Genetic Analysis and Functional Assessment of a TGFBR2 Variant in Micrognathia and Cleft Palate.

Cleft lip and cleft palate are among the most common congenital anomalies and are the result of incomplete fusion of embryonic craniofacial processes or palatal shelves, respectively. We know that genetics play a large role in these anomalies but the list of known causal genes is far from complete. As part of a larger sequencing effort of patients with micrognathia and cleft palate we identified a candidate variant in transforming growth factor beta receptor 2 (TGFBR2) which is rare, changing a highly conserved amino acid, and predicted to be pathogenic by a number of metrics. The family history and population genetics would suggest this specific variant would be incompletely penetrant, but this gene has been convincingly implicated in craniofacial development. In order to test the hypothesis this might be a causal variant, we used genome editing to create the orthologous variant in a new mouse model. Surprisingly, Tgfbr2V387M mice did not exhibit craniofacial anomalies or have reduced survival suggesting this is, in fact, not a causal variant for cleft palate/ micrognathia. The discrepancy between in silico predictions and mouse phenotypes highlights the complexity of translating human genetic findings to mouse models. We expect these findings will aid in interpretation of future variants seen in TGFBR2 from ongoing sequencing of patients with congenital craniofacial anomalies.

Sync for Genes Phase 5: Computable artifacts for sharing dynamically annotated FHIR-formatted genomic variants.

Introduction: Variant annotation is a critical component in next-generation sequencing, enabling a sequencing lab to comb through a sea of variants in order to hone in on those likely to be most significant, and providing clinicians with necessary context for decision-making. But with the rapid evolution of genomics knowledge, reported annotations can quickly become out-of-date. Under the ONC Sync for Genes program, our team sought to standardize the sharing of dynamically annotated variants (e.g., variants annotated on demand, based on current knowledge). The computable biomedical knowledge artifacts that were developed enable a clinical decision support (CDS) application to surface up-to-date annotations to clinicians. Methods: The work reported in this article relies on the Health Level 7 Fast Healthcare Interoperability Resources (FHIR) Genomics and Global Alliance for Genomics and Health (GA4GH) Variant Annotation (VA) standards. We developed a CDS pipeline that dynamically annotates patient's variants through an intersection with current knowledge and serves up the FHIR-encoded variants and annotations (diagnostic and therapeutic implications, molecular consequences, population allele frequencies) via FHIR Genomics Operations. ClinVar, CIViC, and PharmGKB were used as knowledge sources, encoded as per the GA4GH VA specification. Results: Primary public artifacts from this project include a GitHub repository with all source code, a Swagger interface that allows anyone to visualize and interact with the code using only a web browser, and a backend database where all (synthetic and anonymized) patient data and knowledge are housed. Conclusions: We found that variant annotation varies in complexity based on the variant type, and that various bioinformatics strategies can greatly improve automated annotation fidelity. More importantly, we demonstrated the feasibility of an ecosystem where genomic knowledge bases have standardized knowledge (e.g., based on the GA4GH VA spec), and CDS applications can dynamically leverage that knowledge to provide real-time decision support, based on current knowledge, to clinicians at the point of care.

ARF1-related disorder: phenotypic and molecular spectrum.

PURPOSE: ARF1 was previously implicated in periventricular nodular heterotopia (PVNH) in only five individuals and systematic clinical characterisation was not available. The aim of this study is to provide a comprehensive description of the phenotypic and genotypic spectrum of ARF1-related neurodevelopmental disorder. METHODS: We collected detailed phenotypes of an international cohort of individuals (n=17) with ARF1 variants assembled through the GeneMatcher platform. Missense variants were structurally modelled, and the impact of several were functionally validated. RESULTS: De novo variants (10 missense, 1 frameshift, 1 splice altering resulting in 9 residues insertion) in ARF1 were identified among 17 unrelated individuals. Detailed phenotypes included intellectual disability (ID), microcephaly, seizures and PVNH. No specific facial characteristics were consistent across all cases, however microretrognathia was common. Various hearing and visual defects were recurrent, and interestingly, some inflammatory features were reported. MRI of the brain frequently showed abnormalities consistent with a neuronal migration disorder. CONCLUSION: We confirm the role of ARF1 in an autosomal dominant syndrome with a phenotypic spectrum including severe ID, microcephaly, seizures and PVNH due to impaired neuronal migration.

