|Ahead of print
Clinical and laboratory profile of pediatric metachromatic leukodystrophies in a tertiary care center from Southern Part of India
Vykuntaraju Kammasandra Gowda1, Srividya G Suryanarayana1, Varunvenkat M Srinivasan1, Sanjay K Shivappa2, Naveen Benakappa2, Maya Bhat3, Yasha Muthane4
1 Department of Pediatric Neurology, Indira Gandhi Institute of Child Health, Bengaluru, India
2 Department of Pediatric Medicine, Indira Gandhi Institute of Child Health, Bengaluru, India
3 Department of Neuroradiology, National Institute of Mental Health and Neurosciences (NIMHANS), Bengaluru, India
4 Department of Neuropathology, National Institute of Mental Health and Neurosciences (NIMHANS), Bengaluru, India
|Date of Submission||27-Dec-2020|
|Date of Decision||11-Feb-2021|
|Date of Acceptance||24-Mar-2021|
|Date of Web Publication||11-Oct-2021|
Vykuntaraju Kammasandra Gowda,
Department of Pediatric Neurology, Indira Gandhi Institute of Child Health, Bengaluru 560029, Karnataka.
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Metachromatic leukodystrophy (MLD) is an autosomal recessive lysosomal disorder due to the deficiency of arylsulfatase A enzyme. Materials and Methods: This is a retrospective analysis of all clinically suspected MLD cases and confirmed by genetics, enzyme assay, and/or nerve biopsy between June 2015 and May 2020 at a tertiary care center. The clinical profile, enzyme levels, nerve conduction studies, MRI of the brain, and variants of ARSA gene causing MLD were analyzed. Results: Out of the 19 cases of MLD, four were juvenile, and the rest were late infantile variants. Eleven were male. The mean age of presentation in the late infantile variant was 28.4 months, and that of the juvenile variant was 44.5 months. The clinical features of the late infantile variant were developmental delay (30.75%), neuroregression (100%), spasticity (66.6%), dystonia (66.6%), small head (60%), whereas the juvenile variant presented with gait difficulties and neuroregression in all (100%), and seizures in 3 (75%). MRI of the brain showed bilateral symmetrical confluent white matter T2 hyperintensities with sparing of subcortical-U-fibers in all cases. The tigroid pattern was seen in two cases. Normal enzyme level was seen in one, but this child had a pathogenic variant PSAP gene resulting in saposin B deficiency. The targeted next-generation sequencing was done in 11/19 cases and showed ARSA gene variant in 10 and PSAP gene in one child. Nerve conduction studies were done in 12 children, and all showed features suggestive of demyelinating motor polyneuropathy. Nerve biopsy showed metachromatic granules in four children. Conclusion: MLD can present with a developmental delay with or without regression, with or without seizures, with normal or small head. MRI and nerve conduction studies are helpful in diagnosis. Normal enzymes do not rule out MLD. Nerve biopsy may be useful in non-affordable families.
Keywords: ARSA gene, arylsulfatase A levels, metachromatic leukodystrophy, nerve conduction studies, saposin B deficiency
|How to cite this URL:|
Gowda VK, Suryanarayana SG, Srinivasan VM, Shivappa SK, Benakappa N, Bhat M, Muthane Y. Clinical and laboratory profile of pediatric metachromatic leukodystrophies in a tertiary care center from Southern Part of India. J Pediatr Neurosci [Epub ahead of print] [cited 2022 Oct 6]. Available from: https://www.pediatricneurosciences.com/preprintarticle.asp?id=327910
| Introduction|| |
Metachromatic leukodystrophy (MLD) is an autosomal recessive lysosomal disorder due to mutations in the ARSA gene resulting in the deficiency of arylsulfatase A (ASA) enzyme. Due to low ASA activity, there is an accumulation of sulfatides in the central and peripheral nervous system leading to demyelination. Based on the age of the first symptoms, MLD is classified into a late-infantile, juvenile, and adult-onset type. The disease type correlates with the kind of ARSA mutation and the degree of residual ASA activity.
