Lysosomal Diseases Research Unit, South Australian Health and Medical Research Institute, Adelaide, SA, Australia
Abstract: Sanfilippo syndrome, mucopolysaccharidosis (MPS) type III, refers to one of five autosomal recessive, neurodegenerative lysosomal storage disorders (MPS IIIA to MPS IIIE) whose symptoms are caused by the deficiency of enzymes involved exclusively durante heparan sulfate degradation. The primary characteristic of MPS III is the degeneration of the central nervous system, resulting durante mental retardation and hyperactivity, typically commencing during childhood. The significance of the order of events leading from heparan sulfate accumulation through to downstream changes durante the levels of biomolecules within the cell and ultimately the (predominantly neuropathological) clinical symptoms is not well understood. The genes whose deficiencies cause the MPS III subtypes have been identified, and their gene products, as well as a selection of disease-causing mutations, have been characterized to varying degrees with respect to both frequency and direct biochemical consequences. A number of genetic and biochemical diagnostic methods have been developed and adopted by diagnostic laboratories. However, there is mai effective therapy available for any form of MPS III, with treatment currently limited to clinical management of neurological symptoms. The availability of animal models for all forms of MPS III, whether spontaneous generated corso gene targeting, has contributed to improved understanding of the MPS III subtypes, and has provided and will deliver invaluable tools to appraise emerging therapies. Indeed, clinical trials to evaluate intrathecally-delivered enzyme replacement therapy durante MPS IIIA patients, and gene therapy for MPS IIIA and MPS IIIB patients are planned underway.
Keywords: lysosomal storage disease, Sanfilippo syndrome, mucopolysaccharidosis III
Lysosomal storage disorders are a group of more than 50 inherited monogenic disorders. Each is caused by a deficiency of an enzyme responsible for the degradation of a metabolic product, whose accumulation results durante lysosomal malfunction and disease.1 Classification is primarily based the nature of stored material, and secondarily by the enzyme whose activity has been impeded. Sopra Australia, the combined incidence of lysosomal storage disorders is at least 12.9 per concludere 100,000 births, thus indicating a significant health and economic impact the family and the community.2
Sanfilippo syndrome, mucopolysaccharidosis (MPS) type III, refers to one of five autosomal recessive, neurodegenerative lysosomal storage disorders whose symptoms are paio to the incomplete lysosomal degradation of heparan sulfate.3 The subtypes are caused by the deficiency of: sulfamidase (MPS IIIA);4 α–acetylglucosaminidase (NAGLU, MPS IIIB);5 heparan acetyl CoA: α-glucosaminide -acetyltransferase (HGSNAT, MPS IIIC);6 -acetylglucosamine 6-sulfatase (GNS, MPS IIID);7 -glucosamine 3–sulfatase (arylsulfatase G ARSG, the currently putative MPS IIIE).8 Phenotypic, enzymatic, and genetic classifications of MPS III subtypes are summarized durante Table 1. A listing of reported prevalence is presented durante Table 2.
Table 1 Summary of the phenotypic, enzymatic, and genetic classification of the subtypes of MPS III
: MPS IIIE is currently a proposed disease insofar as ARSG deficiency durante humans has yet to be uncovered. As such, it has not been assigned an MIM number for its phenotype. The EC number for -glucosamine 3–sulfatase has not been updated beyond 3.1.6.
Abbreviations: MIM, Mendelian Inheritance durante Man; NA, not applicable; EC, Enzyme Commission; MPS, mucopolysaccharidosis; NAGLU, α–acetylglucosaminidase; HGSNAT, heparan acetyl CoA: α-glucosaminide -acetyltransferase; GNS, -acetylglucosamine 6-sulfatase; ARSG, arylsulfatase G; SGSH, N-sulfoglucosamine sulfohydrolase.
Table 2 Summary of reported prevalence studies of MPS III
Note: Note that ARSG deficiency manifested as MPS IIIE has yet to be detected durante humans.
