Lysosomal Storage Diseases, Focusing On Gaucher Disease: Perspectives And Principles

Lysosomal Storage Diseases

Lysosomal storage diseases (LSDs) are genetic disorders caused by deficiencies in single lysosomal hydrolases. Deficiencies result in cellular and organ damage due to subsequent accumulation of a specific substrate for that particular enzyme, hence the term “storage” disease. However, the diverse role of the lysosome in cellular metabolism means that the pathological consequences of enzyme deficiency extend beyond substrate accumulation.

Although individually rare, the true prevalence of these disorders is likely to be much higher. Overall, 60 or so monogenic disorders have a combined frequency of approximately 1:7000 live births, and are probably increased due to consanguinity rates among different populations. All are autosomally inherited except for Fabry, Danon and mucopolysaccharidoses II (MPS II), which are sex-linked. The classification is in accordance with the biochemical nature of the primary storage material and pathogenic mechanisms ( Table 54.1 ).

Hers was the first to describe enlarged and abnormally shaped lysosomes resulting from primary accumulation of storage material in a patient with Pompe disease (α-glucosidase deficiency), thus delineating the first LSD. Many LSDs have an early age of onset, associated with genetic mutations which lead to absent, or very low, enzyme levels and a correspondingly severe clinical course. However, several also have an attenuated form with later age of onset, genetic mutations associated with reduced or mutant enzyme production and reduced (but not absent) catalytic activity. LSDs are characterized by broad systemic involvement of multiple tissues and organs (typically involving bone, viscera, connective tissue, skin, kidney, and heart). Crucially, the majority have extensive neurological involvement manifesting as intellectual disability, dementia, seizures, movement disorders and sensory organ abnormalities.

Accumulation of storage material compromises the functional capacity of the lysosome and initiates a cascade of secondary downstream pathologic events. These include impaired autophagy (see Chapter 10 ), apoptosis and signal transduction, which precipitate cellular death and neurodegeneration. There is deficient supply, recycling and transport of key membrane and intracellular receptors, and macromolecules leading to synaptic failure and energy depletion. Cellular damage induces inflammation as an initial protective response but chronic induction is deleterious. Abnormal calcium homeostasis disturbs intracellular fusional and signaling events, and alters the potential of hydrogen (pH) to cause trafficking defects affecting storage and egress of cholesterol, sphingomyelin, and glycosphingolipids (GSLs). Macrophage activation, possibly mediated by cytokine release, has been demonstrated in Gaucher disease (GD) and other LSDs. Accumulation of lysosphingolipids (i.e., GSLs that do not have N-acylated fatty acids) may be of pathogenic significance in Krabbe, Gaucher, and Fabry diseases.

The biochemical composition of the accumulated macromolecules distinguishes the main categories of LSD: the glycosphingolipidoses (GPS), the mucopolysaccharidoses (MPS) and carbohydrate storage diseases. The pattern of storage of the accumulated lipid substrate in LSDs often mirrors the normal distribution of the molecule in the body, giving rise to organ-specific pathology. Thus, accumulation of GSLs in the central nervous system (CNS) leads to neurodegeneration, whereas storage in visceral cells leads to organomegaly, skeletal abnormalities, pulmonary infiltration, and other manifestations. In the MPS disorders, the accumulating lipid substrates (also known as glycosaminoglycans [GAGs]) are found predominantly within connective tissues to cause cartilage and bone abnormalities. The CNS may also be affected, particularly in diseases (e.g., MPS type I) where the GAG heparan sulfate accumulates. In contrast, in Maroteaux-Lamy disease (MPS type VI), only dermatan sulfate, which is not usually found in the brain, accumulates, and CNS manifestations do not occur; while in MPS IV, the accumulation of keratan sulfate leads to a predominant skeletal phenotype. (See box on Illustrative Mucopolysaccharidoses Case History.)

Lysosomal Protein Biosynthesis and Sorting

All lysosomal enzymes are translated on the endoplasmic reticulum (ER)-bound polyribosomes and contain a leader sequence. They enter the ER through the ER-specific translocons, and then lose the leader sequence. During translation the protein sequence, already within the ER is glycosylated on asparagine residues, which are part of the consensus sequence: Asparagine-X-Serine/Threonine (N-X-S/T; “X”

can be any amino-acid except proline). These N-linked glycan trees (dubbed N-glycans) consist of nine mannose residues and three glucose residues. The enzymes are then folded, and, if recognized by the ER quality control machinery as correctly folded, they exit the ER in specific trafficking vesicles (called COPII vesicles) toward the Golgi network. They undergo further modifications on their N-linked glycan trees, which lose the glucose residues and three to four mannose residues. Most lysosomal enzymes are phosphorylated by the enzyme N-acetylglucosaminyl-1-phosphotransferase on a terminal mannose and as such are recognized by the mannose 6-phosphate receptor, which shuttles them, on specific late endosomal vesicles to the lysosomes. Mutations in the gene encoding this enzyme lead to severe LSD (I-cell disease), characterized by the abnormal secretion of all lysosomal enzymes whose lysosomal targeting depend on the M6P recognition signal.

