Identification of rare genetic variants for rotator cuff tearing and repair in high-risk pedigrees.

Background: Common genetic variants with small effect sizes have been associated with rotator cuff tearing although very few rare, highly penetrant variants have been identified. The purpose of this pilot study was to identify dominant coding variants that segregated with affected individuals in pedigrees at high risk for rotator cuff tears (RCTs). We hypothesize that rare variants contribute to symptomatic RCTs and that they can be identified in related cases with a full-thickness tear requiring surgical management. Methods: We used the Utah Population Database to identify pedigrees that exhibited a significant excess of individuals who had undergone surgical repair of a full-thickness RCT. We analyzed whole exome sequence analysis to identify rare coding variants in 9 independent affected cousin pairs (first or second cousins) who had undergone arthroscopic surgery for repair of a full-thickness RCT (mean age at diagnosis 68 years). Validation of association of the candidate variants with risk for rotator cuff tearing was accomplished utilizing data from the UK Biobank and a separate cohort of unrelated cases of full-thickness RCTs. Results: A total of 82 rare (minor allele frequency <0.005) coding variants were identified as shared in at least one cousin pair affected with full-thickness rotator cuff tearing belonging to a high-risk pedigree, which included variants in RUNX1, ADAM12, TGFBR2, APBB1, PDLIM7, LTBP1, MAP3K4, and MAP3K1. Analysis of 39 of these variants with data available in the UK Biobank (3899 cases with rotator cuff injury and 11,697 matched controls; mean case age 59.9 years) identified a significant association with the APBB1 gene (OR = 2.37, P = .007, uncorrected). The PDLIM7 allele was found to be in significant excess in RCT cases in a separate cohort of Utah patients with full-thickness RCTs (10 carriers out of 458 independent, unrelated patients; minor allele frequency of 0.022) compared to a minor allele frequency of 0.0058 for the European (non-Finnish) control population rate (749 carriers out of 128612 tested) (chi-square test: 19.3 [P < .001]). Discussion: The analysis of closely related individuals with confirmed full-thickness RCTs from high-risk pedigrees has identified 82 rare, shared candidate genetic predisposition coding variants. Association of the PDLIM7 allele with risk for tear was confirmed in an independent cohort of RCTs. Further analysis of the variant alleles is required for confirmation of these genes in rotator cuff tearing.

The Association of Social Media Use and Eating Behaviour of Belgian Adolescent Girls Diagnosed with Anorexia Nervosa-A Qualitative Approach.

BACKGROUND: Social media have become integral in adolescents' lives, presenting both opportunities and risks, especially concerning psychiatric issues like eating disorders, prevalent in this vulnerable age group. METHODS: This qualitative study employed semi-structured interviews with seven adolescent girls (aged 15-17) diagnosed with eating disorders. Interviews covered seven predefined topics, recorded and transcribed for thematic analysis. RESULTS: Participants identified four key themes: exposure to selective content, biased interpretation, behavioural adaptation, and evolving perspectives during recovery. They highlighted social media's role in exacerbating body dissatisfaction and altering behaviours related to eating disorders. CONCLUSIONS: This research underscores the critical need for awareness and guidance in adolescents' social media use to mitigate negative impacts, emphasizing the potential link between exposure to specific content and cognitive-behavioural changes in those with eating disorders. Further investigation is warranted to deepen our comprehension of these dynamics.

Detection of tumor-derived cell-free DNA in cerebrospinal fluid using a clinically validated targeted sequencing panel for pediatric brain tumors.

