Our Research

PDE pyridoxine-dependent epilepsy

PDE is a rare disease (1:65000) characterized by intractable neonatal seizures, responsive to pyridoxine (vitamin B6) supplementation. Intellectual disability and a broad spectrum of accompanying neurobehavioral symptoms affect the majority of PDE patients, and are not prevented by life-long B6 treatment which often causes a peripheral neuropathy. More than half a century after PDE was first clinically described in 1954 our Dutch group uncovered its etiology; PDE is caused by autosomal recessive mutations in ALDH7A1, which results in a deficiency of the enzyme α-aminoadipic semialdehyde (AASA) dehydrogenase, responsible for catalyzing the third step in the lysine oxidation pathway. In PDE, accumulating AASA (P6C) inactivates the active B6 vitamer pyridoxal-5-phosphate, an important cofactor for neurotransmitter synthesis. B6 supplementation can replace this loss and improve seizure control; however more than 75% of patients still suffer intellectual disability and neurologic abnormalities, due to toxicity of accumulated highly reactive upstream metabolites (primarily AASA and P6C).

GA1 glutarica ciduria type 1

GA1 is caused by autosomal recessive deficiency of glutaryl-CoA dehydrogenase(GCDH), a mitochondrial flavoprotein that catalyses the oxidative decarboxylation of glutaryl-CoA to crotonyl-CoA in the converging degradative pathways of Llysine, L-hydroxylysine, and L-tryptophan. It is an ultra-rare disease (1:100000). The vast majority (90-95%) of untreated GA1 babies develop irreversible striatal damage and a complex movement disorder with predominant dystonia. About half of dystonic individuals never reach adulthood because of secondary complications. C5DC-based Newborn Screening for GA1 has been successfully established in a growing number of countries worldwide and is a prerequisite of a favourable disease outcome. Early diagnosis and initiation of intense metabolic therapy including low lysine diet, arginine supplementation, and carnitine supplementation can prevent the onset of severe striatal injury in about 2/3 of GA1 patients. Despite early and aggressive management, many patients do not obtain benefit, and available therapies do not prevent the manifestation of chronic kidney disease and the progression of white matter changes in adolescence and adulthood (18-20). Finally, late start and poor adherence to therapy might increase the risk of brain neoplasms. To date, the complex pathogenic mechanisms underlying GA1 are still incompletely understood. However, it is clear that potentially toxic metabolites accumulate in the brain with age. Metabolite-induced inhibition of the 2-oxoglutarate dehydrogenase complex and the NC3 transporter, and inhibition or glutarylation of glutamate dehydrogenase all synergise in reducing flux through the TCA cycle, causing bioenergetic impairment and brain dysfunction.


State of the art Research
The Netherlands
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The International PDE Consortium, established and chaired by our CHARLIE coordinator Prof Van Karnebeek unites the expertise of over 50 scientists and clinicians to address gaps in disease insights, early diagnosis, targeted therapies and patient care. The PDE Consortium published consensus recommendations for diagnosis and treatment, developed digital tools for dietary adherence, and manages a digital PDE registry that includes genotype, phenotype, and clinical outcome data for more than 250 PDE patients. Our CHARLIE coordinator was the first to target the newly unveiled genetic and biochemical pathophysiology, leading studies of dietary lysine reduction in addition to B6 therapy for PDE patients. AASA levels decreased in body fluids; however, the effect on the primary clinical outcomes of epilepsy, development, and cognition was insufficient. Additionally, partner Coene and her Radboudumc team discovered 2-oxopropylpiperidine- 2-carboxylic acid (2-OPP) in PDE patient blood. (Fig.2*) This novel biomarker meets all criteria for newborn screening panel inclusion and together with 6-oxopipecolic acid (6-oxoPIP) is suitable for diagnostic and therapeutic monitoring.

CANADA
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Our Canadian partner Prof. Leavitt recently generated the Aldh7a1 deficient mouse to study disease pathophysiology, and as a PDE model for pre-clinical therapeutic development. Extensive phenotypic characterization revealed that Aldh7a1-/- mice accurately recapitulate the PDE patients’ biochemical and clinical phenotypes; developing high levels of the key PDE biomarker metabolites and neurobehavioral deficits (Fig. 3**). Aldh7a1-/- mice subjected to high-lysine diet exhibited vigorous seizures and died within 2 days. Mice on a low-lysine diet had improved seizure control, but developed neurobehavioral deficits and accumulated lysine metabolites similar to patients. An aldh7a1-null zebrafish model was also generated by our CHARLIE partners and displays a spontaneous epilepsy phenotype even without lysine fortification. The zebrafish model replicates other hallmark PDE features such as B6-responsiveness and P6C and PIP accumulation. These genetically accurate PDE animal models are a crucial asset for pre-clinical testing of our innovative therapeutic approaches.

