The rapidly evolving view of lysosomal storage diseases

Review18 January 2021Open Access The rapidly evolving view of lysosomal storage diseases Giancarlo Parenti Giancarlo Parenti orcid.org/0000-0002-6287-5748 Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Department of Translational Medical Sciences, Section of Pediatrics, Federico II University, Naples, Italy Search for more papers by this author Diego L Medina Diego L Medina orcid.org/0000-0002-7347-2645 Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Department of Translational Medical Sciences, Section of Pediatrics, Federico II University, Naples, Italy Search for more papers by this author Andrea Ballabio Corresponding Author Andrea Ballabio [email protected] orcid.org/0000-0003-1381-4604 Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Department of Translational Medical Sciences, Section of Pediatrics, Federico II University, Naples, Italy Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Jan and Dan Duncan Neurological Research Institute, Texas Children Hospital, Houston, TX, USA SSM School for Advanced Studies, Federico II University, Naples, Italy Search for more papers by this author Giancarlo Parenti Giancarlo Parenti orcid.org/0000-0002-6287-5748 Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Department of Translational Medical Sciences, Section of Pediatrics, Federico II University, Naples, Italy Search for more papers by this author Diego L Medina Diego L Medina orcid.org/0000-0002-7347-2645 Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Department of Translational Medical Sciences, Section of Pediatrics, Federico II University, Naples, Italy Search for more papers by this author Andrea Ballabio Corresponding Author Andrea Ballabio [email protected] orcid.org/0000-0003-1381-4604 Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Department of Translational Medical Sciences, Section of Pediatrics, Federico II University, Naples, Italy Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Jan and Dan Duncan Neurological Research Institute, Texas Children Hospital, Houston, TX, USA SSM School for Advanced Studies, Federico II University, Naples, Italy Search for more papers by this author Author Information Giancarlo Parenti1,2, Diego L Medina1,2 and Andrea Ballabio *,1,2,3,4,5 1Telethon Institute of Genetics and Medicine, Pozzuoli, Italy 2Department of Translational Medical Sciences, Section of Pediatrics, Federico II University, Naples, Italy 3Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA 4Jan and Dan Duncan Neurological Research Institute, Texas Children Hospital, Houston, TX, USA 5SSM School for Advanced Studies, Federico II University, Naples, Italy *Corresponding author. Tel: +39 081 19230607; E-mail: [email protected] EMBO Mol Med (2021)13:e12836https://doi.org/10.15252/emmm.202012836 See the Glossary for abbreviations used in this article. PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Lysosomal storage diseases are a group of metabolic disorders caused by deficiencies of several components of lysosomal function. Most commonly affected are lysosomal hydrolases, which are involved in the breakdown and recycling of a variety of complex molecules and cellular structures. The understanding of lysosomal biology has progressively improved over time. Lysosomes are no longer viewed as organelles exclusively involved in catabolic pathways, but rather as highly dynamic elements of the autophagic-lysosomal pathway, involved in multiple cellular functions, including signaling, and able to adapt to environmental stimuli. This refined vision of lysosomes has substantially impacted on our understanding of the pathophysiology of lysosomal disorders. It is now clear that substrate accumulation triggers complex pathogenetic cascades that are responsible for disease pathology, such as aberrant vesicle trafficking, impairment of autophagy, dysregulation of signaling pathways, abnormalities of calcium homeostasis, and mitochondrial dysfunction. Novel technologies, in most cases based on high-throughput approaches, have significantly contributed to the characterization of lysosomal biology or lysosomal dysfunction and have the potential to facilitate diagnostic processes, and to enable the identification of new therapeutic targets. Glossary Autophagy A multistep and regulated pathway that removes unnecessary or dysfunctional cellular components and allows for delivery of cargo materials to lysosomes, where they are degraded and recycled. High-content imaging Cell-based technologies based on automated microscopy and complex image algorithms to extract multidimensional information on cell morphology, fluorescence intensity, or distribution of fluorescent markers within cells. Genome editing and CRISPR-Cas9 An RNA-guided targeted genome editing tool: This methodology which allows to introduce different specific genetic changes such as gene