Biomarkers and genetic modulators of cerebral vasculopathy in sub-Saharan ancestry children with sickle cell anemia.

We investigated biomarkers and genetic modulators of the cerebral vasculopathy (CV) subphenotype in pediatric sickle cell anemia (SCA) patients of sub-Saharan African ancestry. We found that one VCAM1 promoter haplotype (haplotype 7) and VCAM1 single nucleotide variant rs1409419_T were associated with stroke events, stroke risk, as measured by time-averaged mean of maximum velocity in the middle cerebral artery, and with high serum levels of the hemolysis biomarker lactate dehydrogenase. Furthermore, VCAM-1 ligand coding gene ITGA4 variants rs113276800_A and rs3770138_T showed a positive association with stroke events. An additional positive relationship between a genetic variant and stroke risk was observed for ENPP1 rs1044498_A. Conversely, NOS3 variants were negatively associated with silent cerebral infarct events (VNTR 4b_allele and haplotype V) and CV globally (haplotype VII). The -alpha3.7kb-thal deletion did not show association with CV. However, it was associated with higher red blood cell and neutrophil counts, and lower mean corpuscular volume, mean corpuscular hemoglobin and red cell distribution width. Our results underline the importance of genetic modulators of the CV sub-phenotype and their potential as SCA therapeutic targets. We also propose that a biomarker panel comprising biochemical, hematological, imaging and genetic data would be instrumental for CV prediction, and prevention.

Genetic modulators of fetal hemoglobin expression and ischemic stroke occurrence in African descendant children with sickle cell anemia.

Sickle cell anemia (SCA) is an autosomal recessive monogenic disease with significant clinical variability. Cerebrovascular disease, particularly ischemic stroke, is one of the most severe complications of SCA in children. This study aimed to investigate the influence of genetic variants on the levels of fetal hemoglobin (Hb F) and biochemical parameters related with chronic hemolysis, as well as on ischemic stroke risk, in ninety-one unrelated SCA patients, children of sub-Saharan progenitors. Our results show that a higher Hb F level has an inverse relationship with the occurrence of stroke, since the group of patients who suffered stroke presents a significantly lower mean Hb F level (5.34 ± 4.57% versus 9.36 ± 6.48%; p = 0.024). Furthermore, the co-inheritance of alpha-thalassemia improves the chronic hemolytic pattern, evidenced by a decreased reticulocyte count (8.61 ± 3.58% versus 12.85 ± 4.71%; p < 0.001). In addition, our findings have confirmed the importance of HBG2 and BCL11A loci in the regulation of Hb F expression in sub-Saharan African SCA patients, as rs7482144_A, rs11886868_C, and rs4671393_A alleles are significantly associated with a considerable increase in Hb F levels (p = 0.019, p = 0.026, and p = 0.028, respectively). Concerning KLF1, twelve different variants were identified, two of them novel. Seventy-three patients (80.2%) presented at least one variant in this gene. However, no correlation was observed between the presence of these variants and Hb F level, severity of hemolysis, or stroke occurrence, which is consistent with their in silico-predicted minor functional consequences. Thus, we conclude that the prevalence of functional KLF1 variants in a sub-Saharan African background does not seem to be relevant to SCA clinical modulation.

Widening the spectrum of deletions and molecular mechanisms underlying alpha-thalassemia.

Inherited deletions of α-globin genes and/or their upstream regulatory elements (MCSs) give rise to α-thalassemia, an autosomal recessive microcytic hypochromic anemia. In this study, multiplex ligation-dependent probe amplification performed with commercial and synthetic engineered probes, Gap-PCR, and DNA sequencing were used to characterize lesions in the sub-telomeric region of the short arm of chromosome 16, possibly explaining the α-thalassemia/HbH disease phenotype in ten patients. We have found six different deletions, in heterozygosity, ranging from approximately 3.3 to 323 kb, two of them not previously described. The deletions fall into two categories: one includes deletions which totally remove the α-globin gene cluster, whereas the other includes deletions removing only the distal regulatory elements and keeping the α-globin genes structurally intact. An indel was observed in one patient involving the loss of the MCS-R2 and the insertion of 39 bp originated from a complex rearrangement spanning the deletion breakpoints. Finally, in another case, no α-globin gene cluster deletion was found and the patient revealed to be a very unusual case of acquired α-thalassemia-myelodysplastic syndrome. This study further illustrates the diversity of genomic lesions and underlying molecular mechanisms leading to α-thalassemia.