Abbreviations: MPS, mucopolysaccharidosis; ARSG, arylsulfatase G.
There is currently mai therapy available for MPS III. Animal models exist for all forms, whether spontaneous generated corso gene targeting.8–18 These have contributed to a better understanding of MPS III, and will continue to deliver invaluable tools to evaluate emerging therapies.
Clinical symptoms of MPS III
Somatic symptoms durante humans can include coarse facial features with broad eyebrows, dark eyelashes, dry and rough hair, and skeletal pathology that affects growth and causes degenerative joint disease, hepatosplenomegaly, macrocrania, and hearing loss.19–22
The primary characteristic of MPS III is however degeneration of the central nervous system (CNS), resulting durante mental retardation and hyperactivity, typically commencing during childhood, although there is some heterogeneity with respect to severity and age of onset.
Pre-natal and early stages of post-natal development are usually normal. The initial stages of disease may begin between the ages of 1 and 3 years, which manifest as delayed cognitive development and/ aggressive behavioral problems, as well as hindered speech development.19,20,23
Behavioral difficulties may become increasingly severe between the ages of 3 and 5 years, commonly manifesting as a combination of hyperactivity, which is often violent and destructive, as well as sleep disturbances.20,24–26
Patients may remain durante this state for between 5 and 10 years, after which there is a regression durante behavioral disturbances. This is associated with a progressive and severe loss of intellectual processes (such as speech) and motor functions (including walking and swallowing). MPS III patients ultimately regress to a vegetative state until death, which can occur anywhere between the early teens durante the most severe scenarios, to as late as the sixth decade durante attenuated forms.19,20,27–30
Biochemistry of genes involved durante heparan sulfate degradation
Heparan sulfate is a negatively charged polysaccharide glycosaminoglycan (GAG) covalently bound to a number of proteins at the cell surface and durante the extracellular matrix. It durante turn consists of repeating disaccharide units of either l-iduronic acid d-glucuronic acid and α-linked glucosamine.3,8,31,32 The -sulfation of any subunit can occur, while the α-glucosamine may be acetylated, -sulfated, unmodified.
Heparan sulfate GAGs exist durante the cell as proteoglycans33 that are catabolized within the lysosome. This step may commence with the action of the endohydrolase heparanase full-length heparan sulfate GAG chains at the plasma membrane, early endosomal vesicles. This activity yields heparan oligosaccharide fragments of approximately 10–20 monosaccharide residues, whose subsequent degradation occurs corso sequential exolytic processes durante the lysosome.34
Depending the non-reducing end of these heparan oligosaccharides, this pathway may commence with the desulfation of 2-sulfated iduronic acid residues corso the action of iduronate 2-sulfatase (Enzyme Commission [EC] 126.96.36.199),35,36 and is followed by the hydrolysis of the subsequent terminal l-iduronic acid by α-l-iduronidase (EC 188.8.131.52).37,38 Both enzymes are also involved durante the degradation of dermatan sulfate and their deficiencies result durante MPS II (MIM 309900) and subtypes of MPS I (MIM 607014, MIM 607015, and MIM 607016), respectively. At this stage, ARSG desulfates 3–sulfated -sulfoglucosamine.8,39 Sulfamidase then hydrolyses the sulfates bound to the amino group of glucosamine,40,41 and the exposed amino group is durante turn acetylated by the activity of HGSNAT.42,43 NAGLU is consequently able to recognize and remove the generated -acetylglucosaminide from the neighboring uronic acid.44,45 Desulfation of any 2–sulfated glucuronic acid residues will then occur with glucoronate 2-sulfatase,46 a deficiency of which is yet to be detected. β-d-glucuronidase (EC 184.108.40.206) then hydrolyzes the terminal glucuronic acid.47 This is also required to degrade dermatan sulfate, and its deficiency causes MPS VII (MIM 253220). Finally, GNS removes the sulfate group from sulfated -acetlyglucosamine residues.48
The catalytic activation of all eukaryotic sulfatases, including those deficient durante MPS IIIA, MPS IIID, and MPS IIIE, undergo post-translational modification durante which a conserved cysteine within the active site is catalytically converted durante the endoplasmic reticulum to a Cα-formylglycine by the enzyme sulfatase modifying factor 1 (SUMF1).49,50 SUMF1 deficiency thus ablates the function of all sulfatases, which manifests as the symptoms of multiple sulfatase deficiency.51
Pathophysiology of MPS III
The order of events from heparan sulfate accumulation through downstream changes durante the levels of other biomolecules within the cell and ultimately the clinical symptoms of MPS III, particularly with respect to CNS degeneration, is probably the least understood aspect.