knock-out, knock-in, insertions, and deletions in cell lines and in vivo. Lysosomes Membrane-limited, ubiquitous, intracellular organelles involved in multiple cellular processes, such as catabolism and recycling of complex molecules and cellular components, signaling, and adaptation to environmental stimuli. Metabolome analyses High-throughput methodologies for the detection of multiple metabolites, mainly based on mass spectrometry or nuclear magnetic resonance-based approaches. Next-generation sequencing A diagnostic tool based on technological platforms that allow for sequencing of millions of small fragments of DNA in parallel. Next-generation sequencing can be used either for ”targeted” sequencing of selected gene panels or for “untargeted” approaches based on whole-exome or genome analysis. MicroRNAs Small non-coding RNAs that that regulate gene expression by targeting messenger RNAs. Introduction Knowledge on lysosomal storage diseases (LSDs) has been evolving for more than a century (Fig 1). The first phenotypes and clinical entities were described in the 19th century (see Mehta et al, 2006 for review), long before the identification of lysosomes and the definition of their biochemistry and pathophysiology. At that time, the identification of these disorders was exclusively based on the characterization of clinical phenotypes and pathology. Figure 1. The evolution of the knowledge on LSDs After the identification of the first clinical phenotypes during of the 19th century, the knowledge on LSDs evolved following the recognition of lysosomes in 1955/56 and the demonstration of the biochemical defects underlying LSDs, starting from 1963. Between the 1970s and 1990s, research in this field was focused on the mannose-6-phosphate receptor pathway and on the mechanisms underlying the sorting of lysosomal enzymes, on the identification of the molecular bases of LSDs, and on the development of tools and strategies to investigate lysosomal biology. The first attempts to treat these disorders by enzyme replacement therapy started in the 1990s. Current research is now focusing on the role of lysosomes as signaling platforms controlling cellular metabolism and on the development of new therapeutic approaches. Download figure Download PowerPoint The biochemical and cellular bases of LSDs were elucidated much later, when Christian de Duve’s work, corroborated by Alex B. Novikoff's electron microscopy observations, led to the identification of lysosomes as cellular catabolic stations (de Duve et al, 1955; Novikoff et al, 1956), and when the biochemical defects underlying some of the previously described clinical entities were discovered. Pompe disease was the first disorder to be identified as an LSD in 1963, when Henri G. Hers demonstrated that this disease is due to the lack of an acidic α-glucosidase, similar to rat liver lysosomal maltase (Hers, 1963), and that this deficiency is responsible for glycogen storage in tissues. He also suggested that other diseases, such as the mucopolysaccharide storage diseases, might be due to enzyme deficiencies. Between 1960 and the mid-70s, the biochemistry of LSDs was further characterized with the identification of the primary storage materials for other LSDs and the recognition of the respective enzyme deficiencies (Van Hoof, 1974). For decades, the biology and function of lysosomes remained associated with their catabolic function, and LSD pathophysiology was seen as a direct consequence of defective degradation and disposal of complex substrates (Vellodi, 2005; Heard et al, 2010). Between the late 1970s and 1990s, research in this field progressed with studies that considerably expanded the knowledge on lysosome biology and on the pathophysiology of LSDs. These studies led to the characterization of the mechanism underlying the sorting of lysosomal enzymes (Sly & Fisher, 1982) and to the identification of the molecular bases of clinical variability of LSDs (Beck, 2001; Kroos et al, 2012). Following the recognition of the mannose-6-phosphate pathway's role in lysosomal enzyme trafficking and the availability of new technologies for the purification and manufacturing of lysosomal enzymes, the early 1990s inaugurated the first attempts to treat these disorders by replacing the defective enzyme activity (Barton et al, 1990; Barton et al, 1991). At the same time, the introduction of novel technologies had a critical role in the study of LSDs. Techniques for targeted gene disruption and generation of knock-out animal models for human LSDs provided tools to better characterize the pathophysiology of these disorders and to develop innovative therapeutic strategies (Pastores et al, 2013). Mass spectrometry technology allowed for lysosomal proteome analyses (e.g., mannose-6-phosphate glycoproteome) (Sleat et al, 2005) that led to the identification of lysosomal proteins and novel molecular bases of some