The Mutation Matters: CSF Profiles of GCase, Sphingolipids, α-Synuclein in PDGBA.

BACKGROUND: With pathway-specific trials in PD associated with variants in the glucocerebrosidase gene (PDGBA ) under way, we need markers that confirm the impact of genetic variants in patient-derived biofluids in order to allow patient stratification merely based on genetics and that might serve as biochemical read-out for target engagement. OBJECTIVE: To explore GBA-pathway-specific biomarker profiles cross-sectionally (TUEPAC-MIGAP, PPMI) and longitudinally (PPMI). METHODS: We measured enzyme activity of the lysosomal glucocerebrosidase, CSF levels of glucosylceramides (upstream substrate of glucocerebrosidase), CSF levels of ceramides (downstream product of glucocerebrosidase), lactosylceramides, sphingosines, sphingomyelin (by-products) and CSF levels of total α-synuclein in PDGBA patients compared to PDGBA_wildtype patients. RESULTS: Cross-sectionally in both cohorts and longitudinally in PPMI: (1) glucocerebrosidase activity was significantly lower in PDGBA compared to PDGBA_wildtype . (2) CSF levels of upstream substrates (glucosylceramides species) were higher in PDGBA compared to PDGBA_wildtype . (3) CSF levels of total α-synuclein were lower in PDGBA compared to PDGBA_wildtype . All of these findings were most pronounced in PDGBA with severe mutations (PDGBA_severe ). Cross-sectionally in TUEPAC-MIGAP and longitudinally in PPMI, CSF levels of downstream-products (ceramides) were higher in PDGBA_severe . Cross-sectionally in TUEPAC-MIGAP by-products sphinganine and sphingosine-1-phosphate and longitudinally in PPMI species of by-products lactosylceramides and sphingomyelin were higher in PDGBA_severe . INTERPRETATION: These findings confirm that GBA mutations have a relevant functional impact on biomarker profiles in patients. Bridging the gap between genetics and biochemical profiles now allows patient stratification for clinical trials merely based on mutation status. Importantly, all findings were most prominent in PDGBA with severe variants. © 2021 International Parkinson and Movement Disorder Society.

SERAC1 deficiency causes complicated HSP: evidence from a novel splice mutation in a large family.

OBJECTIVE: To demonstrate that mutations in the phosphatidylglycerol remodelling enzyme SERAC1 can cause juvenile-onset complicated hereditary spastic paraplegia (cHSP) clusters, thus adding SERAC1 to the increasing number of complex lipid cHSP genes. METHODS: Combined genomic and functional validation studies (whole-exome sequencing, mRNA, cDNA and protein), biomarker investigations (3-methyl-glutaconic acid, filipin staining and phosphatidylglycerols PG34:1/PG36:1), and clinical and imaging phenotyping were performed in six affected subjects from two different branches of a large consanguineous family. RESULTS: 5 of 6 affected subjects shared cHSP as a common disease phenotype. Three subjects presented with juvenile-onset oligosystemic cHSP, still able to walk several miles at age >10-20 years. This benign phenotypic cluster and disease progression is strikingly divergent to the severe infantile phenotype of all SERAC1 cases reported so far. Two family members showed a more multisystemic juvenile-onset cHSP, indicating an intermediate phenotype between the benign oligosystemic cHSP and the classic infantile SERAC1 cluster. The homozygous splice mutation led to loss of the full-length SERAC1 protein and impaired phosphatidylglycerol PG34:1/PG36:1 remodelling. These phosphatidylglycerol changes, however, were milder than in classic infantile-onset SERAC1 cases, which might partially explain the milder SERAC1 phenotype. CONCLUSIONS: Our findings add SERAC1 to the increasing list of complex lipid cHSP genes. At the same time they redefine the phenotypic spectrum of SERAC1 deficiency. It is associated not only with the severe infantile-onset 'Methylglutaconic aciduria, Deafness, Encephalopathy, Leigh-like' syndrome (MEGDEL syndrome), but also with oligosystemic juvenile-onset cHSP as part of the now unfolding SERAC1 deficiency spectrum.

