Alison Skinner RCPath self help 6 th November 2008

1. How can aberrant methylation give rise to disease? Provide examples of disorders where methylation changes are important and describe the current diagnostic methods used to detect methylation status Alison Skinner RCPath self help 6th November 2008

2. How Can Aberrant Methylation Give Rise to Disease

3. DNA Methylation • Epigenetic modification • In humans methylation occurs at CpG dinucleotides • It involves the addition of a methyl group to the C5 position of CpG dinucleotides creating 5-methylcytosine

4. DNA Methylation Methyltransferases catalyse the transfer of a methyl group from S-adenosylmethionine (SAM) to the C5 position of cytosine creating 5-methylcytosine

5. How can Aberrant DNA Methylation Give Rise to Disease? • DNA methylation usually occurs in repetitive genomic regions such as SINEs, LINEs and endogenous retroviruses / transposons • Prevents the use of transposon promoters • Helps to maintain genomic stability as methylated DNA is more tightly condensed around histones which protects against illegitimate recombination

6. How can Aberrant DNA Methylation Give Rise to Disease? • Aberrent (hypo)methylation in these repetitive regions can cause disease by: • Allowing transcription of genes that should be silenced through the use of ‘unmasked’ promoters • Generating antisense transcripts through the use of ‘unmasked’ promoters, effectively silencing other genes that should normally be transcribed and translated • Causing genomic instability as the hypomethylated repetitive regions become more prone to recombination

7. How can Aberrant DNA Methylation Give Rise to Disease? • CpG islands around promoters are generally unmethylated as methylation represses transcription by preventing the binding of transcription factors and recruiting chromatin remodelling complexes • There are exceptions to this and some promoters are methylated in an allele specific manner, these are know as differentially methylated regions (DMRs); the dosage of these gene products is usually important for development. • Genes whose expression is controlled by these DMRs are referred to as imprinted

8. How can Aberrant DNA Methylation Give Rise to Disease? • Aberrant methylation at imprinted loci can cause disease by: • Changing the dosage of these gene products resulting in either too much gene product or too little / no gene product • Causes of aberrant methylation in imprinted loci are usually due to either imprinting control region (ICR) mutations, uniparental disomy (UPD) or a deletion / duplication of one allele which alters the dosage of the gene product

9. Provide examples of disorders where methylation changes are important

10. Fragile X • FMR1/FMRP is important for development of neurones, and people who have fragile X syndrome often have mental retardation • The promoter of FMR1 is unmethylated in normal and premutation individuals with <200 repeats allowing the FMRP protein to be transcribed and translated • In individuals with fragile X syndrome (>~200 repeats), FMR1 is not transcribed as the repeated sequence in the promoter is methylated

11. Fragile X

12. Cancer • Genomes of cancer cells are often hypomethylated in the repetitive regions of the genome – predisposing to genomic instability, a hallmark of many types of cancer • Genes involved in cell-cycle regulation and DNA repair often have hypermethylation at their promoters causing the gene to be silenced (this is often the first ‘hit’) An example of this is hypermethylation at the hMLH1 promoter in HNPCC.

13. Cancer – Loss of Imprinting (LOI) • LOI activating the usually silent allele occurs in growth promoting imprinted genes increases the expression of the gene product leading to a growth advantage • An example of this is the LOI at the IGF2 / H19 locus which was first discovered in Wilms’ tumour • LOI silencing the active allele of growth-inhibitory genes may lead to deregulated cell growth • An example of this is LOI at the p57KIP2 which also occurs in some Wilms’ tumours

14. Imprinted Regions – Beckwith-Wiedemann Syndrome (BWS) • BWS is a overgrowth syndrome • Two imprinting control regions: ICR1 is in the H19 region which controls IGF2 expression, and ICR2 is in the KCNQ1 (KvDMR) region which controls CDKN1C (p57KIP2) • BWS is caused by hypermethylation at ICR1 and/or hypomethylation at ICR2 • Gain of methylation at H19 can cause BWS by increasing the dosage of IGF2 (a growth factor) • Loss of methylation at KvDMR can cause BWS by decreasing the dosage of the p57KIP2 (CDKN1C), a growth inhibitor

15. Imprinted Regions - TNDM • Transient neonatal diabetes mellitus • Caused by loss of the maternally imprinted allele at 6q24 leading to increased expression of the ZAC (PLAGL1 / LOT1) gene and reduced expression of the ZAC antisense which is also involved in ZAC regulation

16. Imprinted Regions – Prader-Willi Syndrome and Angelman Syndrome • Prader-Willi syndrome is characterised by failure to thrive in the first year followed by rapid weight gain thereafter • Prader-Willi syndrome is cause by the loss of the paternal unmethylated allele at the PWS-ICR • This results in the loss of transcription of many genes in the 15q11-13 region including SNURF/SNRPN, NDN and ZNF127

17. Imprinted Regions – Prader-Willi Syndrome and Angelman Syndrome • Angelman syndrome is characterised by severe learning disability, lack of speech, abnormal EEG, seizures, happy demeanour and movement / balance disorder • Angelman syndrome is caused by the loss of maternally expressed UBE3A

