Molecular and Phenotypic Characterization of the RORB-Related Disorder.

BACKGROUND AND OBJECTIVES: Heterozygous variants in RAR-related orphan receptor B (RORB) have recently been associated with susceptibility to idiopathic generalized epilepsy. However, few reports have been published so far describing pathogenic variants of this gene in patients with epilepsy and intellectual disability (ID). In this study, we aimed to delineate the epilepsy phenotype associated with RORB pathogenic variants and to provide arguments in favor of the pathogenicity of variants. METHODS: Through an international collaboration, we analyzed seizure characteristics, EEG data, and genotypes of a cohort of patients with heterozygous variants in RORB. To gain insight into disease mechanisms, we performed ex vivo cortical electroporation in mouse embryos of 5 selected variants, 2 truncating and 3 missense, and evaluated on expression and quantified changes in axonal morphology. RESULTS: We identified 35 patients (17 male, median age 10 years, range 2.5-23 years) carrying 32 different heterozygous variants in RORB, including 28 single-nucleotide variants or small insertions/deletions (12 missense, 12 frameshift or nonsense, 2 splice-site variants, and 2 in-frame deletions), and 4 microdeletions; de novo in 18 patients and inherited in 10. Seizures were reported in 31/35 (89%) patients, with a median age at onset of 3 years (range 4 months-12 years). Absence seizures occurred in 25 patients with epilepsy (81%). Nineteen patients experienced a single seizure type: absences, myoclonic absences, or absences with eyelid myoclonia and focal seizures. Nine patients had absence seizures combined with other generalized seizure types. One patient had presented with absences associated with photosensitive occipital seizures. Three other patients had generalized tonic-clonic seizures without absences. ID of variable degree was observed in 85% of the patients. Expression studies in cultured neurons showed shorter axons for the 5 tested variants, both truncating and missense variants, supporting an impaired protein function. DISCUSSION: In most patients, the phenotype of the RORB-related disorder associates absence seizures with mild-to-moderate ID. In silico and in vitro evaluation of the variants in our cohort, including axonal morphogenetic experiments in cultured neurons, supports their pathogenicity, showing a hypomorphic effect.

Symptoms and signs of lung cancer prior to diagnosis: case-control study using electronic health records from ambulatory care within a large US-based tertiary care centre.

OBJECTIVE: Lung cancer is the most common cause of cancer-related death in the USA. While most patients are diagnosed following symptomatic presentation, no studies have compared symptoms and physical examination signs at or prior to diagnosis from electronic health records (EHRs) in the USA. We aimed to identify symptoms and signs in patients prior to diagnosis in EHR data. DESIGN: Case-control study. SETTING: Ambulatory care clinics at a large tertiary care academic health centre in the USA. PARTICIPANTS, OUTCOMES: We studied 698 primary lung cancer cases in adults diagnosed between 1 January 2012 and 31 December 2019, and 6841 controls matched by age, sex, smoking status and type of clinic. Coded and free-text data from the EHR were extracted from 2 years prior to diagnosis date for cases and index date for controls. Univariate and multivariable conditional logistic regression were used to identify symptoms and signs associated with lung cancer at time of diagnosis, and 1, 3, 6 and 12 months before the diagnosis/index dates. RESULTS: Eleven symptoms and signs recorded during the study period were associated with a significantly higher chance of being a lung cancer case in multivariable analyses. Of these, seven were significantly associated with lung cancer 6 months prior to diagnosis: haemoptysis (OR 3.2, 95% CI 1.9 to 5.3), cough (OR 3.1, 95% CI 2.4 to 4.0), chest crackles or wheeze (OR 3.1, 95% CI 2.3 to 4.1), bone pain (OR 2.7, 95% CI 2.1 to 3.6), back pain (OR 2.5, 95% CI 1.9 to 3.2), weight loss (OR 2.1, 95% CI 1.5 to 2.8) and fatigue (OR 1.6, 95% CI 1.3 to 2.1). CONCLUSIONS: Patients diagnosed with lung cancer appear to have symptoms and signs recorded in the EHR that distinguish them from similar matched patients in ambulatory care, often 6 months or more before diagnosis. These findings suggest opportunities to improve the diagnostic process for lung cancer.