LSDs. In the past decade, a number of studies have expanded our knowledge of lysosomal biology and provided new and important insights on LSD pathophysiology. These studies identified the lysosomes as highly dynamic organelles involved in multiple cellular functions, including signaling, and able to adapt to environmental stimuli. (Settembre et al, 2013; Perera & Zoncu, 2016; Ballabio & Bonifacino, 2020). Significant advancements have also facilitated greater understanding on the molecular and metabolic mechanisms underlying LSDs and the development of new therapeutic strategies for these diseases (Parenti et al, 2015; Platt et al, 2018; Ren & Wang, 2020). This review will focus on new tools and technologies to study LSDs, on emerging aspects of lysosomal biology, and on recent discoveries on the cellular and organismal consequences of lysosomal dysfunction. The biology of lysosomes, old concepts and new views Lysosomes are membrane-limited, ubiquitous, intracellular organelles involved in multiple cellular processes (Saftig & Klumperman, 2009; Ballabio & Bonifacino, 2020). More than two hundred lysosomal-resident proteins contribute to the biology and function of these organelles. Approximately 60 of them are acidic hydrolases (Lubke et al, 2009; Schroder et al, 2010). Most of them act as exoglycosidases or sulfatases and are localized to the lysosomal lumen. The others are localized at the lysosomal membrane and have multiple functions such as formation of a glycocalyx-like layer, transport across the membrane, acidification, membrane stability, and mediating interaction between lysosomes and other cellular structures (Saftig & Klumperman, 2009; Ballabio & Bonifacino, 2020). In addition to lysosomal-resident proteins, other proteins interact with the lysosome and participate to lysosomal function by being dynamically recruited to the lysosomal surface under certain conditions, for example, the transcription factor EB (TFEB), the mechanistic target of rapamycin complex 1 (mTORC1) (Perera & Zoncu, 2016; Ballabio & Bonifacino, 2020; Yim & Mizushima, 2020), the mTORC1 regulator tuberous sclerosis complex (TSC) (Dibble & Cantley, 2015), folliculin (FLCN) and FLCN-interacting protein (FNIP) (Lawrence et al, 2019), the energy-sensing complex AMP-activated kinase (AMPK) (Zhang et al, 2014), and the signal transducer and activator of transcription-3 (STAT3) (Liu et al, 2018). The first function of normal lysosomes to be recognized is turnover of cellular constituents. Lysosomes are involved in the degradation of a broad variety of structurally diverse compounds, such as proteins, glycosaminoglycans, sphingolipids, oligosaccharides, glycogen, nucleic acids, and complex lipids. Cellular and extracellular materials and substrates destined for degradation reach lysosomes through different routes (endocytosis, phagocytosis, autophagy), or by direct transport. In this respect, lysosomes are part of a more complex pathway, referred to as the autophagy–lysosomal pathway (ALP). Autophagy plays a crucial role in cell homeostasis by controlling intracellular clearance and recycling of a variety of molecules and cellular components and also by sustaining cellular energy metabolism. Autophagy is a multistep pathway that involves autophagosome formation, cargo recruitment, and autophagosome–lysosome fusion. Importantly, autophagic function is entirely dependent on the ability of the lysosome to degrade and recycle autophagy substrates (Yim & Mizushima, 2020). Much attention has been paid in recent years to the nutrient-sensing function of lysosomes. Lysosomes are able to monitor the nutrient status of cells and to adjust their metabolism to changing energetic conditions. When nutrients are available mTORC1 is dynamically recruited to the lysosomal surface where it becomes active and promotes cellular anabolic processes (Saxton & Sabatini, 2017). Recent studies have provided compelling evidence that lysosomal biogenesis and autophagy are controlled by the master transcriptional regulator transcription factor EB (TFEB) (Sardiello et al, 2009; Settembre et al, 2011). In addition to TFEB, another member of the MiT-TFE family of transcription factors, TFE3, has a partially redundant function and is regulated in a similar manner (Martina et al, 2014; Raben & Puertollano, 2016). TFEB subcellular localization and function is regulated by nutrient-induced mTORC1-mediated phosphorylation of specific serine residues (Settembre et al, 2012; Roczniak- Ferguson et al, 2012; Martina et al, 2012). On phosphorylation by mTORC1, TFEB is retained in the cytoplasm. A variety of stimuli leading to mTORC1 inactivation, such as starvation, induce TFEB dephosphorylation and nuclear translocation. Thus, the mTORC1-TFEB regulatory axis enables lysosomes to adapt their function to environmental cues, such as nutrient availability (Ballabio & Bonifacino, 2020). Several additional