Pantoea Species Bacteremia in a Child With Sickle Cell Disease: Looking for a Culprit.

Pantoea agglomerans has been classically associated with cellulitis or synovitis secondary to penetrating trauma by vegetation. It is an infrequent cause of systemic infections. We describe the case of a 5-year-old girl with sickle cell disease with P. agglomerans bacteremia and review its potential causes.

Prolonged thrombocytopenia in a child with severe neonatal alloimmune reaction and Noonan syndrome.

Fetomaternal alloimmune thrombocytopenia (FMAIT) caused by maternal antibodies is the leading cause of severe neonatal thrombocytopenia. A 1-month-old Caucasian girl was referred to our Hematology Clinic for persistent thrombocytopenia diagnosed after a bleeding episode. Diagnostic tests suggested FMAIT. Mild thrombocytopenia persisted for 18 months, and subsequent findings of dysmorphic facies, short stature and mild pulmonary stenosis led to the hypothesis of Noonan syndrome (NS), which was confirmed by genetic test. Other hematological abnormalities were excluded and she had no further bleeding episodes. This case illustrates the possibility of different diagnoses with the same clinical manifestations. The persistence of thrombocytopenia longer than expected associated with typical physical features led to the diagnosis of NS.

Cold Agglutinin Syndrome and Hemophagocytic Lymphohistiocytosis: An Unusual Combination Caused by Epstein-Barr Virus Infection.

Autoimmune hemolytic anemia (AIHA) and hemophagocytic lymphohistiocytosis (HLH) are rare complications of infectious mononucleosis. The authors describe a 12-year-old male with acute infectious mononucleosis, hepatitis, cholestasis, and an autoimmune hemolytic disorder caused by cold agglutinins IgM (anti-i specificity). Clinical deterioration with persistent fever, anemia, and hepatosplenomegaly was consistent with cold AIHA plus concomitant HLH. The patient was treated with corticosteroids and acyclovir, with an uneventful recovery. Although rare, cold agglutinin syndrome and HLH can complicate infectious mononucleosis and should be considered in a patient with clinical deterioration. Corticosteroids are the mainstay treatment of HLH and may be beneficial in infection-associated cold agglutinin syndrome.

Linezolid in the treatment of multidrug-resistant/extensively drug-resistant tuberculosis in paediatric patients: experience of a paediatric infectious diseases unit.

Linezolid has been used in the treatment of multidrug-resistant/extensively drug-resistant tuberculosis in adults with encouraging results, however experience in children is scarce. We describe our experience with the use of linezolid as part of a multidrug regimen in the treatment of 4 patients who had persistent positive cultures, despite prolonged combined therapy.

Haemophilia A: health and economic burden of a rare disease in Portugal.

BACKGROUND: Haemophilia A is a hereditary bleeding disorder, which has been considered rare and chronic. The burden of this disease in Portugal remains unknown. The aim of this study was to estimate the annualized cost and health burden of haemophilia A in Portugal. METHODS: Data were extracted from a Portuguese expert panel, from official data and national literature. Annual costs were calculated from the perspective of the society including direct and indirect costs. Unitary costs were extracted from 2017 national official sources and are expressed in euros. Health burden was expressed in disability adjusted life years (DALYs) based on incidence and quality of life questionnaires. Estimates are presented for the overall population and stratified by severity, age group (< 18 years vs. adults) and inhibitor status. RESULTS: The yearly average cost per patient is estimated to range from €39,654/patient without inhibitors and €302,189/patient with inhibitors, representing a 7.6 fold difference. Amongst patients without inhibitors, the annual average cost was €401 in mild, €5327 in moderate and €85,805 in severe disease. Average cost per child and adult is €72,287 and €51,737, respectively. Direct costs represent approximately 95% of all costs, of which almost the totality accounts for clotting factor replacement therapy and bypassing agents. The total annual cost of haemophilia A for the Portuguese society was estimated to be €42,66 million, one third of which was related to the treatment of patients with inhibitors. It is estimated that haemophilia A is responsible for 3878 DALYs in Portugal (497 DALYs in mild, 524 DALYs in moderate, 2031 DALYs in severe patients without inhibitors and 784 DALYs in patients with inhibitors) for the cohort of 2017 (750 patients) or 5.2 DALY/patient during lifetime. CONCLUSIONS: Despite being rare, the economic and health burden of haemophilia A is remarkable. The main cost driver is clotting factor replacement therapy. Moreover, haemophilia A is more costly in children than in adults and rises exponentially with disease severity.

