20^th Meeting of European Society of Pediatric Clinical Research (ES-PCR, previously known as Pediatric Research of Central European Countries)

Brno, 1. – 2. 7. 2011   

International Scientific Advisory Board

András Arató (Hungary)

Nataša Marčun Varda (Slovenia)

Milan Bayer (Czech Republic)

Jan Janda (Czech Republic)

László Kovács (Slovakia)

Ingrid Brucknerová (Slovakia)

Susane Greber-Platzer (Austria)

Radvan Urbanek (Germany)

Gábor Kovács (Hungary) 

Local Organizing Committee

Hana Hrstkova

Daniela Kodytkova

Jaroslav Sterba 

Antibodies against complement factor H in aHUS: 1 year follow up data from the international Innsbruck HUS NET aHUS registry 

Hofer J¹, Riedl M¹, Giner T¹, Rosales A¹,   Jeller V¹, Würzner R², Jungraithemayr T¹

¹Department für Pädiatrie I, Medizinische Universität Innsbruck, Austria

²Department für Hygiene, Mikrobiologie und Sozialmedizin, Medizinische Universität Innsbruck, Austria 

Introduction: Antibodies against complement factor H (FH Ab) have been reported in aHUS patients. The role of FH Ab in disease onset, progression and treatment is of critical interest for physicians and patients dealing with this unsolved problem. At present, evidence based therapy recommendations are missing.

Methods: We comment on 16 patients with FH Ab associated aHUS from the Innsbruck HUS-Net registry (www.hus-online.at). Patients were followed from the beginning of the acute phase, with recording on patient’s therapy and clinical progression over a period of 1 year.

Results: Patients show a median age at disease onset of 7 years. All patients presented with hemolytic anemia (mean hemoglobin: 5,8 g/l), thrombocytopenia (mean platelet count: 33,2x10⁹/µl) and elevated creatinine levels (mean: 458 µmol/l). Only 37% of the patients showed decreased C3 levels and 15% showed decreased Factor H levels. Within the follow up period of 1 year 25% of the patients developed renal insufficiency, 33% showed ESRD, and 67% showed at least one disease recurrence. Using supportive therapy without plasmatherapy or immunosuppression 2/2 patients showed disease recurrence, 6/7 patients recurred under plasmatherapy without additional immunosuppression and only 2/7 patients with plasmatherapy followed by immunosuppression developed recurrences.

Conclusion: CFH Ab positivity is a distinct pathogenetic aHUS subgroup mainly of pediatric patients. Testing for CFH Ab as soon as possible is mandatory, as in positive cases this has important impact on prognosis and the recommended therapy. Following our results and the literature a recommendation for the use of plasmatherapy as induction therapy followed by a maintenance therapy using immunosuppressive agents can be given. Nevertheless, treatment responses are heterogeneous and the different alternative immunosuppressive agents, the used dosages and the timing of initiation and withdrawal are still a matter of speculation. 

Proteomic analysis of renal ischaemia/reperfusion injury: search for new targets

Sziksz E^1,2, Himer L^1,2, Kékesi KA³,  Juhász GD³, Simor A³, Gulyássy P³, Darula Z⁴, Szebeni B¹, Reusz G², Szabó A⁵, Tulassay T^1,3, Vannay A^1,2

¹Research Group for Pediatrics and Nephrology, Semmelweis University and Hungarian Academy of Sciences, Budapest, Hungary

²First Department of Pediatrics, Semmelweis University, Budapest, Hungary

³Laboratory of Proteomics, Eötvös Loránd University, Budapest, Hungary

⁴HAS Biological Research Center, Institute of Biochemistry, Szeged, Hungary

 ⁵Second Department of Pediatrics, Semmelweis University, Budapest, Hungary 

Background: Ischaemia/reperfusion (I/R) injury is a leading cause of acute kidney failure (ARF), which is observed most frequently in patients after major surgery, burns, severe hypovolemia, and renal transplantation. Mortality rate and treatment of ARF means a serious problem in the intensive health care. Ischaemic preconditioning (IP) may induce tissue adaptation to stress and protect it from a subsequent severe I/R insult.

Aim: Because the complex molecular pathomechanism of renal I/R injury is not fully understood, our present aim is to analyse I/R and IP induced protein changes in the kidney to find potential key molecules and therapeutic targets.

Methods: Kidneys of ischemized, preconditioned/ischemized and control rats were analyzed using two dimensional gel electrophoresis and mass spectrometry. Functional protein analysis was performed by Pathway Studio software.

Results: 108 proteins were altered after insult. They were ranked into functional groups: components of cytoskeleton, elements of different metabolism, proteolysis, DNA/RNA processing, signaling and miscellaneous. All proteins were manually validated and searched in the literature. Some protein of interest were choosen for further experiments.

Conclusion: Here we investigate the alteration in the complex interaction of different proteins of kidney during ARF. Our results may help the identification of new biomarkers of I/R injury leading to kidney damage. Determination of the protective IP induced proteomic changes will help us to find new targets for therapeutic intervention.     

Characterisation of uropathogenic escherichia coli from children with urinary tract infection in different countries

Jankó V, Ramosa NL, Ngoc Dzung DT, Stopsack K, Pourshafie MR, Katouli M, Kovács L, Brauner A

Faculty of Science, Health and Education, University of the Sunshine Coast, Maroochydore, Queensland, Australia

Department of Microbiology, Tumour and Cell Biology, Division of Clinical Microbiology, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden

Department of Biochemistry, Hanoi Medical University and Hanoi Medical University Hospital, Hanoi, Vietnam

2nd Department of Pediatrics, Comenius University Medical School, University Children´s Hospital, Bratislava, Slovakia

Department of Microbiology, Pasteur Institute of Iran, Tehran, Iran 

Purpose: Uropathogenic Escherichia coli (UPEC) carry many virulence factors, including those involved in long-term survival in the urinary tract. However, their prevalence and role among UPEC causing urinary tract infection (UTI) in children is not well studied. To further understand the virulence characteristics of these bacteria, we investigated the prevalence of antibiotic resistance, antigen 43 genes, curli and cellulose among UPEC from children from different countries.

Methods: Isolates (n=337) from 5 countries were tested for antibiotic susceptibility, phylogenetic groups, prevalence of flu, fluACFT073, fluBCFT073, curli and cellulose.

Results: High prevalence of multidrug resistance and extended spectrum beta lactamase production was found among Iranian and Vietnamese isolates. Resistance was associated with phylogenetic group D while group B2 was associated with fluACFT073 and fluBCFT073. Fewer Iranian isolates carried fluACFT073, curli and cellulose. fluBCFT073 was most prevalent among Slovak isolates.

Ampicillin and amoxicillin/clavulanic acid resistance was prevalent among fluACFT073- and fluBCFT073-positive Australian, Iranian and Swedish isolates. Lack of curli and cellulose was associated with resistance among Vietnamese isolates.

Conclusions: We conclude that major differences exist in the prevalence of antibiotic resistence among UPEC from different countries. Associations observed between resistance and virulence factors may, in different ways, promote the long-term survival of UPEC in the urinary tract.

Role of IL-17 in the pathomechanism of renal fibrosis

Szebeni B¹, Himer L¹, Sziksz E¹, Saijo S², Kis E³, Prókai Á³, Bánki NF³, Kardos M⁴, Degrell R⁵, Fekete A³, Rusai K³, Szabó AJ³, Szabó A⁶, Reusz G², Tulassay T^1,3, Vannay Á^1,3

 ¹Research Laboratory of Paediatrics and Nephrology, Hungarian Academy of Sciences, Budapest, Hungary

 ²Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, Japan

³First Department of Paediatrics, Semmelweis University, Budapest, Hungary

 ⁴Second Department of Pathology Semmelweis University, Budapest, Hungary

⁵Second Department of Medicine and Nephrological Center, University of Pécs, Hungary

⁶Second Department of Paediatrics, Semmelweis University, Budapest, Hungary 

Background: Renal fibrosis is a typical example of organ fibrosis leading to end stage kidney disease and increased morbidity and mortality. Regardless of the initiating cause, the mechanism of organ fibrosis is similar in the different chronic renal failures and always have a major inflammatory component. Recently, a new Th cell lineage, termed Th17 cells, has been identified based on their capacity to produce interleukin (IL)-17A but not the classic Th1 or Th2 cytokines. IL-17A recruits neutrophils and macrophages and stimulates the production of pro-inflammatory cytokines. Interestingly, less attention has been paid to the impact of Th17 cells on non immune cells such as epithelial cells, although IL-17A receptors (IL-17RA) are intensively expressed in the kidney, especially on the tubular epithelial cells.

Aims: Our present work investigates the role of IL-17A on renal tubular epithelial cells and renal fibrosis.

Methods: We evaluated the renal level and localization of IL-17A and IL-17RA in a mouse model of ureteric obstruction (UUO). For this purpose real-time PCR, immunhistochemical staining and flow cytometry were used. The role of IL-17A on epithelial to mesenchymal transition (EMT) and on activated signaling pathways was tested in vitro using HK-2 renal tubular epithelial cells by flow cytometry. The impact of the selective modulation of IL-17A on fibrosis was studied in vivo by Western blot determination of αSMA levels after UUO using IL-17A knock-out and wild-type animals.

Results: The number of IL-17A producing T-cells and IL17RA-positive epithelial cells elevated 5 days after UUO. After IL-17A treatment of HK-2 cells we found increased phosphorylation of Erk1/2, Jnk1/2, Smad2/3 signaling pathways. At the same time, we found increased number of αSMA - positive HK-2 cells. Finally, after UUO the level of αSMA was less increased in the kidney of IL-17KO mice compared to that of control mice.

