Next-generation Diagnostics for Fragile X disorders

Transcription

1 Next-generation Diagnostics for Fragile X disorders Overview Bio-Link presents novel diagnostic technology that offers compelling clinical, technical and commercial advantages over existing tests for fragile X syndrome (FXS) and related conditions. Researchers at the Murdoch Children s Research Institute (MCRI) in Melbourne, Australia have recently discovered and validated new epigenetic biomarkers designated Fragile X-related Epigenetic Elements (FREE). The analysis of methylation in FREE biomarkers provides unique diagnostic utility for FXS, autism spectrum disorder (ASD), and other FX-related disorders. FREE methylation analysis overcomes several major difficulties associated with current FXS diagnostic methods, and can be incorporated as a complementary approach with the existing tests. Over time, as the unique clinical utility and reliability of FREE analysis become more widely accepted, it has the potential to be implemented as a stand-alone test in many applications as the definitive, next-generation assay for FX-related disorders. The applications of FREE analysis range from targeted testing to newborn screening to potential companion diagnostics. In addition to the FREE biomarkers, MCRI have developed an advanced assay platform based on methylation specific-quantitative melt analysis (MS-QMA). The platform offers an ultra-low detection limit for methylation, and is broadly applicable for genetic analysis beyond only FMR1-related conditions. On behalf of MCRI, Bio-Link is seeking to license the technology for commercial development of advanced clinical diagnostics in a global market with the potential to exceed $500 million. Background Fragile X Syndrome (FXS) is a severe neurodevelopmental disorder, and is one of the major inherited conditions co-morbid with autistic behaviors. FXS is caused by the expansion of the repetitive sequence of the CGG trinucleotide in the fragile X mental retardation 1 (FMR1) gene. An expansion of more than 200 CGG repeats is termed full mutation (FM), and is usually associated with the shut down of FMR1 gene expression and loss of fragile X mental retardation protein (FMRP), its protein product. FMRP is essential for normal neurodevelopment. Smaller expansions designated premutation (PM, 55 to 199 CGG repeats) and grey zone (GZ, 44 to 54 CGG repeats) are more common. While the incidence of FM is about 1 in 4000, that of PM is about 1 in 300, and of GZ about 1 in 30 individuals. PM and GZ have been primarily associated with the late onset conditions such as Fragile X-associated Tremor Ataxia Syndrome (FXTAS) and premature ovarian failure (POF). Fragile X testing is recommended for all females who have reproductive or fertility problems before the age of 40, and as part of prenatal testing for all female carriers of the expanded alleles.

2 Problem Fragile X syndrome testing has evolved from the original cytogenetic assays of the late 1970s, in which fragile sites in the X chromosome were visualized under a microscope, to more sensitive PCR techniques developed in the 1990s. To an extent, the early PCR methods enabled rapid assessment of the number of CGG repeats, or expansion sizing. However, the Southern blot method was also required in many cases for further analysis of sizing and methylation. Southern blot is a time-consuming and laborious method that requires relatively large quantities of high quality DNA. Improved PCR sizing assays such as triplet repeat primed (TP PCR) introduced around 2010 by Quest Diagnostics, Celera, Asuragen and others, have reduced but not eliminated the need for Southern blot. More importantly, even these newer assays have limited diagnostic utility. There is a great unmet medical need for improvement of the diagnostic performance of current FXS tests, in terms of sensitivity, specificity, and positive and negative predictive values across the range of clinical phenotypes and genotypes for both males and females with FMR1 expansions. For example, currently available PCR tests for FXS are specific for FM males, but none can predict specific cognitive and behavioral impairments in female carriers with expanded FMR1 alleles. As a result of this and other performance shortcomings of existing tests and their continued reliance upon Southern Blot, the feasibility of wide-scale screening for FXS remains limited by technical, practical, economic, and ethical considerations. Solution MCRI s novel FREE biomarkers are inversely correlated with levels of FMRP and cognitive status in both males and females with FMR1 expansions, overcoming the limitations of currently available PCR tests for FXS. In earlier studies based upon mass spectrometry methods, MCRI showed that FREE methylation analysis is superior to methylationsensitive Southern blot and FMRP immuno-staining in blood as a predictor of cognitive impairment, and that it is related to X-inactivation skewing in FM females. MCRI subsequently developed a new real-time PCR method named Methylation Specific- Quantitative Melt Analysis (MS-QMA), which combines the qualitative strengths of high resolution melt and the high-throughput, quantitative real-time PCR standard curve to provide accurate quantification of DNA methylation in a single assay. After analyzing thousands of samples with reference methods, MCRI completed a large scale validation of MS-QMA FREE methylation analysis in a cohort of 685 individuals, and compared its performance with that of the reference methods including Southern blot and mass spectrometry. In male and female newborn blood spots MS-QMA differentiated FM from control alleles, with sensitivity, specificity, positive and negative predictive values between 92 and 100%. MS-QMA has an immediate application in FXS diagnostics, with a potential use of its quantitative methylation output for prognosis in both sexes. Bio-Link 2

