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Expert Recommendations for the Laboratory Diagnosis of
Neuronal Ceroid Lipofuscinosis Type 2 (CLN2 disease): Diagnostic Al...
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2016 BDSRA Fietz, Giugliani, AlSayed, Burke, Cohen-Pfeffer, Cooper, de Halec, Dvorakova, Izzo, Jahnova, Lukacs, Mole, Pearce, Schulz, Specchio, Xin, Miller

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Expert Recommendations for the Laboratory Diagnosis of Neuronal Ceroid Lipofuscinosis Type 2 (CLN2 disease): Diagnostic Algorithm and Best Practice Guidelines for a Timely Diagnosis

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2016 BDSRA Fietz, Giugliani, AlSayed, Burke, Cohen-Pfeffer, Cooper, de Halec, Dvorakova, Izzo, Jahnova, Lukacs, Mole, Pearce, Schulz, Specchio, Xin, Miller

  1. 1. Expert Recommendations for the Laboratory Diagnosis of Neuronal Ceroid Lipofuscinosis Type 2 (CLN2 disease): Diagnostic Algorithm and Best Practice Guidelines for a Timely Diagnosis Michael Fietz1* , Roberto Giugliani2* , Moeenaldeen AlSayed3 , Derek Burke4 , Jessica Cohen-Pfeffer5 , Jonathan D Cooper6 , Ines Noher de Halac7 , Lenka Dvořáková8 , Emanuela Izzo5 , Helena Jahnová8 , Zoltan Lukacs9 , Sara E. Mole10 , David A. Pearce11 , Angela Schulz12 , Nicola Specchio13 , Winnie Xin14 , and Nicole Miller5 * Co- first authors 1 Department of Diagnostic Genomics, PathWest Laboratory Medicine WA, Nedlands, Australia, 2 Serviço de Genética Médica Hospital de Clinicas de Porto Alegre, Departamento de Genética Universidade Federal do Rio Grande do Sul and National Institute of Medical Genetics Population, Porto Alegre, Rio Grande do Sul, Brazil, 3 Department of Medical Genetics, Alfaisal University and King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia, 4 Chemical Pathology, Camelia Botnar Laboratories, Great Ormond Street Hospital, London, UK, 5 BioMarin Pharmaceutical Inc., Novato, California, USA, 6 Institute of Psychiatry, Psychology, & Neuroscience, King’s College London, London, UK, 7 Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, Córdoba, Argentina and National Research Council-CONICET, Argentina, 8 Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Prague, Czech Republic, 9 Newborn screening and Metabolic Diagnostics unit, Hamburg University Medical Center, Hamburg, Germany, 10 MRC Laboratory for Molecular Cell Biology and UCL Institute of Child Health, University College London, London, UK, 11 Sanford Children’s Health Research Center, Sioux Falls, South Dakota, USA, 12 Children’s Hospital, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 13 Department of Neuroscience, Bambino Gesù Children’s Hospital, Rome, Italy, 14Neurogenetics DNA Diagnostic Lab, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA ABSTRACT The neuronal ceroid lipofuscinoses (NCLs) are a heterogeneous group of lysosomal storage disorders that include the rare autosomal recessive neurodegenerative disorder, CLN2 disease. CLN2 disease is caused by mutations in the TPP1/CLN2 gene, resulting in tripeptidyl peptidase-1 (TPP1) enzyme deficiency. Classic late-infantile CLN2 disease phenotype has a pediatric onset with initial symptoms of language delay and seizures, followed by progressive dementia, motor and visual deterioration, and early death. Variant phenotypes of CLN2 disease occur more rarely. Diagnosis of CLN2 disease is based on laboratory testing following clinical suspicion; however, delays in diagnosis are common due to low disease awareness, non-specific initial symptoms, and limited diagnostic testing access in some regions. With the advent of new therapies, early CLN2 disease diagnosis is key to ensure optimal clinical care and outcomes. In May 2015, a panel of international experts met to recommend best laboratory practices for early diagnosis of CLN2 disease. When NCL is suspected due to the presence of suggestive clinical signs, a TPP1 enzyme activity test should be the first diagnostic test performed (along with palmitoyl protein