Sickle cell anemia - Nitric oxide related genetic modifiers of hematological and biochemical parameters.

BACKGROUND: Sickle cell anemia (SCA) is an inherited blood disorder. SCA patients present clinical and hematologic variability that cannot be only explained by the single mutation in the beta-globin gene. Others genetic modifiers and environmental effects are important for the clinical phenotype. SCA patients present arginine deficiency that contributes to a lower nitric oxide (NO) bioactivity. OBJECTIVE: The aim of this work is to determine the association between hematological and biochemical parameters and genetic variants from eNOS gene, in pediatric SCA patients. METHODS: 26 pediatric SCA patients were genotyped using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) techniques in three important eNOS gene polymorphisms - rs2070744, rs1799983 and intron 4 VNTR. RESULTS: Results from this study show a significant statistical association between some parameters and genetic variants: an increased reticulocyte count and high serum lactate dehydrogenase levels were associated with both the rs2070744_TT and the rs1799983_GG genotypes at eNOS gene and high levels of neutrophils were associated with the eNOS4a allele at intron 4 VNTR. CONCLUSIONS: Our results reinforce the importance of NO bioactivity in SCA. We presume that NO, and its precursors might be used as therapy to improve the quality of life of SCA patients.

Hemorheological alterations in sickle cell anemia and their clinical consequences - The role of genetic modulators.

Sickle cell anemia (SCA) is an autosomal recessive disease caused by the HBB:c.20A>T mutation that leads to hemoglobin S synthesis. The disease presents with high clinical heterogeneity characterized by chronic hemolysis, recurrent episodes of vaso-oclusion and infection. This work aimed to characterize by in silico studies some genetic modulators of severe hemolysis and stroke risk in children with SCA, and understand their consequences at the hemorheological level.Association studies were performed between hemolysis biomarkers as well as the degree of cerebral vasculopathy and the inheritance of several polymorphic regions in genes related with vascular cell adhesion and vascular tonus in pediatric SCA patients. In silico tools (e.g. MatInspector) were applied to investigate the main variant consequences.Variants in vascular adhesion molecule-1 (VCAM1) gene promoter and endothelial nitric oxide synthase (NOS3) gene were significantly associated with higher degree of hemolysis and stroke events. They potentially modify transcription factor binding sites (e.g. VCAM1 rs1409419_T allele may lead to an EVI1 gain) or disturb the corresponding protein structure/function. Our findings emphasize the relevance of genetic variation in modulating the disease severity due to their effect on gene expression or modification of protein biological activities related with sickled erythrocyte/endothelial interactions and consequent hemorheological abnormalities.

Genotype-phenotype correlation in a cohort of Portuguese patients comprising the entire spectrum of VWD types: impact of NGS.

The diagnosis of von Willebrand disease (VWD), the most common inherited bleeding disorder, is characterised by a variable bleeding tendency and heterogeneous laboratory phenotype. The sequencing of the entire VWF coding region has not yet become a routine practice in diagnostic laboratories owing to its high costs. Nevertheless, next-generation sequencing (NGS) has emerged as an alternative to overcome this limitation. We aimed to determine the correlation of genotype and phenotype in 92 Portuguese individuals from 60 unrelated families with VWD; therefore, we directly sequenced VWF. We compared the classical Sanger sequencing approach and NGS to assess the value-added effect on the analysis of the mutation distribution in different types of VWD. Sixty-two different VWF mutations were identified, 27 of which had not been previously described. NGS detected 26 additional mutations, contributing to a broad overview of the mutant alleles present in each VWD type. Twenty-nine probands (48.3 %) had two or more mutations; in addition, mutations with pleiotropic effects were detected, and NGS allowed an appropriate classification for seven of them. Furthermore, the differential diagnosis between VWD 2B and platelet type VWD (n = 1), Bernard-Soulier syndrome and VWD 2B (n = 1), and mild haemophilia A and VWD 2N (n = 2) was possible. NGS provided an efficient laboratory workflow for analysing VWF. These findings in our cohort of Portuguese patients support the proposal that improving VWD diagnosis strategies will enhance clinical and laboratory approaches, allowing to establish the most appropriate treatment for each patient.