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  • 1. Rare diseases in Greece: Experience of a referral center M. Speletas, A.E. Germenis DEPARTMENT OF IMMUNOLOGY & HISTOCOMPATIBILITY SCHOOL OF MEDICINE – UNIVERSITY OF THESSALY – Larissa, GR www.immunology.edu.gr
  • 2. Our identity • Est. 2003 • Health care services - 700 beds - 2 m population • Outsourcing research services • Research - Tumor-specific CTLs and Immunosenescence - Regulation of Inflammation - Molecular study of PID DEPARTMENT OF IMMUNOLOGY & HISTOCOMPATIBILITY SCHOOL OF MEDICINE – UNIVERSITY OF THESSALY – Larissa, GR www.immunology.edu.gr
  • 3. Rare diseases in Greece Hereditary angioedema Mutational analysis of 3 Greek families with type I HAE • Family A: 4 members • Family B: 3 members • Family C: 4 members Speletas M et al. Hum Immunol (submitted) DEPARTMENT OF IMMUNOLOGY & HISTOCOMPATIBILITY SCHOOL OF MEDICINE – UNIVERSITY OF THESSALY – Larissa, GR www.immunology.edu.gr
  • 4. Rare diseases in Greece Hereditary angioedema Nonsense and missense mutations found in Greek HAE patients Speletas M et al. Hum Immunol (submitted) DEPARTMENT OF IMMUNOLOGY & HISTOCOMPATIBILITY SCHOOL OF MEDICINE – UNIVERSITY OF THESSALY – Larissa, GR www.immunology.edu.gr
  • 5. Rare diseases in Greece Hereditary angioedema SERPING1 molecular analysis – Family A F225X Speletas M et al. Hum Immunol (submitted) DEPARTMENT OF IMMUNOLOGY & HISTOCOMPATIBILITY SCHOOL OF MEDICINE – UNIVERSITY OF THESSALY – Larissa, GR www.immunology.edu.gr
  • 6. Rare diseases in Greece Hereditary angioedema Speletas M et al. Hum Immunol (submitted) DEPARTMENT OF IMMUNOLOGY & HISTOCOMPATIBILITY SCHOOL OF MEDICINE – UNIVERSITY OF THESSALY – Larissa, GR www.immunology.edu.gr
  • 7. Rare diseases in Greece Hereditary angioedema Dinucleotide substitution is an extremely rare mutagenesis mechanism in eukaryotes and usually results in a missense mutation • SERPING1: 8502_03TC>AA  V233E Roche et al, Human Mutation 2005 • factor IX gene: TA>C,  S365G Giannelli et al, Nucleic Acid Res 1992 • factor IX gene: AT>CA  I90H, CTG>TTT  C124F, TGG>AGA  L273Q and E274K Hemophilia B Mutation Database • ret proto-ocnogene: GC>TT  A883F, causing MEN-2B Gimm et al, J Clin Endocrinol Metabol 1997 • Keratin 10: CG>GA  R83E, causing annular epidermolytic ichthyosis Joh et al, J Invest Dermatol 1997 DEPARTMENT OF IMMUNOLOGY & HISTOCOMPATIBILITY SCHOOL OF MEDICINE – UNIVERSITY OF THESSALY – Larissa, GR www.immunology.edu.gr
  • 8. Rare diseases in Greece Hereditary angioedema Conclusion  We described a novel alteration in SERPING1 that constitutes a novel mutagenesis mechanism in HAE  Given a frequency of HAE of 1:50,000 population, the diagnosis of the disease is underestimated in Greece  There is still place for molecular analysis in HAE  The molecular analysis can help to an appropriate diagnosis and treatment DEPARTMENT OF IMMUNOLOGY & HISTOCOMPATIBILITY SCHOOL OF MEDICINE – UNIVERSITY OF THESSALY – Larissa, GR www.immunology.edu.gr
  • 9. Rare diseases in Greece CVID and sIgAD TACI expression and mutational status Transmembrane Activator, calcium modulator and Cyclophilin ligand Interactor a transmembrane receptor mediating isotype switching in B cells Mutations in TNFRSF13B, encoding TACI, were recently reported in 5-20% of patients with CVID, implying their contribution in disease pathogenesis and/or phenotype DEPARTMENT OF IMMUNOLOGY & HISTOCOMPATIBILITY SCHOOL OF MEDICINE – UNIVERSITY OF THESSALY – Larissa, GR www.immunology.edu.gr
