Introduction

Metachromatic leukodystrophy (MLD) (OMIM #250100) is an autosomal recessive lysosomal storage disorder. It is caused by the variations in ARSA gene (OMIM *607574), which results in the deficiency of Arylsulfatase A enzyme and accumulation of cerebroside-3-sulfate in the lysosomes of the tissues of central and peripheral nervous system. Intralysosomal storage of galactosylsulfatides leads to progressive demyelination and a variety of neurological symptoms like gait disturbance and cognitive regression. Though the disease occurs pan-ethnically with an estimated frequency of 1 in 40,000 [1] the incidence in India is not yet known.

Three different clinical forms of MLD have been characterized based on age at onset of symptoms: late infantile (onset before 30 months), juvenile (30 months–16 years) and adult (after 16 years). In the late infantile and juvenile forms, patients have gait disturbances, blindness, loss of speech, quadriparesis, peripheral neuropathy, and seizures. In the adult patients, the major signs are behavioral disturbances and dementia with a slow progression over several decades [2].

The ARSA gene located on chromosome 22q13 consists of eight exons and spans a genomic region of 3.2 kb. The ARSA cDNA hybridizes to three different mRNA species of 2.1, 3.7, and 4.8 kb and codes for a 507 aminoacids protein that contains three potential N-glycosylation sites [3]. About 186 variations causing MLD have been identified so far in ARSA gene [4]. In this study, we attempted to analyze the variation spectrum of ARSA gene in Indian patients with MLD.

Methods

Patient recruitment

The patients described in this study were unrelated and belonged to different regions in India. The patients were recruited to the study after obtaining informed consent. In case of children, consent was obtained from parents or guardians. The Institute Ethics Committee approved the study. A total of 51 families with at least one affected individual with MLD were included in the study. In 41 families where an affected individual was available for evaluation, a detailed clinical examination was done by a Clinical Geneticist. Magnetic resonance imaging (MRI) of brain was done whenever possible and assay of Arylsulfatase enzyme was done in all patients with clinical and radiological features of MLD. A total of 4 ml blood was collected in ethylenediaminetetraacetic acid vacutainer for all these individuals. In families where previous affected individuals were not available for evaluation, from the available medical records, a diagnosis of MLD was made. Blood samples of parents were collected in such families for molecular genetic analysis.

Arylsulfatase A enzyme assay

Arylsulfatase A enzyme activity in patients was measured in leukocytes obtained from the whole blood sample, using the substrate 10 mM P-Nitrocatechol, in 0.5 M sodium acetate–acetic acid buffer with pH 5.2 and containing 0.023% sodium pyrophosphate and 9.94% sodium chloride. Protein concentration in the lysate was determined by the Lowry method [5] and the ideal concentration was 0.2–0.3 mg of protein. Totally, 200 μl substrate and 200 μl leukocyte homogenate were incubated for 1 h at 37 °C. Then the absorbance was read at 515 nm using Shimadzu UV 2450 spectrophotometer. The enzymatic activity was expressed as nmol of substrate hydrolyzed per mg of protein per hour. For standardization of reference range of enzymes, 20 normal control samples were used. For testing each sample, another control was used. The reference enzyme activity for leukocytes was in the range of 25–80 nmol/h/mg and for fibroblasts was in the range of 190–415 nmol/h/mg. Enzyme assay was performed in our center in 37 out of the 41 patients. In ten families, the index patient was no longer alive. In the remaining four patients, ARSA enzyme levels were not available, but molecular diagnosis was performed based on clinical and radiological features.

Genetic analysis

The genomic DNA was isolated from whole blood using the phenol chloroform extraction method. Primers for the eight exons and the flanking intron–exon boundaries of ARSA were designed by Primer 3 software Input version 0.4.0 and NCBI primer BLAST software. Polymerase chain reaction (PCR) was carried out in the Bio-Rad DNA Engine Peltier Thermal Cycler (Bio-Rad Laboratories, Inc., Hercules, CA) for all 8 exons of ARSA with specific primers, for 25 cycles, with each cycle consisting of denaturation at 94 °C for 1 min, annealing temperature specific to the primers for 55 s and extension at 72 °C for 50 s, with an initial denaturation for 8 min at 96 °C and final extension for 7 min at 72 °C. Bidirectional sequencing was carried out on all the purified PCR products by capillary electrophoresis on ABI 3130 automated genetic analyzer (Applied Biosystems, Foster City, CA). The sequencing data were analyzed using the software EMBOSS [6] and Chromas Lite. The Human Genome Variation Society nomenclature guidelines were used for reporting the identified sequence variations [7]. Nucleotide numbering for ARSA variations is based on cDNA sequence of Ensembl gene transcript ID ENST00000547307. In 41 families, where the proband samples were available, sequencing was first done for the proband, followed by parents, if samples were available. In ten families where proband samples were not available, sequencing of the gene in parents was performed.

