Background
Spinal muscular atrophy (SMA) is a neuromuscular disease affecting nerves and skeletal muscles by progressive degeneration of motor neurons in the spinal cord and brain stem. It includes a heterogeneous group of disorders, which may be classified into two subtypes each depending on its own genes:
SMN1 and
SMN2 genes are involved in about 95% of cases and define the so called “5q SMA” group. The other one is called “non-5q SMA”, and is caused by mutations in 20 genes including
ASAH1 [
1].
ASAH1 is a relatively small gene containing 14 exons. It is located on 8p22 chromosome and encodes the acid ceramidase enzyme (aCDase) which breaks down ceramide into sphingosine and free fatty acid. Its alternative splicing results in multiple transcripts, among which at least one encodes a proteolytically processed preproprotein. This generates a protein, consisting of a non-glycosylated alpha and a glycosylated beta subunits, which is cleaved to the mature lysosomal enzyme [
2,
3]. Since
ASAH1 is ubiquitously expressed, mutations in this gene result in lysosomal accumulation of ceramides in various tissues which eventually causes the phenotypical manifestations [
4].
So far, 107 pathogenic and 49 likely pathogenic variants have been reported for this gene in ClinVar (
https://www.ncbi.nlm.nih.gov/clinvar/), among which the most frequent are missense. These mutations lead to aCDase deficiency mainly causing Farber disease and rarely the phenotype of spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME, OMIM 159950). Farber is usually a neonatal or early infantile disease characterized by a hoarse voice or a weak cry, small lumps of fat under the skin and in other tissues (lipogranulomas), and painful contractures of joints [
5]. Affected individuals may also have difficulty in breathing, hepatosplenomegaly, and developmental delay [
6]. SMA-PME was first described by Jankovic and Rivera in 1978 but the disease phenotype was linked to the
ASAH1 gene in 2012 [
7]. It is an autosomal recessive disorder representing a heterogeneous group of epilepsies, usually starting within 2–6 years of age with muscle atrophy, difficulty in walking and tremors, and later developing into myoclonic epilepsy usually during late childhood. Since the first symptoms of SMA-PME may overlap with those of“5q SMA”, it is usually necessary to carry out genetic testing for diagnosis confirmation and to rule out 5q SMA disease [
8]. No successful treatment has been reported until now, and patients are managed by symptomatic multidisciplinary treatments [
9].
Since only 37 genetically confirmed SMA-PME patients have been reported to date [
6,
8,
10‐
17], most of which show the p.T42M mutation, new cases and mutations can significantly contribute to a better understanding of the disease. Herein, we present 3 cases of SMA-PME with homozygous missense mutations, and briefly compare them with previously reported missense mutations of
ASAH1 causing SMA-PME.
Discussion
SMA is one of the most common neuromuscular disorders with pediatric lethality [
26]. In about 95% of cases, SMA is caused by deletions in exons 7 and 8 of SMN1 gene. This gene is on chromosome 5 (5q13), so, the relevant SMA is called 5q SMA. Other less prevalent types of SMA which are not due to mutations in this gene, are called non-5q SMA. Mutations in 20 genes including
ASAH1 have been identified to be responsible for “non-5q SMA”, about half of
ASAH1-related mutations are T42A and T42M occurring in exon 2 [
14]. The number of SMA-PME and SMA-PME like patients reported up to 2018 were respectively 23 and 20, accounting for a total of 43 cases [
14]. Moreover, so far, 58 cases of SMA-PME have been reported, 37 of which have been confirmed by genetic testing. In this study, we investigated 3 patients from unrelated families by clinical examination and genetic testing carrying two pathogenic or likely pathogenic variants in the
ASAH1 gene. Mutations in
ASAH1 cause Farber disease and SMA with progressive myoclonic epilepsy (SMA-PME). Farber disease is severe, showing infancy onset and a median survival period of about 3 years [
27]. Its classic features include the triad of subcutaneous nodules, joint swellings/arthritis and hoarse or weak voice [
14]. Typically, activity of the aCDase is more markedly reduced in Farber disease than SMA-PME [
14]. Zielonka et al. showed that a higher residual activity of aCDase is associated with later onset and longer survival of Farber patients [
27]. A similar pattern seems to be true also for SMA-PME which is an ultrarare (Prevalence:
<1/1 000 000
) [
28‐
30] childhood neurological condition leading to muscle weakness and atrophy. It also manifests seizures and uncontrollable myoclonic epilepsy [
8]. The main cause of death in SMA-PME patients is respiratory insufficiency, usually occurring 5 to 15 years after the onset of the disease. All our patients are alive.