  • 10. Rare diseases in Greece CVID and sIgAD TACI expression and mutational status  42 unrelated patients (M/F 22/20, mean age at diagnosis 13.7 yrs (range 1-64) • 24 CVID • 13 sIgAD • 3 sIgG4D • 2 transient hypogammaglobulinemia  108 healthy controls (M/F 44/64, mean age 42.6 yrs (range 7-93) were also analyzed for TACI expression and for the presence of the two most common missense polymorphisms (V220A, P251L) Speletas M et al (paper in preparation) DEPARTMENT OF IMMUNOLOGY & HISTOCOMPATIBILITY SCHOOL OF MEDICINE – UNIVERSITY OF THESSALY – Larissa, GR www.immunology.edu.gr
  • 11. Rare diseases in Greece CVID and sIgAD TACI expression and mutational status WT WT Her Heter V220A Homoz P251L Speletas M et al (paper in preparation) DEPARTMENT OF IMMUNOLOGY & HISTOCOMPATIBILITY SCHOOL OF MEDICINE – UNIVERSITY OF THESSALY – Larissa, GR www.immunology.edu.gr
  • 12. Rare diseases in Greece CVID and sIgAD TACI expression and mutational status  Immunodeficient patients • B cell TACI expression was unrelated with the presence of TNFRSF13B/TACI polymorphisms • B cell TACI expression was found significantly lower than that detected in normal controls (18.9±20.8 vs 34.8±17.2, p=0.018)  Normal controls • The presence of polymorphisms was not associated with hypogammaglobulinemia • Negative correlation was observed between the P251L polymorphism and TACI expression Speletas M et al (paper in preparation) DEPARTMENT OF IMMUNOLOGY & HISTOCOMPATIBILITY SCHOOL OF MEDICINE – UNIVERSITY OF THESSALY – Larissa, GR www.immunology.edu.gr
  • 13. Rare diseases in Greece CVID and sIgAD Conclusion  The TNFRSF13B/TACI coding region does not seem to dispose disease-associated mutations in Greek patients with PID  Observed differences in TACI expression levels indicate a possible functional role of common TNFRSF13B/TACI polymorphisms DEPARTMENT OF IMMUNOLOGY & HISTOCOMPATIBILITY SCHOOL OF MEDICINE – UNIVERSITY OF THESSALY – Larissa, GR www.immunology.edu.gr
  • 14. Rare diseases in Greece Hereditary hemochromatosis type IV (ferroportin disease)  A form of iron overload caused by mutations in the SLC40A1 gene, encoding Fpn  Heterogeneous clinical presentation • hyperferritinemia • macrophage iron deposition • normal transferrin saturation • borderline anemia with low tolerance to phlebotomy • abnormalities similar to classical hemochromatosis (elevated transferrin saturation and iron deposition in hepatocytes) DEPARTMENT OF IMMUNOLOGY & HISTOCOMPATIBILITY SCHOOL OF MEDICINE – UNIVERSITY OF THESSALY – Larissa, GR www.immunology.edu.gr
  • 15. Rare diseases in Greece Hereditary hemochromatosis type IV (ferroportin disease)  Dominant genetic inheritance (unlike other types of hemochromatosis)  20 missence mutations of the SLC40A1 have been reported in individuals from diverse ethnic groups • the vast majority leads to hyperferritinemia and iron overload • for 6 mutations (H174I, D270V, G399D, L384M, L384V, R561G) there are no clinical or laboratory data, and the Q248H mutation may be a functional polymorphism with a high incidence in African- Americans DEPARTMENT OF IMMUNOLOGY & HISTOCOMPATIBILITY SCHOOL OF MEDICINE – UNIVERSITY OF THESSALY – Larissa, GR www.immunology.edu.gr
