TP53-mediated clonal hematopoiesis confers increased risk for incident atherosclerotic disease.

Somatic mutations in blood indicative of clonal hematopoiesis of indeterminate potential (CHIP) are associated with an increased risk of hematologic malignancy, coronary artery disease, and all-cause mortality. Here we analyze the relation between CHIP status and incident peripheral artery disease (PAD) and atherosclerosis, using whole-exome sequencing and clinical data from the UK Biobank and Mass General Brigham Biobank. CHIP associated with incident PAD and atherosclerotic disease across multiple beds, with increased risk among individuals with CHIP driven by mutation in DNA Damage Repair (DDR) genes such as TP53 and PPM1D. To model the effects of DDR-induced CHIP on atherosclerosis, we used a competitive bone marrow transplantation strategy, and generated atherosclerosis-prone Ldlr-/- chimeric mice carrying 20% p53-deficient hematopoietic cells. The chimeric mice were analyzed 13-weeks post-grafting and showed increased aortic plaque size and accumulation of macrophages within the plaque, driven by increased proliferation of p53-deficient plaque macrophages. In summary, our findings highlight the role of CHIP as a broad driver of atherosclerosis across the entire arterial system beyond the coronary arteries, and provide genetic and experimental support for a direct causal contribution of TP53-mutant CHIP to atherosclerosis.

Clonal hematopoiesis and atherosclerotic cardiovascular disease: A primer / Hematopoyesis clonal y enfermedad aterosclerótica cardiovascular: un iniciador

Despite current standards of care, a considerable risk of atherosclerotic cardiovascular disease remains in both primary and secondary prevention. In this setting, clonal hematopoiesis driven by somatic mutations has recently emerged as a relatively common, potent and independent risk factor for atherosclerotic cardiovascular disease and other cardiovascular conditions. Experimental studies in mice suggest that mutations in TET2 and JAK2, which are among the most common in clonal hematopoiesis, increase inflammation and are causally connected to accelerated atherosclerosis development, which may explain the link between clonal hematopoiesis and increased cardiovascular risk. In this review, we provide an overview of our current understanding of this emerging cardiovascular risk factor.(AU)

A pesar de los estándares de los cuidados actuales, persiste un riesgo considerable de enfermedad cardiovascular aterosclerótica tanto en la prevención primaria como secundaria. En este contexto, la hematopoyesis clonal impulsada por mutaciones somáticas ha emergido recientemente como un factor de riesgo relativamente común, potente e independiente, para la enfermedad cardiovascular aterosclerótica y otras situaciones cardiovasculares. Los estudios experimentales en ratones sugieren que las mutaciones en TET2 y JAK2, que se encuentran dentro de las hematopoyesis clonales más comunes, incrementan la inflamación y están causalmente conectadas con el desarrollo de aterosclerosis acelerado, lo cual puede explicar el vínculo entre la hematopoyesis clonal y el incremento del riesgo cardiovascular. En esta revisión, aportamos una visión general de nuestra comprensión actual de este factor de riesgo cardiovascular.(AU)

Clonal hematopoiesis and atherosclerotic cardiovascular disease: A primer.

Despite current standards of care, a considerable risk of atherosclerotic cardiovascular disease remains in both primary and secondary prevention. In this setting, clonal hematopoiesis driven by somatic mutations has recently emerged as a relatively common, potent and independent risk factor for atherosclerotic cardiovascular disease and other cardiovascular conditions. Experimental studies in mice suggest that mutations in TET2 and JAK2, which are among the most common in clonal hematopoiesis, increase inflammation and are causally connected to accelerated atherosclerosis development, which may explain the link between clonal hematopoiesis and increased cardiovascular risk. In this review, we provide an overview of our current understanding of this emerging cardiovascular risk factor.

Emerging Role of Acquired Mutations and Clonal Hematopoiesis in Atherosclerosis　- Beyond Conventional Cardiovascular Risk Factors.

