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AIM: Diagnosing and classifying heart failure (HF) in the oldest-old patients has technical and interpretation issues, especially in the acute setting. We assessed the usefulness of both N-terminal pro-brain natriuretic peptide (NT-proBNP) and lung ultrasound (LUS) for confirming HF diagnosis and predicting, among hospitalized HF patients, those with reduced ejection fraction (HFrEF). METHODS: We performed a cross-sectional study on 148 consecutive patients aged ≥ 80 years admitted to our Internal Medicine and Geriatrics ward with at least one symptom/sign compatible with HF and NT-proBNP ≥ 125 pg/mL. We measured serum NT-proBNP levels and performed LUS and transthoracic echocardiography (TTE) on admission before diuretic therapy. We divided our cohort into three subgroups according to the left ventricular ejection fraction (LVEF): reduced (LVEF ≤ 40%), mildly-reduced (LVEF = 41-49%) and preserved (LVEF ≥ 50%). RESULTS: The mean age was 88±5 years. Male prevalence was 42%. Patients with HFrEF were 19%. Clinical features and laboratory parameters did not differ between the three subgroups, except for higher NT-proBNP in HFrEF patients, which also had a higher number of total B-lines and intercostal spaces of pleural effusion at LUS. Overall, NT-proBNP showed an inverse correlation with LVEF (r = -0.22, p = 0.007) and a direct correlation with age, total pulmonary B-lines, and intercostal spaces of pleural effusion. According to the ROCs, NT-proBNP levels, pulmonary B-lines and pleural effusion extension were poorly predictive for HFrEF. The best-performing cut-offs were 9531 pg/mL for NT-proBNP (SP 0.70, SE 0.50), 13 for total B-lines (SP 0.69, SE 0.85) and one intercostal space for pleural effusion (SP 0.55, SE 0.89). Patients with admission NT-proBNP ≥ 9531 pg/mL had a 2-fold higher risk for HFrEF (OR 2.5, 95% CI 1.3-4.9), while we did not find any association for total B-lines ≥ 13 or pleural effusion ≥ 1 intercostal space with HFrEF. A significant association with HFrEF emerged for the combination of NT-proBNP ≥ 9531 pg/mL, total B-lines ≥ 13 and intercostal spaces of pleural effusion ≥ 1 (adjusted OR 4.3, 95% CI 1.5-12.9). CONCLUSIONS: Although NT-proBNP and LUS help diagnose HF, their accuracy in discriminating HFrEF from non-HFrEF was poor in our real-life clinical study on oldest-old hospitalized patients, making the use of TTE still necessary to distinguish HF phenotypes in this peculiar setting. These data require confirmation in more extensive and longer prospective studies.
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Insuficiência Cardíaca , Derrame Pleural , Humanos , Masculino , Idoso de 80 Anos ou mais , Insuficiência Cardíaca/diagnóstico por imagem , Insuficiência Cardíaca/epidemiologia , Peptídeo Natriurético Encefálico , Volume Sistólico , Estudos Transversais , Estudos Prospectivos , Função Ventricular Esquerda , Biomarcadores , Fragmentos de Peptídeos , Pulmão/diagnóstico por imagemRESUMO
BACKGROUND: Coronavirus disease COVID-19 is a heterogeneous condition caused by SARS-CoV-2 infection. Generally, it is characterized by interstitial pneumonia that can lead to impaired gas-exchange, acute respiratory failure, and death, although a complex disorder of multi-organ dysfunction has also been described. The pathogenesis is complex, and a variable combination of factors has been described in critically ill patients. COVID-19 is a particular risk for older persons, particularly those with frailty and comorbidities. Blood bacterial DNA has been reported in both physiological and pathological conditions and has been associated with some haematological and laboratory parameters but, to date, no study has characterized it in hospitalized old COVID-19 patients The present study aimed to establish an association between blood bacterial DNA (BB-DNA) and clinical severity in old COVID-19 patients. RESULTS: BB-DNA levels were determined, by quantitative real-time PCRs targeting the 16S rRNA gene, in 149 hospitalized older patients (age range 65-99 years) with COVID-19. Clinical data, including symptoms and signs of infection, frailty status, and comorbidities, were assessed. BB-DNA was increased in deceased patients compared to discharged ones, and Cox regression analysis confirmed an association between BB-DNA and in-hospital mortality. Furthermore, BB-DNA was positively associated with the neutrophil count and negatively associated with plasma IFN-alpha. Additionally, BB-DNA was associated with diabetes. CONCLUSIONS: The association of BB-DNA with mortality, immune-inflammatory parameters and diabetes in hospitalized COVID-19 patients suggests its potential role as a biomarker of unfavourable outcomes of the disease, thus it could be proposed as a novel prognostic marker in the assessment of acute COVID-19 disease.