Conclusion: Our work contributes to the better understanding of the role of IL-17A in renal fibrosis. Our results show that IL-17A induces EMT through the activation of Erk1/2, Jnk1/2, Smad2/3 signaling pathways in vitro. IL-17A alters renal fibrosis in vivo as well. However, further works are needed to elucidate its exact effects on renal injury.   

„In vitro“ effectiveness of probiotics on E. Coli in comparison with commonly used antibiotics in treatment of urinary tract infections in children

Meštrović Popovič K¹, Cencič A^2,3,  Ander MT², Eneko M², Gorenjak M³, Gradišnik L³, Marčun Varda N¹

¹University Medical Centre Maribor, Department of Paediatrics, Maribor, Slovenia

²University of Maribor, Faculty of Agriculture and Life Sciences, Maribor, Slovenia

³ University of Maribor, Faculty of Medicine, Maribor, Slovenia 

The aim of the study: Management of recurrent urinary tract infections (UTI) with antibiotic prophylaxis is well defined. Because of rising resistance of bacteria to antibiotics, questionable cost/effectiveness and parental dissaproval of long term antibiotic prophylaxis, better options are looked for. One of the expanding areas of research is possible preventive role of probiotics. The aim of our study was to determine the inhibitory effect of some probiotics vs. antibiotics on E. coli in preclinical study. Probiotic with the best inhibitory results will be used in future clinical trial.

Methods: Clinical isolates of E. coli and a control E. coli strain (ATCC 11105) were tested for inhibition by probiotic strains and some antibiotics using well diffusion method. We used L. plantarum strains (PCS20, PCS22, PCS25, PCS26) from Slovenian local cheese and 3 commercial  probiotics (Probio Junior, Prolife and BioGaia) in comparison to the widely used L. rhamnosus LGG strain (“the golden standard”). We also tested gentamycin (40 mg/ml) and cefuroxime (100 mg/ml), two commonly used antibiotics in treatment of UTI.

Results: We found out that all probiotics have lower activity against E. coli (approximately 30% less) than both antibiotics, cefuroxime and gentamycin. Most of the tested probiotic strains exert resembling activity against uropathogenic E. coli. Prolife in yeast potato dextrose broth, encouraging growth of yeasts, in this case Saccharomyces cerevisae, produced significant inhibition of growth of most E. coli strains, especially the ATCC control strain, and isolate 6497. Such activity was comparable to that of gentamycin, but significantly lower than that of cefuroxime.

Conclusion: Antimicrobial effects of some probiotics on E.coli clinical strains show promissing results for possible prevention of UTI. Although Saccharomyces cerevisae showed very good results, we believe that yeasts as possible opportunistic pathogens should not be used  in clinical practice. However, further validation is necessary.   

NPHP1 homozygous deletion rate in Hungarian children with juvenile nephronophthisis

Kerti A¹, Antalfi R¹, Balogh E¹, Bernáth M¹, Moriniere V², Szabó A¹, Sallay P¹, Wagner L³, Reusz G¹, Saunier S², Antignac C², Tory K¹

¹Ist Department of Pediatrics, Semmelweis University, Budapest, Hungary

²INSERM U983, Necker Hospital, University Paris Descartes, Paris, France

³Department of Transplantation and Surgery, Semmelweis University, Budapest, Hungary 

Juvenile nephronophthisis (NPH) is responsible for 6-10% of ESRD in childhood. The homozygous deletion of the first nephronophthisis gene (NPHP1) is responsible for 22-45% of NPH cases, but has not yet been screened in Hungary.

Within the cohort of 67 Hungarian children treated at the I^st Department of Pediatrics, Semmelweis University with cystic kidney disease or chronic renal failure without hematuria, proteinuria or urinary tract malformation, 22 patients have been diagnosed as NPH based on clinical symptoms, recessive inheritance and renal morphology. Five of these patients presented with Joubert syndrome and 1 patient with Senior-Loken syndrome (SLS).

The 22 patients with NPH have been screened for the NPHP1 homozygous deletion by the amplification of exons 7 and 19, and introns 2 and 18. An extragenic region was amplified as a control.

Eight of 22 patients with NPH (36%) have been found to carry NPHP1 homozygous deletion. They have all developed ESRD at the median age of 14 years (range: 13-18). Out of them, 4 patients presented with isolated NPH, 2 patients with Joubert syndrome, 1 with SLS and 1 with NPH and sensorineural hearing impairment. The clinical phenotype was particular in three cases. The retinopathy of the patient with SLS is more severe than expected in patients with NPHP1 mutations; his visual acuity at the age of 29 years is lost one one side and 0,25 on the other. One patient with Joubert syndrome was treated with autism, which is not exceptional in Joubert syndrome, but has not been reported in patients carrying NPHP1 mutations. Finally, one patient was treated with sensorineural hearing impairment which is rarely associated to NPH.

The rate of NPHP1 homozygous deletion is similar in Hungarian children to that found in world-wide cohorts. The associated extra-renal symptoms can be more extreme than previously found. 

Rapamycin sensitivity and mTOR activity in lymphoma/leukemia cells

Váradi Zs^1,2, Sebestyén A², Nemes K^1,2, Márk Á², Hajdú M², Kovács G¹, Kopper L², Csóka M¹

¹2nd Dept. of Pediatrics, Semmelweis University, Budapest, Hungary

 ²1st Dept. of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary 

Acute lymphoblastic leukemia (ALL) is the most common malignancy diagnosed in children, representing nearly one third of all pediatric cancers. Recently, the mammalian target of rapamycin (mTOR) has gained more attention as a potential target in different tumors including hematological malignancies.

The serine-threonine kinase mTOR is a central mediator of several signals, regulates cell growth, protein translation, cellular proliferation and survival. mTOR can form two distinct complexes termed mTORC1 and mTORC2 with different rapamycin sensitivity. Molecular biological studies suggest that mTOR inhibition may be a useful therapeutic strategy in different human malignancies. However, there are limited data about the activity of mTOR in different lymphomas, specially in pediatric lymphomas/leukemias.

In the present work we studied the expression of mTOR activity in different lymphoma/leukemia cell lines, in bone marrow/blood samples from pediatric ALL patients and in isolated normal PMNC-, B- and T-cells. The expression of mTOR activity dependent proteins (p-4EBP1, p- mTOR, p-S6, p-p70S6K) was examined. We statistically analyzed the expression results in clinical samples during the therapy and at the possible relapses based on minimum 2 years follow up. The effect of rapamycin treatment and the expression of mTOR complexes were also studied by flow cytometry and immunocytochemistry.

Leukemia and lymphoma cell lines showed increased mTOR activity compared to normal lymphoid cells. More than 70 bone marrow samples from pediatric ALL and from non leukemic patients were analyzed. The p-4EBP1 expression was significantly higher (20–58x) in all ALL samples than in normal cells. The p-4EBP1 expression level showed significant correlation with the prognosis of the patients. According to the in vitro rapamycin treatment and the mTOR related protein expression studies, in cases where p-4EBP1 expression level was decreased by in vitro rapamycin treatment, apoptosis induction could also be occured. We also found that rictor/raptor expression can influence the rapamycin sensitivity of malignant lymphoid cells.

Our results confirm that pediatric ALL cells have high mTOR activity. This could be detected by p-4EBP1 expression. The recent results suggest that mTOR activity measurments could be useful method to identify ALL patients with worse prognosis at diagnosis and to recognize early relapses. However, further studies are required to define the best way to sort ALL and other lymphoma patients for future rapalog treatments based on rapalog sensitivity prediction. 

Supported by OTKA K81624, K84262.   

Absence of IDH polymorphisms in bone tumours

Bacsi K^1,2, Amary MF², Maggiani F², Halai D², Flanagan AM²

¹Second Department of Paediatrics, Semmelweis University, Budapest, Hungary

²Histopathology, Royal National Orthopaedic Hospital NHS Trust, Stanmore, Middlesex HA7 4LP, UK 

The aim of the study: The isoforms of isocitrate dehydrogenase (IDH), IDH1 and IDH2 are responsible for α-ketoglutarate and NADPH production as essential substrates for cell respiration. In the case of somatic mutations, these enzymes dramatically change: their affinity for α-ketoglutarate decreases and as a gain-function-event a new enzyme product, 2-hydroxyglutarate appears in abundance. 2-hydroxyglutarate as an oncometabolite was shown led to carcinogenesis through the altered DNA methylation profile and the stabilized hypoxia-inducible factor 1 connecting an increased angiogenesis in cancers.

IDH1 R132, IDH2 R140 and R172 mutations were found in 60-90% of secondary gliomas and 5% of primary gliomas. Other tumours such as acute myeloid leukemias (12–18%), prostate cancers (3%), acute lymphoblastic leukemias (2%) also harboured these polymorphisms. Since patients with multiple enchondromas have occasionally been reported to have these conditions, we hypothesized that the same mutations would occur in other mesenchymal tumours such as PNET/Ewing sarcoma and osteosarcoma.

Methods: DNA was extracted from a paraffin-embedded tumour, which was at least 60% tumour-rich. The majority of the bone tumour samples had been decalcified in EDTA, a minority in formic acid. Osteosarcoma samples from 222 patients, 19 osteosarcoma cell lines and PNET/Ewing sarcoma samples from 25 patients were screened for IDH1 R132 mutations by Sequenom high-throughput MassARRAY platform. 64.4% (143 samples) of the osteosarcoma samples were also analysed for IDH2 R140 and R172 mutations by capillary sequencing. A cut-off of 10% of allelic frequency was generally used. Looking for germline mutations, non-neoplastic tissue (skeletal muscle, bone marrow, blood) from 30 patients was investigated by restriction digestion and DNA sequencing.

Results: Neither IDH1 nor IDH2 mutations were detected in osteosarcomas or in PNET/Ewing sarcoma samples. No germline mutations were found.