Enzymatic characterization of novel arylsulfatase A variants using human arylsulfatase A-deficient immortalized mesenchymal stromal cells.

Metachromatic leukodystrophy (MLD) is an autosomal-recessive lysosomal storage disease caused by mutations in the ARSA gene leading to arylsulfatase A (ARSA) deficiency and causing sulfatide accumulation. Main symptoms of the disease are progressive demyelination, neurological dysfunction, and reduced life expectancy. To date, more than 200 different ARSA variants have been reported in MLD patients. Here, we report the biochemical characterization of seven novel pathogenic variants (c.98T > C, c.195delC, c.229G > C, c.545C > G, c.674A > G, c.852T > A, and c.1274A > G), which were found when sequencing a cohort of 31 German MLD families. For that purpose, the ARSA cDNAs carrying the respective mutations inserted by site-directed mutagenesis were cloned into a MigR1 (MSCV, IRES, GFP, retrovirus-1) vector. The constructs were overexpressed using retroviral gene transfer in immortalized, human multipotent mesenchymal stromal cells prepared from a patient deficient in ARSA activity (late infantile MLD). In this novel ARSA-/- cell system, the seven ARSA mutants showed ARSA activity of less than 10% when compared with wild type, which is evidence for the pathogenicity of all seven variants. In conclusion, the system of ARSA-/- -immortalized MSC turned out to be a helpful novel tool for the biochemical characterization of ARSA variants.

Rare Variant of GM2 Gangliosidosis through Activator-Protein Deficiency.

GM2 gangliosidosis, AB variant, is a very rare form of GM2 gangliosidosis due to a deficiency of GM2 activator protein. We report on two patients with typical clinical features suggestive of GM2 gangliosidosis, but normal results for hexosaminidase A and hexosaminidase B as well as their corresponding genes. Genetic analysis of the gene encoding the activator protein, the GM2A gene, elucidated the cause of the disease, adding a novel mutation to the spectrum of GM2 AB variant. This report points out that in typical clinical constellations with normal enzyme results, genetic diagnostic for activator protein defects should be performed.

Clinical, Imaging, Genetic, and Disease Course Characteristics in Patients With GM2 Gangliosidosis: Beyond Age of Onset

BACKGROUND AND OBJECTIVES: GM2 gangliosidoses, a group of autosomal-recessive neurodegenerative lysosomal storage disorders, result from ß-hexosaminidase (HEX) deficiency with GM2 ganglioside as its main substrate. Historically, GM2 gangliosidoses have been classified into infantile, juvenile, and late-onset forms. With disease-modifying treatment trials now on the horizon, a more fine-grained understanding of the disease course is needed. METHODS: We aimed to map and stratify the clinical course of GM2 gangliosidoses in a multicenter cohort of pediatric and adult patients. Patients were stratified according to age at onset and age at diagnosis. The 2 resulting GM2 disease clusters were characterized in-depth for respective disease features (detailed standardized clinical, laboratory, and MRI assessments) and disease evolution. RESULTS: In 21 patients with GM2 gangliosidosis (17 Tay-Sachs, 2 GM2 activator deficiency, 2 Sandhoff disease), 2 disease clusters were discriminated: an early-onset and early diagnosis cluster (type I; n = 8, including activator deficiency and Sandhoff disease) and a cluster with very variable onset and long interval until diagnosis (type II; n = 13 patients). In type I, rapid onset of developmental stagnation and regression, spasticity, and seizures dominated the clinical picture. Cherry red spot, startle reactions, and elevated AST were only seen in this cluster. In type II, problems with balance or gait, muscle weakness, dysarthria, and psychiatric symptoms were specific and frequent symptoms. Ocular signs were common, including supranuclear vertical gaze palsy in 30%. MRI involvement of basal ganglia and peritrigonal hyperintensity was seen only in type I, whereas predominant infratentorial atrophy (or normal MRI) was characteristic in type II. These types were, at least in part, associated with certain genetic variants. DISCUSSION: Age at onset alone seems not sufficient to adequately predict different disease courses in GM2 gangliosidosis, as required for upcoming trial planning. We propose an alternative classification based on age at disease onset and dynamics, predicted by clinical features and biomarkers, into type I-an early-onset, rapid progression cluster-and type II-a variable onset, slow progression cluster. Specific diagnostic workup, including GM2 gangliosidosis, should be performed in patients with combined ataxia plus lower motor neuron weakness to identify type II patients.