Accumulating evidence suggests that conventional cardiovascular risk factors are incompletely predictive of cardiovascular disease, as a substantial risk remains even when these factors are apparently managed well. In this context, clonal hematopoiesis has emerged as a new and potent risk factor for atherosclerotic cardiovascular disease and other cardiometabolic conditions. Clonal hematopoiesis typically arises from somatic mutations that confer a competitive advantage to a mutant hematopoietic stem cell, leading to its clonal expansion in the stem cell population and its progeny of blood leukocytes. Human sequencing studies and experiments in mice suggest that clonal hematopoiesis, at least when driven by certain mutations, contributes to accelerated atherosclerosis development. However, the epidemiology, biology and clinical implications of this phenomenon remain incompletely understood. Here, we review the current understanding of the connection between clonal hematopoiesis and atherosclerosis, and highlight knowledge gaps in this area of research.

Brown adipose tissue dysfunction promotes heart failure via a trimethylamine N-oxide-dependent mechanism.

Low body temperature predicts a poor outcome in patients with heart failure, but the underlying pathological mechanisms and implications are largely unknown. Brown adipose tissue (BAT) was initially characterised as a thermogenic organ, and recent studies have suggested it plays a crucial role in maintaining systemic metabolic health. While these reports suggest a potential link between BAT and heart failure, the potential role of BAT dysfunction in heart failure has not been investigated. Here, we demonstrate that alteration of BAT function contributes to development of heart failure through disorientation in choline metabolism. Thoracic aortic constriction (TAC) or myocardial infarction (MI) reduced the thermogenic capacity of BAT in mice, leading to significant reduction of body temperature with cold exposure. BAT became hypoxic with TAC or MI, and hypoxic stress induced apoptosis of brown adipocytes. Enhancement of BAT function improved thermogenesis and cardiac function in TAC mice. Conversely, systolic function was impaired in a mouse model of genetic BAT dysfunction, in association with a low survival rate after TAC. Metabolomic analysis showed that reduced BAT thermogenesis was associated with elevation of plasma trimethylamine N-oxide (TMAO) levels. Administration of TMAO to mice led to significant reduction of phosphocreatine and ATP levels in cardiac tissue via suppression of mitochondrial complex IV activity. Genetic or pharmacological inhibition of flavin-containing monooxygenase reduced the plasma TMAO level in mice, and improved cardiac dysfunction in animals with left ventricular pressure overload. In patients with dilated cardiomyopathy, body temperature was low along with elevation of plasma choline and TMAO levels. These results suggest that maintenance of BAT homeostasis and reducing TMAO production could be potential next-generation therapies for heart failure.

Clonal Hematopoiesis and Risk of Progression of Heart Failure With Reduced Left Ventricular Ejection Fraction.

BACKGROUND: Clonal hematopoiesis driven by somatic mutations in hematopoietic cells, frequently called clonal hematopoiesis of indeterminate potential (CHIP), has been associated with adverse cardiovascular outcomes in population-based studies and in patients with ischemic heart failure (HF) and reduced left ventricular ejection fraction (LVEF). Yet, the impact of CHIP on HF progression, including nonischemic etiology, is unknown. OBJECTIVES: The purpose of this study was to assess the clinical impact of clonal hematopoiesis on HF progression irrespective of its etiology. METHODS: The study cohort comprised 62 patients with HF and LVEF <45% (age 74 ± 7 years, 74% men, 52% nonischemic, and LVEF 30 ± 8%). Deep sequencing was used to detect CHIP mutations with a variant allelic fraction >2% in 54 genes. Patients were followed for at least 3.5 years for various adverse events including death, HF-related death, and HF hospitalization. RESULTS: CHIP mutations were detected in 24 (38.7%) patients, without significant differences in all-cause mortality (p = 0.151). After adjusting for risk factors, patients with mutations in either DNA methyltransferase 3 alpha (DNMT3A) or Tet methylcytosine dioxygenase 2 (TET2) exhibited accelerated HF progression in terms of death (hazard ratio [HR]: 2.79; 95% confidence interval [CI]: 1.31 to 5.92; p = 0.008), death or HF hospitalization (HR: 3.84; 95% CI: 1.84 to 8.04; p < 0.001) and HF-related death or HF hospitalization (HR: 4.41; 95% CI: 2.15 to 9.03; p < 0.001). In single gene-specific analyses, somatic mutations in DNMT3A or TET2 retained prognostic significance with regard to HF-related death or HF hospitalization (HR: 4.50; 95% CI: 2.07 to 9.74; p < 0.001, for DNMT3A mutations; HR: 3.18; 95% CI: 1.52 to 6.66; p = 0.002, for TET2 mutations). This association remained significant irrespective of ischemic/nonischemic etiology. CONCLUSIONS: Somatic mutations that drive clonal hematopoiesis are common among HF patients with reduced LVEF and are associated with accelerated HF progression regardless of etiology.

