Robert Dobrovolny

Leber′s Hereditary Optic Neuropathy (LHON) is a maternally inherited mitochondrial disorder characterised by painless, progressive, and sequential visual failure. Most cases of LHON are driven by mitochondrial DNA mutations which cause dysfunction of respiratory Complex I, triggering retinal ganglion cell loss. Retinal ganglion cell degeneration in LHON is thought to be linked to reduced production of metabolic intermediates and adenosine triphosphate, and enhanced reactive oxygen species production. Thus, decades of research have focussed on LHON as a disease of the retinal ganglion cells, which has considerably improved our understanding of the pathology but has yielded few therapeutic interventions. In addition, some LHON-associated phenomena remain unclear. In particular, we still do not fully understand the mechanisms underlying the recorded phenomenon of spontaneous visual recovery, in which patients experience measurable increases in visual acuity following onset of LHON vision loss. Understanding this phenomenon may be critical for developing new therapeutic approaches for LHON. Moreover, the contribution of non-neuronal cell populations to LHON pathology remains poorly understood despite a growing appreciation for the roles played by these cells in other neurodegenerative conditions. Astrocytes are a highly heterogeneous group of glial cells, found throughout the central nervous system including the retina and optic nerve, and are well-known for their role as key homeostatic mediators. In recent years, our appreciation for the role played by astrocytes in neurodegenerative diseases has expanded considerably, and we are now aware that astrocytes undergo significant loss of their homeostatic functions in neurodegenerative disease, acting as key mediators of neuronal loss. Importantly, the contributions astrocytes make toward mediating LHON pathology and visual recovery remain unclear, and provide promising ground for potentially novel therapeutic angles and enhanced understanding of this complex pathology. Here, we leverage human iPSC-derived astrocytes from patients with the LHON m.14484T>C genotype, to explore the role astrocytes play in LHON pathology, stratifying cells by their visual recovery status. We report that astrocytes undergo significant morphological and bioenergetic compromise in LHON, and that differences between ′recovery′ and ′non-recovery′ astrocytes may explain individual capacity for visual recovery, potentially opening novel therapeutic approaches.

Mucopolysaccharidosis type II (MPSII) is a rare X‐linked lysosomal storage disorder caused by mutations in the iduronate‐2‐sulfatase (IDS) gene (IDS, Xq28). MPSII is characterized by skeletal deformities, hearing loss, airway obstruction, hepatosplenomegaly, cardiac valvular disease, and progressive neurological impairment. At the cellular level, IDS deficiency leads to lysosomal storage of glycosaminoglycans (GAGs), dominated by accumulation of dermatan and heparan sulfates. Human induced pluripotent stem cells (iPSC) represent an alternative system that complements the available MPSII murine model. Herein we report on the reprogramming of peripheral white blood cells from male and female MPSII patients into iPSC using a non‐integrating protocol based on the Sendai virus vector system. We differentiated the iPSC lines into IDS deficient and GAG accumulating β‐Tubulin III⁺ neurons, GFAP⁺ astrocytes, and CNPase⁺ oligodendrocytes. The lysosomal system in these cells displayed structural abnormalities reminiscent of those previously found in patient tissues and murine IDS deficient neuronal stem cells. Furthermore, quantitative determination of GAGs revealed a moderate increase in GAG levels in IDS deficient neurons and glia. We also tested the effects of recombinant IDS and found that the exogenous enzyme was internalized from the culture media and partially decreased the intracellular GAG levels in iPSC‐derived neural cells; however, it failed to completely prevent accumulation of GAGs. In summary, we demonstrate that this human iPSC based model expresses the cellular and biochemical features of MPSII, and thus represents a useful experimental tool for further pathogenesis studies as well as therapy development and testing.