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Herpesviridae reactivation such as cytomegalovirus (CMV) has been described in severe COVID-19 (COronaVIrusDisease-2019). This study aimed to understand if CMV reactivation in older COVID-19 patients is associated with increased inflammation and in-hospital mortality. In an observational single-center cohort study, 156 geriatric COVID-19 patients were screened for CMV reactivation by RT-PCR. Participants underwent a comprehensive clinical investigation that included medical history, functional evaluation, laboratory tests and cytokine assays (TNF-α, IFN-α, IL-6, IL-10) at hospital admission. In 19 (12.2%) of 156 COVID-19 patients, CMV reactivation was detected. Multivariate Cox regression models showed that in-hospital mortality significantly increased among CMV positive patients younger than 87 years (HR: 9.94, 95% CI: 1.66-59.50). Other factors associated with in-hospital mortality were C-reactive protein (HR: 1.17, 95% CI: 1.05-1.30), neutrophil count (HR: 1.20, 95% CI: 1.01-1.42) and clinical frailty scale (HR:1.54, 95% CI: 1.04-2.28). In patients older than 87 years, neutrophil count (HR: 1.13, 95% CI: 1.05-1.21) and age (HR: 1.15, 95% CI: 1.01-1.31) were independently associated with in-hospital mortality. CMV reactivation was also correlated with increased IFN-α and TNF-α serum levels, but not with IL-6 and IL-10 serum changes. In conclusion, CMV reactivation was an independent risk factor for in-hospital mortality in COVID-19 patients younger than 87 years old, but not in nonagenarians.
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COVID-19 , Infecções por Citomegalovirus , Idoso de 80 Anos ou mais , Humanos , Idoso , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/complicações , Interleucina-10 , Estudos de Coortes , Interleucina-6 , Fator de Necrose Tumoral alfa , COVID-19/complicações , Ativação Viral , Estudos RetrospectivosRESUMO
A dysregulation of the renin-angiotensin system (RAS) has been involved in the genesis of lung injury and acute respiratory distress syndrome from different causes, including several viral infections. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of pneumocytes, the hallmark of the pandemic coronavirus disease 2019 (COVID-19) involving both alveolar interstitium and capillaries, is linked to angiotensin-converting enzyme 2 (ACE2) binding and its functional downregulation. ACE2 is a key enzyme for the balance between the two main arms of the RAS: the ACE/angiotensin (Ang) II/Ang II type 1 receptor axis ("classic RAS") and the ACE2/Ang(1-7)/Mas receptor (MasR) axis ("anti-RAS"). The ACE2 downregulation, as a result of SARS-coronaviruses binding, enhances the classic RAS, leading to lung damage and inflammation with leaky pulmonary blood vessels and fibrosis, when the attenuation mediated by the anti-RAS arm is reduced. ACE inhibitors (ACE-I) and Ang II type 1 receptor blockers (ARB), effective in cardiovascular diseases, were found to prevent and counteract acute lung injury in several experimental models by restoring the balance between these two opposing arms. The evidence of RAS arm disequilibrium in COVID-19 and the hypothesis of a beneficial role of RAS modulation supported by preclinical and clinical studies are the focus of the present review. Preclinical and clinical studies on drugs balancing RAS arms might be the right way to counter COVID-19.
