The c.-265G>A GLA gene promoter variant causes Fabry disease: The hidden culprit identified.

Here, we report the identification and functional characterization of a novel GLA variant, not detectable by routine molecular tests, in a family with FD suspicion.

Deficiency of Glucocerebrosidase Activity beyond Gaucher Disease: PSAP and LIMP-2 Dysfunctions.

Glucocerebrosidase (GCase) is a lysosomal enzyme that catalyzes the breakdown of glucosylceramide in the presence of its activator saposin C (SapC). SapC arises from the proteolytical cleavage of prosaposin (encoded by PSAP gene), which gives rise to four saposins. GCase is targeted to the lysosomes by LIMP-2, encoded by SCARB2 gene. GCase deficiency causes Gaucher Disease (GD), which is mainly due to biallelic pathogenetic variants in the GCase-encoding gene, GBA1. However, impairment of GCase activity can be rarely caused by SapC or LIMP-2 deficiencies. We report a new case of LIMP-2 deficiency and a new case of SapC deficiency (missing all four saposins, PSAP deficiency), and measured common biomarkers of GD and GCase activity. Glucosylsphingosine and chitotriosidase activity in plasma were increased in GCase deficiencies caused by PSAP and GBA1 mutations, whereas SCARB2-linked deficiency showed only Glucosylsphingosine elevation. GCase activity was reduced in fibroblasts and leukocytes: the decrease was sharper in GBA1- and SCARB2-mutant fibroblasts than PSAP-mutant ones; LIMP-2-deficient leukocytes displayed higher residual GCase activity than GBA1-mutant ones. Finally, we demonstrated that GCase mainly undergoes proteasomal degradation in LIMP-2-deficient fibroblasts and lysosomal degradation in PSAP-deficient fibroblasts. Thus, we analyzed the differential biochemical profile of GCase deficiencies due to the ultra-rare PSAP and SCARB2 biallelic pathogenic variants in comparison with the profile observed in GBA1-linked GCase deficiency.

Distribution of Exonic Variants in Glycogen Synthesis and Catabolism Genes in Late Onset Pompe Disease (LOPD).

Pompe disease (PD) is a monogenic autosomal recessive disorder caused by biallelic pathogenic variants of the GAA gene encoding lysosomal alpha-glucosidase; its loss causes glycogen storage in lysosomes, mainly in the muscular tissue. The genotype-phenotype correlation has been extensively discussed, and caution is recommended when interpreting the clinical significance of any mutation in a single patient. As there is no evidence that environmental factors can modulate the phenotype, the observed clinical variability in PD suggests that genetic variants other than pathogenic GAA mutations influence the mechanisms of muscle damage/repair and the overall clinical picture. Genes encoding proteins involved in glycogen synthesis and catabolism may represent excellent candidates as phenotypic modifiers of PD. The genes analyzed for glycogen synthesis included UGP2, glycogenin (GYG1-muscle, GYG2, and other tissues), glycogen synthase (GYS1-muscle and GYS2-liver), GBE1, EPM2A, NHLRC1, GSK3A, and GSK3B. The only enzyme involved in glycogen catabolism in lysosomes is α-glucosidase, which is encoded by GAA, while two cytoplasmic enzymes, phosphorylase (PYGB-brain, PGL-liver, and PYGM-muscle) and glycogen debranching (AGL) are needed to obtain glucose 1-phosphate or free glucose. Here, we report the potentially relevant variants in genes related to glycogen synthesis and catabolism, identified by whole exome sequencing in a group of 30 patients with late-onset Pompe disease (LOPD). In our exploratory analysis, we observed a reduced number of variants in the genes expressed in muscles versus the genes expressed in other tissues, but we did not find a single variant that strongly affected the phenotype. From our work, it also appears that the current clinical scores used in LOPD do not describe muscle impairment with enough qualitative/quantitative details to correlate it with genes that, even with a slightly reduced function due to genetic variants, impact the phenotype.

First experience of combined enzyme replacement therapy and hematopoietic stem cell transplantation in alpha-mannosidosis.

We describe the first case of bridge therapy in alpha-mannosidosis (AM) in an infant diagnosed at only 5 months of life who underwent enzyme replacement therapy (ERT) in the pre- and peri-transplant phases. Eight ERT infusions were administered before hematopoietic stem cell transplantation (HSCT) and continued for additional 90 days until complete engraftment. The clinical and laboratory data after 3 years post-HSCT show that the early combined intervention may reduce the disease progression and the urine and plasma content of mannosyl-oligosaccharides (OS) monitored by liquid chromatography tandem mass spectrometry (LC-MS/MS). This report highlights that early diagnosis and prompt initiation of such treatments in AM are the best chance to minimize the progression of symptoms.

