Visual circuits get the VIP treatment.

Behavioral state, specifically locomotion, has been shown to enhance sensory responses in primary visual cortex. In this issue of Cell, Fu et al. reveal the circuit elements that mediate this plasticity and suggest that these circuits may serve a general modulatory function across primary sensory areas.

Sensory pollutants alter bird phenology and fitness across a continent.

Expansion of anthropogenic noise and night lighting across our planet1,2 is of increasing conservation concern3-6. Despite growing knowledge of physiological and behavioural responses to these stimuli from single-species and local-scale studies, whether these pollutants affect fitness is less clear, as is how and why species vary in their sensitivity to these anthropic stressors. Here we leverage a large citizen science dataset paired with high-resolution noise and light data from across the contiguous United States to assess how these stimuli affect reproductive success in 142 bird species. We find responses to both sensory pollutants linked to the functional traits and habitat affiliations of species. For example, overall nest success was negatively correlated with noise among birds in closed environments. Species-specific changes in reproductive timing and hatching success in response to noise exposure were explained by vocalization frequency, nesting location and diet. Additionally, increased light-gathering ability of species' eyes was associated with stronger advancements in reproductive timing in response to light exposure, potentially creating phenological mismatches7. Unexpectedly, better light-gathering ability was linked to reduced clutch failure and increased overall nest success in response to light exposure, raising important questions about how responses to sensory pollutants counteract or exacerbate responses to other aspects of global change, such as climate warming. These findings demonstrate that anthropogenic noise and light can substantially affect breeding bird phenology and fitness, and underscore the need to consider sensory pollutants alongside traditional dimensions of the environment that typically inform biodiversity conservation.

Cellular Therapy for Children with Central Nervous System Tumors: Mining and Mapping the Correlative Data.

PURPOSE OF REVIEW: Correlative studies should leverage clinical trial frameworks to conduct biospecimen analyses that provide insight into the bioactivity of the intervention and facilitate iteration toward future trials that further improve patient outcomes. In pediatric cellular immunotherapy trials, correlative studies enable deeper understanding of T cell mobilization, durability of immune activation, patterns of toxicity, and early detection of treatment response. Here, we review the correlative science in adoptive cell therapy (ACT) for childhood central nervous system (CNS) tumors, with a focus on existing chimeric antigen receptor (CAR) and T cell receptor (TCR)-expressing T cell therapies. RECENT FINDINGS: We highlight long-standing and more recently understood challenges for effective alignment of correlative data and offer practical considerations for current and future approaches to multi-omic analysis of serial tumor, serum, and cerebrospinal fluid (CSF) biospecimens. We highlight the preliminary success in collecting serial cytokine and proteomics from patients with CNS tumors on ACT clinical trials.

Childhood Cancer Information-Seeking: Findings from the National Cancer Institute's Cancer Information Service.

The benefits of cancer information-seeking may be particularly salient to individuals impacted by childhood cancer, including patients, caregivers, health professionals, and advocates. The purpose of this study was to explore information-seeking patterns for childhood cancer through the National Cancer Institute's Cancer Information Service (CIS), a multi-channel, bilingual resource for cancer information. The study team conducted descriptive analyses on secondary data characterizing 1820 caregivers, health professionals, organizations, and members of the general public who contacted the CIS about childhood cancer between September 2018 and June 2022. Almost 80% of inquiries about childhood cancer were initiated by caregivers, followed by the public, health professionals, and organizations. Although English was the primary language used by individuals to reach the CIS when discussing childhood cancer, there were variations in points of access (i.e., telephone, instant messaging, email, social media) across the four user groups. Most childhood cancer inquiries were about staging and treatment, and the primary cancer sites discussed by CIS users were neurologic or brain, hematologic, and musculoskeletal cancers. Discussion topics included managing and coping with cancer, clinical trials, and treatment side effects. Just over half (54%) of CIS contacts about childhood cancer resulted in a health professional referral. Findings provide direction for the CIS and other public health organizations to deliver, prioritize, and tailor their services to support the information needs of childhood cancer survivors and their families-as well as those who care and advocate for them-who may have a significant need for credible cancer information.

Rare pathogenic variants in WNK3 cause X-linked intellectual disability.