In silico characterization of identifies variants

Common polymorphisms and natural variants were filtered out using dbSNP, 1000 Genome browser, ExAC genome browser and in house database. By searching databases like ClinVar and Human Genome Mutation Database (HGMD), known pathogenic variants were identified. The disease causing potential of novel missense pathogenic variants was predicted using the mutation pathogenicity prediction softwareslike MutationTaster [8], HANSA [9], SIFT [10], PROVEAN [11], and PolyPhen-2 [12]. Evolutionary conservation of the mutated amino acidresidues was checked across different species. Mendelian segregation pattern of the identified novel sequence variants in the respective families was studied wherever feasible.

In silico characterization of the effects of novel variants on protein sequence and structure

Homologous sequences of P15289 (protein product of ARSA gene) [13] were obtained by running three rounds of PSIBLAST [14] against the non-redundant database. Multiple sequence alignments were generated using homologous sequences having sequence coverage of at least 95% of the query sequence with ClustalW-2.1 [15] running on default parameters. Gribskov’s score was calculated using the PROPHECY program of EMBOSS by incorporating the BLOSUM62 matrix [6]. The X-ray structure of the P15289 protein was also used for further analysis (PDB code 1AUK) [16]. The percent solvent accessible surface areas of the residues were calculated using PSA program of the JOY suite [17] TRANSEQ program of EMBOSS was used to obtain translated products of the deletion mutant c.576 del C and insertion mutants c.188_189 ins A, and c.445_446 ins T6. PyMOL [18] and Swiss-PdbViewer4.0.4 [19] were used for performing structural analysis and rendering figures. Prediction of the domains affected by the variations was performed using the Uniprot [13] and PROSITE databases [20].

Prenatal diagnosis

Chorion villus sampling (CVS) was done around 11–12 weeks gestation and enzyme assay and sequencing of ARSA were done on the fetal sample. Enzyme assay was done directly in the obtained CVS sample without any prior culturing. Targeted variation analysis was done in CVS DNA based on the variations identified in the proband or in the carrier parents.

Results

Clinical and biochemical evaluation

Of the 41 probands, 20 were males and 21 were females. The age of presentation ranged from 18 months to 26 years. A total of 80% (33/41) of patients had late infantile form of MLD, 14% (6/41) had juvenile type, and 4% (2/41) had adult onset type of MLD. All of them showed evidence of leukodystrophy on MRI brain. The clinical features and molecular spectrum are depicted in Table 1. Consanguinity in parents was present in 39 out of 51 families (76%). In all the consanguineous families, homozygous variations were found. In 13 families without consanguinity, homozygous variations were found in 7 families and compound heterozygous variations were found in 6 families.

Table 1 Clinical and molecular spectrum of patients with metachromatic leukodystrophy
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The arylsulfatase enzyme activity of the patients tested in our laboratory ranged from 0 to 19 nmol/h/mg. The reference enzyme activity was in the range of 25–80 nmol/h/mg. Enzyme activity was not measurable in eight patients.

ARSA variations

A total of 36 variations were identified out of which 16 were novel variants. The variations included 23 missense variants, 3 nonsense variants, and 6 frameshift variations (3 single base deletions and 3 single base duplications), 1 indel, one 3 bp deletion, and 2 splice site variations [Supplementary Table S1 (30−40)]. The exon wise distribution of the variations is shown in Fig. 1. Nine out of 36 variants were identified in exon 5 (25%). In the ten families, where the proband was not available, in two families, both spouses had known variations and in one family one spouse had a known variation and the other had a novel variant.

Fig. 1
figure 1

Exon and intron wise distribution of variants. Blue boxes numbered from 1 to 8 indicate exons. Exon 5 has the maximum number of variants. The novel variants are depicted in red and known variants in black. ^ Indicates number of alleles

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In silico characterization of novel variants

All the 16 novel variants were predicted to be disease causing by MutationTaster, SIFT, Polyphen-2, PROVEAN, and HANSA (Supplementary Table S2). None of these variants were present in population database 1000 Genome and pathogenic variant databases like Clinvar and HGMD. Two variants, c.1174 G > A and c.379 G > A, were present in one and two heterozygotes, respectively in ExAC database.

Characterization of the effects of novel variants on protein structure and function by in silico analysis

The human ARSA protein has 507 aminoacids and has a single sulfatase domain. The distribution of the variations in relation to the 8 exons and 507 aminoacids is shown in Fig. 1. Multiple sequence alignments indicate that all the residues at the affected sites are highly conserved and any changes to these positions are expected to cause deleterious effects (Supplementary Figure S1). A total of seven novel nonsynonymous missense variations viz.p.Gly34Glu(c.101 G > A), p.Gln139Lys(c.415 C > A), p.Gly127Arg(c.379 G > A), p.Pro180Gln(c.539 C > A), p.Arg299Trp (c.896 G > C), p.Gly392Arg(c.1174 G > A), and p.Phe399Ser(c.1196 T > C) were identified (Supplementary Table S2) and the effects of these novel variations were analysed on the ARSA protein structure (P15289) (Supplementary Figure S2). In the protein, the residues Gln at 139 and Pro at 180 are buried where as the residues Gly at 34 and Arg at 299 are exposed or solvent accessible (Supplementary Table S3).