Mechanism of KMT5B haploinsufficiency in neurodevelopment in humans and mice.

Pathogenic variants in KMT5B, a lysine methyltransferase, are associated with global developmental delay, macrocephaly, autism, and congenital anomalies (OMIM# 617788). Given the relatively recent discovery of this disorder, it has not been fully characterized. Deep phenotyping of the largest (n = 43) patient cohort to date identified that hypotonia and congenital heart defects are prominent features that were previously not associated with this syndrome. Both missense variants and putative loss-of-function variants resulted in slow growth in patient-derived cell lines. KMT5B homozygous knockout mice were smaller in size than their wild-type littermates but did not have significantly smaller brains, suggesting relative macrocephaly, also noted as a prominent clinical feature. RNA sequencing of patient lymphoblasts and Kmt5b haploinsufficient mouse brains identified differentially expressed pathways associated with nervous system development and function including axon guidance signaling. Overall, we identified additional pathogenic variants and clinical features in KMT5B-related neurodevelopmental disorder and provide insights into the molecular mechanisms of the disorder using multiple model systems.

Intersectionality of Systemic Disadvantage on Mortality and Care Following TBI.

BACKGROUND: People of color (POC), especially those who also hold social identities associated with disadvantage (non-English-speaking, female, older, lower socioeconomic level), continue to be underserved in the health system, which can result in poorer care and worsened health outcomes. Most disparity research in traumatic brain injury (TBI) focuses on the impact of single factors, which misses the compounding effect of belonging to multiple historically marginalized groups. OBJECTIVE: To examine the intersectional impact of multiple social identities vulnerable to systemic disadvantage following TBI on mortality, opioid usage during acute hospitalization, and discharge location. METHODS: Retrospective observational design utilizing electronic health records merged with local trauma registry data. Patient groups were defined by race and ethnicity (POC or non-Hispanic White), age, sex, type of insurance, and primary language (English-speaking vs non-English-speaking). Latent class analysis (LCA) was performed to identify clusters of systemic disadvantage. Outcome measures were then assessed across latent classes and tested for differences. RESULTS: Over an 8-year period, 10 809 admissions with TBI occurred (37% POC). LCA identified a 4-class model. Groups with more systemic disadvantage had higher rates of mortality. Classes with older populations had lower rates of opioid administration and were less likely to discharge to inpatient rehabilitation following acute care. Sensitivity analyses examining additional indicators of TBI severity demonstrated that the younger group with more systemic disadvantage had more severe TBI. Controlling for more indicators of TBI severity changed statistical significance in mortality for younger groups. CONCLUSION: Results demonstrate significant health inequities in the mortality and access to inpatient rehabilitation following TBI along with higher rates of severe injury in younger patients with more social disadvantages. While many inequities may be related to systemic racism, our findings suggested an additive, deleterious effect for patients who belonged to multiple historically disadvantaged groups. Further research is needed to understand the role of systemic disadvantage for individuals with TBI within the healthcare system.

Payment strategies for behavioral health integration in hospital-affiliated and non-hospital-affiliated primary care practices.

Recent value-based payment reforms in the U.S. called for empirical data on how primary care practices of varying characteristics fund their integrated behavioral health services. To describe payment strategies used by U.S. primary care practices to fund behavioral health integration and compare strategies between practices with and without hospital affiliation.Baseline data were used and collected from 44 practices participating in a cluster-randomized, pragmatic trial of behavioral health integration. Data included practice characteristics and payment strategies-fee-for-service payment, pay-for-performance incentives, grants, and graduate medical education funds. Descriptive and comparative analyses using Fisher's exact tests and independent T-tests were conducted. The sample had 26 (59.1%) hospital-affiliated (hospital/health system-owned, academic medical centers and hospital-affiliated practices) and 18 (40.9%) non-hospital-affiliated practices (community health centers/federally qualified health centers and privately-owned practices). Most practices (88.6%) received payments through fee-for-service; 63.6% received pay-for-performance incentives; 31.8% received grant funds. Collaborative Care Management billing (CPT) codes were used in six (13.6%) practices. Over half (53.8%) of hospital-affiliated practices funded their behavioral health services through fee-for-service and pay-for-performance incentives only, as opposed to two-thirds (66.7%) of non-hospital-affiliated practices required additional support from grants and/or general medical education funds. Primary care practices support behavioral health integration through diverse payment strategies. More hospital-affiliated practices compared to non-hospital-affiliated practices funded integrated behavioral health services through fee-for-service and pay-for-performance incentives. Practices without hospital affiliation relied on multiple funding streams including grants and/or general medical education funds, suggesting their approach to financial sustainment may be more precarious or challenging, compared to hospital-affiliated practices.