Conclusion: IDH1 and IDH2 somatic mutations did not contribute to the pathogenesis of osteosarcoma and PNET/Ewing sarcoma suggesting the existence of a more complex regulation in the case of these tumours.   

When thrombocytopenia is not immune thrombocytopenic purpura

Sulovska L, Pospisilová D, Flögelova H,  Stejskalova I, Novak Z, Hrachovinova I¹

Department of Pediatrics, Faculty of Medicine of Palacky University and University Hospital, Olomouc, Czech Republic

¹Institute of Hematology and Blood Transfusion, Prague, Czech Republic 

Introduction: Thrombotic thrombocytopenic purpura (TTP) is a rare disease in childhood. Pathogenesis of TTP is associated with deficiency of cleaving metalloproteasis known as ADAMTS 13  („a disintegrine-like and metalloprotease with thrombospondine repeats“). In healthy individuals, this proteases degrades ultralarge multimers of von Willebrand factor (vWF) into inactive monomers, which are not able to react with platelets. The deficiency of ADAMTS 13 causes abnormal platelet aggregation and results in microvascular thrombosis. TTP is characterised by consumptive thrombocytopenia, microangiopatic hemolytic anemia and organ dysfunction. We present an unusual manifestatation of TTP in 17 years old girl with bruises and petechiae.

Case report: Seventeen years old girl was admitted with severe thrombocytopenia (8x10⁹/l platelets). In the previous year she underwent two courses of high dose imunoglobulin and corticosteroid therapy for severe immune thrombocytopenia. Remission lasted for 6 months in both cases. Four days after admission, the patient developed headache, microangiopathic hemolytic anemia (with high LDH and free hemoglobin levels) and deterioration of renal function (serum creatinine and urea both increased to twice). The peripheral-blood smear showed schistocytes – fragmented red blood cells. Diagnosis of TTP was made and plasma exchanged therapy started. The patient underwent a total of 26 cycles of plasmapheresis. ADAMTS 13 activity was below 1% and a high titer inhibitor of metalloprotease was found. Due to non-complete response to plasmapheresis, vincristine and rituximab were added. Treatment was complicated by recurrent allergic reactions to plasma and rituximab.

Conclusions: Thrombotic thrombocytopenic purpura in children is extremely rare, but  serious disease. There are two forms of TTP: acquired and congenital. The congenital form – also known as Upshaw-Schulman syndrom – is a less common type. It is caused by mutation in ADAMTS 13 gene. The acquired type is caused by anti-ADAMTS13 autoantibodies, which inhibit the cleaving function of this metalloprotease. Patients with severe ADAMTS13 deficiency are at high risk of relapse, so our patient must be still periodically observed.

The aim of this case report is to highlight the wide spectrum of symptoms of TTP and to note, that not all diagnostic kriteria are always present. In a retrospective view of previous attacs, we found a mild anemia with hemoglobin 115 g/l. We suppose that the previous episodes of thrombocytopenia have to be the first manifestation of incompletely expressed TTP. Treatment of thrombotic thrombocytopenic purpura can caused many complications and, in some cases, plasmapheresis only is not able to induce a remission. 

This work was supported by Internal grant of the Faculty of Medicine, Palacky University, Olomouc, Czech Republic, no. LF-2011-006.         

Extracorporal membrane oxygenation (ECMO) in a pediatric intensive care unit

Cortina G¹, Beck C¹, Ojinaga V¹, Schönlaub J¹, Zimmerhackl LB¹, Geiger R¹, Schweigmann U¹, Schermer E¹, Neu N¹, Frühwirth M¹

¹Department of Pediatrics, Medical University Innsbruck, Austria 

Introduction: Extracorporal membrane oxygenation (ECMO) is an invasive supportive technique used in patients with potentially reversible cardiac and/or respiratory failure in whom conventional therapies have failed. Here we report the frequency of usage, patient demographics, and outcomes in children treated with ECMO in our pediatric intensive care unit (PICU).

Patients: 31 pediatric patients treated with ECMO at the PICU of the Medical University Innsbruck, Austria between 1996 and 2010 were retrospectively analyzed. Indications for ECMO were either hemodynamic (58,6%) or respiratory (41,6%) failure. 5 patients underwent ECMO insertion under rescucitation.

Results: ECMO Duration was significally higher in patients with respiratory support (9,3±6,3 d) than in patients with hemodynamic support (3,6±3 d) and highest in oncologic patients with ARDS (11,9±5,2 d). All but one (30/31) patient needed a mean of 2 vasopressors (range: 2–4) during ECMO. Renal replacment therapy (RRT) is not performed routinely with ECMO in our center. In 15 patients (48%) RRT had to be performed because of fluid overload. Mean requirment of blood products was 12±9 Packed red blood cells and 9±12 Platelet concentrates. Bleeding complications occured in 16 (52%) patients and was associated with the lenght of ECMO duration. Severe bleedings occured in 6 patients (4 lung, 2 intracerebral haemorrhage). Overall survival was 59% (18/31) and was significantly higher in patients with cardiac disease (70,6%) and lowest in oncologic patients (40%). Bleeding complications and need for RRT were associated with higher mortality.

Conclusion: In this analysis overall survival is comparable to those in the literature and ECMO can be lifesaving in these patients. Survival was best in patients with cardiac disease and worst in oncologic patients with ARDS. Bleeding complications and need for RRT were associated with higher mortality. According to the ILCOR guidelines on PALS 2010 Extracorporal cardiac life support (ECLS) may be considered in selceted patients with cardiac arrest, if cardiac arrest occurs in an evironment with appropriate expertise and protocols for ECLS.

The use of simple genetic methods as a second tier in the screening for congenital adrenal hyperplasia screening is complicated

Malikova J¹, Votava F², Cinek O¹, Lebl J¹

¹University Hospital Motol and Second Faculty of Medicine, Charles University in Prague, Department of Pediatrics, Prague, Czech Republic

²University Hospital Kralovske Vinohrady and Third Faculty of Medicine, Charles University in Prague, Department of Pediatrics, Prague, Czech Republic 

Background: The neonatal screening for congenital adrenal hyperplasia (CAH) leads to identification of a certain proportion of newborns with moderately increased 17-hydroxyprogesterone (17-OHP) who require a follow-up until normalization of 17-OHP. Among these individuals, we aimed to assess the number of copies of the CYP21A2 gene and its pseudogene and the prevalence of point mutations.

Patients and methods: We analyzed 754 randomly chosen neonatal dried blood spots from subjects with moderately elevated 17-OHP, who decreased to normal 17-OHP levels thereafter, 70 samples with known genotypes and 191 control blood samples of healthy children. The CYP21A2 gene was sequenced. The copy number ratio between the CY21A2 gene and its CYP21P pseudogene was tested by two methods utilizing the difference in the sequence of exon 3 (8bp deletion): two-colored real-time quantification, and fragment analysis. The samples clustered into four major clusters according to the gene/pseudogene ratio, representative samples from each group were analyzed by the multiplex ligation-dependent probe amplification (MLPA).

Results: Among the samples with moderately elevated 17-OHP, we identified 22 (3%) subjects with a known heterozygous point mutations and 10 (1.4%) samples with substitutions of unknown functional significance. Furthermore, we observed high proportions of samples deviate from the usual 1:1 ratio of the CYP21A2 to its pseudogene in dried blood spots with moderately elevated 17-OHP and also in control blood samples. Over 66 samples (9%) could not be classified due to failure of one or more methods because of the low quality of DNA obtained from the dried blood spots.

Conclusion: Using a second tier based on detection of pathogenic CYP21A2 variants, we would exclude clinical CAH caused by steroid 21-hydroxylase deficiency in 688 samples (91%). But the efforts towards definite confirmation of no less than one functional allele of the CYP21A2 gene would be far too costly and laborious as compared to the now utilized recall measurements of 17-OHP.

Pre-term newborns bone metabolism

Kanioková P, Pecková A¹, Bayer M

Dept. of Paediatrics, Charles University, Medical Faculty, Hradec Kralove, Czech Republic

¹Dept. of Gynecology and Obstetrics, Charles University, Medical Faculty, Hradec Kralove, Czech Republic 

Introduction: Growth and development of bone structure during intrauterine life have predictive quality for development during childhood until the adulthood. From the studies with term borned newborns we know that the leptin influence the maturation of chondrocytes and osteoblasts. There is positive correlation between the cord blood level of leptin and bone density (term newborns). There are little information about pre-term newborns, who are in higher risk of low bone density.

Methods: Prospective study. We investigate 60 pre-term newborns (slight and mild immaturity). There was fill in the questionnaire – medication during pregnancy, weight gain during pregnancy, diabetes during pregnancy, smoking, meassuring of the placenta – average, weight, longitude of umbilical, cord blood acid base balance, newborn - gestation age, delivery type,  time of delivery, Apgar score, newborns weight and lenght, minerals in urine. Cord blood analyses – Ca, P, ALP, AST, ALT, 25-OH D, leptin, osteocalcin. Hypothesis – there is correlation between cord blood analytes and newborns an placental description attributes.

Results: Analyses of the results are still running, preliminary results will be available at the time of the symposium.   

Screening strategies for the identification of the first degree relatives of T1D patients with the highest risk of progression to clinical onset of type one diabetes

Petruzelkova L, Vcelakova J, Stechova K, Lebl J, Vyhnankova M, Kolouskova S

Pediatric Dpt., University Hospital Motol, 2nd Medical Faculty, Charles University, Prague, Czech Republic 

Aim: The first degree relatives of patient with T1D are at 4-8 fold increased risk for diabetes compared with the general population. We asked whether combined examination of risk HLA genotype and pancreatic islet autoantibodies considering their titer, type and the number of them would improve the selection of the children under the highest risk for clinical onset of T1D.