Monosialic gangliosides GM2 and GM3 accumulate durante lysosomes and other organelles (such as mitochondria and Golgi bodies) secondary to heparan sulfate accumulation,18,52–55 either by direct GAG-mediated inhibition of lysosomal enzymes responsible for ganglioside degradation,56 , alternatively, by deregulated trafficking synthesis of gangliosides.53
Not only is the reason for GM2 and GM3 accumulation still a matter of debate, but also their contribution to neurodegenerative aspects of MPS III. It is speculated from studies with MPS III animal models that both heparan sulfate and gangliosides initiate an inflammatory response that may exacerbate damage within the brain.12,18,57–59 This may occur as heparan sulfate released from lysosomes (probably corso exocytosis) as well as ganglioside-engorged and damaged neurons are phagocytosed by microglia (immunologically active cells involved durante CNS defense to damage).
The overall degradative function of lysosomes also relies the capacity of the lysosomal membranes to associate with target membranes, such as those of autophagic vacuoles.60 Interfering with the composition of lysosomal membranes has been shown to affect trafficking and fusion within the endo-lysosomal rete televisiva privata. Using embryonic fibroblasts derived from mouse models of MPS IIIA and multiple sulfatase deficiency, it has been demonstrated that cholesterol abnormally accumulates durante the endo-lysosomal membrane as a consequence of disease. This sequesters soluble -ethylmaleimide-sensitive factor attachment protein receptors, which are durante turn required for the function of the fusion machinery durante these membranes, thus inhibiting this function.61 This impairment may have wide reaching consequences, such as the pathology of the mitochondrial system observed durante the MPS IIIC mouse, caused by the accumulation of dysfunctional mitochondria otherwise eliminated by normal autophagy and lysosomal function.18
Genetics of MPS III
The mutations that have been sequenced durante the alleles of the causative genes durante patients diagnosed with MPS III are summarized durante Table 3. A commentary of those deemed of interest paio to frequency and/ phenotypic consequences follows.
Table 3 Summary of mutation types found durante the alleles of the causative genes of patients diagnosed with MPS III
: ©2015 Cardiff University. All rights reserved. Adapted with permission from the Human Gene Mutation Database (HGMD Professional 2014.2) (http://www.hgmd.cf.ac.uk/ac/index.php).152 Patronato from Stenson et al.152 Accessed October 1, 2014. Sopra this table, small refers to deletions and/ insertions involving 20 less postazione pairs, while gross describes deletions insertions involving 21 more postazione pairs.
Abbreviations: MPS, mucopolysaccharidosis; , α–acetylglucosaminidase; , heparan acetyl CoA: α-glucosaminide -acetyltransferase; , -acetylglucosamine 6-sulfatase; , -sulfoglucosamine sulfohydrolase.
The first patient known to be diagnosed with symptoms associated with an MPS caused by heparan sulfate accumulation was described by Sanfilippo et al.62
Later, it was observed that medium conditioned by the fibroblasts of a particular MPS III patient was able to reverse mucopolysaccharide accumulation durante some but not all of a cohort of other MPS III fibroblasts.63 This cross-correction was repeated with urine from these patients. This suggested that the disease could be caused by the absence of one of two factors, and as such was arbitrarily classified as types A B, based which agent was evidently deficient.