thioesterase [PPT1] enzyme to exclude CLN1). However, since initial suspicion of CLN2 disease and NCLs is often challenging, where available, the use of epilepsy gene panels for the investigation of unexplained seizures in childhood is endorsed. These panels should include TPP1/CLN2 as well as genes for NCLs that lack diagnostic biochemical tests. Diagnostic TPP1 enzyme testing in leucocytes is well established and robust and in DBS is considered diagnostic if followed by molecular confirmatory testing. Future methods to measure TPP1 activity via tandem mass spectrometry may improve DBS-based TPP1 enzyme testing sensitivity. Experts recommend that to confirm specific clinical suspicion of CLN2 disease, the gold standard for laboratory diagnosis is demonstrating deficient TPP1 enzyme activity and/or detecting causative mutations in each allele of the TPP1/CLN2 gene. Addendum: After abstract submission, the gold standard statement was revised to the following to reflect a more clear recommendation for laboratory diagnosis: “The gold standard for laboratory diagnosis is demonstrating deficient TPP1 enzyme activity and detecting causative mutations in each allele of the TPP1/CLN2 gene.” BACKGROUND ■■ Since early symptoms of CLN2 disease are not specific, prior to ordering specific laboratory tests for diagnosis of CLN2 disease, there are 3 (often sequential) phases through which many clinicians will pass: (1) suspicion of an unknown neurological disorder, (2) general suspicion of an NCL disorder, and finally, (3) specific suspicion of CLN2 disease. –– Diagnosis of CLN2 often involves lengthy diagnostic odysseys (Figure 1). Figure 1. Typical CLN2 Disease Diagnostic Odyssey 1- to 4-year delay Further symptom progression: seizures, language loss, developmental milestone loss, visual deterioration, others Symptoms progress: eizures, motor symptoms, developmental milestone regression, others Early symptoms: unexplained new onset seizures Clinical observations Onset of symptoms Specific confirmatory lab tests: TPP1 enzyme activity defect and gene mutations Diagnostic investigations: clinical tests, generic EEG, MRI, etc Epilepsy or developmental delay gene panels NCL gene panels, enzyme activity or electron microscopy EEG photic stimulation - s panels, light TPP1/CLN2 Unknown neurological disorder Is it linked to a genetic disorder? Clinical/laboratory tests Low suspicion High suspicion CLN2 disease Neuronal ceroid lipofuscinosis The above is a description of typical diagnostic practice. The specific tests ordered, approaches and techniques may vary among different laboratories, clinicians and centers worldwide depending on local diagnostic testing availability and disease awareness. Figure 2. Typical Delays in the Diagnosis of CLN2 Disease Results from survey of NCL clinical experts (N = 12) responding to the question, From your own experience, for patients not initially seen by NCL specialists, what is the time from onset of CLN2 disease symptoms until: 0 10 20 30 40 50 Months from onset of CLN2 symptoms until: 1 year: 82% 1 year: 100% From left to right: minimum, mean, maximum Referral to an NCL specialist Diagnosis with CLN2 disease ■■ Delays in CLN2 disease diagnosis are common, but diagnosis is rapid once patients are referred to appropriate specialists. METHODS ■■ International NCL experts met in May 2015 to discuss and recommend best laboratory practices to support early diagnosis of CLN2 disease. ■■ The algorithms and recommendations here reflect the consensus of the experts. ■■ TPP1 enzyme activities are reported as observed by individual diagnostic laboratories in samples from unaffected individuals, heterozygous carriers, and individuals with CLN2 disease. Laboratories utilized modifications of the fluorogenic TPP1 assay.1-6 The data presented here are not generated from BioMarin-sponsored clinical trial patient samples. APPROACH TO SUSPICION AND DIAGNOSIS ■■ Any unexplained seizure in a child 1 to 9 years of age,7 particularly when associated with a history