Argentinian clinical genomics in a leukodystrophies and genetic leukoencephalopathies cohort: Diagnostic yield in our first 9 years.

INTRODUCTION AND OBJECTIVES: Leukodystrophies and genetic leukoencephalopathies constitute a vast group of pathologies of the cerebral white matter. The large number of etiopathogenic genes and the frequent unspecificity on the clinical-radiological presentation generate remarkable difficulties in the diagnosis approach. Despite recent and significant developments, molecular diagnostic yield is still less than 50%. Our objective was to develop and explore the usefulness of a new diagnostic procedure using standardized molecular diagnostic tools, and next-generation sequencing techniques. MATERIALS AND METHODS: A prospective, observational, analytical study was conducted in a cohort of 46 patients, evaluated between May 2008 and December 2016, with a suspected genetic leukoencephalopathy or leukodystrophy. A diagnostic procedure was set up using classical monogenic tools in patients with characteristic phenotypes, and next-generation techniques in nonspecific ones. RESULTS: Global diagnostic procedure yield was 57.9%, identifying the etiological pathogenesis in 22 of the 38 studied subjects. Analysis by subgroups, Sanger method, and next-generation sequencing showed a yield of 64%, and 46.1% respectively. The most common pathologies were adrenoleukodystrophy, cerebral autosomal-dominant arteriopathy with subcortical infarcts (CADASIL), and vanishing white matter disease. CONCLUSIONS: Our results confirm the usefulness of the proposed diagnostic procedure expressed in a high diagnostic yield and suggest a more optimal cost-effectiveness in an etiological analysis phase.

Brain MRI and biological diagnosis in five Tunisians MLD patients.

Metachromatic leukodystrophy (MLD) is a recessive autosomal disease which is characterized by an accumulation of sulfatides in the central and peripheral nervous system. It is due to the enzyme deficiency of the sulfatide sulfatase i.e. arylsulfatase A (ASA). we studied 5/200 cases of MLD and clearly distinguished three clinical forms. One of them presented the juvenile form; two presented the late infantile form; and two other presented the adult form. The Magnetic Resonance Imaging (MRI) of these patients showed a diffuse, bilateral and symmetrical demyelination. The biochemical diagnosis of MLD patients evidencing the low activity of ASA and sulfatide accumulation. PATIENTS AND METHODS: We studied 5/200 MLD patients addressed to us for behavioral abnormalities and progressive mental deterioration. All of them were diagnosed at first by brain MRI evidencing a bilateral demyelination, then the measurement of ASA activity using P-nitrocathecol sulfate as substrate, finally the sulfatiduria was performed using thin-layer chromatography using alpha-naphtol reagent. RESULTS: In this study, from 200 patients presenting behavioral abnormalities and a progressive mental deterioration, we reported just 2 patients were diagnosed as late-infantile form of MLD. Only1 case presented as the juvenile form; and 2 patients with the adult-type of MLD. The brain magnetic resonance imaging (MRI) of all patients showed characteristic lesions of MLD with extensive demyelination. Biochemical investigations of these patients detected a low level of ASA activity at 0°C and 37°C; the excess of sulfatide in sulfatiduria. CONCLUSION: MRI is required to orient the diagnosis of MLD patients; the latter must be confirmed by the biochemical investigations which is based on the measurement of ASA activity and the excess of sulfatide showed in the sulfatiduria. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here:http://www.diagnosticpathology.diagnomx.eu/vs/1791578262610232.

Development and reliability of a classification system for gross motor function in children with metachromatic leucodystrophy.

AIM: Motor deterioration is a key feature of late infantile and juvenile metachromatic leucodystrophy (MLD). Assessment of the disease course implies the need for a standardized description of motor decline. The aim of this study was to establish a classification system for gross motor function in MLD and to assess its interrater reliability. METHOD: The Gross Motor Function Classification in MLD (GMFC-MLD) was modelled analogous to the Gross Motor Function Classification System in cerebral palsy. Motor data from 59 individuals (27 male; 32 female) with MLD (21 late infantile; 38 juvenile) born between 1970 and 2007 were gathered from a nationwide survey and classified by six independent raters. Median age at onset was 17 months (range 9-27 mo) for the late infantile group and 74 months (35-168 mo) for the juvenile group. RESULTS: The GMFC-MLD consists of seven levels and is applicable from the age of 18 months. It represents all clinically relevant stages from normal (level 0) to loss of all gross motor function (level 6). The kappa coefficient was 0.90 for overall rater agreement. There were no significant differences between level-specific kappa coefficients. INTERPRETATION: The GMFC-MLD is a highly reliable, feasible tool for standardized assessment of gross motor function in MLD which can be used for the description of the natural course of the disease and for evaluation of therapeutic options such as stem cell transplantation and enzyme replacement, both of which are topics of current research.