One of the variants here reported is a missense mutation, carried by the patient belonging to family A, who showed progressive muscle weakness, seizures, fasciculation, and scleral telangiectasia. Previous studies indicate all these symptoms related to SMA-PME patients except for the last one [
4,
14,
31]. The present finding of ocular telangiectasia may expand the clinical phenotype of the disease. T42M is the variant harbored by the other 2 patients of our study. Since various mutations diversly affect structure and function of the protein, a number of studies indicate that the substitution of threonine by methionine at position 42 of the encoded protein causes a milder effect on the final product so that acid ceramidase activity is partially preserved reaching 30% [
32]. As expected, the present patients with T42M mutation have started to show muscular weakness with a slowly progressive pattern, reflecting their likely less severe type of mutation.
Previously one patient has been reported from Iran with p.T42M mutation in
ASAH1 and classic symptoms of SMA-PME [
12]. Here, we describe 3 new cases. Two of our patients are born by consanguineous parents, but the patient related to family A, whose parents belong to a small city in west of Iran, was born by a non-consanguineous marriage. Regarding the latter case, it should be noted that ancient consanguinity, especially in such a small town cannot be excluded [
33]. The average rate of consanguineous marriage in Iran is 37.4% which puts it among the highest rates in the world [
34,
35]. Therefore the rate of recessively inherited disorders are expected to be higher than western countries with very low rates of consanguineous marriages [
36]. In accordance with previous studies showing higher rates of congenital abnormalities including inborn errors of metabolism in presence of parental consanguinity [
37], most of the reported SMA-PME cases as well as 2/3 of our patients are born by consanguineous parents [
6,
31,
35].
For the confirmation of SMA-PME it is worth to assess aCDase enzyme activity. However, one limitation of our study was unavailability of this test in Iran. So, we have confirmed the SMA-PME cases based on clinical, primary laboratory and subsequent genetic evaluations. However, we showed that the aCDase structural stability could be deteriorated with the destabilizing (ΔΔG > 0) p.Pro37Thr mutation. The other variant (p.Thr42Met) was a slightly stabilizing (ΔΔG < 0) mutation. Although the structural prediction scheme classified the current mutation as a stabilizing change, it is noteworthy to mention here that the thermodynamic stability of proteins is not the conclusive determining factor of mutation effects. Point mutations can contribute to the development of human diseases by disturbing protein-protein interaction (PPI) networks [
38]. Such mutations, even located far from the active site, can also induce biophysical mechanisms that affect the affinity of substrate-enzyme interaction resulting in the malfunction of the enzyme [
39].
In silico prediction for both of the substitutions with SIFT showed the deleteriousness of them. The PolyPhen2 analysis indicated these changes as ‘probably damaging’ and Mutation taster program predicted them as disease-causing. Also, they had relatively high CADD Phred scores. These bioinformatic analyses were in accordance with the causality of these two mutations in our patients. Moreover, these bioinformatic analyses were performed for all previously reported missense mutations of
ASAH1 known to cause SMA-PME (Table
3). While these prediction tools cannot fully substitute the functional assays for assessment of genetic variants, they can provide a broad insight into their pathogenicity.
Conclusion
We performed MLPA and Whole Exome Sequencing (WES) for 3 patients affected with SMA-PME, and found a rare mutation (c.109 C > A; [p.Pro37Thr]) in 1 patient and a previously well-known mutation (c.125 C > T [p.Thr42Met]) in the other 2 patients. Both of them cause SMA-PME disease, due to deficiency of the aCDase enzyme. The patient with the rarer mutation in the ASAH1 gene also manifested ocular telangiectasia, further expanding the clinical phenotype of SMA-PME.
Next-generation sequencing (NGS) is a robust genetic method for analyzing human genome, and WES as a more cost-effective version of NGS techniques, significantly accelerates the detection of disease-causing genetic variations [
40,
41]. This powerful technique aids clinicians in the diagnostic process of genetic disorders with clinical and/ or genetic heterogeneity, and can help in family planning through genetic counselling about recurrence risk and primary prevention options including prenatal diagnosis (PND) or preimplantation genetic diagnosis (PGD) [
42,
43]. Finally, it is noteworthy that NGS may help to formulate more accurate prognosis evaluation and individualized follow-up, based for each single case on the specific genomic profile [
44].
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