Danko Milošević

Genetic analysis – a diagnostic tool for primary hyperoxaluria type I

We read with great interest the article “Genetic analysis: a diagnostic tool for primary hyperoxaluria type 1 (PH1)”, published online in Pediatric Nephrology on 9 October 2002 by Danko Milosevic and colleagues, but disagree with some of the conclusions. The authors correctly point out that the diagnosis “is confirmed by the measurement of decreased alanine:glyoxylate aminotransferase activity in a liver sample”. They further state that this “enzymatic assay is not readily available to pediatric nephrologists in many parts of the world”, since “shipment of a frozen liver sample abroad is cumbersome and expensive” and propose, as an alternative, mutation analysis by sequencing of the entire coding region of the AGXT gene. We feel strongly that this approach deserves further comment. Clinical diagnosis of PH1 and its distinction from type 2 and other, as yet unclassified, forms of primary hyperoxaluria [1, 2] is difficult and, despite extensive clinical and laboratory work-up of urine metabolites (glycollate/L-glycerate) can, in some cases, be inconclusive without liver biopsy [3]. Milosevic et al. correctly recommend combined liver and kidney transplantation as treatment of the severe forms of PH1. With such a major step, all transplant centers throughout the world should demand a definite diagnosis of PH1 (or PH2) and offer the means to ship a frozen liver sample to a specialized laboratory. Genetic analysis in PH1 is a diagnostic tool that we also frequently use for family studies when definite diagnosis of an index patient has been performed by liver enzyme assay. In these cases either mutation analysis of the more frequent mutations, such as G630A, C156ins, or T853C, or linkage analysis using microsatellites is feasible [4, 5, 6]. However, screening for these common mutations has a sensitivity of less than 50% [4] and, in the majority of patients, will not lead to a definite diagnosis of PH1 due to the great number of mutations described. Exceptions to this may occur in populations with a high prevalence of single mutations, when screening for this particular mutation may lead to a high diagnostic sensitivity and specificity. Direct cycle sequencing of the entire coding region, as recommended by Milosevic et al., is by no means readily available to pediatric nephrologists in many parts of the world, and is mainly performed in centers with a scientific interest in this disease who may therefore not charge for this expensive laboratory method. It has the additional disadvantage that even this approach does not have 100% sensitivity, as illustrated by the finding of only a single mutation in family S described by Milosevic and colleagues. This suggests that mutations in the promoter region or intronic regions of the gene may be overlooked. Finally, in such a polymorphic gene, the Authors’ reply to this letter is available at http://dx.doi.org/ 10.1007/s00467-003-1108-2

Value of the urinary stone promoters/inhibitors ratios in the estimation of the risk of urolithiasis

An imbalance between urinary-promoting and -inhibiting factors has been suggested as more important in urinary stone formation than a disturbance of any single substance. To investigate the value of promoter/inhibitor ratios for estimation of the risk of urolithiasis, urinary citrate/calcium, magnesium/calcium oxalate, and oxalate/citrate x glycosaminoglycans ratios were determined in 30 children with urolithiasis, 36 children with isolated hematuria, and 15 healthy control children. The cutoff points between normal children and children with urolithiasis, accuracy, specificity, and sensitivity for each ratio were determined and compared with those of the 24-h urine calcium and oxalate excretion and urine saturation calculated with the computer program EQUIL 2. The neural network application (aiNET Artificial Neural Network, version 1.25) was used for the determination of the cutoff points for the classification of normal children and the urolithiasis group. The best test for differentiating stone formers from non-stone formers proved the aiNET determined cutoff values of oxalate/citrate x glycosaminoglycans ratio. The method showed 97.78% accuracy, 100% sensitivity, and 93.33% specificity. Two cutoff points between normal and urolithiasis groups were found showing that the children with urolithiasis had ratio values either above 34.00 or less than 10.16. Increased oxalate excretion was linked to the first cutoff value (34.00), and decreased glycosaminoglycans excretion was typical of the second cutoff value (10.16).

Determination of urine saturation with computer program EQUIL 2 as a method for estimation of the risk of urolithiasis.

To investigate the risk for the development of urolithiasis in 30 children with urolithiasis, 36 children with isolated hematuria, and 15 healthy control children, 24-h urinary excretion of calcium, sodium, oxalate, citrate, sulfate, phosphate, magnesium, urate, chloride, ammonium, and glycosaminoglycans was determined and urine saturation for calcium oxalate was calculated with the computer program EQUIL 2. Compared with controls, children with urolithiasis had significantly increased calcium excretion, oxalate excretion, and urine saturation, whereas children with isolated hematuria had significantly increased calcium excretion only. The best estimation of the relative risk of urolithiasis can be made after urine saturation, using logistic regression. The percentage of patients correctly classified after urine saturation is 85.41% in comparison with 80.95% and 73.81% when the estimation was done by calcium excretion and oxalate excretion, respectively. Using the breakpoint value of 4.29 for urine saturation, it was possible to separate children with increased risk of urolithiasis development from the group of children with isolated hematuria.

