Introduction

Metachromatic leukodystrophy (MLD) is an autosomal recessive neurodegenerative disorder with progressive demyelination and dysfunction of the central and peripheral nervous system. The disease is caused by mutations in the ARSA gene which encodes the lysosomal enzyme arylsulfatase A, or, more rarely, by mutations in the PSAP gene affecting function of saposin B. The PSAP gene (MIM #176801, 10q22.1, 14 exons, GenBank accession numbers NM_002778.2 and NP_002769.1) encodes a 524 amino-acid precursor protein prosaposin (pSap), that is cleaved to four small glycoproteins—saposins (SapA, B, C, and D) (Fig. 1a) (Kishimoto et al. 1992). Saposins are sphingolipid activator proteins required for the function of specific lysosomal hydrolases. Mutations in the PSAP gene can cause a deficiency of either the entire pSap protein (null alleles) or an individual saposin. Patients with two null alleles present with pSap deficiency and fatal infantile lysosomal storage disease (Elleder et al. 2005; Motta et al. 2016). Patients with at least one allele with a mutation impairing only isolated saposins present with disorders resembling impairment of particular hydrolases. Defects of SapB result in MLD due to impaired degradation and accumulation of cerebroside-3-sulfate (sulfatide), which is in the normal state catabolized by the arylsulfatase A (Wrobe et al. 2000). Defects in SapA and SapC lead to atypical Krabbe and Gaucher diseases, respectively (Spiegel et al. 2005; Vaccaro et al. 2010). SapD deficiency was reported only in mice and resembled Farber disease (Matsuda et al. 2004).

Fig. 1
figure 1

a Variants reported in patients with MLD, Krabbe disease, Gaucher disease, or prosaposin deficiency. Variants in shaded frame result in a null allele. Underlined variants were identified in this study. b Diffuse white matter changes, hypersignal intensity in T2-weighted images (a, b, c – black arrows) and FLAIR (d, e, f – black arrows) localized supra - and infratentorially on both sides, saving the juxtacortical region and U-fibers, pathological findings in the dorsal capsula interna (white arrows a), corpus callosum (white arrows d, e)

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While more than 250 ARSA causative variants were described to date (Bohringer et al. 2017; Cesani et al. 2016; Golchin et al. 2017; Shahzad et al. 2017; Stoeck et al. 2016), only 10 PSAP pathogenic variants found in 26 MLD patients were published in the literature (reviewed in (Cesani et al. 2016)). Majority of them, 17 patients, presented with the late infantile form, 6 with juvenile–onset disease, and 1 with adult MLD; 2 patients were reported as being still asymptomatic.

Here, we describe a case of a patient with late infantile MLD due to two novel compound heterozygote mutations, one in the SapB portion of the PSAP gene and the other in a SapD encoding exon, but resulting in a null allele, presumably.

Case Description

We report a boy from the first pregnancy of non-consanguineous parents. The mother suffered from gallbladder colics during gestation. He was born on term, birth weight 3380 g, length 50 cm; post-natal adaptation period and psychomotor development until the age of 22 months were nearly normal, including speech and walking development (he walked clumsily but without support). At the age of 2 years, he was examined by a neurologist for the first time for walking disturbance. Findings suggesting spastic diparesis were dominant in neurological examination, evolving into tetraparesis with psychomotor regression over the next 10 months. Brain findings included diffuse hypersignal intensity changes in T2-weighted and FLAIR images supra- and infratentorially on both sides, saving the juxtacortical region and U-fibers. Pathological findings were in the dorsal capsula interna, corpus callosum, and white matter of both cerebral hemispheres. These findings were consistent with MRI changes in MLD (Fig. 1b).

Cerebrospinal fluid examination revealed mildly increased protein content of 0.696 g/l (reference range 0.190–0.310) and slightly elevated lactate—2.55 mmol/l (reference range 1.20–2.10); other parameters were within reference values. Electromyography examination showed combined axonal-demyelinating polyneuropathy; abdomen ultrasound was normal. We focused on the determination of enzymatic levels of arylsulphatase A activity in leukocytes, which was 60.9 nmol/15 h/mg (reference ranges 50.0–125.0 nmol/15 h/mg). Urinary excretion of sulfatides was clearly increased (qualitative analysis—26.0 nmol/l (reference ranges 6.36–13.55), quantitative analysis 5623 nmol/l (reference ranges 73–326). The patient was strongly suspect of SapB protein deficiency.