Soon after, the increased mucopolysaccharide durante MPS IIIA was purified from disease fibroblasts and shown to migrate with the mobility of heparan sulfate. Sopra addition, a compound capable of reducing the accumulation of mucopolysaccharide was purified from the urine of healthy individuals and demonstrated to display -sulfoglucosamine sulfohydrolase (, sulfamidase) activity,64 an absence of which was observed durante fibroblasts, leucocytes, and lymphoblasts from MPS IIIA patients.4,65 Eventually, the gene for MPS IIIA was identified.41 The gene for sulfamidase () is localized to chromosome 17 q25.3 and contains eight exons.41,66 The cDNA codes for a product of approximately 55 kDa (excluding the signal peptide sequence). Within this, there are up to five asparagine-linked glycosylation sites.41
Specific sulfamidase mutations have been found durante high frequencies durante distinct geographical locations. The missense mutation p.R74C, which replaces a large, positively charged R group durante the active site with one that is small and non-polar, occurs at a frequency of 56% of the Polish population.67 The p.R245H amino acid change is found durante 31% of mutant alleles durante Australia, 35% of those durante Germany, and 58% of those durante the Netherlands.67,68 This residue is not conserved among human sulfatases, and has little consequence specific activity, but it does however affect the stability of the protein.69,70 The p.S66W missense mutation is found durante 29% of alleles durante a cohort of Italian patients;71 this is not conserved among sulfatases, but is close to the conserved cysteine within the active site that is catalytically converted to a Cα-formylglycine by SUMF1.49,50 A substitution of the small R group of serine with the aromatic R group of tryptophan would thus be predicted to interfere with the active site, and studies of this variant demonstrated a compromise between stability and activity.69,70
Soon after the genetically heterogeneous nature of MPS III63 and the factor deficient durante MPS IIIA were discovered,64 it was shown that MPS IIIB was caused by a critically reduced activity of α-2-acetamido-2-deoxy-d-glucoside acetamidodeoxyglucohydrolase, (α–acetylglucosaminidase, ).72
The gene for MPS IIIB was cloned around the same time as that for MPS IIIA.45,44 is chromosome 17q21.1 and contains six exons. The cDNA codes for a polypeptide of 743 amino acids, which contains a cleavable signal sequence and six asparagine-linked glycosylation sites. The mature protein’s 720 amino acids yield a molecular mass of approximately 80 kDa.
Unlike MPS IIIA, there are mai common mutations durante MPS IIIB. Rather, most of the known mutant alleles durante MPS IIIB patients occur at low frequencies not more than once. However, the p.F48L, p.G69S, p.S612G, and p.R643C missense mutations have been associated with a later-onset phenotype.73–75
Primarily paio to the biochemical properties of the deficient protein, the gene for MPS IIIC remained more elusive. Originally, it was demonstrated that skin fibroblasts from MPS IIIC patients displayed a lack of HGSNAT activity.6 The responsible enzyme was then localized to the lysosomal membrane and shown to catalyze a transmembrane acetylation of the terminal glucosamine residue of intra-lysosomal heparan sulfatase.76–79 Over 20 years later, the gene was identified.42,43 is chromosome 8p11.1 and consists of 18 exons. The cDNA encodes a polypeptide of 635 amino acids, which contains eleven transmembrane domains and five asparagine-linked glycosylation sites. The amino-terminal 42 amino acids form a signal peptide crucial for integration into the lysosomal membrane, where it is then post-translationally modified into a 27 kDa α chain and a 44 kDa β chain.80,81
There is contrasting but compelling evidence for two different models of the mechanism of HGSNAT activity (Figure 1). One proposes that the enzyme binds acetyl CoA from the cytoplasmic side of the lysosomal membrane, and is itself acetylated at an active site histidine. A conformational change allows for the transfer of the acetyl group into the lysosome. Once heparan sulfate interacts with the active site, the terminal glucosamine acquires the acetyl group, thus forming -acetylglucosaminide.76–78,80 A second model suggests that catalysis occurs corso a random ternary order complex, durante one step, and with mai direct intermediary acetylation of the enzyme.79,81
Figure 1 Proposed models for the mechanism of heparan acetyl CoA: α-glucosaminide -acetyltransferase (HGSNAT) activity.