PURPOSE: WNK3 kinase (PRKWNK3) has been implicated in the development and function of the brain via its regulation of the cation-chloride cotransporters, but the role of WNK3 in human development is unknown. METHOD: We ascertained exome or genome sequences of individuals with rare familial or sporadic forms of intellectual disability (ID). RESULTS: We identified a total of 6 different maternally-inherited, hemizygous, 3 loss-of-function or 3 pathogenic missense variants (p.Pro204Arg, p.Leu300Ser, p.Glu607Val) in WNK3 in 14 male individuals from 6 unrelated families. Affected individuals had ID with variable presence of epilepsy and structural brain defects. WNK3 variants cosegregated with the disease in 3 different families with multiple affected individuals. This included 1 large family previously diagnosed with X-linked Prieto syndrome. WNK3 pathogenic missense variants localize to the catalytic domain and impede the inhibitory phosphorylation of the neuronal-specific chloride cotransporter KCC2 at threonine 1007, a site critically regulated during the development of synaptic inhibition. CONCLUSION: Pathogenic WNK3 variants cause a rare form of human X-linked ID with variable epilepsy and structural brain abnormalities and implicate impaired phospho-regulation of KCC2 as a pathogenic mechanism.

Alignment of practices for data harmonization across multi-center cell therapy trials: a report from the Consortium for Pediatric Cellular Immunotherapy.

Immune effector cell (IEC) therapies have revolutionized our approach to relapsed B-cell malignancies, and interest in the investigational use of IECs is rapidly expanding into other diseases. Current challenges in the analysis of IEC therapies include small sample sizes, limited access to clinical trials and a paucity of predictive biomarkers of efficacy and toxicity associated with IEC therapies. Retrospective and prospective multi-center cell therapy trials can assist in overcoming these barriers through harmonization of clinical endpoints and correlative assays for immune monitoring, allowing additional cross-trial analysis to identify biomarkers of failure and success. The Consortium for Pediatric Cellular Immunotherapy (CPCI) offers a unique platform to address the aforementioned challenges by delivering cutting-edge cell and gene therapies for children through multi-center clinical trials. Here the authors discuss some of the important pre-analytic variables, such as biospecimen collection and initial processing procedures, that affect biomarker assays commonly used in IEC trials across participating CPCI sites. The authors review the recent literature and provide data to support recommendations for alignment and standardization of practices that can affect flow cytometry assays measuring immune effector function as well as interpretation of cytokine/chemokine data. The authors also identify critical gaps that often make parallel comparisons between trials difficult or impossible.

Unique Spatial Integration in Mouse Primary Visual Cortex and Higher Visual Areas.

Neurons in the visual system integrate over a wide range of spatial scales. This diversity is thought to enable both local and global computations. To understand how spatial information is encoded across the mouse visual system, we use two-photon imaging to measure receptive fields (RFs) and size-tuning in primary visual cortex (V1) and three downstream higher visual areas (HVAs: LM (lateromedial), AL (anterolateral), and PM (posteromedial)) in mice of both sexes. Neurons in PM, compared with V1 or the other HVAs, have significantly larger RF sizes and less surround suppression, independent of stimulus eccentricity or contrast. To understand how this specialization of RFs arises in the HVAs, we measured the spatial properties of V1 inputs to each area. Spatial integration of V1 axons was remarkably similar across areas and significantly different from the tuning of neurons in their target HVAs. Thus, unlike other visual features studied in this system, specialization of spatial integration in PM cannot be explained by specific projections from V1 to the HVAs. Further, the differences in RF properties could not be explained by differences in convergence of V1 inputs to the HVAs. Instead, our data suggest that distinct inputs from other areas or connectivity within PM may support the area's unique ability to encode global features of the visual scene, whereas V1, LM, and AL may be more specialized for processing local features.SIGNIFICANCE STATEMENT Surround suppression is a common feature of visual processing whereby large stimuli are less effective at driving neuronal responses than smaller stimuli. This is thought to enhance efficiency in the population code and enable higher-order processing of visual information, such as figure-ground segregation. However, this comes at the expense of global computations. Here we find that surround suppression is not equally represented across mouse visual areas: primary visual cortex has substantially more surround suppression than higher visual areas, and one higher area has significantly less suppression than two others examined, suggesting that these areas have distinct functional roles. Thus, we have identified a novel dimension of specialization in the mouse visual cortex that may enable both local and global computations.

Artificial night light and anthropogenic noise interact to influence bird abundance over a continental scale.