TET2-Loss-of-Function-Driven Clonal Hematopoiesis Exacerbates Experimental Insulin Resistance in Aging and Obesity.

Human aging is frequently accompanied by the acquisition of somatic mutations in the hematopoietic system that induce clonal hematopoiesis, leading to the development of a mutant clone of hematopoietic progenitors and leukocytes. This somatic-mutation-driven clonal hematopoiesis has been associated with an increased incidence of cardiovascular disease and type 2 diabetes, but whether this epidemiological association reflects a direct, causal contribution of mutant hematopoietic and immune cells to age-related metabolic abnormalities remains unexplored. Here, we show that inactivating mutations in the epigenetic regulator TET2, which lead to clonal hematopoiesis, aggravate age- and obesity-related insulin resistance in mice. This metabolic dysfunction is paralleled by increased expression of the pro-inflammatory cytokine IL-1ß in white adipose tissue, and it is suppressed by pharmacological inhibition of NLRP3 inflammasome-mediated IL-1ß production. These findings support a causal contribution of somatic TET2 mutations to insulin resistance and type 2 diabetes.

Tet2-mediated clonal hematopoiesis in nonconditioned mice accelerates age-associated cardiac dysfunction.

Clonal hematopoiesis of indeterminate potential is prevalent in elderly individuals and associated with increased risks of all-cause mortality and cardiovascular disease. However, mouse models to study the dynamics of clonal hematopoiesis and its consequences on the cardiovascular system under homeostatic conditions are lacking. We developed a model of clonal hematopoiesis using adoptive transfer of unfractionated ten-eleven translocation 2-mutant (Tet2-mutant) bone marrow cells into nonirradiated mice. Consistent with age-related clonal hematopoiesis observed in humans, these mice displayed a progressive expansion of Tet2-deficient cells in multiple hematopoietic stem and progenitor cell fractions and blood cell lineages. The expansion of the Tet2-mutant fraction was also observed in bone marrow-derived CCR2+ myeloid cell populations within the heart, but there was a negligible impact on the yolk sac-derived CCR2- cardiac-resident macrophage population. Transcriptome profiling revealed an enhanced inflammatory signature in the donor-derived macrophages isolated from the heart. Mice receiving Tet2-deficient bone marrow cells spontaneously developed age-related cardiac dysfunction characterized by greater hypertrophy and fibrosis. Altogether, we show that Tet2-mediated hematopoiesis contributes to cardiac dysfunction in a nonconditioned setting that faithfully models human clonal hematopoiesis in unperturbed bone marrow. Our data support clinical findings that clonal hematopoiesis per se may contribute to diminished health span.

Tet2-Mediated Clonal Hematopoiesis Accelerates Heart Failure Through a Mechanism Involving the IL-1ß/NLRP3 Inflammasome.

