Tuesday, August 30, 2016

Exciting new bloodspot screening



(The following is a summary from James O'Brien, IPWSO Board member, from Australia, who attended the recent IPWSO Conference in Toronto)

A Novel FMR1/SNRPN methlation test for Fragile X Syndrome and chromosome 15 imprinting disorder screening of symptomatic children and newborns.

By David Godler, Cyto-Molecular Diagnostics Research Laboratory, Victorian Clinical Genetics Services, Murdoch Children’s Research Institute, Melbourne, Australia.


David Godler spoke about his research into newborn bloodspot screening over the past 18 months, primarily into Fragile X diagnostic. Recent funding from the Foundation for Prader-Willi Research, USA, has enabled David to expand his work with fragile X to include the Chromosome 15 imprinting disorders: Prader-Willi Syndrome (PWS), Angelman Syndrome and 15Q Duplication Syndrome.

The presentation was about David’s two new methods of testing blood collected in a tube and blood spots from adults, children and newborns. A newborn blood spot is the tiny spot of dried blood on blotting paper that is already taken through a heel pin prick for almost all newborns within the first few days of life in many countries. The tests currently performed on this material identify 30 or so conditions including phenylketonuria, hypothyroidism and cystic fibrosis, but not PWS.

Initially David and his team developed a new Methylated Specific Quantitate Melt Analysis (MS-QMA) test for Fragile X. To enable him to test 100,000 newborn bloodspots he also developed technologies to automate the process and to confirm positive findings. He is now able to test 100 samples in 90 minutes rather than the old 2 to 3 day turnaround for each test. At the same time his team developed new software to monitor both the results and accuracy of the test, and this method had been published for Fragile X. Accuracy of this test appears to be nearing 100%.

The beauty of his new test is that it can be performed on blood spot material left over from other routine testing. This means that the babies suspected of PWS would not be required to undergo collection of another blood sample required for more traditional diagnostic methods. It would also mean that the PWS babies that may not be tested for PWS and initially missed because they may be premature or may not have typical PWS, would be picked up by this new test. This would be because newborn blood spot testing reaches virtually all babies born irrespective of symptoms, but current PWS testing is only performed on babies that show symptoms that are recognized by doctors to be associated with PWS.

David’s first step in PWS test development was to analyse blood DNA samples from patients that had PWS symptoms, with only 9 patients confirmed to have PWS by standard DNa testing. From the first 30 symptomatic referral samples tested, his MS-QMA test confirmed 100% agreement for samples positive by standard testing. In addition to these  3 previously undiagnosed cases of PWS were identified. In order to prove that his new test was not simply showing false positives, David needed to develop an even more accurate second line confirmation test.

His second test he has called CINQ Droplet Digital PCR, which looks at individual methylated and unmethylated chromosomes suspended in miniature droplets. Exact details of how this new test works (or what the letters even stand for) are not yet published, however, its accuracy appears to be much higher again than standard testing, able to detect abnormal methylation down to an unprecedented less than 0.1% in the PWS deletion group. Furthermore, the new test has confirmed positive MS-QMA results missed by standard testing, and is showing that a number of previously diagnosed Uni Paternal Disomy (UPD) individuals may actually be Triasomi 15. The unexpressed male 15 chromosome appears to be hiding in the background rather than showing up on previous more traditional tests.

Furthermore, a new group of Mosaic PWS is likely to emerge. David is doing some further work on this subset. (With mosacizm PWS may not be present in every cell in the body. Depending on which cells in the body have the PWS variations may result in changes in how the characteristics of the Syndrome present, especially in UPD).

So, what does this mean for children with PWS?


  • ·       David and his team have come up with two new and seemingly very accurate methods of testing for PWS using blood spots and other sample types.
  • ·       The tests appear to be picking up previously unconfirmed cases of PWS.
  • ·       A new subset of T15 within the UPD group seems likely.
  • ·       A new phenotype of Mosacizm is possible.
  • ·       Bloodspots can be taken at newborn or at any age, resulting in a less invasive, more accessible and sensitive method of obtaining a PWS DNA test than is currently available.

 Where to from here?

 In 2015 David and his team have been awarded a large NHMRC grant to test 100,000 newborns for fragile X (the world’s largest fragile X prevalence study), and is hoping to use the remaining materials and expensive infrastructure initially developed for Fragile X, for PWS testing. Funding is currently an issue for this larger project, and David is now applying to multiple granting bodies to make this possible. In a less expensive project, over the next 3 years David and his team has started recruiting and assessing PWS patients to perform analysis of how the low level mosaicism identified by his two new tests related to variation in severity of PWS, especially in UPD. Concurrently, PWSA Australia has commissioned a Health Economics Report to analyse the financial benefit of early diagnosis. If the test is proven accurate, David and PWSA Australia will approach State or National Newborn Bloodspot Screening Programs with his two tests and our Health Economics Report to apply for the inclusion of PWS in their testing programs for all babies born in Australia.

If admission to the NBS programs is successful:
·       We should start to see a diagnosis within a few days of birth avoiding unnecessary multiple tests and prolonged family stress when waiting for a diagnosis.
·       Doctors, specialists and scientists will be able to develop a more detailed understanding of the various phenotypes of PWS, possibly enabling better targeted clinical interventions.
·       Testing of all babies will give a definitive answer regarding the prevalence of PWS live births in Australia.

David cited individual colleges that are assisting in the research from Cyto-Molecular Diagnostic Research Group, Victorian Clinical Genetics Services, Royal Children’s Hospital, Victorian and New South Wales Newborn and Metabolic Screening laboratories, Genetics and Learning Disabilities Services (Newcastle and The Children’s Hospital (Westmead, Sydney).

David also acknowledged assistance from a number of organisations including VCGS, Royal Children’s Hospital Foundation, The Marion and EH Flick Trust, Australian Government, Thrasher Research Fund, and Foundation for Prader Willi Research

Note: This report is James O’Brien’s understanding of David Golder’s presentation and may contain inaccuracies in its interpretation. 4 August 2016

No comments:

Post a Comment