The 2019-2020 Dengue Fever Epidemic: Genomic Markers Indicating Severity in Dominican Republic Children.

We performed an observational cohort study to assess associations between genetic factors of dengue fever (DF) severity in children in the Dominican Republic. A total of 488 participants had serologically confirmed DF. We replicated the association between the IFIH1 gene (rs1990760) and severe DF (n = 80/488, p = 0.006) and identified novel associations needing further investigation.

Introducing HL7 FHIR Genomics Operations: a developer-friendly approach to genomics-EHR integration.

OBJECTIVE: Enabling clinicians to formulate individualized clinical management strategies from the sea of molecular data remains a fundamentally important but daunting task. Here, we describe efforts towards a new paradigm in genomics-electronic health record (HER) integration, using a standardized suite of FHIR Genomics Operations that encapsulates the complexity of molecular data so that precision medicine solution developers can focus on building applications. MATERIALS AND METHODS: FHIR Genomics Operations essentially "wrap" a genomics data repository, presenting a uniform interface to applications. More importantly, operations encapsulate the complexity of data within a repository and normalize redundant data representations-particularly relevant in genomics, where a tremendous amount of raw data exists in often-complex non-FHIR formats. RESULTS: Fifteen FHIR Genomics Operations have been developed, designed to support a wide range of clinical scenarios, such as variant discovery; clinical trial matching; hereditary condition and pharmacogenomic screening; and variant reanalysis. Operations are being matured through the HL7 balloting process, connectathons, pilots, and the HL7 FHIR Accelerator program. DISCUSSION: Next-generation sequencing can identify thousands to millions of variants, whose clinical significance can change over time as our knowledge evolves. To manage such a large volume of dynamic and complex data, new models of genomics-EHR integration are needed. Qualitative observations to date suggest that freeing application developers from the need to understand the nuances of genomic data, and instead base applications on standardized APIs can not only accelerate integration but also dramatically expand the applications of Omic data in driving precision care at scale for all.

PPP2R1A neurodevelopmental disorder is associated with congenital heart defects.

Protein phosphatase 2A (PP2A) is a heterotrimeric serine/threonine phosphatase that regulates numerous biological processes. PPP2R1A encodes the scaffolding "Aα" subunit of PP2A. To date, nearly 40 patients have been previously reported with 19 different pathogenic PPP2R1A variants, with phenotypes including intellectual disability, developmental delay, epilepsy, infant agenesis/dysgenesis of the corpus callosum, and dysmorphic features. Apart from a single case, severe congenital heart defects (CHD) have not been described. We report four new unrelated individuals with pathogenic heterozygous PPP2R1A variants and CHD and model the crystal structure of several variants to investigate mechanisms of phenotype disparity. Individuals 1 and 2 have a previously described variant (c.548G>A, p.R183Q) and similar phenotypes with severe ventriculomegaly, agenesis/dysgenesis of the corpus callosum, and severe CHD. Individual 3 also has a recurrent variant (c.544C>T, p.R182W) and presented with agenesis of corpus callosum, ventriculomegaly, mild pulmonic stenosis, and small patent foramen ovale. Individual 4 has a novel variant (c.536C>A, p.P179H), ventriculomegaly, and atrial septal defect. To conclude, we propose expansion of the phenotype of PPP2R1A neurodevelopmental disorder to include CHD. Further, the R183Q variant has now been described in three individuals, all with severe neurologic abnormalities, severe CHD, and early death suggesting that this variant may be particularly deleterious.

vcf2fhir: a utility to convert VCF files into HL7 FHIR format for genomics-EHR integration.