PURPOSE: Clinical sequencing of tumor DNA is necessary to render an integrated diagnosis and select therapy for children with primary central nervous system (CNS) tumors, but neurosurgical biopsy is not without risk. In this study, we describe cell-free DNA (cfDNA) in blood and cerebrospinal fluid (CSF) as sources for "liquid biopsy" in pediatric brain tumors. METHODS: CSF samples were collected by lumbar puncture, ventriculostomy, or surgery from pediatric patients with CNS tumors. Following extraction, CSF-derived cfDNA was sequenced using UW-OncoPlex™, a clinically validated next-generation sequencing platform. CSF-derived cfDNA results and paired plasma and tumor samples concordance was also evaluated. RESULTS: Seventeen CSF samples were obtained from 15 pediatric patients with primary CNS tumors. Tumor types included medulloblastoma (n = 7), atypical teratoid/rhabdoid tumor (n = 2), diffuse midline glioma with H3 K27 alteration (n = 4), pilocytic astrocytoma (n = 1), and pleomorphic xanthoastrocytoma (n = 1). CSF-derived cfDNA was detected in 9/17 (53%) of samples, and sufficient for sequencing in 8/10 (80%) of extracted samples. All somatic mutations and copy-number variants were also detected in matched tumor tissue, and tumor-derived cfDNA was absent in plasma samples and controls. Tumor-derived cfDNA alterations were detected in the absence of cytological evidence of malignant cells in as little as 200 µl of CSF. Several clinically relevant alterations, including a KIAA1549::BRAF fusion were detected. CONCLUSIONS: Clinically relevant genomic alterations are detectable using CSF-derived cfDNA across a range of pediatric brain tumors. Next-generation sequencing platforms are capable of producing a high yield of DNA alterations with 100% concordance rate with tissue analysis.

A Rare Variant in MDH2 (rs111879470) Is Associated with Predisposition to Recurrent Breast Cancer in an Extended High-Risk Pedigree.

A significant fraction of breast cancer recurs, with lethal outcome, but specific genetic variants responsible have yet to be identified. Five cousin pairs with recurrent breast cancer from pedigrees with a statistical excess of recurrent breast cancer were sequenced to identify rare, shared candidate predisposition variants. The candidates were tested for association with breast cancer risk with UKBiobank data. Additional breast cancer cases were assayed for a subset of candidate variants to test for co-segregation. Three-dimensional protein structure prediction methods were used to investigate how the mutation under consideration is predicted to change structural and electrostatic properties in the mutated protein. One hundred and eighty-one rare candidate predisposition variants were shared in at least one cousin pair from a high-risk pedigree. A rare variant in MDH2 was found to segregate with breast-cancer-affected relatives in one extended pedigree. MDH2 is an estrogen-stimulated gene encoding the protein malate dehydrogenase, which catalyzes the reversible oxidation of malate to oxaloacetate. The molecular simulation results strongly suggest that the mutation changes the NAD+ binding pocket electrostatics of MDH2. This small sequencing study, using a powerful approach based on recurrent breast cancer cases from high-risk pedigrees, identified a set of strong candidate variants for inherited predisposition for breast cancer recurrence, including MDH2, which should be pursued in other resources.

High-Risk Pedigree Study Identifies LRBA (rs62346982) as a Likely Predisposition Variant for Prostate Cancer.

There is evidence for contribution of inherited factors to prostate cancer, and more specifically to lethal prostate cancer, but few responsible genes/variants have been identified. We examined genetic sequence data for 51 affected cousin pairs who each died from prostate cancer and who were members of high-risk prostate cancer pedigrees in order to identify rare variants shared by the cousins as candidate predisposition variants. Candidate variants were tested for association with prostate cancer risk in UK Biobank data. Candidate variants were also assayed in 1195 additional sampled Utah prostate cancer cases. We used 3D protein structure prediction methods to analyze structural changes and provide insights into mechanisms of pathogenicity. Almost 4000 rare (<0.005) variants were identified as shared in the 51 affected cousin pairs. One candidate variant was also significantly associated with prostate cancer risk among the 840 variants with data in UK Biobank, in the gene LRBA (p = 3.2 × 10-5; OR = 2.09). The rare risk variant in LRBA was observed to segregate in five pedigrees. The overall predicted structures of the mutant protein do not show any significant overall changes upon mutation, but the mutated structure loses a helical structure for the two residues after the mutation. This unique analysis of closely related individuals with lethal prostate cancer, who were members of high-risk prostate cancer pedigrees, has identified a strong set of candidate predisposition variants which should be pursued in independent studies. Validation data for a subset of the candidates identified are presented, with strong evidence for a rare variant in LRBA.

