Semiautomated approach focused on new genomic information results in time and effort-efficient reannotation of negative exome data.

Most rare disease patients (75-50%) undergoing genomic sequencing remain unsolved, often due to lack of information about variants identified. Data review over time can leverage novel information regarding disease-causing variants and genes, increasing this diagnostic yield. However, time and resource constraints have limited reanalysis of genetic data in clinical laboratories setting. We developed RENEW, (REannotation of NEgative WES/WGS) an automated reannotation procedure that uses relevant new information in on-line genomic databases to enable rapid review of genomic findings. We tested RENEW in an unselected cohort of 1066 undiagnosed cases with a broad spectrum of phenotypes from the Mayo Clinic Center for Individualized Medicine using new information in ClinVar, HGMD and OMIM between the date of previous analysis/testing and April of 2022. 5741 variants prioritized by RENEW were rapidly reviewed by variant interpretation specialists. Mean analysis time was approximately 20 s per variant (32 h total time). Reviewed cases were classified as: 879 (93.0%) undiagnosed, 63 (6.6%) putatively diagnosed, and 4 (0.4%) definitively diagnosed. New strategies are needed to enable efficient review of genomic findings in unsolved cases. We report on a fast and practical approach to address this need and improve overall diagnostic success in patient testing through a recurrent reannotation process.

Thoracic Aortic Aneurysmal Disease: Comprehensive Recommendations for the Primary Care Physician.

Thoracic aortic aneurysm (TAA) is a commonly encountered disease that is defined as aortic dilation with an increase in diameter of at least 50% greater than the expected age- and sex-adjusted size. Thoracic aortic aneurysms are described by their size, location, morphology, and cause. Primary care clinicians and other noncardiologists are often the first point of contact for patients with TAA. This review is intended to provide them with basic information on the differential diagnosis, diagnostic evaluation, and medical and surgical management of TAAs. Management decisions depend on having as precise a diagnosis as possible. Fortunately, this can often be achieved with a stepwise diagnostic approach that incorporates imaging and targeted genetic testing. Our review includes recommendations. In this review, we discuss these issues at a basic level and include recommendations for patients considering pregnancy.

Impact of Genetic Disorders in the Surgical Management of Congenital Heart Disease.

The congenital heart surgeon frequently encounters patients with various genetic disorders requiring surgical intervention. Although the specifics of the genetics for these patients and their families lie in the purview of specialists in genetics, the surgeon is well-served to be familiar with aspects of specific syndromes that impact surgical management and perioperative care. This aids in counseling families in expectations for the hospital course and recovery as well as can impact intraoperative and surgical management. This review article summarizes key characteristics for the congenital heart surgeon to be familiar with for common genetic disorders as they help coordinate care.

Vitamin C epigenetically controls osteogenesis and bone mineralization.

Vitamin C deficiency disrupts the integrity of connective tissues including bone. For decades this function has been primarily attributed to Vitamin C as a cofactor for collagen maturation. Here, we demonstrate that Vitamin C epigenetically orchestrates osteogenic differentiation and function by modulating chromatin accessibility and priming transcriptional activity. Vitamin C regulates histone demethylation (H3K9me3 and H3K27me3) and promotes TET-mediated 5hmC DNA hydroxymethylation at promoters, enhancers and super-enhancers near bone-specific genes. This epigenetic circuit licenses osteoblastogenesis by permitting the expression of all major pro-osteogenic genes. Osteogenic cell differentiation is strictly and continuously dependent on Vitamin C, whereas Vitamin C is dispensable for adipogenesis. Importantly, deletion of 5hmC-writers, Tet1 and Tet2, in Vitamin C-sufficient murine bone causes severe skeletal defects which mimic bone phenotypes of Vitamin C-insufficient Gulo knockout mice, a model of Vitamin C deficiency and scurvy. Thus, Vitamin C's epigenetic functions are central to osteoblastogenesis and bone formation and may be leveraged to prevent common bone-degenerating conditions.

TRAPPC9-CDG: A novel congenital disorder of glycosylation with dysmorphic features and intellectual disability.

