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, University Children’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) No. of patients Mean age at diagnosis (y)Range (y) Mean F/u period (y) Range of F/u (y) Slovenia103.11.2 – 7.417.91.0 – 34.3 Czech Republik102.80.7 – 8.09.91.9 – 29.0 Serbia83.30.2 – 7.913.74.3 – 25.5 Slovakia68.31.0 – 12.514.13.0 – 22.0

7. Clinical manifestations - presentation Presenting infection SLO (n)CZ (n)SRB (n)SK (n) Median age4m12m13m4.7y Lymphadenitis14 (45%)4 (3xBCG) 3 (3xBCG) 4 (2xBCG) 3 (2xBCG) Septicemia4 (13%)112 Respiratory disease4 (13%)112 Skin disease3 (10%)21 GI disease3 (10%)21 Liver abcess1 (3%)1 Other (otitis, sistits) 2 (6%)2 Clinical data for 31/34 patients Mean age at presentation 18.4m (4 - 208m) BCG lympadenitis was most common presenting infection (32%) n=9 n=6n=7

8. Clinical manifestations – infections No. of Episodes No. of affected Pt. SLOCZSRBSK RT infection and abscesses5119148929224/31 (77%) GI infections and abscesses30192126320/31 (65%) Lymphadenitis and lymph node abscesses161812146027/31 (87%) Dermatitis and skin abscesses229433815/31 (48%) Ear, nose, throat infections209073613/31 (42%) Urinary tract infections410054/31 (13%) Osteomyelitis922114147/31 (23%) Septicemia158213133813/31 (42%) Total No. of episodes16716785363658 345 345 different severe infectious episodes in 458.5y of F/u (0.8 severe infection per year)

9. Infectious organisms Affected patients Relative frequency (% of 184 isolates) Staphylococcus aureus22 / 31 ( 71%)30 Aspergillus sp.15 / 31 (49%)15 Streptococcus sp.8 / 31 (26%)8 Salmonella sp.7 / 31 (23%)8 Candida albicans7 / 31 (23%)7 Haemophilus influenzae3 / 31 (10%)4 Burkholderia cepacia3 / 31 (10%)2 Proteus sp.2 / 31 (6%)3 Serratia marcescens2 / 31 (6%)3 Klebsiella sp.2 / 31 (6%)1 Other1/31 (3%) 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. I II H III H IV HVHHVH VI N C 8 2944122168225267 6997191203290 membrane cytosol extracellular space NADPH FAD S8 S9 sK4, sK5 S3 sB6 533delGly, S3 674+4A>C, sK4, sK5 45+1G>A, S8 Leu66Pro, S9 gp91 phox sB3 Arg54_Ala55del, sB6 His338Gln, sB3 sB7 483+1G>T, sB7 C2 Ser112Pro, C2 S10 Cys64Arg, S10

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,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 PrahaBeograd Aleš Janda Srđan Pašić Andrea Poloučková Anna Šedivá BrnoBratislava Tomáš FreibergerPeter Čižnár

16. povabilo v SLO

19. Results of molecular diagnostics mutation P5ex1 ds+1G>A C3R43X S1R54-A55del P6L66P P3R73X C1R91X C2S112P Sl1R159fsX170 P7,P 8 R290X S2H338Q P1P346fsX384 S3W361X P2533delG Serb Slovene Czech Slovak patient mother P5 CYBB exon 1 ds+1G>A de novo mutation P6 patient mother sister, donor of BM Mutation: CYBB exon 3 197 T>C L66P C2 patient mother sister Mutation: CYBB exon 4 334 T>C S112P Sl1 patient mother sister Mutation: CYBB exon 5 474-481del7nt R159fsX170 del AATAAAG patient mother S3 CYBB exon 9 1083G>A, W361X G