Exploring the Pathophysiologic Cascade Leading to Osteoclastogenic Activation in Gaucher Disease Monocytes Generated via CRISPR/Cas9 Technology.

Gaucher disease (GD) is caused by biallelic pathogenic variants in the acid ß-glucosidase gene (GBA1), leading to a deficiency in the ß-glucocerebrosidase (GCase) enzyme activity resulting in the intracellular accumulation of sphingolipids. Skeletal alterations are one of the most disabling features in GD patients. Although both defective bone formation and increased bone resorption due to osteoblast and osteoclast dysfunction contribute to GD bone pathology, the molecular bases are not fully understood, and bone disease is not completely resolved with currently available specific therapies. For this reason, using editing technology, our group has developed a reliable, isogenic, and easy-to-handle cellular model of GD monocytes (GBAKO-THP1) to facilitate GD pathophysiology studies and high-throughput drug screenings. In this work, we further characterized the model showing an increase in proinflammatory cytokines (Interleukin-1ß and Tumor Necrosis Factor-α) release and activation of osteoclastogenesis. Furthermore, our data suggest that GD monocytes would display an increased osteoclastogenic potential, independent of their interaction with the GD microenvironment or other GD cells. Both proinflammatory cytokine production and osteoclastogenesis were restored at least, in part, by treating cells with the recombinant human GCase, a substrate synthase inhibitor, a pharmacological chaperone, and an anti-inflammatory compound. Besides confirming that this model would be suitable to perform high-throughput screening of therapeutic molecules that act via different mechanisms and on different phenotypic features, our data provided insights into the pathogenic cascade, leading to osteoclastogenesis exacerbation and its contribution to bone pathology in GD.

Acid Sphingomyelinase Deficiency: A Clinical and Immunological Perspective.

Acid sphingomyelinase deficiency (ASMD) is a lysosomal storage disease caused by deficient activity of acid sphingomyelinase (ASM) enzyme, leading to the accumulation of varying degrees of sphingomyelin. Lipid storage leads to foam cell infiltration in tissues, and clinical features including hepatosplenomegaly, pulmonary insufficiency and in some cases central nervous system involvement. ASM enzyme replacement therapy is currently in clinical trial being the first treatment addressing the underlying pathology of the disease. Therefore, presently, it is critical to better comprehend ASMD to improve its diagnose and monitoring. Lung disease, including recurrent pulmonary infections, are common in ASMD patients. Along with lung disease, several immune system alterations have been described both in patients and in ASMD animal models, thus highlighting the role of ASM enzyme in the immune system. In this review, we summarized the pivotal roles of ASM in several immune system cells namely on macrophages, Natural Killer (NK) cells, NKT cells, B cells and T cells. In addition, an overview of diagnose, monitoring and treatment of ASMD is provided highlighting the new enzyme replacement therapy available.

Accurate Molecular Diagnosis of Gaucher Disease Using Clinical Exome Sequencing as a First-Tier Test.

Gaucher disease (GD) is an autosomal recessive lysosomal disorder due to beta-glucosidase gene (GBA) mutations. The molecular diagnosis of GD is complicated by the presence of recombinant alleles originating from a highly homologous pseudogene. Clinical exome sequencing (CES) is a rapid genetic approach for identifying disease-causing mutations. However, copy number variation and recombination events are poorly detected, and further investigations are required to avoid mis-genotyping. The aim of this work was to set-up an integrated strategy for GD patients genotyping using CES as a first-line test. Eight patients diagnosed with GD were analyzed by CES. Five patients were fully genotyped, while three were revealed to be homozygous for mutations that were not confirmed in the parents. Therefore, MLPA (multiplex ligation-dependent probe amplification) and specific long-range PCR were performed, and two recombinant alleles, one of them novel, and one large deletion were identified. Furthermore, an MLPA assay performed in one family resulted in the identification of an additional novel mutation (p.M124V) in a relative, in trans with the known p.N409S mutation. In conclusion, even though CES has become extensively used in clinical practice, our study emphasizes the importance of a comprehensive molecular strategy to provide proper GBA genotyping and genetic counseling.

