Deletion patterns of dystrophin gene in Hungarian patients with Duchenne/Becker muscular dystrophies

Deletion pattern analysis of the dystrophin gene was performed in 159 Hungarian patients with Duchenne/Becker muscular dystrophy. In 116 cases (73% of total patients), exon deletions were detected by PCR amplification. In 37 patients (31.9% of patients with a deletion) one exon was deleted, while five or more exons were missing in 40 children (34.4%). With respect to the proximal-distal distribution of the deletions, 90 children (77.6%) had deletions exclusively at the 3′ end of the gene, 21 deletions (18.1%) affected only the 5′ end, and in five patients (4.3%) large-scale deletions were detected, which affected both regions. Analysis of the breakpoint distribution pattern in the dystrophin gene showed that, similarly to that observed in several Western European populations, intron 44 was involved most frequently (n=35, 15.1%) as a starting breakpoint. In the Hungarian population introns 50 and 52 were the second (n=30, 12.9%) and third (n=29, 12.5%) most frequently observed hot spots at the 3′ end; these seem to be characteristic for the Hungarian patients. At the 5′ end the breakpoint peak (n=6, 2.58%) was in intron two. As it was proposed by previous national studies, our findings also suggest that certain intronic sequences, characteristic for a population, probably determine the development of a preferential breakpoint profile in this disease.     

Endothelial nitric oxide synthase gene interactions and the risk of ischaemic stroke

OBJECTIVE: Endothelial nitric oxide synthase (eNOS), which produces NO, plays an important role in the endothelial function under a wide range of physiological conditions. eNOS exon 7 polymorphism (Glu298Asp, G894T) has been considered to influence the risk of coronary artery disease. Alone, however, it has not been shown to be a genetic risk factor for ischaemic stroke. With the assumption of additive interactions, we examined whether the eNOS G894T or eNOS 894TT genotypes in combination with the methylenetetrahydrofolate reductase 677TT (MTHFR 677TT) or angiotensin-converting enzyme (ACE) D/D genotype could contribute to acute ischaemic stroke. MATERIAL AND METHODS: The data on 407 consecutive patients with acute ischaemic stroke who had never suffered a previous stroke event were analysed. As a control group, 295 stroke and neuroimaging alteration-free Caucasian subjects were examined. With the use of the PCR technique, the eNOS G894T, eNOS 894TT, MTHFR 677TT and ACE D/D mutations, as unfavourable common genotypes were determined in the participants. Logistic regression models were used to evaluate the roles of the genotypes and their combinations in the development of ischaemic stroke. RESULTS: The MTHFR C677TT genotype combined with the eNOS G894T or eNOS 894TT genotypes occurred significantly more frequently in the subjects with ischaemic stroke (7.1%; P < 0.025) than in the control group (3.1%). The co-occurrence of the ACE D/D genotype and eNOS G894T or eNOS 894TT was calculated to be more frequent in the ischaemic stroke group (20.9%, P < 0.0001) than in the control group (5.4%). CONCLUSION: The eNOS G894T or eNOS 894TT genotypes in combination with the MTHFR 677TT or ACE D/D genotype increases the risk of ischaemic stroke.     

Effect of ACE gene polymorphism on carbohydrate metabolism, on oxidative stress and on end-organ damage in type-2 diabetes mellitus

