Jana Lukac-Bajalo

Mutations in galactose-1-phosphate uridyltransferase gene in patients with idiopathic presenile cataract

Summary: Impaired activity of the enzyme galactose-1-phosphate uridyltransferase (GALT) has been proposed as a risk factor for idiopathic presenile cataract. A study was undertaken to determine the prevalence of the three most common mutations in the GALT gene (Q188R, K285N and N314D, including its variant Duarte-2) in a group of Slovenian patients with idiopathic presenile cataract. GALT activity was determined in the erythrocytes of 30 cataract patients. DNA was isolated from their blood and analysed for Q188R, K285N and N314D mutations and IVS5−24G>A intronic variation by means of polymerase chain reaction and digestion with restriction enzymes. The average GALT activity of the cataract group was 19.5±4.9 U/g Hb, which is lower than the normal range (p = 0.034). Frequencies of Q188R, K285N, N314D and Duarte-2 alleles in the cataract group were 0.00%, 5.0%, 11.7% and 3.3%, respectively. Only the frequency of the K285N mutation was significantly higher in the patient group than in the control group (p = 0.0244). Our results support the reported association of decreased GALT activity with idiopathic presenile cataract. Molecular analysis indicates that, in the Slovenian population, this association is linked to the K285N mutation, which is neonatally benign in heterozygotes.

Altered distribution of urinary glycosaminoglycans in diabetic subjects

Abstract. We studied the influence of type 1 and type 2 diabetes mellitus with similar duration on the urinary excretion of total glycosaminoglycans and alteration of urinary glycosaminoglycan distribution pattern. Investigations were performed in the 24-hour urine samples of 31 children with type 1 diabetes mellitus, 36 adults with type 2 diabetes mellitus and in 30 age-matched controls for each group. We found that type 2 diabetes mellitus also induced an increased urinary excretion of total glycosaminoglycans and that both type 1 and type 2 diabetes alter the urinary distribution of heparan sulphate and dermatan sulphate. Observed changes correlate with duration of the disease. Microalbuminuria was detected in 9 of 36 type 2 adult diabetics (25%). The microalbuminic group had a significanlty higher heparan sulphate and/or dermatan sulphate excretion rate. To clarify whether an altered urinary distribution of heparan sulphate and dermatan sulphate may precede the development of microalbuminuria, it is necessary to performed a prospective study in which urinary glycosaminoglycans and microalbuminuria are measured year by year starting from the diagnosis of diabetes.

Cell-free DNA in the peritoneal effluent of peritoneal dialysis solutions.

The beneficial effects of novel peritoneal dialysis solutions low in glucose degradation products regarding peritoneal cell apoptosis and necrosis are well established in vitro, however in vivo data is lacking. Cell‐free DNA quantification is a possible method to determine cell damage through apoptosis and necrosis in vivo. We performed a prospective, cross‐over study on 26 stable continuous ambulatory peritoneal dialysis (CAPD) patients, treating each patient for 3 months in a randomized order with a conventional, lactate‐buffered, acidic solution (solution D) and a novel, bicarbonate/lactate‐buffered neutral solution (solution P). The timed overnight peritoneal effluent was sampled for cell‐free DNA quantification using a fluorometric assay. The effluent samples of eighteen patients were finally available for DNA quantification. The concentration range of cell‐free DNA in the peritoneal effluents was 1.8–9.5 µg/L. The coefficient of intrapatient variation in overnight effluent cell‐free DNA appearance was 15.6 ± 12.4%. Cell‐free DNA peritoneal appearance using solutions D and P was 14.9 ± 6.8 µg and 11.8 ± 3.4 µg, respectively (P = 0.02), with the average difference of 3.1 µg (95% CI, 0.7–5.6 µg). Our results show that cell‐free DNA is present in the overnight peritoneal effluent of stable CAPD patients. A significant decrease in the cell‐free DNA appearance with solution P was found; however, before accepting this as an indicator of a more biocompatible profile causing less peritoneal membrane cell necrosis and apoptosis, confirmatory data on larger patient samples are needed. Our results indicate the potential future role of cell‐free DNA in the diagnosis and prognosis of therapy‐related peritoneal membrane degeneration.

Frequencies of Q188R and N314D mutations and IVS5-24g>A intron variation in the galactose-1-phosphate uridyl transferase (GALT) gene in the Slovenian population.