Enzyme, cell and gene-based therapies for metachromatic leukodystrophy.

Metachromatic leukodystrophy (MLD) is a demyelinating storage disease caused by deficiency of the lysosomal enzyme arylsulfatase A (ARSA). Lack of ARSA activity leads to the accumulation of galactosylceramide-3-O-sulfate (sulfatide) in the central and peripheral nervous systems. Based on the age at onset, the disease is usually classified into three forms: the late-infantile form, which manifests in the second year of life; the juvenile variants (onset between 4 and 12 years), which are subdivided into early-juvenile (EJ, onset before 6 years) and late-juvenile (LJ, onset after 6 years); and the adult form (onset after 12 years of age). Currently, there is no efficient therapy for the late-infantile form of MLD (50% of the patients), death occurring within a few years after onset of neurological symptoms. Allogeneic haematopoietic cell transplantation (HCT), when performed at a very early stage of the disease, may improve selected patients with juvenile or adult forms of MLD. As with other lysosomal storage diseases, the physiopathology of MLD is poorly understood. Demyelination is the main pathological finding, but substantial storage of sulfatides in neurons also occurs, and may contribute to the clinical phenotype. The physiopathological process leading to neuronal and glial cell degeneration and apoptosis involves accumulation of undegraded sulfatides but also secondary abnormalities (storage/mislocalization of unrelated lipids, inflammatory processes). This review summarizes the recent advances in the understanding of the physiopathology of MLD and the new therapeutic perspectives currently under preclinical investigation, including enzyme replacement therapy, gene therapy and cell therapy.

Motor and psycho-cognitive clinical types in adult metachromatic leukodystrophy: genotype/phenotype relationships?

Metachromatic leukodystrophy (MLD) is a recessive autosomal disease which is biochemically characterized by an accumulation of sulfatides (sulfogalactosylceramides) mainly in oligodendrocytes and macrophages/microglia. The deficient enzyme is a lysosomal hydrolase, cerebroside sulfate sulfatase (arylsulfatase A). MLD is both a dysmyelinating and a demyelinating disease. The main clinical forms are infantile or juvenile, but some forms appear at adulthood. This disease involves also neuronal cells as sulfatides are also present in neurons in which the defect in degradation occurs also. We have studied 12 cases of adult MLD and clearly distinguished two clinical forms. One of them was characterized by mainly central nervous system motor signs (pyramidal, cerebellar, and seldom dystonia) and a peripheral neuropathy. The other form always started by behavioural abnormalities with modifications of mood, peculiar social reactions; a progressive mental deterioration occurred also. The diagnosis of schizophrenia was often mentioned. Most of these patients remained for many years without any neurological symptoms, and the diagnosis was only made when neurological signs appeared, or when Magnetic Resonance Imaging (MRI) was performed. MRI showed a diffuse demyelination, bilateral and often symmetrical, which could be temporarily limited to the periventricular areas. The diagnosis of adult MLD was biochemical, evidencing the low activity of arylsulfatase A (ASA) and sulfatide accumulation. To determine the respective participation of neurons and glial cells in the physiopathology of both the motor forms and the psycho-cognitive forms, our first approach was to search for mutations differing according to the clinical status. Motor forms involved the major adult ASA mutation P426L in a homozygote form in contrast to psycho-cognitive forms which involved as a compound heterozygote a specific I179S mutation.

Metachromatic leukodystrophy: subtype genotype/phenotype correlations and identification of novel missense mutations (P148L and P191T) causing the juvenile-onset disease.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease resulting from the deficient activity of arylsulfatase A (ASA) and the accumulation of sulfatides. The disease is characterized by several subtypes, designated by age at onset: the late-infantile-, juvenile-, and adult-onset variants. Mutation analysis of genomic DNA from a proband with each variant was performed to identify and characterize their causative ASA mutations. Two sisters with the infantile-onset disease were homoallelic for the missense mutation D335V, a juvenile-onset proband was heteroallelic for two novel missense mutations, P148L and P191T, and an adult-onset patient was heteroallelic for the H397Y and P426L mutations. The novel mutations were not identified in 108 normal alleles indicating that these base substitutions were not common polymorphisms. To further characterize the mutant gene products, the mutant enzymes were partially purified from cultured fibroblasts and their molecular weights and charges were compared by immunoblotting following SDS-PAGE or isoelectric focusing (IEF). Normal fibroblast ASA had a single, broad band at 54 kDa. The enzyme from the late-infantile-onset patient had distinct bands of 36 and 78 kDa, but lacked the normal 54-kDa species. The juvenile- and adult-onset patients each had a faint band of 54 kDa and several other bands ranging from 29 to 64 kDa. IEF revealed several bands for the partially purified normal enzyme with a relatively narrow pH range around 4.0, whereas numerous bands with a wider range of isoelectric points were observed with the enzymes from the juvenile- and adult-onset fibroblasts. In contrast, the enzyme from the late-infantile-onset proband had four bands with more acidic isoelectric points, none corresponding to those of the normal enzyme. These results document changes in both size and charge of the mutant enzymes from patients with different mutations and MLD subtypes.