Changes in global gene expression indicate disordered autophagy, apoptosis and inflammatory processes and downregulation of cytoskeletal signalling and neuronal development in patients with Niemann-Pick C disease.

Changes in gene expression profiles were investigated in 23 patients with Niemann-Pick C1 disease (NPC). cDNA expression microarrays with subsequent validation by qRT-PCR were used. Comparison of NPC to control samples revealed upregulation of genes involved in inflammation (MMP3, THBS4), cytokine signalling (MMP3), extracellular matrix degradation (MMP3, CTSK), autophagy and apoptosis (CTSK, GPNMB, PTGIS), immune response (AKR1C3, RCAN2, PTGIS) and processes of neuronal development (RCAN2). Downregulated genes were associated with cytoskeletal signalling (ACTG2, CNN1); inflammation and oxidative stress (CNN1); inhibition of cell proliferation, migration and differentiation; ERK-MAPK pathway (COL4A1, COL4A2, CPA4); cell adhesion (IGFBP7); autophagy and apoptosis (CDH2, IGFBP7, COL4A2); neuronal function and development (CSRP1); and extracellular matrix stability (PLOD2). When comparing NPC and Gaucher patients together versus controls, upregulation of SERPINB2 and IL13RA2 and downregulation of CSRP1 and CNN1 were characteristic. Notably, in NPC patients, the expression of PTGIS is upregulated while the expression of PLOD2 is downregulated when compared to Gaucher patients or controls and potentially could serve to differentiate these patients. Interestingly, in NPC patients with (i) jaundice, splenomegaly and cognitive impairment/psychomotor delay-the expression of ACTG2 was especially downregulated; (ii) ataxia-the expression of ACTG2 and IGFBP5 was especially downregulated; and (iii) VSGP, dysarthria, dysphagia and epilepsy-the expression of AKR1C3 was especially upregulated while the expression of ACTG2 was downregulated. These results indicate disordered apoptosis, autophagy and cytoskeleton remodelling as well as upregulation of immune response and inflammation to play an important role in the pathogenesis of NPC in humans.

Impact of COVID-19 related healthcare crisis on treatments for patients with lysosomal storage disorders, the first Italian experience.

The direct and indirect effects of Coronavirus Disease-19 (COVID-19) pandemic, on Italian patients with lysosomal storage disorders receiving therapy, were analyzed by a phone questionnaire. No proved COVID-19 emerged among 102 interviewed. No problems were reported by patients receiving oral treatments. Forty-nine% of patients receiving enzyme replacement therapy in hospitals experienced disruptions, versus 6% of those home-treated. The main reasons of missed infusions were fear of infection (62.9%) and re-organization of the infusion centers (37%).

Mechanistic convergence and shared therapeutic targets in Niemann-Pick disease.

Niemann-Pick disease type C (NPC) and Tangier disease are genetically and clinically distinct rare inborn errors of metabolism. NPC is caused by defects in either NPC1 or NPC2; whereas Tangier disease is caused by a defect in ABCA1. Tangier disease is currently without therapy, whereas NPC can be treated with miglustat, a small molecule inhibitor of glycosphingolipid biosynthesis that slows the neurological course of the disease. When a Tangier disease patient was misdiagnosed with NPC and treated with miglustat, her symptoms improved. This prompted us to consider whether there is mechanistic convergence between these two apparently unrelated rare inherited metabolic diseases. In this study, we found that when ABCA1 is defective (Tangier disease) there is secondary inhibition of the NPC disease pathway, linking these two diseases at the level of cellular pathophysiology. In addition, this study further supports the hypothesis that miglustat, as well as other substrate reduction therapies, may be potential therapeutic agents for treating Tangier disease as fibroblasts from multiple Tangier patients were corrected by miglustat treatment.

CRISPR/Cas9 Editing for Gaucher Disease Modelling.

Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the acid ß-glucosidase gene (GBA1). Besides causing GD, GBA1 mutations constitute the main genetic risk factor for developing Parkinson's disease. The molecular basis of neurological manifestations in GD remain elusive. However, neuroinflammation has been proposed as a key player in this process. We exploited CRISPR/Cas9 technology to edit GBA1 in the human monocytic THP-1 cell line to develop an isogenic GD model of monocytes and in glioblastoma U87 cell lines to generate an isogenic GD model of glial cells. Both edited (GBA1 mutant) cell lines presented low levels of mutant acid ß-glucosidase expression, less than 1% of residual activity and massive accumulation of substrate. Moreover, U87 GBA1 mutant cells showed that the mutant enzyme was retained in the ER and subjected to proteasomal degradation, triggering unfolded protein response (UPR). U87 GBA1 mutant cells displayed an increased production of interleukin-1ß, both with and without inflammosome activation, α-syn accumulation and a higher rate of cell death in comparison with wild-type cells. In conclusion, we developed reliable, isogenic, and easy-to-handle cellular models of GD obtained from commercially accessible cells to be employed in GD pathophysiology studies and high-throughput drug screenings.

