This survey study assesses intensivists' and parents' perceptions of the utility of rapid genomic testing for critically ill neonates.

Improved detection sensitivity from combined Long-Range PCR (LR-PCR), Next-Generation Sequencing (NGS), and droplet digital PCR (ddPCR) to identify multiple large-scale mtDNA deletions (MLSMD) and quantify deletion heteroplasmy have introduced clinical interpretation challenges. We sought to evaluate clinical, biochemical, and histopathological phenotypes of a large clinical cohort harboring MLSMD in muscle to better understand their significance across a range of clinical phenotypes. A single-site retrospective study was performed of 212 diagnostic muscle biopsies obtained from patients referred for Primary Mitochondrial Disease (PMD) evaluation with muscle mitochondrial (mt)DNA sequencing performed at our institution, including electronic medical record (EMR) review of symptoms, biochemical results, and Mitochondrial Myopathy Composite Assessment Tool (MM-COAST) scores. MLSMD were identified in 50 of 212 (24%) diagnostic tissue biopsies, and were universally present. in subjects ≥50 years (n = 18/18). In 45 of 50 (90%) subjects with MLSMD, no definitive genetic etiology was identified, despite clinical whole exome sequencing (WES) and/or whole genome sequencing (WGS). MLSMD heteroplasmy levels quantified by ddPCR ranged from 0% to 33%, exceeding 10% heteroplasmy in 5/45 (11%). Subjects with MLSMD (n = 45) were more likely to demonstrate mitochondrial abnormalities on histopathology, upregulation (≥150% of control mean) of one or more electron transport chain (ETC) complex enzyme activities, and reduced citrate synthase indicative of mitochondrial depletion (<60% of control mean) relative to subjects without MLSMD (n = 155). As clinical phenotypes varied across the MLSMD cohort, Bernier diagnostic criteria major/minor symptoms were used to discriminate 13 of 45 subjects with "suspected" PMD having unrevealing WES/WGS results and 32 of 45 subjects scored as "less likely" to have PMD. Relative to the "less likely" cohort, a significantly higher frequency of biochemical and muscle histopathological abnormalities (ragged red and COX negative fibers) were observed in the "suspected" cohort, further supporting a higher index of suspicion for PMD, p < 0.05. MLSMD in skeletal muscle tissue were a common molecular finding (24%) in our cohort and consistently present in subjects ≥50 years. Among those with genetically undiagnosed MLSMD (n = 45), the "suspected" PMD subset (n = 13/45) represent a promising cohort for novel gene discoveries.

Kabuki syndrome (KS) is a multisystem disorder characterized by facial dysmorphic features, growth delays, skeletal anomalies, and variable intellectual disability (ID) due to pathogenic variants in KMT2D and KDM6A. Significant phenotypic variability has been reported in patients with KS. To further characterize the variability observed in the genomic sequencing era, comprehensive genotypic and phenotypic information from 36 patients with KS and likely pathogenic or pathogenic KMT2D or KDM6A variants at the Children's Hospital of Philadelphia (CHOP) was collected. Dysmorphic features, growth restriction, and developmental delays were commonly reported, as expected. Hyperinsulinism (HI) was seen more frequently than in previously published cohorts. Patients diagnosed with HI were more likely to require neonatal intensive care unit admission and feeding tube(s). Intellectual disability was variable in severity and less frequent than previously reported. This study highlights the wide phenotypic spectrum of KS and expands our knowledge of the diagnostic process for KS. This study is limited by potential ascertainment bias as CHOP is a HI Center of Excellence, however, our cohort is unique as many were ascertained with disease-agnostic testing. Screening for hyperinsulinism, including consideration of diagnostic fast, at time of KS diagnosis is warranted to prevent long-term neurologic effects of untreated hypoglycemia.

