Dr Gigliola Di Matteo Dr Andrea Finocchi Prof. Paolo Rossi

1. Molecular Analysis of Chronic Granulomatous Disease (CGD).Applications for a rapid method of diagnosis using DHPLC Dr Gigliola Di Matteo Dr Andrea Finocchi Prof. Paolo Rossi

2. Chronic Granulomatous Disease (CGD) is an immunodeficiency that affects phagocytes of the innate immune system and is characterized by a greatly increased susceptibility to severe bacterial and fungal infections of the subcutaneous tissues, lungs, lymphonodes, liver and bones. Patients with Chronic Granulomatous Disease are defective in the enzymatic system of the phagocytes known as NADPH oxidase that acts mainly by transferring electrons from NADPH to molecular oxygen in order to form superoxide. The main goal is to kill ingered microorganisms in particular catalase-positive.

3. Flavocytocrome b 558 outside Fe Fe Rac GTP gp91 gp91 p22 p22 Fe Fe FAD FAD p67 p47 p40 Rac GDP Rho GDI p67 p47 inside p40 MODEL OF PHAGOCYTE NADPH OXIDASE (RESPIRATORY BURST) ACTIVATION O2 - O2 outside e activation membrane e P P P P P P P P NADP+ + H+ inside Heyworth PG et al., 2003 Current Opinion in Immunology 15:578

4. CGD p22-phox Rac2 CYBA gene p47-phox p67-phox Ncf-1 gene Ncf-2 gene Gp91-phox CYBB gene

5. Classification 95% OF CGD MUTATIONS RESULT IN A COMPLETE ABSENCE OR GREATLY DIMINISHED LEVEL OF PROTEIN

6. CYBB GENE and gp91 protein

7. METHODS • DHPLC (Denaturating High Performance Liquid Chromatography) • SEQUENCING

8. DHPLC (Denaturating High Performance Liquid Chromatography) DNA is allowed to bind a hydrophobic column in a buffer of triethyl ammonium acetate and is eluted with an increasing gradient of acetonitrile, under certain key parameters of temperature and buffer concentration, partial denaturation of the double stranded DNA occurs. IF THE SAMPLE CONTAINS HETERODUPLEX MOLECULES (PRESENCE OF MUTATIONS OR POLYMORPHISMS), THESE WILL BE VISUALIZED AS A PEAK OR PEAKS WITH SHORTER RETENTION TIMES THAN HOMODUPLEXES.

9. DETECTION OF MUTATIONBY DHPLC Denaturation and Rinaturation • PROTOCOL: • AMPLIFICATION OF SAMPLES AND CONTROLS. • DENATURATION AND RINATURATION OF PCR PRODUCTS MIXED IN EQUIMOLAR • QUANTITY OF SAMPLE AND CONTROL (FORMATION OF HETERODUPLEX AND • HOMODUPLEX MOLECULES) • CHROMATOGRAPHIC ANALISYS AT DIFFERENT CONDITIONS OF TEMPERATURE AND • BUFFER CONCENTRATION.

10. CGD PATIENTS

11. Frameshift Phe62X66 MOLECULAR ANALYSIS Ex 3 CGD1 t184del Ex 9 g1123>t Glu375X CGD3

12. Ex 3 Splicing defect CGD4 g252>a Ishibashi et al. Blood 2001 CGD10 Ex11 t1357>a CGD7-8 Trp453>Arg

13. Ex 9 CGD5 g1006>t Glu336X Ex 9 g1076>c Gly359Ala CGD9 Ex 11

15. NCF-1 GENE (CHROMOSOME 7q11.23) GTGT Intron 2 c ex1 ex2 ex11 NCF-1 gene 20 bp DGT YNCF-1 t ex1 ex11 20 bp X 2 GTGT YNCF-1 typeII t ex1 ex11 20 bp X 2 Unaffected controls (non-CGD) 2:1 1:1 1:2 A47° CGD carriers A47° patient 5:1 2:1

16. CGD6 PATIENT

17. CONCLUSIONS ADVANCES IN PRENATAL DIAGNOSIS AND GENETIC COUNSELLING ANALYSIS OF THE OTHER COMPONENTS OF THE NADPH OXIDASE SYSTEM STUDIES OF GENOTYPE-PHENOTYPE CORRELATION