SEMA6B variants cause intellectual disability and alter dendritic spine density and axon guidance.

Intellectual disability (ID) is a neurodevelopmental disorder frequently caused by monogenic defects. In this study, we collected 14 SEMA6B heterozygous variants in 16 unrelated patients referred for ID to different centers. Whereas, until now, SEMA6B variants have mainly been reported in patients with progressive myoclonic epilepsy, our study indicates that the clinical spectrum is wider and also includes non-syndromic ID without epilepsy or myoclonus. To assess the pathogenicity of these variants, selected mutated forms of Sema6b were overexpressed in Human Embryonic Kidney 293T (HEK293T) cells and in primary neuronal cultures. shRNAs targeting Sema6b were also used in neuronal cultures to measure the impact of the decreased Sema6b expression on morphogenesis and synaptogenesis. The overexpression of some variants leads to a subcellular mislocalization of SEMA6B protein in HEK293T cells and to a reduced spine density owing to loss of mature spines in neuronal cultures. Sema6b knockdown also impairs spine density and spine maturation. In addition, we conducted in vivo rescue experiments in chicken embryos with the selected mutated forms of Sema6b expressed in commissural neurons after knockdown of endogenous SEMA6B. We observed that expression of these variants in commissural neurons fails to rescue the normal axon pathway. In conclusion, identification of SEMA6B variants in patients presenting with an overlapping phenotype with ID and functional studies highlight the important role of SEMA6B in neuronal development, notably in spine formation and maturation and in axon guidance. This study adds SEMA6B to the list of ID-related genes.

Expanding the genotypic and phenotypic spectrum in a diverse cohort of 104 individuals with Wiedemann-Steiner syndrome.

Wiedemann-Steiner syndrome (WSS) is an autosomal dominant disorder caused by monoallelic variants in KMT2A and characterized by intellectual disability and hypertrichosis. We performed a retrospective, multicenter, observational study of 104 individuals with WSS from five continents to characterize the clinical and molecular spectrum of WSS in diverse populations, to identify physical features that may be more prevalent in White versus Black Indigenous People of Color individuals, to delineate genotype-phenotype correlations, to define developmental milestones, to describe the syndrome through adulthood, and to examine clinicians' differential diagnoses. Sixty-nine of the 82 variants (84%) observed in the study were not previously reported in the literature. Common clinical features identified in the cohort included: developmental delay or intellectual disability (97%), constipation (63.8%), failure to thrive (67.7%), feeding difficulties (66.3%), hypertrichosis cubiti (57%), short stature (57.8%), and vertebral anomalies (46.9%). The median ages at walking and first words were 20 months and 18 months, respectively. Hypotonia was associated with loss of function (LoF) variants, and seizures were associated with non-LoF variants. This study identifies genotype-phenotype correlations as well as race-facial feature associations in an ethnically diverse cohort, and accurately defines developmental trajectories, medical comorbidities, and long-term outcomes in individuals with WSS.

Partners in care.

This festschrift contribution, written for my colleague and mentor John Graham, reflects on geneticist-genetic counselor interactions in clinical care, samples of alternative models of care for pediatric and general genetic counselors, and avenues for expanding access to genetic healthcare services utilizing genetic counselors.

SPEN haploinsufficiency causes a neurodevelopmental disorder overlapping proximal 1p36 deletion syndrome with an episignature of X chromosomes in females.

