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.

A heparin cofactor II mutation (HCII Rimini) combined with factor V Leiden or type I protein C deficiency in two unrelated thrombophilic subjects.

305 patients with juvenile thromboembolic episodes were screened for the presence of heparin cofactor II deficiency. The heterozygous deletion of two bases was found in the exon 5 of the heparin cofactor II gene in two unrelated patients, very likely due to a founder effect. This molecular lesion, causing a frameshift and elongated translation, affects the core of the molecule and should cause the complete unfolding of the protein, which is in accordance with the observed type I deficiency. The corresponding region of antithrombin III gene is affected by a cluster of frameshift mutations suggesting that heparin cofactor II and antithrombin III could share similar mutational patterns. The heparin cofactor II gene alteration was associated with, in one patient, the factor V Leiden mutation and, in the other, type I protein C deficiency. The tracing of the single defects in several family members indicated that the mutations became clinically manifest only when present in the doubly heterozygous condition. This study provides two examples, based on molecular findings, of the interplay of risk factors which is potentially useful to define a role for heparin cofactor II deficiency in inherited thrombophilia.

Mutations in the R2 FV gene affect the ratio between the two FV isoforms in plasma.

Molecular genetics and biochemical studies were performed in homozygotes for the R2 allele (4070G) in the factor V gene, most of them affected by coronary artery disease. Novel polymorphisms (G642T, 156Ser; T1328C, 385Met/Thr), among which a functional candidate (A6755G, 2194Asp/Gly) located in the C2 domain of FV, were identified in the R2 gene. In chromatographic studies R2 FV appeared qualitatively identical to normal FV. However, a relative increase of the more thrombogenic and more glycosylated FV isoform (FV1) was observed in plasma of 2194Gly homozygotes (mean FV1/FV2 ratio 0.71, 95% CI 0.66-0.77) as compared to R2-free controls (0.37, 95% CI 0.34-0.40). We conclude that carriership of the R2 FV gene is associated with an imbalance between the two functionally different FV isoforms, and propose that genetically determined differential glycosylation of FV could represent a novel mechanism of thrombotic disease.

Further cytogenetic evidence for a multistep pathogenesis of Ph-positive chronic myelogenous leukemia.

A case of Ph-positive chronic myelogenous leukemia in blastic crisis was studied extensively by means of cytogenetic techniques. Karyotypic features, as well as growth patterns, kinetic data, and rates of sister chromatid exchange, were examined in bone marrow, blood, and pleural effusion cells. The data provide strong evidence for a multistep pathogenesis of the disease, the development of which appears to be linked to mechanisms of clonal selection and genetic imbalance in the malignant cell population.

Cytogenetically distinct leukemic cell lines displaying in vitro specific proliferative and differentiation capacities may account for early disease relapse in the blast phase of CML.

The cytogenetic features and the proliferative and differentiation capabilities of blast cell fractions purified on a density gradient were studied in one patient with chronic myeloid leukemia (CML) in blast crisis, both at the emergence and at relapse of the disease. The results show that relapse was due to the appearance of a new leukemic cell line that was characterized by peculiar chromosomal, growth, and differentiation features, which seemingly accounted for early refractoriness to therapy and disease progression.

Normal bone marrow karyotype in paroxysmal nocturnal hemoglobinuria--a cooperative European study.

Sixteen patients with paroxysmal nocturnal hemoglobinuria (PNH) from five European centers have been submitted to chromosome analysis. All of them had a normal bone marrow karyotype. The associations between some chromosomally abnormal cases reported in the literature and "typical" PNH or PNH phenomenon during the course of other hematological disorders are discussed.

Idiopathic central retinal vein occlusion in a thrombophilic patient with the heterozygous 20210 G/A prothrombin genotype.

PURPOSE: To report the occurrence of monolateral central retinal vein occlusion in a patient with heterozygous 20210 G/A prothrombin genotype, known to be associated with high thrombophilic risk. METHODS: A monolateral central retinal vein occlusion was diagnosed in a 71-year-old woman, who had suffered from a deep vein thrombosis in her left leg at the age of 36 years. Mutations of the genes involved in the coagulation process were investigated by DNA polymerase chain reaction. RESULT: DNA analysis showed the patient to be heterozygous for the prothrombin 20210 G/A genetic variation. CONCLUSION: The 20210 G/A prothrombin gene mutation may be associated with central retinal vein occlusion.

New dosimetric approach for multidimensional dose evaluation in gamma knife radiosurgery. Technical note.