Human multipotent mesenchymal stromal cells use galectin-1 to inhibit immune effector cells.

Human multipotent mesenchymal stromal cells (MSCs) suppress proliferation and alloreactivity of T cells. Several signaling molecules and enzymes contribute to this effect. We focused on carbohydrate-protein interactions and investigated whether lectins are involved in immune modulation by MSC. Gene expression profiling of MSCs revealed that one of the most important lectins in this setting, galectin-1, was highly expressed. Galectin-1 protein was detected intracellularly and on the cell surface of MSCs. In addition, galectin-1 was released into the cell culture supernatant by MSCs. To analyze the functional role of galectin-1, a stable knockdown of galectin-1 in MSCs with use of a retroviral transfection system was established. Galectin-1 knockdown in MSCs resulted in a significant loss of their immunomodulatory properties, compared with MSCs infected with nontargeting control sequences. The galectin-1 knockdown partially restored the proliferation of CD4(+) and CD8(+) T cells. By contrast, the effect of MSCs on nonalloreactive natural killer (NK) cells was unaffected by down-regulation of galectin-1 expression. Furthermore, MSC-derived galectin-1 significantly modulated the release of cytokines involved in graft-versus-host disease (GVHD) and autoimmunity (eg, tumor necrosis factor-α [TNFα], IFNγ, interleukin-2 [IL-2], and IL-10. These results identify galectin-1 as the first lectin mediating the immunomodulatory effect of MSCs on allogeneic T cells.

Chitotriosidase is a biomarker for adult-onset leukoencephalopathy with axonal spheroids and pigmented glia.

Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) leads to rapidly progressive dementia and is caused by mutations in the gene CSF1R. Neurodegeneration is driven by dysfunction of microglia, the predominant cell type expressing CSF1R in the brain. We assessed chitotriosidase, an enzyme secreted by microglia, in serum and cerebrospinal fluid of patients with ALSP. Chitotriosidase activity was highly increased in cerebrospinal fluid of patients and correlated inversely with disease duration. Of interest, presymptomatic CSF1R mutation carriers did not show elevated chitotriosidase levels. This makes chitotriosidase a promising new biomarker of disease activity for this rare disease.

Identification of neurodegeneration indicators and disease progression in metachromatic leukodystrophy using quantitative NMR-based urinary metabolomics.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by a deficiency of the arylsulfatase A (ARSA). ARSA deficiency leads to an accumulation of sulfatides primarily in the nervous system ultimately causing demyelination. With evolving therapeutic options, there is an increasing need for indicators to evaluate disease progression. Here, we report targeted metabolic urine profiling of 56 MLD patients including longitudinal sampling, using 1H (proton) nuclear magnetic resonance (NMR) spectroscopy. 1H-NMR urine spectra of 119 MLD samples and 323 healthy controls were analyzed by an in vitro diagnostics research (IVDr) tool, covering up to 50 endogenous and 100 disease-related metabolites on a 600-MHz IVDr NMR spectrometer. Quantitative data reports were analyzed regarding age of onset, clinical course, and therapeutic intervention. The NMR data reveal metabolome changes consistent with a multiorgan affection in MLD patients in comparison to controls. In the MLD cohort, N-acetylaspartate (NAA) excretion in urine is elevated. Early onset MLD forms show a different metabolic profile suggesting a metabolic shift toward ketogenesis in comparison to late onset MLD and controls. In samples of juvenile MLD patients who stabilize clinically after hematopoietic stem cell transplantation (HSCT), the macrophage activation marker neopterin is elevated. We were able to identify different metabolic patterns reflecting variable organ disturbances in MLD, including brain and energy metabolism and inflammatory processes. We suggest NAA in urine as a quantitative biomarker for neurodegeneration. Intriguingly, elevated neopterin after HSCT supports the hypothesis that competent donor macrophages are crucial for favorable outcome.