ACE gene insertion/deletion (I/D) polymorphism is a well-known risk factor of hypertension, cardiovascular diseases and progression of diabetic nephropathy. In carriers of allele D, serum level of angiotensin-II is higher, which can be associated with increased oxidative stress and subsequent endothelial damage. Albuminuria is a sensitive marker of endothelial damage, while serum activity of the enzyme gamma-glutamyl transferase--that plays important role in the antioxidant defense--may refer to the level of oxidative stress. The present paper reports on a cross-sectional clinical study, where authors have examined on the relation between ACE gene insertion/deletion polymorphism and carbohydrate metabolism, hypertension as well as albuminuria in type 2 diabetics (n = 145). In patients carrying allele D, fructosamine levels were significantly higher (p = 0.007) than in carriers of allele I. Patients with II + ID genotypes and those who were treated with insulin took more antihypertensive drugs than the ones with II genotype or orally treated (p = 0.015). They found a significant association between genotype and fructosamine level (p = 0.023). Association between genotype or modality of treatment of diabetes (oral vs, insulin) and combined treatment of hypertension (number of antihypertensive drugs) was of borderline significance. They found that fructosamin level of patients receiving ACE inhibitor was lower than that of patients not receiving ACE inhibitors. In patients with allele D, they have also found higher activity of gamma-GT and higher albuminuria. From this results and data of the literature the authors conclude that because of insulin resistance (in connection with the presence of allele D), these patients tend to have a worse metabolic state, more advanced glycation products, due to which oxidative stress and endothelial cell damage may develop. As albuminuria and activity of gamma-GT were both found higher in patients with allele D, and our patients did not suffer of any hepatic disease, authors take the consequence that gamma-GT is a marker of the oxidative stress caused by allele D. Endothelial damage may explain that these patients take a higher number of antihypertensive combination. Based on this, D allele may contribute--via increased glycation and oxidative stress--to the target organ damage in type 2 diabetes.     

Sequence heterogeneity among human picobirnaviruses detected in a gastroenteritis outbreak

Summary. Human picobirnaviruses characterised in this study were serendipitously detected in a non-bacterial gastroenteritis outbreak when specimens were examined for the presence of human rotaviruses using polyacrylamide gel electrophoresis. Of ten stool samples sent for virological examination, two, three, and one specimens were positive for human caliciviruses, picobirnaviruses, and both viruses, respectively. Partial sequences of the RNA-dependent RNA polymerase gene were determined for three picobirnavirus-positive samples. The sequence identity among these three strains was 60% to 65% for the nucleic acid and 64% to 70% for the deduced amino acid sequences. Phylogenetic analysis revealed that each of the three strains clustered with strains identified in geographically separate areas. In contrast, human calicivirus strains co-incidentally identified, showed complete nucleotide sequence identity. These findings demonstrate a lack of common exposure to or point of source for picobirnavirus infection, suggesting that the outbreak was caused by human caliciviruses. Further studies are needed to determine the etiologic role and to establish the taxonomic basis of picobirnaviruses.     

The Goose Reovirus Genome Segment Encoding the Minor Outer Capsid Protein, σ1/σC,is Bicistronic and Shares Structural Similarities with its Counterpart in Muscovy Duck Reovirus

Reoviruses have recently been shown to be associated with disease in young geese and to be involved in epizooties of severe outcome in Hungary. To assess the genetic variability among these pathogenic goose reoviruses (GRVs), we sequenced the S4 genome segment of five GRV strains isolated from different diseased flocks. We found that the GRV S4 genome segment, consisting of two partially overlapping open reading frames (ORFs), shares substantial structural similarity with its counterpart in muscovy duck reoviruses (DRVs). ORF1 is predicted to encode a polypeptide highly similar to the p10 polypeptide of DRV, and ORF2 supposedly encodes the minor outer capsid protein, σ1/σC. In one of the five GRV strains examined, we identified a single uracil base insertion close to the middle of ORF2. This insertion resulted in a frameshift and in concomitant acquisition of a termination codon (UAA) a few codons downstream, apparently causing truncation of the C-terminal part of the protein. The functional consequences of this assumed mutation, which would result in loss of more than a half of the protein, have yet to be determined. Nonetheless, the sequence and structural similarities between the genome segment encoding σl/σC in GRVs and DRVs suggest that these viruses belong to a species distinct from other established species within subgroup 2 of orthoreoviruses.     

Giant cystic pheochromocytoma located in the renal hilus

A malignant tumor in the past medical history of a patient often makes the differential diagnosis of a second tumor more difficult, especially if one of the tumors does not show its characteristic features. The authors report a case of a 55-year-old male who presented with a malignant melanoma on his left shoulder. A retroperitoneal giant cystic mass, 200 mm in diameter, was found incidentally. Adrenal origin was ruled out by imaging techniques. The absence of typical clinical symptoms made a correct preoperative diagnosis unlikely, and severe cardiovascular complications set in during surgery. Considering the characteristics of the cutaneous malignant melanoma, the metastatic origin of the giant retroperitoneal tumor was not likely either. During surgery the left kidney, with a cystic tumor located in the hilus, was removed. The postoperative pathologic diagnosis was pheochromocytoma located in the hilus of the left kidney.     