Purpose: We developed a genome sequencing-based test (Rapid Targeted Analysis of the Genome for Infants [rTAG-I]) to minimize turnaround time while maximizing diagnostic yield and access to rapid sequencing for critically ill infants. We sought to create a system of predicting which infants would have a molecular finding. Methods: We performed a prospective observational study of infants referred for genetics consult who received rTAG-I testing, which analyzes 3183 curated genes with phenotype-agnostic prioritization of pathogenic and likely pathogenic variants. Infants were stratified by perceived likelihood of a diagnostic result and divided into “Likely,” “Uncertain,” and “Not Likely.” We also assessed whether reportable findings correlated with patient phenotypes. Results: We identified reportable findings in 133/400 (33%) infants. Access to rapid testing increased from 1% to 20% of all infants hospitalized in the neonatal/infant intensive care unit and cardiac intensive care unit, with a median turnaround time of 4.9 days. rTAG-I performed as well as exome/genome sequencing. Clinically associated results were identified in 59% of the “Likely” group and 9% of the “Not Likely” group. Conclusion: rTAG-I produced a high rate of reportable findings with a rapid turnaround time. Our ability to predict infants who would benefit most was imperfect, reinforcing that broad access to genome-based testing is still required.

The need for education focusing on genomic technologies and variant interpretation for genetic counseling trainees has prompted genetic counseling training programs to alter their curricula to incorporate this content. Given students' diverse experiences prior to matriculation into genetic counseling training programs, students' familiarity with these topics may vary. Following receipt of feedback from trainees at a large genetic counseling program regarding an existing course focused on molecular technologies, a three-part asynchronous module series was created as a prerequisite to this course as an opportunity to align knowledge. Designed to be completed by students on their own time and at their own pace, the modules allowed for additional instruction without increases in classroom time or credit hours. Content included a refresh on genetics concepts and an introduction to available genetics resources for developing a differential diagnosis as well as variant interpretation framework. Modules utilized a clinical scenario to anchor learning with interactive content, allowing students to progress at their own pace and explore content as they found necessary. Completion of this asynchronous module series was required by incoming first-year students prior to the start of the academic semester. Following completion, students were asked to provide feedback on the module series. Reviews were primarily positive with students indicating that while the content was not entirely new, they found the review valuable and would be likely to reference the modules later in their genetic counseling training. Areas identified for improvement included additional detail regarding genetic testing methods as well as adjusting the interactive content to ensure accessibility for all students and systems. Taken together, the development and implementation of this asynchronous series as an additional component to genetic counseling training was considered a success and this approach can be considered to address additional topics dependent on a programs' needs.

PURPOSE:Klinefelter syndrome, a sex chromosome aneuploidy (SCA), is associated with a 47,XXY chromosomal complement and is diagnosed in ∼1:600 live male births. Individuals with a 46,XX cell line, in addition to 47,XXY, are less common with a limited number of published case reports. METHODOLOGY:To better understand the implications of a 47,XXY/46,XX karyotype, we conducted a retrospective, multicenter analysis of the cytogenetic findings and associated clinical records of 34 patients diagnosed with this SCA across 14 institutions. RESULTS:Presence of the XX cell line ranged from 5% to 98% in patient specimens. Phenotypes also exhibited significant heterogeneity with some reporting a single reason for referral and others presenting with a constellation of symptoms, including ambiguous genitalia and ovotestes. Ovotestes were present in 12% of individuals in this cohort, who had a significantly higher percentage of XX cells. Notably, 2 patients were assigned female sex at birth. CONCLUSION:These findings highlight the variability of the clinical phenotypes associated with this SCA, as well as the challenges of clinical management for this population. Karyotype or fluorescence in situ hybridization analysis, which offer single-cell resolution, rather than chromosomal microarray or molecular testing, is the ideal test strategy in these instances as mosaicism can occur at low levels.