Abstract Numerous mutations in the galactose-1-phosphate uridyl transferase (GALT) gene have been found to impair GALT activity to different extent, causing galactosemia. This disorder exhibits considerable allelic heterogeneity in different populations and ethnic groups. The Q188R mutation accounts for 60–70% of classical galactosemia alleles in the Caucasian population. Individuals homoallelic for Q188R have a severe phenotype with complete loss of enzyme activity. Another form of GALT deficiency is Duarte galactosemia with N314D mutation associated alleles (Duarte-2). Although heterozygotes for classical galactosemia are asymptomatic at birth and Duarte galactosemia appears to be quite benign, there are some indications that these disorders can increase the risk of developing certain diseases later in life. The aim of our study was to analyze a healthy Slovenian population for the frequencies of Q188R and N314D mutations, and for the Duarte-2 indicative intronic variation IVS5-24G>A. DNA samples from 174 healthy subjects were analyzed for all three mutations by polymerase chain reaction and digestion with restriction enzymes. Allele frequencies for Q188R and N314D mutations and IVS5-24G>A intron variation were found to be 0.29%, 8.0% and 5.7%, respectively. These results correlate well with those reported for most other healthy Caucasian populations.

Relationship between the sialic acid content of low-density lipoprotein (LDL) and autoantibodies to oxidized LDL in the plasma of healthy subjects and patients with atherosclerosis.

Abstract To determine whether the sialic acid (SA) content of the low-density lipoprotein (LDL) is related to the plasma concentration of autoantibodies to oxidized LDL (oxLDL), we measured the SA content of LDL and the concentrations of oxLDL and autoantibodies to oxLDL in plasma of 20 apparently healthy subjects and 20 patients with advanced coronary atherosclerosis. In the healthy subjects the SA content of LDL correlated positively with plasma concentration of autoantibodies to oxLDL. In agreement with the literature the decreased SA content of LDL was associated with an increased fraction of oxLDL; a decreased fraction of oxLDL was associated with an increased plasma concentration of autoantibodies to oxLDL. In the patients the SA content of LDL and plasma concentrations of oxLDL and autoantibodies to oxLDL were not related. We conclude that the SA content of LDL correlates positively with plasma concentration of autoantibodies to oxLDL in healthy subjects. However, this association may vary depending on the stage of atherogenesis. Although our results suggest dependence of LDL SA content on the clearance of oxidatively modified (desialylated and oxidized) LDL from blood by autoantibodies to oxLDL, the mechanisms regulating the SA content of LDL await further studies.

Optimization of Purification of Human Cell‐Free mRNA from Plasma

Cell‐free RNA has a potential for diagnosis and prognosis of many diseases. Our aim was to optimize a commercially available QIAamp UltraSens Virus Kit (Qiagen, Hilden, Germany) for isolation of mRNA from human plasma. The amount of carrier RNA to bind plasma mRNA, the centrifugal force to pellet the mRNA–carrier RNA complex, the incubation time for proteolysis, and the centrifugal force to bind mRNA to the silica gel membrane were modified in order to maximize the yield of isolated mRNA. The isolated cell‐free mRNA was detected for cycA using TaqMan real‐time quantitative RT‐PCR. The lowest threshold cycle (Ct) was obtained with the use of 4.0 μL of carrier RNA. Pelleting with a centrifugal force of 1500 ×g yielded the lowest Ct, but caused difficulties in resuspending the pellet. Therefore, we suggest using a lower centrifugal force of 1300 ×g. The duration of proteolysis had no effect on Ct. To bind mRNA to the silica gel membrane, a centrifugal force of 5000 ×g is recommended. Our results show that the UltraSens Virus Kit is an appropriate choice for isolating mRNA from human plasma, with imprecision expressed as coefficient of variation below 2%.

Synergistic effect of high lactase activity genotype and galactose-1-phosphate uridyl transferase (GALT) mutations on idiopathic presenile cataract formation

OBJECTIVES To evaluate the possible synergistic role of partial galactose metabolism defects, high lactase (LPH) genotype and lactose and galactose ingestion in presenile cataract formation. DESIGN AND METHODS 51 patients with idiopathic presenile cataracts and 172 healthy cataract-free subjects were genotyped to determine their galactose-1-phosphate uridyl transferase (GALT) and LPH status. Whole milk, skimmed milk and yoghurt consumption was recorded in 19 cataract patients and 172 controls by questionnaire. RESULTS GALT mutations and whole milk consumption increased the risk of cataract formation in high LPH genotype group, but not in lactose intolerant subjects. Logistic regression analysis showed the synergistic effect of GALT and LPH mutations on cataract formation. CONCLUSIONS High lactase activity genotypes and mutations in galactose-1-phosphate uridyl transferase have a synergistic effect on presenile cataract formation.