Assessment of the functional impact on the pre-mRNA splicing process of 28 nucleotide variants associated with Pompe disease in GAA exon 2 and their recovery using antisense technology.

Glycogen storage disease II (GSDII), also called Pompe disease, is an autosomal recessive inherited disease caused by a defect in glycogen metabolism due to the deficiency of the enzyme acid alpha-glucosidase (GAA) responsible for its degradation. So far, more than 500 sequence variants of the GAA gene have been reported but their possible involvement on the pre-messenger RNA splicing mechanism has not been extensively studied. In this work, we have investigated, by an in vitro functional assay, all putative splicing variants within GAA exon 2 and flanking introns. Our results show that many variants falling in the canonical splice site or the exon can induce GAA exon 2 skipping. In these cases, therefore, therapeutic strategies aimed at restoring protein folding of partially active mutated GAA proteins might not be sufficient. Regarding this issue, we have tested the effect of antisense oligonucleotides (AMOs) that were previously shown capable of rescuing splicing misregulation caused by the common c.-32-13T>G variant associated with the childhood/adult phenotype of GSDII. Interestingly, our results show that these AMOs are also quite effective in rescuing the splicing impairment of several exonic splicing variants, thus widening the potential use of these effectors for GSDII treatment.

Shortened primary cilium length and dysregulated Sonic hedgehog signaling in Niemann-Pick C1 disease.

The Niemann-Pick type C1 (NPC1) disease is a neurodegenerative lysosomal storage disorder due to mutations in the NPC1 gene, encoding a transmembrane protein related to the Sonic hedgehog (Shh) receptor, Patched, and involved in intracellular trafficking of cholesterol. We have recently found that the proliferation of cerebellar granule neuron precursors is significantly reduced in Npc1-/- mice due to the downregulation of Shh expression. This finding prompted us to analyze the formation of the primary cilium, a non-motile organelle that is specialized for Shh signal transduction and responsible, when defective, for several human genetic disorders. In this study, we show that the expression and subcellular localization of Shh effectors and ciliary proteins are severely disturbed in Npc1-deficient mice. The dysregulation of Shh signaling is associated with a shortening of the primary cilium length and with a reduction of the fraction of ciliated cells in Npc1-deficient mouse brains and the human fibroblasts of NPC1 patients. These defects are prevented by treatment with 2-hydroxypropyl-ß-cyclodextrin, a promising therapy currently under clinical investigation. Our findings indicate that defective Shh signaling is responsible for abnormal morphogenesis of the cerebellum of Npc1-deficient mice and show, for the first time, that the formation of the primary cilium is altered in NPC1 disease.

microRNAs as biomarkers in Pompe disease.

PURPOSE: We studied microRNAs as potential biomarkers for Pompe disease. METHODS: We analyzed microRNA expression by small RNA-seq in tissues from the disease murine model at two different ages (3 and 9 months), and in plasma from Pompe patients. RESULTS: In the mouse model we found 211 microRNAs that were differentially expressed in gastrocnemii and 66 in heart, with a different pattern of expression at different ages. In a preliminary analysis in plasma from six patients 55 microRNAs were differentially expressed. Sixteen of these microRNAs were common to those dysregulated in mouse tissues. These microRNAs are known to modulate the expression of genes involved in relevant pathways for Pompe disease pathophysiology (autophagy, muscle regeneration, muscle atrophy). One of these microRNAs, miR-133a, was selected for further quantitative real-time polymerase chain reaction analysis in plasma samples from 52 patients, obtained from seven Italian and Dutch biobanks. miR-133a levels were significantly higher in Pompe disease patients than in controls and correlated with phenotype severity, with higher levels in infantile compared with late-onset patients. In three infantile patients miR-133a decreased after start of enzyme replacement therapy and evidence of clinical improvement. CONCLUSION: Circulating microRNAs may represent additional biomarkers of Pompe disease severity and of response to therapy.