During the past two decades, the progress in computerized treatment planning systems has led to more accurate imaging and therapy by using the gamma knife, especially with the smallest collimators (4 mm). However, the ionization chambers that have been used to calibrate the gamma knife are not useful with the smallest collimators because the chambers are too big compared with the irradiated volume. Therefore, it is important to develop more suitable dosimeters. This study proposes a new dosimeter method. The FriXyGel method proposed here is based on a phantom dosimeter, an acquisition chain, and dedicated software. This dosimeter uses an agarose gel into which a ferrous sulphate solution (Fricke solution) and a metal ion indicator (xylenol orange) are incorporated. The absorbed dose is detected through measurements of visible light transmission, imaged by means of a charge-coupled device camera provided with a suitable optical filter. Gel layers are imaged before and after irradiation, and the differences in light absorption are related to the absorbed dose. By choosing convenient thickness of gel layers and by building up a phantom with different gel slices, it is possible to obtain a three-dimensional (3D) representation of the absorbed dose. The final 3D representation is reached after several mathematical processes have been applied to the images. The first step identifies and reduces all factors that could alter the original data, such as nonuniformity in illumination. Then, after calibration procedures, it is possible to obtain absorbed dose values and to discover their 3D representation. This goal has been reached by developing appropriate software that performs all the calculations necessary for spatial representation routines and prompt comparison with theoretical calculations.

Stereotactic radiosurgery for the treatment of arteriovenous malformations in childhood.

BACKGROUND: The main techniques and results in stereotactic radiosurgical treatment of endocranial AVM's are described and compared. The authors also report their preliminary experience in the treatment of 6 consecutive pediatric patients with intracerebral vascular malformations using gamma knife (GK) radiosurgery. METHODS: The various stereotactic radiosurgery methods currently used (charged-particle beam, modified linear accelerator, and GK) are described. At the Department of Neurosurgery in Verona, from February 1993 to February 1996, stereotactic GK radiosurgery was performed on 721 patients, including 20 of pediatric age (3%). Of the 78 AVMs, 7 (9%) were diagnosed in children. One patient was lost at follow-up. Among the remaining 6 children, there were 3 males and 3 females with a mean age of 12.3 years (range, 5-16 years). Treatment general anesthesia was needed only in 1 case. The AVM volume was always less than 10 cc. After completion of the procedure, children were discharged from the hospital the following day. RESULTS: The follow-up period ranged from 4 months to 29 months (median 18.8 months). The angiographic confirmed total obliteration is used as the end point of an AVM treated radiosurgically, and usually requires 2 to 3 years. All the patients are alive; four of the treated children are neurologically normal and one patient has clinically improved to a normal neurological status. The sixth patient has fixed neurological deficits that existed prior to treatment. Among the three cases with a follow-up period of over 2 years, complete obliteration has been angiographically confirmed in 2 patients and subtotal in 1 patient. In the three remaining patients with follow-up periods less than 2 years, serial MR images suggest subtotal obliteration in 2 cases and no significant change in one patient who had undergone treatment within the current year. To date, neither persistent GK radiosurgery-related complications nor bleeding following stereotactic radiosurgery has been described. CONCLUSIONS: The review of literature and our preliminary results suggest that also in children, as in adults, the use of stereotactically delivered irradiation represents a safe and effective technique obtaining complete obliteration of AVMs previously considered surgically inaccessible due to their location and poor response to resection and/or embolization.

Management of kidney transplantation in a factor VII-deficient patient: case report.

Transplantation in patients with congenital bleeding disorders is a challenge requiring an integrated approach of various specialists. Renal transplantation, the most frequent type of solid organ transplantation, is rarely performed in individuals with congenital hemorrhagic disorders. We performed a renal transplantation in a 53-year-old man with end-stage renal disease and congenital coagulation factor VII deficiency, a rare bleeding disorder with a peculiar clinical picture requiring replacement therapy in surgical interventions. Perioperative bleeding was successfully prevented by administration of recombinant activated factor VII. Treatment schedule, administration rate, and long-term follow-up are reported in detail. Our report confirmed the feasibility and safety of recombinant activated factor VII in major surgical procedures like solid organ transplantations. Success requires evaluation of doses and therapeutic schedules as well as a multidisciplinary approach.

Antimicrobial activity of garlic against oral streptococci.

The antimicrobial activity of two garlic clones' (1: purple and 2: white) crude extracts against oral microbiota was evaluated in vitro (study 1) and in vivo (study 2). Study 1 consisted of the evaluation of minimum inhibitory (MIC) and bactericidal (MBC) concentrations against nine streptococci strains. In study 2, a 2.5% garlic (clone 2) solution was used as a mouthwash in a 5-week study by 30 subjects. Blood agar and Mitis Salivarius Bacitracin agar were inoculated with subjects' saliva to quantify oral microorganisms and mutans streptococci. Study 1 showed MIC ranging from 0.5 to 32.0 mg ml(-1) for clone 2 and from 8 to 64.0 mg ml(-1) for clone 1. MBC ranged from 1.0 to 128.0 mg ml(-1) and from 8.0 to 128.0 mg ml(-1) regarding clones 2 and 1 respectively. Study 2 showed that 2.5% garlic mouthwash solution had good antimicrobial activity against mutans streptococci and oral microorganisms. Maintenance of reduced salivary levels of streptococci was observed after 2 weeks at the end of mouthwash use. Unpleasant taste (100%), halitosis (90%) and nausea (30%) were reported by subjects after the end of the study. It was concluded that the garlic clones have antimicrobial properties in vitro against streptococci and anticariogenic properties against oral microorganism in spite of its adverse effects.