The extent of artificial night light and anthropogenic noise (i.e., "light" and "noise") impacts is global and has the capacity to threaten species across diverse ecosystems. Existing research involving impacts of light or noise has primarily focused on noise or light alone and single species; however, these stimuli often co-occur and little is known about how co-exposure influences wildlife and if and why species may vary in their responses. Here, we had three aims: (1) to investigate species-specific responses to light, noise, and the interaction between the two using a spatially explicit approach to model changes in abundance of 140 prevalent bird species across North America, (2) to investigate responses to the interaction between light exposure and night length, and (3) to identify functional traits and habitat affiliations that explain variation in species-specific responses to these sensory stimuli with phylogenetically informed models. We found species that responded to noise exposure generally decreased in abundance, and the additional presence of light interacted synergistically with noise to exacerbate its negative effects. Moreover, the interaction revealed negative emergent responses for several species that only reacted when light and noise co-occurred. Additionally, an interaction between light and night length revealed 47 species increased in abundance with light exposure during longer nights. In addition to modifying behavior with optimal temperature and potential foraging opportunities, birds might be attracted to light, yet suffer inadvertent physiological consequences. The trait that most strongly related to avian response to light and noise was habitat affiliation. Specifically, species that occupy closed habitat were less tolerant of both sensory stressors compared to those that occupy open habitat. Further quantifying the contexts and intrinsic traits that explain how species respond to noise and light will be fundamental to understanding the ecological consequences of a world that is ever louder and brighter.

False-Negative Rate of Direct Immunofluorescence on Lower Extremities in Bullous Pemphigoid.

ABSTRACT: Bullous pemphigoid (BP) is the most common autoimmune blistering disorder of the skin. It is typified by tense blisters with a subepidermal split and mixed dermal inflammatory infiltrate on histology. Biopsy of the perilesional skin for direct immunofluorescence (DIF) has become the gold standard in the diagnosis of BP. Currently there is a pervasive clinical opinion that the lower extremity is a site with a high false-negative rate (FNR) for DIF in the diagnosis of BP. This notion is primarily based on 2 early studies from the 1980s without more recent confirmatory studies. To readdress this question regarding the lower extremities, a retrospective study from 2012 to 2018 was performed in our institution that evaluated the FNR of DIF by an anatomical site in the diagnosis of BP. Cases of BP were identified using standard criteria (clinical and histological data reviewed in cases with negative DIF), and overall, 79 patients were included in the study. A total of 4 false-negative DIF biopsies were verified. Two negative DIF were from the lower extremity yielding a FNR of 10% compared with 4% on the trunk and 3% from the upper extremity, with no statistically significant difference by anatomical sites. Our study fails to demonstrate a high FNR of DIF from the lower extremity in the diagnosis of BP.

Variants in HNRNPH2 on the X Chromosome Are Associated with a Neurodevelopmental Disorder in Females.

Via whole-exome sequencing, we identified six females from independent families with a common neurodevelopmental phenotype including developmental delay, intellectual disability, autism, hypotonia, and seizures, all with de novo predicted deleterious variants in the nuclear localization signal of Heterogeneous Nuclear Ribonucleoprotein H2, encoded by HNRNPH2, a gene located on the X chromosome. Many of the females also have seizures, psychiatric co-morbidities, and orthopedic, gastrointestinal, and growth problems as well as common dysmorphic facial features. HNRNPs are a large group of ubiquitous proteins that associate with pre-mRNAs in eukaryotic cells to produce a multitude of alternatively spliced mRNA products during development and play an important role in controlling gene expression. The failure to identify affected males, the severity of the neurodevelopmental phenotype in females, and the essential role of this gene suggests that male conceptuses with these variants may not be viable.

Germline Loss-of-Function Mutations in EPHB4 Cause a Second Form of Capillary Malformation-Arteriovenous Malformation (CM-AVM2) Deregulating RAS-MAPK Signaling.

