Late infantile and adult‐onset metachromatic leukodystrophy due to novel missense variants in the PSAP gene: Case report from India

Abstract Metachromatic leukodystrophy (MLD) due to Sap‐B deficiency is a rare autosomal recessive disorder caused due to biallelic variants in the PSAP gene. The PSAP gene encodes a precursor protein prosaposin, which is subsequently cleaved to form four active glycoproteins: Sap‐A, Sap‐B, Sap‐C, and Sap‐D. In case of deficiency of the sphingolipid activator protein Sap‐B, there is a gradual accumulation of cerebroside‐3‐sulfate in the myelin of the nervous system resulting in progressive demyelination. Only 12 variants have been reported in the PSAP gene causing Sap‐B deficiency to date. Here, we report two cases of MLD due to Sap‐B deficiency (late‐infantile and adult‐onset form) harboring two novel missense variants c.688T > G and c.593G > A in the PSAP gene respectively. This study reports the third case of adult‐onset MLD due to Sap‐B deficiency in the world. The proband, a 3‐year‐old male child presented with complaints of hypotonia, lower limb tremors and global developmental delay. His MRI showed hyperintense signals in the bilateral cerebellar white matter. Overall, the findings were suggestive of metachromatic leukodystrophy. The second case was a 19‐year‐old male child with clinical features of regression of speech, gait ataxia and bilateral tremors referred to our clinic. MRI data suggested metachromatic leukodystrophy. Normal enzyme activity of arylsulfatase‐A led to a suspicion of saposin B deficiency. For both cases, targeted sequencing was performed. This identified homozygous variant c.688T > G (p.Cys230Gly) and c.593G > A (p.Cys198Tyr) in exon 6 of the PSAP gene, respectively.


| INTRODUCTION
Metachromatic leukodystrophy (MLD) is an autosomal recessive neurodegenerative disorder. It is characterized by psychomotor regression, gait disturbances, ataxia, spastic paraparesis, and visual disturbances. 1 The breakdown of the substrate is catalyzed by the lysosomal enzyme Arylsulfatase-A (E.C.3.1.6.8) in presence of a sphingolipid activator protein, saposin-B (Sap-B). 1,2 The gradual accumulation of cerebroside-3-sulfate particularly in the myelin of the nervous system results in progressive demyelination and dysfunction of the central nervous system and the peripheral nervous system respectively. Any defect in the genes ARSA or PSAP which encode the Arylsulfatse-A enzyme and Sap-B protein respectively can result in MLD. The majority of the MLD cases are due to pathogenic variants in the ARSA gene causing MLD due to ARSA deficiency (OMIM#250100). A small group of 30 MLD patients due to deficiency of the activator protein Sap-B has been reported (OMIM#249900). The key distinguishing feature between these two groups is that arylsulfatase-A enzyme levels in Sap-B patients are within the normal range as opposed to that in ARSA deficiency patients. However, in both conditions, there is an abnormal excretion of urine sulfatide. 3 The PSAP gene (MIM#176801) located on chromosome 10 consisting of 14 coding exons encodes a 524 amino acid precursor protein prosaposin (pSap) which is subsequently cleaved to form four active glycoproteins (Sap-A, Sap-B, Sap-C, and Sap-D). Mutations in the PSAP gene can lead to a deficiency of the entire pSap protein or individual saposins. 3 MLD is due to a defect in Sap-B whereas Sap-A and Sap-C defects cause atypical Krabbe disease and Gaucher disease respectively. [4][5][6] Sap-D deficiency has been reported only in mice and it resembles Farber disease. 7 To date, a total of 12 variants in the PSAP gene have been reported in 30 MLD patients worldwide. 1 In India, two cases of MLD due to Sap-B deficiency have been reported. 8,9 Both were late infantile forms and showed the presence of the variant c.679_681delAAG in exon 6 of the PSAP gene. The severity of the condition is governed by the type of variant. 1 Clinically, the most severe prosaposin deficit is due to two PSAP null alleles. Of all the Sap-B deficient MLD patients reported, the majority of them presented with the late infantile form (17 patients) followed by 6 cases of juvenile-onset and two cases of the adult-onset form. 10,11 The three most common PSAP-MLD alleles reported are c.645C > A, c.722G > C, and c.577-1G > T, and these account for about 67% of the total PSAP-MLD alleles. 1 Overall, 4 missense variants have been reported in the PSAP gene and they have been associated with both lateinfantile as well as juvenile forms of MLD due to Sap-B deficiency. Here, we report two cases: a late-infantile form and an adult-onset form of MLD due to novel missense variants in the PSAP gene. To the best of our knowledge, this study describes the third report in the world and the first from India, of an adult-onset MLD phenotype resulting from Sap-B deficiency.