of language delay and/or developmental milestone regression, should be suspected as linked to a genetic disorder or to an NCL disorder. –– Atypical forms with alternative presentations exist, eg, juvenile forms and autosomal recessive spinocerebellar ataxia 7 (SCAR7 disease, also caused by mutations in the TPP1/CLN2 gene).8 ■■ When a genetic neurological disorder is suspected, a molecular gene panel test to investigate genetic causes of childhood-onset epilepsy or developmental delay is recommended. –– Experts recommend that the TPP1/CLN2 gene should be included in such gene panels. ■■ When NCL disorders in general are suspected, targeted NCL gene panels may help in reaching a diagnosis of CLN2 disease, as well as other types of NCLs. ■■ EEG analysis under specific intermittent photic stimulation (1 to 2 Hz) may be of use as a clinical test to reach a suspicion of CLN2 disease.9-11 –– EEG spikes in the posterior region in response to photic stimulation at 1-2 Hz are characteristic of CLN2 disease and some other late-infantile NCLs (eg, CLN6 and CLN8 diseases). ■■ Early testing of TPP1 and PPT1 enzyme deficiency can rapidly rule out or confirm suspicion of CLN2 or CLN1 disease, respectively. –– Assays for TPP1 and PPT1 enzyme activity are widely available. –– CLN2 and CLN1 disease are the most prevalent NCL disorders among the late infantile NCL types.10 –– Enzyme activity assay exists also for cathepsin D (CLN10 disease), but routine laboratory testing for this enzyme is currently less common. Figure 3. Algorithm for Suspicion and Diagnosis of CLN2 Disease Suspicion of a genetic disorder Suspicion of an NCL disorder Observation of common initial symptoms at late-infantile age (2 to 4 years; classic phenotype) or at juvenile age (> 4 years; atypical phenotype): • New-onset seizures, in association with • A history of language delay and/or developmental milestone regression Intermittent photic stimulation (1 to 2 Hz) or screening of TPP1 and PPT1 enzymesa Disease or symptom gene panelb or screening of TPP1 and PPT1 enzymesa NCL gene panelb or screening of TPP1 and PPT1 enzymesa TPP1 enzyme activity analysis Leukocytes or fibroblasts or DBSc Molecular analysis of the TPP1/CLN2 geneb Diagnosis Suspicion Clinical testing and differential diagnosis Suspicion of CLN2 disease Laboratory confirmatory diagnosis of CLN2 disease CLN2 disease diagnosis DBS, dried blood spot. a Early screening of TPP1 and PPT1 deficiency (eg, in DBS) can rule out or confirm CLN2 suspicion. b Molecular testing alone may be diagnostic if 2 pathogenic mutations consistent with clinical presentation are detected in trans. c A finding of deficient TPP1 enzyme activity in leukocytes or fibroblasts may be considered diagnostic if consistent with clinical presentation, but molecular analysis is recommended. ■■ Following clinical suspicion, diagnosis of CLN2 disease is based on specific well-established laboratory tests. ■■ TPP1 enzyme activity can be assessed in several sample types. ■■ Molecular analysis of the TPP1/CLN2 gene can confirm a diagnosis and assist genetic counseling. The gold standard for diagnosis of CLN2 disease is: ■■ Demonstration of decreased or deficient TPP1 enzyme activity (together with normal activity of appropriate controls); and ■■ Detection of 1 deleterious mutation in each allele of the TPP1/CLN2 gene ■■ If it is not possible to perform both the TPP1 enzyme assay and molecular testing due to local restrictions: –– A finding of deficient TPP1 enzyme activity in leukocytes or fibroblasts is diagnostic when consistent with the clinical presentation of CLN2 disease. –– Detection of two pathogenic TPP1/CLN2 mutations in trans is diagnostic in the absence of TPP1 enzyme testing results when consistent with clinical presentation. RECOMMENDATIONS FOR LABORATORY DIAGNOSIS Analysis of TPP1 Enzyme Activity ■■ The TPP1 enzyme is a lysosomal exopeptidase that cleaves N-terminal tripeptides.1-3,12 ■■ Diagnostic laboratory assessment of TPP1 enzyme activity utilizes the fluorogenic substrate Ala-Ala-Phe-7-amido-4-methylcoumarin.2,3,12 –– A substrate for assessment of TPP1 enzyme activity by tandem mass spectrometry has been developed13 and may support future screening of newborn DBS samples. 