Deletion 1p36 (del1p36) syndrome is the most common human disorder resulting from a terminal autosomal deletion. This condition is molecularly and clinically heterogeneous. Deletions involving two non-overlapping regions, known as the distal (telomeric) and proximal (centromeric) critical regions, are sufficient to cause the majority of the recurrent clinical features, although with different facial features and dysmorphisms. SPEN encodes a transcriptional repressor commonly deleted in proximal del1p36 syndrome and is located centromeric to the proximal 1p36 critical region. Here, we used clinical data from 34 individuals with truncating variants in SPEN to define a neurodevelopmental disorder presenting with features that overlap considerably with those of proximal del1p36 syndrome. The clinical profile of this disease includes developmental delay/intellectual disability, autism spectrum disorder, anxiety, aggressive behavior, attention deficit disorder, hypotonia, brain and spine anomalies, congenital heart defects, high/narrow palate, facial dysmorphisms, and obesity/increased BMI, especially in females. SPEN also emerges as a relevant gene for del1p36 syndrome by co-expression analyses. Finally, we show that haploinsufficiency of SPEN is associated with a distinctive DNA methylation episignature of the X chromosome in affected females, providing further evidence of a specific contribution of the protein to the epigenetic control of this chromosome, and a paradigm of an X chromosome-specific episignature that classifies syndromic traits. We conclude that SPEN is required for multiple developmental processes and SPEN haploinsufficiency is a major contributor to a disorder associated with deletions centromeric to the previously established 1p36 critical regions.

Evaluating co-created patient-facing materials to increase understanding of genetic test results.

Patients often have difficulty understanding genetic test reports. Technical language and jargon can impede comprehension and limit patients using results to act on findings. One potential way to improve patient understanding of genetic test reports is to provide patient-facing materials. This study aimed to examine understandability and actionability of co-created patient-facing materials for genetic test results in a research context. We combined interprofessional perspectives and patient engagement to co-create patient-facing materials for patients undergoing research genetic testing for congenital hypogonadotropic hypogonadism (Kallmann syndrome). The iterative development process was guided by principles of health literacy and human-centered design (i.e., design thinking). Readability was assessed using eight validated algorithms. Patients and parents evaluated materials using a web-based survey. The gold standard Patient Education Materials Assessment Tool for print materials (PEMAT-P) was employed to measure understandability (content, style, use of numbers, organization, design, use of visual aids) and actionability. PEMAT-P scores >80% were considered high quality. Results were analyzed descriptively and correlations performed to identify relationships between education/health literacy and PEMAT-P ratings. A consensus score of eight algorithms indicated the materials were an 8th -9th grade reading level. Our findings are consistent with the U.S. Department of Health and Human Services 'average difficulty' classification (i.e., 7th-9th grade). In total, 61 patients/parents evaluated the materials. 'Visual Aids' received the lowest mean PEMAT-P rating (89%). All other parameters scored 90%-97%. PEMAT-P scores did not differ according to educational attainment (less than college vs. college or more, p = 0.28). Participants with adequate health literacy were more likely to approve of the 'organization' of information (p < 0.05). Respondents with low health literacy had more favorable views of 'visual aids' (p < 0.01). Involving patients in a co-creation process can produce high-quality patient-facing materials that are easier to understand.

Defining and measuring core processes and structures in integrated behavioral health in primary care: a cross-model framework.

A movement towards integrated behavioral health (IBH) in primary care continues to grow, among an accumulating evidence base for its effectiveness for improving care. However, healthcare organizations struggle to navigate where to target their limited resources for improving integration. We evaluated a cross-model framework of IBH core processes and structures. We used a mixed-methods approach for evaluation of the framework, which included (a) an evaluation survey of national experts and stakeholders, (b) crosswalks with common IBH measures, and (c) a real-world usability test. Five core IBH principles, mapping to 25 processes, and nine clinic structures were defined. Survey responses from 29 IBH domain and policy experts and stakeholders resulted in uniformly high ratings of importance and variable levels of feasibility for measurement, particularly with respect to electronic health record (EHR) systems. A real-world usability test resulted in good uptake and use of the framework across a state-wide effort. An IBH Cross-Model Framework of core principles, processes, and structures generated good acceptability and showed good real-world utility in a state-wide effort to improve IBH across disparate levels of integration in diverse primary care settings. Findings identify feasible areas of measurement, particularly with EHR systems. Next steps include testing the relationship between the individual framework components and patient outcomes to help guide clinics towards prioritizing efforts focused on improving integration.