The late-infantile form presents before the age of 30 months with psychomotor regression, irritability, ataxia, peripheral neuropathy, dysphagia, and seizures. Death occurs within a few years after the onset of the disease. In the juvenile variant, the onset is between 30 months and 16 years of age, and presents with features of cognitive deterioration and behavioral changes, later followed by central and peripheral motor deterioration and epilepsy. The disease duration is variable. The adult-onset variant has an insidious onset after 16 years with cognitive and behavioral changes and later polyneuropathy. Disease progression is generally slower with death occurring after decades. MRI of the brain shows bilateral symmetrical hyperintensities on T2-weighted imaging, starting in the corpus callosum and progressing to the periventricular white matter, initially sparing the subcortical fibers.,,, Typically, there is a pattern of radiating stripes with normal signal intensity within the abnormal white matter. The overall incidence of MLD varies from 1 in 40 000 to 1 in 170 000 in different populations. Jabbehdari et al. reported a series of 18 Iranian cases with MLD of which 12 patients were late infantile and 6 were juvenile variants. There is a paucity of studies done exclusively on MLD overall and especially in India. Hence, we planned this study to increase the awareness on various phenotype and genotype of MLD.
| Materials and Methods|| |
The medical records of children diagnosed with MLD who were either admitted or attending the pediatric neurology outpatient department of a tertiary care center in south India were reviewed from June 2015 to May 2020. The clinically suspected cases of MLD were subjected to arylsuphatase A enzyme assay, nerve biopsy, and/or genetics. MLD was confirmed with either low ARSA enzyme assay, nerve biopsy showing metachromatic granules, and/or variants in ARSA/PSAP gene. For imaging of the brain, 1.5 Tesla MRI was used and T1 weighted images, T2 weighted images, FLAIR, and DWI images of axial, coronal, and sagittal views of the patients were used to evaluate the pattern of white matter involvement in the brain.
Enzyme study was carried out from leukocytes using preferential cleavage of 4-nitro catechol sulfate a specific substrate for the ASA enzyme. NCS in motor nerves were measured in the median, ulnar, tibial, and peroneal nerves and sensory nerves from the median, ulnar, and sural nerves. The distal latency, conduction velocity, and amplitude were recorded for each nerve. For nerve biopsy, a 2cm length of the sural nerve was biopsied along the lateral malleolus, and one part of it was processed for paraffin wax embedding. The sections were routinely stained with hematoxylin and eosin, Kulchitsky Pal (for myelin), and Maison’s trichrome (for stromal elements). The other segment of the biopsy was cryosectioned and stained with Cresyl violet to demonstrate the presence of metachromatic material.
Genetic testing was done by targeted next-generation sequencing and confirmed by Sanger sequencing. For targeted gene sequencing by next-generation sequencing, DNA extracted from blood was used to perform targeted gene capture using a custom capture kit. The libraries were sequenced to mean >80–100X coverage on the Illumina sequencing platform as per the manufacturer protocol. Sequences obtained were aligned to the human reference genome (GRCh37/hg19), QC, data mapping, variant calling, and annotation of variants with external and internal data sources were achieved with a customized GATK framework. Gene/variant annotation was achieved using the VEP program against the Ensemble release 91 human gene model. Each transcript listed, the analyzed region includes coding exons and ±10 base pairs of flanking intronic region on both sides of each exon. Clinically relevant mutations were annotated using published variants in the literature and a set of diseases databases—ClinVar, OMIM (updated on 21 November 2018), GWAS, HGMD (v2018.3), and SwissVar. Common variants are filtered based on allele frequency in 1000 Genome Phase 3, ExAC (v1.0), gnomAD (v2.1), EVS, dbSNP (v151), 1000 Japanese Genome, and Indian population database. The non-synonymous variant effect was calculated using multiple algorithms such as PolyPhen-2, SIFT, MutationTaster2, and LRT. Only non-synonymous and splice site variants found in the targeted gene capture were used for clinical interpretation and synonymous, and deep intronic variants without being reported elsewhere were not considered for the analysis. QIAGEN Ingenuity Variant Analysis was used to identify variants that are relevant to the clinical indication and were classified as per the ACMG guidelines. The variants were classified based on the standard guidelines of ACMG-AMP and were scored based on the evidence and strength of each criterion as described in the ACMG guidelines.