Children’s Hospital of Philadelphia (CHOP) is committed to improving diagnostics and therapeutics for children by leveraging genomic data. One project, “Baby Eagle”, utilizes the CHOP-developed rapid Targeted Analysis of the Genome for Infants (rTAG-I) test, a rapid genomic test for infants in the neonatal intensive care units (NICU) to allow for early diagnosis, implementation of targeted management, recurrence risk counseling and reduced overall costs. A quality improvement framework was utilized to develop Baby Eagle’s primary aim: To decrease the time from genetic consult to disclosure of preliminary actionable genomic sequencing results to under 7 days, while expanding the cohort of NICU patients eligible for rapid genomic sequencing, regardless of race and ethnicity, by December 2023.

Objective To evaluate factors influencing the diagnostic yield of comprehensive gene panel testing (CGPT) for hearing loss (HL) in children and to understand the characteristics of undiagnosed probands. Study design This was a retrospective cohort study of 474 probands with childhood-onset HL who underwent CGPT between 2016 and 2020 at a single center. Main outcomes and measures included the association between clinical variables and diagnostic yield and the genetic and clinical characteristics of undiagnosed probands. Results The overall diagnostic yield was 44% (209/474) with causative variants involving 41 genes. While the diagnostic yield was high in the probands with congenital, bilateral, and severe HL, it was low in those with unilateral, noncongenital, or mild HL; cochlear nerve deficiency; preterm birth; neonatal intensive care unit admittance; certain ancestry; and developmental delay. Follow-up studies on 49 probands with initially inconclusive CGPT results changed the diagnostic status to likely positive or negative outcomes in 39 of them (80%). Reflex to exome sequencing on 128 undiagnosed probands by CGPT revealed diagnostic findings in 8 individuals, 5 of whom had developmental delays. The remaining 255 probands were undiagnosed, with 173 (173/255) having only a single variant in the gene(s) associated with autosomal recessive HL and 28% (48/173) having a matched phenotype. Conclusion CGPT efficiently identifies the genetic etiologies of HL in children. CGPT-undiagnosed probands may benefit from follow-up studies or expanded testing.

PURPOSE:This study aimed to establish variants in CBX1, encoding heterochromatin protein 1β (HP1β), as a cause of a novel syndromic neurodevelopmental disorder. METHODS:Patients with CBX1 variants were identified, and clinician researchers were connected using GeneMatcher and physician referrals. Clinical histories were collected from each patient. To investigate the pathogenicity of identified variants, we performed in vitro cellular assays and neurobehavioral and cytological analyses of neuronal cells obtained from newly generated Cbx1 mutant mouse lines. RESULTS:In 3 unrelated individuals with developmental delay, hypotonia, and autistic features, we identified heterozygous de novo variants in CBX1. The identified variants were in the chromodomain, the functional domain of HP1β, which mediates interactions with chromatin. Cbx1 chromodomain mutant mice displayed increased latency-to-peak response, suggesting the possibility of synaptic delay or myelination deficits. Cytological and chromatin immunoprecipitation experiments confirmed the reduction of mutant HP1β binding to heterochromatin, whereas HP1β interactome analysis demonstrated that the majority of HP1β-interacting proteins remained unchanged between the wild-type and mutant HP1β. CONCLUSION:These collective findings confirm the role of CBX1 in developmental disabilities through the disruption of HP1β chromatin binding during neurocognitive development. Because HP1β forms homodimers and heterodimers, mutant HP1β likely sequesters wild-type HP1β and other HP1 proteins, exerting dominant-negative effects.

Identifying the underlying etiology of a genetic disease with phenotypic and/or genetic heterogeneity can be tackled using either targeted gene panels or by exome sequencing (ES). Gene panel testing is conducted to thoroughly evaluate all of the genes associated with a phenotype and report all diagnostic, partially diagnostic, and non-diagnostic findings, whereas ES is performed to identify and report diagnostic/partially diagnostic variants tailored to the individual's phenotype. Historically, gene panels have been deemed most suitable for well-defined phenotypes related to a limited number of genes, while ES has been mostly used for non-specific phenotypes for which little initial speculation could be made about their genetic etiology.