3 Program Highlights MCRI researcher Dr David Godler and colleagues have developed and clinically validated new epigenetic biomarkers for FXS, and adapted the assay to a rapid, cost-effective and commercially viable platform based upon MS-QMA. The biomarkers are located within novel regions of the FMR1 gene locus designated Fragile X-related Epigenetic Elements 1 (FREE1) and 2 (FREE2). Highlights of the technology program are listed below. The discovery, development, and clinical validation of the FREE biomarkers and MS-QMA platform is based upon the collective analysis of several thousand patient samples and large cohorts with associated detailed clinical phenotype data. Dr Godler s work has been supported by more than A$2,500,000 in competitive grant funding from Australian and international sources, resulting in publication of over 11 research articles in top-tier journals in the field (see references, p. 8). In 2014 Dr Godler was the recipient of an A$50,000 prize from MCRI and Royal Childrens Hospital Foundation for the clinical and commercial translation of his research, and he has received increasing recognition in the commercial press and in science & medical news (see references, p. 8). The research was conducted at MCRI s subsidiary, Victorian Clinical Genetics Services Limited (VCGS), accredited under Australia s National Association of Testing Authorities (NATA, Accreditation Number 3171). VCGS is a Category S (Specialised) facility fully compliant with the requirements of ISO 15189:2007. MS-QMA Technology Platform Though FREE biomarkers were discovered on the MALDI-TOF mass spectrometry platform, MCRI has adapted the assay to run on MS-QMA as a simpler, faster platform more widely available in both research and clinical diagnostics settings. Transfer from mass spectrometry to MS-QMA required the development of innovative methods that are proprietary to MCRI widely applicable for epigenetic analysis. MCRI also developed proprietary software applications to automate set-up, calibration, and data analysis. Highlights of the MS-QMA platform are presented below. The MS-QMA platform has broad diagnostic applications to other disease indications beyond only FMR1-related disorders. Applicability to various sample types including venous blood, NBS, buccal swab, saliva or chorionic villus sampling. Ultra-low limit of detection of methylation at 2% quantitative, and 1% qualitative (see Figure 1). The requirement for ~100 fold less DNA quantity than Southern blot analysis, with reduced reliance upon the quality of DNA samples. Closed-tube format minimizes the risk of contamination. Total turn-around-time of between 6-9 hours (2.5 to 3 hours post-bisulfite conversion). Data analysis from real-time PCR and HRM is performed simultaneously (in less than 1 minute) using proprietary algorithm incorporated into Q-MAX application. Bio-Link 3

4 (G) HRM methylation standard Figure curve 1. HRM analysis Methylation standard curve AFU62 AFU42 AFU4 78C 100% methylated 50% methylated 0% methylated Expected Methylationo Ratio Coefficient of correlation (R 2 ) = MS-QMA Aligned Fluorescence Unit (AFU) at Melting Temperature of 78C Figure 1. HRM methylation standard curve automatically obtained from 100% methylated and completely unmethylated control samples spiked at different ratios. Error bars represent two standard deviations from the mean for 16 separate spiking experiments. FREE2 MS-QMA Test Performance and Clinical Applications The following summary focuses on the development and clinical validation of FREE2 biomarkers using venous blood samples or newborn blood spots (NBS) on the MS-QMA platform. The superior performance of FREE2 MS-QMA was proven in clinical studies that demonstrated: Large scale validation in a cohort of 685 males and females using venous blood and NBS demonstrated that FREE2 MS-QMA differentiated FM from control alleles, with sensitivity, specificity, positive and negative predictive values between 92 and 100%. Unprecedented diagnostic utility for FMR1-related disorders including FXS, autism spectrum disorder (ASD), and other FXS-related conditions. Suitability for both targeted testing and wide-scale (e.g. newborn) screening. Application 1 (targeted testing): Identification of only those individuals likely to be cognitively impaired as reflected by low verbal IQ (<70) at >5 years of age, with analyses performed any time after birth (see Figure 2). Application 2 (newborn screening): Identification of most FM males and females in newborn blood spots or venous blood with analyses performed in the 1st year of life (see Figures 3 and 4). FREE2 MS-QMA correlates with the reference methods and provides superior prediction of cognitive impairment in FXS compared to the gold-standard Southern Blot and to FMRP immuno-staining in blood. FREE2 biomarkers are also related to X-inactivation skewing in FM females. In venous blood of FM females, FREE MS-QMA correlated most strongly with verbal IQ impairment (see Figure 2). FREE2 MS-QMA of buccal samples provides information about tissue mosaicism, and can identify low-level FM mosaic individuals missed by existing FXS assays such TP PCR (poster presented in May 2014 at 13th International Child Neurology Conference in Brazil; awarded Best Platform Presentation of the Development, Cognition and Psychiatry session). Bio-Link 4