BACKGROUND: VCF formatted files are the lingua franca of next-generation sequencing, whereas HL7 FHIR is emerging as a standard language for electronic health record interoperability. A growing number of FHIR-based clinical genomics applications are emerging. Here, we describe an open source utility for converting variants from VCF format into HL7 FHIR format. RESULTS: vcf2fhir converts VCF variants into a FHIR Genomics Diagnostic Report. Conversion translates each VCF row into a corresponding FHIR-formatted variant in the generated report. In scope are simple variants (SNVs, MNVs, Indels), along with zygosity and phase relationships, for autosomes, sex chromosomes, and mitochondrial DNA. Input parameters include VCF file and genome build ('GRCh37' or 'GRCh38'); and optionally a conversion region that indicates the region(s) to convert, a studied region that lists genomic regions studied by the lab, and a non-callable region that lists studied regions deemed uncallable by the lab. Conversion can be limited to a subset of VCF by supplying genomic coordinates of the conversion region(s). If studied and non-callable regions are also supplied, the output FHIR report will include 'region-studied' observations that detail which portions of the conversion region were studied, and of those studied regions, which portions were deemed uncallable. We illustrate the vcf2fhir utility via two case studies. The first, 'SMART Cancer Navigator', is a web application that offers clinical decision support by linking patient EHR information to cancerous gene variants. The second, 'Precision Genomics Integration Platform', intersects a patient's FHIR-formatted clinical and genomic data with knowledge bases in order to provide on-demand delivery of contextually relevant genomic findings and recommendations to the EHR. CONCLUSIONS: Experience to date shows that the vcf2fhir utility can be effectively woven into clinically useful genomic-EHR integration pipelines. Additional testing will be a critical step towards the clinical validation of this utility, enabling it to be integrated in a variety of real world data flow scenarios. For now, we propose the use of this utility primarily to accelerate FHIR Genomics understanding and to facilitate experimentation with further integration of genomics data into the EHR.

Deleterious Variants in ABCC12 are Detected in Idiopathic Chronic Cholestasis and Cause Intrahepatic Bile Duct Loss in Model Organisms.

BACKGROUND & AIMS: The etiology of cholestasis remains unknown in many children. We surveyed the genome of children with chronic cholestasis for variants in genes not previously associated with liver disease and validated their biological relevance in zebrafish and murine models. METHOD: Whole-exome (n = 4) and candidate gene sequencing (n = 89) was completed on 93 children with cholestasis and normal serum γ-glutamyl transferase (GGT) levels without pathogenic variants in genes known to cause low GGT cholestasis such as ABCB11 or ATP8B1. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 genome editing was used to induce frameshift pathogenic variants in the candidate gene in zebrafish and mice. RESULTS: In a 1-year-old female patient with normal GGT cholestasis and bile duct paucity, we identified a homozygous truncating pathogenic variant (c.198delA, p.Gly67Alafs∗6) in the ABCC12 gene (NM_033226). Five additional rare ABCC12 variants, including a pathogenic one, were detected in our cohort. ABCC12 encodes multidrug resistance-associated protein 9 (MRP9) that belongs to the adenosine 5'-triphosphate-binding cassette transporter C family with unknown function and no previous implication in liver disease. Immunohistochemistry and Western blotting revealed conserved MRP9 protein expression in the bile ducts in human, mouse, and zebrafish. Zebrafish abcc12-null mutants were prone to cholangiocyte apoptosis, which caused progressive bile duct loss during the juvenile stage. MRP9-deficient mice had fewer well-formed interlobular bile ducts and higher serum alkaline phosphatase levels compared with wild-type mice. They exhibited aggravated cholangiocyte apoptosis, hyperbilirubinemia, and liver fibrosis upon cholic acid challenge. CONCLUSIONS: Our work connects MRP9 with bile duct homeostasis and cholestatic liver disease for the first time. It identifies a potential therapeutic target to attenuate bile acid-induced cholangiocyte injury.

Systemic Juvenile Idiopathic Arthritis-Associated Lung Disease: Characterization and Risk Factors.