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Antagonistas de Receptores de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Infecções por Coronavirus/patologia , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/patologia , Alvéolos Pulmonares/patologia , Síndrome do Desconforto Respiratório/patologia , Células Epiteliais Alveolares/virologia , Enzima de Conversão de Angiotensina 2 , Betacoronavirus/metabolismo , COVID-19 , Infecções por Coronavirus/tratamento farmacológico , Regulação para Baixo , Humanos , Lesão Pulmonar/tratamento farmacológico , Lesão Pulmonar/prevenção & controle , Lesão Pulmonar/virologia , Pandemias , Pneumonia Viral/tratamento farmacológico , Proto-Oncogene Mas , Alvéolos Pulmonares/irrigação sanguínea , Alvéolos Pulmonares/virologia , Receptores de Angiotensina/efeitos dos fármacos , Sistema Renina-Angiotensina/efeitos dos fármacos , Síndrome do Desconforto Respiratório/virologia , SARS-CoV-2RESUMO
Cardiovascular (CV) comorbidities in patients with chronic obstructive pulmonary disease (COPD) are associated with increased morbidity and mortality, especially in old and very old subjects. The question if long-acting beta-agonist and long-acting muscarinic antagonist could be associated with the increased prevalence of CV-related adverse effects has puzzled, particularly in the past, specialists involved in the management of respiratory diseases. The safety of these compounds has scarcely been tested in patients aged ≥ 65 years with CV comorbidities, since randomized controlled trials rarely include this subpopulation. However, the fixed combination indacaterol/glycopyrronium has shown a favorable CV safety profile in both healthy volunteers and COPD patients. Thus, we aimed to assess the CV safety pro
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Agonistas de Receptores Adrenérgicos beta 2/efeitos adversos , Doenças Cardiovasculares/induzido quimicamente , Glicopirrolato/efeitos adversos , Indanos/efeitos adversos , Antagonistas Muscarínicos/efeitos adversos , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Quinolonas/efeitos adversos , Administração por Inalação , Idoso de 80 Anos ou mais , Quimioterapia Combinada , Eletrocardiografia , Feminino , Humanos , MasculinoRESUMO
We report two families, members of which are carriers of a novel hemoglobin (Hb) variant that was named Hb Olivet [α13(A11)AlaâThr (α1) (GCC > ACC); HBA1: c.40G > A; p.Ala14Thr]. The analysis of these cases allowed a clear description of this anomaly that behaves as a silent Hb. In the first family, of Portuguese ethnicity living in France, the proband, a 24-year-old male and his 57-year-old mother, both appeared to be carriers. The son presented with borderline mean corpuscular volume (MCV), while the mother was normocytic and normochromic. Hemoglobin separation on capillary electrophoresis (CE) was normal, while a slightly asymmetric peak was observed on high performance liquid chromatography (HPLC). In a second family, originally from Surinam but living in The Netherlands, the proband, a 6-year-old girl, showed a mild microcytosis at low ferritin levels. The abnormal Hb was inherited from the mother who was clearly iron depleted, was not present in the sister and brother of the proband. The microcytic hypochromic anemia was only shown in two out of a total of four carriers. It therefore seems likely that iron depletion is causative as two carriers are completely normal. Characterization and genotype/phenotype correlation are briefly described.
