Raphael Schiffmann M.D.

Vanderver, A., C. Simons, G. Helman, J. Crawford, N. I. Wolf, G. Bernard, A. Pizzino, J. L. Schmidt, A. Takanohashi, D. Miller, A. Khouzam, V. Rajan, E. Ramos, S. Chowdhury, T. Hambuch, K. Ru, G. J. Baillie, S. M. Grimmond, L. Caldovic, J. Devaney, M. Bloom, S. H. Evans, J. L. Murphy, N. McNeill, B. L. Fogel, R. Schiffmann, M. S. van der Knaap and R. J. Taft (2016). “Whole exome sequencing in patients with white matter abnormalities.” Ann Neurol 79(6): 1031-1037.

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Here we report whole exome sequencing (WES) on a cohort of 71 patients with persistently unresolved white matter abnormalities with a suspected diagnosis of leukodystrophy or genetic leukoencephalopathy. WES analyses were performed on trio, or greater, family groups. Diagnostic pathogenic variants were identified in 35% (25 of 71) of patients. Potentially pathogenic variants were identified in clinically relevant genes in a further 7% (5 of 71) of cases, giving a total yield of clinical diagnoses in 42% of individuals. These findings provide evidence that WES can substantially decrease the number of unresolved white matter cases.

Meng, X. L., T. S. Day, N. McNeill, P. Ashcraft, T. Frischmuth, S. H. Cheng, Z. P. Liu, J. S. Shen and R. Schiffmann (2016). “Molecular basis for globotriaosylceramide regulation and enzyme uptake in immortalized aortic endothelial cells from fabry mice.” J Inherit Metab Dis 39(3): 447-455.

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Fabry disease is caused by deficient activity of alpha-galactosidase A and subsequent intracellular accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3). Vascular endothelial cells may play important roles in disease pathogenesis, and are one of the main target cell types in therapeutic interventions. In this study, we generated immortalized aortic endothelial cell lines from a mouse model of Fabry disease. These cells retained endothelial cell-specific markers and functions. Gb3 expression level in one of these clones (referred to as FMEC2) was highly susceptible to culture media, and appeared to be regulated by glucosylceramide synthase. Results also showed that Gb3 could be upregulated by hydrocortisone. FMEC2 express the mannose 6-phosphate receptor and sortilin but not the mannose receptor. Uptake studies suggested that sortilin plays a role in the binding and internalization of mammalian cell-produced alpha-galactosidase A. Moss-aGal (a plant-made enzyme) was endocytosed by FMEC2 via a receptor other than the aforementioned receptors. In conclusion, this study suggests that glucosylceramide synthase and hydrocortisone may play important roles in modulating Gb3 levels in Fabry mouse aortic endothelial cells, and that endocytosis of recombinant alpha-galactosidase A involves a combination of multiple receptors depending on the properties of the enzyme.

Mochel, F., E. Hainque, D. Gras, I. M. Adanyeguh, S. Caillet, B. Heron, A. Roubertie, E. Kaphan, R. Valabregue, D. Rinaldi, S. Vuillaumier, R. Schiffmann, C. Ottolenghi, J. Y. Hogrel, L. Servais and E. Roze (2016). “Triheptanoin dramatically reduces paroxysmal motor disorder in patients with glut1 deficiency.” J Neurol Neurosurg Psychiatry 87(5): 550-553.