BACKGROUND: Recent studies have shown that hematopoietic stem cells can undergo clonal expansion secondary to somatic mutations in leukemia-related genes, thus leading to an age-dependent accumulation of mutant leukocytes in the blood. This somatic mutation-related clonal hematopoiesis is common in healthy older individuals, but it has been associated with an increased incidence of future cardiovascular disease. The epigenetic regulator TET2 is frequently mutated in blood cells of individuals exhibiting clonal hematopoiesis. OBJECTIVES: This study investigated whether Tet2 mutations within hematopoietic cells can contribute to heart failure in 2 models of cardiac injury. METHODS: Heart failure was induced in mice by pressure overload, achieved by transverse aortic constriction or chronic ischemia induced by the permanent ligation of the left anterior descending artery. Competitive bone marrow transplantation strategies with Tet2-deficient cells were used to mimic TET2 mutation-driven clonal hematopoiesis. Alternatively, Tet2 was specifically ablated in myeloid cells using Cre recombinase expressed from the LysM promoter. RESULTS: In both experimental heart failure models, hematopoietic or myeloid Tet2 deficiency worsened cardiac remodeling and function, in parallel with increased interleukin-1beta (IL-1ß) expression. Treatment with a selective NLRP3 inflammasome inhibitor protected against the development of heart failure and eliminated the differences in cardiac parameters between Tet2-deficient and wild-type mice. CONCLUSIONS: Tet2 deficiency in hematopoietic cells is associated with greater cardiac dysfunction in murine models of heart failure as a result of elevated IL-1ß signaling. These data suggest that individuals with TET2-mediated clonal hematopoiesis may be at greater risk of developing heart failure and respond better to IL-1ß-NLRP3 inflammasome inhibition.

Acute and Chronic Increases of Circulating FSTL1 Normalize Energy Substrate Metabolism in Pacing-Induced Heart Failure.

BACKGROUND: FSTL1 (follistatin-like protein 1) is an emerging cardiokine/myokine that is upregulated in heart failure (HF) and is found to be cardioprotective in animal models of cardiac injury. We tested the hypothesis that circulating FSTL1 can affect cardiac function and metabolism under baseline physiological conditions and in HF. METHODS AND RESULTS: FSTL1 was acutely (10 minutes) or chronically (2 weeks) infused to attain clinically relevant blood levels in conscious dogs with cardiac tachypacing-induced HF. Dogs with no cardiac pacing and FSTL1 infusion served as control. 3H-oleate and 14C-glucose were infused to track the metabolic fate of free fatty acids and glucose. Cardiac uptake of lactate and ketone bodies and systemic respiratory quotient were also measured. HF caused a shift from prevalent cardiac and systemic fat to carbohydrate oxidation. Although acute FSTL1 administration caused minimal hemodynamic changes at baseline, in HF dogs it enhanced cardiac oxygen consumption and transiently reversed the changes in free fatty acid and glucose oxidation and systemic respiratory quotient. In HF, chronic FSTL1 infusion stably normalized cardiac free fatty acid, glucose, ketone body consumption, and systemic respiratory quotient, while moderately improving diastolic and contractile function. Consistently, FSTL1 prevented the downregulation of medium-chain acyl-CoA dehydrogenase-a representative enzyme of the free fatty acid oxidation pathway. Complementary in vitro experiments in primary cardiac and skeletal muscle myocytes showed that FSTL1 stimulated oxygen consumption through AMPK (AMP-activated kinase) activation. CONCLUSIONS: These findings support a novel function for FSTL1 and provide the first direct evidence that a circulating cardiokine/myokine can alter myocardial and systemic energy substrate metabolism, in vivo.

Activation of non-canonical WNT signaling in human visceral adipose tissue contributes to local and systemic inflammation.