Activation of mitochondrial ATP-sensitive potassium channels prevents neuronal cell death after ischemia in neonatal rats

Activation of mitochondrial ATP-sensitive potassium channels (mK(ATP)) has been shown to protect against cell death following ischemia/reperfusion in the heart but not in brain. We examined whether mK(ATP) activation with diazoxide (DIZ) prevents neuronal cell death following hypoxia-ischemia (HI) in 7-day-old rat pups. Rat pups were subjected to HI (left carotid ligation; 8% O(2); 2.5 h), following administration of vehicle, 1.9 mg/kg DIZ, 3.8 mg/kg DIZ or DIZ plus 10 mg/kg 5-hydroxydecanoic acid (mK(ATP) antagonist). Total infarct volume was reduced from 99.8+/-2.7% in vehicle animals to 80.6+/-4.2% in 3.8 mg/kg DIZ treated animals (n=85, P<0.05). Western blotting showed K(ATP) subunits concentrated in mitochondria. Fluorescent studies indicated DIZ directly depolarized the mitochondria. In conclusion, selective opening of mK(ATP) prior to HI results in neuroprotection in immature rats.     

DETECTION OF N-MYC GENE AMPLIFICATION IN NEUROBLASTOMA BY COMPARATIVE, IN SITU,AND REAL-TIME POLYMERASE CHAIN REACTION

We have used semiquantitative comparative and real-time quantitative polymerase chain reactions (PCR) to detect n-myc gene-amplification in 20 frozen neuroblastoma biopsies and IMR 32 cell line to predict biological behavior of the tumors. Two primer pairs were used for the semiquantitative method to co-amplify a 520-bp fragment of the β-globin gene--used as a single copy reference standard--and a 258-bp fragment of the n-myc gene. After 30 cycles the PCR products were electrophoresed through an agarose gel and were compared to each other with use of a gel-densitometer. Real-time quantitative analyses were performed in a LightCycler instrument. A single primer pair was used to amplify a 120-bp fragment of the n-myc oncogene and a LC640-labeled fluorescent probe pair to detect the product. Calibration curve, set up from a serial dilution including samples with 1, 2, 10, 13, 25-fold n-myc oncogene amplification, was used for quantitative analysis. The semiquantitative method did not show distinct difference between tumor groups with no amplification and less than 10-fold amplification, whereas quantitative LightCycler analysis was able to detect even 2-fold amplification. Differentiated neuroblastomas seldom show n-myc amplification. In spite of this, we have found two partly differentiated tumor samples that contained n-myc amplification. In these cases in situ PCRs were performed to examine the tumor heterogeneity. We used biotinated ATP labeling and the same primer pair as for the LightCycler analysis. In both cases differentiated cells did not show n-myc gene amplification, whereas considerable amplification was detected in the neuroblasts.     

Cytochrome oxidase deficiency affecting the structure of the myofibre and the shape of mitochondrial cristae membrane

Cytochrome oxidase deficiency was detected in the skeletal muscle of a newborn floppy child. There was a significant decrease in the quantity of subunit 5 and 6 of cytochrome oxidase as showed in Western blot with cytochrome oxidase antibody. By contrast, the NADH: cytochrome c oxidoreductase activity was normal. Electron microscopic studies revealed serious distortion in the myofibres with broken Z-bands and disorganized fibers. The relative molecular mass of actin in the myopathic muscle was smaller than in control. The diffuse actin band in Western blot suggested a proteolytic degradation of F-actin in the myopathic muscle. There was also a serious distortion in the mitochondrial structure. Cytochrome oxidase has a direct role in the formation of cristae and mutation in its components may be directly responsible for the abnormal structure.