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.

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.

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.

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.

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.

Histone H4 Methyltransferase Suv420h2 Maintains Fidelity of Osteoblast Differentiation.

Osteogenic lineage commitment and progression is controlled by multiple signaling pathways (e.g., WNT, BMP, FGF) that converge on bone-related transcription factors. Access of osteogenic transcription factors to chromatin is controlled by epigenetic regulators that generate post-translational modifications of histones ("histone code"), as well as read, edit and/or erase these modifications. Our understanding of the biological role of epigenetic regulators in osteoblast differentiation remains limited. Therefore, we performed next-generation RNA sequencing (RNA-seq) and established which chromatin-related proteins are robustly expressed in mouse bone tissues (e.g., fracture callus, calvarial bone). These studies also revealed that cells with increased osteogenic potential have higher levels of the H4K20 methyl transferase Suv420h2 compared to other methyl transferases (e.g., Suv39h1, Suv39h2, Suv420h1, Ezh1, Ezh2). We find that all six epigenetic regulators are transiently expressed at different stages of osteoblast differentiation in culture, with maximal mRNAs levels of Suv39h1 and Suv39h2 (at day 3) preceding maximal expression of Suv420h1 and Suv420h2 (at day 7) and developmental stages that reflect, respectively, early and later collagen matrix deposition. Loss of function analysis of Suv420h2 by siRNA depletion shows loss of H4K20 methylation and decreased expression of bone biomarkers (e.g., alkaline phosphatase/Alpl) and osteogenic transcription factors (e.g., Sp7/Osterix). Furthermore, Suv420h2 is required for matrix mineralization during osteoblast differentiation. We conclude that Suv420h2 controls the H4K20 methylome of osteoblasts and is critical for normal progression of osteoblastogenesis. J. Cell. Biochem. 118: 1262-1272, 2017. © 2016 Wiley Periodicals, Inc.

Epigenetic Control of Skeletal Development by the Histone Methyltransferase Ezh2.

Epigenetic control of gene expression is critical for normal fetal development. However, chromatin-related mechanisms that activate bone-specific programs during osteogenesis have remained underexplored. Therefore, we investigated the expression profiles of a large cohort of epigenetic regulators (>300) during osteogenic differentiation of human mesenchymal cells derived from the stromal vascular fraction of adipose tissue (AMSCs). Molecular analyses establish that the polycomb group protein EZH2 (enhancer of zeste homolog 2) is down-regulated during osteoblastic differentiation of AMSCs. Chemical inhibitor and siRNA knockdown studies show that EZH2, a histone methyltransferase that catalyzes trimethylation of histone 3 lysine 27 (H3K27me3), suppresses osteogenic differentiation. Blocking EZH2 activity promotes osteoblast differentiation and suppresses adipogenic differentiation of AMSCs. High throughput RNA sequence (mRNASeq) analysis reveals that EZH2 inhibition stimulates cell cycle inhibitory proteins and enhances the production of extracellular matrix proteins. Conditional genetic loss of Ezh2 in uncommitted mesenchymal cells (Prrx1-Cre) results in multiple defects in skeletal patterning and bone formation, including shortened forelimbs, craniosynostosis, and clinodactyly. Histological analysis and mRNASeq profiling suggest that these effects are attributable to growth plate abnormalities and premature cranial suture closure because of precocious maturation of osteoblasts. We conclude that the epigenetic activity of EZH2 is required for skeletal patterning and development, but EZH2 expression declines during terminal osteoblast differentiation and matrix production.

The effects of polymorphisms on human gene targeting.

DNA mismatches that occur between vector homology arms and chromosomal target sequences reduce gene targeting frequencies in several species; however, this has not been reported in human cells. Here we demonstrate that even a single mismatched base pair can significantly decrease human gene targeting frequencies. In addition, we show that homology arm polymorphisms can be used to direct allele-specific targeting or to improve unfavorable vector designs that introduce deletions.

High-resolution molecular validation of self-renewal and spontaneous differentiation in clinical-grade adipose-tissue derived human mesenchymal stem cells.