conditions, such as infection, inflammation, physical exercise, endoplasmic reticulum stress, and mitochondrial damage, also promote TFEB nuclear translocation, highlighting the complexity of TFEB regulation (reviewed in Puertollano et al, 2018; Cinque et al, 2015). Recent findings have added further complexity to the mechanisms by which mTORC1 phosphorylates TFEB (Napolitano et al, 2020). Unlike other substrates of mTORC1, TFEB is known to interact with RagGTPases (Martina & Puertollano, 2013). Due to this interaction, TFEB phosphorylation occurs through an mTORC1 substrate-specific mechanism that is strictly dependent on the amino acid-induced activation of RagC and RagD GTPases but is insensitive to Rheb activity induced by growth factors. This allows mTORC1 activity to be differentially regulated by different stimuli (Napolitano et al, 2020). This substrate-specific regulation of TFEB by the mTORC1 pathway has a crucial role in Birt–Hogg–Dubé syndrome, a disorder characterized by benign skin tumors, lung, and kidney cysts and renal cell carcinoma (Kauffman et al, 2014; Calcagnì, et al, 2016) and caused by mutations in the lysosomal RagC/D activator folliculin (FLCN) (Napolitano et al, 2020). Lysosomes are emerging as calcium (Ca2+) storage organelles. The concentration of free Ca2+ within the lysosome is around 500 µM, and therefore comparable to endoplasmic reticulum (ER) Ca2+ levels (Christensen et al, 2002). Ca2+ channels, such as transient receptor potential mucolipin-1 (TRPML-1, Mucolipin 1, MCOLN1) and the two-pore channel (TPC), reside on the lysosomal membrane and have been shown to mediate local Ca2+ signals from intracellular compartments (e.g., mitochondria) (Xu et al, 2015; Xu & Ren, 2015). Lysosomal Ca2+ signaling participates in multiple cellular processes such as lysosomal acidification, the fusion of lysosomes with other cellular organelles, membrane trafficking and repair, autophagy, and formation of contact sites between the lysosome and the endoplasmic reticulum (Kilpatrick et al, 2013; Lloyd-Evans & Waller-Evans, 2019). Furthermore, lysosomal Ca2+ signaling is involved in the regulation of lysosomal biogenesis and autophagy through the activation of TFEB. Upon starvation, lysosomal Ca2+ release through TRPML1 activates the Ca2+-dependent serine/threonine phosphatase calcineurin (CaN), which binds and dephosphorylates TFEB, thus promoting its nuclear translocation (Medina et al, 2015). TRPML1 also induces autophagic vesicle biogenesis through the generation of phosphatidylinositol 3-phosphate (PI3P) and the recruitment of essential PI3P-binding proteins to the nascent phagophore in a TFEB-independent manner (Scotto-Rosato et al, 2019). The nosography of LSDs LSDs are multisystem disorders that are associated with a broad range of clinical manifestations affecting multiple organs and systems and causing visceral, ocular, hematologic, skeletal, and neurological signs. These manifestations are often highly debilitating, causing progressive physical and neurological disabilities. In general, LSD presentations show broad variability (Beck, 2001), ranging from early-onset (in some cases neonatal), severe clinical forms that often result in premature death of patients, to late-onset, attenuated phenotypes that have a lesser impact on patient health and lifespan. Albeit individually rare, their cumulative incidence is estimated in approximately 1 in 5,000–7,500 births, with higher rates in specific populations. It is noteworthy that newborn screening programs for LSDs, now active in some countries, may in the future significantly change these estimates and will likely provide a more precise figure of LSD incidence (Spada et al, 2006; Hopkins et al, 2018; Wasserstein et al, 2019). The nosography of LSDs has evolved over time, reflecting the advancements in the knowledge of lysosomal function and the cellular consequences of its dysfunction. The traditional classification based on the classes of stored substrates (glycosaminoglycans in the mucopolysaccharidoses, glycosphingolipids in the glycosphingolipidoses, glycoproteins in the oligosaccharidosis, etc) largely reflects the vision of lysosomes as catabolic organelles and is centered on the disease biochemistry. Accurate and exhaustive information on LSD classification and nosography can be found elsewhere (Platt et al, 2018). With the improved knowledge on the molecular and cellular bases of LSDs, an alternative way of classifying LSDs has been proposed, based on the process that is defective in the biogenesis of lysosomal enzymes, rather than on the stored substrate (Platt, 2018). The majority of these disorders is due to deficiencies of soluble hydrolases that are involved in the sequential degradation of a specific substrate. Other disorders are due to deficiencies in upstream processes, such as post-translational modifications (multiple sulfatase deficiency, MSD, due