clinical characteristics and molecular analysis of 34 central european patients with chronic...
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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)
• 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 inheritanceNCF1 gene (p47phox) NCF2 gene (p67phox) CYBA gene (p22phox)
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
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
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).
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
patientsMean age at diagnosis (y) Range (y)
Mean F/u period (y)
Range of F/u (y)
Slovenia 10 3.1 1.2 – 7.4 17.9 1.0 – 34.3
Czech Republik 10 2.8 0.7 – 8.0 9.9 1.9 – 29.0
Serbia 8 3.3 0.2 – 7.9 13.7 4.3 – 25.5
Slovakia 6 8.3 1.0 – 12.5 14.1 3.0 – 22.0
Clinical manifestations - presentation
Presenting infection
SLO (n) CZ (n) SRB (n) SK (n)
Median age 4m 12m 13m 4.7y
Lymphadenitis 14 (45%) 4 (3xBCG) 3 (3xBCG) 4 (2xBCG) 3 (2xBCG)
Septicemia 4 (13%) 1 1 2
Respiratory disease 4 (13%) 1 1 2
Skin disease 3 (10%) 2 1
GI disease 3 (10%) 2 1
Liver abcess 1 (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=9 n=6n=7
Clinical manifestations – infections
No. of Episodes
No. of affected Pt.
SLO CZ SRB SK
RT infection and abscesses 51 19 14 8 92 24/31 (77%)
GI infections and abscesses 30 19 2 12 63 20/31 (65%)
Lymphadenitis and lymph node abscesses 16 18 12 14 60 27/31 (87%)
Dermatitis and skin abscesses 22 9 4 3 38 15/31 (48%)
Ear, nose, throat infections 20 9 0 7 36 13/31 (42%)
Urinary tract infections 4 1 0 0 5 4/31 (13%)
Osteomyelitis 9 2 2 1 14 7/31 (23%)
Septicemia 15 8 2 13 38 13/31 (42%)
Total No. of episodes 167 85 36 58 345
345 different severe infectious episodes in 458.5y of F/u (0.8 severe infection per year)
Infectious organismsInfectious organisms Affected patients
Relative frequency
(% of 184 isolates)
Staphylococcus aureus 22 / 31 ( 71%) 3030
Aspergillus sp. 15 / 31 (49%) 1515
Streptococcus sp. 8 / 31 (26%) 88
Salmonella sp. 7 / 31 (23%) 88
Candida albicans 7 / 31 (23%) 77
Haemophilus influenzae 3 / 31 (10%) 44
Burkholderia cepacia 3 / 31 (10%) 22
Proteus sp. 2 / 31 (6%) 33
Serratia marcescens 2 / 31 (6%) 33
Klebsiella sp. 2 / 31 (6%) 11
Other 1/31 (3%)
Other bacterial infections: Nocardia, Pseudomonas aer., Enterobacter, Acinetobacter, Propionibacter
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
I IIHIIIH
IVHVH
VI
N
C
8
29 44 122 168 225 267
69 97 191 203 290
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
gp91phox
sB3
Arg54_Ala55del, sB6
His338Gln, sB3
sB7 483+1G>T, sB7
C2
Ser112Pro, C2
S10
Cys64Arg, S10
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)
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.
Contact mail
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)
Acknowledgement
Praha Beograd
Aleš Janda Srđan Pašić
Andrea Poloučková
Anna Šedivá
Brno Bratislava
Tomáš Freiberger Peter Čižnár
povabilo v SLO
Results of molecular diagnosticsmutation
P5 ex1 ds+1G>A
C3 R43X
S1 R54-A55del
P6 L66P
P3 R73X
C1 R91X
C2 S112P
Sl1 R159fsX170
P7,P8
R290X
S2 H338Q
P1 P346fsX384
S3 W361X
P2 533delG
Serb
SloveneCzech
Slovakpatient
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