BACKGROUND: Most arteriovenous malformations (AVMs) are localized and occur sporadically. However, they also can be multifocal in autosomal-dominant disorders, such as hereditary hemorrhagic telangiectasia and capillary malformation (CM)-AVM. Previously, we identified RASA1 mutations in 50% of patients with CM-AVM. Herein we studied non-RASA1 patients to further elucidate the pathogenicity of CMs and AVMs. METHODS: We conducted a genome-wide linkage study on a CM-AVM family. Whole-exome sequencing was also performed on 9 unrelated CM-AVM families. We identified a candidate gene and screened it in a large series of patients. The influence of several missense variants on protein function was also studied in vitro. RESULTS: We found evidence for linkage in 2 loci. Whole-exome sequencing data unraveled 4 distinct damaging variants in EPHB4 in 5 families that cosegregated with CM-AVM. Overall, screening of EPHB4 detected 47 distinct mutations in 54 index patients: 27 led to a premature stop codon or splice-site alteration, suggesting loss of function. The other 20 are nonsynonymous variants that result in amino acid substitutions. In vitro expression of several mutations confirmed loss of function of EPHB4. The clinical features included multifocal CMs, telangiectasias, and AVMs. CONCLUSIONS: We found EPHB4 mutations in patients with multifocal CMs associated with AVMs. The phenotype, CM-AVM2, mimics RASA1-related CM-AVM1 and also hereditary hemorrhagic telangiectasia. RASA1-encoded p120RASGAP is a direct effector of EPHB4. Our data highlight the pathogenetic importance of this interaction and indicts EPHB4-RAS-ERK signaling pathway as a major cause for AVMs.

Barrett's oESophagus trial 3 (BEST3): study protocol for a randomised controlled trial comparing the Cytosponge-TFF3 test with usual care to facilitate the diagnosis of oesophageal pre-cancer in primary care patients with chronic acid reflux.

BACKGROUND: Early detection of oesophageal cancer improves outcomes; however, the optimal strategy for identifying patients at increased risk from the pre-cancerous lesion Barrett's oesophagus (BE) is not clear. The Cytosponge, a novel non-endoscopic sponge device, combined with the biomarker Trefoil Factor 3 (TFF3) has been tested in four clinical studies. It was found to be safe, accurate and acceptable to patients. The aim of the BEST3 trial is to evaluate if the offer of a Cytosponge-TFF3 test in primary care for patients on long term acid suppressants leads to an increase in the number of patients diagnosed with BE. METHODS: The BEST3 trial is a pragmatic multi-site cluster-randomised controlled trial set in primary care in England. Approximately 120 practices will be randomised 1:1 to either the intervention arm, invitation to a Cytosponge-TFF3 test, or the control arm usual care. Inclusion criteria are men and women aged 50 or over with records of at least 6 months of prescriptions for acid-suppressants in the last year. Patients in the intervention arm will receive an invitation to have a Cytosponge-TFF3 test in their general practice. Patients with a positive TFF3 test will receive an invitation for an upper gastro-intestinal endoscopy at their local hospital-based endoscopy clinic to test for BE. The primary objective is to compare histologically confirmed BE diagnosis between the intervention and control arms to determine whether the offer of the Cytosponge-TFF3 test in primary care results in an increase in BE diagnosis within 12 months of study entry. DISCUSSION: The BEST3 trial is a well-powered pragmatic trial testing the use of the Cytosponge-TFF3 test in the same population that we envisage it being used in clinical practice. The data generated from this trial will enable NICE and other clinical bodies to decide whether this test is suitable for routine clinical use. TRIAL REGISTRATION: This trial was prospectively registered with the ISRCTN Registry on 19/01/2017, trial number ISRCTN68382401 .

Gain-of-function variants in the ODC1 gene cause a syndromic neurodevelopmental disorder associated with macrocephaly, alopecia, dysmorphic features, and neuroimaging abnormalities.

Polyamines serve a number of vital functions in humans, including regulation of cellular proliferation, intracellular signaling, and modulation of ion channels. Ornithine decarboxylase 1 (ODC1) is the rate-limiting enzyme in endogenous polyamine synthesis. In this report, we present four patients with a distinct neurometabolic disorder associated with de novo heterozygous, gain-of-function variants in the ODC1 gene. This disorder presents with global developmental delay, ectodermal abnormalities including alopecia, absolute or relative macrocephaly, and characteristic facial dysmorphisms. Neuroimaging variably demonstrates white matter abnormalities, prominent Virchow-Robin spaces, periventricular cysts, and abnormalities of the corpus callosum. Plasma clinical metabolomics analysis demonstrates elevation of N-acetylputrescine, the acetylated form of putrescine, with otherwise normal polyamine levels. Therapies aimed at reducing putrescine levels, including ODC1 inhibitors, dietary interventions, and antibiotics to reduce polyamine production by gastrointestinal flora could be considered as disease-modifying therapies. As the ODC1 gene has been implicated in neoplasia, cancer surveillance may be important in this disorder.

Loss-of-function variants in NFIA provide further support that NFIA is a critical gene in 1p32-p31 deletion syndrome: A four patient series.