PURPOSE: TRAPPC9 deficiency is an autosomal recessive disorder mainly associated with intellectual disability (ID), microcephaly, and obesity. Previously, TRAPPC9 deficiency has not been associated with biochemical abnormalities. METHODS: Exome sequencing was performed in 3 individuals with ID and dysmorphic features. N-Glycosylation analyses were performed in the patients' blood samples to test for possible congenital disorder of glycosylation (CDG). TRAPPC9 gene, TRAPPC9 protein expression, and N-glycosylation markers were assessed in patient fibroblasts. Complementation with wild-type TRAPPC9 and immunofluorescence studies to assess TRAPPC9 expression and localization were performed. The metabolic consequences of TRAPPC9 deficiency were evaluated using tracer metabolomics. RESULTS: All 3 patients carried biallelic missense variants in TRAPPC9 and presented with an N-glycosylation defect in blood, consistent with CDG type I. Extensive investigations in patient fibroblasts corroborated TRAPPC9 deficiency and an N-glycosylation defect. Tracer metabolomics revealed global metabolic changes with several affected glycosylation-related metabolites. CONCLUSION: We identified 3 TRAPPC9 deficient patients presenting with ID, dysmorphic features, and abnormal glycosylation. On the basis of our findings, we propose that TRAPPC9 deficiency could lead to a CDG (TRAPPC9-CDG). The finding of abnormal glycosylation in these patients is highly relevant for diagnosis, further elucidation of the pathophysiology, and management of the disease.

Early and Late Outcomes of Cardiovascular Surgery in Patients With Ehlers-Danlos Syndrome.

BACKGROUND: Cardiovascular surgical outcomes reports are few for vascular type IV of Ehlers- Danlos Syndrome (vEDS) compared to non-vascular types I-III (nEDS). METHODS: To define cardiovascular surgical outcomes among adult patients (≥18 years) with EDS types, a review of our institution's in-house STS Adult Cardiac Surgery Database-compliant software and electronic medical records from Mayo Clinic (1993-2019) was performed. Outcomes were compared for vEDS patients and nEDS patients. Demographics, baseline characteristics, operative, in-hospital complications and follow-up vital status were analyzed. RESULTS: Over the study time frame, 48 EDS patients underwent surgery (mean age 52.6 ± 14.6 years; 48% females). Of these, 17 patients had vEDS and 31 patients had nEDS. Six patients (12.5%) underwent prior sternotomy. Urgent or emergent surgery was performed in 10 patients (20.8%). Aortic (vEDS 76.5% vs. nEDS 16.1%) and mitral procedures (vEDS 11.8% vs. nEDS 48.4%) were the two most common cardiovascular surgeries performed (p < .01 and p = .007, respectively). Cardiopulmonary bypass time (CPB) (165 ± 18 vs. 90 ± 13 min; p = .015) and aortic cross clamp times (140 ± 14 vs. 62 ± 10 min; p < .001) were longer for vEDS patients. There was 1 (2.1%) early and 7 (14.6%) late deaths; 6 among vEDS and 2 among nEDS patients. Survival at 5 (80% vs. 93%), 10 (45% vs. 84%) and 15 years (45% vs. 84%) was lower in patients with vEDS (p = .015 for each comparison). CONCLUSION: Cardiovascular surgeries are significantly more complex with longer bypass and cross clamp times for type IV vEDS compared to nEDS patients. Reduced overall survival underscores the complexity and fragility of vEDS patients.

Three Patient Kindred with a Novel Phenotype of Osteogenesis Imperfecta due to a COL1A1 Variant

Osteogenesis imperfecta (OI) is characterized by fractures and progressive bone deformities. Fracture rates peak during the toddler and adolescent years and decline during adulthood but do not stop entirely. We describe a kindred, the affected members of which were the mother and two sons, who presented with an apparently unique phenotype of OI. Our patients demonstrated a pattern of prenatal bone deformities followed by multiple, nontraumatic long bone fractures within the first two years of life and then an absence of nontraumatic fractures thereafter. No extra-skeletal manifestations have been noted to date. The mother did not receive bisphosphonate therapy but had no nontraumatic fractures after the age of five months. Intravenous bisphosphonate therapy was started for both sons within two months of birth, with the most recent infusions at age 18 months and 28 months in Patients 2 and 3, respectively. Two patients harbored a variant of uncertain significance in the COL1A1 gene. This heterozygous variant, c.3548C>T; p.(Pro1183Leu), is listed in the OI Variant Database as affecting only one other individual with osteopenia. We describe three family members with a unique presenting phenotype of OI, characterized by cessation of nontraumatic fractures after the first two years of life.

Impact of integrated translational research on clinical exome sequencing.