143 ■■ Several variants on the fluorogenic TPP1 enzyme assay have been published.4-6 ■■ It is essential to assay the activity of control enzymes. –– PPT1 (mutated in CLN1 disease) and β-galactosidase are appropriate control enzymes. Figure 4. TPP1 Enzyme Activity in Leukocyte Samples, by Diagnostic Laboratory 1 2 3 4 5 0 100 200 Patient with CLN2 disease Laboratory TPP1 Enzyme Activity, nmol/h/mg protein Carrier Unaffected individuals 300 400 ■■ Different assay implementations can distinguish affected from unaffected individuals. –– Laboratories must establish reference ranges for their individual implementation. Molecular Analysis of the TPP1/CLN2 Gene ■■ Mutations have been reported throughout the TPP1/CLN2 gene.14 ■■ Two deleterious mutations are frequently reported: c.509-1GC and c.622CT (p.Arg208*). –– These two mutations together represent 57% of all reported mutations. –– At least 1 of these 2 mutations is reported in 89% of patients with CLN2 disease.14 ■■ Sequencing should evaluate the entire coding region and associated splice junctions. ■■ A database of mutations associated with the TPP1/CLN2 gene, and other NCL genes, is maintained at http://www.ucl.ac.uk/ncl/mutation. Figure 5. Reported Frequency of TPP1/CLN2 Mutant Alleles14 c.509-1GC 156 p.Arg208* 149 At least 1 reported in 89% of patients Missense Splice site Nonsense/stop p.Gly284Val p.Gln509* p.Gln422His 35 18 10 176 0 25 50 75 100 Mutation Reported in Kousi et al, 2012 125 150 175 200 84 mutations reported 10 times CONCLUSIONS ■■ CLN2 disease is a rare, progressive, and devastating disease that can be difficult to identify clinically at an early stage. –– Key initial symptoms in many patients are new-onset seizures, often in combination with a history of early language delay. –– Atypical forms of disease—with alternative presentations—caused by mutations in the TPP1/CLN2 gene also exist, such as SCAR7.8 ■■ Delays in diagnosis are common due to the time necessary to come to suspicion of CLN2 disease. Valuable tools to help speed time to diagnosis include: –– EEG intermittent photic stimulation (1 to 2 Hz). –– Symptom- or disease-based gene panels that include the TPP1/CLN2 gene. –– Early screening for TPP1 enzyme deficiency (together with PPT1 for CLN1). ■■ Diagnostic laboratory testing for CLN2 disease is well established. ■■ The gold standard for laboratory diagnosis is demonstration of deficient TPP1 enzyme activity and molecular analysis that detects 1 pathogenic mutation on each allele of TPP1/CLN2 gene. REFERENCES 1. Sohar I, et al. J Neurochem. 1999;73:700-711. 2. Vines D, Warburton MJ. Biochim Biophys Acta. 1998;1384:233-242. 3. Vines D, Warburton MJ. FEBS Lett. 1999;443:131-135. 4. Muller VJ, et al. Eur J Paediatr Neurol. 2001;5(suppl A):197-200. 5. Young EP, et al. Prenat Diagn. 2000;20:337-339. 6. Van Diggelen OP et al. Eur J Paediatr Neurol. 2001;5(suppl A):189-200. 7. Kohan R et al. Biochim Biophys Acta. 2013;1852:2301-2311. 8. Sun Y et al. Hum Mutat. 2013;34:706-713. 9. Binelli S, et al. Neurol Sci. 2000;21:S83-S87. 10. Mole SE, et al. The Neuronal Ceroid Lipofuscinoses (Batten disease). 2nd edition. Oxford, UK: Oxford University Press; 2011. 11. Naqvi SZ, et al. Pediatr Neurol. 1998;19:395-398. 12. Sleat DE, et al. Science. 1997;277:1802-1805. 13. Barcenas M, et al. Anal Chem. 2014;86:7962-7968. 14. Kousi M, et al. Hum Mutat. 2012;33:42-63. ACKNOWLEDGMENTS The authors thank Karl Zawadzki, PhD, and Evelyn Rose, PharmD (Health Interactions), for assistance with the preparation of this poster, which was funded by BioMarin Pharmaceutical Inc. ©2016 BioMarin Pharmaceutical Inc. All rights reserved.Presented at the 12th Annual WORLD Symposium: 29 February – 4 March, 2016, San Diego, CA http://www.biomarin.com/pdf/WORLD2016p7.pdf

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