The association between 1p32-p31 contiguous gene deletions and a distinct phenotype that includes anomalies of the corpus callosum, ventriculomegaly, developmental delay, seizures, and dysmorphic features has been long recognized and described. Recently, the observation of overlapping phenotypes in patients with chromosome translocations that disrupt NFIA (Nuclear factor I/A), a gene within this deleted region, and NFIA intragenic deletions has led to the hypothesis that NFIA is a critical gene within this region. The wide application and increasing accessibility of whole exome sequencing (WES) has helped identify new cases to support this hypothesis. Here, we describe four patients with loss-of-function variants in the NFIA gene identified through WES. The clinical presentation of these patients significantly overlaps with the phenotype described in previously reported cases of 1p32-p31 deletion syndrome, NFIA gene disruptions and intragenic NFIA deletions. Our cohort includes a mother and daughter as well as an unrelated individual who share the same nonsense variant (c.205C>T, p.Arg69Ter; NM_001145512.1). We also report a patient with a frameshift NFIA variant (c.159_160dupCC, p.Gln54ProfsTer49). We have compared published cases of 1p32-p31 microdeletion syndrome, translocations resulting in NFIA gene disruption, intragenic deletions, and loss-of-function mutations (including our four patients) to reveal that abnormalities of the corpus callosum, ventriculomegaly/hydrocephalus, macrocephaly, Chiari I malformation, dysmorphic features, developmental delay, hypotonia, and urinary tract defects are common findings. The consistent overlap in clinical presentation provides further evidence of the critical role of NFIA haploinsufficiency in the development of the 1p32-p31 microdeletion syndrome phenotype.

Using Lean Six Sigma Methodology to Improve Quality of the Anesthesia Supply Chain in a Pediatric Hospital.

Six Sigma and Lean methodologies are effective quality improvement tools in many health care settings. We applied the DMAIC methodology (define, measure, analyze, improve, control) to address deficiencies in our pediatric anesthesia supply chain. We defined supply chain problems by mapping existing processes and soliciting comments from those involved. We used daily distance walked by anesthesia technicians and number of callouts for missing supplies as measurements that we analyzed before and after implementing improvements (anesthesia cart redesign). We showed improvement in the metrics after those interventions were implemented, and those improvements were sustained and thus controlled 1 year after implementation.

Pathogenetics of alveolar capillary dysplasia with misalignment of pulmonary veins.

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal lung developmental disorder caused by heterozygous point mutations or genomic deletion copy-number variants (CNVs) of FOXF1 or its upstream enhancer involving fetal lung-expressed long noncoding RNA genes LINC01081 and LINC01082. Using custom-designed array comparative genomic hybridization, Sanger sequencing, whole exome sequencing (WES), and bioinformatic analyses, we studied 22 new unrelated families (20 postnatal and two prenatal) with clinically diagnosed ACDMPV. We describe novel deletion CNVs at the FOXF1 locus in 13 unrelated ACDMPV patients. Together with the previously reported cases, all 31 genomic deletions in 16q24.1, pathogenic for ACDMPV, for which parental origin was determined, arose de novo with 30 of them occurring on the maternally inherited chromosome 16, strongly implicating genomic imprinting of the FOXF1 locus in human lungs. Surprisingly, we have also identified four ACDMPV families with the pathogenic variants in the FOXF1 locus that arose on paternal chromosome 16. Interestingly, a combination of the severe cardiac defects, including hypoplastic left heart, and single umbilical artery were observed only in children with deletion CNVs involving FOXF1 and its upstream enhancer. Our data demonstrate that genomic imprinting at 16q24.1 plays an important role in variable ACDMPV manifestation likely through long-range regulation of FOXF1 expression, and may be also responsible for key phenotypic features of maternal uniparental disomy 16. Moreover, in one family, WES revealed a de novo missense variant in ESRP1, potentially implicating FGF signaling in the etiology of ACDMPV.

Climate change is projected to outpace rates of niche change in grasses.

Climate change may soon threaten much of global biodiversity, especially if species cannot adapt to changing climatic conditions quickly enough. A critical question is how quickly climatic niches change, and if this speed is sufficient to prevent extinction as climates warm. Here, we address this question in the grass family (Poaceae). Grasses are fundamental to one of Earth's most widespread biomes (grasslands), and provide roughly half of all calories consumed by humans (including wheat, rice, corn and sorghum). We estimate rates of climatic niche change in 236 species and compare these with rates of projected climate change by 2070. Our results show that projected climate change is consistently faster than rates of niche change in grasses, typically by more than 5000-fold for temperature-related variables. Although these results do not show directly what will happen under global warming, they have troubling implications for a major biome and for human food resources.