A rare FGF5 candidate variant (rs112475347) for predisposition to nonsquamous, nonsmall-cell lung cancer.

A unique approach with rare resources was used to identify candidate variants predisposing to familial nonsquamous nonsmall-cell lung cancers (NSNSCLC). We analyzed sequence data from NSNSCLC-affected cousin pairs belonging to high-risk lung cancer pedigrees identified in a genealogy of Utah linked to statewide cancer records to identify rare, shared candidate predisposition variants. Variants were tested for association with lung cancer risk in UK Biobank. Evidence for linkage with lung cancer was also reviewed in families from the Genetic Epidemiology of Lung Cancer Consortium. Protein prediction modeling compared the mutation with reference. We sequenced NSNSCLC-affected cousin pairs from eight high-risk lung cancer pedigrees and identified 66 rare candidate variants shared in the cousin pairs. One variant in the FGF5 gene also showed significant association with lung cancer in UKBiobank. This variant was observed in 3/163 additional sampled Utah lung cancer cases, 2 of whom were related in another independent pedigree. Modeling of the predicted protein predicted a second binding site for SO4 that may indicate binding differences. This unique study identified multiple candidate predisposition variants for NSNSCLC, including a rare variant in FGF5 that was significantly associated with lung cancer risk and that segregated with lung cancer in the two pedigrees in which it was observed. FGF5 is an oncogenic factor in several human cancers, and the mutation found here (W81C) changes the binding ability of heparan sulfate to FGF5, which might lead to its deregulation. These results support FGF5 as a potential NSNSCLC predisposition gene and present additional candidate predisposition variants.

A likely HOXC4 predisposition variant for Chiari malformations.

OBJECTIVE: Inherited variants predisposing patients to type 1 or 1.5 Chiari malformation (CM) have been hypothesized but have proven difficult to confirm. The authors used a unique high-risk pedigree population resource and approach to identify rare candidate variants that likely predispose individuals to CM and protein structure prediction tools to identify pathogenicity mechanisms. METHODS: By using the Utah Population Database, the authors identified pedigrees with significantly increased numbers of members with CM diagnosis. From a separate DNA biorepository of 451 samples from CM patients and families, 32 CM patients belonging to 1 or more of 24 high-risk Chiari pedigrees were identified. Two high-risk pedigrees had 3 CM-affected relatives, and 22 pedigrees had 2 CM-affected relatives. To identify rare candidate predisposition gene variants, whole-exome sequence data from these 32 CM patients belonging to 24 CM-affected related pairs from high-risk pedigrees were analyzed. The I-TASSER package for protein structure prediction was used to predict the structures of both the wild-type and mutant proteins found here. RESULTS: Sequence analysis of the 24 affected relative pairs identified 38 rare candidate Chiari predisposition gene variants that were shared by at least 1 CM-affected pair from a high-risk pedigree. The authors found a candidate variant in HOXC4 that was shared by 2 CM-affected patients in 2 independent pedigrees. All 4 of these CM cases, 2 in each pedigree, exhibited a specific craniocervical bony phenotype defined by a clivoaxial angle less than 125°. The protein structure prediction results suggested that the mutation considered here may reduce the binding affinity of HOXC4 to DNA. CONCLUSIONS: Analysis of unique and powerful Utah genetic resources allowed identification of 38 strong candidate CM predisposition gene variants. These variants should be pursued in independent populations. One of the candidates, a rare HOXC4 variant, was identified in 2 high-risk CM pedigrees, with this variant possibly predisposing patients to a Chiari phenotype with craniocervical kyphosis.

Engaging Institutional Stakeholders to Develop and Implement Guidelines for Recruiting Participants in Research Studies Using Social Media: Mixed Methods, Multi-Phase Process.