The data were presented as percentages or mean, defined as appropriate for qualitative and quantitative variables. The clinical parameters were compared between the infantile and juvenile forms of MLD. Ethical clearance was obtained from the institutional ethical committee.
| Results|| |
A total of 19 cases of MLD were studied. Four children had the juvenile variants, and the rest had a late infantile variant of MLD. Eleven out of the 19 children were male. All children were born to consanguineous couples, except case one. A family history of MLD was present in two families. The mean age of late infantile MLD children at the time of presentation to the hospital was 28.6 months (range: 18–46 months), while the mean age at presentation to the hospital of juvenile MLD children was 44.5 months (range 35–52 months). The various clinical and laboratory findings are mentioned in [Table 1].
The various features in the late infantile variant of MLD at the time of presentation are developmental delay (30.75%), neuroregression 15/15 (100%), spasticity and dystonia (66.6%), microcephaly (60%), strabismus (46.6%), nystagmus (13.3%), seizures 9/15 (60%), hypotonia (20%), and ataxia (6.6%). In a juvenile variant, clinical features include gait difficulties 4/4 (100%), regression 4/4 (100%), and seizures (75%) (one child had fever triggered generalized seizures and the other two had myoclonic seizures). The other rare presentations were coarse facies 1/4 (25%) in late infantile variant and presenting as peripheral neuropathy one case each in late infantile and juvenile-onset.
MRI of the brain was done in all the children. It showed bilateral symmetrical confluent white matter T2- and fluid-attenuated inversion recovery (FLAIR) hyperintensities with sparing of subcortical-U-fibers. Posterior limb of the internal capsule involvement was seen in six children, and cerebral atrophy was seen in five children. The “tigroid” pattern was appreciated in two children, and the brainstem long tracts were involved in one child. Basal ganglia and cerebellum were normal in all children. [Figure 1] is an MRI of the brain on T2 weighted images showing diffuse involvement of periventricular lobar white matter in frontal and parietal lobes with sparing of subcortical U fibers and Tigroid appearance. [Figure 2] is an MRI of the brain with axial T2 weighted images showing hyperintensities of middle and superior cerebellar peduncle and periventricular white matter.
|Figure 1: (A and B) Axial T2 weighted images showing diffuse involvement of periventricular lobar white matter in frontal and parietal lobes with sparing of subcortical U fibers. The tigroid appearance of white matter is also noted|
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|Figure 2: (A and B) Axial T2 weighted images showing hyperintensities of middle and superior cerebellar peduncle and periventricular white matter|
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Nerve conduction studies were available for 12 children. In all cases, NCS was suggestive of demyelinating motor polyneuropathy. Enzyme assays of ASA were found to be low in 14/15 (93.3%). The ARSA gene mutations on genetic studies were present in 10 patients and in PSAP gene in one child as shown in [Table 2]. In one child, the enzyme level was normal, but they had pathogenic variants in PSAP gene on the genetic study and urinary sulfatide was increased 120 nmol/mg lipid (normal 0–2 nmol/mg lipid) in a child with normal ASA level. One child who presented with motor delay and visual impairment had normal enzyme levels, but a genetic study showed mutations in the PSAP gene resulting in a deficiency of a saposin B. We were not able to do genetic testing and enzyme levels in all children because of financial constraints.
Metachromatic granules on nerve biopsy were seen in four children, out of which two children had normal enzyme levels. [Figure 3] shows a single nerve fascicle of sural nerve biopsy on lower magnification (A), on higher magnification with hematoxylin and eosin shows degraded material in macrophages (B), myelinated fibers on Kulchitsky Pal stain (C), and metachromatic granules on cryosection with Cresyl violet stain staining (D).
|Figure 3: (A) Single nerve fascicle of sural nerve biopsy on lower magnification, (B) higher magnification Hematoxylin and Eosin (H&E) showing degraded material in macrophages, (C) Kulchitsky Pal stain showing thinly myelinated fibers, (D) Cryosection on Cresyl violet stain showing metachromatic granules|
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| Discussion|| |
MLD is one of the most common demyelinating leukodystrophies in the pediatric age group, the overall incidence in India is unknown, most of the time it gets wrongly diagnosed as cerebral palsy especially in the late infantile type due to associated developmental delay. Only a few similar studies of pediatric-onset MLD have been done.