18. Imprinted Regions – Other Disorders Associated with Aberrant Methylation • Russell-Silver syndrome can be caused by maternal UPD7 (exact locus is unknown at present) or loss of methylation at the H19 (ICR1) locus at 11p15.5 • Pseudohypoparathyroidism type 1b is caused by the loss of the maternally imprinted region at 20q13 (GNAS locus) • 14q32.2 has a DMR that causes different disease phenotypes depending on whether the maternally or paternally imprinted allele is lost • Recently, constitutive mutations in the gene ZFP57 have been shown to cause a hypomethylation syndrome at multiple loci (including TNDM and BWS loci)

19. Describe the current diagnostic methods used to detect methylation status

20. Bisulphite Treatment of DNA • DNA is treated with sodium bisulphite • This causes all the unmethylated cytosine residues to change to uracil • Bisulphite treated DNA is used as a template in methylation status assays • Primers must be designed in order that they complement the bisulphite treated DNA and must be specific to the methylated or unmethylated allele

21. Methylation Sensitive PCR • Uses bisulphite treated DNA • One common primer which pairs with either the maternal specific primer or paternal specific primer • The maternal and paternal PCR products should be different sizes • The ratio of maternal to paternal alleles can be determined if a reduced number of PCR cycles is used (as with QF PCR) • Does not identify the cause of the methylation defect

22. Paternal 221-bp product PAT Maternal 313-bp product MAT COMMON AS N AS PWS Normal PWS Methylation Sensitive PCR

23. Methylation Sensitive MLPA • Does not require bisulphite treated DNA • Probes are hybridised to genomic DNA • Genomic DNA is cleaved with a methylation sensitive restriction enzyme (only methylated regions stay intact) • Ligation occurs on probes bound to intact DNA • This method can identify deletions/duplications of DMRs as well as aberrant methylation not caused by gross deletions or duplications, i.e. differentiates between UPD and copy number changes

24. Methylation Sensitive MLPA AS (UPD) 2 alleles unmethylated PWS (UPD) 2 alleles methylated Normal control

25. High Resolution Melt Analysis • Requires bisulphite treated DNA • Initial PCR amplification using non-selective primers (not differentially methylated CpG islands), PCR mixture contains dye (such as LCgreen) • Slowly increasing temperature makes dsDNA melt releasing the dye, decreasing the amount of fluorescence • Methylated DNA melts (GC rich) at a higher temperature than unmethylated DNA (AT rich)

26. High Resolution Melt Analysis

27. High Resolution Melt Analysis Normal controls 2.5 fold LOM 4.5 fold LOM 15 fold LOM 5 fold LOM 18 fold LOM 8 fold LOM Total LOM Quantification of the loss of methylation was determined by MS-PCR

28. Pyrosequencing • Requires bisulphite treated DNA • Fragment containing differentially methylated CpG islands is PCR’d • Pyrosequencing is performed on single stranded PCR product • The C/T ratio at many sites is quantified • Does not tell you the cause of the methylation defect, so further work is required to identify this

29. C:34.8% C:0.0% C:35.8% C:29.4% C:32.7% Normal C:100% C:0.0% C:100% C:85.9% C:84.4% PWS C:0.0% C:0.0% C:0.0% C:0.0% C:0.0% AS Pyrosequencing

30. COBRA – combined bisulphite restriction analysis

31. Bisulphite Sequencing • Bisulphite DNA • PCR to make paternal and maternal specific PCR products • Clone PCR products into plasmids • Sequence using ddNTP approach • Cannot ascertain ratio of maternal to paternal products as there is an excess of methylated clones (unmethylated clones are AT rich and therefore unstable) • This method identifies all the differentially methylated CpG islands in the region

32. Methylation sensitive RFLPs and blots • Traditional method of identifying methylation defects using a double digest • Uses a methylation sensitive and a methylation insensitive enzyme • Methylation sensitive enzyme cuts in different places depending on the methylation status of each strand • Digested products are separated by electrophoresis • Southern blotting undertaken using a probe specific to the area of interest • Size of the methylated and unmethylated digest products are different therefore methylation status can be identified on the basis of size

33. MALDI-TOF Methylation Analysis

34. References • Hypomethylation of multiple imprinted loci in individuals with transient neonatal diabetes is associated with mutations in ZFP57. Mackay D., et al. Nat Genet. 40 (8) 949-951 (2008) • DNA methylation and human disease. Robertson KD. Nat Rev Genet. 6 (8) 597-610 (2005) • Beckwith-Wiedemann syndrome. Weksberg R., et al. Am J Med Genet. 137C 12-23 (2005) • Methylation-Specific MLPA (MS-MLPA): simultaneous detection of CpG methylation and copy number changes of up to 40 sequences. Nygren A., et al. Nuc Acid Res. 33 (14) e128 (2005) • COBRA: a sensitive and quantitative DNA methylation assay. Xiong Z., et al. Nuc Acid Res. 25 (12) 2532-2534 (1997) Acknowledgements: • Jonathan Callaway • Anne-Marie Reuther • Stacey Sandell • Dr Helen White