| CASE REPORT
Case 1 is a 3-year-old male child born to consanguineous parents. On presentation to the clinician, global developmental delay, hypotonia, lower limb tremors and poor feeding was noted. There was a history of recurrent fever, cold, and cough since 1 year of age. The parents complained that the child had become more irritable in the last 6 months. He was able to walk with support and speak monosyllables at the age of 2 years. Thereafter, parents noticed regression of learned skills. Brain MRI studies of the proband showed moderate confluent FLAIR/T2 hyperintense signal in the periventricular and deep white matter of bilateral frontal, parietal, and occipital lobes. In addition, FLAIR/T2 hyperintense signal was noted in corpus callosum involving genu and splenium. A mild FLAIR hyperintense signal was also noted in the bilateral deep cerebellar white matter. Overall, the MRI findings were suggestive of metachromatic leukodystrophy (MLD).
For molecular confirmation, genomic DNA was extracted from peripheral blood using salting out technique and was subjected to a targeted exome sequencing study as mentioned in the online supplementary file. Variant filtration and prioritization analysis revealed a homozygous variant c.688T > G (p.Cys230Gly) in exon 6 of the PSAP gene (NM_002778.4). This variant has not been reported in the 1000 genomes 12 and gnomAD databases. 13 The in-silico prediction of the variant is damaging by SIFT, PolyPhen-2, PROVEAN, and MutationTaster. The variant was classified as likely pathogenic as per the ACMG-AMP guidelines and ClinGen framework with the

Synopsis
We describe two cases of MLD due to novel pathogenic variants in the PSAP gene from India. Overall, we conclude that MLD due to Sap-B deficiency should be considered as a differential diagnosis in adult patients presenting with speech regression, gait, and bilateral tremors.
following criteria-PP3 (strong) and PM2 (moderate). 14,15 This established the diagnosis of MLD due to Sap-B deficiency in the proband. As the child's sample was not available for Sanger confirmation study, we performed parental segregation analysis. We found both parents to be heterozygous (carrier) for the variant c.688T > G in exon 6 of the PSAP gene ( Figure S1).
Case 2 is a 19-year-old male child born to a phenotypically healthy and an endogamous couple from Rajasthan, presented with gait ataxia, difficulty in walking and nystagmus. His psychomotor development including speech was normal until 14 years of age. At the age of 13 years, his parents noticed him having difficulty in speech. Following this, he started having trouble in walking. He also had a history of febrile illness. His MRI brain revealed confluent and symmetrical areas of T2W/FLAIR hyperintensities in the periventricular and deep white matter of bilateral cerebral hemispheres, centrum semiovale and corpus callosum. Mild thinning of the corpus callosum was also seen. No other abnormality was noted in the cerebral parenchyma, cerebellum, brainstem, and pituitary gland. The MRI results led to an initial suspicion of a case of MLD.
The proband was referred to our Centre at the age of 19 years. In addition to difficulty in walking and speech, he also had a history of tremors in both hands. In order to rule out MLD, enzyme levels of arylsulfatase-A activity were assessed from leukocytes. Briefly, the arylsulfatase-A assay was carried out using p-nitrocatechol as the substrate. The assay mixture was incubated at 0 C for a period of 16 h followed by the addition of 1 N NaOH to stop the reaction. The extent of activity was then measured spectrophotometrically. 16 He showed a normal enzyme activity of 2.9 nmol/ h/mg protein (Normal range: 0.6-4.99). Thus, MLD was ruled out; however, considering the clinical indications, a plausible differential diagnosis of saposin B deficiency was suggested. We then subjected the DNA of the proband to a targeted gene panel study based on the single molecule molecular inversion probes (smMIPs) for 23 genes associated with common lysosomal storage disorders in India. [17][18][19] A detailed methodology is mentioned in the online supplementary file. This revealed the presence of a homozygous novel missense variant c.593G > A (p.Cys198Tyr) in exon 6 of the PSAP (NM_002778.4) gene ( Figure S2). This variant has not been reported in the 1000 genomes 12 and gnomAD databases. 13 The in-silico prediction of the variant is damaging by MutationTaster, PROVEAN, LRT, DANN, and SIFT. The variant was classified as likely pathogenic as per the ACMG guidelines and ClinGen framework with the following criteria-PP3 (strong), PM1 (supporting), PM2 (supporting). 14,15 This confirmed the diagnosis of MLD due to Sap-B deficiency in the proband. Sanger sequencing in the proband confirmed the presence of the homozygous variant c.593G > A in the PSAP gene ( Figure S3). However, the family was lost to follow-up and hence there is a paucity of MRI images and parental segregation studies.