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Estudos de Associação Genética , Hemoglobinopatias/patologia , Hemoglobinas Anormais/genética , Mutação/genética , Criança , Família , Feminino , França/epidemiologia , Hemoglobinopatias/sangue , Hemoglobinopatias/genética , Heterozigoto , Humanos , Deficiências de Ferro , Masculino , Pessoa de Meia-Idade , Países Baixos/epidemiologia , Portugal/etnologia , Suriname/etnologia , Adulto JovemRESUMO
The objective of this study was to expand and study the molecular spectrum of ß-thalassemia (ß-thal) mutations in Oman by examining cases from seven different regions and comparing the prevalence with neighboring countries. A total of 446 cases of ß hemoglobinopathies was obtained and analyzed to determine the frequency and distribution of the different ß alleles. The molecular spectrum of ß-thal in Oman revealed the presence of 32 mutations from different origins and 11 alleles are reported for the first time in the Omani population. The wide heterogeneous spectrum of ß-thal mutations found can be associated with the history of trade and migration as well as the past domination from other countries. The presented data will facilitate the development of a comprehensive prevention strategy in Oman.
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Mutação , Globinas beta/genética , Talassemia beta/epidemiologia , Talassemia beta/genética , Alelos , Éxons , Frequência do Gene , Genótipo , Humanos , Íntrons , Omã/epidemiologiaRESUMO
We report two examples showing how problematic it can be to define the phenotype of new or rare globin genes mutations. We describe two mutations observed for the first time in the Omani population: the first was found in the consanguineous parents of a deceased newborn with hepatomegaly, cardiomegaly and severe hemolytic anemia, putatively homozygous for the rare Hb Lansing (HBA2: c.264C > G) variant. The second is a novel ß-globin gene promoter mutation [-52 (G > T)] observed in four independent patients. Two with borderline/elevated Hb A2, α-thalassemia (α-thal) and hypochromic red cell indices, and two heterozygotes for Hb S (HBB: c.20A > T), α-thal and with Hb A/Hb S ratios possibly indicating a very mild ß(+)-thalassemia (ß(+)-thal) mutation.
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Hemoglobina A2/genética , Mutação , Regiões Promotoras Genéticas , Globinas beta/genética , Adulto , Alelos , Análise Mutacional de DNA , Evolução Fatal , Feminino , Estudos de Associação Genética , Genótipo , Heterozigoto , Homozigoto , Humanos , Recém-Nascido , Masculino , Omã , FenótipoRESUMO
We describe a case of Hb H disease associated with homozygosity for a two nucleotide deletion in the polyadenylation signal of the α2-globin gene (HBA2: c.*93_*94delAA). The patient, a 27-year-old son of a consanguineous couple, needs regular blood transfusions every 6 months.
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Homozigoto , Mutação , Poli A , Poliadenilação/genética , RNA Mensageiro/genética , alfa-Globinas/genética , Talassemia alfa/genética , Adulto , Idoso , Transfusão de Sangue , Análise Mutacional de DNA , Índices de Eritrócitos , Feminino , Hemoglobina H/genética , Humanos , Irã (Geográfico) , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/química , Talassemia alfa/diagnóstico , Talassemia alfa/terapiaRESUMO
We describe the molecular characterization of α-globin gene defects in a cohort of 634 Omani patients. A total of 21 different α gene mutations were found in 484 subjects. Overall, we identified three different large deletions, three small deletions, 11 point mutations [two on the α2 polyadenylation signal (polyA) (HBA2: c.*94A>G), and nine α chain variants], three ααα(anti 3.7) triplication, a 21 nucleotide (nt) duplication on the α1 gene and two novel (presumed) polymorphisms on the α 3.7 kb hybrid gene, namely -5 (C>T) and +46 (C>A). Of these defects, 15 have not been previously reported in the Omani population. This large heterogeneity of α-thalassemia (α-thal) observed in the Omani population could be expected in neighboring Arab countries. The high frequency of α-thal, solely or in association with ß-globin gene defects, emphasize the necessity of adding α-thal testing to pre marital programs for accurate genetic counseling.