OBJECTIVE: Systemic juvenile idiopathic arthritis (JIA) is associated with a recently recognized, albeit poorly defined and characterized, lung disease (LD). The objective of this study was to describe the clinical characteristics, risk factors, and histopathologic and immunologic features of this novel inflammatory LD associated with systemic JIA (designated SJIA-LD). METHODS: Clinical data collected since 2010 were abstracted from the medical records of patients with systemic JIA from the Cincinnati Children's Hospital Medical Center. Epidemiologic, cellular, biochemical, genomic, and transcriptional profiling analyses were performed. RESULTS: Eighteen patients with SJIA-LD were identified. Radiographic findings included diffuse ground-glass opacities, subpleural reticulation, interlobular septal thickening, and lymphadenopathy. Pathologic findings included patchy, but extensive, lymphoplasmacytic infiltrates and mixed features of pulmonary alveolar proteinosis (PAP) and endogenous lipoid pneumonia. Compared to systemic JIA patients without LD, those with SJIA-LD were younger at the diagnosis of systemic JIA (odds ratio [OR] 6.5, P = 0.007), more often had prior episodes of macrophage activation syndrome (MAS) (OR 14.5, P < 0.001), had a greater frequency of adverse reactions to biologic therapy (OR 13.6, P < 0.001), and had higher serum levels of interleukin-18 (IL-18) (median 27,612 pg/ml versus 5,413 pg/ml; P = 0.047). Patients with SJIA-LD lacked genetic, serologic, or functional evidence of granulocyte-macrophage colony-stimulating factor pathway dysfunction, a feature that is typical of familial or autoimmune PAP. Moreover, bronchoalveolar lavage (BAL) fluid from patients with SJIA-LD rarely demonstrated proteinaceous material and had less lipid-laden macrophages than that seen in patients with primary PAP (mean 10.5% in patients with SJIA-LD versus 66.1% in patients with primary PAP; P < 0.001). BAL fluid from patients with SJIA-LD contained elevated levels of IL-18 and the interferon-γ-induced chemokines CXCL9 and CXCL10. Transcriptional profiling of the lung tissue from patients with SJIA-LD identified up-regulated type II interferon and T cell activation networks. This signature was also present in SJIA-LD human lung tissue sections that lacked substantial histopathologic findings, suggesting that this activation signature may precede and drive the lung pathology in SJIA-LD. CONCLUSION: Pulmonary disease is increasingly detected in children with systemic JIA, particularly in association with MAS. This entity has distinct clinical and immunologic features and represents an uncharacterized inflammatory LD.

Prevalence of abnormal glucose metabolism in pediatric acute, acute recurrent and chronic pancreatitis.

Type 3C Diabetes, or diseases of the exocrine pancreas has been reported to occur in approximately 30% of adult patient with pancreatitis. The incidence of glucose abnormalities or risk factors that may predict the development of abnormal glucose in the pediatric pancreatitis population is not known. We performed a retrospective chart review from 1998-2016 for patients who carry the diagnosis of acute pancreatitis (AP), acute recurrent pancreatitis (ARP), and chronic pancreatitis (CP). We extracted glucose values, HbA1c%, and data from oral glucose tolerance and mixed meal testing with timing in relation to pancreatic exacerbations. Patient characteristic data such as age, gender, body proportions, family history of pancreatitis, exocrine function and genetic mutations were also assessed. Abnormal glucose was based on definitions put forth by the American Diabetes Society for pre-diabetes and diabetes. Fifty-two patients had AP and met criteria. Of those, 15 (29%) had glucose testing on or after the first attack, 21 (40%) were tested on or after the second attack (in ARP patients) and 16 (31%) were tested after a diagnosis of CP. Of the patients tested for glucose abnormalities, 25% (13/52) had abnormal glucose testing (testing indicating pre-DM or DM as defined by ADA guidelines. A significantly higher proportion of the abnormal glucose testing was seen in patients (85%, 11/13) with a BMI at or greater than the 85th percentile compared to the normal glucose patients (28%, 11/39) (p = 0.0007). A significantly higher proportion of the abnormal glucose patients (77%, 10/13) had SAP during the prior AP episode to testing compared to the 10% (4/39) of the normal glucose patients (p<0.0001). Older age at DM testing was associated with a higher prevalence of abnormal glucose testing (p = 0.04). In our patient population, a higher proportion of glucose abnormalities were after the second episode of pancreatitis, however 62% (8/13) with abnormalities was their first time tested. We identified obesity and having severe acute pancreatitis (SAP) during the prior AP episode to testing could be associated with abnormal glucose. We propose that systematic screening for abnormal glucose after the first episode of acute pancreatitis in order to better establish the timing of diabetes progression.