Methods: The siblings and offspring of T1D patients were recruited by the Czech T1D Prediction Programme where they are prospectively followed up for T1D development and which includes estimation of risk HLA genotype (DQA1, DQB1) and regularly examination of T1D associated autoantibodies (GADA, IA2A, IAA by radiobinding assay). In Ab positive children under the high risk of T1D development we performed IVGTT (41 children). Diabetes status of all siblings and offspring was defined.

Results: Of the 1062 first degree relatives, 152 (14,3%) were positive for at least one Ab and 53 (4.9%) developed diabetes during follow up (median follow up 3 years, range 1–12). The prevalence of GADA or IA2A or combination of GADA and IA2A in Ab positive children was 72 (47.3%), 34 (22.3%) and 43 (28.2%). The study showed a strong association among T1D risk and HLA (p<0.05), Ab titer (p<0.01), Ab type (p<0.05) and Ab number (p<0.01). The highest risk were connected with risk genotype DQA1*05-DQB1*0201/DQA1*03-DQB1* 0302, high-titer of IAA, IA2A (p<0.01). We proved association between FPIR and length of Ab positivity, but no association between FPIR and Ab titers. As the secondary result MODY diabetes was proven in 13 cases of Ab negative diabetic patients.

Conclusion: HLA and Ab screening is able to define children at actual risk of diabetes progression. On the other hand the estimation of the period to T1D manifestation is very difficult because the length of beta cells destruction is too variable as shown FPIR and we should focused on the others cofactors.   

Renin-Angiotensin-Aldosterone-System (RAAS) blockers in diabetic nephropathy (DN)

Bánki NF, Wagner L, Prókai Á, Vér Á, Vannay Á, Degrell P, Szabó AJ, Fekete A 1^st

Dep. of Pediatrics, Semmelweis University, Budapest, Hungary

Dep. of Transplant Surgery, Semmelweis University, Budapest, Hungary

Dep. of Med. Chem. Mol. Biol. and Pathobiochem., Semmelweis University, Budapest, Hungary

2nd Dep. of Internal Medicine and Nephrology Center, Pécs, Hungary 

Objectives and study: Renal RAAS is clearly activated in diabetes, which is the main cause of end-stage renal failure. Previously in a rat model of DN we found impaired renal function parallel with elevated expression and translocation of the renal Na/K ATPase (NKA). Heat-shock protein (HSP) 72 stabilized membrane associated NKA and preserved enzyme function. Here, we analyze the effect of various RAAS inhibitors on diabetes or hyperglycemia induced renal damage.

Methods: In vivo – Diabetes was induced in male Wistar rats with iv. Streptozotocin After 5 weeks of diabetes rats were treated daily with ACE inhibitor enalapril, ARB losartan and aldosteron antagonists spironolactone and eplerenone for 2 weeks p.o. Vehicle-treated diabetic, and treated non-diabetic animals served as controls (n=6/group). Renal histology, kidney function, protein levels and intrarenal localization of NKA and HSP72 were evaluated. In vitro - HK-2 cells were cultured under 5mM (C) and 35mM (G) glucose concentrations and in 35mM mannitol (M). G cells were treated with the same RAAS blockers for 72 hours. NKA and HSP72 protein levels were measured.

Results: Diabetes resulted in impaired renal histological and functional parameters, elevated and translocated renal NKA and HSP72. RAAS inhibitors ameliorated histological damage, while kidney function was mostly preserved by aldosterone antagonists. Each RAAS treatment -except enalapril - lowered renal NKA and HSP72 and prevented NKA translocation. Hyperglycemia induced elevation of tubular NKA was decreased by each treatment, except losartan.

Conclusion: Aldosteron antagonists, even in monotherapy might be beneficial in the treatment of DN beside ACE inhibitors and ARBs. RAAS inhibitors might help to stabilize NKA and thereby sodium homeostasis, which could be an additional explanation for their renoprotective effect. 

Supported by grants OTKA-NNF78846-K71730 and TÁMOP-4.2.2-08/1/KMR-2008-0004.   

Genetic deficiency of tartrate-resistant acid phosphatase is associated with skeletal dysplasia, cerebral calcifications and autoimmunity

Lausch E¹, Janecke A², Bros M³, Unger S^1,4, Zabel B¹, Superti-Furga A^1,4

¹Centre for Pediatric and Adolescent Medicine, University of Freiburg, Freiburg, Germany

²Department of Pediatrics II and Division of Human Genetics, Innsbruck Medical University, Innsbruck, Austria

³Department of Dermatology, University Medical Center of the Johannes Gutenberg University, Mainz, Germany

⁴Department of Medical Genetics, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland 

Aims: Monogenetic autoimmune diseases hold the promise to elucidate complex dysregulation observed in common disorders like systemic lupus erythematosus (SLE) and multiple sclerosis (MS). Vertebral and metaphyseal dysplasia, spasticity with cerebral calcifications, and strong predisposition to autoimmune diseases are the hallmarks of the genetic disorder spondyloenchondrodysplasia (SPENCD).

Methods and results: We mapped a locus in five consanguineous families to chromosome 19p13 and identified mutations in ACP5, which encodes tartrate-resistant phosphatase (TRAP), in 14 affected individuals and showed that these mutations abolish enzyme function in the serum and cells of affected individuals. Phosphorylated osteopontin, a protein involved in bone reabsorption and in immune regulation, accumulates in serum, urine and cells cultured from TRAP-deficient individuals. Patient-derived dendritic cells exhibit an altered cytokine profile and are more potent than matched control cells in stimulating allogeneic T cell proliferation in mixed lymphocyte reactions.

Conclusions: These findings shed new light on the role of osteopontin and its regulation by TRAP in the pathogenesis of common autoimmune disorders. Pharmacologic modulation osteopontin may thus provide a novel therapeutic angle for diseases like SLE and MS.   

A novel mutation of the 25-hydroxyvitamin D-1 alpha hydroxylase gene (CYP27B1) in a boy with vitamin D-dependent rickets type 1

 Ertl A¹, Streubel B³, Zeitlhofer P², Raimann A¹, Sagmeister S¹, Haeusler G¹

¹Medical University of Vienna, Department of Paediatrics and Adolescent Medicine, Department of Pediatric and Adolescent Medicine, Div. of Pediatric Pulmonology, Allergology and Endocrinology, Austria

²Medgen Vienna, Austria

³Medical University of Vienna, Clinical Institute for Pathology and Genetics, Austria 

Case report: A male patient (parents unrelated) presented at one year of age with radiological and biochemical signs of rickets despite regular prophylactic administration of 25-hydroxyl-vitamin D3 (25-OH VitD3). Whereas 25-OH VitD3 administration failed to improve severe hypocalcaemia, therapy with 1,25 (OH)₂- cholecalciferol and calcium was followed by rapid improvement of clinical and biochemical findings. During childhood, serum calcium levels were stable, but during adolescent growth, very high levels of parathormone (298 ng/ml, normal 10-60 pg/ml), with normal serum calcium and alkaline phosphatase indicated the need for dosage adjustment. Interestingly, the patient was completely asymptomatic during a period of severe hypocalcaemia, which can be attributed to his chronic adjustment. Genetic analysis had not been available at the time of presentation, and was performed by sequencing analysis of the CYP27B1 gene. Our patient proved to be compound heterozygous for two new mutations, namely NM_000785.3:c.[217G>A];[790+2T>G] (nt 1 corresponds to the A of the ATG initiation codon). The first one is located in exon 2 and causes the amino acid substitution p.Gly73Arg, whereas the second one is a potential splice site mutation (intron 4).

Discussion: Vitamin D-dependent rickets type 1 is an extremely rare disorder (36 mutations in 54 patients reported), usually found in consanguineous families. In our patient, the asymptomatic parents had a heterozygous mutation, each of which has so far not been described to lead to functional defects.

Perspective: In order to test the relevance of the mutations, we will first use a structure prediction program (PSIPRED, Protein Structure Prediction Server) to predict the change of protein secondary structure, and, afterwards, we will characterize the functional consequence of these mutations on 1 alpha-hydroxylase activity in cell culture.   

Heparin Co-Factor II Thrombin Complex (HCII-T) – a new biomarker for MPS

Ratschmann R, Clarke L, Herkner K, Kasper D, Kircher SG, Konstantopoulou V, Moeslinger D, Pollak A

Medical University of Vienna, Department of Paediatrics and Adolescent Medicine, Austria 

The mucopolysaccharidoses are a heterogeneous group of multisystemic and progressive lysosomal storage disorders caused by a deficiencies of specific enzymes in the glycosaminoglycans pathway.

Storage of glycosaminoglycans also influences physiological processes within the body like inflammatory reactions or haemostasis. The pathological storage leads to an activation of serine protease inhibitors, like Heparin Co-Factor II. Due to activation HCII inhibits thrombin by forming a covalent linkage.

Randall et al. discover a correlation between the well known influence of GAGs to the formation of HCII-T-complex and MPS. A 630 fold increase of the HCII-T complex level was demonstrated in the mouse model.

In patients suffering from MPS I/ MPS II/ MPS VI it was also possible to demonstrate an elevated HCII-T level.

Langford- Smith et al. inject dermatan sulfate (MPS I/ MPS II/MPS VI) into a mouse and observe an increase of HCII-T.

Neither injections of heparan sulfate (MPS III) nor of keratan sulfate (MPS IV) provoke an increase of HCII-T. Interestingly, in samples from patients suffering from MPS III and MPS IV the HCII-T level was increased.

We did measurements in 7 male patients suffering from MPS II before and during enzyme replacement therapy. Before starting the ERT the HCII-T level was highly elevated and dropped down during therapy.

In our group it was not possible to find a correlation between the level and the severity of the disease.   

