Increased prevalence of factor V Leiden mutation in premature but not in full-term infants with grade I intracranial haemorrhage.

OBJECTIVES: In the current prospective study our aim was to analyse the distribution of the factor V Leiden (G1691A) mutation in preterm and full-term neonates with grade I intraventricular haemorrhage and in control neonates. STUDY METHOD: A group of 125 individually selected neonates with grade I intraventricular haemorrhage and 128 controls were investigated. RESULTS: The allele frequency was 7.2% in the total population of affected infants while it was 3.9% in the controls (p < 0.05); the latter corresponds to an average European allele frequency in healthy populations. When the infants were grouped as premature (<2,500 g and < or =36 weeks of gestational age) and appropriate for gestational age full-term infants the statistical analysis revealed an increased prevalence of the mutation in the premature group (10% allele frequency vs. 4.8% in the controls, p < 0.05), and a normal prevalence in the mature group (4.6 vs. 3.1%, respectively); therefore, the overall increase was due to the increase of incidence rate in preterm neonates. CONCLUSIONS: These data confirm our previous results and suggest that as the preterm and term infants differ from each other in haemorrhage susceptibility in many clinical particulars, carrying of the mutation has probably also a different impact in premature and in full-term infants with respect to the intraventricular haemorrhage.

High prevalence of factor V Leiden mutation in mothers of premature neonates.

The prevalence of the Leiden mutation was tested in 50 mothers of premature infants and in 56 mothers of intrauterine-growth-retarded neonates. The prevalence of the Leiden mutation was 7.2% in the mothers of growth-retarded neonates and 18% in the group of mothers of premature infants, the latter being significantly higher than the 6.3% prevalence of this mutation in healthy Hungarian subjects (p < 0.01). In spite of the relatively small number of mothers examined, the unexpected finding may call attention to a hitherto unknown relationship.

[Incidence of factor V G1681A (Leiden) mutation in samplings from the Hungarian population]. / A faktor V G1691A (Leiden) mutáció gyakorisága magyar népességmintákban.

Thromboembolic disorders affect 0.1% of the adult population. Two main groups of the underlying predisposition factors can be identified: environmental factors (e.g. dietary habits, physical activity, surgical interventions, pregnancy etc.) and several genetic predispositions (e.g. inherited anticoagulant defects). After the discovery of the genetic mutation of factor V, called Leiden mutation, it turned out, that this mutation is responsible for the development of resistance to activated protein C in majority of the cases. The importance of the Leiden mutation is further emphasised by population based investigations, which makes it the most frequent thrombosis risk factor known today. In our study we have identified 43 heterozygotes and 3 homozygotes with Leiden mutation in total of 665 samples. The 6.47% heterozigocy is in the range of earlier reports from Europe. The homozygote/heterozygote distribution deviated from the value predicted by the Hardy-Weinberg law.

[Delta-F508 screening of infants at the Perinatal Intensive Care Center of the city if Pécs]. / Delta-F508 szúrés a pécsi Perinatalis Intenzív Centrumban ápolt újszülöttekben.

Cystic fibrosis (CF) is one of the most frequent (1:2500), potentially lethal autosomal recessive diseases among Caucasians. Molecular genetic examination has become the most valuable method used for diagnosis or population screening. 300 newborns treated in the Perinatal Intensive Care Unit were examined for the mutation delta F508. The results showed that the frequency of affected deltaF508 homozygotes was 1:100, which is significantly higher than found in the general population, but the frequency of carriers (1:33) is similar to the overall value.

[Kabuki syndrome]. / Kabuki-syndroma.

Kabuki syndrome is characterised by a peculiar face resembling the make-up of actors in Kabuki, the traditional Japanese theatre, postnatal growth deficiency, mild to moderate mental retardation, unusual dermatoglyphic patterns, and various skeletal and visceral anomalies. The author would like to draw attention to this less known condition in Hungary by a case-report of a 23 months old female patient with Kabuki syndrome.

Overexpression of human methylmalonyl CoA mutase in mice after in vivo gene transfer with asialoglycoprotein/polylysine/DNA complexes.