Newborn screening for lysosomal storage disorders by tandem mass spectrometry in North East Italy.

BACKGROUND: Lysosomal storage diseases (LSDs) are inborn errors of metabolism resulting from 50 different inherited disorders. The increasing availability of treatments and the importance of early intervention have stimulated newborn screening (NBS) to diagnose LSDs and permit early intervention to prevent irreversible impairment or severe disability. We present our experience screening newborns in North East Italy to identify neonates with Mucopolysaccharidosis type I (MPS I) and Pompe, Fabry, and Gaucher diseases. METHODS: Activities of acid ß-glucocerebrosidase (ABG; Gaucher), acid α-glucosidase (GAA; Pompe), acid α-galactosidase (GLA; Fabry), and acid α-L-iduronidase (IDUA; MPS-I) in dried blood spots (DBS) from all newborns during a 17-month period were determined by multiplexed tandem mass spectrometry (MS/MS) using the NeoLSD® assay system. Enzymatic activity cutoff values were determined from 3500 anonymous newborn DBS. In the screening study, samples were retested if the value was below cutoff and a second spot was requested, with referral for confirmatory testing and medical evaluation if a low value was obtained. RESULTS: From September 2015 to January 2017, 44,411 newborns were screened for the four LSDs. We recalled 40 neonates (0.09%) for collection of a second DBS. Low activity was confirmed in 20, who had confirmatory testing. Ten of 20 had pathogenic mutations: two Pompe, two Gaucher, five Fabry, and one MPS-I. The incidences of Pompe and Gaucher diseases were similar (1/22,205), with Fabry disease the most frequent (1/8882) and MPS-I the rarest (1/44411). The combined incidence of the four disorders was 1/4411 births. CONCLUSIONS: Simultaneously determining multiple enzyme activities by MS/MS, with a focus on specific biochemical markers, successfully detected newborns with LSDs. The high incidence of these disorders supports this screening program.

Glycogen Reduction in Myotubes of Late-Onset Pompe Disease Patients Using Antisense Technology.

Glycogen storage disease type II (GSDII) is a lysosomal disorder caused by the deficient activity of acid alpha-glucosidase (GAA) enzyme, leading to the accumulation of glycogen within the lysosomes. The disease has been classified in infantile and late-onset forms. Most late-onset patients share a splicing mutation c.-32-13T > G in intron 1 of the GAA gene that prevents efficient recognition of exon 2 by the spliceosome. In this study, we have mapped the splicing silencers of GAA exon 2 and developed antisense morpholino oligonucleotides (AMOs) to inhibit those regions and rescue normal splicing in the presence of the c.-32-13T > G mutation. Using a minigene approach and patient fibroblasts, we successfully increased inclusion of exon 2 in the mRNA and GAA enzyme production by targeting a specific silencer with a combination of AMOs. Most importantly, the use of these AMOs in patient myotubes results in a decreased accumulation of glycogen. To our knowledge, this is the only therapeutic approach resulting in a decrease of glycogen accumulation in patient tissues beside enzyme replacement therapy (ERT) and TFEB overexpression. As a result, it may represent a highly novel and promising therapeutic line for GSDII.

In vitro recapitulation of the site-specific editing (to wild-type) of mutant IDS mRNA transcripts, and the characterization of IDS protein translated from the edited mRNAs.

The transfer of genomic information into the primary RNA sequence can be altered by RNA editing. We have previously shown that genomic variants can be RNA-edited to wild-type. The presence of distinct "edited" iduronate 2-sulfatase (IDS) mRNA transcripts ex vivo evidenced the correction of a nonsense and frameshift variant, respectively, in three unrelated Hunter syndrome patients. This phenomenon was confirmed in various patient samples by a variety of techniques, and was quantified by single-nucleotide primer extension. Western blotting also confirmed the presence of IDS protein similar in size to the wild-type. Since preliminary experimental evidence suggested that the "corrected" IDS proteins produced by the patients were similar in molecular weight and net charge to their wild-type counterparts, an in vitro system employing different cell types was established to recapitulate the site-specific editing of IDS RNA (uridine to cytidine conversion and uridine deletion), and to confirm the findings previously observed ex vivo in the three patients. In addition, confocal microscopy and flow cytometry analyses demonstrated the expression and lysosomal localization in HEK293 cells of GFP-labeled proteins translated from edited IDS mRNAs. Confocal high-content analysis of the two patients' cells expressing wild-type or mutated IDS confirmed lysosomal localization and showed no accumulation in the Golgi or early endosomes.