[Table 3] shows a comparison of various features in the current study with previously reported studies. The majority of the children in Gulati et al.’s were late infantile variants (91.6%), similar to our study (79%). However, in the Raina et al. study, the majority of the children were of the Juvenile variant (66.6%). A high rate of consanguineous marriage (94.7%) was noted in our study compared with 66.6% in Gulati et al., 41.6% in Raina et al., and 80% in Jabbehdari et al. Because consanguineous marriages are common in south India. Clinical features of neuroregression, spasticity, and cognitive decline in our study were similar to Gulati et al.and Raina et al. in the late infantile form.
A family history of MLD disease was positive for four patients in Jabbehdari et al., whereas it was present in two families in the current study. Twelve patients had a late infantile form of this disorder and six patients had a juvenile form which was comparable to the current study with 15 cases being late infantile and 5 being juvenile variants. MRI in all patients showed the leukodystrophy pattern as arcuate fibers sparing and subcortical rim in white matter and periventricular involvement similar results were seen in the current study.
Motor delay alone was noted in four of our patients, which was not seen in studies by Gulati et al. and Raina et al. Other features of visual involvement, microcephaly, and seizures were similar to Gulati et al. The ataxia which was seen in one of our case which was not seen in the other studies.
Most of the children with juvenile variants in our study had similar presentation of gait difficulties (100%), neuroregression (100%), seizures (75%) as in Gulati et al., but one child each had peripheral neuropathy and coarse facies.
As seen in our study and both, Gulati et al. and Raina et al. studies, bilateral near symmetrical confluent white matter involvement with sparing of subcortical-U-fibers usually point towards MLD. Posterior limb of the internal capsule involvement and brainstem long tracts may be supporting features. The “tigroid” pattern generally considered may not be there always as we noted only in two cases in our study, and in seven children (58.3%) by Gulati et al.
Nerve conduction studies showed demyelinating motor axonal polyneuropathy, similar to Gulati et al., Jabbehdari et al., and Bindu et al. The histopathologically confirmed cases of MLD showed evidence of severe demyelinating and length-dependent sensory-motor neuropathy in Bindu et al., similar findings are observed in this study, further highlighting the role of electrophysiology and histopathology as an ancillary tool for diagnosis of MLD.
In one child, the ASA enzyme level was normal, but they had pathogenic variant in PSAP gene. In the study by Narayanan et al., the enzyme levels were decreased in all (37) of the patients in whom the testing was performed. In the study by Bindu et al., the ASA enzyme levels were normal in 13 of the 40 patients studied. The probable reasons for normal ASA levels are described below. A variant of MLD has been described in which the patients have normal concentrations of ASA, but increased urinary sulfatide excretion. It is known that certain missense mutations, changing an amino acid, may affect the way the enzyme handles the substrate. With some mutations, the activity is completely knocked out, leaving little or no residual activity, but in some cases, the amino acid change may make the enzyme work less efficiently. This may be partially overcome by raising the substrate concentration. So, at lower levels of the substrate, the effect of the mutation may be more obvious as noted by Doherty et al. MLD due to Saposin B deficiency caused by homozygous or compound heterozygous mutation in the Prosaposin (PSAP) gene on chromosome 10q22 will also have normal ASA enzyme levels.
Absence of history of perinatal insult, presence of consanguinity (94.7%) of cases in our study, regression, or worsening of symptoms over a period of time and neuroimaging, nerve conduction studies give clues to the diagnosis of MLD and can be confirmed by enzyme assay or genetic testing.
The limitations of our study are, we have not done enzyme assay, genetic testing, and nerve conduction studies in all children due to financial issues.
| Conclusion|| |
One should think of MLD in a child with a regression with or without developmental delay, presence of consanguinity, and family history of MLD. Ataxia, visual involvement, microcephaly, with or without seizures especially in early childhood can be present. The MRI of the brain and nerve conduction studies are useful tests and confirmed by genetic testing or enzyme assay or nerve biopsy. Normal enzymes do not rule out MLD, hence in a high degree of suspicion, one must do genetic testing. Peripheral neuropathy can be a presenting feature of MLD, where nerve biopsy is still useful to test in resource constraint settings.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]