The accumulation of visceral adiposity is strongly associated with systemic inflammation and increased cardiometabolic risk. WNT5A, a non-canonical WNT ligand, has been shown to promote adipose tissue inflammation and insulin resistance in animal studies. Among other non-canonical pathways, WNT5A activates planar cell polarity (PCP) signaling. The current study investigated the potential contribution of non-canonical WNT5A/PCP signaling to visceral adipose tissue (VAT) inflammation and associated metabolic dysfunction in individuals with obesity. VAT and subcutaneous adipose tissue (SAT) samples obtained from subjects undergoing bariatric surgery were analyzed by qRT-PCR for expression of WNT/PCP genes. In vitro experiments were conducted with preadipocytes isolated from VAT and SAT biopsies. The expression of 23 out of 33 PCP genes was enriched in VAT compared to SAT. Strong positive expression correlations of individual PCP genes were observed in VAT. WNT5A expression in VAT, but not in SAT, correlated with indexes of JNK signaling activity, IL6, waist-to-hip ratio and hsCRP. In vitro, WNT5A promoted the expression of IL6 in human preadipocytes. In conclusion, elevated non-canonical WNT5A signaling in VAT contributes to the exacerbated IL-6 production in this depot and the low-grade systemic inflammation typically associated with visceral adiposity.

Genetic deficiency of Wnt5a diminishes disease severity in a murine model of rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a common autoimmune disease characterized by chronic inflammation of the joints, leading to bone erosion and joint dysfunction. Despite the recent successes of disease-modifying anti-rheumatic drugs (DMARDs), there is still clinical need for understanding the development and molecular etiology of RA. Wnts are developmental morphogens whose roles in adult pathology are poorly characterized. Wnt5a is a member of the non-canonical family of Wnts that modulates a wide range of cell processes, including differentiation, migration, and inflammation. Wnt5a has been implicated as a possible contributor to arthritis and it is upregulated in synovial fibroblasts from RA patients. METHODS: We investigated the role of endogenous Wnt5a in RA. Tamoxifen-inducible, Wnt5a knockout (Wnt5a cKO) mice and littermate controls were monitored for arthritis development and joint pathology using the K/BxN serum transfer-induced arthritis (STIA) model. To explore a role of Wnt5a in osteoclast fusion, bone marrow-derived monocytes (BMDMs) were differentiated in vitro. RESULTS: Wnt5a cKO mice were resistant to arthritis development compared to control littermates as assessed by ankle thickness and histologic measurements. Some parameters of inflammation were reduced in the Wnt5a cKO mice, including the extent of polymononuclear cell infiltration and extra-articular inflammation. Wnt5a cKO mice also exhibited less cartilage destruction and a reduction in osteoclast activity with concomitant reduction in tartrate-resistant acid phosphatase (TRAP), cathepsin K (CTSK), macrophage colony-stimulating factor (MCSF), matrix metalloproteinase (MMP)2 and MMP9 in the arthritic joints. Treatment of BMDMs with Wnt5a enhanced osteoclast fusion and increased the expression of dendrocyte-expressed seven transmembrane protein (DCSTAMP) and MMP9, that are necessary for osteoclast formation and activity. CONCLUSIONS: These data suggest that Wnt5a modulates the development of arthritis by promoting inflammation and osteoclast fusion, and provide the first mouse genetic evidence of a role for endogenous Wnt5a in autoimmune disease.

Humans and Mice Display Opposing Patterns of "Browning" Gene Expression in Visceral and Subcutaneous White Adipose Tissue Depots.

Visceral adiposity is much more strongly associated with cardiometabolic disease in humans than subcutaneous adiposity. Browning, the appearance of brown-like adipocytes in the white adipose tissue (WAT), has been shown to protect mice against metabolic dysfunction, suggesting the possibility of new therapeutic approaches to treat obesity and type 2 diabetes. In mice, subcutaneous WAT depots express higher levels of browning genes when compared with visceral WAT, further suggesting that differences in WAT browning could contribute to the differences in the pathogenicity of the two depots. However, the expression of browning genes in different WAT depots of human has not been characterized. Here, it is shown that the expression of browning genes is higher in visceral than in subcutaneous WAT in humans, a pattern that is opposite to what is observed in mice. These results suggest that caution should be applied in extrapolating the results of murine browning gene expression studies to human pathophysiology.