BACKGROUND: Limited regulatory guidance surrounding the use of social media channels for participant recruitment is an interdisciplinary challenge. Establishing stakeholder-informed procedures is essential for ethical and effective use of social media for participant recruitment. OBJECTIVE: This study aims to provide replicable procedures for developing and implementing guidelines for using social media to recruit participants in research studies. METHODS: Social media use cases at the university were used to identify institutional stakeholders for the initiative. After establishing workflow procedures, a scoping review of web-based materials about recruitment and research on the internet and social media from 19 peer institutions and 2 federal agencies was conducted to inform the structure of the policies and procedures. End users (investigators and study coordinators; N=14) also provided feedback on the policies and procedures and implementation. RESULTS: Representatives (n=7) from 5 institutional offices and 15 subject-matter experts from 5 areas were identified as stakeholders in the development of policies and procedures. Peers with web-based materials (n=16) identified in the scoping review revealed 4 themes that served as a basis for developing our policies and procedures. End user feedback further informed the policies and procedures and implementation. A centrally managed social media account for communicating with participants and hosting advertising campaigns on social media was also established and, when combined with the policies and procedures, resulted in 39 advertising campaigns, and 2846 participants were enrolled in health and clinical research studies. CONCLUSIONS: Our policies and procedures allow research teams to harness the potential of social media to increase study recruitment and participation; the transparent, stakeholder-informed process can be replicated by institutional administrators to establish policies and procedures that meet the interests and needs of their research community.

A Rare Variant in ERF (rs144812092) Predisposes to Prostate and Bladder Cancers in an Extended Pedigree.

Pairs of related bladder cancer cases who belong to pedigrees with an excess of bladder cancer were sequenced to identify rare, shared variants as candidate predisposition variants. Candidate variants were tested for association with bladder cancer risk. A validated variant was assayed for segregation to other related cancer cases, and the predicted protein structure of this variant was analyzed. This study of affected bladder cancer relative pairs from high-risk pedigrees identified 152 bladder cancer predisposition candidate variants. One variant in ERF (ETS Repressing Factor) was significantly associated with bladder cancer risk in an independent population, was observed to segregate with bladder and prostate cancer in relatives, and showed evidence for altering the function of the associated protein. This finding of a rare variant in ERF that is strongly associated with bladder and prostate cancer risk in an extended pedigree both validates ERF as a cancer predisposition gene and shows the continuing value of analyzing affected members of high-risk pedigrees to identify and validate rare cancer predisposition variants.

An intronic variant in the CELF4 gene is associated with risk for colorectal cancer.

BACKGROUND: Germline predisposition variants associated with colorectal cancer (CRC) have been identified but all are not yet identified. We sought to identify the responsible predisposition germline variant in an extended high-risk CRC pedigree that exhibited evidence of linkage to the 18q12.2 region (TLOD = +2.81). METHODS: DNA from two distantly related carriers of the hypothesized predisposition haplotype on 18q12.2 was sequenced to identify candidate variants. The candidate rare variants shared by the related sequenced subjects were screened in 3,094 CRC cases and 5x population-matched controls from UKBiobank to test for association. Further segregation of the variant was tested via Taqman assay in other sampled individuals in the pedigree. RESULTS: Analysis of whole genome sequence data for the two related hypothesized predisposition haplotype carriers, restricted to the shared haplotype boundaries, identified multiple (n = 6) rare candidate non-coding variants that were tested for association with CRC risk in UKBiobank. A rare intronic variant ofCELF4 gene, rs568643870, was significantly associated with CRC (p = 0.004, OR = 5.0), and segregated with CRC in other members of the linked pedigree. CONCLUSION: Evidence of segregation in a high-risk pedigree, case-control association in an external dataset, and identification of additional CRC-affected carriers in the linked pedigree support a role for a rareCELF4 intronic variant in CRC risk.

A role for the MEGF6 gene in predisposition to osteoporosis.