A Mutation-Agnostic Hematopoietic Stem Cell Gene Therapy for Metachromatic Leukodystrophy.

Metachromatic leukodystrophy (MLD) is a rare genetic disorder caused by mutations in the Arylsulfatase-A (ARSA) gene. The enzyme plays a key role in sulfatide metabolism in brain cells, and its deficiency leads to neurodegeneration. The clinical manifestations of MLD include stagnation and decline of motor and cognitive function, leading to premature death with limited standard treatment options. Here, we describe a mutation-agnostic hematopoietic stem and progenitor cell (HSPC) gene therapy using CRISPR-Cas9 and AAV6 repair template as a prospective treatment option for MLD. Our strategy achieved efficient insertions and deletions (>87%) and a high level of gene integration (>47%) at the ARSA locus in human bone marrow-derived HSPCs, with no detectable off-target editing. As a proof of concept, we tested our mutation-agnostic therapy in HSPCs derived from two MLD patients with distinct mutations and demonstrated restoration of ARSA enzyme activity (>30-fold improvement) equivalent to healthy adults. In summary, our investigation enabled a mutation-agnostic therapy for MLD patients with proven efficacy and strong potential for clinical translation.

Hematopoietic Stem Cell Transplantation with Mesenchymal Stromal Cells in Children with Metachromatic Leukodystrophy.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder primarily affecting the white matter of the nervous system that results from a deficiency of the arylsulfatase A (ARSA). Mesenchymal stem cells (MSCs) are able to secrete ARSA and have shown beneficial effects in MLD patients. In this retrospective analysis, 10 pediatric MLD patients [mesenchymal stem cell group (MSCG)] underwent allogeneic hematopoietic stem cell transplantation (HSCT) and received two applications of 2 × 106 MSCs/kg bodyweight at day +30 and +60 after HSCT between 2007 and 2018. MSC safety, occurrence of graft-versus-host disease (GvHD), blood ARSA levels, chimerism, cell regeneration and engraftment, magnetic resonance imaging (MRI) changes, and the gross motor function were assessed within the first year of HSCT. The long-term data included clinical outcomes and safety aspects of MSCs. Data were compared to a control cohort of seven pediatric MLD patients [control group (CG)] who underwent HSCT only. The application of MSC in pediatric MLD patients after allogeneic HSCT was safe and well tolerated, and long-term potentially MSC-related adverse effects up to 13.5 years after HSCT were not observed. Patients achieved significantly higher ARSA levels (CG: median 1.03 nmol·10-6 and range 0.41-1.73 | MSCG: median 1.58 nmol·10-6 and range 0.44-2.6; P < 0.05), as well as significantly higher leukocyte (P < 0.05) and thrombocyte (P < 0.001) levels within 365 days of MSC application compared to CG patients. Statistically significant effects on acute GvHD, regeneration of immune cells, MRI changes, gross motor function, and clinical outcomes were not detected. In conclusion, the application of MSCs in pediatric MLD patients after allogeneic HSCT was safe and well tolerated. The two applications of 2 × 106/kg allogeneic MSCs were followed by improved engraftment and hematopoiesis within the first year after HSCT. Larger, prospective trials are necessary to evaluate the impact of MSC application on engraftment and hematopoietic recovery.

Association of Age at Onset and First Symptoms With Disease Progression in Patients With Metachromatic Leukodystrophy.