PURPOSE: Exome sequencing often identifies pathogenic genetic variants in patients with undiagnosed diseases. Nevertheless, frequent findings of variants of uncertain significance necessitate additional efforts to establish causality before reaching a conclusive diagnosis. To provide comprehensive genomic testing to patients with undiagnosed disease, we established an Individualized Medicine Clinic, which offered clinical exome testing and included a Translational Omics Program (TOP) that provided variant curation, research activities, or research exome sequencing. METHODS: From 2012 to 2018, 1101 unselected patients with undiagnosed diseases received exome testing. Outcomes were reviewed to assess impact of the TOP and patient characteristics on diagnostic rates through descriptive and multivariate analyses. RESULTS: The overall diagnostic yield was 24.9% (274 of 1101 patients), with 174 (15.8% of 1101) diagnosed on the basis of clinical exome sequencing alone. Four hundred twenty-three patients with nondiagnostic or without access to clinical exome sequencing were evaluated by the TOP, with 100 (9% of 1101) patients receiving a diagnosis, accounting for 36.5% of the diagnostic yield. The identification of a genetic diagnosis was influenced by the age at time of testing and the disease phenotype of the patient. CONCLUSION: Integration of translational research activities into clinical practice of a tertiary medical center can significantly increase the diagnostic yield of patients with undiagnosed disease.

Generation of Induced Pluripotent Stem Cells.

Induced pluripotent stem cells (iPSCs) are generated from somatic cells that have been reprogrammed by the ectopic expression of defined embryonic transcription factors. This technology has provided investigators with a powerful tool for modeling disease and developing treatments for human disorders. This chapter will provide the researcher with some background on iPSCs and details on how to produce MEF-conditioned medium, prepare mitotically arrested mouse embryonic fibroblasts (MEFs), create iPSCs using viral vectors, passage iPSCs, and cryopreserve iPSCs. The methods offered here have been used in many laboratories around the world and the reader can initially follow these methods. However, not all cell types are easily transduced using viral vectors and other methods of delivering the reprogramming transcription factors may need to be tested.

Functional expression of ZNF467 and PCBP2 supports adipogenic lineage commitment in adipose-derived mesenchymal stem cells.

Bone marrow-derived mesenchymal stromal/stem cells (BMSCs) have the potential to be employed in many different skeletal therapies. A major limitation to utilizing BMSCs as a therapeutic strategy in human disease and tissue regeneration is the low cell numbers obtained from initial isolation necessitating multiple cell passages that can lead to decreased cell quality. Adipose-derived mesenchymal stromal/stem cells (AMSCs) have been proposed as an alternative cell source for regenerative therapies; however the differentiation capacity of these cells differs from BMSCs. To understand the differences between BMSCs and AMSCs, we compared the global gene expression profiles of BMSCs and AMSCs and identified two genes, PCBP2 and ZNF467 that were differentially expressed between AMSCs and BMSCs. We demonstrate that PCBP2 and ZNF467 impact adipogenic but not osteogenic differentiation, further supporting evidence that AMSCs and BMSCs appear to be adapted to their microenvironment.

Case Report: Early-Onset Behavioral Variant Frontotemporal Dementia in Patient With Retrotransposed Full-Length Transcript of Matrin-3 Variant 5.

Frontotemporal dementia (FTD) rarely occurs in individuals under the age of 30, and genetic causes of early-onset FTD are largely unknown. The current report follows a 27 year-old patient with no significant past medical history presenting with two years of progressive changes in behavior, rushed speech, verbal aggression, and social withdrawal. MRI and FDG-PET imaging of the brain revealed changes maximally in the frontal and temporal lobes, which along with the clinical features, are consistent with behavioral variant FTD. Next generation sequencing of a panel of 28 genes associated with dementia and amyotrophic lateral sclerosis (ALS) initially revealed a duplication of exon 15 in Matrin-3 (MATR3). Whole genome sequencing determined that this genetic anomaly was, in fact, a sequence corresponding with full-length MATR3 variant 5 inserted into chromosome 12, indicating retrotransposition from a messenger RNA intermediate. To our knowledge, this is a novel mutation of MATR3, as the majority of mutations in MATR3 linked to FTD-ALS are point mutations. Genomic DNA analysis revealed that this mutation is also present in one unaffected first-degree relative and one unaffected second-degree relative. This suggests that the mutation is either a disease-causing mutation with incomplete penetrance, which has been observed in heritable FTD, or a benign variant. Retrotransposons are not often implicated in neurodegenerative diseases; thus, it is crucial to clarify the potential role of this MATR3 variant 5 retrotransposition in early-onset FTD.

Oncolytic Measles Virotherapy and Opposition to Measles Vaccination.