to the lack of an enzyme converting a cysteine into a formylglycine residue in the catalytic site of sulfatases) (Cosma et al, 2002; Dierks et al, 2002), or to defective sorting of lysosomal enzymes to lysosomes (mucolipidoses—ML—II and III, with deficient generation of mannose-6-phosphate) (Hickman & Neufeld, 1972). Others are due to mutations of non-enzymatic activator proteins (saposin activator protein, SAP, deficiencies) (Tamargo et al, 2012), of solute carriers (cystinosis, infantile sialic acid storage disease) (Gahl et al, 1982; Verheijen et al, 1999) and other lysosomal membrane proteins (Danon disease, due to LAMP2 defective function) (Nishino et al, 2000; Tanaka et al, 2000), or are the consequence of defects in assembly and stability of multienzymatic complexes (galactosialidosis, due to cathepsin A deficiency) (d’Azzo et al, 1982). New technologies and cellular modeling to study lysosomal function in health and disease Novel technologies have had significant impacts on the characterization of lysosome biology, the development of diagnostic tools for patients with a suspicion of LSD, and the identification and validation of new therapeutic targets (Fig 2) (Table 1). In several cases, these approaches are based on high-throughput techniques combined with bioinformatic analysis of a large body of information (metabolomic, genomic, proteomic approaches). Novel approaches also include automated robotic-based, miniaturized or cell-based procedures (high-content imaging) as well as innovative techniques that allow for manipulation of genetic information and generation of in vitro and in vivo models of disease. Figure 2. New technologies to study lysosomal function and biology New technologies, in most cases based on high-throughput techniques combined with bioinformatic analysis, have been exploited for the diagnostic work-up in patients with a suspected LSD, for the identification of new disease genes, for the search of disease biomarkers, for the characterization of lysosome biology and disease mechanisms, and for the identification and validation of new therapeutic targets. Download figure Download PowerPoint Table 1. Examples of application of novel technologies for LSDs. Technology Applications Examples of successful applications Genomic sequencing Diagnosis of LSD patients and identification of mutations of known genes Identification of mutations in genes not associated with LSD Identification of new LSDs caused by mutation of the VPS33A (Pavlova et al, 2019) and VPS16 (Steel et al, 2020) genes Transcriptomic analysis Information on pathways involved in disease pathophysiology Response to environmental conditions /pharmacological manipulations Similarities between the microglia expression profiles of LSDs (mucolipidosis type IV mouse and Niemann-Pick disease type C1) with common neurodegenerative disorders (Cougnoux et al, 2019) Genome-wide association studies Identification of modifying factors Information on disease pathophysiology Identification of a c.510C > T variant that may be predictive of clinical course and outcome in late-onset Pompe disease patients (Bergsma et al, 2019) microRNA sequencing Identification of disease biomarkers that correlate with disease severity and assist in monitoring disease progression and efficacy of therapies Identification of pathways involved in disease pathophysiology Identification of differentially expressed microRNAs potentially predictive of disease severity in Pompe disease (Tarallo et al, 2019) Biochemical and metabolomic analyses Support and validation of diagnosis Identification of disease biomarkers that correlate with disease severity, monitoring disease progression, monitoring efficacy of therapies Newborn screening Identification of pathways involved in disease pathophysiology Identification of disease biomarkers for several LSDs (Boutin & Auray-Blais, 2015; Reunert et al, 2015; Polo et al, 2019) Development of methods for simultaneous detection of multiple enzyme activities in dried blood spots suitable for newborn screening programs for several LSDs (Anderson, 2018; Donati et al, 2018; Kumar et al, 2019; Lukacs et al, 2019; Scott et al, 2020) Cell-based assays and high-content imaging technologies Identification of pathways involved in disease pathophysiology Screening for correctors and therapeutic agents Development of multiplex staining assays that allow screening of FDA-approved compounds and identification of correctors for cellular phenotypes of LSDs (Pipalia et al, 2006; Pugach et al, 2018) Targeted gene knock-out and genome editing—iPSc Identification of pathways involved in disease pathophysiology Screening and validation of therapeutic agents Gene editing of mutant genes to correct disease-causing mutations CRISPR-Cas9-mediated generation of knock-out models of LSDs, such as sphingolipidoses and Niemann-Pick disease type C (Santos & Amaral, 2019) Organellar