Improving the effectiveness of adipose-tissue derived human mesenchymal stromal/stem cells (AMSCs) for skeletal therapies requires a detailed characterization of mechanisms supporting cell proliferation and multi-potency. We investigated the molecular phenotype of AMSCs that were either actively proliferating in platelet lysate or in a basal non-proliferative state. Flow cytometry combined with high-throughput RNA sequencing (RNASeq) and RT-qPCR analyses validate that AMSCs express classic mesenchymal cell surface markers (e.g., CD44, CD73/NT5E, CD90/THY1, and CD105/ENG). Expression of CD90 is selectively elevated at confluence. Self-renewing AMSCs express a standard cell cycle program that successively mediates DNA replication, chromatin packaging, cyto-architectural enlargement, and mitotic division. Confluent AMSCs preferentially express genes involved in extracellular matrix (ECM) formation and cellular communication. For example, cell cycle-related biomarkers (e.g., cyclins E2 and B2, transcription factor E2F1) and histone-related genes (e.g., H4, HINFP, NPAT) are elevated in proliferating AMSCs, while ECM genes are strongly upregulated (>10-fold) in quiescent AMSCs. AMSCs also express pluripotency genes (e.g., POU5F1, NANOG, KLF4) and early mesenchymal markers (e.g., NES, ACTA2) consistent with their multipotent phenotype. Strikingly, AMSCs modulate expression of WNT signaling components and switch production of WNT ligands (from WNT5A/WNT5B/WNT7B to WNT2/WNT2B), while upregulating WNT-related genes (WISP2, SFRP2, and SFRP4). Furthermore, post-proliferative AMSCs spontaneously express fibroblastic, osteogenic, chondrogenic, and adipogenic biomarkers when maintained in confluent cultures. Our findings validate the biological properties of self-renewing and multi-potent AMSCs by providing high-resolution quality control data that support their clinical versatility.

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.

Normal collagen and bone production by gene-targeted human osteogenesis imperfecta iPSCs.

Osteogenesis imperfecta (OI) is caused by dominant mutations in the type I collagen genes. In principle, the skeletal abnormalities of OI could be treated by transplantation of patient-specific, bone-forming cells that no longer express the mutant gene. Here, we develop this approach by isolating mesenchymal cells from OI patients, inactivating their mutant collagen genes by adeno-associated virus (AAV)-mediated gene targeting, and deriving induced pluripotent stem cells (iPSCs) that were expanded and differentiated into mesenchymal stem cells (iMSCs). Gene-targeted iMSCs produced normal collagen and formed bone in vivo, but were less senescent and proliferated more than bone-derived MSCs. To generate iPSCs that would be more appropriate for clinical use, the reprogramming and selectable marker transgenes were removed by Cre recombinase. These results demonstrate that the combination of gene targeting and iPSC derivation can be used to produce potentially therapeutic cells from patients with genetic disease.

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.

Nonintegrating foamy virus vectors.

Foamy viruses (FVs), or spumaviruses, are integrating retroviruses that have been developed as vectors. Here we generated nonintegrating foamy virus (NIFV) vectors by introducing point mutations into the highly conserved DD35E catalytic core motif of the foamy virus integrase sequence. NIFV vectors produced high-titer stocks, transduced dividing cells, and did not integrate. Cells infected with NIFV vectors contained episomal vector genomes that consisted of linear, 1-long-terminal-repeat (1-LTR), and 2-LTR circular DNAs. These episomes expressed transgenes, were stable, and became progressively diluted in the dividing cell population. 1-LTR circles but not 2-LTR circles were found in all vector stocks prior to infection. Residual integration of NIFV vectors occurred at a frequency 4 logs lower than that of integrase-proficient FV vectors. Cre recombinase expressed from a NIFV vector mediated excision of both an integrated, floxed FV vector and a gene-targeted neo expression cassette, demonstrating the utility of these episomal vectors. The broad host range and large packaging capacity of NIFV vectors should make them useful for a variety of applications requiring transient gene expression.

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.

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.

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.

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.

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.