.2%) for the neurological plus other organ systems group, 30/83 (36.1%; 95% CI, 26.1%-47.5%) for the specific neurological group, and 49/244 (20.1%; 95% CI, 15.6%-25.8%) for the nonneurological group. The Mendelian disease patterns of the 527 molecular diagnoses included 280 (53.1%) autosomal dominant, 181 (34.3%) autosomal recessive (including 5 with uniparental disomy), 65 (12.3%) X-linked, and 1 (0.2%) mitochondrial. Of 504 patients with a molecular diagnosis, 23 (4.6%) had blended phenotypes resulting from 2 single gene defects. About 30% of the positive cases harbored mutations in disease genes reported since 2011. There were 95 medically actionable incidental findings in genes unrelated to the phenotype but with immediate implications for management in 92 patients (4.6%), including 59 patients (3%) with mutations in genes recommended for reporting by the American College of Medical Genetics and Genomics. Conclusions and Relevance Whole-exome sequencing provided a potential molecular diagnosis for 25% of a large cohort of patients referred for evaluation of suspected genetic conditions, including detection of rare genetic events and new mutations contributing to disease. The yield of whole-exome sequencing may offer advantages over traditional molecular diagnostic approaches in certain patients.

Chromodomain helicase DNA-binding protein 4 (CHD4) is an ATP-dependent chromatin remodeler involved in epigenetic regulation of gene transcription, DNA repair, and cell cycle progression. Also known as Mi2β, CHD4 is an integral subunit of a well-characterized histone deacetylase complex. Here we report five individuals with de novo missense substitutions in CHD4 identified through whole-exome sequencing and web-based gene matching. These individuals have overlapping phenotypes including developmental delay, intellectual disability, hearing loss, macrocephaly, distinct facial dysmorphisms, palatal abnormalities, ventriculomegaly, and hypogonadism as well as additional findings such as bone fusions. The variants, c.3380G>A (p.Arg1127Gln), c.3443G>T (p.Trp1148Leu), c.3518G>T (p.Arg1173Leu), and c.3008G>A, (p.Gly1003Asp) (GenBank: NM_001273.3), affect evolutionarily highly conserved residues and are predicted to be deleterious. Previous studies in yeast showed the equivalent Arg1127 and Trp1148 residues to be crucial for SNF2 function. Furthermore, mutations in the same positions were reported in malignant tumors, and a de novo missense substitution in an equivalent arginine residue in the C-terminal helicase domain of SMARCA4 is associated with Coffin Siris syndrome. Cell-based studies of the p.Arg1127Gln and p.Arg1173Leu mutants demonstrate normal localization to the nucleus and HDAC1 interaction. Based on these findings, the mutations potentially alter the complex activity but not its formation. This report provides evidence for the role of CHD4 in human development and expands an increasingly recognized group of Mendelian disorders involving chromatin remodeling and modification.

Gravin, a multivalent A-kinase anchoring protein (AKAP), localizes to the cell periphery in several cell types and is postulated to target PKA and other binding partners to the plasma membrane. An N-terminal myristoylation sequence and three regions rich in basic amino acids are proposed to mediate this localization. Reports indicating that phorbol ester affects the distribution of SSeCKS, the rat orthologue of gravin, further suggest that PKC may also regulate the subcellular distribution of gravin, which in turn may affect PKA distribution. In this study, quantitative confocal microscopy of cells expressing full-length and mutant gravin–EGFP constructs lacking the proposed targeting domains revealed that either the N-myristoylation site or the polybasic regions were sufficient to target gravin to the cell periphery. Moreover, phorbol ester treatment induced redistribution of gravin–EGFP from the cell periphery to a juxtanuclear vesicular compartment, but this required the presence of the N-myristoylation site. Confocal microscopy further revealed that not only did gravin–EGFP target a PKA RII-ECFP construct to the cell periphery, but PKC activation resulted in redistribution of the gravin and PKA constructs to the same subcellular site. It is postulated that this dynamic response by gravin to PKC activity may mediate PKC dependent control of PKA activity.