Congenital heart defects caused by FOXJ1.

FOXJ1 is expressed in ciliated cells of the airways, testis, oviduct, central nervous system and the embryonic left-right organizer. Ablation or targeted mutation of Foxj1 in mice, zebrafish and frogs results in loss of ciliary motility and/or reduced length and number of motile cilia, affecting the establishment of the left-right axis. In humans, heterozygous pathogenic variants in FOXJ1 cause ciliopathy leading to situs inversus, obstructive hydrocephalus and chronic airway disease. Here, we report a novel truncating FOXJ1 variant (c.784_799dup; p.Glu267Glyfs*12) identified by clinical exome sequencing from a patient with isolated congenital heart defects (CHD) which included atrial and ventricular septal defects, double outlet right ventricle (DORV) and transposition of the great arteries. Functional experiments show that FOXJ1 c.784_799dup; p.Glu267Glyfs*12, unlike FOXJ1, fails to induce ectopic cilia in frog epidermis in vivo or to activate the ADGB promoter, a downstream target of FOXJ1 in cilia, in transactivation assays in vitro. Variant analysis of patients with heterotaxy or heterotaxy-related CHD indicates that pathogenic variants in FOXJ1 are an infrequent cause of heterotaxy. Finally, we characterize embryonic-stage CHD in Foxj1 loss-of-function mice, demonstrating randomized heart looping. Abnormal heart looping includes reversed looping (dextrocardia), ventral looping and no looping/single ventricle hearts. Complex CHDs revealed by histological analysis include atrioventricular septal defects, DORV, single ventricle defects as well as abnormal position of the great arteries. These results indicate that pathogenic variants in FOXJ1 can cause isolated CHD.

Dnmt3a deficiency in the skin causes focal, canonical DNA hypomethylation and a cellular proliferation phenotype.

DNA hypomethylation is a feature of epidermal cells from aged and sun-exposed skin, but the mechanisms responsible for this methylation loss are not known. Dnmt3a is the dominant de novo DNA methyltransferase in the skin; while epidermal Dnmt3a deficiency creates a premalignant state in which keratinocytes are more easily transformed by topical mutagens, the conditions responsible for this increased susceptibility to transformation are not well understood. Using whole genome bisulfite sequencing, we identified a focal, canonical DNA hypomethylation phenotype in the epidermal cells of Dnmt3a-deficient mice. Single-cell transcriptomic analysis revealed an increased proportion of cells with a proliferative gene expression signature, while other populations in the skin were relatively unchanged. Although total DNMT3A deficiency has not been described in human disease states, rare patients with an overgrowth syndrome associated with behavioral abnormalities and an increased risk of cancer often have heterozygous, germline mutations in DNMT3A that reduce its function (Tatton-Brown Rahman syndrome [TBRS]). We evaluated the DNA methylation phenotype of the skin from a TBRS patient with a germline DNMT3AR882H mutation, which encodes a dominant-negative protein that reduces its methyltransferase function by ∼80%. We detected a focal, canonical hypomethylation phenotype that revealed considerable overlap with hypomethylated regions found in Dnmt3a-deficient mouse skin. Together, these data suggest that DNMT3A loss creates a premalignant epigenetic state associated with a hyperproliferative phenotype in the skin and further suggest that DNMT3A acts as a tumor suppressor in the skin.

Remethylation of Dnmt3a-/- hematopoietic cells is associated with partial correction of gene dysregulation and reduced myeloid skewing.

Mutations in the DNA methyltransferase 3A (DNMT3A) gene are the most common cause of age-related clonal hematopoiesis (ARCH) in older individuals, and are among the most common initiating events for acute myeloid leukemia (AML). The most frequent DNMT3A mutation in AML patients (R882H) encodes a dominant-negative protein that reduces methyltransferase activity by ∼80% in cells with heterozygous mutations, causing a focal, canonical DNA hypomethylation phenotype; this phenotype is partially recapitulated in murine Dnmt3a-/- bone marrow cells. To determine whether the hypomethylation phenotype of Dnmt3a-/- hematopoietic cells is reversible, we developed an inducible transgene to restore expression of DNMT3A in transplanted bone marrow cells from Dnmt3a-/- mice. Partial remethylation was detected within 1 wk, but near-complete remethylation required 6 mo. Remethylation was accurate, dynamic, and highly ordered, suggesting that differentially methylated regions have unique properties that may be relevant for their functions. Importantly, 22 wk of DNMT3A addback partially corrected dysregulated gene expression, and mitigated the expansion of myeloid cells. These data show that restoring DNMT3A expression can alter the epigenetic "state" created by loss of Dnmt3a activity; this genetic proof-of-concept experiment suggests that this approach could be relevant for patients with ARCH or AML caused by loss-of-function DNMT3A mutations.

Genetic Variants Associated With Cancer Therapy-Induced Cardiomyopathy.