OBJECTIVE: To compare disease progression between different onset forms of metachromatic leukodystrophy (MLD) and to investigate the influence of the type of first symptoms on the natural course and dynamic of disease progression. METHODS: Clinical, genetic, and biochemical parameters were analyzed within a nationwide study of patients with late-infantile (LI; onset age ≤2.5 years), early-juvenile (EJ; onset age 2.6 to <6 years), late-juvenile (LJ; onset age 6 to <16 years), and adult (onset age ≥16 years) forms of MLD. First symptoms were categorized as motor symptoms only, cognitive symptoms only, or both. Standardized clinical endpoints included loss of motor and language functions, as well as dysphagia/tube feeding. RESULTS: Ninety-seven patients with MLD were enrolled. Patients with LI (n = 35) and EJ (n = 18) MLD exhibited similarly rapid disease progression, all starting with motor symptoms (with or without additional cognitive symptoms). In LJ (n = 38) and adult-onset (n = 6) patients, the course of the disease was as rapid as in the early-onset forms, when motor symptoms were present at disease onset, while patients with only cognitive symptoms at disease onset exhibited significantly milder disease progression, independently of their age at onset. A certain genotype-phenotype correlation was observed. CONCLUSIONS: In addition to age at onset, the type of first symptoms predicts the rate of disease progression in MLD. These findings are important for counseling and therapy. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that in patients with MLD, age at onset and the type of first symptoms predict the rate of disease progression.

Long-term disease course of two patients with multiple sulfatase deficiency differs from metachromatic leukodystrophy in a broad cohort.

Multiple sulfatase deficiency (MSD) is a lysosomal storage disease caused by a deficiency of formylglycine-generating enzyme due to SUMF1 defects. MSD may be misdiagnosed as metachromatic leukodystrophy (MLD), as neurological and neuroimaging findings are similar, and arylsulfatase A (ARSA) deficiency and enhanced urinary sulfatide excretion may also occur. While ARSA deficiency seems a cause for neurological symptoms and later neurodegenerative disease course, deficiency of other sulfatases results in clinical features such as dysmorphism, dysostosis, or ichthyosis. We report on a girl and a boy of the same origin presenting with severe ARSA deficiency and neurological and neuroimaging features compatible with MLD. However, exome sequencing revealed not yet described homozygosity of the missense variant c.529G > C, p.Ala177Pro in SUMF1. We asked whether dynamics of disease course differs between MSD and MLD. Comparison to a cohort of 59 MLD patients revealed different disease course concerning onset and disease progression in both MSD patients. The MSD patients showed first gross motor symptoms earlier than most patients with juvenile MLD (<10th percentile of Gross-Motor-Function in MLD [GMFC-MLD] 1). However, subsequent motor decline was more protracted (75th and 90th percentile of GMFC-MLD 2 (loss of independent walking) and 75th percentile of GMFC-MLD 5 (loss of any locomotion)). Language decline started clearly after 50th percentile of juvenile MLD and progressed rapidly. Thus, dynamics of disease course may be a further clue for the characterization of MSD. These data may contribute to knowledge of natural course of ultra-rare MSD and be relevant for counseling and therapy.

Optimization of Enzyme Essays to Enhance Reliability of Activity Measurements in Leukocyte Lysates for the Diagnosis of Metachromatic Leukodystrophy and Gangliosidoses.

(1) Lysosomal storage diseases are rare inherited disorders with no standardized or commercially available tests for biochemical diagnosis. We present factors influencing the quality of enzyme assays for metachromatic leukodystrophy (MLD) and gangliosidoses (GM1; GM2 variants B and 0) and validate the reliability and stability of testing in a retrospective analysis of 725 samples. (2) Patient leukocytes were isolated from ethylene-diamine-tetra-acetic acid (EDTA) blood and separated for subpopulation experiments using density gradient centrifugation or magnetic cell separation. Enzyme activities in whole leukocyte lysate and leukocyte subpopulations were determined. (3) The enzyme activities in leukocyte subpopulations differed significantly. Compared to lymphocytes, the respective enzyme activities were 2.31-4.57-fold higher in monocytes and 1.64-2.81-fold higher in granulocytes. During sample preparation, a considerable amount of the lysosomal enzymes was released from granulocytes. Nevertheless, with the sample preparation method used here, total leukocyte count proved to be more accurate than total protein amount as a reference unit for enzyme activities. Subsequent analysis of 725 individuals showed clear discrimination of enzyme activities in patient samples (48 MLD; 21 gangliosidoses), with a sensitivity of 100% and specificity of 98-99%.