Clinical exome sequencing (CES) aids in the diagnosis of rare genetic disorders. Herein, we report the molecular diagnostic yield and spectrum of genetic alterations contributing to disease in 700 pediatric cases analyzed at the Children's Hospital of Philadelphia. The overall diagnostic yield was 23%, with three cases having more than one molecular diagnosis and 2.6% having secondary/additional findings. A candidate gene finding was reported in another 8.4% of cases. The clinical indications with the highest diagnostic yield were neurodevelopmental disorders (including seizures), whereas immune- and oncology-related indications were negatively associated with molecular diagnosis. The rapid expansion of knowledge regarding the genome's role in human disease necessitates reanalysis of CES samples. To capture these new discoveries, a subset of cases (n = 240) underwent reanalysis, with an increase in diagnostic yield. We describe our experience reporting CES results in a pediatric setting, including reporting of secondary findings, reporting newly discovered genetic conditions, and revisiting negative test results. Finally, we highlight the challenges associated with implementing critical updates to the CES workflow. Although these updates are necessary, they demand an investment of time and resources from the laboratory. In summary, these data demonstrate the clinical utility of exome sequencing and reanalysis, while highlighting the critical considerations for continuous improvement of a CES test in a clinical laboratory.

Clinical exome sequencing (CES) aids in the diagnosis of rare genetic disorders. Herein, we report the molecular diagnostic yield and spectrum of genetic alterations contributing to disease in 700 pediatric cases analyzed at the Children's Hospital of Philadelphia. The overall diagnostic yield was 23%, with three cases having more than one molecular diagnosis and 2.6% having secondary/additional findings. A candidate gene finding was reported in another 8.4% of cases. The clinical indications with the highest diagnostic yield were neurodevelopmental disorders (including seizures), whereas immune- and oncology-related indications were negatively associated with molecular diagnosis. The rapid expansion of knowledge regarding the genome's role in human disease necessitates reanalysis of CES samples. To capture these new discoveries, a subset of cases (n = 240) underwent reanalysis, with an increase in diagnostic yield. We describe our experience reporting CES results in a pediatric setting, including reporting of secondary findings, reporting newly discovered genetic conditions, and revisiting negative test results. Finally, we highlight the challenges associated with implementing critical updates to the CES workflow. Although these updates are necessary, they demand an investment of time and resources from the laboratory. In summary, these data demonstrate the clinical utility of exome sequencing and reanalysis, while highlighting the critical considerations for continuous improvement of a CES test in a clinical laboratory.

The current study was designed to investigate the role of serotonin (5-HT) and nuclear factor-kappa beta (NF-κB) in the ameliorative effect of ginger on acetic acid (AA)-induced colitis rat model. Colitis was induced by intra-colonic instillation of 3% AA, preceded or followed by daily administration of ginger (400 mg/kg) by gavage for 5 days. Colons were assessed macroscopically and microscopically and the expression of NF-κB was evaluated by immunohistochemistry. Colonic tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10), total peroxide (TP), and serum 5-HT levels were assessed. Administration of ginger ameliorated the effects of AA-induced colitis by plummeting colon weight-to-length ratio, macroscopic and microscopic scores. These effects were further supported by down-regulation of NF-κB and reduction of colonic TNF-α, IL-10, TP and serum 5-HT levels. Moreover, there were significant positive correlations between serum 5-HT and macroscopic, microscopic, immunoreactivity scores and colonic TNF-α level. In conclusion, ginger ameliorated AA-induced colitis not only through its anti-inflammatory and anti-oxidant properties, but also through the reduction of 5-HT which may contribute to the down-regulation of NF-κB-dependent TNF-α expression and the reduction of lipid peroxidation and tissue damage. In addition, the therapeutic effect of ginger was more pronounced than its preventive effect.