Clonal hematopoiesis associated with TET2 deficiency accelerates atherosclerosis development in mice.

Human aging is associated with an increased frequency of somatic mutations in hematopoietic cells. Several of these recurrent mutations, including those in the gene encoding the epigenetic modifier enzyme TET2, promote expansion of the mutant blood cells. This clonal hematopoiesis correlates with an increased risk of atherosclerotic cardiovascular disease. We studied the effects of the expansion of Tet2-mutant cells in atherosclerosis-prone, low-density lipoprotein receptor-deficient (Ldlr-/-) mice. We found that partial bone marrow reconstitution with TET2-deficient cells was sufficient for their clonal expansion and led to a marked increase in atherosclerotic plaque size. TET2-deficient macrophages exhibited an increase in NLRP3 inflammasome-mediated interleukin-1ß secretion. An NLRP3 inhibitor showed greater atheroprotective activity in chimeric mice reconstituted with TET2-deficient cells than in nonchimeric mice. These results support the hypothesis that somatic TET2 mutations in blood cells play a causal role in atherosclerosis.

WNT5A-JNK regulation of vascular insulin resistance in human obesity.

Obesity is associated with the development of vascular insulin resistance; however, pathophysiological mechanisms are poorly understood. We sought to investigate the role of WNT5A-JNK in the regulation of insulin-mediated vasodilator responses in human adipose tissue arterioles prone to endothelial dysfunction. In 43 severely obese (BMI 44±11 kg/m2) and five metabolically normal non-obese (BMI 26±2 kg/m2) subjects, we isolated arterioles from subcutaneous and visceral fat during planned surgeries. Using videomicroscopy, we examined insulin-mediated, endothelium-dependent vasodilator responses and characterized adipose tissue gene and protein expression using real-time polymerase chain reaction and Western blot analyses. Immunofluorescence was used to quantify endothelial nitric oxide synthase (eNOS) phosphorylation. Insulin-mediated vasodilation was markedly impaired in visceral compared to subcutaneous vessels from obese subjects (p<0.001), but preserved in non-obese individuals. Visceral adiposity was associated with increased JNK activation and elevated expression of WNT5A and its non-canonical receptors, which correlated negatively with insulin signaling. Pharmacological JNK antagonism with SP600125 markedly improved insulin-mediated vasodilation by sixfold (p<0.001), while endothelial cells exposed to recombinant WNT5A developed insulin resistance and impaired eNOS phosphorylation (p<0.05). We observed profound vascular insulin resistance in the visceral adipose tissue arterioles of obese subjects that was associated with up-regulated WNT5A-JNK signaling and impaired endothelial eNOS activation. Pharmacological JNK antagonism markedly improved vascular endothelial function, and may represent a potential therapeutic target in obesity-related vascular disease.

Corrigendum: Ionizing Particle Radiation as a Modulator of Endogenous Bone Marrow Cell Reprogramming: Implications for Hematological Cancers.

[This corrects the article on p. 231 in vol. 5, PMID: 26528440.].

Ionizing Particle Radiation as a Modulator of Endogenous Bone Marrow Cell Reprogramming: Implications for Hematological Cancers.