Bohring-Opitz syndrome (BOS) with a new ASXL1 pathogenic variant: Review of the most prevalent molecular and phenotypic features of the syndrome.

Bohring-Opitz syndrome (BOS) was first described by Bohring et al. [1999]. The authors reported four cases which had several features in common, including a prominent metopic suture, hypertelorism, exophthalmos, cleft lip and palate, limb anomalies, as well as difficulty feeding with severe developmental delays. In almost 50% of cases that meet the clinical criteria for BOS, de novo frameshift and nonsense mutations in the ASXL1 gene have been detected, suggesting that loss of function of this gene is a major cause. We report on the clinical characterization of one young female patient who was evaluated because of severe developmental delays, failure to thrive, and multiple minor anomalies and was clinically diagnosed with BOS. Whole exome sequencing analysis detected one novel disruptive frameshift mutation in the ASXL1 gene and we were also able to confirm the presence of two CFTR mutations associated with her chronic pancreatitis with acute severe breakthrough attacks requiring multiple ICU admissions. This latter complication of pancreatitis further contributed to the complexity of the clinical presentation and represents an independent genetic finding. Our case report emphasizes the importance of highly specific phenotypic characterization of patients with complex phenotypes before proceeding with molecular studies. That approach will lead to more accurate molecular data interpretation and better clinical genetic diagnosis, particularly for those patients with rare, difficult-to-diagnose disorders.

New Insights into the Genetics of Fetal Megacystis: ACTG2 Mutations, Encoding γ-2 Smooth Muscle Actin in Megacystis Microcolon Intestinal Hypoperistalsis Syndrome (Berdon Syndrome).

OBJECTIVE: To identify the molecular basis for prenatally suspected cases of megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) (MIM 249210) in 3 independent families with clinical and radiographic evidence of MMIHS. METHODS: Whole-exome sequencing (WES) and Sanger sequencing of the ACTG2 gene. RESULTS: We identified a novel heterozygous de novo missense variant in ACTG2 c.770G>A (p.Arg257His) encoding x03B3;-2 smooth muscle actin (ACTG2) in 2 siblings with MMIHS, suggesting gonadal mosaicism of one of the parents. Two additional de novo missense variants (p.Arg257Cys and p.Arg178His) in ACTG2 were identified in 2 additional MMHIS patients. All of our patients had evidence of fetal megacystis and a normal or slightly increased amniotic fluid volume. Additional findings included bilateral renal hydronephrosis, an enlarged fetal stomach, and transient dilated bowel loops. ACTG2 immunostaining of the intestinal tissue showed an altered muscularis propria, a markedly thinned longitudinal muscle layer, and a reduced amount and abnormal distribution of ACTG2. CONCLUSION: Our study demonstrates that de novo mutations in ACTG2 are a cause of fetal megacystis in MMIHS and that gonadal mosaicism may be present in a subset of cases. These findings have implications for the counseling of families with a diagnosis of fetal megacystis with a preserved amniotic fluid volume and associated gastrointestinal findings.

The usefulness of whole-exome sequencing in routine clinical practice.

PURPOSE: Reports of the use of whole-exome sequencing in clinical practice are limited. We report our experience with whole-exome sequencing in 115 patients in a single center and evaluate its feasibility and clinical usefulness in clinical care. METHODS: Whole-exome sequencing was utilized based on the judgment of three clinical geneticists. We describe age, gender, ethnicity, consanguinity, indication for testing, family history, insurance, laboratory results, clinician interpretation of results, and impact on patient care. RESULTS: Most patients were children (78.9%). The most common indications for testing were birth defects (24.3%) and developmental delay (25.2%). We identified four new candidate human disease genes and possibly expanded the disease phenotypes associated with five different genes. Establishing a diagnosis led to discontinuation of additional planned testing in all patients, screening for additional manifestations in eight, altered management in fourteen, novel therapy in two, identification of other familial mutation carriers in five, and reproductive planning in six. CONCLUSION: Our results show that whole-exome sequencing is feasible, has clinical usefulness, and allows timely medical interventions, informed reproductive choices, and avoidance of additional testing. Our results also suggest phenotype expansion and identification of new candidate disease genes that would have been impossible to diagnose by other targeted testing methods.