Osteoporosis is a common skeletal disorder characterized by deterioration of bone tissue. The set of genetic factors contributing to osteoporosis is not completely specified. High-risk osteoporosis pedigrees were analyzed to identify genes that may confer susceptibility to disease. Candidate predisposition variants were identified initially by whole exome sequencing of affected-relative pairs, approximately cousins, from 10 pedigrees. Variants were filtered on the basis of population frequency, concordance between pairs of cousins, affecting a gene associated with osteoporosis, and likelihood to have functionally damaging, pathogenic consequences. Subsequently, variants were tested for segregation in 68 additional relatives of the index carriers. A rare variant in MEGF6 (rs755467862) showed strong evidence of segregation with the disease phenotype. Predicted protein folding indicated the variant (Cys200Tyr) may disrupt structure of an EGF-like calcium-binding domain of MEGF6. Functional analyses demonstrated that complete loss of the paralogous genes megf6a and megf6b in zebrafish resulted in significant delay of cartilage and bone formation. Segregation analyses, in silico protein structure modeling, and functional assays support a role for MEGF6 in predisposition to osteoporosis.

The HOXB13 p.Gly84Glu variant observed in an extended five generation high-risk prostate cancer pedigree supports risk association for multiple cancer sites.

HOXB13 p.Gly84Glu is recognized as a rare variant associated with increased risk for prostate cancer; risk association for other cancers is uncertain. This HOXB13 variant was originally reported in several 3-generation prostate cancer pedigrees and has been reported to be associated with increased risk for bladder and colorectal cancer and leukemia in GWAS. A HOXB13 pGly84Glu variant carrier was identified in a set of Utah individuals born more than 100 years ago who were members of high-risk cancer pedigrees. The proband carrier was diagnosed with colon cancer and is a member of a high-risk prostate cancer pedigree. The HOXB13 pGLY84Glu variant was assayed in other sampled relatives in the pedigree and was observed to segregate in relatives of the proband carrier in the extended pedigree; this pedigree showed significant excess of prostate cancer, cervical cancer, leukemia, colorectal cancer, and gastric cancer among descendants. Multiple additional variant carriers were identified, diagnosed with prostate, bladder, and colon cancers in the 5-generation high-risk cancer pedigree. This study shows the power and efficiency of a biorepository of samples with known genealogy from extended high-risk pedigrees for definition of cancer-associated risks. Association of HOXB13 p.Gly84Glu with risk of colon and bladder cancers in this extended pedigree confirms previous reports for risk association for both cancers.

A hyperactivating proinflammatory RIPK2 allele associated with early-onset osteoarthritis.

Osteoarthritis (OA) is a common debilitating disease characterized by abnormal remodeling of the cartilage and bone of the articular joint. Ameliorating therapeutics are lacking due to limited understanding of the molecular pathways affecting disease initiation and progression. Notably, although a link between inflammation and overt OA is well established, the role of inflammation as a driver of disease occurrence is highly disputed. We analyzed a family with dominant inheritance of early-onset OA and found that affected individuals harbored a rare variant allele encoding a significant amino acid change (p.Asn104Asp) in the kinase domain of receptor interacting protein kinase 2 (RIPK2), which transduces signals from activated bacterial peptidoglycan sensors through the NF-κB pathway to generate a proinflammatory immune response. Functional analyses of RIPK2 activity in zebrafish embryos indicated that the variant RIPK2104Asp protein is hyperactive in its signaling capacity, with augmented ability to activate the innate immune response and the NF-κB pathway and to promote upregulation of OA-associated genes. Further we show a second allele of RIPK2 linked to an inflammatory disease associated with arthritis also has enhanced activity stimulating the NF-κB pathway. Our studies reveal for the first time the inflammatory response can function as a gatekeeper risk factor for OA.

A Nonsynonymous Variant in the GOLM1 Gene in Cutaneous Malignant Melanoma.

