Prevalence and aetiology of PWS low level mosaicism in UPD undetected by standard testing

Prader-Willi syndrome (PWS) is a severe neurodevelopmental disorder found in ~1 in 15,000 to 20,000 births. PWS phenotype caused by the loss of function of several genes located on chromosome 15. These genes are usually ‘switched on’ on the chromosome 15 that is inherited from the father and ‘switched off’ on the chromosome 15 inherited from the mother. The mechanism of this ‘switch’ is a chemical change called ‘methylation’. Most commonly PWS is caused by either a deletion on the paternal chromosome 15, or to both chromosome 15s coming from the mother (maternal uniparental disomy 15). In this study we will use several new state of the art techniques which we have developed that can measure methylation, number of copies and gene expression on chromosome 15 with very great precision. This type of analysis has never been done in PWS. In our preliminary studies we have identified a subset of individuals with PWS in whom there are small numbers of cells in which some of the PWS genes on chromosome 15 remain active ‘switched on’ / unmethylated. We will undertake further studies in 140 PWS patients to determine how common this phenomenon is in two patient cohorts (one from Australia and another from USA), and whether it correlates with gene expression the type of PWS and the severity of symptoms.

Importance of research, next steps, and downstream projects:
This proposal will lead to the development of new tests that can accurately quantify subtle differences in methylation in PWS individuals. The results of these studies will:
a) open new avenues to explain the cause of PWS and the variability in symptoms between different PWS individuals
b) identify new sensitive biomarkers that will be able to be used in clinical trials to help analyse and predict response to various treatments
c) allow the development of tests that will help predict future health problems in young children with PWS
d) identify more sensitive ways to screen drug libraries for compounds that may re-activate genes that are switched off in PWS.

Research Outcomes: Public Summary

PWS phenotype caused by the loss of function of several genes located on chromosome 15. These genes are usually ‘switched on’ on the chromosome 15 that is inherited from the father and ‘switched off’ on the chromosome 15 inherited from the mother. The mechanism of this ‘switch’ is a chemical change called ‘methylation’. This DNA methylation is abnormally increased in PWS, and is largely restricted to a regulatory region of a gene called SNRPN. Most commonly subnormal SNRPN methylation in PWS is caused by either a deletion on the paternal chromosome 15, or to both chromosome 15s coming from the mother (maternal uniparental disomy 15). In the 1st year of the project study we have used several new state of the art techniques which we have developed that can determine DNA methylation levels, number of gene copies, DNA sequence and gene expression profile on chromosome 15, and on other chromosomes with very great precision. This type of multi-level analysis (called omics) has never been done in PWS. In year 1 we have identified with 2 or more techniques that 4% of individuals that has an extra copy of the whole paternal chromosome 15 in approximately 10% of cells that had unmethylated SNRPN, while 90% had methylated SNRPN. This was done in two patient cohorts (one from Australia and another from USA). However, using the most sensitive technique in our toolbox, called droplet digital PCR (ddPCR), examining a larger DNA region than what is usually examined, variability in methylation was detected in up to 16% of PWS study participants. While the level of SNRPN methylation detected by ddPCR was significantly higher in the UPD and the imprinting centre mutation groups compared to deletions, heterogeneity in methylation signatures was also detected in the deletion group. To determine what this previously undetected variability in methylation means, we also explored gene expression profiles as well as characterized the type of PWS and the severity of symptoms in our participants. By comparing expression of genes outside the imprinted region it was hoped that we would be able to show excess gene expression across the whole chromosome 15 in UPD, but not in deletion cases. As expected variability in gene expression dose within and outside of the imprinted locus on chromosome 15 reflected variability in SNRPN methylation between deletion and non-deletion groups in PWS. However, this variability for specific clusters of imprinted and non-imprinted genes in peripheral blood was significantly associated with phenotype severity in PWS. Furthermore, we have discovered novel significant genotype-phenotype relationships for genes located outside of the imprinted region, not previously examined for chromosome 15 imprinting disorders. While expression levels for the genes significantly correlated in PWS with behavioural abnormalities, as well as, the level of intellectual functioning (p<0.05; n=22), a number of important relationships fell just short of significance e.g. food seeking behaviours for PWS (Age = 6 years) (R= -0.484; p=0.068 n=15). Based on the already observed effect sizes, the larger sample size of 60 participants planned for the second year of the project should be sufficient to reach significance for some of the most important relationships (e.g. with food seeking behaviours). While exciting, these preliminary data should be treated with caution, until confirmed in larger independent cohorts planned for the second year of the study.

Research Outcomes: Publications

Exploring autism symptoms in an Australian cohort of patients with Prader-Willi and Angelman syndromes. Baker EK, Godler D, Bui M, Hickerton C, Rogers C, Field M, Amor D, and Bretheton B. Journal of Neurodevelopmental Disorders. 2018,10:24