Hereditary pancreatitis in children – possibilities of molecular-genetic testing

Töröková K, Čierna I, Kovács L

2nd Department of Pediatrics, Comenius University Medical School, University Children´s Hospital, Bratislava, Slovakia 

Hereditary pancreatitis is a very rare disease with early onset, slow progress and variable clinical presentation. Inheritance is autosomal dominant with incomplete, approximately 80 percent penetrance and variable expressivity. Mutation of the PRSS1 gene (cationic trypsinogen gene) increases the autocathalytic conversion of trypsinogen to active trypsin that increases the autodestruction of the pancreas leading to chronic inflammatory changes. Other genes associated with chronic pancreatitis are PRSS2 (anionic trypsinogen gene), SPINK1 (serin protease inhibitor – Kazal type 1) and CFTR (cystic fibrosis transmembrane conductance regulator).

Our aim is to point out the therapeutical pitfalls and possibilities of genetic diagnosis with case studies of three patients with chronic pancreatitis. The onset of the disease was at ages 8, 14 and 16 with strong abdominal pain from acute exacerbation of until that time asymptomatic chronic pancreatitis. Chronic inflammatory changes led to pancreatic fibrosis, pseudocysts and calcifications in the parenchyma and along the pancreatic duct. All of them required Sumery according to fulminant course of the acute exacerbation. DNA analysis was indicated to confirm the inherited form of chronic pancreatitis with autosomal dominant inheritance from mutations in genes PRSS1 and SPINK1. One of the patients had mutation of PRSS1 gene – R122H and in two patiens mutation of SPINK1 gene – N34S in heterozygous form was revealed.

These case studies draw attention to the importance of diagnostic of inhereted forms of chronic pancreatitis. After exclusion of other causes included cystic fibrosis, the indication for PRSS1 and SPINK1 mutation testing in symptomatic patients should be one of the following criteria:

a) recurrent unexplained attacks of acute pancreatitis and a positive family history (in two of our patients acute pancreatitis appeared in a first-degree family members in young adulthood), b) unexplained chronic pancreatitis and a positive family history, c) unexplained chronic pankreatitis without a positive family history after exclusion of other causes, d) unexplained pancreatitis episode in children.

Patients with confirmed hereditary pancreatitis are at risk for developing exocrine and

endocrine insufficiency and are at 50-fold increased risk of pancreatic cancer; therefore the prevention of the complications is possible with early diagnostic, follow-up and education of these children.   

DNAH5 and DNAI1 mutations in patients with primary ciliary dyskinesia detected by sequencing of selected exons

Djakow J¹, Svobodová T¹, Cinek O¹, Uhlík J², Pohunek P¹

¹Department of Pediatrics, 2^nd Faculty of Medicine, Charles University in Prague and University Hospital Motol, Prague, Czech Republic

²Department of Histology and Embryology, 2^nd Faculty of Medicine, Charles University in Prague, Czech Republic 

Introduction and the aim of the study: Primary ciliary dyskinesia (PCD) is a rare genetically heterogenous condition. Mutations in DNAH5 or DNAI1 genes can be found in about a third of the patients with PCD. Frequent mutations were described in some exons of these structurally complicated genes. The objective of the study was to determine a prevalence of the DNAH5 or DNAI1 mutations in the patients with PCD using sequencing of selected exons and to evaluate whether this approach can be used as a first-step genetic testing.

Methods: Based on clinical findings and diagnostic tests on the ciliated epithelium, 31 paediatric patients with PCD were identified. Twenty-six patients from 23 families agreed on genetic testing. Direct sequencing of 9 exons of DNAH5 and 4 exons of the DNAI1 was performed.

Results: In total, 6 mutated alleles were found in 5 of the patients. One patient, a 17-year old male with PCD caused by outer dynein arm (ODA) defect and completely immotile cilia, was a compound heterozygote for DNAH5 mutations, while in other four individuals we found only one mutation. Four of the mutations found were previously described, two mutations were novel substitutions.

Conclusions: Selected exon sequencing is likely to detect at least one mutated allele in most of the patients who are expected to carry DNAH5 or DNAI1 mutations. Targeted and tiered genetic testing may be a reasonable approach to detect mutations in PCD patients, especially if their phenotype is taken into account.   

Genetic, epigenetic, RNA and protein analysis of myocardial tissue samples in congenital heart defects – recommendation on preparation and long-term storage

Diener K-H¹, Dangl A², Greber-Platzer S¹

¹Division of Pediataric Pulmonology, Allergology and Endocrinology, Medical University of Vienna, Department of Paediatrics and Adolescent Medicine, Austria

²Division of Pediatric Cardiology, Medical University of Vienna, Department of Paediatrics and Adolescent Medicine, Austria 

Aim of the study: In congenital heart defects (CHD) several mutations in different cardiac specific genes are discussed to increase the risk for malformations of the heart during embryogenesis. This can cause deficiency, absence or malfunction of the produced protein leading to disruption of the normal function of cardiac-specific pathways in the developing heart.

Therefore we plan to analyze genetic sequences of cardiac specific genes in myocardial tissue of patients with CHD. Afterwards, for better understanding of the disordered function, mRNA expression, epigenetic evaluation and analysis of protein quantity and quality of genes with detected mutations should be performed. Myocardial samples are taken during cardiac surgery after informed consent of the patients with CHD or their parents.

To ensure a maximum stability of RNA, DNA and protein in myocardial tissue, optimal procedures of handling and storage are required. With this study, the best practices for handling and storage of the extracted myocardial samples should be worked out to guarantee best quality and quantity for a long time.

So we compared the conventional technique of myocardial samples storage at -80°C for DNA analysis and at liquid nitrogen for measurements of RNA and proteins with a new stabilizing reagent (QIAGEN AllProtect) to improve storage condition and duration at -80°C for all tissue metabolites.

Methods: Immediately after surgical extraction the myocardial samples are divided in small, equally sized pieces, which are used to analyse baseline levels of RNA, DNA and protein in freshly drawn specimen and for further storage using the conventional technique or after treatment with the stabilizing reagent at -80°C. Further analysis will be performed after 4, 12 and 24 months storage. Isolation is prepared by QIAGEN AllPrep DNA/RNA/Protein-Mini Kit, which allows the extraction out of one myocardial piece. Quantification and qualitative analysis are performed by use of the Nanodrop Spectrophotometer ND-1000 and Agilent Bioanalyzer.   

Severity of hypertension in obese children and adolescents

Babinská K¹, Kovács L¹, Jankó V¹, Dallos T.¹, Vitáriušová E¹, Feber J²

¹2nd Department of Pediatrics, Comenius University Medical School, University Children´s Hospital, Bratislava, Slovakia

²Division of Nephrology, Department of Pediatrics, Children’s Hospital of Eastern Ontario, Otawa, Canada 

The aim of the study: Childhood obesity represents a major problem, at present time affects in Slovakia 15% children and is characterized with early occurrence of complications, including hypertension. The ambulatory blood pressure monitoring (ABPM) enables precise detection of arterial hypertension, assessment of blood pressure load and blood pressure dipping during the night. The goal of our study was to analyze the impact of obesity on blood pressure, detect the severity of hypertension and characterize the type of hypertension in obese children and adolescents.

Methods: Blood pressure was assessed with ambulatory blood pressure monitoring. The severity of hypertension was determined according the BP load and corrupted night blood pressure dipping. One hundred-nine patients with primary obesity aged 7 to18 years (mean±SD age = 14.1±3.1) were enrolled in the study. Patients were divided into3 groups according to BMI Z scores: Group 1 (n=27): BMI >1.65 and <3.28 SDS; Group 2 (n=55): BMI >3.29 and <4.91 SDS; Group 3 (n=27): BMI >4.92 SDS. Anthropometric, laboratory and blood pressure data were analyzed.

Results: Only 26 patients (24%) had ambulatory normotension, 27 (25%) had ambulatory prehypertension, 3 (3%) had hypertension and 53 (48%) had severe ambulatory hypertension. The severity of hypertension increased significantly with the degree of obesity (Chi square test for trend, p=0.0027). Daytime systolic, diastolic and mean arterial pressures (SBP, DBP, MAP) increased significantly with increased BMI (shift to the right), whereas the nighttime pressure remained elevated regardless of the degree of obesity. Isolated nighttime hypertension was observed in 25% of patients, 38% were classified as non-dippers.

Conclusion: Almost 50% of children with obesity and hypertension suffer from severe ambulatory hypertension detected on ABPM. Obesity has a major impact not only on incidence but also on the severity of hypertension. Nighttime blood pressure is elevated in early stage of obesity, in severe obese patients raises the daytime blood pressure.   

Assessment of arterial stiffness in hypertensive children and adolescents

Štelcar A, Miksić M, Gregorič A, Marčun Varda N

Department of Paediatrics, University Medical Centre Maribor, Slovenia 

The aim of the study: The important part of investigations in hypertensive children is to establish the effects of elevated blood pressure on the hypertensive target organs. One potential early marker of this damage is the increase of arterial stiffness now recognized as a major driver of cardiovascular disease. The aim of our study was to investigate if there are any differences between normotensive and hypertensive children according to the measures of arterial stiffness.

Methods: Twenty normotensive and 20 hypertensive children of both sexes, matched by age and sex, were included in the study. Carotid-radial pulse wave velocity (PWV) was measured in hypertensive children as an index of arterial stiffness using the applanation tonometry-SphygmoCor and compared to the values in normotensive children. Central aortic systolic pressure (AoSP), aortic pulse pressure (AoPP), augmentation pressure (AP) and the augmentation index (AIx@HR75) were also compared between the two groups.

Results: The hypertensives presented higher mean values of PWV (6.30 m/s), AoSP (131.9 mmHg), AP (0.35 mmHg) and the AIx@HR75 (2.80) than the controls (PWV (6.09 m/s), AoSP (125.5 mmHg), AP (-2.30 mmHg) and the AIx@HR75 (-0.75)). The AoPP was higher in the normotensive group (55.9 vs. 51.1 mmHg). No significant differences in PWV (p=0.57), AoSP (p=0.24), AP (p=0.37), the AIx@HR75 (p=0.34) and AoPP (p=0.26) were found.