| DISCUSSION
MLD is a storage disorder characterized by the gradual accumulation of sulfatides that trigger demyelination. MLD caused due to sulfatide activator protein deficiency encoded by the PSAP gene is relatively rare with only 12 causative variants reported globally (Table 1). 1,10 The clinical presentation of these patients is similar to patients affected with MLD except that the latter show deficiency of ARSA enzyme activity. The present study describes two cases of MLD due to Sap-B deficiency from India with novel missense variants in the PSAP gene. Also, this is the first report from India describing an adult-onset MLD phenotype because of Sap-B deficiency.
The initial symptoms at onset in most of the Sap-B deficient patients include difficulty in walking, gait disturbance, speech problems and tremors, which are similar to that seen in both cases. The majority of cases reported to date are of patients with late infantile onset where the symptoms developed after 2 years of age which was also observed in case 1. In the second case, however, the proband had normal development and started presenting early signs of difficulty in walking only after 14 years of age, which suggested an adult-onset presentation of the condition. The brain MRI results seen in both of our patients are consistent with that seen in MLD patients. Generally, brain MRI findings include signs of demyelination, diffuse white matter changes, and impairment of basal ganglia. 20 It is crucial to note that the MRI information of patients can aid in the early diagnosis of MLD. For proband in case 2, activity of the arylsulfatase-A enzyme was observed within the normal range. This is in concordance with previous observations whereby high excretion levels of urinary sulfatides couple with normal activity of arylsulfatase-A enzyme is observed. 10 Testing of urinary sulfatide is therefore important to confirm Saposin-B deficiency in such cases. However, as the proband was lost to the follow-up, investigation of urinary sulfatides could not be carried out post genetic test.
To date, different types of mutations including missense, nonsense, splicing, frameshift, deletion and start loss have been reported in the PSAP gene. Both our patients showed the presence of novel missense variants in exon 6 of the PSAP gene. The variants c.688T > G and c.593G > A identified in the present study have been submitted in the ClinVar database under the submission ID, SUB12964537 and SUB12964504 respectively. In addition to this, three missense variants and a 3-bp deletion have been previously reported in exon 6 of the PSAP gene in  Note: The bold values represent case specific genetic findings identified in the two patients presented here and provide a contrast against other known cases. Abbreviations: NA, not applicable; ND, not done; NR, not reported.
patients with Sap-B deficiency. Out of the missense variants reported, c.645C > A is one of the common PSAP-MLD alleles. This variant was first reported in an Italian patient and was shown to abolish the N-glycosylation site of saposin B. 21 Overall, we find maximum variants reported in exon-6 of the PSAP gene suggesting it to be a hotspot region. There have been two previous reports of MLD due to Sap-B deficiency from India. In both cases, the variant c.679_681delAAG in exon 6 of the PSAP gene was identified. 8,9 Both these patients presented with the lateinfantile form. Interestingly, this variant has also been reported in a compound heterozygous state in a late infantile MLD patient. 10 All patients harboring the c.679_681delAAG variant have shown a similar clinical course. However, it might be essential to analyze the effect of different variant types on the pSap protein in order to relate it to the clinical course of the patient. As both variants identified in this study are novel missense variants, we predicted their effect on the protein by using Missense3D (http://missense3d.bc.ic.ac.uk/missense3d/ , accessed on November 28, 2022). Interestingly, in both cases the wild-type amino acid that was substituted was cysteine. For case 1, the mutant allele resulted in the substitution of cysteine by glycine. For case 2, there was the substitution of cysteine by tyrosine. The wild-type cysteine residues at positions 230 and 198 are involved in disulphide bond formation with the cysteine residues at the 47th and 36th positions respectively. Thus, the substitution in both cases disrupts this bond, thereby disturbing the stability of the protein (Figures 1A,B). 22 Although, the predicted effect of the mutant residue on the protein is similar for both cases, yet there was a difference in the age of onset as well as phenotypic heterogeneity was seen. The former case had an early onset with severe presentation while the latter case had an adult onset. This suggests that there are likely to be other factors that influence the protein function and functional studies can aid in understanding the genotype-phenotype correlation.
In conclusion, the present study adds two novel variants in the existing mutation list for the PSAP gene and suggests that exon 6 of the PSAP gene is likely to be a hotspot region for mutation. It also highlights the need to consider MLD due to Sap-B deficiency in the differential diagnosis of patients presenting with speech regression, gait, and bilateral tremors.

AUTHOR CONTRIBUTIONS
Conceived and designed experiments: Jayesh Sheth, Aadhira Nair and Harsh Sheth. Patient recruitment and clinical analysis: Jayesh Sheth, Naresh Tayade, Frenny Sheth and Heli Shah. Enzyme study: Riddhi Bhavsar. Sequencing data analysis and interpretation: Harsh Sheth and Aadhira Nair. Write first draft of the manuscript: Aadhira Nair and Jayesh Sheth. Made critical revisions and approved final version: Jayesh Sheth, Harsh Sheth, C. Ratna Prabha. All authors reviewed and approved the final manuscript.

ACKNOWLEDGMENTS
We are grateful to the family of the patient for their kind co-operation and permission.

FUNDING INFORMATION
We sincerely acknowledge research funding from the Department of Biotechnology (BT/PR39587/MED/12/ F I G U R E 1 (A) Predicted 3D structure of the PSAP protein due to the variant c.688T > G (Blue: wildtype residue: Cys, Red: mutant residue: Gly) and (B) Predicted 3D structure of the PSAP protein due to the variant c.593G > A (Blue: wildtype residue: Cys, Red: mutant residue: Tyr).