RESUMO
Celocentesis is a new sampling tool for prenatal diagnosis available from 7 weeks in case of couples at risk for genetic diseases. In this study, we reported the feasibility of earlier prenatal diagnosis by celocentesis in four cases of cystic fibrosis and one case of cystic fibrosis and ß-thalassemia co-inherited in the same fetus. Celomic fluids were aspired from the celomic cavity between 8+2 and 9+3 weeks of gestation and fetal cells were picked up by micromanipulator. Maternal DNA contamination was tested and target regions of fetal DNA containing parental pathogenetic variants of CFTR and HBB genes were amplified and sequenced. Four of the five fetuses resulted as being affected by cystic fibrosis and, in all cases, the women decided to interrupt the pregnancy. In the other case, the fetus presented a healthy carrier of cystic fibrosis. The results were confirmed in three cases on placental tissue. In one case, no abortive tissue was obtained. In the last case, the woman refused the prenatal diagnosis to confirm the celocentesis data; the pregnancy is ongoing without complications. This procedure provides prenatal diagnosis of monogenic diseases at least four weeks earlier than traditional procedures, reducing the anxiety of patients and providing the option for medical termination of the affected fetus at 8-10 weeks of gestation, which is less traumatic and safer than surgical termination in the second trimester.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Diagnóstico Pré-Natal , Humanos , Fibrose Cística/genética , Fibrose Cística/diagnóstico , Feminino , Gravidez , Diagnóstico Pré-Natal/métodos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Adulto , Talassemia beta/genética , Talassemia beta/diagnóstico , FetoRESUMO
ß-Thalassemias are inherited anemias that are caused by the absent or insufficient production of the ß chain of hemoglobin. Here we report 6-8-year follow-up of four adult patients with transfusion-dependent ß-thalassemia who were infused with autologous CD34+ cells transduced with the TNS9.3.55 lentiviral globin vector after reduced-intensity conditioning (RIC) in a phase 1 clinical trial ( NCT01639690) . Patients were monitored for insertional mutagenesis and the generation of a replication-competent lentivirus (safety and tolerability of the infusion product after RIC-primary endpoint) and engraftment of genetically modified autologous CD34+ cells, expression of the transduced ß-globin gene and post-transplant transfusion requirements (efficacy-secondary endpoint). No unexpected safety issues occurred during conditioning and cell product infusion. Hematopoietic gene marking was very stable but low, reducing transfusion requirements in two patients, albeit not achieving transfusion independence. Our findings suggest that non-myeloablative conditioning can achieve durable stem cell engraftment but underscore a minimum CD34+ cell transduction requirement for effective therapy. Moderate clonal expansions were associated with integrations near cancer-related genes, suggestive of non-erythroid activity of globin vectors in stem/progenitor cells. These correlative findings highlight the necessity of cautiously monitoring patients harboring globin vectors.
Assuntos
Terapia Genética/métodos , Vetores Genéticos , Globinas/genética , Lentivirus/genética , Condicionamento Pré-Transplante/métodos , Talassemia beta/terapia , Adolescente , Adulto , Antígenos CD34/genética , Transfusão de Sangue , Feminino , Humanos , Masculino , Transdução Genética , Adulto JovemRESUMO
Rasburicase is a recombinant urate oxidase enzyme indicated for tumor lysis syndrome, a potential life-threatening oncologic emergency that occurs most commonly during initial chemotherapy for hematological malignancies. As a result of the defects in the physiological antioxidant pathway, erythrocytes of patients with glucose-6-phosphate dehydrogenase deficiency are not protected against the oxidizing stress exerted by hydrogen peroxide generated with the administration of rasburicase. The authors report a 14-year-old patient, diagnosed with T-cell acute lymphoblastic leukemia, who developed methemoglobinemia and hemolytic anemia with low oxygen saturation after starting steroids, hyperhydratation, and rasburicase administration. The complications resolved with supportive therapy only.