Dominant-negative IKZF1 mutations cause a T, B, and myeloid cell combined immunodeficiency.

Ikaros/IKZF1 is an essential transcription factor expressed throughout hematopoiesis. IKZF1 is implicated in lymphocyte and myeloid differentiation and negative regulation of cell proliferation. In humans, somatic mutations in IKZF1 have been linked to the development of B cell acute lymphoblastic leukemia (ALL) in children and adults. Recently, heterozygous germline IKZF1 mutations have been identified in patients with a B cell immune deficiency mimicking common variable immunodeficiency. These mutations demonstrated incomplete penetrance and led to haploinsufficiency. Herein, we report 7 unrelated patients with a novel early-onset combined immunodeficiency associated with de novo germline IKZF1 heterozygous mutations affecting amino acid N159 located in the DNA-binding domain of IKZF1. Different bacterial and viral infections were diagnosed, but Pneumocystis jirovecii pneumonia was reported in all patients. One patient developed a T cell ALL. This immunodeficiency was characterized by innate and adaptive immune defects, including low numbers of B cells, neutrophils, eosinophils, and myeloid dendritic cells, as well as T cell and monocyte dysfunctions. Notably, most T cells exhibited a naive phenotype and were unable to evolve into effector memory cells. Functional studies indicated these mutations act as dominant negative. This defect expands the clinical spectrum of human IKZF1-associated diseases from somatic to germline, from haploinsufficient to dominant negative.

Brief Report: Novel UNC13D Intronic Variant Disrupting an NF-κB Enhancer in a Patient With Recurrent Macrophage Activation Syndrome and Systemic Juvenile Idiopathic Arthritis.

OBJECTIVE: Macrophage activation syndrome (MAS) is a life-threatening complication of systemic juvenile idiopathic arthritis (JIA) and has pathologic similarity to hemophagocytic lymphohistiocytosis (HLH). Intronic variants in UNC13D are found in patients with familial HLH type 3 (FHLH3), but the role of noncoding variants in MAS is unknown. The objective of this study was to identify deep intronic UNC13D variants in patients with MAS. METHODS: A custom enrichment library was constructed to sequence a genomic region of ~1 Mb flanking UNC13D in 24 patients with systemic JIA, recurrent MAS, and negative results of prior genetic (exon/coding) testing. The functional consequences of intronic variants were assessed using quantitative polymerase chain reaction in patient-derived peripheral blood mononuclear cells (PBMCs), electromobility shift assay, in vitro transcriptional enhancer assays, and natural killer (NK) cell degranulation assays. RESULTS: We evaluated a patient with systemic JIA and recurrent MAS in whom a novel functional intronic variant in UNC13D, c.117+143A>G, was observed. This variant occurred in a proposed regulatory region that drives lymphocyte-specific UNC13D expression and is associated with reduced transcript levels in patient PBMCs. This variant also disrupted NF-κB binding to a functional transcriptional enhancer, leading to reduced enhancer activity in vitro. Partial knockdown of UNC13D expression also led to impaired NK cell degranulation. An additional patient was identified with a previously described UNC13D intronic variant, for a total noncoding variant hit rate of 8.3% (2 of 24). CONCLUSION: These findings highlight the notion that intronic variants in key regulatory regions may be associated with MAS in patients with systemic JIA and support deep sequencing approaches when causative coding variants are not identified.