BACKGROUND: Cancer therapy-induced cardiomyopathy (CCM) is associated with cumulative drug exposures and preexisting cardiovascular disorders. These parameters incompletely account for substantial interindividual susceptibility to CCM. We hypothesized that rare variants in cardiomyopathy genes contribute to CCM. METHODS: We studied 213 patients with CCM from 3 cohorts: retrospectively recruited adults with diverse cancers (n=99), prospectively phenotyped adults with breast cancer (n=73), and prospectively phenotyped children with acute myeloid leukemia (n=41). Cardiomyopathy genes, including 9 prespecified genes, were sequenced. The prevalence of rare variants was compared between CCM cohorts and The Cancer Genome Atlas participants (n=2053), healthy volunteers (n=445), and an ancestry-matched reference population. Clinical characteristics and outcomes were assessed and stratified by genotypes. A prevalent CCM genotype was modeled in anthracycline-treated mice. RESULTS: CCM was diagnosed 0.4 to 9 years after chemotherapy; 90% of these patients received anthracyclines. Adult patients with CCM had cardiovascular risk factors similar to the US population. Among 9 prioritized genes, patients with CCM had more rare protein-altering variants than comparative cohorts ( P≤1.98e-04). Titin-truncating variants (TTNtvs) predominated, occurring in 7.5% of patients with CCM versus 1.1% of The Cancer Genome Atlas participants ( P=7.36e-08), 0.7% of healthy volunteers ( P=3.42e-06), and 0.6% of the reference population ( P=5.87e-14). Adult patients who had CCM with TTNtvs experienced more heart failure and atrial fibrillation ( P=0.003) and impaired myocardial recovery ( P=0.03) than those without. Consistent with human data, anthracycline-treated TTNtv mice and isolated TTNtv cardiomyocytes showed sustained contractile dysfunction unlike wild-type ( P=0.0004 and P<0.002, respectively). CONCLUSIONS: Unrecognized rare variants in cardiomyopathy-associated genes, particularly TTNtvs, increased the risk for CCM in children and adults, and adverse cardiac events in adults. Genotype, along with cumulative chemotherapy dosage and traditional cardiovascular risk factors, improves the identification of patients who have cancer at highest risk for CCM. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov . Unique identifiers: NCT01173341; AAML1031; NCT01371981.

Detailed phenotyping reveals distinct trajectories of cardiovascular function and symptoms with exposure to modern breast cancer therapy.

BACKGROUND: Breast cancer therapies are associated with a risk of cardiac dysfunction, most commonly defined by changes in left ventricular ejection fraction (LVEF). Recently, the authors identified 3 classes of LVEF change after exposure to anthracyclines and/or trastuzumab using latent class growth modeling. The objective of the current study was to characterize the clinical, biochemical, and functional profiles associated with LVEF trajectory class membership. METHODS: Transthoracic echocardiography and biomarker assessments were performed and questionnaires were administered at standardized intervals in a longitudinal cohort of 314 patients with breast cancer who were treated with anthracyclines and/or trastuzumab. Univariable and multivariable multinomial regression analyses evaluated associations between baseline variables and LVEF trajectory class membership. Generalized estimating equations were used to define mean changes in cardiovascular measures over time within each class. RESULTS: Among the 3 distinct subgroups of LVEF changes identified (stable [class 1]; modest, persistent decline [class 2]; and significant early decline followed by partial recovery [class 3]), higher baseline LVEF, radiotherapy, and sequential therapy with anthracyclines and/or trastuzumab were associated with class 2 or 3 membership. Sustained abnormalities in longitudinal strain and N-terminal pro-B-type natriuretic peptide (NT-proBNP) were observed in patients in class 2, as were heart failure symptoms. Similar abnormalities were observed in patients in class 3, but there was a trend toward recovery, particularly for longitudinal strain. CONCLUSIONS: Patients with modest, persistent LVEF declines experienced sustained abnormalities in imaging and biochemical markers of cardiac function and heart failure symptoms. Further investigation is needed to characterize the long-term risk of heart failure, particularly in those with modest LVEF declines.

A cross-sectional study of environmental, dog, and human-related risk factors for positive canine leptospirosis PCR test results in the United States, 2009 to 2016.

BACKGROUND: Canine leptospirosis is a reemerging zoonotic disease concern in North America, and a better understanding of its epidemiology is needed. Wide-scale use and subsequent analyses of polymerase chain reaction (PCR) testing may provide additional insight into leptospirosis. This study aimed to describe temporal trends, to descriptively map, and to identify environmental, dog, and human-level factors associated with positive canine leptospirosis PCR test results in the United States. RESULTS: Data obtained from IDEXX Laboratories, Inc. on 40,118 canine leptospirosis PCR tests run in the United States between 2009 and 2016 were evaluated. Climate and socioeconomic (e.g. urban influence, income) data were obtained from public databases. Choropleth maps were created to identify high test-positive proportion areas and a cross-sectional analysis was completed with generalized (univariable, followed by multivariable) mixed logistic regression models accounting for county within state to identify significant predictors for a positive test. Overall test-positive proportion was 5.4% across the United States, with the regional point estimate highest in the southwest (8.1%). In the final multivariable model, the odds of a positive test were greater for male dogs (Odds Ratio [OR] = 1.28) and dogs 0-4 years of age (ORs ranged from 0.35-0.71 for the other age groups). The odds of a positive test were greater for dogs living in areas with wet environmental conditions (OR = 1.24). Season and temperature, as well as the interaction between them, were significant predictors of a positive test. Dogs had a greater probability of testing positive during cool temperatures (< 4 °C) compared to the other temperature categories in the fall season. CONCLUSIONS: These findings based on PCR testing allow for an improved understanding of factors influencing a positive canine leptospirosis PCR test and will assist targeted education and prevention efforts.