GERMANY

For almost two decades, the Heidelberg institution has conducted comprehensive studies in large patient cohorts (>98.3% of patients identified by newborn screening, Germany) to elucidate natural history, safety and efficacy of current therapies, newborn screening, short and long-term health outcomes of GA1 patients. These studies have critically assessed the limitations of current therapies. Since 2003, the institution has been coordinating the development of evidence-based guidelines by an international group of experts, which has become the gold standard of clinical care for GA1 patients. In addition, the Heidelberg colleagues have coordinated the EU funded project “European registry and network for intoxication type metabolic diseases” (E-IMD), a web based modular registry (follow-up data of 1500 IMD patients) including >250 GA1 patients, and the EU funded project “Unified registry of inherited metabolic diseases” (U-IMD), which is the official registry of the European reference network for Hereditary Metabolic Disorders (MetabERN) and maps onto the European Rare Disease Registry Infrastructure; an important basis for future inter-ventional studies, fostering the identification of meaningful endpoints and patient recruitment.

SPAIN
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Research Partner Dr.Fillat and her team studied the Gcdh-/-mice neuro-pathological phenotype and response to high lysine diet. They developed tools to study this model, i.e. a new method to measure GCDH activity in brain and liver based on blue native gel electrophoresis. Also, an AAV-GCDH virus has been generated for gene therapy in Gcdh-/- mice. Six months after AAV-GCDH delivery in adult mouse, a significant reduction in serum glutarylcarnitine was observed, suggesting functional Rescue.(Fig 4***)


Our research is divided into 6 workpackages:

Workpackage 1
  • Patients in the lead
  • In this workpackage our patient expert Hanka Dekker and Prof Van Karnebeek are prioritizing and communicating patients’ needs and perspectives for integration in therapy development process & trial readiness.
Workpackage 2
  • Therapy development
  • Dr Fillat and Prof Leavitt are efficiently targeting and decreasing AASS in brain using ASO and AAV-shRNA approaches. Prof Linster and Prof La Marca are focusing on identification of small molecule inhibitors for AASS by library high-throughput screening and evaluation.
Workpackage 3
  • Model systems
  • In this workpackage representative PDE and GA1 disease models are being developed:
  • Prof Linster and her team at Luxembourg University using zebrafish models
  • Prof Van Karnebeek and her team in Radboudumc via iPSC cells
  • Prof Hrabé and his team in the German Mouse Clinic using mouse models
  • Dr Dimitrov in Heidelberg University Hospital also via mouse models
Workpackage 4
  • Proof-of-concept studies
  • In this workpackage Prof Leavitt’s team and Prof Hrabé’s team are testing efficacy of AASS inhibition on behavioural, biochemical, EEG, and neuropathologic endpoints in PDE and GA1 disease models in pre-clinical therapeutic studies.
  • The team of Dr Fillat will focus on efficient biochemical and behavioural normalization in PDE and GA1 mouse models with Gcdh and Aldh7a1 gene therapy.
Workpackage 5
  • Companion Diagnostics
  • Dr Coene and Prof La Marca started identification of efficacy biomarkers for PDE and GA1.
  • Prof Van Gool took over from Dr. Coene. He and Prof La Marca will continue to look for novel insights into and translatability of lysine metabolism from model systems to humans.
Workpackage 6
  • Coordination & knowledge dissemination
  • Prof Karnebeek and Prof Leavitt will focus on the Management structure to monitor deliverables and timelines for effective therapy development, Exploitation, Industrialization, Risk Mitigation and Follow-on Funding.