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Hemoglobinas Anormais/genética , Mutação , alfa-Globinas/genética , Talassemia alfa/genética , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Omã/epidemiologia , Talassemia alfa/epidemiologia , Globinas beta/genéticaRESUMO
Although δ-thalassemia (δ-thal) is not categorized as a severe disease, it is essential to know the molecular spectrum of the δ gene mutations frequently occurring in specific areas, particularly if these areas are characterized by a high rate of ß-thalassemia (ß-thal) such as Oman. This is because coinherited δ-globin gene defects can interfere with the basic diagnosis of a ß-thal carrier when this is based upon the measurement of the Hb A2 only. Because of that, we have investigated 33 patients with low Hb A2 levels, collected from different hospitals in Oman. Some cases had a second Hb A2 fraction, while others had only significantly lower Hb A2 levels. Among these patients, 20 did carry a δ-globin gene mutation, the rest were carriers of α thalassemia (α-thal) defects or could be iron depleted or both. In total, eight different known mutations and two novel δ variants were found. The characterization of the δ-globin gene mutation spectrum will improve carrier diagnostics and genetic counseling in the Omani population screened for ß-thal.
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Hemoglobina A2/metabolismo , Mutação , Globinas delta/genética , Talassemia delta/sangue , Talassemia delta/genética , Cromatografia Líquida de Alta Pressão , Códon , Análise Mutacional de DNA , Feminino , Genótipo , Hemoglobina A2/química , Humanos , Masculino , Omã , Talassemia delta/diagnósticoRESUMO
We report a new silent ß-globin gene variant found in a family from Angola living in the north eastern Italian city of Ferrara. The probands, two young sisters, presented with hematological parameters compatible with a ß-thalassemia (ß-thal) minor but with normal Hb A2 levels and normal hemoglobin (Hb) separation on high performance liquid chromatography (HPLC). Molecular analyses revealed a homozygosity for the common -α(3.7) (rightward) deletion and heterozygosity for a novel transition (GCT > ACT) at codon 135 of the ß-globin gene, leading to an Ala â Thr single amino acid substitution that was inherited from the healthy father.
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Hemoglobinas Anormais/genética , Mutação Puntual , Talassemia alfa/genética , Globinas beta/genética , Substituição de Aminoácidos , Angola/etnologia , Pré-Escolar , Códon , Pai , Feminino , Deleção de Genes , Hemoglobinas Anormais/análise , Hemoglobinas Anormais/química , Heterozigoto , Homozigoto , Humanos , Itália , Índice de Gravidade de Doença , Irmãos , Talassemia alfa/sangue , Talassemia alfa/fisiopatologia , Globinas beta/análise , Globinas beta/químicaRESUMO
Genomic DNA of 3 patients, born as healthy carriers and developing a late-onset severe transfusion-dependent beta-thalassemia major was studied by high-density genome wide SNP array analysis. A mosaic loss of heterozygosity for almost the entire 11p was found, not attributable to deletions but involving mosaicism for segmental paternal isodisomy of 11p. Mitotic recombination leading to mosaic segmental uniparental isodisomy on chromosome 11p in multiple tissues has been described as a molecular disease mechanism for a subset of sporadic Beckwith-Wiedemann syndrome cases. A similar mechanism also seems to be involved in causing late-onset disease in carriers of recessive mutations in other genes located in 11p, such as late-onset beta-thalassemia major and sickle cell disease. We suggest that the loss of maternally imprinted IGF-2 and H19 genes may account for the selective advantage of hematopoietic cells containing this segmental paternal isodisomy of 11p carrying the ß-thalassemia mutation.