5 Figure 2. FREE2 MS-QMA correlation with verbal IQ (targeted testing) Figure 2. The median FREE2 MS-QMA methylation ratio (MR) was significantly increased in FM females with scores <70 for verbal IQ (VIQ) compared to females with VIQ scores >70, PM females and controls. Figure 3. FREE2 MS-QMA correlation with FM in males (newborn screening) (A) % Male controls Male FM 0.1 Figure 3. FREE2 MS-QMA analysis of methylation ratio of CpG sites 2-12 in NBS from 87 control males (with CGG <40) and 13 FM males. Bio-Link 5

6 Figure 4. FREE2 MS-QMA correlation with FM in females (newborn screening) *** 93% % 0.47 Female controls Female FM Figure 4. FREE2 MS-QMA analysis of methylation ratio of CpG sites 2-12 in NBS from 95 control females (with CGG <40) and 15 FM females. Market Potential: $500 Million While the incidence of FXS (affected cases) is low (1/3,800 males to 1/5,000 females); the prevalence of patients with PM and GZ is considerably higher (1 in 813 males and 1 in 259 females; and 1/25 males or females, respectively). Besides profound FX-related mental retardation, a broader range of disease phenotypes or conditions is becoming increasingly recognised as associated with milder anomalies in the FMR1 gene. These include, among others, reproductive problems in women, social and cognitive difficulties in both sexes, and developmental delay or autism. Widescale newborn and/or prenatal population screening has been much debated, but has remained heretofore ethically and economically unfeasible. MCRI s test addresses this widened scope of FX-related genetic implications, and creates the potential for greatly expanded market opportunities in clinical diagnostics. FXS-related diagnostics generally fall into two categories, either targeted testing or widescale screening. Asuragen is currently selling its targeted test for US$100. Government supported screening tests would have a much lower selling price of US$3-5. Based upon the statistics presented below, the market for targeted FXS testing in the US (at $100 selling price) is nearly $100 million. The market for wide-scale screening may be comparable based upon a selling price of only US$1. The total market potential for FREE2 MS-QMA parallels that of cystic fibrosis testing in the range of $500 million worldwide. Targeted testing Children with developmental difficulties and their families. US Incidence: >520,000 All women under 40 with reproductive problems, possibly suffering from FX-related premature ovarian insufficiency (FXPOI). US Prevalence: ~250,000 Patients and families with FX-associated tremor/ataxia syndrome (FXTAS). US Prevalence: >100,000 Potential companion diagnostics for new FXS drugs. US Prevalence: ~75,000 Bio-Link 6