If recognized as misfolded by the ER quality control machinery, lysosomal proteins undergo several folding attempts. If unsuccessful, the misfolded protein is translocated from the ER to undergo polyubiquitination and proteasomal degradation. This is known as ER-associated degradation (ERAD; Fig. 54.1 ). Persistent retention of misfolded molecules in the ER provokes ER stress and induces the Unfolded Protein Response, known as UPR. UPR monitors the conditions in the ER by sensing insufficiency in protein folding capacity and translating this information into gene expression by a signal transduction.

Several lysosomal enzymes and proteins (including the hexosaminidase A alpha and beta chains, mutated in Tay-Sachs disease and in Sandhoff disease, respectively) undergo proteolytic cleavage in the lysosomes as the last stage of their maturation process.

A large number of mutant lysosomal enzymes undergo ERAD and, therefore, lysosomal diseases may be regarded as misfolding or conformational diseases.

Genetics of Lysosomal Storage Diseases

Lysosomal genes are regarded as “housekeeping,” that is, they are expressed in the majority of cell types and are essential for cellular function, such that complete absence of most of the enzymes is incompatible with life; Fabry disease is an important exception. All LSDs are inherited in an autosomal recessive fashion except for Fabry, MPS II and Danon, which are X-linked. One lysosomal gene, SMPD1 encoding acid sphingomyelinase (ASM), is known to be “paternally imprinted” (i.e., preferentially expressed from the maternal chromosome). Diverse mutations underly individual LSDs, and the majority of these are unique (“private”) to individual pedigrees. Broad genotype-phenotype relationships have been established. GD is a good example—major organizational disruptions of the gene are associated with a more severe phenotype.

In general, heterozygous “carriers” of single mutations in a lysosomal gene do not develop clinical symptoms of the disorder, except in the X-linked disorders. For example, in Fabry disease X-inactivation patterns can lead to clusters of cells without enzyme activity, and heterozygous females often develop disease-related pathology primarily as a result of unbalanced X chromosome inactivation. Heterozygous carriers of glucocerebrosidase gene ( GBA1 ) mutations have a much increased risk of developing Parkinson disease (PD).

Some LSD-specific populations have recurrent mutations caused by founder effects and/or consanguinity. This facilitates the use of DNA-based screening methods for their detection. This has been translated into clinical use in the Ashkenazi Jewish population and has led to the establishment of a DNA-based “Jewish Genetic Disease” screening panel. Evolving cost-effective, high-throughput sequencing methods are likely to open other populations and disorders to these screening approaches. However, DNA-based methods will generate large numbers of GVUS (genetic variants of uncertain significance) and should not be used to predict clinical outcomes in patients unless the biochemical consequences of the mutations are fully established. An example of a mutation which was formerly a GVUS, but has recently been demonstrated to undoubtedly be associated with late-onset hypertophic cardiomyopathy in Fabry disease, is c.472 G>A; however, detailed studies have shown that although this variant is a necessary requirement for Fabry cardiomyopathy in affected individuals, it is not sufficient and other disease mechanisms are important for disease manifestations to develop.

Diagnosis of Lysosomal Storage Diseases

The key to diagnosis is clinical suspicion of the disease. Clinical and laboratory diagnosis should be coordinated—once there is clinical suspicion, laboratory diagnosis is usually straightforward. These are multi-system disorders with few signature manifestations which occur among all ethnicities. The carrier rate among Ashkenazi Jews is about 1 in 15 for GD, 1 in 30 for Tay-Sachs, and 1 in 80 to 100 for Niemann-Pick type A.

Accurate clinical history and physical examination are crucial. The history must include a detailed family history and inquiry about consanguinity within the pedigree. Delay in diagnosis is a common problem and often leads to inappropriate investigations, delay in instituting specific treatment, and avoidable complications. A particular pitfall in diagnosis for pediatric and adult hematologists is that LSDs may have features that are shared by hematologic conditions (e.g., anemia, thrombocytopenia, hepatosplenomegaly, bone lesions). A survey of hematologists in the USA who have a clinical history of classic type 1 GD revealed that the overwhelming majority were initially considered to have a hematologic malignancy. The majority of Gaucher patients make their initial presentation to a hematologist and report a delay in diagnosis.

