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Markelj G, Debeljak M, Avčin T Department of Allergology, Rheumatology and Clinical Immunology,

Clinical characteristics and molecular analysis of 34 Central European patients with chronic granulomatous disease. Markelj G, Debeljak M, Avčin T Department of Allergology, Rheumatology and Clinical Immunology, University Children’s Hospital, Ljubljana. Chronic Granulomatous Disease (CGD).

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Markelj G, Debeljak M, Avčin T Department of Allergology, Rheumatology and Clinical Immunology,

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  1. Clinical characteristics and molecular analysis of 34 Central European patients with chronic granulomatous disease Markelj G, Debeljak M, Avčin T Department of Allergology, Rheumatology and Clinical Immunology, UniversityChildren’s Hospital, Ljubljana

  2. Chronic Granulomatous Disease (CGD) • Rare inherited disorder (1/ 100 000 - 250 000)of the innate immune system characterized by the failure of phagocytic cells to produce superoxide and its toxic derivates • defects in any of 4 genes encoding protein components of the phagocyte NADPH oxidase • Type of mutation • X-Linked inheritance (65% patients) • CYBB gene (gp91phox) • Autosomal inheritance • NCF1 gene (p47phox) • NCF2 gene (p67phox) • CYBA gene (p22phox)

  3. Objectives • Analyze epidemiological, clinical and genetic features in Central European CGD population • Investigate genotype - phenotype correlation • Compare features of CE CGD population with other CGD patients

  4. Methods • Patients diagnosed with CGD from Central European countries • Clinical data were provided by the clinicians that take care of the CGD patients. • Detailed data abstraction form • epidemiological information • clinical information: • presenting symptoms • organ specific infections and isolated MO

  5. Methods • Genetic analysis of CYBB gene: • Genomic DNA was isolated from whole blood stored in EDTA or was sent to our laboratory from patients’hospitals already isolated • PCR amplification of all exons and the exon-intron boundaries of the gene. • Directly sequenced and sequences were compared a normal gene sequence from the Genebank • Novel mutations were identified and named starting numbering from AUG codon (Gene Bank Access No. AF469757).

  6. Results as of 1.4. 2009 • 34 patients, all male • 4 different central European countries (Slovenia, Czech Republic, Serbia, Slovakia) • Mean age 14.1y (0.2 – 34.3y) • Mean age of diagnosis 4.2y (0.2 – 12.5y) • Mean follow up period 13.8y (1.0 – 34.3y)

  7. Clinical data for 31/34 patients Clinical manifestations - presentation Mean age at presentation 18.4m (4 - 208m) BCG lympadenitis was most common presenting infection (32%) n=9 n=9 n=7 n=6

  8. Clinical manifestations – infections 345 different severe infectious episodes in 458.5y of F/u (0.8 severe infection per year)

  9. Infectious organisms Other bacterial infections: Nocardia, Pseudomonas aer., Enterobacter, Acinetobacter, Propionibacter

  10. Results of molecular diagnostics • 33 samples of DNA (could not perform the testing on 4 samples due to amount and quality of the DNA) • 29 CYBB genes: in 3 patients we did not find mutation on CYBB gene • 22 different mutations, 9 not yet described

  11. gp91phox sB7 Cys64Arg, S10 483+1G>T, sB7 extracellular space sK4, sK5 Arg54_Ala55del, sB6 Leu66Pro, S9 29 44 122 168 225 267 II 674+4A>C, sK4, sK5 I H III H IV H V H VI sB6 membrane C2 S8 8 69 97 191 203 290 S10 S9 N 45+1G>A, S8 Ser112Pro, C2 C cytosol NADPH FAD sB3 S3 His338Gln, sB3 533delGly, S3

  12. Work in progress • Include additional patients from other central European countries • Include the epidemiological information on all known patients diagnosed with CGD in participating countries • Protein expression analysis in patients with normal CYBB gene, and further genetic analysis of other CGD genes (CYBA, NCF1, NCF2)

  13. Conclusion • Patients included in our study have similar frequencies of infections and infecting microorganisms as patients described in other series • We have tested 33 patients for mutations on CYBB gene • We have found 22 different mutations, 9 so far not yet described. Each familiy has its own specific mutation. • In 3 patients mutations are not yet determined.

  14. Contact mail • gmarkelj@gmail.com, • tadej.avcin@kclj.si  Genetic analysis available free of charge (Grant of the Slovenian Research Agency) • isolated DNA CYBB 10μg of DNA, other CGD genes 40μg of DNA • EDTA blood sample (5 ml)

  15. Acknowledgement Praha Beograd Aleš Janda Srđan Pašić Andrea Poloučková Anna Šedivá Brno Bratislava Tomáš FreibergerPeter Čižnár

  16. povabilo v SLO

  17. Mutation: CYBB exon 3 197 T>C L66P P6 Mutation: CYBB exon 4 334 T>C S112P C2 Mutation: CYBB exon 5 474-481del7nt R159fsX170 P5 CYBB exon 1 ds+1G>A de novo mutation S3 CYBB exon 9 1083G>A, W361X Sl1 Slovene Czech Serb Slovak del AATAAAG patient patient patient patient patient mother mother mother G sister, donor of BM mother mother sister sister Results of molecular diagnostics

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