Early clinical course after hematopoietic stem cell transplantation in children with juvenile metachromatic leukodystrophy.

BACKGROUND: Long-term outcomes of hematopoietic stem cell transplantation (HSCT) in children with juvenile metachromatic leukodystrophy (MLD) have been investigated systematically, while short-term effects of HSCT on the course of the disease remain to be elucidated. RESULTS: In this study, the clinical course was evaluated over the first 24 months following HSCT, conducted at our center in 12 children with juvenile MLD (mean follow-up 6.75 years, range 3-13.5) and compared with 35 non-transplanted children with juvenile MLD. Motor function (GMFM-88 and GMFC-MLD), cognitive function (FSIQ), peripheral neuropathy (tibial nerve conduction velocity), and cerebral changes (MLD-MR severity score) were tested prospectively. Seven children remained neurologically stable over a long period, five exhibited rapid disease progression over the first 12 to 18 months after transplantation. In the latter, time from first gross motor symptoms to loss of independent walking was significantly shorter compared with non-transplanted patients at the same stage of disease (p < 0.02). Positive prognostic factors were good motor function (GMFM = 100%, GMFC-MLD = 0) and a low MR severity score (≤ 17) at the time of HSCT. CONCLUSIONS: Our results show that if disease progression occurs, this happens early on after HSCT and proceeds faster than in non-transplanted children with juvenile MLD, indicating that HSCT may trigger disease progression.

Measurement of recombinant human arylsulfatase A and leukocyte sulfatase activities by analytical isotachophoresis.

Metachromatic Leukodystrophy (MLD) and Multiple Sulfatase Deficiency (MSD) are rare and ultra-rare lysosomal storage diseases. Due to enzyme defects, patients are unable to split the sulfategroup from the respective substrates. In MSD all sulfatases are affected due to a defect of the Sulfatase Modifying Factor 1 (SUMF1) gene coding for the formylglycine generating enzyme (FGE) necessary for the modification of the active site of sulfatases. In MLD mutations in the arylsulfatase A (ARSA) gene cause ARSA deficiency with subsequent accumulation of 3-sulfogalactocerebroside especially in oligodendrocytes. The clinical consequence is demyelination and a devastating neurological disease. Enzyme replacement therapy (ERT) with recombinant human arylsulfatase A (rhARSA), gene therapy, and stem cell transplantation are suggested as new therapeutic options. The aim of our study was to characterize rhARSA concerning its substrate specificity using analytical isotachophoresis (ITP). Substrate specificity could be demonstrated by sulfate splitting from the natural substrates 3-sulfogalactocerebroside and ascorbyl-2-sulfate and the artificial substrate p-nitrocatecholsulfate, whereas galactose-6-sulfate, a substrate of galactose-6­sulfurylase, was totally resistant. In contrast, leukocyte extracts of healthy donors were able to split sulfate also from galactose-6-sulfate. The ITP method allows therefore a rapid and simple differentiation between samples of MLD and MSD patients and healthy donors. Therefore, the isotachophoretic diagnostic assay from leukocyte extracts described here provides a fast and efficient way for the diagnosis of MLD and MSD patients and an elegant system to differentiate between these diseases in one assay.

Generation of two iPSC lines derived from two unrelated patients with Gaucher disease.

Gaucher disease is the most common autosomal recessive lysosomal storage disorder, caused by mutations in the ß-glucocerebrosidase gene GBA. Here we describe generation of iPSC from skin-derived fibroblasts from two unrelated individuals with neuronopathic forms of Gaucher disease. The donor for line iPSC-GBA-1, a 21 month old girl, carried the recurring GBA mutation c.1448 T > C, p.Leu483Pro at homozygous state; fibroblasts for line iPS-GBA-2 were obtained from a 4 year old girl compound heterozygous for the GBA mutations c.667 T > C, p.Trp223Arg and c.1226A > G, p.Asn409Ser. iPSCs were developed using integration free episomal vectors (OCT4, KLF4; L-MYC, SOX2 (OSKM) and LIN28). Resource table.

