A cura di Michele Marra e Matilde Ghibaudi
The mucopolysaccharidoses (MPSs) are a group of rare genetic disorders of glycosaminoglycan (GAG) catabolism that affect manly children. Nine types of MPS are classified; each one is caused by a deficiency in the activity of a single, specific lysosomal enzyme required for GAG degradation (Fig. 1). These diseases are biochemically characterized by an accumulation of partially degraded GAG within lysosomes and the elevation of GAG fragment in urine, blood and cerebral spinal fluid (Fig. 2). The GAG accumulation results in progressive cellular damage, which can affect multiple organ systems and lead to organ failure, cognitive impairment, and reduced life expectancy. In addition, skeletal and joint abnormalities are a prominent feature of many of the MPS disorders; patients often present with skeletal dysplasia, decreased joint mobility, short stature. With the exception of the MPSII, the MPS disorders are inherited in a autosomal recessive pattern and affect both males and females equally. Instead, MPSII is a X-linked recessive disorder that generally affect only males, although rare female patients with MPSII have been described. This can be caused by X-autosome translocation and non-random X-chromosome inactivation in a carrier female. More than 50 metabolic storage diseases are currently known, caused by genetic blocks in the breakdown of macromolecules in the lysosomes. These diseases comprise ~14% of all inherited diseases of metabolism and, although individually rare, together affect one out of every 7.700 newborn children.
Overview of the mucopolysaccharidoses,2011
Pathophysiology of the MPS disorders:
The MPSs are chronic and progressive syndromes that produce a spectrum of signs and symptoms in multiple organ systems. MPS diseases are due to a deficiency of different lysosomal enzymes activity but have similar clinical features. For example, MPS I,II and VII present both somatic and cognitive involvement. Skeletal abnormalities result in profound loss of joint range of motion, restricted mobility, growth slowing or arrest in childhood and short stature. Hand and wrist involvement are also common and include decreased wrist range of motion, stiffening of the IP joint and curved finger. These abnormalities cause the hands to take on a claw-like appearance and can result in loss of hand function. Other signs and symptoms include coarse facial features, vision loss, hearing loss, decreased pulmonary function and obstructive sleep apnoea, frequent and recurrent respiratory infections, cardiac disease, hepatomegaly and splenomegaly, umbilical and inguinal hernias, chronic diarrhoea, carpal tunnel syndrome, communicating hydrocephalus and spinal cord compression. Unique among the MPS disorders, MPS II patients may have a distinctive skin lesion (pebbling), which is described as ivory-white papules that are 2-10mm in diameter, often coalescing to form ridges (Fig. 3).
In addition to the somatic manifestations, children with severe MPS I, II or VII have extensive cognitive impairment, characterized early in the disease course by globally delayed developmental milestones and a plateau in development, followed by progressive and inexorable regression. Instead, MPS IV and VI have no primary cognitive involvement; however, the somatic manifestations are similar to those seen with the previous syndromes. MPS IX and III are slightly different. Only four cases of MPS IX have been described in the literature which present, in addition to the previous symptoms , an unusual trait: synovial biopsies were notable for an infiltration of macrophages with abundant cytoplasm filled with faintly basophilic vacuoles. In the end, MPS III are the most common mucopolysaccharidoses. It presents the highest incidence among newborn children and has a dramatic impact on patients family life due to its deep cognitive and neurological implications. MPSIII presents cognitive and neurological impairment with little or no somatic involvement. These disorder may be recognized in childhood by developmental delays, behavioural difficulties, sleep disturbances and dementia. The mental retardation can be profound in patients with severe disease, with a lack of development of social or communicative skills in early childhood. Such patients eventually enter a vegetative state and generally only live into their second or third decade. Because of its strong impact, especially in childhood, MPS III is the most critical and well studied syndrome among all mucopolysaccharidoses.
Mucopolysaccharidosis. Nosology--clinical aspects--therapeutic approaches,1991
Mucopolysaccharidoses type III (MPS III, Sanfilippo syndrome) is the most frequently occurring type of the mucopolysaccharidoses. Birth prevalence of 0.28–4.1 per 100 000 have been reported. However, this may well underestimate the true prevalence owing to the large phenotypic variability and often relatively mild somatic features. To date, the birth prevalence of 1.89 and 1.52, represent the best prediction of the true prevalence, since in the Netherlands and Australia only a few diagnostic centres are involved in the diagnosis of MPS III. Type A is the most common type of MPS III in north-west Europe, while type B is the most frequent type in south-east Europe. Types C and D appear to be much rarer (prevalence)
Sanfilippo sydrome is an autosomal recessive disorder, caused by a deficiency in one of the four enzymes involved in the lysosomal degradation of the glycosaminoglycan heparan sulfate. Based on the enzyme deficiency, four different subtypes, MPS IIIA, B, C, and D, are recognized.