A case of chronic myelogenous leukemia with 11q- in blast crisis with monoblastic differentiation.

We report a case of chronic myeloid leukemia in monoblastic blast crisis in which a deletion of chromosome 11 (11q-) was detected. Such a finding seems to provide further support to a relationship between this chromosome anomaly and monocytic proliferation.

Gamma knife radiosurgery in meningiomas of the posterior fossa. Experience with 62 treated lesions.

OBJECTIVES: This study was undertaken to assess the role of the gamma knife (GK) in the treatment of meningiomas of the posterior cranial fossa (PCF) and to statistically analyze the predictability of arbitrarily-selected prognostic factors in such treatment. METHODS: From February 1993 to November 1998, 57 patients underwent GK treatment for 62 meningiomas of the PCF (19 M/38 F; average age, 57.5 years, ranging from 25 - 82 years). Tumor sites included: foramen jugular-petrous bone (26/62), petroclival (23/62), cerebellar convexity (6/62), tentorium (6/62), and foramen magnum (1/62). Single lesions were treated in 44/62 cases while meningiomatosis was treated in the remaining 18. Post-operative residual or recurrent tumor was found in 27/62 patients and, in 7/27, histology documented characteristics of biological aggressiveness (GII/III). Indications for radiosurgery included: advanced age, high operative risk, tumor volume < 20 ml, inoperable or refused for additional surgery. The prognostic factors statistically analyzed included: meningiomatosis (yes/no), radiosurgery as primary or adjuvant treatment, GI vs. GII/III histology, and tumor volume (< or = 5 ml vs. > 5 ml). RESULTS: The observation periods varied from 6 to 64.3 months (median 28.7 months). At the end of the study, 53/57 patients were alive and reported to be in stable or improved neurological condition. The cause of death for the remaining 4 patients included: 2 deaths associated with tumor progression, while 2 died due to causes unrelated to the disease. Neuroradiological evaluation documented the disappearance or reduction of the meningioma mass in 34/62 (55 %) cases, a stable imaging picture in 25/62 (40 %), and a progression only in 3/62 (5 %). To date, there have been no reported cases of post-GK permanent morbidity or mortality. Side effects observed were of a transient nature due to post-radiosurgical edema (6.5 %). With regard to statistical analysis, the only factor to appear to significantly influence efficacy of radiosurgery for tumor growth control (TGC) was the biological nature of the meningioma (chi(2) = 2.708). The presence of meningiomatosis, SR as a primary or adjuvant treatment nor tumor volume were shown to statistically influence tumor behavior after GK. CONCLUSIONS: The excellent results obtained for TGC with minimal associated side effects suggest that GK is an effective therapeutic tool also for treatment of PCF meningiomas.

The heterozygous 20210 G/A genotype prevalence in patients affected by central and branch retinal vein occlusion: a pilot study.

BACKGROUND: Several inherited conditions have been associated with an increased or decreased incidence of retinal vein occlusion (RVO). The A allele in the 20210 G/A prothrombin gene has been found to be associated with systemic venous thrombosis. The aim of this study has been to verify the prevalence of this mutation in patients affected by central RVO (CRVO) or branch RVO (BRVO). METHODS: A retrospective study was carried out on 100 consecutive patients suffering from RVO, more than 50 years old, unaffected by systemic diseases known to be associated with markedly increased RVO occurrence. We determined the frequency of this mutation by performing mutagenised amplification of exon 14 followed by restriction analysis of the amplified DNA fragment. RESULTS: The overall frequency of prothrombin 20210A allele in RVO patients was 6.0%. All heterozygous patients had suffered from CRVO. In this study subgroup, the frequency of the 20210 G/A prothrombin heterozygosis was 12.0%. The difference in the frequency of this the genetic variant between the CRVO and BRVO groups was statistically significant. None of the conventional RVO risk factors were statistically related to the occurrence of the disease in either the CRVO or the BRVO subgroup. CONCLUSION: The prevalence of the prothrombin 20210A mutation observed in CRVO patients is significantly higher than in the normal Italian population. Moreover, the prevalence is significantly greater in CRVO than in BRVO patients. These results raise the possibility that the prothrombin 20210A variant may be considered as a risk factor for CRVO.

Visual control of hand-reaching movement: activity in parietal area 7m.

The activity of single neurons was studied in parietal area 7m while monkeys performed an instructed-delay reaching task to visual targets under normal light conditions and in darkness. The task was aimed at assessing the influence of vision of hand position on the neural activity of 7m related either to static posture and movement of the hand or to eye position in the orbit. The results show the existence of preparatory, movement-related and postural activity for the control of reaching, all of which are strongly modulated by vision. The activity of many 7m neurons, otherwise insensitive to pure visual stimuli, seems to reflect complex interactions between gaze angle and hand position in the visual field.