Prader-Willi (PWS) and Angelman (AS) syndromes are different genetic disorders caused by opposite effects of DNA methylation at the same genomic location. Both have a reported frequency of approximately 1 in 15,000 births and are associated with intellectual disability. PWS is the most common genetic cause of life-threatening obesity; importantly, it can be effectively treated with hormone therapy and restricted diet if detected early. There is a strong case for including PWS and AS into newborn screening programs as there are clear benefits for affected infants and their families through early diagnosis and intervention. A key requirement for any new condition to be included in newborn screening is a test with high sensitivity and low cost ($1 to $3 per individual). The test must be ethical and ideally based on one or two 3 mm punches of dried blood spot material. Although DNA methylation testing of the SNRPN gene regulatory region detects most PWS (99%) and AS (78%) cases, use of one 3 mm punch of newborn blood spot material has not been validated. Dr Godler’s team has developed a novel, low-cost methylation test named Methylation Specific Quantitative Melt Analysis (MS-QMA) and shown that methylation testing of FMR1 can be used to effectively diagnose Fragile X syndrome (FXS), another relatively common genetic disorder (1 in 4000 in the general population). This test has been applied in more than 3000 cases using DNA from blood, cheek cells, saliva and newborn blood spots. Two important questions will be asked in this study: (1) Is MS-QMA a feasible approach for combined FMR1/SNRPN newborn screening? (2) Does SNRPN methylation change over time, and if so, in what proportion of patients? The first question will be addressed using 5000 newborn blood spots from control babies from the general population, 50 PWS, 50 AS and 50 FXS babies. A combined FMR1/SNRPN methylation test performed on the same 3 mm blood spot punch would have reagent costs of only $2 per test per condition. This would be far more cost-effective than testing for each condition separately. The next step would be inclusion of the SNRPN methylation test into a large scale FXS (50,000 babies) prospective newborn screening pilot being planned for late 2016 to early 2017. The second question will be addressed by examining the correlations between MS-QMA methylation data in dried blood spots of affected children of age 1 to 10 years and that in their newborn blood spots retrieved from Victorian Clinical Genetic Services repositories. The significance of question 2 is related to improved understanding of the epigenetic changes as children with AS and PWS grow up. Methylation at some genomic locations is stable from birth over time, while methylation at others changes with the child’s development. This has not been studied for the PWS and AS and is relevant for determining prognosis for AS and PWS babies.
As part of this project we have developed a new low-cost methylation test named Methylation Specific Quantitative Melt Analysis (MS-QMA) to perform combined analysis of methylation for FMR1 and SNRPN genes. This involved development of a new software called Q-MAX and modified chemistry to make the test more efficient when performed on poor quality and low quantity DNA samples. We have since shown that this a feasible and cost effective method to detect four neurodevelopmental disorders from a single sample including fragile X syndrome (FXS), Prader-Willi syndrome (PWS), Angelman syndrome (AS) and chromosome 15q duplication syndrome (matC15dDup) as part of this project. While matC15qDup syndrome was not part of the original proposal, it was included in this study because matC15dDup DNA samples are SNRPN methylation mosaics (two maternal copies of the methylated SNRPN promoter; and one unmethylated paternal copy). There was 100% concordance with the expected genotype and with MS-QMA results for these venous blood samples; including 62 FXS, 44 PWS, 15 AS, and nine C15qDup samples; and 44 typically developing controls. We then tested dried blood spots (DBS) made at time of the recruitment and newborn blood spots (NBS) retrospectively retrieved from the 1st year’s participants, and found 100% concordance for the expected genotype for MS-QMA between venous blood, NBS and DBS samples. Finally we developed another second line test called CINQ droplet digital PCR, show to be even more sensitive than MS-QMA (detects abnormal methylation in less than 1% of cells). We found that this was essential for future newborn screening studies as the standard testing (MS-MLPA and microarray) did not work well (high reaction failure rate) on DBS and NBS material, due to DNA quality and quantity issues. There was limited material available to us for this and future studies (three 3mm punches for each NBS) which was insufficient for standard testing. Provided that the 1st year findings are confirmed on the samples planned for the second year of the project, the next step would be to determine prevalence of PWS (including SNRPN mosaicism) on materials left over from the world’s largest FXS prevalence study (100,000 babies) / prospective newborn screening pilot that we have initiated in 2016 funded by the National Health and Medical Research Council of Australia. Other important question that will be addressed once all planned samples have been collected in the second year will be whether low level mosaicism for SNRPN methylation found by the newly developed protocol in some PWS participants is stable from birth over time (by comparing NBS and DBs data). An important future direction will also include characterization of the relationship between this low level mosaicism with gene expression and child’s development especially in PWS cause by maternal uniparental disomy. This has not been studied for the PWS, and may be relevant for determining prognosis for PWS babies that have UPD.