Methylmalonic acidemia resulting from genetic deficiency of methylmalonyl CoA mutase (MCM) is an often fatal metabolic disease. Somatic gene therapy for this disorder may require gene replacement in the liver. We describe overexpression of MCM in the liver of mice after in vivo gene delivery using asialoglycoprotein/polylysine/DNA (ASO/PL/DNA) targeted delivery to the liver of plasmids expressing recombinant MCM. After intravenous administration of the ASO/PL/DNA complex, the vector sequences are cleared from the blood with t1/2 = 2.5 min and > 95% of the vector is taken up by the liver. Vector sequences are cleared from the liver with t1/2 = 1.0-1.3 hr. MCM enzyme activity in the liver increases to levels 30-40% over baseline 6-24 hr after injection. No acute or chronic toxicity was observed. This net level of expression is likely to be therapeutic for MCM if the complex could be administered repetitively to treat acute episodes of life-threatening acidosis or establish a steady-state level of MCM activity. Repetitive administration of the ASO/PL/DNA complexes in mice was associated with formation of antibodies against asialo-orosomucoid and the asialo-orosomucoid complex but not against DNA.

Cloning of functional alpha propionyl CoA carboxylase and correction of enzyme deficiency in pccA fibroblasts.

Propionyl CoA carboxylase (PPC) is a heteromeric enzyme composed of alpha subunits (PCCA) and beta (PCCB) subunits. We describe cDNA clones expressing human PCCA and complementation of the genetic defect in pccA fibroblasts by DNA-mediated gene transfer. Two cDNA clones were constructed. The first corresponds to the previously reported, putatively full-length, open reading frame. The second encodes a chimera composed of the mitochondrial leader sequence of human methylmalonyl CoA mutase and the mature PCCA protein. Both clones reconstitute propionate flux to normal levels in fibroblasts from patients genetically deficient in PCCA (pccA). The maximal level of propionate flux approached, but never exceeded, the levels seen in control plates of normal cells. In contrast, the maximal level of PPC holoenzyme activity reached only 10%-20% that of normal controls, which corresponded roughly to the fraction of cells actually transformed with the recombinant gene. These data suggest that the level of PCCA expression in fibroblasts does not normally limit PCC holoenzyme activity or propionate flux. The fact that a small fraction of cells reconstitutes propionate flux to normal levels suggests that metabolic cooperation between cells is capable of increasing the metabolic capacity of recombinant enzyme in a subpopulation of cells. These factors may have important implications for the rational design of somatic gene therapy for PCCA deficiency.

Propionate metabolism in cultured human cells after overexpression of recombinant methylmalonyl CoA mutase: implications for somatic gene therapy.

Strategies for somatic gene therapy must consider the metabolic consequences of expressing the recombinant gene product in addition to methods for gene transfer and expression. We describe studies of propionate metabolism in cultured cells transfected with methylmalonyl CoA mutase (MCM), the enzyme deficient in mut methylmalonic acidemia. Transfection of MCM into mut fibroblasts restores propionate metabolism to normal levels in a dose-dependent manner. Overexpression of MCM, or the addition of excess propionate, carnitine, or cobalamin, does not increase propionate metabolism in normal human fibroblasts, lymphoblasts, or hepatoma cells, although hepatic cells exhibit > 10-fold higher levels of propionate metabolism. Significantly, the restoration of propionate metabolism in mut fibroblasts is disproportionately greater than the efficiency of transfection, suggesting the presence of a cooperative phenomenon between cells. Intercellular participation in propionate metabolism is evident in cocultures of MCM-deficient and propionyl CoA carboxylase-deficient cells. We conclude that the liver is the preferred target for gene therapy of MCM deficiency because of its greater capacity for propionate metabolism and that cooperation between cells could enhance the biological effect of a subpopulation of cells transformed with recombinant MCM.

A highly sensitive one-step method for silver intensification of the nickel-diaminobenzidine endproduct of peroxidase reaction.

We have developed a new technique which makes silver intensification of the oxidatively polymerized diaminobenzidine (DAB), the endproduct of peroxidase reaction, less laborious without any loss in selectivity or sensitivity. The new technique is based on two strategies: (a) increasing the argyrophilia of the DAB by modifying its polymerization with Ni ions, and (b) decreasing tissue argyrophila by using a mildly acidic physical developer instead of the alkaline one previously presented. Because the nickel modification takes place in the DAB substrate solution, i.e., in the final step of the peroxidase reaction, only one additional step, the physical development, must be carried out if intensification is needed.

Oxidation catalyzed by H+ ions improves the silver intensification of 3,3'-diaminobenzidine staining by strongly suppressing tissue argyrophilia.

We found that a 15-min treatment of tissue sections with 2 M/l H2O2 dissolved in 6 M/l sulphuric acid strongly suppresses the argyrophilia of tissue elements, thus facilitating selective silver intensification in histochemical procedures. When applied inserted between the 3,3'-diaminobenzidine reaction and physical development, this treatment allows the selective and sensitive visualization of very low levels of peroxidase activity.