Biochemically, Sanfilippo syndrome is characterized by the accumulation of undegraded heparan sulfate (HS;) in lysosomes and the excretion of this polysaccharide, or fragments derived from it, in body fluids. In MPSIII, HS degradation proceeds in a stepwise fashion by one sulfamidase, one glucosaminidase, one acetyltransferase and a sulfatase (Fig. 4). Sulfamidase (SGSH); heparan N-sulfatase; EC 220.127.116.11), the enzyme deficient in MPS IIIA, releases sulfate groups linked to the amino group of glucosamine. N-Acetyl-a-glucosaminidase (NAGLU; EC 18.104.22.168) hydrolyses the a, 1Y 4 linkage between N-acetylglucosamine and the neighbouring uronic acid and this enzyme is deficient in MPS IIIB. Acetyl CoA: a-glucosaminide N-acetyltransferase (HGSNAT; EC 22.214.171.124) is deficient in MPS IIIC. It catalyses acetylation of the free amino groups of glucosamine that either already exist in HS the N-unsubstituted form or have been produced by sulfamidase. HGSNAT is a transmembrane protein and the only biosynthetic lysosomal enzyme known. Recent studies have proposed a model for HGSNAT function in which the acetyl group of cytoplasmic acetyl-CoA is first transferred to a histidine residue in HGSNAT, followed by the translocation of the acetylated histidine into the lysosome and the transfer of the acetyl moiety to the terminal glucosamine residue of heparan sulfate. Finally, N-acetylglucosamine 6-sulfatase (GNS; EC 126.96.36.199) is required to remove sulfate from the C6-hydroxyl moiety of Nacetylglucosamine residues. This enzyme is deficient in MPS IIID. An enzyme with glucuronate 2 sulfatase activity has been described, which is distinct from iduronate 2-sulfatase since it is normally present in fibroblasts of patients with MPS II. Glucuronate 2-sulfatase deficiency (resulting in a putative Sanfilippo type E syndrome) has not yet been detected. This may be because this assay is performed by few laboratories. Finally, glucosamine 3-sulfatase is also involved in degradation of HS. However, this enzyme has not been characterized nor has the encoding gene been identified.
Overview of the mucopolysaccharidoses,2011
Sanfilippo syndrome: A mini-review,2007
Mutations underlying the different enzyme deficiencies are numerous and an increasing number of mutations are still being reported:
The gene encoding sulfamidase was identified in 1995 and localized to chromosome 17q25.3.
The 502-amino-acid sulfamidase protein contains five potential N-glycosylation sites.
So far, 68 different mutations have been reported, which include 48 missense mutations, four nonsense mutations, one splice site mutation, eight small deletions, and seven small insertions (Fig.5). Molecular characterization of MPS-IIIA patients has identified common mutations in different geographical regions. Establishing a genotype–phenotype correlation in MPS IIIA is complicated by the presence of several polymorphisms. It is not known whether these polymorphisms influence the clinical phenotype by modifying the residual activity of mutant SGSH.
The gene for human a-N-acetylglucosaminidase (NAGLU) was identified in 1996 and localized to chromosome 17q21.1. The enzyme cDNA encodes a 720-amino-acid protein that has six potential N-glycosylation sites. Furthermore, over 100 different mutations have been reported: the largest number are missense mutations, but nonsense mutations, deletions, insertions and splice-site mutations also occur. Allele frequencies of the different mutations are very low and the majority of mutations is unique to individual families so the allelic heterogeneity is assumed to contribute to the wide spectrum of clinical phenotypes in MPS IIIB. As in MPS IIIA, prediction of genotype-phenotype relation in MPS IIIB is complicated by numerous polymorphisms potentially modifying disease severity.
In 2006 the gene encoding enzyme acetyl–CoA : a-glucosaminide N-acetyltransferase (HGSNAT) was cloned and localized to chromosome 8p11.1. In total 43 different mutations have already been reported, including 8 frameshift mutations, 10 splice-site mutations, 19 missense mutations and 6 nonsense mutations. Indeed ,so far, no studies have been reported addressing the effect of detected mutations in the HGSNAT gene locus on the enzyme activity, stability, localization, processing, and ability to interact with other proteins. Such studies, especially important in the case of missense mutations, will allow the clarification of the pathogenic mechanism of MPS IIIC, establish the correlations between the type of mutation and severity of the biochemical and clinical phenotype useful for clinical prognosis, and may also suggest novel therapeutic approaches (Fig. 6). Such studies may also shed light on the molecular structure and function of the enzyme since naturally-occurring mutations are known to cause clinically significant deficiency of the enzyme and therefore appear in functionally important areas. In the vast majority of patients at least one missense mutation affects an amino acid residue in or adjacent to the transmembrane domains of HGSNAT, probably interfering with the proper folding of the enzyme.