Recent measles epidemics in US and European cities where vaccination coverage has declined are providing a harsh reminder for the need to maintain protective levels of immunity across the entire population. Vaccine uptake rates have been declining in large part because of public misinformation regarding a possible association between measles vaccination and autism for which there is no scientific basis. The purpose of this article is to address a new misinformed antivaccination argument-that measles immunity is undesirable because measles virus is protective against cancer. Having worked for many years to develop engineered measles viruses as anticancer therapies, we have concluded (1) that measles is not protective against cancer and (2) that its potential utility as a cancer therapy will be enhanced, not diminished, by prior vaccination.

Fracture incidence in Ehlers-Danlos syndrome - A population-based case-control study.

BACKGROUND: The differential diagnosis of non-accidental injury during childhood includes medical conditions that predispose to skeletal fragility. Ehlers-Danlos syndrome (EDS) has been proposed as one such condition despite little objective evidence in the medical literature. OBJECTIVE: To investigate if EDS causes increased bone fragility during infancy and childhood. PARTICIPANTS AND SETTING: Residents of an 8-county region in southern Minnesota using the Rochester Epidemiology Project (REP) medical records-linkage system. METHODS: This retrospective, population-based, case-control study identified subjects with EDS from 1976 to 2015 who had complete records for at least their first year of life. Validity of diagnosis was ascertained using the 2017 International Classification of the Ehlers-Danlos Syndromes. Records were reviewed for fracture diagnoses that were characterized by age, location, type and mechanism. RESULTS: Of 219 potential cases, 21 had complete records for the first year of life and sufficient evidence in the medical record to support an EDS diagnosis. Of these 21, there were 14 hypermobile, 2 classical, 4 vascular, and 1 arthrochalasia EDS subtypes. 11 of 21 EDS cases (52.4%) and 15 of 63 controls (23.8%) had one or more fractures during childhood. No fractures were identified in the first year of life. Comparing cases to controls, EDS was associated with having any fractures during childhood with an odds ratio of 3.4 (95% CI: 1.20-9.66). CONCLUSIONS: We found no evidence that infants with common forms of EDS are predisposed to more frequent fractures. Ambulatory subjects with these EDS subtypes may have a higher incidence of fractures during childhood.

Correction: Arterial tortuosity syndrome: 40 new families and literature review.

In the published version of this paper the author Neus Baena's name was incorrectly given as Neus Baena Diez. This has now been corrected in both the HTML and PDF versions of the paper.

Validation of Osteogenic Properties of Cytochalasin D by High-Resolution RNA-Sequencing in Mesenchymal Stem Cells Derived from Bone Marrow and Adipose Tissues.

Differentiation of mesenchymal stromal/stem cells (MSCs) involves a series of molecular signals and gene transcription events required for attaining cell lineage commitment. Modulation of the actin cytoskeleton using cytochalasin D (CytoD) drives osteogenesis at early timepoints in bone marrow-derived MSCs and also initiates a robust osteogenic differentiation program in adipose tissue-derived MSCs. To understand the molecular basis for these pronounced effects on osteogenic differentiation, we investigated global changes in gene expression in CytoD-treated murine and human MSCs by high-resolution RNA-sequencing (RNA-seq) analysis. A three-way bioinformatic comparison between human adipose tissue-derived MSCs (hAMSCs), human bone marrow-derived MSCs (hBMSCs), and mouse bone marrow-derived MSCs (mBMSCs) revealed significant upregulation of genes linked to extracellular matrix organization, cell adhesion and bone metabolism. As anticipated, the activation of these differentiation-related genes is accompanied by a downregulation of nuclear and cell cycle-related genes presumably reflecting cytostatic effects of CytoD. We also identified eight novel CytoD activated genes-VGLL4, ARHGAP24, KLHL24, RCBTB2, BDH2, SCARF2, ACAD10, HEPH-which are commonly upregulated across the two species and tissue sources of our MSC samples. We selected the Hippo pathway-related VGLL4 gene, which encodes the transcriptional co-factor Vestigial-like 4, for further study because this pathway is linked to osteogenesis. VGLL4 small interfering RNA depletion reduces mineralization of hAMSCs during CytoD-induced osteogenic differentiation. Together, our RNA-seq analyses suggest that while the stimulatory effects of CytoD on osteogenesis are pleiotropic and depend on the biological state of the cell type, a small group of genes including VGLL4 may contribute to MSC commitment toward the bone lineage.

Aortic Root Dilation: Do Patients With Marfan Syndrome Fare Worse Than Those With Marfanoid Features?