Screening Programs

Screening programs aim to reduce diagnostic delay by institution of coordinated community diagnosis. Newborn screening (NBS) is already performed worldwide for certain metabolic diseases where early intervention is widely available and effective at preventing long term complications (e.g., dried blood spot [DBS] tests for phenylketonuria and hypothyroidism). Many states in the USA and countries in Europe and worldwide have embarked on these programs. Most of the laboratories engaged in LSD NBS carry out first-tier testing by measurement of lysosomal enzymatic activities in DBS on NBS cards using either tandem mass spectrometry (MS/MS) or fluorimetry (either digital microfluidics [DMF-F] or a standard plate reader). Confirmatory enzymatic and DNA analysis is performed on positive samples. An emerging problem is that the programs are generally associated with a significant false positive rate. Furthermore, for some conditions (e.g., Fabry) neonatal screening based on gene sequencing has revealed prevalence rates which are far higher than previously suspected (e.g., 1:3200 for Fabry), but the majority of detected variants are of uncertain significance (GVUS), raising important ethical and procedural issues.

Targeted screening is the approach whereby clinical algorithms are used to identify subjects and family members considered to be at increased risk of suffering an LSD who can then be offered accelerated laboratory diagnosis. Such clinical algorithms have been devised for GD based on expert guidance. A multi-national multi-center targeted screening study of 500 subjects with splenomegaly attending hematologists in Europe yielded a diagnosis of GD among 15 subjects. Other examples of successful targeted screening programs include screening for Fabry disease among subjects with left ventricular hypertrophy and premature “cryptogenic” stroke.

Laboratory Diagnosis

DBS samples are increasingly used, replacing anticoagulated whole blood and, in some cases urine, and referred to specialty laboratories so that common pitfalls in laboratory diagnosis are avoided. Full clinical details and family history should be provided. Tissue biopsy is not indicated. Blood film morphology may give important clues (see below). Specific enzyme activity will then be measured in white blood cells, lymphocytes (such as T cells), plasma or DBS. Cultured fibroblasts are required to measure the activity of certain enzymes, for example, neuraminidase. Mass spectrometric assays allow simultaneous analysis of activity of several enzymes from DBS. Positive and negative controls are often included. Deficient activity in a clinically normal individual may also indicate “pseudodeficiency” due to the presence of a polymorphism—for example, as is commonly found with arylsulfatase A. A false-negative result may indicate a laboratory error but could rarely occur due to deficiency of a sphingolipid activator protein (SAP, e.g., the GM-2 activator protein or saposins A–D). Enzymatic activity from a heterozygous female will be normal or only marginally reduced if the condition is X-linked. Confirmatory DNA analysis should always be performed Laboratory diagnosis should also include confirmatory tests, for example, measurement of substrate level and relevant biomarkers.

Baseline and Sequential Assessment

A genetic counselor can help with the interpretation of the results, the organization of pedigree analysis, and guidance with respect to treatment. A biomarker is a laboratory analyte that reflects the presence and/or extent of a biological process, and should be directly linked to the clinical manifestations and treatment outcome. Quantifiable biomarkers, which are specific, sensitive, reliable, and reproducible, are of great value in the management of several LSDs.

The diversity of clinical manifestations means that multidisciplinary clinical assessment should be conducted to document structure, function, and patient-reported elements at diagnosis and at specified time intervals thereafter, irrespective of whether or not specific treatments are applied. Specific therapy should improve the disease manifestations, yet in many LSDs the impact of the intervention is much more limited, and may only amount to structural or functional reduction in the natural rate of decline. Reliable data on untreated individuals is crucial. Regulatory and funding authorities increasingly regard patient-reported outcome (PRO) as good measures reflecting the quality of life (QOL) and the overlay of psychological, social, societal concerns and stressors which are unique to LSD patients.

Specific Treatment

Cellular Therapy, Including Gene Therapy

Cell therapy is aimed at engraftment of cells (autologous or allogeneic) which can produce endogenous enzymes (see Chapter 103 ). Advantages of hemopoietic stem cells (HSCs) include their potential to self-propagate and to differentiate into multiple stem and progenitor cell lineages, which could potentially repopulate diverse organs and tissues. Furthermore, the application of this technology in subjects with non-malignant metabolic diseases does not impose a need to eliminate host hematopoiesis; and advances in molecular techniques increasingly offer ways of bioengineering the grafted cells. However, intensive immunosuppressive pre-conditioning with chemotherapeutic agents, with its associated high morbidity and potential mortality, is still required to achieve effective engraftment; and an allogeneic donor raises the specter of graft-versus-host disease. Donor availability is improved with the availability of cord blood banks, and haploidentical and even sibling carrier donors; but despite improved transplantation techniques which have reduced the risks of stem cell transplantation, the possibility of confounding the Hippocratic injunction to “do no harm” remains high.

