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Aug 17, 2016
Expert Recommendations for the Laboratory Diagnosis of Neuronal Ceroid Lipofuscinosis Type 2 (CLN2 disease): Diagnostic Algorithm and Best Practice Guidelines for a Timely Diagnosis
Expert Recommendations for the Laboratory Diagnosis of
Neuronal Ceroid Lipofuscinosis Type 2 (CLN2 disease): Diagnostic Algorithm
and Best Practice Guidelines for a Timely Diagnosis
, 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
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,
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,
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,
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,
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
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
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.”
■■ 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
eizures, motor symptoms,
unexplained new onset
Onset of symptoms
Specific confirmatory lab tests:
TPP1 enzyme activity defect and
clinical tests, generic EEG,
Epilepsy or developmental
delay gene panels
NCL gene panels,
or electron microscopy
EEG photic stimulation
Unknown neurological disorder
Is it linked to a genetic disorder?
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
■■ Delays in CLN2 disease diagnosis are common, but diagnosis is rapid once patients are referred to
■■ 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
Suspicion of an
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
screening of TPP1 and PPT1 enzymesa
NCL gene panelb
screening of TPP1 and PPT1 enzymesa
TPP1 enzyme activity analysis
Leukocytes or fibroblasts or DBSc
Molecular analysis of the TPP1/CLN2 geneb
Clinical testing and
diagnosis of CLN2 disease
CLN2 disease diagnosis
DBS, dried blood spot.
Early screening of TPP1 and PPT1 deficiency (eg, in DBS) can rule out or confirm CLN2 suspicion.
Molecular testing alone may be diagnostic if 2 pathogenic mutations consistent with clinical presentation are detected in trans.
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
■■ 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
–– 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
–– A substrate for assessment of TPP1 enzyme activity by tandem mass spectrometry has been
and may support future screening of newborn DBS samples.
■■ 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
0 100 200
Patient with CLN2 disease
TPP1 Enzyme Activity,
Carrier Unaffected individuals
■■ 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
Figure 5. Reported Frequency of TPP1/CLN2 Mutant Alleles14
At least 1 reported in
89% of patients
0 25 50 75 100
Mutation Reported in Kousi et al, 2012
125 150 175 200
84 mutations reported 10 times
■■ 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
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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.
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