Currently, at the age of 4 years, the boy suffers from profound psychomotor regression with minimal motor activity, pronounced spastic paraparesis, and no verbal expression; he is relatively well responding to speech and sounds and has no swallowing difficulties. There are no epileptic seizures present.

Genetic testing by Sanger sequencing of the patient’s DNA revealed the presence of compound heterozygous variants c.679_681delAAG (p.Lys227del) and c.1268delT (p.Leu423Argfs*40) in the PSAP gene (Supplementary Figure). Segregation analysis in both parents confirmed variant c. 679_681delAAG in the father, and variant c.1268delT in the mother.

The variant c.679_681delAAG (p.Lys227del) is located in exon 6 and causes deletion of lysine 227 in SapB protein. This variant was found only in one heterozygous individual in the GO ESP project database (Exome Variant Server) but is absent from ExAC and gnomAD databases (Exome Variant Server; Lek et al. 2016). It has not been described in relation to a disease. In silico tool PROVEAN capable of assessment of deletions (Choi and Chan 2015) predicts this variant to be deleterious (score − 9.19, cutoff − 2.5). Additionally, as we have found this variant in trans with another pathogenic allele and the patient’s phenotype corresponds with MLD caused by mutations in the PSAP gene, it can be classified as likely pathogenic according to ACMG guidelines (Richards et al. 2015).

The variant c.1268delT is located in exon 11 encoding the SapD protein. It causes the p.Leu423Argfs*40 frameshift resulting in a premature termination codon. This variant leads to a null allele as such aberrant transcript should be degraded in the process of nonsense-mediated decay. This variant has not been previously reported in patients nor publicly available population databases. Together with the patient’s specific phenotype, there is enough evidence to classify this variant as pathogenic (classification according to (Richards et al. 2015)).

Both variants were added to the locus-specific database LOVD (www.lovd.nl/PSAP, IDs PSAP_000017 and PSAP_000018) and submitted to ClinVar (www.ncbi.nlm.nih.gov/clinvar/, accession numbers SCV000809015 and SCV000809016).

Discussion

The patient discussed in this report was highly suspected of MLD due to deficiency of saposin B protein according to clinical findings and biochemical studies. This diagnosis was confirmed by sequencing analysis of the PSAP gene where we have found two variants.

In the published cohort of 26 PSAP – MLD patients (Cesani et al. 2016), only two cases were compound heterozygotes carrying a SapB impairing variant and a null allele: p.Asn215Lys + p.Met1Val (Deconinck et al. 2008) and p.Asp193_Ile240del; p.Asp193_Gln199del + p.Lys275fs (Kuchar et al. 2009) with the former one being the only case with a mutation outside the SapB encoding exons 6–8. Here, we describe another such case, one single amino acid deletion located in exon 6 encoding SapB and one frameshift variant located in the exon 11 encoding SapD. The phenotype of the patient confirms that the frameshift variant c.1268delT (p.Leu423fs) in exon 11 results in a null allele and he suffers from an isolated SapB deficiency due to the p.Lys227del allele in exon 6. Importantly, further PSAP null alleles were described in prosaposin or SapC deficiency (Table 1) and could be possibly involved also in MLD if combined with a mutation within the SapB portion of the gene. On the other side, some of the splicing mutations in the PSAP gene cause only isolated saposin deficit due to skipping of exons causing in-frame deletions (Table 1, Fig. 1). Therefore, it might be necessary to analyze the effect of novel splicing mutations on the mRNA level to confirm the genetic etiology of the disease definitively.

Table 1 Characteristics of the PSAP alleles up to date
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Furthermore, it might be noteworthy that all three reported patients with one null allele presented with the late infantile form of MLD, as well as the majority of patients with both mutations impairing selectively the SapB protein. The clinical course of their disease was similarly severe, without substantial differences.

In conclusion, our data update the PSAP gene mutation list and show a new example of how a mutation in a non-SapB exon can contribute to isolated SapB deficiency. Awareness of this might be crucial to prevent delayed diagnosis or misdiagnosis and to promptly provide an accurate genetic counseling to families.