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Mosaicismo , Dissomia Uniparental , Talassemia beta/genética , Talassemia beta/metabolismo , Adolescente , Adulto , Fatores Etários , Alelos , Criança , Cromossomos Humanos Par 11 , Feminino , Frequência do Gene , Genótipo , Humanos , Masculino , Mutação , Linhagem , Polimorfismo de Nucleotídeo Único , Adulto Jovem , Globinas beta/genética , Talassemia beta/diagnósticoRESUMO
BACKGROUND: Autologous cord blood (CB) red blood cells (RBCs) can partly substitute transfusion needs in premature infants suffering from anemia. To explore whether expanded CB cells could provide additional autologous cells suitable for transfusion, we set up a simple one-step protocol to expand premature CB cells. STUDY DESIGN AND METHODS: CB buffy coat cells and isolated CD34-positive (CD34(pos) ) cells from premature and full-term CB and adult blood were tested with several combinations of growth factors while omitting xenogeneic proteins from the culture medium. Cell differentiation was analyzed serially during 21 days using flow cytometry, progenitor assays, and high-performance liquid chromatography. RESULTS: Expanded CB buffy coat cells resulted in a threefold higher number of erythroblasts than the isolated CD34(pos) cells. However, the RBCs contaminating the buffy coat remained present during the culture with uncertain quality. Premature and full-term CB CD34(pos) cells had similar fold expansion capacity and erythroid differentiation. With the use of interleukin-3, stem cell factor, and erythropoietin, the fold increases of all CD34(pos) cell sources were similar: CB 3942 ± 1554, adult peripheral mobilized blood 4702 ± 1826, and bone marrow (BM) 4143 ± 1908. The proportion of CD235a expression indicating erythroblast presence on Day 21 was slightly higher in the adult CD34(pos) cell sources: peripheral blood stem cells (96.7 ± 0.8%) and BM (98.9 ± 0.5%) compared to CB (87.7 ± 2.7%; p = 0.002). We were not able to induce further erythroid maturation in vitro. CONCLUSION: This explorative study showed that fairly pure autologous erythroid-expanded cell populations could be obtained by a simple culture method, which should be optimized. Future challenges comprise obtaining ex vivo enucleation of RBCs with the use of a minimal manipulating approach, which can add up to autologous RBCs derived from CB in the treatment of anemia of prematurity.
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Anemia/terapia , Transfusão de Sangue/métodos , Células Eritroides/citologia , Sangue Fetal/citologia , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Células Eritroides/metabolismo , Eritropoetina/metabolismo , Humanos , Interleucina-3/metabolismo , Fator de Células-Tronco/metabolismoRESUMO
We report a new hemoglobin (Hb) variant, found in a North-East Italian family living in the city of Treviso. The proband, a non anemic 60-year-old male with a history of chronic rhinitis, allergy to Parietaria and suspected obstructive sleep apnea syndrome, was referred for blood gas analysis. Determination of the oxygen affinity revealed a p50 of 32.5 mmHg (control 27.5 mmHg) indicating a moderate decrease in oxygen affinity. An abnormal pattern compatible with an α Hb variant was observed on high performance liquid chromatography (HPLC); direct sequencing revealed a transition at codon 91 of the α2 gene (HBA2: c.274C>T) changing leucine into phenylalanine. Characterization and phenotype studies are reported.
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Hemoglobina A2/genética , Hemoglobinas Anormais/genética , Mutação de Sentido Incorreto , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Análise Mutacional de DNA , Hemoglobina A2/metabolismo , Hemoglobinas Anormais/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Oxigênio/metabolismoRESUMO
The aim of our study was to assess the lung sequelae and clinical consequences 3 and 6 months after hospitalization for COVID-19 pneumonia in older patients. An observational study was conducted on 55 patients aged 65 years and older. Activities of daily living (ADL) and clinical frailty scale (CFS) were assessed at baseline and after 3 months. Both quantitative assessment at chest high-resolution computed tomography (CT) and semi-quantitative severity score (CTSS) were performed at baseline and after 3 and 6 months. Mean age: 82.3 ± 7.1 years. Male prevalence: 56.4%. After 6 months, ground-glass opacities (GGO) were still detectable in 22% of subjects, while consolidations were no longer appreciable. During follow-up, CTSS reached an overall median score of zero after 6 months. Fibrotic-like changes were found in 40% of subjects with an overall median score of 0 (0-5) points, being more prevalent in males. Patients reporting worsening ADL and CFS were 10.9% and 45.5%, respectively. They were associated with the burden of comorbidities, especially history of heart failure and chronic obstructive pulmonary disease at baseline. Amnesic disorders, exertional dyspnea, and fatigue were the most relevant symptoms reported. No association emerged between persistent or new-onset symptoms and evidence of fibrotic-like changes. The typical chest CT abnormalities of the COVID-19 pneumonia acute phase resolved in most of our older patients. Mild fibrotic-like changes persisted in less than half of the patients, especially males, without significantly affecting the functional status and frailty condition, which instead were more likely associated with pre-existing comorbidities.