Background: Statistically significant linkage of melanoma to chromosome 9q21 was previously reported in a Danish pedigree resource and independently confirmed in Utah high-risk pedigrees, indicating strong evidence that this region contains a melanoma predisposition gene. Methods: Whole-exome sequencing of pairs of related melanoma case subjects from two pedigrees with evidence of 9q21 linkage was performed to identify the responsible predisposition gene. Candidate variants were tested for association with melanoma in an independent set of 454 unrelated familial melanoma case subjects and 396 unrelated cancer-free control subjects from Utah, and 1534 melanoma case subjects and 1146 noncancer control subjects from Texas (MD Anderson) via a two-sided Fisher exact test. Results: A rare nonsynonymous variant in Golgi Membrane Protein 1 (GOLM1), rs149739829, shared in two hypothesized predisposition carriers in one linked pedigree was observed. Segregation of this variant in additional affected relatives of the index carriers was confirmed. A statistically significant excess of carriers of the variant was observed among Utah case subjects and control subjects (odds ratio [OR] = 9.81, 95% confidence interval [CI] = 8.35 to 11.26, P < .001) and statistically significantly confirmed in Texas case subjects and control subjects (OR = 2.45, 95% CI = 1.65 to 3.25, P = .02). Conclusion: These findings support GOLM1 as a candidate melanoma predisposition gene.

Germline but not somatic de novo mutations are common in human congenital diaphragmatic hernia.

OBJECTIVES: Congenital diaphragmatic hernia (CDH) is a developmental defect of the diaphragm that causes high newborn morbidity and mortality. CDH is considered to be a multifactorial disease, with strong evidence implicating genetic factors. Although recent studies suggest the biological role of deleterious germline de novo variants, the effect of gene variants specific to the diaphragm remains unclear, and few single genes have been definitively implicated in human disease. METHODS: We performed genome sequencing on 16 individuals with CDH and their unaffected parents, including 10 diaphragmatic samples. RESULTS: We did not detect damaging somatic mutations in diaphragms, but identified germline heterozygous de novo functional mutations of 14 genes in nine patients. Although the majority of these genes are not known to be associated with CDH, one patient with CDH and cardiac anomalies harbored a frameshift mutation in NR2F2 (aka COUP-TFII), generating a premature truncation of the protein. This patient also carried a missense variant predicted to be damaging in XIRP2 (aka Myomaxin), a transcriptional target of MEF2A. Both NR2F2 and MEF2A map to chromosome 15q26, where recurring de novo deletions and unbalanced translocations have been observed in CDH. CONCLUSIONS: Somatic variants are not common in CDH. To our knowledge, this is the second case of a germline de novo frameshift mutation in NR2F2 in CDH. Since NR2F2 null mice exhibit a diaphragmatic defect, and XIRP2 is implicated in cardiac development, our data suggest the role of these two variants in the etiology of CDH, and possibly cardiac anomalies.

Mutual proximity graphs for improved reachability in music recommendation.

This paper is concerned with the impact of hubness, a general problem of machine learning in high-dimensional spaces, on a real-world music recommendation system based on visualisation of a k-nearest neighbour (knn) graph. Due to a problem of measuring distances in high dimensions, hub objects are recommended over and over again while anti-hubs are nonexistent in recommendation lists, resulting in poor reachability of the music catalogue. We present mutual proximity graphs, which are an alternative to knn and mutual knn graphs, and are able to avoid hub vertices having abnormally high connectivity. We show that mutual proximity graphs yield much better graph connectivity resulting in improved reachability compared to knn graphs, mutual knn graphs and mutual knn graphs enhanced with minimum spanning trees, while simultaneously reducing the negative effects of hubness.

5'-methylschweinfurthin G reduces chondrosarcoma tumor growth .

New treatment options are urgently required in the field of chondrosarcoma, particularly of chondrosarcomas with a well-differentiated hyaline cartilage-like extracellular matrix (e.g., collagen II and proteoglycan-rich) phenotype, notoriously resistant to drug penetration, and having potential of progression towards higher grade. We investigated the feasibility of using 5'-methylschweinfurthin G (MeSG) as a tumor suppressor agent in the Swarm rat chondrosarcoma, an intermediate- to high-grade chondrosarcoma model, having a hyaline cartilage-like phenotype. Tumor cell culture studies were performed to identify their proliferative and cytotoxicity sensitivity to MeSG. Tumor burden mice were treated with MeSG and analyzed for tumor growth, morphology and regression. The chondrosarcoma tumor cells had a half maximum cytotoxicity concentration (IC50 ) of 35 nM MeSG; approximately 300-fold less than freshly isolated rat chondrocytes (IC50 of 11 µM). Multiple injections of MeSG (20 mg/kg, body weight) resulted in reduced/eliminated tumor growth over a 17-day period in mice, and an 83% reduction (p = 0.023) in tumor mass. Three out of ten MeSG treated mice had complete elimination of tumor. Tumors of treated mice had a decrease in chondrosarcoma cell proliferation (p = 0.012) and an increase in cell death (p = 0.030) compared with tumors of control mice. These findings in an animal model demonstrate the effectiveness of MeSG for treatment of rat chondrosarcomas, and may have the potential use as a therapeutic option for the difficult-to-treat intermediate-to high-grade hyaline cartilage-like chondrosarcoma. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1283-1293, 2018.