Conclusion: We found no significant differences of arterial stiffness between normotensive and hypertensive children. Nevertheless, we believe that with increasing size of studied population the difference would become more significant.   

Nonalcoholic fatty liver disease (NAFLD) in children and adolescents

Gombošová K¹, Nováková B², Schuszter A¹, Kuchta M¹

¹IInd Department of Children and Adolescents, Children Faculty Hospital, P. J. Š. University, Košice, Slovakia

²IIIrd Internal Clinic, University Hospital of L. Pasteur, Košice, Slovakia 

Non-alcoholic fatty liver disease is  now considered as one of the commonest liver diseases in developed countries. It is recognised as an increasing clinical problem in children and adolescents. Risk factors include obesity, insulin resistance, and disorders of lipid metabolism.

The aim: of our study was to evaluate basic characteristics of NAFLD – its prevalence, diagnosis based on laboratory parameters and ultrasound examination of the liver among children and adolescents with identified disorder of lipid metabolism.

Methods: The retrospective study included 50 children and adolescents (27 mail and 23 female) aged from 1 to 19 years (mean age was 12,5±4,48) with impaired lipid metabolism. Serum aminotransferases, total bilirubin, glucose levels and parameters of lipid metabolism were analyzed from fasting blood samples. Ultrasound examinations were realized on ACUSON Sequoia Echo C512, using convex probe. In order to achieve greater objectivity of ultrasound evaluation, ultrasound images were quantified by programe Adobe Photoshop 5.5 function Histogram.

Results: Antrophometric parameters (body weight, BMI, waist circumference, hip circumference and WHR – waist to hip ratio) were significantly higher in the group of children with NAFLD, compared with the group without NAFLD. There was a significant association between activity of aminotransferases (AST and ALT) and body weight, BMI and level of triglycerides in the group with NAFLD. Patients with NAFLD had significantly higher echogenity of liver parenchyma, what was objectivised and quantified by the density of pixels in usg image. There was a statistically significant correlation between PO index (ratio: liver parenchyma/cortex of the right kidney) and antrophometric parameters (body weight, BMI, waist, hip circumference, WHR).

Conclusions: Non invasive diagnostic methods, such as measurement of antrophometric parameters, ultrasound examination – mainly objectivisation of the currently used ultrasound liver examination by using the Adobe Photoshop quantification utility, allow to predict presence of nonalcoholic fatty liver disease.   

Improvement in biomarkers of bone formation during 54-week infliximab therapy in pediatric patients with Crohn´s disease

Molnár K^1,2, Szabó D¹, Kővári ɹ, Vannay Á², Dezsőfi A¹, Arató A¹, Tulassay T¹, Veres G¹

¹First Department of Pediatrics, Semmelweis University, Budapest, Hungary

²Research Group for Pediatrics and Nephrology, Semmelweis University and Hungarian Academy of Sciences, Budapest, Hungary 

The aim of the study: Treatment with infliximab (IFX) may improve growth and disturbed bone metabolism in pediatric patients with Crohn disease (CD), but the characteristics of bone formation and resorption factors under IFX treatment are not well known. This study examined changes in bone formation (osteocalcin/OC, bone-specific alkaline phosphatase/bALP) and resorption (beta-crosslaps/bCL) under IFX treatment. Moreover, associations between bone biomarkers, and CRP, vitamin D level, disease activity index (Pediatric Crohn’s Disease Activity Index, PCDAI), and dual energy x-ray absorptiometry (DEXA) after 54 weeks of IFX therapy were analyzed.

Methods: Twenty-eight subjects with moderate to severe CD received IFX induction (5 mg/kg/dose) at weeks 0, 2, and 6. Maintenance therapy was given all patients every 8 weeks. Serum OC, bCL, bALP, and vitamin D were collected at baseline, 6 weeks, 30 weeks and 54 weeks, in addition CRP and PCDAI were determined. DEXA z-scores were assessed at baseline, 30 weeks and 54 weeks.

Results: Serum levels of bone formation OC increased significantly after IFX induction treatment. bALP increased significantly between baseline and weeks 6 (mean, 110U/L, 161U/L, respectively, P=0.002). There were no significant differences concerning bCL and vitamin D at different time points. Nevertheless, both z-score of the lumbar spine and femoral neck improved after 54 weeks when compared with baseline respectively. Increment of bone forming OC correlated negatively with decrement of CRP and PCDAI (week 0 vs. weeks 6, week 30, and weeks 54).

Conclusion: Clinical response to IFX therapy was associated with an increased level of bone forming osteocalcin in pediatric patients with 54-week treatment of IFX. Bone resorption marker (bCL) was not increased suggesting a bone forming effect of IFX treatment.   

An interventional study on mass hysteria in children

Pushpa Prasad Sharma¹, Ajaya Kumar Jha², Anish Joshi², Ritesh Lamsal²

¹Department of Psychiatry

²Department of Paediatrics, Dhulikhel Hospital, Kathmandu University Teaching Hospital, Nepal 

Background: A Mass conversion dissociative disorder, also known as “Mass hysteria”, has become a common phenomenon in rural and urban school going children. This study has been carried out to find out the socio-demographic pattern of this disorder, explore factors that may be contributory to this problem and the utility of psychological intervention, carried out on the affected children, their family members and teachers, on the recurrence of this disease.

Materials and methods: This is a retrospective study of 67 children, who suffered from episodes of pseudo-seizures in the community school. A detailed questionnaire form was used, which was specifically prepared to evaluate these children. Detailed mental status examination was performed by a psychiatrist. Psychological intervention was done in which group therapy and individual counseling were carried out.

After six months of prospective follow up, all children who had recurrence of the episode were re-interviewed. Similar type of group therapy and individual counseling were carried out in children with repeated attacks. In both the occasions, in addition to the children, teachers and parents were also counseled separately.

Results: Most of the affected children were in the 12 to 14 years age group and majorities (91%) were females and were from 6^th to 9^th grades. Family history of similar attacks was found in 25% of the cases. Prevalence of the attack was found to be commoner in children with lower socio-economic status. Some form of mental illness like anxiety, depressive disorder was found in 13% of cases. These were all determined by using the criteria in DSM IV. Alcohol abuse and disturbed family dynamics were associated in 33% of the cases. Majority of the affected children were found to have poor scholastic performance. More than 50% of the children with attack were found to have witnessed or been involved in violent activities and in 15% of the cases there was a history of corporal punishment.

After six months of prospective follow up, nearly 20% had recurrent episodes of the pseudo-seizure while 80% had no recurrence. All children who had a repeat-attack were females. School dropout was found in 5% of the cases. Overall outcome after psychological intervention was found to be satisfactory and it was found to be very effective in decreasing the recurrence of pseudo-seizures.

Conclusion: Effective intervention and prospective follow up has shown to be very cost effective in decreasing the recurrence of mass pseudo-seizures. We recommend comparison of similar problems in rural versus urban areas.  Broader community participation and prolonged follow up is strongly suggested.   

Barorefles sensitivity and analysis of pulse wave in young diabetic patients

Šťastná J¹, Petrová A², Pekař M², Nováková Z², Brázdová L³, Pejchlová M¹, Hrstková H¹, Závodná E², Honzíková N^2,1

¹1st Department of Paediatrics, Faculty Hospital in Brno and Faculty of Medicine, Masaryk University, Czech Republic

²Department of Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic

³Merciful Brothers’ Hospital in Brno, Czech Republic 

The aim of the study: Type 1 diabetes mellitus is a chronic endocrine and metabolic disease, which usually starts in childhood. The insufficient compensation of glycaemia may result in cardiovascular disorder. The aim of the study was to evaluate the baroreflex sensitivity (BRS) and the pulse-wave velocity (PWV) and augmentation index (AIx) in young diabetic patients.

Methods: We examined 15 patients with type 1 diabetes mellitus without clinical signs of an autonomous disorder (group D, age: 15±3.2 yrs, duration of the disease 6.33±2.94 years, glycated haemoglobin HbA1c 7.29±1.35%) and 18 healthy individuals (group C, age: 16.7±0.5 years). We used a 5-min continuous non-invasive blood pressure (BP) measurement (Finometer, FMS, The Netherlands). BRS was calculated by beat-to-beat values of BP and inter-beat intervals by spectral analysis and expressed in ms/mmHg. The applanation tonometry was used for detection of pulse-wave velocity, the augmentation index was estimated by pulse-wave analysis (Sfygmocor, AtCorMedical, Australia). The Mann‑Whitney test was used for statistical evaluation (STATISTICA 9.CZ, StatSoft).

Results: Comparing group D with group C, we found a significant increase of the PWV values (D vs. K: 7.33±0.60 vs. 6.36±0.53 m/s; p<0.05), AIx values (D vs. K: 0.30±10.35 vs.-1.63±1.42 %; p<0.05) and heart rate (76.65±10.35 vs. 66.76±8.50 bpm, p<0.05), and a significant decrease of the BRS (7.84±4.11 vs. 11.91±4.72 ms/mmHg; p<0.05).

Conclusion: The study showed that an increase of pulse-wave velocity, augmentation index and a decrease of baroreflex sensitivity can be found in young patients with type 1 diabetes mellitus who do not have any clinical signs of an autonomous disorder during some years of duration of this disease. 

Supported by grant: MSM 0021622402.   

Neonatal abstinence syndrome – our experience

Žikavská T, Brucknerová I, Červeňová O

1^st Department of Paediatrics, Medical School, Comenius University, Bratislava, Slovakia 

In infants, who were exposed to addictive substances during pregnancy, neonatal abstinence syndrome (NAS) can develop. Distinctive signs and symptoms of central and autonomic nervous system regulatory dysfunction are presented.