In parallel to this approach, comparison of the hydrophobicity of normal and mutated protein sequences of variations Gly34Glu, Pro180Gln, and Arg299Trp showed a wide range of alterations in the overall hydrophobicity at the sites of variation, suggesting the protein structure instability (Supplementary Figure S3).

The deletion and insertion variations c.188_189 ins A, c.576 del C, and c.445_446 ins T induce frameshift variations and premature termination of the C-terminal domains (Supplementary Figure S4).

Pseudodeficiency alleles

In our study the following polymorphisms were identified in 21 patients: c.1049 A > G(p.Asn350Ser) [pseudodeficiency allele], c.1172 C > G(p.Thr391Ser), c.220 G > C(p.Ala74Pro), and p.Try190Cys in addition to the putative pathogenic variations.

Prenatal diagnosis

Prenatal diagnosis was done for 11 families based on proband diagnosis. Of the 11 fetuses, 3 were affected and 8 were unaffected (Table 1).

Discussion

Metachromatic leukodystrophy due to Arylsulfatase A deficiency should be suspected in any patient with progressive neurological deterioration, white matter changes on MRI of brain and a low level of arylsulfatase enzyme. However, decreased activity of arylsulfatase enzyme is not sufficient to diagnose MLD because of the presence of pseudodeficiency allele where the enzyme activity could be 5–20% of normal controls. Hence, for establishing the diagnosis of MLD molecular testing is essential. It is also essential in carrier status detection, especially in families with pseudodeficiency alleles.

In our study, majority of the patients had late infantile form of MLD (80%), which was comparable to the results from a previous study conducted in India [21] where 55% of the enrolled subjects (11/20) had late infantile type of disease.

In a variation update of ARSA and PSAP genes causing MLD by Cesani et al. [22], 200 distinct ARSA-MLD alleles were identified. A total of 66.5% of those variants were missense, 7.5% were nonsense, 6.5% were splice site variations and 12% were frameshift variations due to deletions, inversions, and duplications. In our study, the most common variations were missense (63.8%; 23/36), followed by frameshift variations (16.6%; 6/36), nonsense variations (8.3%; 3/36), and splicing variations (5.5%; 2/36).

The most common variant identified in our cohort was c.931 C > T (p.Arg311*), which is a known disease causing variant. This variant was identified in 14/122 alleles, which were checked, accounting for 11.4% of the disease causing variants, followed by c.459 + 1 G > A, which was seen in 9.8% of all alleles tested. The most common disease causing variation in patients with late infantile form from European ancestry is c.459 + 1 G > A [23]. In a previous study done in India, c.459 + 1 G > A was the most common disease causing variant identified [21], but the sample size of that study was limited. In adult onset or juvenile phenotype, the most common reported variants were the missense variants c.1277 C > T and c.536 T > G [22]. Only one patient in our cohort had the variant c.1277 C > T in a compound heterozygous state. He had adult onset disease and the other variant he had, was a known disease causing variant, c.1288 G > T. The variation profile identified in our study was distinct from the variation spectrum in the previous Indian study [22] and studies from other populations [24, 25, 26, 27]. In our study, 44% (16/36) were novel variants, implying the uniqueness of variation spectrum in Indian population. Identification of a common variation in Indian population would help in designing a low-cost targeted variation testing in patients with suspected MLD before resorting to costlier methods for molecular diagnosis.

Previous studies have shown that majority of ARSA-MLD alleles clustered around exon 2 and exon 5 [22]. In our study 25% of variants were in exon 5, but only 11% of variants were in exon 2. Hence, exon 5 can be considered as a “common mutated exon” for variations in Indian patients and thus this information can be used to screen for exon 5 in Indian patients followed by sequencing of all exons.

In our cohort, 76% of families were consanguineous. But in the 13 nonconsanguineous families, more than 50% had homozygous variations. This is not an uncommon phenomenon in India where inbreeding is prevalent and can result in autosomal recessive diseases in offspring and this has been documented in previous molecular studies from India [28, 29].

The c.459 + 1 G > A is a null allele and makes no protein and hence causes late infantile type of MLD. In our study, the three probands who had homozygous c.459 + 1 G > A had no detectable arylsulfatase enzyme and had a late infantile type of MLD. However, we were unable to derive any correlation between the phenotype, genotype, and biochemical parameters.

Treatment for MLD is mainly supportive even though hematopeotic stem cell transplantation (HSCT) has been suggested as a curative therapy especially in presymptomatic or early symptomatic juvenile or adult onset disease. HSCT is not recommended for patients with late infantile MLD. Majority of the patients in our cohort had late infantile MLD and were managed by supportive care. None of the patients in with juvenile or adult onset MLD in our cohort underwent hematopeotic stem cell transplant (HSCT).

To conclude, this study helped in identifying the variation spectrum of ARSA gene in Indian patients with MLD and aided in identifying the most common type of variation seen in Indian patients. This study stresses the need for molecular diagnosis in all patients with MLD, so that carrier testing, genetic counseling, and prenatal diagnosis can be provided for families with MLD.