Recurrent seizures in metachromatic leukodystrophy.

The unusual presentation of juvenile onset metachromatic leukodystrophy (MLD) and frequent complex partial seizures in a patient led us to perform a retrospective study of 18 patients with MLD to identify the prevalence and type of recurrent seizures during the first 2 years of the disease. Five of 17 patients (29%) had developed recurrent seizures within 12 months of the onset of symptoms, and one patient was lost to follow-up. By 24 months after onset of symptoms, 5 patients were lost to follow-up, and 6 of the remaining 13 patients (46%) had developed recurrent seizures. In all, 7 patients, 4 with late infantile-onset and 3 with juvenile-onset disease, developed recurrent seizures. Four patients, including 3 with juvenile-onset disease had complex partial seizures. We conclude that recurrent seizures are common in MLD and may occur at any stage of the disease, particularly in patients with juvenile onset. Generalized seizures are more frequent in patients with late infantile-onset, whereas partial seizures are more common in those with juvenile-onset disease.

Demyelinating hereditary neuropathies in children: a morphometric and ultrastructural study.

Twenty-three cases of hereditary demyelinating neuropathies are reported, 13 with different types of hereditary motor and sensory neuropathy (HMSN) and 9 with globoid cell or meta-chromatic leucodystrophies. Ultrastructural and morpho-metric studies showed some critical pathological features emphasizing: 1) the variability of the recessive forms of HMSN; 2) the morphological distinction between HMSN type I and type III; and 3) differences between these types of HMSN and other "onion bulb" neuropathies such as those found in leucodystrophies, which account for distinct underlying mechanisms.

Lipidoses detected in Poland through 1993.

It is estimated that 70-100 children suffering from a lysosomal storage disease are born in Poland every year. From 1975 to 1993, the activity of various lysosomal enzymes was determined in the leukocytes, cultured skin fibroblasts, or hair roots from 5,594 patients, mainly children, in whom the diagnosis of a lipidosis was suspected. In that material 162 cases of a lipidosis were diagnosed. Metachromatic leukodystrophy seems to be the most frequent of the lipidoses; GM1 gangliosidosis is more frequent than GM2 gangliosidosis and Gaucher and Niemann-Pick diseases appear to be almost as frequent as the former.

[Molecular analysis of Japanese patients with metachromatic leukodystrophy].

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by a deficiency of arylsulfatase A (ASA). Three forms of the disease can be distinguished according to the age at onset: late-infantile, juvenile and adult. To investigate the molecular basis of Japanese patients with MLD, we examined ten Japanese patients for the presence of three known ASA mutations. The results indicate quite different distribution of these mutations between Caucasian and Japanese patients with MLD. We have performed further analysis on ASA gene of Japanese MLD patients, and identified two new mutations. Phenotype-genotype relationship in Japanese patients with MLD is discussed.

Partial seizures in two cases of metachromatic leukodystrophy: electrophysiologic and neuroradiologic findings.

This report concerns two cases of metachromatic leukodystrophy presenting partial seizures. One was a 2-year-old boy with a late infantile type and the other a 17-year-old girl with a juvenile type. The former had tonic-clonic seizures on the left with concomitant twitching of the left side of the face and adversive conjugate deviation of the eyes. After a while, his interictal sleep electroencephalogram (EEG) showed spikes in the right central area. The second case had hemiconvulsions on the right side, consisting mainly of tonic flexion of the upper limb followed by clonic flexions, and accompanied by adversive conjugate deviation of the head and eyes. Her ictal EEG showed rhythmic 6- to 7-Hz wave bursts in the left frontal area. To this date, no report has given a detailed discussion of the type of seizures and ictal EEG in metachromatic leukodystrophy. In addition, there have been few detailed reports of magnetic resonance imaging (MRI) in the juvenile type. It is interesting that typical partial seizures were observed in a hereditary metabolic disorder characterized by diffuse demyelination of the white matter, and the pathophysiology is discussed here mainly in relation to MRI findings of the case with the juvenile type.