7 Children (<21 years old) with ASD. US Prevalence ~750,000; Incidence >35,000 Children with developmental disability ranging from mild (e.g. speech and language impairments) to serious (e.g. intellectual disabilities, cerebral palsy, and autism). US Prevalence: 13% of children (with >6,000,000 under age 11) Wide-scale (Newborn, Infant and Early Childhood) screening >4 million births in the US; 8 million in Europe each year >125 million infants screened for genetic diseases annually worldwide Asia (78M births, 20% screened); Latin America (11M, 60%); Africa (30M, 0%) Intellectual Property MCRI has secured a robust intellectual property position with a suite of patent families directed toward the use of FREE2 and other associated regions, as well as a broader patent application covering the MS-QMA technology. Murdoch Childrens Research Institute MCRI is an independent non-profit research institute based at the Royal Children's Hospital and affiliated with the University of Melbourne in Australia. The Institute has approximately 1500 researchers including 100 postgraduate students, and is the largest medical research institute specialising in adolescent and child health in Australia. MCRI s areas of research include cerebral palsy, cancer, genetics, muscular dystrophy, diabetes, asthma, allergies, deafness, infectious diseases, depression and behavioural problems. MCRI also leads genetic ethics research and community debate of controversial issues such as stem cell research. The Institute receives over AUD$90 million annually for laboratory, clinical, and public health research. Indicative of the MCRI s commitment to conduct research of the highest quality, the Institute publishes over 600 internationally peerreviewed articles each year and holds over 60 patent applications. Commercial Opportunity Bio-Link is an international business development consultancy that works with Australian biotech companies and biomedical research institutions to facilitate licensing and commercial partnering of innovative biotechnologies. The present opportunity provides novel epigenetic biomarkers for FMR1-related disorders and a next-generation diagnostic assay which enables more rapid, cost-effective, and clinically informative analysis of epigenetic contributions to fragile X syndrome. On behalf of MCRI, Bio-Link is seeking to license the technology to companies for commercial development of advanced medical diagnostics, as well as for research applications. Interested parties please contact Christopher Boyer, Executive Director, Bio-Link. Christopher Boyer, Executive Director Level 2, Birdwood Offices, Prospect St. Box Hill, VIC 3128 Australia Ph Fax c.boyer@bio-link.com Bio-Link 7

8 Research Articles 1. Inaba Y, Schwarz CE, Bui QM, Li X, Skinner C, Field M, Wotton T, Hagerman RJ, Francis D, Amor DJ, Hopper JL, Loesch DZ, Bretherton L, Slater HR, and Godler DE. Early Detection of Fragile X Syndrome: Applications of a Novel Approach for Improved Quantitative Methylation Analysis in Venous Blood and Newborn Blood Spots. Clin Chem May; 60:7. 2. Godler DE, Inaba Y, Shi EZ, Skinner C, Bui QM, Francis D, et al. Relationships between age and epi-genotype of the fmr1 exon 1/intron 1 boundary are consistent with non-random x-chromosome inactivation in fm individuals, with the selection for the unmethylated state being most significant between birth and puberty. Hum Mol Genet 2013; 22(8): Inaba Y, Herlihy AS, Schwartz CE, Skinner C, Bui QM, Cobb J, et al. Fragile x-related element 2 methylation analysis may provide a suitable option for inclusion of fragile x syndrome and/or sex chromosome aneuploidy into newborn screening: A technical validation study. Genetics in Medicine 2013; 15: Godler DE, Slater HR, Bui QM, Storey E, Ono M, Gehling F, Inaba Y, Francis D, Hopper J, Kinsella G, Amor D, Hennerich D, Taylor AK, Hagerman RJ, Loesch DZ. Fragile X Mental Retardation 1 (FMR1) Intron 1 Methylation in Blood Predicts Verbal Cognitive Impairment in Female Carriers of Expanded FMR1 Alleles: Evidence from a Pilot Study. Clin Chem Jan; 58: Godler DE, Slater HR, Bui QM, Ono M, Gehling F, Francis D, Amor DJ, Hopper JL, Hennerich D, Taylor AK, Hagerman R, and Loesch DZ. FMR1 intron 1 methylation predicts FMRP expression in blood of female carriers of expanded FMR1 alleles. J Mol Diagnostics. 2011; 13(5): Godler DE, Tassone F, Loesch DZ, Taylor AK, Gehling F, Hagerman RJ, Burgess T, Ganesamoorthy D, Hennerich D, Gordon L, Evans A, Choo KHA, and Slater HR. Methylation of novel markers of Fragile X alleles is inversely correlated with FMRP expression and FMR1 activation ratio. Hum Mol Genet Apr 15; 19(8): Godler DE, Slater HR., Amor D, and Loesch DZ. Methylation analysis of Fragile X Related Epigenetic Elements may provide a suitable newborn screening test for Fragile X Syndrome. Genetics in Medicine Sep; 12(9):595. Commercial Press and Health & Science News 1. Johnson M. (2014, May 8). Australian Team Develops Melt Curve Methylation Assay for Fragile X Detection, Potential Screening. GenomeWeb PCR Insider. 2. O Connell B. (2013, August 14). X Test Boost Autism Gene Family Hope. Herald Sun, p Yamine E. (2012, October 16). Babies test for fragile X - Early diagnosis aids treatment. Daily Telegraph, p. 12. Bio-Link 8