1. Liu B, Du H, Rutkowski R, Gartner A, Wang X. LAAT-1 is the lysosomal lysine/arginine transporter
that maintains amino acid homeostasis. Science. 2012;337(6092):351-4.PMC3432903
LAAT-1 is the lysosomal lysine/arginine transporter that maintains amino acid homeostasis - PubMed (nih.gov)

2. Houten SM, Te Brinke H, Denis S, Ruiter JP, Knegt AC, de Klerk JB, et al. Genetic basis of
hyperlysinemia. Orphanet J Rare Dis. 2013;8:57.PMC3626681
Genetic basis of hyperlysinemia - PubMed (nih.gov)

3. Coughlin CR, 2nd, Swanson MA, Spector E, Meeks NJL, Kronquist KE, Aslamy M, et al. The
genotypic spectrum of ALDH7A1 mutations resulting in pyridoxine dependent epilepsy: A common epileptic
encephalopathy. J Inherit Metab Dis. 2019;42(2):353-61.PMC6345606
The genotypic spectrum of ALDH7A1 mutations resulting in pyridoxine dependent epilepsy: A common epileptic encephalopathy - PubMed (nih.gov)

4. Stockler S, Plecko B, Gospe SM, Jr., Coulter-Mackie M, Connolly M, van Karnebeek C, et al.
Pyridoxine dependent epilepsy and antiquitin deficiency: clinical and molecular characteristics and
recommendations for diagnosis, treatment and follow-up. Mol Genet Metab. 2011;104(1-2):48-60
Pyridoxine dependent epilepsy and antiquitin deficiency: clinical and molecular characteristics and recommendations for diagnosis, treatment and follow-up - PubMed (nih.gov)

5. van Karnebeek CD, Tiebout SA, Niermeijer J, Poll-The BT, Ghani A, Coughlin CR, 2nd, et al.
Pyridoxine-Dependent Epilepsy: An Expanding Clinical Spectrum. Pediatr Neurol. 2016;59:6-12
Pyridoxine-Dependent Epilepsy: An Expanding Clinical Spectrum - PubMed (nih.gov)

6. Mills PB, Struys E, Jakobs C, Plecko B, Baxter P, Baumgartner M, et al. Mutations in antiquitin in
individuals with pyridoxine-dependent seizures. Nat Med. 2006;12(3):307-9
Mutations in antiquitin in individuals with pyridoxine-dependent seizures - PubMed (nih.gov)

7. Ho G, Ueda K, Houben RF, Joa J, Giezen A, Cheng B, et al. Metabolic Diet App Suite for inborn
errors of amino acid metabolism. Mol Genet Metab. 2016;117(3):322-7
Metabolic Diet App Suite for inborn errors of amino acid metabolism - PubMed (nih.gov)

8. Wempe MF, Kumar A, Kumar V, Choi YJ, Swanson MA, Friederich MW, et al. Identification of a
novel biomarker for pyridoxine-dependent epilepsy: Implications for newborn screening. J Inherit Metab
Dis. 2019;42(3):565-74
Identification of a novel biomarker for pyridoxine-dependent epilepsy: Implications for newborn screening - PubMed (nih.gov)

9. Jansen LA, Hevner RF, Roden WH, Hahn SH, Jung S, Gospe SM, Jr. Glial localization of antiquitin:
implications for pyridoxine-dependent epilepsy. Ann Neurol. 2014;75(1):22-32.PMC3945410
Glial localization of antiquitin: implications for pyridoxine-dependent epilepsy - PubMed (nih.gov)

10. van Karnebeek CD, Stockler-Ipsiroglu S, Jaggumantri S, Assmann B, Baxter P, Buhas D, et al.
Lysine-Restricted Diet as Adjunct Therapy for Pyridoxine-Dependent Epilepsy: The PDE Consortium
Consensus Recommendations. JIMD Rep. 2014;15:1-11.PMC4270869
Lysine-Restricted Diet as Adjunct Therapy for Pyridoxine-Dependent Epilepsy: The PDE Consortium Consensus Recommendations - PubMed (nih.gov)

11. Coughlin CR, 2nd, van Karnebeek CD, Al-Hertani W, Shuen AY, Jaggumantri S, Jack RM, et al.
Triple therapy with pyridoxine, arginine supplementation and dietary lysine restriction in pyridoxinedependent
epilepsy: Neurodevelopmental outcome. Mol Genet Metab. 2015;116(1-2):35-43
Triple therapy with pyridoxine, arginine supplementation and dietary lysine restriction in pyridoxine-dependent epilepsy: Neurodevelopmental outcome - PubMed (nih.gov)