Results of hematopoietic stem-cell transplantation (HSCT) for LSDs have been variable. The capacity of HSCs to repopulate different tissues is not uniform throughout the body, and is limited with respect to the skeleton (cartilage and bone) and the CNS. Permanent tissue damage is not reversible and transplantation is best undertaken early in the course of the disease. Liver transplantation has offered interesting perspectives which may have lessons for future gene therapy strategies using adenoviral constructs which infect hepatocytes. The procedure has been undertaken to treat end-stage cirrhosis complicating GD and long-term results show excellent recovery of liver function and continued absence of substrate accumulation in the liver. However, skeletal remodeling does not occur, and subjects continue to require ERT to treat systemic disease outside the liver. Ex vivo gene therapy approaches (see Chapter 104 ) for a range of LSDs are being investigated whereby the genetic construct is introduced into autologous HSCs which are then transplanted. A pilot study (prematurely closed to recruitment in January 2022) has reported autologous infusion of lentivirus transduced HSCs in five male patients with Fabry disease with up to 3 years of follow-up; all patients produced α-gal A to near normal levels within one week and three patients have discontinued ERT. However, safety concerns have led to discontinuation of the trial in January 2022. (Also see box on Fabry Disease: Specific Treatment).

The long-term results of this and other studies are awaited with interest, amid continuing concern about future risk of side-effects related to the chemotherapeutic conditioning regimen. Exciting developments have also been reported with regard to the potential for cellular therapy with induced neural stem cells.

Small Molecule Therapy

Small molecules have important advantages over ERT. They can be taken orally, they have the potential to cross the BBB and access the CNS, antibody formation is not anticipated, production costs are lower, and small molecules are likely to have a higher volume of distribution in the body and to thereby access tissues such as bone, kidney, lungs, and the myocardium.

Two important approaches are SRT and PC. Eliglustat (see below) is established as an important alternative to ERT for many patients with type 1 GD. Neurologic pathology in Niemann-Pick disease

(NPD), more specifically NPD type C (NPDC), is associated with the secondary accumulation of a particular glycolipid, GM2 ganglioside. Miglustat inhibits glycolipid biosynthesis and results in reduction of GM2 storage and partial neurologic improvements.

Pharmacological chaperones (PCs) are usually specific inhibitors of the corresponding misfolded mutant proteins, and facilitate their delivery to lysosomes by enhancing residual activity; they are currently undergoing clinical trials for several LSDs. Migalastat is licensed for use in Fabry patients with “amenable” mutations. It is a PC which stabilizes and facilitates trafficking of amenable mutant forms of alpha galactosidase A from the endoplasmic reticulum to lysosomes and increases its lysosomal activity. Oral migalastat is the first pharmacological chaperone approved for treating patients ≥ 18 years in the US and Canada or ≥ 16 years in other countries with Fabry disease who have a migalastat-amenable alpha galactosidase A mutation. In patients with migalastat-amenable alpha galactosidase A mutations, relative to placebo, migalastat treatment significantly reduced the mean number of GL-3 inclusions/KIC and plasma lyso-globotriaosylsphingosine levels at 6 months. Among evaluable patients, migalastat maintained renal function and reduced cardiac mass after ≤24 months’ therapy. Renal function was maintained during 18 months of migalastat or ERT; however, migalastat significantly reduced cardiac mass compared with ERT. Migalastat is generally well tolerated. Given that it can be administered orally and that limited therapeutic options are available, migalastat is an important treatment option for Fabry disease in patients with migalastat-amenable alpha galactosidase A mutations. Numerous other small molecule approaches are being evaluated in the various LSD animal models, including targeting inflammatory pathways and stem cell-based therapies. A combination of approaches, targeted at individual diseases and organs, may well be optimal.

General Aspects of Treatment

The advent of treatment for LSDs is a recent phenomenon. Specialty centers are best able to assess patients and families in a multidisciplinary fashion. Each patient should have a designated “lead” specialist, who not only provides specialist input into their own discipline (e.g., a hematologist working within a center for GD; pediatricians trained in metabolic medicine), but will also coordinate care across a team of specialist physicians and health care professionals. Centers of excellence focus on providing holistic care, not only to the index patient, but to families and care providers. Specialty centers should work alongside patient advocacy groups and the pharmaceutical industry to organize active research programs, extending beyond the clinical trials. It is a mandatory requirement for manufacturers of many LSD drugs to provide post-marketing information regarding the long-term safety and efficacy of their products, and this is often done through Registries.