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COVID-19 , Fragilidade , Humanos , Masculino , Idoso , Idoso de 80 Anos ou mais , COVID-19/complicações , COVID-19/epidemiologia , Atividades Cotidianas , Estado Funcional , SARS-CoV-2 , Pulmão/diagnóstico por imagem , Progressão da Doença , HospitalizaçãoRESUMO
Our study aimed to identify clusters of hospitalized older COVID-19 patients according to their main comorbidities and routine laboratory parameters to evaluate their association with in-hospital mortality. We performed an observational study on 485 hospitalized older COVID-19 adults (aged 80+ years). Patients were aggregated in clusters by a K-medians cluster analysis. The primary outcome was in-hospital mortality. Medical history and laboratory parameters were collected on admission. Frailty, defined by the Clinical Frailty Scale (CFS), referred to the two weeks before hospitalization and was used as a covariate. The median age was 87 (83-91) years, with a female prevalence (59.2%). Three different clusters were identified: cluster 1 (337), cluster 2 (118), and cluster 3 (30). In-hospital mortality was 28.5%, increasing from cluster 1 to cluster 3: cluster 1 = 21.1%, cluster 2 = 40.7%, and cluster 3 = 63.3% (p < 0.001). The risk for in-hospital mortality was higher in clusters 2 [HR 1.96 (95% CI: 1.28-3.01)] and 3 [HR 2.87 (95% CI: 1.62-5.07)] compared to cluster 1, even after adjusting for age, sex, and frailty. Patients in cluster 3 were older and had a higher prevalence of atrial fibrillation, higher admission NT-proBNP and C-reactive protein levels, higher prevalence of concurrent bacterial infections, and lower estimated glomerular filtration rates. The addition of CFS significantly improved the predictive ability of the clusters for in-hospital mortality. Our cluster analysis on older COVID-19 patients provides a characterization of those subjects at higher risk for in-hospital mortality, highlighting the role played by cardio-renal impairment, higher inflammation markers, and frailty, often simultaneously present in the same patient.
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Implementation of multiplex ligation-dependent probe amplification (MLPA) for thalassemia causing deletions has lead to the detection of new rearrangements. Knowledge of the exact breakpoint sequences should give more insight into the molecular mechanisms underlying these rearrangements, and would facilitate the design of gap-PCRs. We have designed a custom fine-tiling array with oligonucleotides covering the complete globin gene clusters. We hybridized 27 DNA samples containing newly identified deletions and nine positive controls. We designed specific primers to amplify relatively short fragments containing the breakpoint sequence and analyzed these by direct sequencing. Results from nine positive controls showed that array comparative genomic hybridization (aCGH) is suitable to detect small and large rearrangements. We were able to locate all breakpoints to a region of approximately 2 kb. We designed breakpoint primers for 22 cases and amplification was successful in 19 cases. For 12 of these, the exact locations of the breakpoints were determined. Seven of these deletions have not been reported before. aCGH is a valuable tool for high-resolution breakpoint characterization. The combination of MLPA and aCGH has lead to relatively cheap and easy to perform PCR assays, which might be of use for laboratories as an alternative for MLPA in populations where only a limited number of specific deletions occur with high frequency.