The N-acetylglucosamine-6-sulfate sulfatase gene was identified by Robertson and colleagues in 1988. The predicted protein contains 552 amino acids and 13 potential N-glycosylation sites.
Sanfilippo syndrome: A mini-review,2007
Sanfilippo Syndrome Type C: Mutation Spectrum in the Heparan Sulfate Acetyl-CoA: a-Glucosaminide N-Acetyltransferase (HGSNAT) Gene,2008
Molecular Genetics of Mucopolysaccharidosis Type IIIA and IIIB: Diagnostic, Clinical, and Biological
Information on the natural course of Sanfilippo syndrome is limited, since only a few studies have been performed addressing this subject. This lack of knowledge, in combination with the extreme heterogeneity found in MPS III patients, even within sibships, makes prediction of the clinical course very difficult in individual patients.
It is frequently assumed that the four MPS III subtypes are clinically indistinguishable. However, the main clinical features have recently been described.
The probable diagnosis of all MPS III subtypes is based on increased concentration of heparan sulfate in the urine. The clinical course of MPS III can be divided into three phases. In the first phase, which usually starts between 1 and 4 years of age, a developmental delay becomes apparent after an initial normal development during the first 1–2 years of life. The second phase generally starts around 3–4 years and is characterized by severe behavioural problems and progressive mental deterioration ultimately leading to severe dementia. In the third and final stage, behavioural problems slowly disappear, but motor retardation with swallowing difficulties and spasticity emerge. Patients usually die at the end of the second or beginning of the third decade of life, although survival into the fourth decade has been reported.
Biochemical diagnosis of mucopolysaccharidoses including Sanfilippo syndrome starts with quantitation of GAGs excretion in urine. Several methods have been described for measuring GAGs, ranging from a semiquantitative spot tests to precise quantitative measurements. Currently the most frequently used assay is the DMB test. Other methods have lower sensitivity and specificity than the DMB test (e.g. turbidity tests, Alcian blue test, spot tests) or are complex (e.g. determination of uronic acids using carbazol). However, the sensitivity of the DMB test alone in detecting Sanfilippo patients may be less than 100%.Therefore, a combination of the DMB test with qualitative electrophoretic separation of GAGs is recommended. When excretion of GAGs is elevated, or in cases of high clinical suspicion, a qualitative test, typically electrophoretic separation of GAGs, is performed to establish the type of GAG that accumulate: heparan sulfate and/or dermatan sulfate, or keratan sulfate. Definitive diagnosis of MPS III and classification of the subtype (A, B, C or D) requires enzyme assays in leukocytes or cultured fibroblasts
and testing for heterozygosity should be performed by mutation analysis.
Novel methods for GAG diagnostic in urine:
2)LC-MS-MS 1-phenyl-3-methyl pyrazolon (PMP) derivatised oligosaccharides
3)GAG degradation by bacterial GAG lyases followed by LC-MS-MS of disaccharides
4)GAG degradation by methanolysis follewed by LC-MS-MS of disaccharides
Heparan sulfate levels in mucopolysaccharidoses and mucolipidoses,2005
Validation of disaccharide compositions derived from dermatan sulfate and heparan sulfate in mucopolysaccharidoses and mucolipidoses II and III by tandem mass spectrometry,2010
The two primary treatment modalities are enzyme replacement therapy (ERT) and haematopoietic stem cell transplantation (HSCT), both of which offer substantial benefit but do not cure the disease (Fig. 7). Due to the progressive nature of these diseases, early diagnosis and early therapeutic intervention is of major importance.Today, ERT and HSCT are the standard of care worldwide for certain MPS diseases. The rationale for both treatments is to provide the patient with active enzyme to replace the enzyme that is deficient. In the case of ERT, it is supplied exogenously through regular infusions, and in the case of HSCT the enzyme is supplied endogenously through synthesis by the transplanted stem cells. An important distinction between these two treatments is that HSCT can treat the brain in some MPS disorders, especially if done early in the course of the disease, as stem cells can engraft and differentiate in the CNS. In contrast, infused ERT is too large a protein to cross the bloodbrain barrier easily.