Regulation of thrombosis and vascular function by protein methionine oxidation.

Redox biology is fundamental to both normal cellular homeostasis and pathological states associated with excessive oxidative stress. Reactive oxygen species function not only as signaling molecules but also as redox regulators of protein function. In the vascular system, redox reactions help regulate key physiologic responses such as cell adhesion, vasoconstriction, platelet aggregation, angiogenesis, inflammatory gene expression, and apoptosis. During pathologic states, altered redox balance can cause vascular cell dysfunction and affect the equilibrium between procoagulant and anticoagulant systems, contributing to thrombotic vascular disease. This review focuses on the emerging role of a specific reversible redox reaction, protein methionine oxidation, in vascular disease and thrombosis. A growing number of cardiovascular and hemostatic proteins are recognized to undergo reversible methionine oxidation, in which methionine residues are posttranslationally oxidized to methionine sulfoxide. Protein methionine oxidation can be reversed by the action of stereospecific enzymes known as methionine sulfoxide reductases. Calcium/calmodulin-dependent protein kinase II is a prototypical methionine redox sensor that responds to changes in the intracellular redox state via reversible oxidation of tandem methionine residues in its regulatory domain. Several other proteins with oxidation-sensitive methionine residues, including apolipoprotein A-I, thrombomodulin, and von Willebrand factor, may contribute to vascular disease and thrombosis.

Protective vascular and cardiac effects of inducible nitric oxide synthase in mice with hyperhomocysteinemia.

Diet-induced hyperhomocysteinemia produces endothelial and cardiac dysfunction and promotes thrombosis through a mechanism proposed to involve oxidative stress. Inducible nitric oxide synthase (iNOS) is upregulated in hyperhomocysteinemia and can generate superoxide. We therefore tested the hypothesis that iNOS mediates the adverse oxidative, vascular, thrombotic, and cardiac effects of hyperhomocysteinemia. Mice deficient in iNOS (Nos2-/-) and their wild-type (Nos2+/+) littermates were fed a high methionine/low folate (HM/LF) diet to induce mild hyperhomocysteinemia, with a 2-fold increase in plasma total homocysteine (P<0.001 vs. control diet). Hyperhomocysteinemic Nos2+/+ mice exhibited endothelial dysfunction in cerebral arterioles, with impaired dilatation to acetylcholine but not nitroprusside, and enhanced susceptibility to carotid artery thrombosis, with shortened times to occlusion following photochemical injury (P<0.05 vs. control diet). Nos2-/- mice had decreased rather than increased dilatation responses to acetylcholine (P<0.05 vs. Nos2+/+ mice). Nos2-/- mice fed control diet also exhibited shortened times to thrombotic occlusion (P<0.05 vs. Nos2+/+ mice), and iNOS deficiency failed to protect from endothelial dysfunction or accelerated thrombosis in mice with hyperhomocysteinemia. Deficiency of iNOS did not alter myocardial infarct size in mice fed the control diet but significantly increased infarct size and cardiac superoxide production in mice fed the HM/LF diet (P<0.05 vs. Nos2+/+ mice). These findings suggest that endogenous iNOS protects from, rather than exacerbates, endothelial dysfunction, thrombosis, and hyperhomocysteinemia-associated myocardial ischemia-reperfusion injury. In the setting of mild hyperhomocysteinemia, iNOS functions to blunt cardiac oxidative stress rather than functioning as a source of superoxide.