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Hibridização Genômica Comparativa/métodos , Análise Mutacional de DNA/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , alfa-Globinas/genética , Talassemia alfa , Globinas beta/genética , Talassemia beta , Pontos de Quebra do Cromossomo , Primers do DNA , Éxons , Rearranjo Gênico , Humanos , Reação em Cadeia da Polimerase , Deleção de Sequência , Talassemia alfa/diagnóstico , Talassemia alfa/genética , Talassemia beta/diagnóstico , Talassemia beta/genéticaRESUMO
OBJECTIVES: To determine the molecular basis in a Greek child suspected of having HbH disease and ß-thalassemia trait. METHODS: Standard hematology, Hb electrophoresis, and HPLC. Multiplex ligation-dependent probe amplification (MLPA), direct sequencing, and breakpoint characterization by NimbleGen fine-tiling array analysis. RESULTS: The index patient showed a moderate microcytic hypochromic anemia with normal ZPP and elevated HbA(2) , indicative for ß-thalassemia trait. However, the moderate microcytic hypochromic anemia along with the observation of HbH inclusions in occasional red blood cells suggested a coexisting α-thalassemia. Molecular analysis indicated that the propositus inherited the ß(+) -thalassemia mutation IVS2-745 (c>g) and a novel α(0) -thalassemia deletion from the mother, and the common non-deletion α-thalassemia allele α(2) (-5nt)α from the father. The α(0) -thalassemia deletion, named -â-(BGS) , is approximately 131.6 kb in length. It removes the major regulatory elements along with the functional α-globin genes but leaves the theta-gene intact. CONCLUSIONS: The compound interaction of a ß-thalassemia defect along with a single functional α-globin gene is quite rare. Although patients with HbH/ß-thal and simple HbH disease have comparable levels of Hb, the absence of free ß-globin chains and thus detectable non-functional HbH means that in HbH/ß-thal, the levels of functional Hb are higher, resulting in a better compensated functional anemia. Rare large deletions as the one described here remain undetected by gap-PCR in routine molecular screening. The introduction of MLPA as a diagnostic screening tool may improve laboratory diagnostics for these defects. The use of NimbleGen fine-tiling arrays may give additional information about the precise location of breakpoints.
Assuntos
alfa-Globinas/genética , Talassemia alfa/genética , Globinas beta/genética , Talassemia beta/genética , Alelos , Pré-Escolar , Cromatografia Líquida de Alta Pressão/métodos , Hibridização Genômica Comparativa , Saúde da Família , Feminino , Deleção de Genes , Grécia , Humanos , Masculino , Análise de Sequência de DNARESUMO
OBJECTIVE: The aim of this study was to develop a pyrophosphorolysis-activated polymerization (PAP) assay for non-invasive prenatal diagnosis (NIPD) of ß-thalassemia major and sickle-cell disease (SCD). PAP is able to detect mutations in free fetal DNA in a highly contaminating environment of maternal plasma DNA. METHODS: Pyrophosphorolysis-activated polymerization primers were designed for 12 informative SNPs, genotyped by melting curve analysis (MCA) in both parents. The PAP assay was tested in a series of 13 plasma DNA samples collected from pregnant women. A retrospective NIPD was performed in a couple at risk for SCD. RESULTS: All PAP reactions were optimized and able to detect <3% target gDNA in a background of >97% wildtype gDNA. In all 13 cases, the paternal allele was detected by PAP in maternal plasma at 10 to 18 weeks of gestation. For the couple at risk, PAP showed presence of the normal paternal SNP allele in maternal plasma, which was confirmed by results of the chorionic villus sampling analysis. CONCLUSIONS: In contrast to other methods used for NIPD, the combined PAP and MCA analysis detecting the normal paternal allele is also applicable for couples at risk carrying the same mutation, provided that a previously born child is available for testing to determine the linkage to the paternal SNPs.