Analysis of Calcium, Oxalate, and Citrate Interaction in Idiopathic Calcium Urolithiasis in Children

The majority of urinary stones in children are composed of calcium oxalate. To investigate the interaction between urinary calcium, oxalate, and citrate as major risk factors for calcium stones formation, their 24-h urinary excretion was determined in 30 children with urolithiasis and 15 normal healthy children. The cutoff points between children with urolithiasis and healthy children, accuracy, sensitivity, and specificity for each risk factor alone as well as for all three taken together were determined. OneR and J4.8 classifiers as parts of the larger data mining software Weka, based on machine learning algorithms, were used for the determination of the cutoff points for differentiation of the children. The decision tree based on J4.8 classifier analysis of all three risk factors together proved to be the best for differentiating stone formers from normal children. In comparison to the accuracy of the differentiation after calcium and oxalate of 80% and 75.6%, respectively, the decision tree showed an accuracy of 97.8%. Even when its stability was tested by the leave-one-out cross-validation procedure, the accuracy remained at a very acceptable percentage of 93.2% correctly classified patients. J4.8 classifier analysis gave a look inside urinary calcium, oxalate, and citrate interaction. Urinary calcium excretion was shown as the most informative in discrimination of the children with urolithiasis from healthy children. However, it was shown that oxalate and citrate excretions might influence the stone formation in a subpopulation of the stone formers. In patients with low urinary calcium, a major role in lithogenesis belongs to oxalate, in some of them alone and in others in conjunction with citrate. Decreased urinary citrate excretion in the presence of increased oxalate excretion may lead to stone formation.

The value of urine citrate/calcium ratio in the estimation of risk of urolithiasis.

The urine saturation is considered as the better parameter for the estimation of risk of urolithiasis than any single urinary constituent. However, the determination of urine saturation is unsuitable for routine clinical practice. To evaluate a simpler and cheaper test than urine saturation for distinguishing stone formers from healthy individuals, urinary citrate/calcium ratio was determined in 30 children with urolithiasis, 36 children with isolated hematuria, and 15 healthy control children. The ratio was significantly lower in urolithiasis group comparing to controls, and significantly higher in hematuria than in urolithiasis group. The cut-off points between normal children and children with urolithiasis, accuracy, specificity and sensitivity were determined and compared with those of the urine saturation calculated with the computer program EQUIL 2. The data mining Weka software was used for the determination of the cut-off points. Children with urolithiasis had citrate/calcium ratio below 1.38 and urine saturation above 5.285. The citrate/calcium ratio showed in comparison to urine saturation similar high accuracy (91.11 vs. 88.89%), somewhat lesser specificity (73.33% vs. 93.33%) and much better sensitivity (100% vs. 86.89%) in discrimination of stone formers from normal children. The advantage in comparison to urine saturation is that it can be easily performed in clinical practice.

Reply to the letter from C. von Schnakenburg et al.

We read the letter of von Schnakenburg et al. with great interest and accept some of the points they raise. We feel, however, that they are hardly applicable to the situation in developing countries. Insisting on performing liver biopsy in these instances would lead to a very low rate of diagnosis regardless of the ultimate therapeutic modality chosen. This is due not only to the economic burden but primarily to the technical difficulty of performing an invasive procedure and the proper preservation and shipment of the specimen abroad.

Genotype-phenotype Correlation of β-thalassemia in Croatian Patients: A Specific hbb Gene Mutations

An analysis of genotype-phenotype correlation was performed for 14 patients with beta-thalassemia who had been registered in Referral Centre for hematology and oncology of the University Hospital Centre, Zagreb, Croatia. HBB gene mutations were determined using a gene-specific Q5 High-Fidelity PCR analysis with direct DNA sequencing of amplified transcripts. Mahidol score index used for classification of thalassemia severity was found to be low for all the patients enrolled in the study, indicating a mild &bgr;-thalassemia phenotype with no signs of disease progression. Most of the patients have already described gene mutations: IVS-II-666 C>T (HBB:c.316-185C>T) and IVS-II-16 G>C (HBB:c.315+16G>C). Each of the aforementioned mutations was found in (11/14; 78,57%) and (10/14; 71,43%) of our patients, respectively. Recently published HBB:c.9T>C mutation was found in 8 of 14 (57,14%) in our study group. IVSII-74 T>G (HBB:c.315+74T>G) is a worldwide mutation found in 6 of 14 (42.86%) of our patients. All these mutations occur among Croatian children with no obvious Indian/Near Eastern/Iranian ancestry. We also identified 7 de novo mutations (c.316-135het_dupT, c.316-133A>G, c.93-54G>A, c.316-68_316-67het_insCGG, c.316-342delA, c.316-312delT, c.316-209delT) of mild severity phenotype according to Mahidol classification score index. We did not find children or adults with thalassemia major severity phenotype.