Detailed Echocardiographic Phenotyping in Breast Cancer Patients: Associations With Ejection Fraction Decline, Recovery, and Heart Failure Symptoms Over 3 Years of Follow-Up.

BACKGROUND: Cardiovascular disease in patients with breast cancer is of growing concern. The longitudinal effects of commonly used therapies, including doxorubicin and trastuzumab, on cardiac remodeling and function remain unknown in this population. We aimed to define the changes in echocardiographic parameters of structure, function, and ventricular-arterial coupling, and their associations with left ventricular ejection fraction (LVEF) and heart failure symptoms. METHODS: In a longitudinal prospective cohort study of 277 breast cancer participants receiving doxorubicin (Dox), trastuzumab (Tras), or both (Dox+Tras), we obtained 1249 echocardiograms over a median follow-up of 2.0 (interquartile range, 1.0-3.0) years. Left ventricular structure, diastolic and contractile function, and ventricular-arterial coupling measures were quantified in a core laboratory blinded to participant characteristics. We evaluated changes in echocardiographic parameters over time, and used repeated-measures regression models to define their association with LVEF decline and recovery. Linear regression models defined the association between early changes in these parameters and subsequent changes in LVEF and heart failure symptoms. RESULTS: Overall, 177 (64%) received Dox, 51 (18%) received Tras, and 49 (18%) received Dox+Tras. With Dox, there was a sustained, modest decrease in LVEF over the follow-up duration (1-year change in LVEF -3.6%; 95% confidence interval [CI], -4.4% to -2.8%; 3-year change -3.8%; 95% CI, -5.1% to -2.5%). With Tras, a similar LVEF decline was observed at 1 year (-4.5%; 95% CI, -6.0% to -2.9%) and 3 years (-2.8%; 95%CI, -5.3 to -0.4%). Participants receiving Dox+Tras demonstrated the greatest declines at 1 year (-6.6%; 95% CI, -8.2 to -5.0%), with partial recovery at 3 years (-2.8%; 95% CI, -4.8 to -0.8%). LVEF declines and recovery were associated primarily with changes in systolic volumes, longitudinal and circumferential strain, and ventricular-arterial coupling indices, effective arterial elastance (Ea) and the coupling ratio Ea/Eessb, without evidence for effect modification across therapies. Early changes in volumes, strain, and Ea/Eessb at 4 to 6 months were associated with 1- and 2-year LVEF changes. Similarly, early changes in strain and Ea were associated with worsening heart failure symptoms at 1 year. CONCLUSIONS: Doxorubicin and trastuzumab resulted in modest, persistent declines in LVEF at 3 years. Changes in volumes, strain, and ventricular-arterial coupling were consistently associated with concurrent and subsequent LVEF declines and recovery across therapies.

Impact of Atrial Fibrillation on Heart Failure in Patients Treated With Anthracycline Chemotherapy.

Atrial fibrillation (AF) and anthracyclines are known risk factors for heart failure (HF). The magnitude of the effect of preexisting AF (preanthracycline AF) and newly developed AF (postanthracycline AF) in patients treated with anthracyclines on the occurrence of HF is unknown. The aim of our study was to characterize the impact of preanthracycline and postanthracycline AF on the subsequent occurrence of HF in patients treated with anthracyclines. In 5,598 patients treated with new anthracycline therapy at a tertiary center between 2008 and 2021, propensity score matching was used to match 204 pairs with or without preanthracycline AF and 135 pairs with or without postanthracycline AF. The primary outcome was new-onset symptomatic HF defined by the American Heart Association/American College of Cardiology guidelines. Patients with and without preanthracycline and postanthracycline AF were well matched for age, gender, medications, and cardiovascular risk factors. A total of 45 patients with preanthracycline AF and 23 matched patients developed HF (5-year cumulative incidence: 29% in the preanthracycline AF group and 13% in the matched group, p = 0.003; hazard ratio 2.1, 95% confidence interval 1.3 to 3.4, p = 0.004). A total of 161 patients (2.9%) developed postanthracycline AF. A total of 39 patients (5-year cumulative incidence: 40%) with postanthracycline AF and 9 matched patients (5-year cumulative incidence: 7%) developed HF (hazard ratio 6.1, 95% confidence interval 3.0 to 12.4, p <0.001). Preanthracycline AF and postanthracycline AF are associated with a high incidence of subsequent HF in patients treated with anthracyclines. Prospective studies of therapies are required to decrease HF in these high-risk patients.