Phenotypic variation between siblings with Metachromatic Leukodystrophy.

BACKGROUND: Metachromatic Leukodystrophy (MLD) is a rare autosomal-recessive lysosomal storage disorder caused by mutations in the ARSA gene. While interventional trials often use untreated siblings as controls, the genotype-phenotype correlation is only partly understood, and the variability of the clinical course between siblings is unclear with some evidence for a discrepant clinical course in juvenile patients. The aim of this study was to systematically investigate the phenotypic variation in MLD siblings in comparison to the variability in a larger MLD cohort and to case reports published in literature. RESULTS: Detailed clinical information was available from 12 sibling-pairs (3 late-infantile, 9 juvenile) and 61 single patients (29 late-infantile, 32 juvenile). Variability of age at onset was similar between the siblings and randomly chosen pairs of the remaining cohort (no statistically different Euclidean distances). However, in children with juvenile MLD both the type of first symptoms and the dynamic of the disease were less variable between siblings compared to the general cohort. In late-infantile patients, type of first symptoms and dynamic of disease were similarly homogeneous between siblings and the whole MLD cohort. Thirteen published case reports of families with affected siblings with MLD are presented with similar findings. CONCLUSIONS: In a systematic analysis of phenotypic variation in families with MLD, siblings with the late-infantile form showed a similar variability as unrelated pairs of children with late-infantile MLD, whereas siblings with juvenile MLD showed a more homogeneous phenotype regarding type of first symptoms and disease evolution in comparison to unrelated children with juvenile MLD, but not regarding their age at onset. These results are highly relevant with respect to the evaluation of treatment effects and for counseling of families with affected siblings.

Late onset Krabbe disease due to the new GALC p.Ala543Pro mutation, with intriguingly high residual GALC activity in vitro.

BACKGROUND: Krabbe disease (KD) is an inherited leukodystrophy due to a defect in the GALC gene which encodes the lysosomal galactosylceramide ß-galactosidase (GALC). About two thirds of patients show the early onset form of KD dominated by cerebral demyelination leading to death in early infancy. Late onset forms include a spectrum of late infantile, juvenile and adult clinical courses. The deficiency of GALC leads to a galactosylceramide lipidosis in which lysosomal storage phenomena are seen almost only at the ultrastructural level. RESULTS: In a 4-year-old boy, the clinical suspicion of KD was high according to neurologic and neuroimaging findings. However, laboratory results were inconclusive; white blood cell GALC activity being at 23 to 25% of the normal level, and GALC genotyping revealing the new homozygous p.Ala543Pro variant which, ex silico, was of unclear significance. Studying a skin biopsy, cultured fibroblasts showed the GALC activity at 21 to 30% of the normal level; ultrastructurally, clearly KD-specific inclusions were seen in the eccrine sweat gland cells, confirming a KD diagnosis. CONCLUSION: The high clinical suspicion combined with the morphologic evidence for KD predict that the p.Ala543Pro variant is pathogenic for (late onset) KD. A hypothesis linked to the proline in the mutant GALC may explain the in vitro effect with high residual GALC activity. This patient would not have been correctly diagnosed, despite the strong clinical criteria of KD, if the electron microscopic results had not been available. The detailed knowledge of neurologic and neuroimaging signs is important in diagnostically problematic KD patients in which also an electron microscopic approach can be crucial.

Long-term Outcome of Allogeneic Hematopoietic Stem Cell Transplantation in Patients With Juvenile Metachromatic Leukodystrophy Compared With Nontransplanted Control Patients.