Late-onset metachromatic leukodystrophy: molecular pathology in two siblings.

We report on a new allele at the arylsulfatase A (ARSA) locus causing late-onset metachromatic leukodystrophy (MLD). In that allele arginine84, a residue that is highly conserved in the arylsulfatase gene family, is replaced by glutamine. In contrast to alleles that cause early-onset MLD, the arginine84 to glutamine substitution is associated with some residual ARSA activity. A comparison of genotypes, ARSA activities, and clinical data on 4 individuals carrying the allele of 81 patients with MLD examined, further validates the concept that different degrees of residual ARSA activity are the basis of phenotypical variation in MLD.

Molecular basis of different forms of metachromatic leukodystrophy.

BACKGROUND: Metachromatic leukodystrophy is an autosomal recessive inherited lysosomal storage disorder caused by a deficiency of arylsulfatase A. Three forms of the disease can be distinguished according to severity and the age at onset: late infantile (1 to 2 years), juvenile (3 to 16), and adult (greater than 16). METHODS AND RESULTS: To understand the molecular basis of the different forms of the disease, we analyzed arylsulfatase A alleles associated with metachromatic leukodystrophy. Two alleles (termed I and A) were identified and accounted for about half of all arylsulfatase A alleles among 68 patients with metachromatic leukodystrophy whom we examined. Sufficient information was available for 66 of the patients to allow classification of their disease. Of the six instances of homozygosity for allele I, all were associated with the late-infantile form of the disease; of the eight instances of homozygosity for allele A, five were associated with the adult form and three with the juvenile form. When both alleles were present, the juvenile form resulted (seven of seven instances). Heterozygosity for allele I (with the other allele unknown) is usually associated with late-infantile disease, and heterozygosity for allele A with a later onset of the disease. The clinical variability can be explained by the different levels of residual arylsulfatase A activity associated with these genotypes. CONCLUSIONS: Like many lysosomal storage disorders, metachromatic leukodystrophy shows clinical heterogeneity that seems to reflect genetic heterogeneity. One of the known alleles (allele I) is associated with earlier and more severe disease than the other (allele A).

Somatic cell hybridization studies on the genetic regulation and allelic mutations in metachromatic leukodystrophy.

Metachromatic leukodystrophy is a hereditary neurodegenerative disease associated with deficient arylsulfatase A activity. Clinical variants differ in onset times and severity of the disease but each breeds true within families. Somatic cell hybridization techniques were used to clarify the genetic relationship among these mutants. Hybrid clones isolated with a nonselective method from fusing fibroblasts of an infantile and a juvenile variant did not show complementation of arylsulfatase A activity. Hence, these clinical variants are allelic mutants. Previous somatic cell hybridization studies suggested that "arylsulfatase A-deficiency" is a dominant phenotype, in contrast to its apparent recessive mode of inheritance. To resolve this discrepancy, hybrid clones from fusing normal and arylsulfatase A-deficient fibroblasts were isolated nonselectively. They continued to express arylsulfatase A activity. Hence, even in vitro, "arylsulfatase A-deficiency" remains as a recessive phenotype.

Multiple molecular forms of arylsulfatase A in different forms of metachromatic leukodystrophy (MLD).

Arylsulfatase A (ARA) can be separated into six to eight individual enzymatic bands of activity by isoelectric focusing on cellulose acetate membranes. The residual ARA activity in juvenile metachromatic leukodystrophy (MLD) has a single band of activity with apI of 5.5, whereas the residual ARA in the late infantile form of MLD has three bands of activity with pI range of from 5.4 to 5.8. The technique of isoelectric focusing on cellulose acetate membranes demonstrates enzymatic differences which can be correlated with the clinical form of the disease.

[Electron microscopical findings in the sural nerve of twins with metachromatic leukodystrophy. An attempt to classify the findings according to distinctive phases of the disease (author's transl)]. / Elektronenmikroskopische Befunde am Nervus suralis von Zwillingskindern mit metachromatischer Leukodystrophie. Versuch einer Zuordnung zu unterschiedlichen Erkrankungsstadien

Sural nerve biopsies taken from a pair of twins with metachromatic leukodystrophy were investigated by electron microscopy. The morphological findings showed distinct alterations. In the first twin the particularity was a large number of granules containing sulfatides. In the second twin, however, the main finding was a damage of myelin sheaths evidently caused by an excessive and morphologically as well as chemically defective myelin formation. According to the grading given by Ulrich the first case corresponds to the stadium one, in which a storage of sulfatides dominates, whereas case two showing a severe damage of myelin sheaths is characteristic of the second stadium of metachromatic leukodystrophy.