1. SPECTRUM OF THE BTK GENE MUTATIONS IN CZECH XLA PATIENTS Tomáš Freiberger Molecular Genetics Lab Centre for Cardiovascular Surgery and Transplantation University Centre for Primary Immunodeficiencies Brno, Czech Republic

2. X - L INKED A GAMMAGLOBULINEMIA u males after 6 M of age u absence of B lymphocytes in PB, severe hypogammaglobulinemia u increased susceptibility to infections y otitis, sinusitis, bronchitis, pneumonia; skin infections; meningitis, sepsis, osteomyelitis (pyogenic encapsulated bacteria) y diarrhea (Salmonella, Giardia lamblia, Campylobacter) y meningoencephalitis (enteroviruses) u prognosis improved after introduction of IVIG ther.

3. X - L INKED A GAMMAGLOBULINEMIA u mutations in the BTK gene u BTK important for B cell development, role in pre- BCR signaling pathway u cytoplasmic protein: 659 AA, 77 kDa PH TH SH3 SH2 KINASE ~ 140 ~ 75 ~ 65 ~ 100 ~ 280 AA

4. Block in B cell development Pro-B PreB-I Pre-B-II Immature B Mature B CD22 CyIgα TdT CD22 CyIgα CD22 CyIgα CD19 TdT ψL CD10++ CD22 CyIgα CD19 TdT ψL CD10+ CD22 CyIgα CD19 ψL CD10+ Cy μ CD22 CyIgα CD19 CD10+ Cy μ CD22 CyIgα CD19 CD10+ SmIgM CD22 CyIgα CD19 CD10+ SmIgM SmIgD Rag+ DHJH Rag+ VLJL Rag+ VHJH Rag- adapted from Noordzij et al., 2002

5. pre-BCR signaling pathway V-preB λ5/14.1 Syk Igα μ NF-κB Igβ Ca ++ influx Lyn BLNK BTK PLCγ

6. BTK mutations u BTK gene: Xq21.3-22 u 19 exons, 37.5 kb u mRNA 2591 bp u mutations scattered all over the gene http://protein.uta.fi/BTKbase

7. XLA: genotype-phenotype correlation ! weak genotype-phenotype correlation ! mutations in the same domain the same type of mutations the same mutation various number of B cells in PB various immunoglobulin levels various clinical features

8. Agammaglobulinemia: one phenotype various gene defects BTK (X)

9. pre-BCR signaling pathway V-preB λ5/14.1 Syk Igα μ NF-κB Igβ Ca ++ influx Lyn BLNK BTK PLCγ

10. Agammaglobulinemia: one phenotype various gene defects BTK (X) μ chain (AR)

11. pre-BCR signaling pathway V-preB λ5/14.1 Syk Igα μ NF-κB Igβ Ca ++ influx Lyn BLNK BTK PLCγ

12. Agammaglobulinemia: one phenotype various gene defects BTK (X) μ chain (AR) λ5/14.1 (AR)

13. pre-BCR signaling pathway V-preB λ5/14.1 Syk Igα μ NF-κB Igβ Ca ++ influx Lyn BLNK BTK PLCγ

14. Agammaglobulinemia: one phenotype various gene defects BTK (X) μ chain (AR) λ5/14.1 (AR) Igα (AR)

15. pre-BCR signaling pathway V-preB λ5/14.1 Syk Igα μ NF-κB Igβ Ca ++ influx Lyn BLNK BTK PLCγ

16. Agammaglobulinemia: one phenotype various gene defects BTK (X) μ chain (AR) λ5/14.1 (AR) Igα (AR) BLNK (AR)

17. pre-BCR signaling pathway V-preB λ5/14.1 Syk Igα μ NF-κB Igβ Ca ++ influx Lyn BLNK BTK PLCγ

18. Agammagobulinemia: candidate genes for causal mutations V-preB λ5/14.1 Syk Igα μ NF-κB Igβ Ca ++ influx Lyn BLNK BTK PLCγ

19. Agammaglobulinemia: one phenotype various gene defects BTK (X) μ chain (AR) λ5/14.1 (AR) Igα (AR) BLNK (AR) Igβ (AR)

20. pre-BCR signaling pathway V-preB λ5/14.1 Syk Igα μ NF-κB Igβ Ca ++ influx Lyn BLNK BTK PLCγ

21. Agammagobulinemia: candidate genes for causal mutations V-preB λ5/14.1 Syk Igα μ NF-κB Igβ Ca ++ influx Lyn BLNK BTK PLCγ

22. MUTATION DETECTION u DNA from 27 unrelated Czech XLA patients u mutation screening (19 exons) y PCR + DGGE and/or SSCP u determination of mutation y direct sequencing u verification of mutation y restriction analysis y second strand sequencing

23. BTK – 11. EXON (p.E301del) DGGE SSCP m wt wt wt wt wt wt m wt

24. SEQUENCING ATG ATT p.M450I

25. RESULTS I y 23 unique mutations detected in 24 unrelated patients 5 affected male relatives 26 female carriers 10 mutations not described previously y No mutation detected in 3 unrelated patients (all regions sequenced) 1x homozygous deletion of the µH gene (JJ van Dongen) 2x analysis in progress

26. BTKbase (http://bioinf.uta.fi) Czech patients RELATIVE FREQUENCY OF MUTATIONS

27. BTKbase (http://bioinf.uta.fi) Czech patients LOCATION OF MUTATIONS PH TH SH3 SH2 KINASE ~ 140 ~ 75 ~ 65 ~ 100 ~ 280 AA

28. RESULTS II y 6x polymorphism 4x previously described: c.908+70t>c; c.980+78g>a; c.1482-29a>g; c.2031c>t 2x novel: c.103-27g>c; c.1763+71c>t

29. SSCP vs. DGGE u both methods used in 13 cases with proven mutation y DGGE positive in 12/13 patients (sensitivity 92%) y SSCP positive in 10/13 patients (sensitivity 77%) y DGGE or SSCP positive in 13/13 patients (= 100%) u DGGE is efficient method for mutation screening of the BTK gene

30. CONCLUSION u Detection of mutations in the BTK gene enables: y confirmation of diagnosis y identification of mutation carriers y prenatal diagnosis in affected families

31. ACKNOWLEDGEMENTS Barbora Ravčuková Lucie Grodecká Jan Nejedlík Jiřina Bartůňková, Anna Šedivá, Radana Zachová, Václava Gutová, Eva Pařízková, Helena Schneiderová, Olga Škopková, Olga Kryštůfková, Vítězslav Novák Jiří Litzman

33. SSCP wt DNA mutant DNA denaturation different migration pattern of denatured DNA single strands (non-denaturing PAGE) wt m

34. DGGE wt DNA mutant DNA denaturation and slow renaturation wt m low denaturant high denaturant primers - GC clamp