Correction: GATAD2B-associated neurodevelopmental disorder (GAND): clinical and molecular insights into a NuRD-related disorder.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

GATAD2B-associated neurodevelopmental disorder (GAND): clinical and molecular insights into a NuRD-related disorder.

PURPOSE: Determination of genotypic/phenotypic features of GATAD2B-associated neurodevelopmental disorder (GAND). METHODS: Fifty GAND subjects were evaluated to determine consistent genotypic/phenotypic features. Immunoprecipitation assays utilizing in vitro transcription-translation products were used to evaluate GATAD2B missense variants' ability to interact with binding partners within the nucleosome remodeling and deacetylase (NuRD) complex. RESULTS: Subjects had clinical findings that included macrocephaly, hypotonia, intellectual disability, neonatal feeding issues, polyhydramnios, apraxia of speech, epilepsy, and bicuspid aortic valves. Forty-one novelGATAD2B variants were identified with multiple variant types (nonsense, truncating frameshift, splice-site variants, deletions, and missense). Seven subjects were identified with missense variants that localized within two conserved region domains (CR1 or CR2) of the GATAD2B protein. Immunoprecipitation assays revealed several of these missense variants disrupted GATAD2B interactions with its NuRD complex binding partners. CONCLUSIONS: A consistent GAND phenotype was caused by a range of genetic variants in GATAD2B that include loss-of-function and missense subtypes. Missense variants were present in conserved region domains that disrupted assembly of NuRD complex proteins. GAND's clinical phenotype had substantial clinical overlap with other disorders associated with the NuRD complex that involve CHD3 and CHD4, with clinical features of hypotonia, intellectual disability, cardiac defects, childhood apraxia of speech, and macrocephaly.

When moments matter: Finding answers with rapid exome sequencing.

BACKGROUND: When time is of the essence in critical care cases, a fast molecular diagnosis is often necessary to help health care providers quickly determine best next steps for treatments, prognosis, and counseling of their patients. In this paper, we present the diagnostic rates and improved quality of life for patients undergoing clinical rapid exome sequencing. METHODS: The clinical histories and results of 41 patients undergoing rapid exome sequencing were retrospectively reviewed. RESULTS: Clinical rapid exome sequencing identified a definitive diagnosis in 13/41 (31.7%) and other relevant findings in 17 of the patients (41.5%). The average time to verbal report was 7 days; to written report was 11 days. CONCLUSIONS: Our observations demonstrate the utility and effectiveness of rapid family-based diagnostic exome sequencing in improving patients care.

De novo variants in PAK1 lead to intellectual disability with macrocephaly and seizures.

Using trio exome sequencing, we identified de novo heterozygous missense variants in PAK1 in four unrelated individuals with intellectual disability, macrocephaly and seizures. PAK1 encodes the p21-activated kinase, a major driver of neuronal development in humans and other organisms. In normal neurons, PAK1 dimers reside in a trans-inhibited conformation, where each autoinhibitory domain covers the kinase domain of the other monomer. Upon GTPase binding via CDC42 or RAC1, the PAK1 dimers dissociate and become activated. All identified variants are located within or close to the autoinhibitory switch domain that is necessary for trans-inhibition of resting PAK1 dimers. Protein modelling supports a model of reduced ability of regular autoinhibition, suggesting a gain of function mechanism for the identified missense variants. Alleviated dissociation into monomers, autophosphorylation and activation of PAK1 influences the actin dynamics of neurite outgrowth. Based on our clinical and genetic data, as well as the role of PAK1 in brain development, we suggest that gain of function pathogenic de novo missense variants in PAK1 lead to moderate-to-severe intellectual disability, macrocephaly caused by the presence of megalencephaly and ventriculomegaly, (febrile) seizures and autism-like behaviour.

A pathogenic CtBP1 missense mutation causes altered cofactor binding and transcriptional activity.