Partial growth hormone insensitivity and dysregulatory immune disease associated with de novo germline activating STAT3 mutations.

Germinal heterozygous activating STAT3 mutations represent a novel monogenic defect associated with multi-organ autoimmune disease and, in some cases, severe growth retardation. By using whole-exome sequencing, we identified two novel STAT3 mutations, p.E616del and p.C426R, in two unrelated pediatric patients with IGF-I deficiency and immune dysregulation. The functional analyses showed that both variants were gain-of-function (GOF), although they were not constitutively phosphorylated. They presented differences in their dephosphorylation kinetics and transcriptional activities under interleukin-6 stimulation. Both variants increased their transcriptional activities in response to growth hormone (GH) treatment. Nonetheless, STAT5b transcriptional activity was diminished in the presence of STAT3 GOF variants, suggesting a disruptive role of STAT3 GOF variants in the GH signaling pathway. This study highlights the broad clinical spectrum of patients presenting activating STAT3 mutations and explores the underlying molecular pathway responsible for this condition, suggesting that different mutations may drive increased activity by slightly different mechanisms.

A comprehensive next-generation sequencing assay for the diagnosis of epidermolysis bullosa.

BACKGROUND: Historically, diagnosis of epidermolysis bullosa has required skin biopsies for electron microscopy, direct immunofluorescence to determine which gene(s) to choose for genetic testing, or both. METHODS: To avoid these invasive tests, we developed a high-throughput next-generation sequencing (NGS)-based diagnostic assay called EBSEQ that allows simultaneous detection of mutations in 21 genes with known roles in epidermolysis bullosa pathogenicity. Mutations are confirmed with traditional Sanger sequencing. RESULTS: We present our EBSEQ assay and preliminary studies on the first 43 subjects tested. We identified 11 cases of epidermolysis bullosa simplex, five cases of junctional epidermolysis bullosa, 11 cases of dominant dystrophic epidermolysis bullosa, 15 cases of recessive dystrophic epidermolysis bullosa, and one case that remains without diagnosis. We also found an additional 52 variants of uncertain clinical significance in 17 of the 21 epidermolysis bullosa-associated genes tested. Three of the variants of uncertain clinical significance were also found in three other patients, for a total of 49 unique variants of uncertain clinical significance. We found the clinical sensitivity of the assay to be 75% to 98% and the analytical sensitivity to be 99% in identifying base substitutions and small deletions and duplications. Turnaround time was 3 to 6 weeks. CONCLUSIONS: EBSEQ is a sensitive, relatively rapid, minimally invasive, comprehensive genetic assay for the diagnosis of epidermolysis bullosa.

Concordance between Research Sequencing and Clinical Pharmacogenetic Genotyping in the eMERGE-PGx Study.

There has been extensive debate about both the necessity of orthogonal confirmation of next-generation sequencing (NGS) results in Clinical Laboratory Improvement Amendments-approved laboratories and return of research NGS results to participants enrolled in research studies. In eMERGE-PGx, subjects underwent research NGS using PGRNseq and orthogonal targeted genotyping in clinical laboratories, which prompted a comparison of genotyping results between platforms. Concordance (percentage agreement) was reported for 4077 samples tested across nine combinations of research and clinical laboratories. Retesting was possible on a subset of 1792 samples, and local laboratory directors determined sources of genotype discrepancy. Research NGS and orthogonal clinical genotyping had an overall per sample concordance rate of 0.972 and per variant concordance rate of 0.997. Genotype discrepancies attributed to research NGS were because of sample switching (preanalytical errors), whereas the majority of genotype discrepancies (92.3%) attributed to clinical genotyping were because of allele dropout as a result of rare variants interfering with primer hybridization (analytical errors). These results highlight the analytical quality of clinically significant pharmacogenetic variants derived from NGS and reveal important areas for research and clinical laboratories to address with quality management programs.