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Mike Oldridge FRC path 17/9/2010

What is an imprinted gene? Using PWS and AS as an example describe the imprinting control centre and explain how the imprint is maintained or erased within the germ line. What are the clinical and diagnostic implications of errors in this process?. Mike Oldridge FRC path 17/9/2010.

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Mike Oldridge FRC path 17/9/2010

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  1. What is an imprinted gene? Using PWS and AS as an example describe the imprinting control centre and explain how the imprint is maintained or erased within the germ line. What are the clinical and diagnostic implications of errors in this process? Mike Oldridge FRC path 17/9/2010

  2. Imprinted genes • An imprinted gene is one in which only one allele is expressed dependent upon its parental origin • Example of epigenetic mechanism • Chromatin modification and DNA methylation • For some chromosomal segments UPD is pathogenic • Mouse models have defined many of these regions • Currently thought to be ~90 human imprinted genes

  3. Imprinted genes • Many involved in controlling growth – paternally expressed genes involved in foetal growth maternally expressed genes limit foetal growth • Theory as to why imprinting exists - conflict of evolutionary interest between mother and father?

  4. Imprinted genes • Imprinting can be confined to certain tissues or stages of development

  5. Common features of imprinted genes • Often found in clusters with both maternally and paternally expressed alleles • Overlapping and oppositely imprinted sense and antisense transcripts only one of which can be expressed at any one time – appears to be act of transcription rather than antisense transcript’s function that prevents transcription on opposite strand • Controlled by one or more small regions that are differentially methylated on the maternal and paternal chromosomes

  6. Imprinting Control Centres (ICs) • Also called Imprinting Control Regions (ICRs) or Differentially Methylated Regions (DMRs) • Cis-acting elements which can have long distance effects • Marked differently in male and female germlines • Maintain imprint in somatic cells • Control imprinting via differential chromatin structure and methylation states on maternal and paternal chromosomes

  7. 2 mechanisms 1. Insulator model – eg IGF2/H19 chrm 11p (BWS) compete for enhancer Binding of CTCF insulator protein to a differentially methylated region determines outcome of the competition

  8. 2 mechanisms • Non-coding RNA model – transcription of nc RNAs regulates (silences) transcription of other genes Good example is the Prader-Willi and Angelman syndrome imprinting region which has a bipartite IC

  9. Prader-Willi (PWS) / Angelman (AS) syndromes • Recurrent microdeletion of 15q11-15q13 -homologous recombination between low copy no. rpts • Leads to 2 different syndromes • Found to be dependent on parental origin of deletion • Paternal deletion – PWS • Maternal deletion – AS • Difference in methylation states at certain loci • PW/AS has provided much insight into imprinting

  10. Clinical features

  11. PW/AS imprinted region

  12. Imprinting Centres (ICs)

  13. Maintaining / erasing imprint • During gametogenesis, de novo methyltransferases (Dnmt3A and 3L) methylate DMRs in a gamete specific manner thereby differentiating parentally derived alleles • After fertilization, maintenance methyltransferases (Dnmt1) maintain the methylation state

  14. Causes of PWS and AS • PWS due to loss of paternally expressed allele • AS due to loss of maternally expressed allele

  15. Clinical implications PW IC del can be passed from female to female with no phenotypic consequence (maternal genes are expressed) Converse for AS IC dels

  16. Clinical implications

  17. Diagnostic implications • Based upon determining methylation status of SNRPN • MS PCR – based on sodium bisulphite treatment of DNA followed by PCR using primers specific for methylated sites within the SNRPN promoter region • MS southern blotting – using methylation sensitive restriction enzymes • MS-MLPA

  18. Normal Results Copy no. Results Methylation Results 100% 50% Digest Controls: Should be absent Digest Controls: Should be present Differentially methylated alleles: Should be 50% Control probes Chr15q11-13 probes: in order down chrm 15q Control probes

  19. Deletion Results Copy no. Results PWS  AS  100% Half heights of probes in PWS/ AS Critical region =  MethylationResults 50% Doubling of methylation signal Loss of methylation signal

  20. UPD/ IC Results Copy no. Results PWS UPD/ IC AS UPD/ IC 100% Equal heights of probes in PWS/ AS Critical region = NO  Methylation Results 50% Doubling of methylation signal Loss of methylation signal

  21. Key words Imprinting Parent of origin Epigenetics Imprinting centres Chromatin modification Methylation PWS/AS

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