The aim of the study: To present a group of neonates with NAS, who were admitted at the Neonatology Unit of 1^st Department of Paediatrics between January 1^st, 2000 and December 31^st, 2010. Risk factors, complications, treatment and consequences are evaluated.

Methods: Group of 76 neonates (35 males and 41 females) with NAS were analyzed. Drug abuse mothers in 95% of cases were positive and maternal anti-epileptic drugs and iatrogenic in 5%. The most frequent drugs were heroin (57%), methadone (53%) and others (marijuana, methamphetamines, benzodiazepines, barbiturates). 28% of mothers smoked. After delivery 7 neonates were asphyxiated (Apgar score ≤7), 18 children were small for gestational age and 30 children were premature.

Results: During the first day of life, 78% of cases were admitted. For initial therapy in 4% of patients oral morphine solution was administered. Other options as chloral hydrate and phenobarbital were also used. 9% of all newborns were without any therapy. These high risks children are threatened with transplacentally transmitted infectious diseases. In our group 3 cases of hepatitis C infection have occured. In other cases passively transferred antibodies were noted (HCV-44, HBV-4, BWR-20, CMV-5). 15% of children had neurological consequences. After complete social investigation 68% of children were discharged home and 4% were moved to regional hospital. 24% from all of them were placed in children’s home and only 4% in foster care.

Conclusion: Drug abuse during pregnancy may lead to fetal damage and have consequences for growth and development of the children. Comprehensive care for newborns includes non-pharmacologic therapy and supportive treatment based on evaluation of the Finnegan Neonatal Abstinence Scoring System. All risk newborns require complex approach involving some professionals and social workers. The most important is to identify the most appropriate social environment.   

Succinate activates the collecting duct renin-angiotensin system

Prókai Á^1,2, Burford J¹, Gevorgyan H¹, Péterfi Z¹, Vargas SL¹, Peti-Peterdi J¹

¹Department of Physiology and Biophysics and Department of Medicine, Zilkha Neurogenetic Institute, University of Southern California, Los Angeles, California, USA

²1st Department of Pediatrics, Semmelweis University, Budapest, Hungary 

Objectives and study: The novel metabolic receptor GPR91 controls renin release, the rate-limiting step of RAS. Furthermore, the renal collecting duct (CD) is the major source of (pro)renin in diabetes. Since the highest GPR91 expression was found in the CD, this study investigated whether succinate, the ligand of GPR91 regulates the local CD RAS.

Methods and results: Western blot analysis of succinate-treated M1 cells (CD cell line) showed a dose-dependent, 2-2.5-fold elevation in pERK½, pp38, COX₂, renin, prorenin and its receptor [(P)RR]. In WT and GPR91^-/- control and STZ-diabetic (DM) mice, CD pERK1/2 (by immunofluorescence) and urinary PGE₂ excretion (measured by ELISA) increased 4-20 fold in DM mice and were GPR91-dependent. Medullary (pro)renin and (P)RR protein expression (by immunoblotting) and renin activity in the CD tubular fluid (visualized in vivo using multiphoton microscopy and a fluorogenic renin substrate delivered by renal micropuncture) increased 4-5 fold in WT DM vs. control mice, which was completely abolished in GPR91^-/- DM mice.

Conclusions: This is the first, direct demonstration of CD renin activity in vivo. Succinate accumulation and GPR91 signaling are novel (patho)physiological regulatory mechanisms that activate the local RAS in the CD via MAP kinases and COX₂, PGE₂ release, and increased (pro)renin and (P)RR synthesis. Succinate and GPR91 may be important regulators of the local CD RAS in DM and may serve as new therapeutic targets in diabetic nephropathy. 

Supported by grants from NIH DK064324; AHA EIA 0640056N; Council on International Educational Exchange (CIEE) and REG_KM_09-1-2009-0016 BAROSS, OTKA NNF78846, Semmelweis University TÁMOP-4.2.1.B-09/1/KMR KUTATÓEGYETEM.   

Familial cases of complete androgen insensivity: germ cell survival and pathology

Pleskacova J¹, Oosterhuis JW², Stoop H², Bruggenwirth H³, Drop SLS⁴, Snajderova M1, Lebl J¹, Looijenga LHJ²

¹Department of Pediatrics, Charles University, 2nd Faculty of Medicine, Prague, Czech Republic

²Department of Pathology, Josephine Nefkens Institute, Erasmus University Medical Center, Daniel den Hoed Cancer Center, Rotterdam, The Netherlands

³Department of Clinical Genetics, Erasmus University Medical Center, Rotterdam, The Netherlands

⁴Division of Pediatric Endocrinology, Eramus University Medical Center, Rotterdam, The Netherlands 

Background: Germ cell decline in gonads of patients with complete androgen insensitivity  syndrome (CAIS) is probably not only dependent on the abdominal location of the testes, but also on the present androgen receptor (AR) mutation. Germ cells with fetal features can be frequently found among cells which are able to survive. These are suspicious for being at risk for malignant transformation, resulting in germ cell cancer.

Aim of the study: To explore whether there might be a link between a level of androgen receptor functionality and germ cell survival and/or abnormal development in CAIS.

Methods: We evaluated gonadal histology focusing on germ cells in four affected sibling pairs. In three families the pathogenic AR mutation resulted in frame-shift, and therefore in a non-functional receptor. In the fourth family point mutation led to residual receptor activity. To further assess germ cell characteristics, immunohistochemical detection of OCT3/4 and KITL was performed.

Results: Germ cells were present in seven out of eight patients, but only in four of them number of settled seminiferous tubules could be calculated (Family 1 with frame-shift: Subj A (1.3 yr) 50 % and Subj B (6 yr) 18%; Family 2 with residual AR activity: Subj A (9.5 yr) 44% and Subj. B (16.5 yr) 20%), in others the number was too small. Germ cells with signs of malignancy (positive for OCT3/4, located within KITL positive area), were identified in four patients from both groups with and without residual activity of AR.

Conclusion: Lesser decline of germ cells was observed in gonads in case of residual AR activity. However, persistence of OCT3/4 positive germ cells is likely influenced by other factors as well, because these cells were detected in gonads of both patients with and without residual AR activity.   

Effect of second-generation antihistamines on lymphocyte ABCB1 protein expression

Potěšil J, Kopřiva F, Džubák P, Kratochvílová R, Radová L, Mihál V, Hajdúch M

University Palacky in Olomouc, Faculty of Medicine/Department of Paediatrics, Czech Republic 

The aim of the study: The purpose of this initial study was to compare expression levels of ABCB1 protein in blood lymphocytes in the course of antihistamines therapy. ABCB1 protein (MDR1 protein or P-glycoprotein), member of the ATP-binding cassette (ABC) transporters family, is a transmembrane efflux pump for removal of xenobiotics from the intracellular compartment, such as drugs. The target group was pediatric patients with allergic disease treated by second-generation antihistamines and healthy paediatric patients who did not use any drugs.

Methods: The evaluation of lymphocyte ABCB1 protein expression was performed on samples of 20 children (14 girls and 6 boys) aged 2–16 years (median 7) with mild, intermittent allergic rhinitis (classification of allergic rhinitis as per ARIA 2008) and no other, treated by cetirizine or levocetirizine (n=10), loratadine or desloratadine (n=10) for at least 3 months, used once or twice a day in an amount recommended by the producer, depending on their age and weight. Reference of lymphocyte ABCB1 expression values were obtained from a group of 45 healthy children without therapy (34 boys and 11 girls) aged 0.2–15 years (median 3). The expression of lymphocyte ABCB1 was determined on fixed and permeabilized blood mononuclear cells using indirect immunofluorescence staining technique by flow cytometry.

Results: Based on the weighted medians for ABCB1 expression in peripheral blood lymphocytes data showed a statistically significant increased expression of ABCB1 in the group treated by second-generation antihistamines compared with the healthy control group without treatment (p<10(-6)).

Conclusion: Second-generation antihistamines therapy can lead to overexpression of ABCB1 proteins on the surface of lymphocytes and thus it can reduce accumulation of concomitant drugs in lymphocytes. But only these drugs that are substrates of ABCB1 protein.   

Incidence of diarrheal disease and associated risk factors among under 5 years old children in Kathmandu University Teaching Hospital

Anish Joshi

Department of Paediatrics, Dhulikhel Hospital, Kathmandu University Teaching Hospital, Nepal 

Background: Five million children under the age of 2 years die from diarrheal disease in developing countries each year and rotavirus infections account for about 20% of these deaths. In Nepal, due to lower socio-economic status and poor hygienic condition of the people, intestinal parasitosis is very much prevalent and intestinal pathogens are important causative agents of diarrhea and are one of the major public health problems of the country. Since few community based studies have addressed the epidemiology or etiology of this condition, this study will undertake prospective diarrheal surveillance among children less than 5 years of age presenting in Kathmandu University Teaching Hospital (KUTH). The result of this study is expected to encourage the health care professionals in terms of diagnosis and treatment plan of childhood diarrhoea.

Materials and methods: This study will be a hospital based, prospective, non-interventional study carried out in KUTH, Department of Paediatrics. All children under 5 years presented with acute onset of watery diarrhoea with or without nausea and vomiting seen in outpatient and those admitted in Inpatient department will be enrolled in the study. Data collection for the study will be over 6 months period.

During the study period the parents will be asked to respond questionnaires to record their age, sex, family size, educational status, mother’s employment and knowledge of Oral Rehydration Solution, clinical history including duration of illness, fever, antibiotics used prior to reporting to the hospital, water source and sanitation, personal hygiene and food habit. Socio-demographic condition, anthropometry, clinical status, degree of dehydration, duration of hospital stay and treatment outcome will also be recorded.