Mucopolysaccharidosis type II (MPS II) is an X-linked lysosomal storage disorder resulting from deficiency of iduronate-2-sulphatase activity. The disease manifests almost exclusively in males; only 16 symptomatic heterozygote girls have been reported so far. We describe the results of X-chromosome inactivation analysis in a 5-year-old girl with clinically severe disease and heterozygous mutation p.Arg468Gln in the IDS gene. X inactivation analysed at three X-chromosome loci showed extreme skewing (96/4 to 99/1) in two patient’s cell types. This finding correlated with exclusive expression of the mutated allele. Induced pluripotent stem cells (iPSC) generated from the patient’s peripheral blood demonstrated characteristic pluripotency markers, deficiency of enzyme activity, and mutation in the IDS gene. These cells were capable of differentiation into other cell types (cardiomyocytes, neurons). In MPS II iPSC clones, the X inactivation ratio remained highly skewed in culture conditions that led to partial X inactivation reset in Fabry disease iPSC clones. Our data, in accordance with the literature, suggest that extremely skewed X inactivation favouring the mutated allele is a crucial condition for manifestation of MPS II in females. This suggests that the X inactivation status and enzyme activity have a prognostic value and should be used to evaluate MPS II in females. For the first time, we show generation of iPSC from a symptomatic MPS II female patient that can serve as a cellular model for further research of the pathogenesis and treatment of this disease.

Fabry disease is an X-linked lysosomal storage disease due to deficient α-galactosidase A (α-Gal A) activity and the resultant lysosomal accumulation of globotriaosylceramide (Gb3) and related lipids primarily in blood vessels, kidney, heart, and other organs. The renal distribution of stored glycolipid species in the α-Gal A knockout mouse model was compared to that in mice to assess relative distribution and absolute amounts of accumulated sphingolipid isoforms. Twenty isoforms of five sphingolipid groups were visualized by mass spectrometry imaging (MSI), and their distribution was compared with immunohistochemical (IHC) staining of Gb3, the major stored glycosphingolipid in consecutive tissue sections. Quantitative bulk lipid analysis of tissue sections was assessed by electrospray ionization with tandem mass spectrometry (ESI-MS/MS). In contrast to the findings in wild-type mice, all three analytical techniques (MSI, IHC, and ESI-MS/MS) revealed increases in Gb3 isoforms and ceramide dihexosides (composed mostly of galabiosylceramides), respectively. To our knowledge, this is the first report of the distribution of individual molecular species of Gb3 and galabiosylceramides in kidney sections in Fabry disease mouse. In addition, the spatial distribution of ceramides, ceramide monohexosides, and sphingomyelin forms in renal tissue is presented and discussed in the context of their biosynthesis. Graphical Abstract

For most Mendelian disorders, targeted genome sequencing is an effective method to detect causative mutations. However, sequencing PCR-amplified exonic regions and their intronic boundaries can miss large deletions or duplications and mutations that lead to PCR failures in autosomal dominant disorders and in heterozygote detection for X-linked diseases. Here, a method is described for detecting large (>50 bp) deletions/duplications in the X-linked α-galactosidase A (GLA) gene, which cause Fabry disease. Briefly, multiplex PCR mixtures were designed to amplify each GLA exon and an unrelated internal control exon to normalize GLA exonic amplicon peak heights. For each normalized GLA amplicon, the normal control female to male peak-height ratios were 1.8 to 2.2 (expected 2.0), whereas the expected ratios for deletions or duplications would be ∼1.0 or 3.0, respectively. Using this method, three novel deletions, c.369+3_547+954del4096insT, c.194+2049_369+773del2619insCG, and c.207_369+651del814ins231, were detected in unrelated women with signs and/or symptoms suggestive of Fabry disease, but no “sequencing-detectable” mutations. The deletions were confirmed by sequencing their respective GLA RT-PCR products. This method can identify gene rearrangements that may be cryptic to genomic DNA sequencing and can be readily adapted to other X-linked or autosomal dominant genes. Hum Mutat 32:1–8, 2011.