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OBJECTIVE: On the basis of our previous work with triheptanoin, which provides key substrates to the Krebs cycle in the brain, we wished to assess its therapeutic effect in patients with glucose transporter type 1 deficiency syndrome (GLUT1-DS) who objected to or did not tolerate ketogenic diets. METHODS: We performed an open-label pilot study with three phases of 2 months each (baseline, treatment and withdrawal) in eight patients with GLUT1-DS (7-47 years old) with non-epileptic paroxysmal manifestations. We used a comprehensive patient diary to record motor and non-motor paroxysmal events. Functional (31)P-NMR spectroscopy was performed to quantify phosphocreatine (PCr) and inorganic phosphate (Pi) within the occipital cortex during (activation) and after (recovery) a visual stimulus. RESULTS: Patients with GLUT1-DS experienced a mean of 30.8 (+/-27.7) paroxysmal manifestations (52% motor events) at baseline that dropped to 2.8 (+/-2.9, 76% motor events) during the treatment phase (p=0.028). After withdrawal, paroxysmal manifestations recurred with a mean of 24.2 (+/-21.9, 52% motor events; p=0.043). Furthermore, brain energy metabolism normalised with triheptanoin, that is, increased Pi/PCr ratio during brain activation compared to the recovery phase (p=0.021), and deteriorated when triheptanoin was withdrawn. CONCLUSIONS: Treatment with triheptanoin resulted in a 90% clinical improvement in non-epileptic paroxysmal manifestations and a normalised brain bioenergetics profile in patients with GLUT1-DS.

Schiffmann, R., M. Fuller, L. A. Clarke and J. M. Aerts (2016). “Is it fabry disease?” Genet Med: May 2016 [Epub ahead of print].

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Fabry disease is caused by mutations in the GLA gene that lower alpha-galactosidase A activity to less than 25-30% of the mean normal level. Several GLA variants have been identified that are associated with relatively elevated residual alpha-galactosidase A. The challenge is to determine which GLA variants can cause clinical manifestations related to Fabry disease. Here, we review the various types of GLA variants and recommend that pathogenicity be considered only when associated with elevated globotriaosylceramide in disease-relevant organs and tissues as analyzed by mass spectrometry. This criterion is necessary to ensure that very costly and specific therapy is provided only when appropriate.

Zhang, J., V. Lachance, A. Schaffner, X. Li, A. Fedick, L. E. Kaye, J. Liao, J. Rosenfeld, N. Yachelevich, M. L. Chu, W. G. Mitchell, R. G. Boles, E. Moran, M. Tokita, E. Gorman, K. Bagley, W. Zhang, F. Xia, M. Leduc, Y. Yang, C. Eng, L. J. Wong, R. Schiffmann, G. A. Diaz, R. Kornreich, R. Thummel, M. Wasserstein, Z. Yue and L. Edelmann (2016). “A founder mutation in vps11 causes an autosomal recessive leukoencephalopathy linked to autophagic defects.” PLoS Genet 12(4): e1005848.

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Genetic leukoencephalopathies (gLEs) are a group of heterogeneous disorders with white matter abnormalities affecting the central nervous system (CNS). The causative mutation in ~50% of gLEs is unknown. Using whole exome sequencing (WES), we identified homozygosity for a missense variant, VPS11: c.2536T>G (p.C846G), as the genetic cause of a leukoencephalopathy syndrome in five individuals from three unrelated Ashkenazi Jewish (AJ) families. All five patients exhibited highly concordant disease progression characterized by infantile onset leukoencephalopathy with brain white matter abnormalities, severe motor impairment, cortical blindness, intellectual disability, and seizures. The carrier frequency of the VPS11: c.2536T>G variant is 1:250 in the AJ population (n = 2,026). VPS11 protein is a core component of HOPS (homotypic fusion and protein sorting) and CORVET (class C core vacuole/endosome tethering) protein complexes involved in membrane trafficking and fusion of the lysosomes and endosomes. The cysteine 846 resides in an evolutionarily conserved cysteine-rich RING-H2 domain in carboxyl terminal regions of VPS11 proteins. Our data shows that the C846G mutation causes aberrant ubiquitination and accelerated turnover of VPS11 protein as well as compromised VPS11-VPS18 complex assembly, suggesting a loss of function in the mutant protein. Reduced VPS11 expression leads to an impaired autophagic activity in human cells. Importantly, zebrafish harboring a vps11 mutation with truncated RING-H2 domain demonstrated a significant reduction in CNS myelination following extensive neuronal death in the hindbrain and midbrain. Thus, our study reveals a defect in VPS11 as the underlying etiology for an autosomal recessive leukoencephalopathy disorder associated with a dysfunctional autophagy-lysosome trafficking pathway.