AB0998 Effects of Rituximab Therapy in 9 Children with Refractory Autoimmune Diseases (Systemic Lupus Erythematosus and ANCA Positive Vasculitis) – Single Centre Expirience

Background There are only few reports of rituximab (RTX) effects in children with autoimmune diseases refractory to conventional therapy protocols. Objectives Authors present their work on the effects of RTX therapy in chidren with systemic lupus erythematosus (SLE) + lupus nephritis (LN) and ANCA positive vasulitis, refractory to standard therapy. Methods Retrospective chart rewiev of all patients treated with RTX at the Departement of Paediatrics, University of Zagreb School of Medicine, University Hospital Centre Zagreb, during the period 2009–2014. Results Nine children were treated with RTX: 6 with SLE + LN (3 boys, 3 girls) and 3 with ANCA positive vasulitis (3 girls). Median age of patients at the disease onset was 11.6 years (5 – 15 years). Conventional therapy included methylprednisolon, cyclophosphamide (CYC), mycophenolate mofetil, azathyoprine, in some cases plasmapheresis. In all cases RTX was introduced due to ineffectivness of conventional therapy in achiving the remission of autoimune disease. Median time between the beginnig of conventional therapy and introduction of RTX was 10.3 months (1 – 48 months). In 8 cases RTX (750 mg/m2, two doses, two weeks apart) was combined with mini pulses of CYC (350 mg/m2), in 1 case RTX was applied in dose of 375 mg/m2 per week, for 4 weeks. After RTX introduction complete, prolonged remission was achived in 6 children. In 2 patients relapses of disease required repeating of RTX therapy. One patient (girl (5 yrs) with ANCA possitive vasculitis) died due to initially severe presentation at disease onset and fulminant, progressive multiorgan failure despite all conventional therapy and early introduction of RTX. Conclusions Complete or partial remission after RTX introduction was achived in 8/9 (88,8%) of our patients with severe autoimmune diseases. To our expirience, RTX represents powerful tool in controlling SLE + LN and ANCA positive vasculitis refractory to conventional therapy protocols. References Yolanda Braun-Moscovici, Yonatan Butbul-Aviel, Ludmila Guralnik et.al. Rituximab: rescue therapy in life-threatening complications of refractory autoimmmune diseases: a single centre expirience. Rheumatol Int (2013) 33:1495-1504. RJ Davies, SR Sangle, NP Jordan et.al. Rituximab in the treatment of resistant lupus nephritis: therapy failure in rapidly progressive crescent lupus nephritis. Lupus (2013) 22, 574-582. D Duxbury, C. Combescure, C Chizzolini. Rituximab in systemic lupus erythematosus: an updated systemic review and meta-analysis. Lupus (2013) 22, 1489-1503. Marc Weidenbusch, Christoph Rommele, Angelika Schrottle, Hnas-Joachim Anders. Beyond the LUNAR trial. Efficacy of rituximab in refractory lupus nephritis. Nephrol Dial Transplant (2013) 28: 106-111. Maria Trachana, Artemis Koutsonikoli et.al. Safety and efficacy of Rituximab in refractory pediatric systemic lupus erythematosus nephritis: a single centre expirience of Northern Greece. Rheumatol Int (2013) 33:809-813. Disclosure of Interest None declared

Urolitijaza - potencijalna komplikacija terapije ceftriaksonom

Svrha studije je bila procijeniti rizik nastanka urolitijaze u djece lijecene ceftriaksonom. Retrospektivno je analizirano 238-ero djece s cistopijelonefritisom, lijecene u 2011. godini, ceftriaksonom u standardnoj dozi od 60 mg/kg/dan kroz 10 dana. Slikovnom obradom u 74-ero djece nađene su anomalije mokracnog sustava, od cega u njih 66-ero vezikoureteralni refluks, a u 7- ero hidronefroza. Ni u jednog djeteta nije nađena urolitijaza. Klinickim pracenjem kroz mjesec dana nije bilo klinickih i/ili laboratorijskih indirektnih znakova urolitijaze. Može se zakljuciti da je lijecenje ceftriaksonom u standardnoj dozi i dužini trajanja sigurno. Oprez je nužan ako su potrebne vise doze i/ili produženo davanje. U tom slucaju preporucujemo pregled mokracnog sustava i žucnjaka, probir na hiperkalciuriju i alkalinizaciju urina.

Vesicoureteral reflux and urodynamic dysfunction.

Introduction: The concept of vesicoureteral reflux (VUR) as a consequence of congenital anomaly of vesicoureteral junction has undergone changes owing to the finding that such children may have lower urinary tract dysfunction, which produces high intravesical pressure and consequently a predisposition for VUR. Patients and Methods: The urodynamics was investigated by pressure-flow-EMG study in 132 children with VUR and 162 refluxing units. Results: Only 33 (25.0%) patients had normal urodynamic finding. The most frequent pathological finding was overactive bladder (OAB), found in 59 (44.7%) children, followed by dysfunctional voiding (DV) in 25 (18.9%) children. Children with VUR grades I and II had a higher percentage of pathological urodynamic findings than children with VUR grades III and IV. OAB was more frequent in children under 5 years of age with unilateral and lower grade VUR. It was found equally in children with and without uroinfections. DV was more frequent in children older than 5 years, with bilateral VUR, higher grade VUR and uroinfections. Conclusions: Children with VUR have a high incidence of urodynamic disorders. The results of the study indicate the possible role of urodynamic dysfunction in the pathogenesis of VUR, especially mild one.