Exposure of individuals to ionizing radiation (IR), as in the case of astronauts exploring space or radiotherapy cancer patients, increases their risk of developing secondary cancers and other health-related problems. Bone marrow (BM), the site in the body where hematopoietic stem cell (HSC) self-renewal and differentiation to mature blood cells occurs, is extremely sensitive to low-dose IR, including irradiation by high-charge and high-energy particles. Low-dose IR induces DNA damage and persistent oxidative stress in the BM hematopoietic cells. Inefficient DNA repair processes in HSC and early hematopoietic progenitors can lead to an accumulation of mutations whereas long-lasting oxidative stress can impair hematopoiesis itself, thereby causing long-term damage to hematopoietic cells in the BM niche. We report here that low-dose (1)H- and (56)Fe-IR significantly decreased the hematopoietic early and late multipotent progenitor (E- and L-MPP, respectively) cell numbers in mouse BM over a period of up to 10 months after exposure. Both (1)H- and (56)Fe-IR increased the expression of pluripotent stem cell markers Sox2, Nanog, and Oct4 in L-MPPs and 10 months post-IR exposure. We postulate that low doses of (1)H- and (56)Fe-IR may induce endogenous cellular reprogramming of BM hematopoietic progenitor cells to assume a more primitive pluripotent phenotype and that IR-induced oxidative DNA damage may lead to mutations in these BM progenitors. This could then be propagated to successive cell lineages. Persistent impairment of BM progenitor cell populations can disrupt hematopoietic homeostasis and lead to hematologic disorders, and these findings warrant further mechanistic studies into the effects of low-dose IR on the functional capacity of BM-derived hematopoietic cells including their self-renewal and pluripotency.

A nuclear ribosomal DNA pseudogene in triatomines opens a new research field of fundamental and applied implications in Chagas disease

A pseudogene, designated as "ps(5.8S+ITS-2)", paralogous to the 5.8S gene and internal transcribed spacer (ITS)-2 of the nuclear ribosomal DNA (rDNA), has been recently found in many triatomine species distributed throughout North America, Central America and northern South America. Among characteristics used as criteria for pseudogene verification, secondary structures and free energy are highlighted, showing a lower fit between minimum free energy, partition function and centroid structures, although in given cases the fit only appeared to be slightly lower. The unique characteristics of "ps(5.8S+ITS-2)" as a processed or retrotransposed pseudogenic unit of the ghost type are reviewed, with emphasis on its potential functionality compared to the functionality of genes and spacers of the normal rDNA operon. Besides the technical problem of the risk for erroneous sequence results, the usefulness of "ps(5.8S+ITS-2)" for specimen classification, phylogenetic analyses and systematic/taxonomic studies should be highlighted, based on consistence and retention index values, which in pseudogenic sequence trees were higher than in functional sequence trees. Additionally, intraindividual, interpopulational and interspecific differences in pseudogene amount and the fact that it is a pseudogene in the nuclear rDNA suggests a potential relationships with fitness, behaviour and adaptability of triatomine vectors and consequently its potential utility in Chagas disease epidemiology and control.

A nuclear ribosomal DNA pseudogene in triatomines opens a new research field of fundamental and applied implications in Chagas disease.

A pseudogene, designated as "ps(5.8S+ITS-2)", paralogous to the 5.8S gene and internal transcribed spacer (ITS)-2 of the nuclear ribosomal DNA (rDNA), has been recently found in many triatomine species distributed throughout North America, Central America and northern South America. Among characteristics used as criteria for pseudogene verification, secondary structures and free energy are highlighted, showing a lower fit between minimum free energy, partition function and centroid structures, although in given cases the fit only appeared to be slightly lower. The unique characteristics of "ps(5.8S+ITS-2)" as a processed or retrotransposed pseudogenic unit of the ghost type are reviewed, with emphasis on its potential functionality compared to the functionality of genes and spacers of the normal rDNA operon. Besides the technical problem of the risk for erroneous sequence results, the usefulness of "ps(5.8S+ITS-2)" for specimen classification, phylogenetic analyses and systematic/taxonomic studies should be highlighted, based on consistence and retention index values, which in pseudogenic sequence trees were higher than in functional sequence trees. Additionally, intraindividual, interpopulational and interspecific differences in pseudogene amount and the fact that it is a pseudogene in the nuclear rDNA suggests a potential relationships with fitness, behaviour and adaptability of triatomine vectors and consequently its potential utility in Chagas disease epidemiology and control.