OBJECTIVE: To discover whether patients with aortic root dilation and leptosomic features but without a diagnosis of Marfan syndrome (MFS) fare similarly to patients with MFS. METHODS: Of 124 patients with aortic root dilation identified from August 1, 1994, through October 31, 2012, 66 had MFS and 58 had leptosomic features but did not meet the Ghent criteria. Genetic testing was performed in 35% of patients (n=43). We compared z scores and aortic root diameters for patients who presented with aortic root dilation with and without an MFS diagnosis and with and without aortic root repair. RESULTS: No difference existed in initial aortic root diameters between groups (P=.15); however, mean ± SD z scores for patients without MFS and with MFS were 3.1±2.3 vs 4.5±3.2 (P=.005). Fourteen of 58 patients (24%) without MFS and 35 (53%) with MFS underwent aortic root operations (P<.05). For both groups who did not have surgery, aortic root diameters and z scores remained similar at follow-up (P=.20), as did 10-year survival: MFS, 100%; no MFS, 94.1% (P=.98). No significant difference was found for mean ± SD root diameter (no MFS, 38.9±7.3 mm; MFS, 35±8.6 mm; P=.06) or z score (no MFS, 2.4±2.0; MFS, 2.1±2.0; P=.53) for patients who underwent surgery. Two patients in each group had aortic root dissections. CONCLUSION: Similar rates of aortic dissection between the 2 groups warrant further study regarding patients with leptosomic features but no diagnosis of MFS. Aortic root dilation progressed similarly in patients who did not undergo surgery.

Arterial tortuosity syndrome: 40 new families and literature review.

PURPOSE: We delineate the clinical spectrum and describe the histology in arterial tortuosity syndrome (ATS), a rare connective tissue disorder characterized by tortuosity of the large and medium-sized arteries, caused by mutations in SLC2A10. METHODS: We retrospectively characterized 40 novel ATS families (50 patients) and reviewed the 52 previously reported patients. We performed histology and electron microscopy (EM) on skin and vascular biopsies and evaluated TGF-ß signaling with immunohistochemistry for pSMAD2 and CTGF. RESULTS: Stenoses, tortuosity, and aneurysm formation are widespread occurrences. Severe but rare vascular complications include early and aggressive aortic root aneurysms, neonatal intracranial bleeding, ischemic stroke, and gastric perforation. Thus far, no reports unequivocally document vascular dissections or ruptures. Of note, diaphragmatic hernia and infant respiratory distress syndrome (IRDS) are frequently observed. Skin and vascular biopsies show fragmented elastic fibers (EF) and increased collagen deposition. EM of skin EF shows a fragmented elastin core and a peripheral mantle of microfibrils of random directionality. Skin and end-stage diseased vascular tissue do not indicate increased TGF-ß signaling. CONCLUSION: Our findings warrant attention for IRDS and diaphragmatic hernia, close monitoring of the aortic root early in life, and extensive vascular imaging afterwards. EM on skin biopsies shows disease-specific abnormalities.

Functional validation reveals the novel missense V419L variant in TGFBR2 associated with Loeys-Dietz syndrome (LDS) impairs canonical TGF-ß signaling.

TGF-ß-related heritable connective tissue disorders are characterized by a similar pattern of cardiovascular defects, including aortic root dilatation, mitral valve prolapse, vascular aneurysms, and vascular dissections and exhibit incomplete penetrance and variable expressivity. Because of the phenotypic overlap of these disorders, panel-based genetic testing is frequently used to confirm the clinical findings. Unfortunately in many cases, variants of uncertain significance (VUSs) obscure the genetic diagnosis until more information becomes available. Here, we describe and characterize the functional impact of a novel VUS in the TGFBR2 kinase domain (c.1255G>T; p.Val419Leu), in a patient with the clinical diagnosis of Marfan syndrome spectrum. We assessed the structural and functional consequence of this VUS using molecular modeling, molecular dynamic simulations, and in vitro cell-based assays. A high-quality homology-based model of TGFBR2 was generated and computational mutagenesis followed by refinement and molecular dynamics simulations were used to assess structural and dynamic changes. Relative to wild type, the V419L induced conformational and dynamic changes that may affect ATP binding, increasing the likelihood of adopting an inactive state, and, we hypothesize, alter canonical signaling. Experimentally, we tested this by measuring the canonical TGF-ß signaling pathway activation at two points; V419L significantly delayed SMAD2 phosphorylation by western blot and significantly decreased TGF-ß-induced gene transcription by reporter assays consistent with known pathogenic variants in this gene. Thus, our results establish that the V419L variant leads to aberrant TGF-ß signaling and confirm the diagnosis of Loeys-Dietz syndrome in this patient.