Assuntos
Metemoglobinemia/induzido quimicamente , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Urato Oxidase/efeitos adversos , Adolescente , Anemia Hemolítica/induzido quimicamente , Anemia Hemolítica/diagnóstico , Humanos , Masculino , Metemoglobinemia/diagnóstico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/diagnóstico , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/uso terapêutico , Urato Oxidase/uso terapêuticoRESUMO
A 59-year-old Italian woman came to our center for revaluation of a previous diagnosis of polycythemia vera. The patient presented with a lifelong history of polycythemia, no increase in white blood cells (WBCs) and platelets, and a negative bone marrow biopsy. Analysis of hemoglobin (Hb) fractions showed an abnormal fast moving Hb component. We aimed to determine if this variant was the cause of polycythemia in this patient. A complete blood count (CBC) was performed by an automated cell counter and Hb fractions were determined by high performance liquid chromatography (HPLC). Standard stability tests and oxygen affinity evaluation were also performed. Genomic DNA was extracted from peripheral blood leukocytes using the phenol chloroform method and the entire ß-globin gene was analyzed by direct sequencing. At the hematological level, no anemia or hemolysis was observed but an abnormal Hb fraction was detected using cation exchange HPLC. Molecular analysis of the ß-globin gene showed heterozygosity for an AAG > ACG substitution at codon 144, resulting in a LysâThr amino acid replacement. We demonstrated that this is a new Hb variant with increased oxygen affinity. Its altered physiology is caused by the reduction of 2,3-diphosphoglycerate (2,3-DPG) effects, due to an amino acid substitution in the central pocket near the C-terminal of the ß chain. We called this new variant Hb San Cataldo for the native city of proband.
Assuntos
Hemoglobinas Anormais/genética , Mutação de Sentido Incorreto , Oxigênio/metabolismo , Policitemia Vera/genética , 2,3-Difosfoglicerato , Diagnóstico Diferencial , Feminino , Hemoglobinopatias/genética , Heterozigoto , Humanos , Pessoa de Meia-Idade , Policitemia Vera/etiologiaRESUMO
Elevated fetal hemoglobin (HbF) ameliorates the clinical severity of hemoglobinopathies such as ß-thalassemia and sickle cell anemia. Currently, the only curative approach for individuals under chronic transfusion/chelation support therapy is allogeneic stem cell transplantation. However, recent analyses of heritable variations in HbF levels have provided a new therapeutic target for HbF reactivation: the transcriptional repressor BCL11A. Erythroid-specific BCL11A abrogation is now actively being sought as a therapeutic avenue, but the specific impact of such disruption in humans remains to be determined. Although single nucleotide polymorphisms in BCL11A erythroid regulatory elements have been reported, coding mutations are scarcer. It is thus of great interest that patients have recently been described with microdeletions encompassing BCL11A. These patients display neurodevelopmental abnormalities, but whether they show increased HbF has not been reported. We have examined the hematological phenotype, HbF levels, and erythroid BCL11A expression in 3 such patients. Haploinsufficiency of BCL11A induces only partial developmental γ-globin silencing. Of greater interest is that a patient with a downstream deletion exhibits reduced BCL11A expression and increased HbF. Novel erythroid-specific regulatory elements in this region may be required for normal erythroid BCL11A expression, whereas loss of separate elements in the developing brain may explain the neurological phenotype.
Assuntos
Proteínas de Transporte/genética , Deleção Cromossômica , Cromossomos Humanos Par 2 , Hemoglobina Fetal/metabolismo , Doenças do Sistema Nervoso/genética , Proteínas Nucleares/genética , Adolescente , Criança , Feminino , Humanos , Masculino , Doenças do Sistema Nervoso/sangue , Proteínas Repressoras , Regulação para CimaRESUMO
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.
Assuntos
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
At 5-12 weeks of gestation the amniotic sac is surrounded by celomic fluid, which contains cells of fetal origin. This fluid can be sampled by celocentesis, which involves the ultrasound-guided insertion of a needle through the vagina. The aim of this study was to examine the feasibility of prenatal diagnosis of haemoglobinopathies from the celomic fluid using a specific protocol. Celocentesis was performed at 7-9 weeks gestation in 26 singleton pregnancies at risk for haemoglobinopathies. In 25 cases more than 30 fetal cells were recovered from the celomic fluid and in all these cases molecular analysis for haemoglobinopathies was possible and the results were confirmed by subsequent chorionic villus sampling or amniocentesis. The results of this study suggest that reliable diagnosis of thalassemia syndromes can be performed from 7 weeks gestation by celocentesis. Further work is necessary to demonstrate the safety of celocentesis before widespread use.