Prenatal diagnosis of homozygous alpha-thalassemia-1 by cell-free fetal DNA in maternal plasma.

OBJECTIVE: To investigate the use of cell-free fetal DNA (cff-DNA) to determine fetal status in pregnant women who were risk for having Hb Bart's. METHODS: Plasma DNA was extracted from 10 mL of maternal blood from couples who both were alpha-thalassemia-1 carriers (SEA deletion). Real time quantitative PCR was performed using fluorescence-labeled probes to monitor wild type (wt) and SEA allele. The quantity of each allele was determined by cycle threshold (Ct). ΔCt (Ct of wt- Ct of SEA) was calculated from each sample. Prenatal diagnosis was performed to determine fetal status. RESULT: There were 62 Hb Bart's, 62 alpha-trait and 34 normal fetuses in this study. Mean ΔCt was 1.04 ± 0.38, 0.21 ± 0.37 and 0.14 ± 0.55 in Hb Bart's, alpha-trait and normal fetuses, respectively. Based on ROC, the best cut-off of ΔCt for predicting Hb Bart's was 0.51, giving 98.4% sensitivity and 20.8% false-positive rate. All but one Hb Bart's (98.4%) had ΔCt above 0.51, whereas 74.2% of alpha-trait and 88.2% of normal fetuses had ΔCt below 0.51. CONCLUSION: There is a positive trend to use cff-DNA in maternal plasma for prenatal diagnosis of homozygous alpha-thalassemia-1. With this technique, invasive prenatal testing and complications can be avoided in 79.2% of unaffected fetuses.

Intracellular readthrough of nonsense mutations by aminoglycosides in coagulation factor VII.

BACKGROUND: Nonsense mutations in coagulation factor (F) VII potentially cause a lethal hemorrhagic diathesis. Readthrough of nonsense mutations by aminoglycosides has been studied in a few human disease models with variable results. OBJECTIVES: We investigated the K316X and W364X FVII mutations, associated with intracranial hemorrhage, and their correction by aminoglycosides. The rare nonsense mutations in FVII represent favorite models to test this strategy, because even tiny increases in the amount of functional full-length protein in patients could ameliorate hemorrhagic phenotypes. RESULTS: A FVII-green fluorescent protein (GFP) chimaera provided us with a fluorescent model of FVII expression in living cells. Appreciable fluorescence in cells transfected with nonsense FVII-GFP mutants was detected upon geneticin treatment, thus demonstrating suppression of premature translation termination. To investigate the rescue of FVII function, nonsense variants of the native FVII without GFP (p316X-FVII and p364X-FVII) were transfected and found to secrete low amounts of FVII (approximately 1% of Wt-FVII activity), thus suggesting a spontaneous stop codon readthrough. Geneticin treatment of cells resulted in a significant and dose-dependent increase of secreted FVII molecules (p316X-FVII, 24 +/- 12 ng mL(-1), 3.6 +/- 0.8% of Wt-FVII activity; p364X-FVII, 26 +/- 10 ng mL(-1), 3.7+/-0.6%) characterized by reduced specific activity, thus indicating the synthesis of dysfunctional proteins. Similar results were observed with gentamicin, a commonly used aminoglycoside of potential interest for patient treatment. CONCLUSIONS: Our approach, extendable to other coagulation factors, represents an effective tool for a systematic study of the effects of aminoglycosides and neighboring sequences on nonsense codon readthrough. These results provide the rationale for a mutation-specific therapeutic approach in FVII deficiency.

Mycotic aneurysm of the left subclavian artery: CT findings.

Mycotic aneurysms caused by aspergillosis are rare. We report a nine-year-old girl with acute lymphoblastic leukaemia who had invasive pulmonary aspergillosis and subsequently developed a left subclavian artery aneurysm. Prior to the aneurysm, computed tomography (CT) of the chest showed a nodule with an air crescent in the left upper lobe, adhering to the mediastinum and the left subclavian artery. The left subclavian artery was ill-defined and had a small lumen, and it was embedded in the wall of the nodule. 37 days after the chest CT, the patient underwent a left thoracotomy because of massive haemoptysis, at which time a false aneurysm in the left subclavian artery was found. Plication of the aneurysm was performed. On a follow-up CT with multiplanar reconstruction six days after surgery, there were the plicated aneurysm and a small amount of pleural effusion in the upper portion of the left hemithorax, adjacent to the plication. In invasive pulmonary aspergillosis, it is important to be aware of the possibility of mycotic aneurysms, particularly in patients with pulmonary lesions adjacent to mediastinal vessels with ill-defined borders and small lumens, since the aneurysms may increase in size and rupture. CT, particularly multidetector CT, helps in visualisation of mycotic aneurysms.

Hematopoietic stem cell transplantation for homozygous ß-thalassemia and ß-thalassemia/hemoglobin E patients from haploidentical donors.

Thalassemia-free survival after allogeneic stem cell transplantation (SCT) is about 80-90% with either matched-related or -unrelated donors. We explored the use of a mismatched-related ('haplo- ') donor. All patients received two courses of pretransplant immunosuppressive therapy (PTIS) with fludarabine (Flu) and dexamethasone (Dxm). After two courses of PTIS, a conditioning regimen of rabbit antithymocyte globulin, Flu and IV busulfan (Bu) was given followed by T-cell-replete peripheral blood progenitor cells. GvHD prophylaxis consisted of cyclophosphamide (Cy) on days SCT +3 and +4 (post-Cy), and on day SCT +5 tacrolimus or sirolimus was started together with a short course of mycophenolate mofetil. Thirty-one patients underwent haplo-SCT. Their median age was 10 years (range, 2-20 years). Twenty-nine patients engrafted with 100% donor chimerism. Two patients suffered primary graft failure. Median time to neutrophil engraftment was 14 days (range, 11-18 days). Five patients developed mild to moderate, reversible veno-occlusive disease, while nine patients developed acute GvHD grade II. Only five patients developed limited-chronic GvHD. Projected overall and event-free survival rates at 2 years are 95% and 94%, respectively. The median follow up time is 12 months (range, 7-33 months).

Hb H hydrops foetalis syndrome: a case report and review of literature.

Haemoglobin H (Hb H) disease is caused by deletion or inactivation of three alpha-globin genes, leaving only one intact and active alpha-globin gene. People with Hb H disease usually have moderate anaemia, but are generally thought to be asymptomatic. Some Hb H disease patients require transfusions, and there are reports of fetuses with Hb H disease who have severe anaemia in utero resulting in fatal hydrops foetalis syndrome. We now report a case of Hb H hydrops foetalis syndrome, caused by the inheritance of a hitherto novel alpha-globin gene point mutation (codon 35 TCC-->CCC or Serine-->Proline) and an alpha-thalassaemia deletion of the Filipino type removing all zeta-alpha-globin genes on the other chromosome 16. The infant was delivered prematurely because of pericardial effusion and fetal distress, and was found to have severe anaemia and congenital anomalies. A review of the relevant literature on this syndrome is presented, and serves to underscore the phenotypic variations of Hb H disease and the need for surveillance for this condition among newborns and genetic counselling in communities with a high proportion of at-risk populations.