Mitochondrial disease diagnosis requires interrogation of both nuclear and mitochondrial (mtDNA) genomes for single-nucleotide variants (SNVs) and copy number alterations, both in the proband and often maternal relatives, together with careful phenotype correlation. We developed a comprehensive mtDNA sequencing test (‘MitoGenome’) using long-range PCR (LR-PCR) to amplify the full length of the mtDNA genome followed by next generation sequencing (NGS) to accurately detect SNVs and large-scale mtDNA deletions (LSMD), combined with droplet digital PCR (ddPCR) for LSMD heteroplasmy quantification. Overall, MitoGenome tests were performed on 428 samples from 394 patients with suspected or confirmed mitochondrial disease. The positive yield was 11% (43/394), including 34 patients with pathogenic or likely pathogenic SNVs (the most common being m.3243A > G in 8/34 (24%) patients), 8 patients with single LSMD, and 3 patients with multiple LSMD exceeding 10% heteroplasmy levels. Two patients with both LSMD and pathogenic SNV were detected. Overall, this LR-PCR/NGS assay provides a highly accurate and comprehensive diagnostic method for simultaneous mtDNA SNV detection at heteroplasmy levels as low as 1% and LSMD detection at heteroplasmy levels below 10%. Inclusion of maternal samples for variant classification and ddPCR to quantify LSMD heteroplasmy levels further enables accurate pathogenicity assessment and clinical correlation interpretation of mtDNA genome sequence variants and copy number alterations.

Mitochondrial DNA (mtDNA) variant pathogenicity interpretation has special considerations given unique features of the mtDNA genome, including maternal inheritance, variant heteroplasmy, threshold effect, absence of splicing, and contextual effects of haplogroups. Currently, there are insufficient standardized criteria for mtDNA variant assessment, which leads to inconsistencies in clinical variant pathogenicity reporting. An international working group of mtDNA experts was assembled within the Mitochondrial Disease Sequence Data Resource Consortium and obtained Expert Panel status from ClinGen. This group reviewed the 2015 American College of Medical Genetics and Association of Molecular Pathology standards and guidelines that are widely used for clinical interpretation of DNA sequence variants and provided further specifications for additional and specific guidance related to mtDNA variant classification. These Expert Panel consensus specifications allow for consistent consideration of the unique aspects of the mtDNA genome that directly influence variant assessment, including addressing mtDNA genome composition and structure, haplogroups and phylogeny, maternal inheritance, heteroplasmy, and functional analyses unique to mtDNA, as well as specifications for utilization of mtDNA genomic databases and computational algorithms.

AbstractPurposeThis study aimed to establish variants inCBX1, encoding heterochromatin protein 1β (HP1β), as a cause of a novel syndromic neurodevelopmental disorder.MethodsPatients withCBX1variants were identified, and clinician researchers were connected using GeneMatcher and physician referrals. Clinical histories were collected from each patient. To investigate the pathogenicity of identified variants, we performedin vitrocellular assays, neurobehavioral and cytological analyses of neuronal cells obtained from newly generatedCbx1mutant mouse lines.ResultsIn three unrelated individuals with developmental delay, hypotonia, and autistic features, we identified heterozygousde novovariants inCBX1. The identified variants were in the chromodomain, the functional domain of HP1 β, which mediates interactions with chromatin.Cbx1chromodomain mutant mice displayed increased latency-to-peak response, suggesting the possibility of synaptic delay or myelination deficits. Cytological and chromatin immunoprecipitation experiments confirmed the reduction of mutant HP1β binding to heterochromatin, while HP1β interactome analysis demonstrated that the majority of HP1β-interacting proteins remained unchanged between the wild-type and mutant HP1β.ConclusionThese collective findings confirm the role ofCBX1in developmental disabilities through the disruption of HP1β chromatin binding during neurocognitive development. As HP1β forms homodimers and heterodimers, mutant HP1β likely sequesters wild-type HP1β and other HP1 proteins, exerting dominant-negative effects.