Background and Purpose— The cause of initial ischemic stroke in up to 30% of young patients remains unclear. Fabry disease, due to deficient α-galactosidase A (α-Gal A) activity, is a vascular endothelial glycosphingolipid storage disease typically presenting in childhood. With advancing age, patients develop renal, cardiac, and cerebrovascular disease and die prematurely. A European study suggested an increased prevalence of unrecognized Fabry disease in patients with cryptogenic stroke. We hypothesized that α-Gal A deficiency is a rare cause of initial early-onset ischemic stroke in men. Methods— The Stroke Prevention in Young Men Study enrolled >550 men (15 to 49 years) with first ischemic stroke in the Baltimore–Washington area in 2004 to 2007. Frozen plasma samples were assayed for α-Gal A activity, and DNA from patients with consistently low plasma α-Gal A activities were sequenced. Results— The study sample consisted of 558 men (42% African-American; median age 44 years). Stroke was cryptogenic in 154 men (40% African-American). In 10 patients with low plasma α-Gal A activities, DNA sequencing identified alterations in the α-Gal A gene in 2 patients. The polymorphism, D313Y, which results in low plasma enzyme activity, but near normal levels of cellular activity was seen in one European-American male. The Fabry disease-causing A143T mutation was seen in an African-American male with cryptogenic stroke (0.18% of all strokes: upper 95% CI=0.53%; 0.65% of cryptogenic strokes: upper 95% CI=1.92%). Conclusions— In this biracial population, unrecognized Fabry disease is a rare but treatable cause of initial ischemic stroke in young men.

Fabry disease (α‐galactosidase A (α‐Gal A, GLA) deficiency) is a panethnic inborn error of glycosphingolipid metabolism. Because optimal therapeutic outcomes depend on early intervention, a pilot program was designed to assess newborn screening for this disease in 171,977 consecutive Taiwanese newborns by measuring their dry blood spot (DBS) α‐Gal A activities and β‐galactosidase/α‐Gal A ratios. Of the 90,288 male screenees, 638 (0.7%) had DBS α‐Gal A activity 10. A second DBS assay reduced these to 91 (0.1%). Of these, 11 (including twins) had 30% (Group‐C) of mean normal leukocyte α‐Gal A activity. All 11 Group‐A, 61 Group‐B, and 1 Group‐C males had GLA gene mutations. Surprisingly, 86% had the later‐onset cryptic splice mutation c.936+919G>A (also called IVS4+919G>A). In contrast, screening 81,689 females detected two heterozygotes. The novel mutations were expressed in vitro, predicting their classical or later‐onset phenotypes. Newborn screening identified a surprisingly high frequency of Taiwanese males with Fabry disease (∼1 in 1,250), 86% having the IVS4+919G>A mutation previously found in later‐onset cardiac phenotype patients. Further studies of the IVS4 later‐onset phenotype will determine its natural history and optimal timing for therapeutic intervention. Hum Mutat 30:1–9, 2009.

The function and intracellular delivery of enzyme therapeutics for Fabry disease were studied in cultured fibroblasts and in the biopsied tissues of two male patients to show diversity of affected cells in response to treatment. In the mutant fibroblasts cultures, the final cellular level of endocytosed recombinant α-galactosidases A (agalsidases, Fabrazyme^TM, and Replagal^TM) exceeded, by several fold, the amount in control fibroblasts and led to efficient direct intra-lysosomal hydrolysis of (³H)Gb3Cer. In contrast, in the samples from the heart and some other tissues biopsied after several months of enzyme replacement therapy (ERT) with Fabrazyme^TM, only the endothelial cells were free of storage. Persistent Gb3Cer storage was found in cardiocytes (accompanied by increase of lipopigment), smooth muscle cells, fibroblasts, sweat glands, and skeletal muscle. Immunohistochemistry of cardiocytes demonstrated, for the first time, the presence of a considerable amount of the active enzyme in intimate contact with the storage compartment. Factors responsible for the limited ERT effectiveness are discussed, namely post-mitotic status of storage cells preventing their replacement by enzyme supplied precursors, modification of the lysosomal system by longstanding storage, and possible relative lack of Sap B. These observations support the strategy of early treatment for prevention of lysosomal storage.