The treatment regimen for ERT involves i.v. infusions of the recombinant human enzyme weekly or every other week. ERT is a life-long therapy, and each infusion takes 14 h depending on the enzyme and the dose. Laronidase was the first ERT approved for treatment of an MPS disorder and has been available in the USA and Europe since 2003. Clinical benefits noted in the drug label include increased distance walked in the 6-min walk test, improved per cent predicted forced vital capacity (FVC), decreased liver volume and decreased (but not normalized) urinary GAG levels. Additional benefits experienced by the majority of patients in the pivotal randomized placebo-controlled trial and extension include stabilized or improved joint range of motion, stabilized or decreased sleep apnoea, decreased left ventricular hypertrophy and improved quality of life. A recombinant form of human I2S, has been commercially available since 2006. Four clinical trials of idursulfase have been conducted in patients with MPS II, encompassing an age range of 5_53 years. No patient in the trials had baseline cognitive impairment. Benefits noted in the drug label are improved walking capacity, along with decreased liver and spleen volume and reduction (but not normalization) of urinary GAG levels. Galsufase, a recombinant form of human arylsulphatase B, has been available since 2005. Clinical benefits noted in the drug label are improvements in walking and stair climbing capacity and reductions (but not normalization) of urinary GAG excretion.
The first successful bone marrow transplant for a patient with MPS I was done in 1980, and since then, hundreds of patients with the severe phenotype of MPS I, Hurler syndrome, have undergone HSCT. HSCT is the recommended treatment for severely affected MPS I patients (Hurler phenotype) under 2 years of age with a developmental quotient 70% of normal. When successful, HSCT significantly prolongs survival from a median of 6.8 years for untreated Hurler patients to 20 years and beyond. Most importantly, HSCT may preserve neurocognition. Although many transplanted patients have some learning issues or deficits, they do not experience the relentless neurocognitive decline that occurs without transplantation. Cognitive outcome is improved with earlier transplant. Somatic improvements include resolution of hepatosplenomegaly, reduction of sleep apnoea and upper airway disease, preservation of hearing and greater mobility of upper extremities. Urinary GAG excretion is also reduced. However, musculoskeletal disease continues to progress, vision usually worsens, cardiac valve disease persists and often progresses and growth is stunted, although a successful transplant may slow down the progression of some of these disease manifestations. For other MPS disorders, HSCT experience is limited and results are mixed.
Both ERT and HSCT, although not cures, have been able to alter the natural history of the disease. Other promising therapeutic approaches are in the pipeline.
Therapy for the mucopolysaccharidoses,2011
AAV-based gene therapy approach
Gene therapy is still largely unexplored as a potential treatment for MPS-IIIA and a lot of preliminar experiments have been conduceted in this sense.
Fraldi et al. describe a gene therapy approach for MPS-IIIA in a mouse model using recombinant adeno-associated virus serotype 5 (AAV2/5) as a vehicle to deliver therapeutic genes to the CNS. SUMF1 (SUlfatase Modifying Factor 1) exhibits an enhancing effect on sulfatase activity when coexpressed with sulfatases. Consistent with these findings, they demonstrated that co-delivery of SUMF1 and SGSH (via an AAV2/5-CMV-SGSH-IRES-SUMF1 vector) resulted in a synergistic increase in SGSH activity, both in primary neural cells and in murine brain (Fig.8). A study aimed at testing the therapeutic efficacy of simultaneous brain administration of SUMF1 and SGSH was then performed by injecting the lateral ventricles of newborn MPS-IIIA/normal mice with either AAV2/5 CMV-SGSH-IRES-SUMF1 or AAV2/5-CMV-GFP vectors. Widespread GFP expression was observed within the GFP-injected brain, and a stable and significant increase of SGSH activity was detected in several brain regions following SGSH-IRES-SUMF1 administration.Treatment with AAV2/5 CMV-SGSH-IRES-SUMF1 vectors resulted in a visible reduction in lysosomal storage and inflammatory markers in transduced brain regions. Finally, the MPS-IIIA mice treated with therapeutic genes displayed an improvement in both motor and cognitive functions. The results of this work suggest that early treatment of CNS lesions by AAV-mediated intraventricular injection of both SGSH and SUMF1 genes may represent a feasible therapy for MPS-IIIA.
In conclusion, early treatment by AAV-mediated intraventricular injection is sufficient to prevent or delay CNS pathology in MPS-IIIA mice in selected brain regions where concurrent stable expression of the transgenes was also present.
Functional correction of CNS lesions in an MPS-IIIA mouse model by intracerebral AAV-mediated
delivery of sulfamidase and SUMF1 genes,2007