We previously reported a pathogenic de novo p.R342W mutation in the transcriptional corepressor CTBP1 in four independent patients with neurodevelopmental disabilities [1]. Here, we report the clinical phenotypes of seven additional individuals with the same recurrent de novo CTBP1 mutation. Within this cohort, we identified consistent CtBP1-related phenotypes of intellectual disability, ataxia, hypotonia, and tooth enamel defects present in most patients. The R342W mutation in CtBP1 is located within a region implicated in a high affinity-binding cleft for CtBP-interacting proteins. Unbiased proteomic analysis demonstrated reduced interaction of several chromatin-modifying factors with the CtBP1 W342 mutant. Genome-wide transcriptome analysis in human glioblastoma cell lines expressing -CtBP1 R342 (wt) or W342 mutation revealed changes in the expression profiles of genes controlling multiple cellular processes. Patient-derived dermal fibroblasts were found to be more sensitive to apoptosis during acute glucose deprivation compared to controls. Glucose deprivation strongly activated the BH3-only pro-apoptotic gene NOXA, suggesting a link between enhanced cell death and NOXA expression in patient fibroblasts. Our results suggest that context-dependent relief of transcriptional repression of the CtBP1 mutant W342 allele may contribute to deregulation of apoptosis in target tissues of patients leading to neurodevelopmental phenotypes.

Non-Cystic Fibrosis-Related Meconium Ileus: GUCY2C-Associated Disease Discovered through Rapid Neonatal Whole-Exome Sequencing.

Meconium ileus is caused by cystic fibrosis; however, mutations in the GUCY2C gene also cause this disease. We report non-cystic fibrosis meconium ileus in an infant of non-Middle Eastern origin with compound heterozygous mutations in GUCY2C.

Variants in TCF20 in neurodevelopmental disability: description of 27 new patients and review of literature.

PURPOSE: To define the clinical characteristics of patients with variants in TCF20, we describe 27 patients, 26 of whom were identified via exome sequencing. We compare detailed clinical data with 17 previously reported patients. METHODS: Patients were ascertained through molecular testing laboratories performing exome sequencing (and other testing) with orthogonal confirmation; collaborating referring clinicians provided detailed clinical information. RESULTS: The cohort of 27 patients all had novel variants, and ranged in age from 2 to 68 years. All had developmental delay/intellectual disability. Autism spectrum disorders/autistic features were reported in 69%, attention disorders or hyperactivity in 67%, craniofacial features (no recognizable facial gestalt) in 67%, structural brain anomalies in 24%, and seizures in 12%. Additional features affecting various organ systems were described in 93%. In a majority of patients, we did not observe previously reported findings of postnatal overgrowth or craniosynostosis, in comparison with earlier reports. CONCLUSION: We provide valuable data regarding the prognosis and clinical manifestations of patients with variants in TCF20.

Novel pathogenic COX20 variants causing dysarthria, ataxia, and sensory neuropathy.

COX20/FAM36A encodes a mitochondrial complex IV assembly factor important for COX2 activation. Only one homozygous COX20 missense mutation has been previously described in two separate consanguineous families. We report four subjects with features that include childhood hypotonia, areflexia, ataxia, dysarthria, dystonia, and sensory neuropathy. Exome sequencing in all four subjects identified the same novel COX20 variants. One variant affected the splice donor site of intron-one (c.41A>G), while the other variant (c.157+3G>C) affected the splice donor site of intron-two. cDNA and protein analysis indicated that no full-length cDNA or protein was generated. These subjects expand the phenotype associated with COX20 deficiency.

FARS2 mutations presenting with pure spastic paraplegia and lesions of the dentate nuclei.

Mutations in FARS2, the gene encoding the mitochondrial phenylalanine-tRNA synthetase (mtPheRS), have been linked to a range of phenotypes including epileptic encephalopathy, developmental delay, and motor dysfunction. We report a 9-year-old boy with novel compound heterozygous variants of FARS2, presenting with a pure spastic paraplegia syndrome associated with bilateral signal abnormalities in the dentate nuclei. Exome sequencing identified a paternal nonsense variant (Q216X) lacking the catalytic core and anticodon-binding regions, and a maternal missense variant (P136H) possessing partial enzymatic activity. This case confirms and expands the phenotype related to FARS2 mutations with regards to clinical presentation and neuroimaging findings.