Disorders of cholesterol metabolism and their unanticipated convergent mechanisms of disease.

Cholesterol plays a key role in many cellular processes, and is generated by cells through de novo biosynthesis or acquired from exogenous sources through the uptake of low-density lipoproteins. Cholesterol biosynthesis is a complex, multienzyme-catalyzed pathway involving a series of sequentially acting enzymes. Inherited defects in genes encoding cholesterol biosynthetic enzymes or other regulators of cholesterol homeostasis result in severe metabolic diseases, many of which are rare in the general population and currently without effective therapy. Historically, these diseases have been viewed as discrete disorders, each with its own genetic cause and distinct pathogenic cascades that lead to its specific clinical features. However, studies have recently shown that three of these diseases have an unanticipated mechanistic convergence. This surprising finding is not only shedding light on details of cellular cholesterol homeostasis but also suggesting novel approaches to therapy.

Cerebrospinal fluid ß-glucocerebrosidase activity is reduced in parkinson's disease patients.

BACKGROUND: Reduced ß-glucocerebrosidase activity was observed in postmortem brains of both GBA1 mutation carrier and noncarrier Parkinson's disease patients, suggesting that lower ß-glucocerebrosidase activity is a key feature in the pathogenesis of PD. The objectives of this study were to confirm whether there is reduced ß-glucocerebrosidase activity in the CSF of GBA1 mutation carrier and noncarrier PD patients and verify if other lysosomal enzymes show altered activity in the CSF. METHODS: CSF ß-glucocerebrosidase, cathepsin D, and ß-hexosaminidase activities were measured in 79 PD and 61 healthy controls from the BioFIND cohort. The whole GBA1 gene was sequenced. RESULTS: Enzyme activities were normalized according to CSF protein content (specific activity). ß-glucocerebrosidase specific activity was significantly decreased in PD versus controls (-28%, P < 0.001). GBA1 mutations were found in 10 of 79 PD patients (12.7%) and 3 of 61 controls (4.9%). GBA1 mutation carrier PD patients showed significantly lower ß-glucocerebrosidase specific activity versus noncarriers. ß-glucocerebrosidase specific activity was also decreased in noncarrier PD patients versus controls (-25%, P < 0.001). Cathepsin D specific activity was lower in PD versus controls (-21%, P < 0.001). ß-Hexosaminidase showed a similar trend. ß-Glucocerebrosidase specific activity fairly discriminated PD from controls (area under the curve, 0.72; sensitivity, 0.67; specificity, 0.77). A combination of ß-glucocerebrosidase, cathepsin D, and ß-hexosaminidase improved diagnostic accuracy (area under the curve, 0.77; sensitivity, 0.71; specificity, 0.85). Lower ß-glucocerebrosidase and ß-hexosaminidase specific activities were associated with worse cognitive performance. CONCLUSIONS: CSF ß-glucocerebrosidase activity is reduced in PD patients independent of their GBA1 mutation carrier status. Cathepsin D and ß-hexosaminidase were also decreased. The possible link between altered CSF lysosomal enzyme activities and cognitive decline deserves further investigation. © 2017 International Parkinson and Movement Disorder Society.

SMPD1 Mutation Update: Database and Comprehensive Analysis of Published and Novel Variants.

Niemann-Pick Types A and B (NPA/B) diseases are autosomal recessive lysosomal storage disorders caused by the deficient activity of acid sphingomyelinase (ASM) because of the mutations in the SMPD1 gene. Here, we provide a comprehensive updated review of already reported and newly identified SMPD1 variants. Among them, 185 have been found in NPA/B patients. Disease-causing variants are equally distributed along the SMPD1 gene; most of them are missense (65.4%) or frameshift (19%) mutations. The most frequently reported mutation worldwide is the p.R610del, clearly associated with an attenuated NP disease type B phenotype. The available information about the impact of 52 SMPD1 variants on ASM mRNA and/or enzymatic activity has been collected and whenever possible, phenotype/genotype correlations were established. In addition, we created a locus-specific database easily accessible at http://www.inpdr.org/genes that catalogs the 417 SMPD1 variants reported to date and provides data on their in silico predicted effects on ASM protein function or mRNA splicing. The information reviewed in this article, providing new insights into the genotype/phenotype correlation, is extremely valuable to facilitate diagnosis and genetic counseling of families affected by NPA/B.