  • 16. Rare diseases in Greece Hereditary hemochromatosis type IV (ferroportin disease)  Mutational analysis of SLC40A1 is currently performed at the genomic DNA level by PCR amplification and direct sequencing (usually in 9 PCR reactions)  We apply a new protocol for the rapid detection of mutations at the mRNA level (two RT-PCR reactions followed by direct sequencing and/or NIRCA (non-isotopic RNase cleavage assay)  We detect SLC40A1 alterations in two female patients and we analysed their family members Speletas M et al. Blood Cells Mol Dis 2008;40:353 DEPARTMENT OF IMMUNOLOGY & HISTOCOMPATIBILITY SCHOOL OF MEDICINE – UNIVERSITY OF THESSALY – Larissa, GR www.immunology.edu.gr
  • 17. Rare diseases in Greece Hereditary hemochromatosis type IV (ferroportin disease) Speletas M et al. Blood Cells Mol Dis 2008;40:353 DEPARTMENT OF IMMUNOLOGY & HISTOCOMPATIBILITY SCHOOL OF MEDICINE – UNIVERSITY OF THESSALY – Larissa, GR www.immunology.edu.gr
  • 18. Rare diseases in Greece Hereditary hemochromatosis type IV (ferroportin disease) Speletas M et al. Blood Cells Mol Dis 2008;40:353 DEPARTMENT OF IMMUNOLOGY & HISTOCOMPATIBILITY SCHOOL OF MEDICINE – UNIVERSITY OF THESSALY – Larissa, GR www.immunology.edu.gr
  • 19. Rare diseases in Greece Idiopathic hypereosinophlic syndrome with FIP1L1-PDGFRA rearrangement  Heterozeneous disease entity (myloproliferative, lymphoproliferative, idiopathic) • Eosinophilia (>1.5×109/L) for more than 6 months • Exclusion of reactive eosinophilia caused by parasitic infections, allergies, or other known causes, as well as eosinophilia associated with neoplasias • Evidence of end-organ damage  Without treatment  bad prognosis • mean survival 9 months • survival after 3 years 12% of patients DEPARTMENT OF IMMUNOLOGY & HISTOCOMPATIBILITY SCHOOL OF MEDICINE – UNIVERSITY OF THESSALY – Larissa, GR www.immunology.edu.gr
  • 20. Rare diseases in Greece Idiopathic hypereosinophlic syndrome with FIP1L1-PDGFRA rearrangement  The most frequent genetic aberration is the cryptic deletion of 4q12, producing the FIP1L1-PDGFRA fusion transcript, which results in an eosinophilic, myeloproliferative disorder (chronic eosinophilic leukemia, CEL)  FIP1L1-PDGFRA is present in approximately 10–15% of patients with HES DEPARTMENT OF IMMUNOLOGY & HISTOCOMPATIBILITY SCHOOL OF MEDICINE – UNIVERSITY OF THESSALY – Larissa, GR www.immunology.edu.gr
  • 21. Rare diseases in Greece Idiopathic hypereosinophlic syndrome with FIP1L1-PDGFRA rearrangement Loules G et al. BMC Blood Disord 2009;9:1 DEPARTMENT OF IMMUNOLOGY & HISTOCOMPATIBILITY SCHOOL OF MEDICINE – UNIVERSITY OF THESSALY – Larissa, GR www.immunology.edu.gr
  • 22. Rare diseases in Greece Idiopathic hypereosinophlic syndrome with FIP1L1-PDGFRA rearrangement in both patients the breakpoints into PDGFRA were located in exon 12 (between two W), interrupting the PDGFRA juxtamembrane region (auto-inhibitory domain) and resulting in a constitutive kinase activation and the leukemic transformation of the affected cells Loules G et al. BMC Blood Disord 2009;9:1 DEPARTMENT OF IMMUNOLOGY & HISTOCOMPATIBILITY SCHOOL OF MEDICINE – UNIVERSITY OF THESSALY – Larissa, GR www.immunology.edu.gr
  • 23. Rare diseases in Greece  Our effort contributes significantly towards the identification and the earlier diagnosis of a series of underdiagnosed cases  The results of the molecular analysis suggest that differences in the genetic background of some RD might exist in our geographical region, indicating that our intended participation in a Balkan initiative could benefit the elucidation of pathogenetic aspects of these diseases DEPARTMENT OF IMMUNOLOGY & HISTOCOMPATIBILITY SCHOOL OF MEDICINE – UNIVERSITY OF THESSALY – Larissa, GR www.immunology.edu.gr