12.
Al-Shekaili H, Petkau T, Pena I, Lengyell T, Verhoeven-Duif N, Ciapaite J, et al. A Novel Mouse
Model for Pyridoxine-Dependent Epilepsy Due to Antiquitin Deficiency. Submitted / under revision to Hum
Mol Genet. 2020.
A novel mouse model for pyridoxine-dependent epilepsy due to antiquitin deficiency - PubMed (nih.gov)

13. Pena IA, Roussel Y, Daniel K, Mongeon K, Johnstone D, Weinschutz Mendes H, et al. Pyridoxine-
Dependent Epilepsy in Zebrafish Caused by Aldh7a1 Deficiency. Genetics. 2017;207(4):1501-
18.PMC5714462
Pyridoxine-Dependent Epilepsy in Zebrafish Caused by Aldh7a1 Deficiency - PubMed (nih.gov)

14. Kolker S, Garbade SF, Greenberg CR, Leonard JV, Saudubray JM, Ribes A, et al. Natural history,
outcome, and treatment efficacy in children and adults with glutaryl-CoA dehydrogenase deficiency. Pediatr
Res. 2006;59(6):840-7
Natural history, outcome, and treatment efficacy in children and adults with glutaryl-CoA dehydrogenase deficiency - PubMed (nih.gov)

15. Perez-Duenas B, De La Osa A, Capdevila A, Navarro-Sastre A, Leist A, Ribes A, et al. Brain injury
in glutaric aciduria type I: the value of functional techniques in magnetic resonance imaging. Eur J Paediatr
Neurol. 2009;13(6):534-40
Brain injury in glutaric aciduria type I: the value of functional techniques in magnetic resonance imaging - PubMed (nih.gov)

16. Kolker S, Christensen E, Leonard JV, Greenberg CR, Burlina AB, Burlina AP, et al. Guideline for the
diagnosis and management of glutaryl-CoA dehydrogenase deficiency (glutaric aciduria type I). J Inherit
Metab Dis. 2007;30(1):5-22
Guideline for the diagnosis and management of glutaryl-CoA dehydrogenase deficiency (glutaric aciduria type I) - Search Results - PubMed (nih.gov)

17. Kolker S, Garbade SF, Boy N, Maier EM, Meissner T, Muhlhausen C, et al. Decline of acute
encephalopathic crises in children with glutaryl-CoA dehydrogenase deficiency identified by newborn
screening in Germany. Pediatr Res. 2007;62(3):357-63
Decline of acute encephalopathic crises in children with glutaryl-CoA dehydrogenase deficiency identified by newborn screening in Germany - PubMed (nih.gov)

18. Couce ML, Lopez-Suarez O, Boveda MD, Castineiras DE, Cocho JA, Garcia-Villoria J, et al. Glutaric
aciduria type I: outcome of patients with early- versus late-diagnosis. Eur J Paediatr Neurol.
2013;17(4):383-9
Glutaric aciduria type I: outcome of patients with early- versus late-diagnosis - PubMed (nih.gov)

19. Kolker S, Boy SP, Heringer J, Muller E, Maier EM, Ensenauer R, et al. Complementary dietary
treatment using lysine-free, arginine-fortified amino acid supplements in glutaric aciduria type I - A decade
of experience. Mol Genet Metab. 2012;107(1-2):72-80
Complementary dietary treatment using lysine-free, arginine-fortified amino acid supplements in glutaric aciduria type I - A decade of experience - PubMed (nih.gov)

20. Harting I, Neumaier-Probst E, Seitz A, Maier EM, Assmann B, Baric I, et al. Dynamic changes of
striatal and extrastriatal abnormalities in glutaric aciduria type I. Brain. 2009;132(Pt 7):1764-82
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Dynamic changes of striatal and extrastriatal abnormalities in glutaric aciduria type I - PubMed (nih.gov)

21. Boy N, Heringer J, Brackmann R, Bodamer O, Seitz A, Kolker S, et al. Extrastriatal changes in
patients with late-onset glutaric aciduria type I highlight the risk of long-term neurotoxicity. Orphanet J Rare
Dis. 2017;12(1):77.PMC5402644
Extrastriatal changes in patients with late-onset glutaric aciduria type I highlight the risk of long-term neurotoxicity - PubMed (nih.gov)

