Identification and Functional Characterisation of a Novel ASAH1 Gene Variant in Two Unrelated Indian Individuals with Childhood-Onset Spinal Muscular Atrophy

Technical Summary: Identification and Functional Characterisation of a Novel ASAH1 Gene Variant in Two Unrelated Indian Individuals with Childhood-Onset Spinal Muscular Atrophy

Problem Statement
Pathogenic variants in the ASAH1 gene, which encodes the lysosomal enzyme acid ceramidase (AC), cause a spectrum of autosomal recessive disorders ranging from severe Farber disease to Spinal Muscular Atrophy with Progressive Myoclonic Epilepsy (SMA-PME). The clinical severity is generally thought to correlate with residual enzymatic activity, where severe deficiency (<10%) leads to Farber disease and partial activity (10–30%) is associated with SMA-PME. However, the precise molecular mechanisms linking specific variants to phenotypic variability, particularly in cases where biochemical markers do not fully align with expected pathogenicity, remain an area of investigation. This study addresses the identification and functional characterization of a novel homozygous missense variant in two unrelated Indian patients presenting with childhood-onset neuromuscular weakness, with one case exhibiting SMA-PME features and the other a pure SMA phenotype.

Methodology
The study employed a multi-disciplinary approach combining clinical genetics, biochemistry, and cell biology:

• Genetic Analysis: Whole-exome sequencing (WES) was performed on two affected individuals. Variants were filtered using standard databases (gnomAD, ClinVar, etc.) and annotated using tools like VEP and Sentieon. Sanger sequencing confirmed the zygosity of the candidate variant in patients and their parents.
• Transcript Analysis: RNA was extracted from Epstein-Barr Virus (EBV)-transformed lymphoblastoid cell lines derived from one patient and his parents to assess for aberrant splicing.
• Functional Assays:
  • Enzymatic Activity: Acid ceramidase activity was measured in patient-derived leukocytes and lymphoblastoid cells using a fluorometric assay.
  • Cellular Models: HEK293T cells and Farber disease fibroblasts (lacking AC activity) were transiently transfected with wild-type (WT) ASAH1 cDNA or the patient-specific c.127T>A (p.Tyr43Asn) variant. A known pathogenic mutant (c.428G>C, p.Cys143Ser) served as a negative control.
  • Protein Analysis: Western blotting was used to detect AC protein expression and precursor cleavage.
  • Metabolic Analysis: Intracellular ceramide and dihydroceramide levels were quantified via LC–MS/MS in transfected cells.
• In Silico Modeling: The variant was analyzed using multiple prediction algorithms (SIFT, PolyPhen-2, REVEL, etc.) and structural modeling tools (HOPE) to assess potential impacts on protein structure and stability.

Key Results

• Variant Identification: WES identified a novel homozygous missense variant, c.127T>A (p.Tyr43Asn), in exon 3 of the ASAH1 gene in both unrelated patients. The variant was absent in public databases (gnomAD) and confirmed in heterozygous state in both sets of parents.
• Clinical Phenotypes:
  • Case 1: A 12-year-old male with childhood-onset proximal muscle weakness, tongue fasciculations, and Gower sign, but no seizures or myoclonus (SMA phenotype).
  • Case 2: A 19-year-old female with similar onset weakness who later developed generalized tonic-clonic seizures, myoclonus, and dystonia (SMA-PME phenotype).
• Biochemical Findings: Patient cells showed reduced, but not null, acid ceramidase activity compared to controls, while parents exhibited intermediate levels, consistent with autosomal recessive inheritance.
• Functional Characterization:
  • Splicing: cDNA analysis confirmed the variant does not cause aberrant mRNA splicing.
  • Protein Expression: Western blots showed that the p.Tyr43Asn mutant protein is expressed and undergoes autocatalytic cleavage into the 14 kDa alpha subunit, unlike the p.Cys143Ser control which failed to cleave.
  • Enzymatic Activity: Contrary to expectations for a pathogenic variant based on patient cell data, ectopic expression of the mutant cDNA in cultured human cells led to acid ceramidase activity similar to that obtained with the wild-type cDNA.
  • Ceramide Levels: Expression of the p.Tyr43Asn mutant in Farber cells successfully reduced intracellular ceramide accumulation to the same extent as the wild-type protein.
• ACMG Classification: Based on the segregation data, absence in controls, presence in two unrelated affected individuals with a consistent phenotype, and in silico predictions, the variant is classified as pathogenic (IIIb) by ACMG guidelines (PS4, PM1, PM2_supporting, PP2, PP4).

Significance and Claims
The authors report the identification of a novel ASAH1 variant (p.Tyr43Asn) associated with a phenotypic spectrum of SMA to SMA-PME in Indian individuals, suggesting a potential founder effect. The study highlights the importance of integrating genetic, enzymatic, and clinical evidence for variant interpretation.

Crucially, the paper claims that the pathogenic mechanism of this specific variant may differ from the classical model of ceramide accumulation. While the variant is associated with reduced enzyme activity in patient-derived cells, functional assays in cultured human cells demonstrated that the mutant protein retains catalytic activity and can correct ceramide accumulation to the same extent as the wild-type. This finding suggests that the primary mechanism underlying the disease in these patients is distinct from simple ceramide accumulation or involves additional mechanisms not captured by the in vitro models used. The study underscores the complexity of ASAH1-related disorders and the necessity of combining diverse lines of evidence to fully understand variant pathogenicity.