Specimen analysis: The fresh stool specimen will be sent for analysis in the laboratory of Dhulikhel Hospital, KUTH. The specimen will be subjected to naked eye examination for consistency, colour, and atypical components (mucus, blood and parasites), then sample will be examined microscopically for the leucocytes, macrophages, RBCs, mucus, reducing substance, fat droplets and parasitic ova and cysts and then the sample will be proceeded by standard microbiology method for culture in order to look for common pathogens. However till date we have not been able to diagnose the viral cause of diarrhoea (eg. Rotavirus). Rotavirus and other viral antigen will be detected using commercially available ELISA kits supported by the Department of Microbiology and Virology from the Medical University of Innsbruck.

Conclusion: The result of this study would help us to understand the etiology of the increasing childhood diarrhea in our hospital and in the surrounding community. This will also help us in the further management of viral diarrhea and also to think about the need of rotavirus immunization in a developing country like Nepal.   

Cartilage hair hypoplasia –  new station

Kanioková P, Xavier BC¹

Charles University, Medical Faculty in Hradec Kralove, Czech Republic

¹CHUV Lausanne; Switzerland/Dept. of Paediatrics; Pédiatrie moleculaire 

Introduction: Cartilage-hair Hypoplasia is a multi-systemic disorder characterized by short stature, blond fine sparse hair, but this may be quite variable, and defective cellular immunity predominantly affecting T-cell mediated responses. Patients may have severe combined immunodeficiency, requiring bone marrow transplantation or they may be asymptomatic. Gastrointestinal dysfunctions are frequently observed such as mal-absorption or Hirschsprung´s disease.

The adult height ranges between 111 and 151 cm in males and between 104 and 137 cm in females. Around 20% of Cartilage Hair Hypoplasia patients exhibit recurrent to severe infections.

RMRP gene is localized 9p21-p12. RMRP is the RNA component of the RNase MRP protein complex.  It functions as a RNA and is not translated into a protein.

The RMRP gene is transcribed by the DNA dependent RNA polymerase III. In bone is more strongly expressed in hypertrophic chondrocytes and pericondrium than in the zone of proliferating chondrocytes. There is also very strong expression in the epiphysis.

Methods: There was analysed DNA from 3 patients (and their parents) with Cartilage-hair Hypoplasia phenotype. There were used following laboratory techniques – DNA amplification by PCR, gel electrophoresis of PCR product, sequencing reactions with fluorescent dyes, capillary elecrophoresis of sequencing reaction product.

Results: There were found mutation and polymorphysm in all 3 families. There were described one new mutation (g.162-166 dup TATCC). There was patient with short statue, seizures, friable hair, dry skin, deep set eyes, epicantic fold.   

Translational research in medulloblastoma: impact for oncoming clinical trials

Zitterbart K¹, Filkova H², Zambo I³, Kren L⁴, Pavelka Z¹, Kuglik P^2,5, Hermanova M^3,4, Veselska R^1,5, Sterba J¹

¹Department of Pediatric Oncology, University Hospital Brno, Czech Republic

²Department of Medical Genetics, University Hospital Brno, Czech Republic

³1st Institute of Pathologic Anatomy, St. Anne’s University Hospital and School of Medicine, Masaryk University, Brno, Czech Republic

⁴Institute of Pathology, University Hospital Brno and School of Medicine, Masaryk University, Brno, Czech Republic

⁵Institute of Experimental Biology, School of Science, Masaryk University, Brno, Czech Republic 

The aim of the study: Medulloblastoma (MB), an embryonal neuroectodermal tumor of the cerebellum, is the most common form of malignant brain tumor of childhood. Identification of biological prognostic markers and cellular targets results in risk-adapted treatment, which is crucial for improving cure rates and reducing long-term sequelae.

Methods: We review the current knowledge of the biological prognostic markers in MB and show the results of our research in this filed.

Results: Several prognostic biomarkers that were identified in retrospective cohorts of medulloblastoma, including MYCC and MYCN amplification, nuclear b-catenin accumulation, and chromosome 17 aberrations, have now been validated in clinical trials as SIOP PNET MB 5 & 6 studies. University Hospital Brno is the National coordinating centre and the National reference centre for biology (MYC status and beta-catenin examination) in these trials.

In our cohort of MB, we revealed limited copy number changes of MYC loci and DeltaNp73 protein overespression as biomarkers associated with poor outcome of patients (Zitterbart et al., Acta Neuropathol 2007, Zitterbart et al., J Neuro-oncol 2011).

Conclusion: We illustrate the importance of further investigations in multicenter trials to better refine the emerging genomic-based prognostic stratification in MB.

We also suggest creating a tissue microarray from a large cohort of MB from Central European region to facilitate translational research that would provide robust data. 

Acknowledgments: This study was supported by grant IGA MZCR NS10218-3/2009.   

Case report: Patient with Down syndrome with Transient Myeloproliferative Disorder complicated with liver fibrosis

Foralová R¹, Mendelová D¹, Zapletal O²

¹Dept. of Pediatric Oncology, University Children´s Hospital in Brno, Czech Republic

²Dept. of Pediatric Hematology, University Childrem´s Hospital in Brno, Czech Republic 

Introduction: Transient myeloproliferative disorder (TMD) is a rare diagnosis of neonates with apparent trisomy of 21st chromosome or its mosaicism, ring formation, Robertsonal translocation and isochromosome 21. TMD occurs in approximately 10% of patients with Down syndrome (Morbus Down - MD) and its usual manifestation hyperleucocytosis and thrombocytopenia, other cytopenias are uncommon. In peripheral blood analysis, the leukemic blasts are present and GATA 1 gene mutation can be demonstrated by molecular methods. In MD spontaneous remission frequently occurs within     3 to 6 months. Unfortunately, 20 to 30% of patients with TMD do not achieve remission or develop Acute Megakaryocytic Leukemia (AMKL- AML/M7) in a few years of their lives. In few cases of TMD or AMKL patients, they develop a rare complication – liver fibrosis, based on expression and secretion of platelet- derived growth factor (PDGF), transforming growth factor beta (TGFβ) and tumor necrosis factor alpha (TNFα) of megakaryoblasts, which is fatal in most cases. The fibrosis can occur even in other organs.

Case report: Our patient was a preterm newborn girl from the 5th pregnancy (2 aborts, 3rd delivery) with no complication, born in the 36th week of pregnancy. The delivery was spontaneous with turbid amniotic fluid, with good postnatal adaptation, Apgar score: 9-9-10, birth weight: 2950 g, birth length 46 cm. The clinical examination brought findings of characteristic stigmatization for Down syndrome and very slight dyspnea, liver and splenic enlargement. The Cardiology showed  normal anatomy and the picture of slight pulmonary hypertension. The 1st postnatal day were found hyperleukocytosis of 140x 10⁹/l and anemia in the peripheral blood, high lactate dehydrogenase and markers of liver function in the biochemistry. The peripheral blood smear (PBS) with 39,7x10⁹ leukocytes with 72% of blast cells led to the suspected diagnosis of transient myeloproliferative disorder (TMD)/AML in Down syndrome with the flowcytometric correlate. In the bone marrow aspiration the infiltration with the blast cells could be detected. The blasts cell count was lower than in the PBS. The diagnosis of TMD was confirmed from PBS in Hannover faculty of medicine- centre of TMD Prevention 2007 study; and the biological marker of GATA1 mutation was confirmed there too. Our patient was enrolled into the TMD Prevention 2007 study and a close follow-up according to the study was recommended. The spontaneous decrease of leukocytes number was visible. The trisomy of the 21st chromosome was confirmed by karyotype: 47,XX+21; and CGH: rev is henh(21)(q11.1qter). 3 weeks later the patient developed the 2nd attack of hyperleukocytosis with 61,5% of blast cells presence in PBS together with liver impairment as hepatomegaly and liver dysfunction. The therapy with low dose ara-c was started - 3 cycles of cytarabine 1,5 mg/kg daily for 7 days was administered. The 1st therapeutic response was shown at week 16 of the study when the cytomorphologic remission was markable, but MRD was still positive. Unfortunately the liver enlargement and even liver dysfunction with obstructive jaundice slowly progressed during the treatment to the life- threatening impairment – suspected liver fibrosis. The dyspnea with pulmonary hypertension got worse and partial respiratory insufficiency and even global respiratory insufficiency occurred. Because of the patient bad general condition there were no liver biopsy had taken.

And because of supposed poor prognosis of liver impairment the life- selvage therapy by corticoids was decided. Patient got 7 pulses of methylprednisolone 5 mg/kg daily, followed by continual therapy with prednisone with dosage of 1 mg/kg. In few days an improvement in overall condition and regression of liver dysfunction was observed. During the next few weeks the patient recovered liver function to normal. The therapy with corticoids was discontinued after 11 weeks of administration. During corticosteroids administration patient surprisingly achieved not only  the cytomorphologic remission (gained after 3. cycle of ara-c) but even the imunophenotypic remission and GATA1 negativity. The patient remains still in complete cytomorphologic, imunophenotypic and molecular remission and her disease free survival is now 28 months.

Conclusion/Discussion: TMD in Down syndrome is a rare diagnosis with supposed good prognosis – 70–80% subsequent remission. An organ impairment – especially liver or pulmonary impairment, with tendency to develop fibrosis decrease outcomes of TMD because the impairment is mostly fatal. The possibilities of reverse of the progressive organ impairment in TMD are practically minimal.  As an inhibitor of PDGF-Rβ receptor for PDGF the imatinib (Gleevec^®, Glivec^®) was considered for our patient. Actually the corticoids were prioritized because of bad condition of the patient and supposition of poor life expectance. A rapid response in liver and pulmonary function helped to return to good overall condition. There still remains a question of corticoids influence for remission attainment. Did we see just the delayed spontaneous remission or the real response to corticoids?