omics Information on lysosome biology Identification of pathways involved in disease pathophysiology Identification of disease biomarkers for correlations with disease severity, monitoring disease progression, monitoring efficacy of therapies Identification of lysosomal proteome and interactome (Sleat et al, 2005; Abu-Remaileh et al, 2017; Thelen et al, 2017; Rabanal-Ruiz & Korolchuk, 2018) Genomic techniques and next-generation sequencing (NGS) A major advancement, particularly in the diagnostic approach to LSDs, was introduced by NGS, a powerful diagnostic tool based on technological platforms that allow sequencing of millions of small fragments of DNA in parallel. This technology has been used both through a targeted strategy with gene panels and by untargeted approaches based on whole-exome sequencing. Given the difficulties in the diagnostic work-up, and due to overlapping clinical phenotypes in LSDs, patients with a clinical suspicion of these disorders are excellent candidates for the application of an NGS-based diagnosis. Before the introduction of NGS, the traditional approach for the diagnosis of LSDs was based on a step-by-step, progressive process starting with physical examination, proceeding to metabolite identification in biological fluids, and leading to the exact diagnosis through the demonstration of an enzymatic deficiency (or the deficiency of a lysosomal function) and the identification of mutations in a specific gene (Winchester, 2014). NGS-based approaches are substantially changing this stepwise process. The molecular analysis and search for mutations in LSD-related genes can be performed immediately after the clinical suspicion of LSD, whereas the functional analysis of the deficient enzyme (or a non-enzymatic protein) offers a complementary approach to definitively confirm disease diagnoses. Such a diagnostic process may prove to be cost-effective and by-pass the need for multiple biochemical analyses or repeated hospital admissions. Some examples of this strategy are already available in the literature, with the development of a gene panel specific for 891 genes involved in the ALP function (Di Fruscio et al, 2015), or the identification of Pompe disease patients in cohorts of unidentified limb-girdle muscular dystrophies (Savarese et al, 2018). NGS-based analysis also has the potential to identify new genes involved in lysosomal disorders, thus expanding the list of LSDs. Indeed, two novel disorders associated with lysosomal abnormalities and impaired vesicle trafficking were recently recognized through whole-exome sequencing. Mucopolysaccharidosis plus (MPS-plus), characterized by typical manifestations of mucopolysaccharidoses such as coarse facial features, skeletal abnormalities, hepatosplenomegaly, respiratory problems, and by remarkable levels of glycosaminoglycans excretion in urines (Kondo et al, 2017), was associated with mutations in the VPS33A gene (Pavlova et al, 2019). Loss-of-function VPS16 gene mutations were found in patients with an early-onset dystonia and with ultrastructural lysosomal abnormalities (Steel et al, 2020). Both VPS33A and VPS16 genes encode for subunits of the homotypic fusion and vacuole protein sorting (HOPS) complex that is essential for lysosome fusion with endosomes and autophagosomes (Wartosch et al, 2015). Genomic approaches may also contribute to the understanding of the complexity and clinical variability of LSDs. The application of these approaches to the study of LSDs is a vast and rapidly expanding field that has been comprehensively reviewed in recent years (Hassan et al, 2017; Davidson et al, 2018). Genome-wide association studies have been performed to identify modifiers in some LSDs, for example, Gaucher disease and Pompe disease. In Gaucher disease, single nucleotide polymorphisms (SNPs) within the CLN8 gene locus were in linkage disequilibrium and associated with disease severity, possibly regulating sphingolipid sensing and/or in glycosphingolipid trafficking (Zhang et al, 2012). In Pompe disease, a c.510C > T variant was identified as a genetic modifier in late-onset patients. This variant negatively influences pre-mRNA splicing in patients carrying the c.-32-13T > G mutation, with significant correlations with residual alpha-glucosidase activity and may be predictive of clinical course and outcome in late-onset patients (Bergsma et al, 2019). Transcriptomic analysis has been performed in several types of LSDs, such as Niemann-Pick disease type C (Martin et al, 2019), mucopolysaccharidoses (Salvalaio et al, 2017; Peck et al, 2019), progranulin deficiency (Evers et al, 2017), Pompe disease (Turner et al, 2016), and Gaucher disease (Dasgupta et al, 2013), mucolipidosis type IV (Cougnoux et al, 2019). Although the animal models and the tissues differed in these analyses, it was possible to recognize a few common patterns in some diseases. In mucolipidosis