Pws Tim

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Pws Tim

  1. 1. NEW TECHNIQUES OF DIAGNOSIS IN PRADER - WILLI SYNDROME Assist. Prof. CRISTINA RUSU, MD PhD “ Gr T Popa” University of Medicine and Pharmacy Medical Genetics Department Iasi, Romania
  2. 2. GENERAL DATA <ul><li>Prader Willi syndrome (PWS) - genetic disorder produced by abnormalities of the 15q11-q13 region; </li></ul><ul><li>CLINICAL PICTURE : </li></ul><ul><ul><li>severe hypotonia, feeding difficulties (early infancy); </li></ul></ul><ul><ul><li>marked increase of appetite -> severe obesity (after 2 years); </li></ul></ul><ul><ul><li>dysmorphic face, small extremities, hypogonadism and developmental delay - frequently associated. </li></ul></ul>
  3. 3. GENERAL DATA (cont.) <ul><li>GENETIC DEFECTS: </li></ul><ul><ul><li>Microdeletions; </li></ul></ul><ul><ul><li>Uniparental disomy (UPD); </li></ul></ul><ul><ul><li>Imprinting defects; </li></ul></ul><ul><ul><li>Deletions; </li></ul></ul><ul><li>INVESTIGATION PROTOCOL : </li></ul><ul><ul><li>FISH test -> microdeletions; </li></ul></ul><ul><ul><li>Methylation study -> UPD, imprinting defects ; </li></ul></ul><ul><ul><li>Karyotype (chromosome study) -> deletions . </li></ul></ul>
  4. 4. MLPA (Multiplex Ligation Probe Amplification) <ul><li>Multiplex PCR-based method; </li></ul><ul><li>Detects deletions/ duplications; </li></ul><ul><li>Up to 50 different DNA/ RNA sequences analysed in the same time; </li></ul><ul><li>Able to distinguish sequences differing in one nucleotide; </li></ul><ul><li>Simple method - requires a thermocycler and capillary electrophoresis equipment; </li></ul><ul><li>Up to 96 samples handled simultaneously; </li></ul><ul><li>Results available in 24 hours. </li></ul>
  5. 5. <ul><li>DNA denaturation and hybridisation of MLPA probes; </li></ul><ul><li>Ligation reaction; </li></ul><ul><li>PCR reaction; </li></ul><ul><li>Separation of amplification products by electrophoresis; </li></ul><ul><li>Data analysis </li></ul>
  6. 6. MLPA - Advantages <ul><li>Cheap; </li></ul><ul><li>Multiplex reaction – checks multiple sequences in the same time; </li></ul><ul><li>May identify very small defects; </li></ul><ul><li>Very different applications, but using the same principle. </li></ul>
  7. 7. MLPA - Applications <ul><li>Prenatal diagnosis (identification of aneuploidies); </li></ul><ul><li>Idiopatic mental retardation (identification of subtelomeric rearrangements); </li></ul><ul><li>Microdeletion syndromes (e.g. PWS, Williams etc); </li></ul><ul><li>Exon deletions for specific genes (e.g. Duchenne, BRCA etc); </li></ul><ul><li>Pharmacogenetics (analysis of specific polymorphisms involved in drug reaction); </li></ul><ul><li>Methylation analysis (e.g. PWS, Wiedemann Beckwith, Fragile X etc); </li></ul><ul><li>mRNA analysis (e.g. inflamation); </li></ul>
  8. 8. MS - MLPA <ul><li>probe–DNA complex is simultaneously ligated and digested by methylation-specific enzymes; </li></ul><ul><li>If the CpG site is methylated -> a normal MLPA product detected; </li></ul><ul><li>If the CpG site is not methylated -> DNA–probe complex digested by the methylation-sensitive enzyme -> no amplification product. </li></ul>
  9. 9. Our protocol <ul><li>Clinical selection (using the diagnostic score); </li></ul><ul><li>Karyotype (identify deletions); </li></ul><ul><li>MLPA (separate kits for microdeletions and for methylation analysis; used as a screening test to identify microdeletions or imprinting defects); </li></ul><ul><li>FISH (confirm microdeletion); </li></ul><ul><li>Methylation analysis (confirm imprinting defect, UPD); </li></ul><ul><li>Epigenetic studies (identify other factors involved; study multiple generations). </li></ul>
  10. 10. THANK YOU ! [email_address]

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