22. Sauer SW, Okun JG, Fricker G, Mahringer A, Muller I, Crnic LR, et al. Intracerebral accumulation of
glutaric and 3-hydroxyglutaric acids secondary to limited flux across the blood-brain barrier constitute a
biochemical risk factor for neurodegeneration in glutaryl-CoA dehydrogenase deficiency. J Neurochem.
2006;97(3):899-910
Intracerebral accumulation of glutaric and 3-hydroxyglutaric acids secondary to limited flux across the blood-brain barrier constitute a biochemical risk factor for neurodegeneration in glutaryl-CoA dehydrogenase deficiency - PubMed (nih.gov)

23. Sauer SW, Okun JG, Schwab MA, Crnic LR, Hoffmann GF, Goodman SI, et al. Bioenergetics in
glutaryl-coenzyme A dehydrogenase deficiency: a role for glutaryl-coenzyme A. J Biol Chem.
2005;280(23):21830-6
Bioenergetics in glutaryl-coenzyme A dehydrogenase deficiency: a role for glutaryl-coenzyme A - PubMed (nih.gov)

24. Lamp J, Keyser B, Koeller DM, Ullrich K, Braulke T, Muhlhausen C. Glutaric aciduria type 1
metabolites impair the succinate transport from astrocytic to neuronal cells. J Biol Chem.
2011;286(20):17777-84.PMC3093853
Glutaric aciduria type 1 metabolites impair the succinate transport from astrocytic to neuronal cells - PubMed (nih.gov)

25. Komatsuzaki S, Ediga RD, Okun JG, Kolker S, Sauer SW. Impairment of astrocytic glutaminolysis in
glutaric aciduria type I. J Inherit Metab Dis. 2018;41(1):91-9
Impairment of astrocytic glutaminolysis in glutaric aciduria type I - PubMed (nih.gov)

26. Schmiesing J, Storch S, Dorfler AC, Schweizer M, Makrypidi-Fraune G, Thelen M, et al. Disease-
Linked Glutarylation Impairs Function and Interactions of Mitochondrial Proteins and Contributes to
Mitochondrial Heterogeneity. Cell Rep. 2018;24(11):2946-56
Disease-Linked Glutarylation Impairs Function and Interactions of Mitochondrial Proteins and Contributes to Mitochondrial Heterogeneity - PubMed (nih.gov)

27. Heringer J, Boy SP, Ensenauer R, Assmann B, Zschocke J, Harting I, et al. Use of guidelines improves the neurological outcome in glutaric aciduria type I. Ann Neurol. 2010;68(5):743-52
Use of guidelines improves the neurological outcome in glutaric aciduria type I - PubMed (nih.gov)

28. Boy N, Mengler K, Thimm E, Schiergens KA, Marquardt T, Weinhold N, et al. Newborn screening: A disease-changing intervention for glutaric aciduria type 1. Ann Neurol. 2018;83(5):970-9
Newborn screening: A disease-changing intervention for glutaric aciduria type 1 - PubMed (nih.gov)

29.   Nikolas Boy, Chris Mühlhausen, Esther M Maier, Diana Ballhausen, Matthias R Baumgartner, Skadi Beblo, Peter Burgard, Kimberly A Chapman, Dries Dobbelaere, Jana Heringer-Seifert, Sandra Fleissner, Karina Grohmann-Held, Gabriele Hahn, Inga Harting, Georg F Hoffmann, Frank Jochum, Daniela Karall , Vassiliki Konstantopoulous , Michael B Krawinkel, Martin Lindner , E M Charlotte Märtner, Jean-Marc Nuoffer, Jürgen G Okun, Barbara Plecko, Roland Posset, Katja Sahm, Sabine Scholl-Bürgi, Eva Thimm , Magdalena Walter, Monique Williams, Stephan Vom Dahl , Athanasia Ziagaki , Johannes Zschocke , Stefan Kölker Recommendations for diagnosing and managing individuals with glutaric aciduria type 1: Third revision
Recommendations for diagnosing and managing individuals with glutaric aciduria type 1: Third revision

30. Imke M.E. Schuurmans a b, Ka M. Wu c, Clara D.M. van Karnebeek b d, Nael Nadif Kasri c, Alejandro Garanto Generation of an induced pluripotent stem cell line carrying biallelic deletions (SCTCi019-B) in ALDH7A1 using CRISPR/Cas9
Generation of an induced pluripotent stem cell line carrying biallelic deletions (SCTCi019-B) in ALDH7A1 using CRISPR/Cas9

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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the EJP RD COFUND-EJP N° 825575.

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