The result of a genetic test showed that our patient was double heterozygote for SMPD1: NM_000543.4:c.[84delC];[96G > A]. The C deletion at position 84 of cDNA promotes a frameshift resulting in an early stop codon, what presumably generates a null allele [NP_000534.3:p.(Gly29AspfsTer48)]. This variation was first described by Sikora et al [5] in 2003 in an double heterozygote Dutch child, being the second mutated allele NM_000543.4:c.748A > C (NP_000534.3:p.Ser250Arg) which causes null enzymatic activity [6]. The child died at the age of 5 years with a mild NPD type A phenotype and hepatosplenic enlargement, psychomotor delay and presence of foam cells in the bone marrow.
The switch of G to A at position 96 of cDNA induces a premature stop codon before the second in-frame initiation codon (NP_000534.3:p.Trp32Ter). This variation was first described by Pittis et al [7]. It is the most frequent mutation in Italian NPD type B patients (18.8% of the alleles). It has been described in 5 Italian patients (1 homozygous patient and 4 heterozygous presenting a second associated mutation, different from the one harboured by our patient). All 5 presented a NPD type B phenotype without neurological involvement regardless the associated mutation, with the exception of a 12-year-old heterozygous girl who developed symptoms during follow-up. All cases had organomegalies and 2 cases presented pulmonary involvement. This variation generates a premature stop codon that presumably would generate a truncated protein and thus should be considered as a null allele, expecting a severe phenotype. In fact, the in vitro functional characterization of the mutation through the expression of mutant SMPD1 in COS-1 cells confirms this hypothesis as nor the expression of protein neither residual enzymatic activity were detected [8]. However, this mutation is associated with a NPD type B phenotype, and residual enzymatic activity in leukocytes and fibroblasts is detected in patients, achieving 20.9% in the leukocytes of a homozygous patient [7], which is a high residual enzymatic value for a mutated protein. The reason of this discrepancy between the in vitro and the in vivo results is unclear, though may be related with differences in the post-translational modification and subcellular trafficking processes.
The SMPD1 gene has the peculiarity of having two in-frame initiation ATG codons at positions 1 and 33 and both can be functional in vitro [9]. Although in vivo translation of wild type SMPD1 initiates from the first initiation codon, the second codon can be functional when the first one has been disabled [7]. To become a mature and functional enzyme, ASM requires several post-translational modifications as well as a proper cellular trafficking [10]. In vitro expression of a mutant ASM lacking the signal peptide results in production of a non-glycosylated, non-secreted, cytosolic protein that lacks enzyme activity in cellular extracts [11]. When the second initiation codon substitutes the first one, the resulting protein misses 32 N-terminal residues of the signal peptide (which comprises 46 residues), so this variant most likely undergoes an abnormal maturing process. Therefore, the dysfunction is more likely to be related to gene expression, maturation and intracellular trafficking than to catalytic activity per se of the enzyme, as is often the case when pathogenic mutations are considered.
In our case, both mutations are located before the second initiation codon and both mutations would result in a null allele if the second codon were not functional, as they introduce early stop codons. Although the underlying mechanism is not fully understood, it is clear that none of the mutations seem to result in a null allele and that the second codon is, at least partially, activated in both alleles. The enzymatic activity of ASM is reduced enough to generate symptoms, but at the same time is high enough to be compatible with a NPD type B phenotype.
This is a unique case with a compound heterozygosity with mutations that induce early stop codons and are located in the same region before the second initiation codon, a circumstance that has not been previously reported in the literature.
The correlation between genotype and phenotype in compound heterozygotes is not evident. Although an equivalent expression of both alleles should normally be expected, due to epigenetic factors and methylation conditions, the maternal inherited allele of the SMPD1 gene may be expressed preferentially [6]. In our case, although we have the family pedigree, we cannot draw any conclusion from this matter since we cannot derive from our data the contribution of each mutation individually and theoretically a similar behaviour may be expected.
NPD can pose a diagnostic challenge. Differential diagnosis includes Wilson disease, Leigh syndrome, adrenoleukodystrophy, arginase deficiency and Gaucher disease. In LAL deficiency, the clinical spectrum can be similar to NPD, including the lipid profile alteration, but lung involvement is not commonly reported in LAL deficiency patients [12]. In Gaucher disease, total cholesterol, as well as LDL and HDL cholesterol are significantly reduced [13]. The lipid profile of our patient led to the suspicion that the initial diagnosis of Gaucher disease was incorrect. However, some authors describe an association between a low HDL-C level and defects in the SMPD1 gene causing Type B NPD [14]. The pathogenesis of the dyslipoproteinemia in these patients is not yet fully understood.
There are no recommendations for the lipid-lowering management in NPD. Some authors report that statins or fibrates may increase transaminases without a substantial reduction of lipid concentrations in NPD type B patients [15, 16]. However, there are cases describing an improvement of lipid profiles and reduction of liver enzymes under treatment with fenofibrate [15]. In our case, our patient received atorvastatin 80 mg and ezetimibe for many years, with a significant reduction of lipid concentrations (almost 70% of total cholesterol and LDL reduction) and liver enzymes. Olipudase alfa, a recombinant human acid sphingomyelinase (ASM), is used as an enzyme replacement therapy for the treatment of non-neurologic manifestations of acid sphingomyelinase deficiency. A 30-month follow-up clinical trial showed statistical significant reductions in liver (31%) and spleen (39%) volumes [17]. There was a mean increase in lung diffusing capacity of 35%, and clinically relevant improvements in infiltrative lung disease parameters, and lipid profiles improved in all 5 patients. Improvements in bone mineral density of the spine were observed in some patients. Lyso-sphingomyelin in dried blood spots decreased with olipudase alfa treatment [17]. We offered our patient to be treated with olipudase alfa, sending her to another hospital to be included in a clinical trial, but she repeatedly refused.
In conclusion, we present a case of NPD type B with a unique genetic mechanism: a compound heterozygosity of mutations that induce a premature stop codon located before the second initiation codon, circumstance that has not been reported in the literature. We also discuss the pharmacological management of the lipid profile, showing that, in our patient, a combination of a high intensity statin with ezetimibe was useful, although more research is needed to broaden the available evidence on this topic.