IMPORTANCE: Allogeneic hematopoietic stem cell transplantation (HSCT) has been the only treatment option clinically available during the last 20 years for juvenile metachromatic leukodystrophy (MLD), reported with variable outcome and without comparison with the natural course of the disease. OBJECTIVE: To compare the long-term outcome of patients who underwent allogeneic HSCT with control patients who did not among a cohort with juvenile MLD. DESIGN, SETTING, AND PARTICIPANTS: Patients with juvenile MLD born between 1975 and 2009 and who received HSCT at a median age of 7 years (age range, 1.5-18.2 years) and nontransplanted patients with juvenile MLD born between 1967 and 2007 were included in this case-control study. The median follow-up after HSCT was 7.5 years (range, 3.0-19.7 years). Patients underwent HSCT at 3 German centers between 1991 and 2012. The analysis was done between July 2014 and August 2015. MAIN OUTCOMES AND MEASURES: Survival and transplantation-related mortality, loss of gross motor function (Gross Motor Function Classification in MLD), loss of any language function, and magnetic resonance imaging (MRI) severity score for cerebral changes. To explore prognostic factors at baseline, patients who underwent HSCT (hereafter, transplanted patients) were a priori divided into stable vs progressive disease, according to gross motor and cognitive function. RESULTS: Participants were 24 transplanted patients (11 boys, 13 girls) and 41 control patients (22 boys, 19 girls) who did not receive transplantation (hereafter, nontransplanted patients) with juvenile MLD. Among the transplanted patients, 4 children died of transplantation-related mortality, and 2 additional children died of rapid MLD progression 1.5 and 8.6 years after HSCT, resulting in a 5-year survival of 79% (19 of 24). Among the nontransplanted patients, 5-year survival after disease onset was 100% (41 of 41). However, 11 died of MLD progression, resulting in similar overall survival within the observation period. Nine of the long-term survivors after HSCT had disease progression, while 11 showed stable disease. Compared with the nontransplanted patients, the transplanted patients were less likely to lose their gross motor or language function and demonstrated significantly lower MRI severity scores at the latest examination. Patients after HSCT were more likely to have a stable disease course when undergoing HSCT at an early stage with no or only mild gross motor deficits (Gross Motor Function Classification in MLD level 0 or 1) and an IQ of at least 85, when age at disease onset was older than 4 years, or when MRI severity scores were low (preferably ≤17). CONCLUSIONS AND RELEVANCE: Among patients with juvenile MLD, patients who underwent HSCT had a better gross motor and language outcome and lower MRI severity scores compared with nontransplanted patients. Transplantation at a presymptomatic or early symptomatic stage of juvenile MLD is associated with a reasonable chance for disease stabilization.

Cerebral gray and white matter changes and clinical course in metachromatic leukodystrophy.

OBJECTIVE: Metachromatic leukodystrophy (MLD) is a rare metabolic disorder leading to demyelination and rapid neurologic deterioration. As therapeutic options evolve, it seems essential to understand and quantify progression of the natural disease. The aim of this study was to assess cerebral volumetric changes in children with MLD in comparison to normal controls and in relation to disease course. METHOD: Eighteen patients with late-infantile MLD and 42 typically developing children in the same age range (20-59 months) were analyzed in a cross-sectional study. Patients underwent detailed genetic, biochemical, electrophysiologic, and clinical characterization. Cerebral gray matter (GM) and white matter (WM) volumes were assessed by multispectral segmentation of T1- and T2-weighted MRI. In addition, the demyelinated WM (demyelination load) was automatically quantified in T2-weighted images of the patients, and analyzed in relation to the clinical course. RESULTS: WM volumes of patients did not differ from controls, although their growth curves were slightly different. GM volumes of patients, however, were on average 10.7% (confidence interval 6.0%-14.9%, p < 0.001) below those of normally developing children. The demyelination load (corrected for total WM volume) increased with disease duration (p < 0.003) and motor deterioration (p < 0.001). CONCLUSION: GM volume in patients with MLD is reduced when compared with healthy controls, already at young age. This supports the notion that, beside demyelination, neuronal dysfunction caused by neuronal storage plays an additional role in the disease process. The demyelination load may be a useful noninvasive imaging marker for disease progression and may serve as reference for therapeutic intervention.