: (A) (1) acquires acetyl CoA from the cytoplasmic side of the lysosomal membrane (2), and is itself acetylated at an active site histidine (3). A conformational change allows for the transfer of the acetyl group into the lysosome (4). Once heparan sulfate interacts with the active site, the terminal glucosamine residue of heparan sulfate (GlcN) acquires the acetyl group (5), thus forming -acetylglucosaminide (6). Patronato from previous studies.76–78,80 (B) HGSNAT (1) catalyzes its reaction corso a random ternary order complex (2), so that the process requires only one step, and mai direct acetylation of the enzyme as an intermediate (3). Patronato from previous studies.79,81
The missense mutations p.R344C and p.S518F account for 22% and 29%, respectively, of mutant alleles durante the Netherlands.20 Furthermore, the deleterious nature of nearly all known missense mutations can be established corso their occurrence durante amino acids present durante a transmembrane domain and/ conservation among orthologs.42,43,82 The protein and activity of the majority of the known missense mutations have been exogenously expressed durante cell culture.83,84 Transfection of 18 of these resulted durante negligible HGSNAT activity, while the remaining four displayed protein and function comparable to those of wild type, and so are therefore currently considered clinically insignificant polymorphisms.
Interestingly, mutations durante HGSNAT have been found durante six patients with the non-syndromic form of retinitis pigmentosa, each of whom was diagnosed durante the third to fifth decade of life.85 These mutations result durante reduced HGSNAT activities compared with healthy controls but sufficiently high to avoid neurodegeneration and other symptoms of MPS IIIC.85 Retinitis pigmentosa was also reported durante the two patients with late-onset MPS IIIC.20,86 Their genotypes were identified as compound heterozygous for two missense mutations (p.G262R and p.S539C), both of which occur durante amino acids predicted to be durante the membrane,42,43,82 and whose separate overexpression durante cell culture resulted durante activity that was essentially undetectable.83,84
Discounting MPS IIIE, which has not yet been described durante humans, MPS IIID was the last to be classified, but the first to have its causative gene discovered. Sopra 1980, skin fibroblasts from two patients with the clinical symptoms of MPS III and high urinary concentrations of heparan sulfate were demonstrated to lack the enzymatic activity required to release sulfate from -acetylglucosamine 6-sulfate linkages. This was thus designated Sanfilippo type D (MPS IIID).7
GNS activity was subsequently purified and characterized48,87 and Edman sequencing of the isolated form permitted cloning of the coding gene.88,89 is found chromosome 12q14 and contains a total of 14 exons. The cDNA encodes for a polypeptide of 552 amino acids, and a protein of 78 kDa. Sopra addition to a cleavable amino-terminal signal peptide of 36 amino acids, post-translational modification also results durante cleavage by internal peptidases into a 32 kDa amino-terminal and 48 kDa carboxy-terminal species. It also contains 13 potential asparagine-linked glycosylation sites.
Similar to MPS IIIB, there are mai common mutations durante MPS IIID. It is noteworthy that there are relatively few missense mutations compared with the other MPS III subtypes (13%, see Table 3), and a dominance of deletions, insertions, and rearrangements.
The gene for MPS IIIE, -glucosamine 3–sulfatase (), is localized to chromosome 17q24.2 and contains eleven exons.8,90,91 The cDNA encodes a protein of 525 amino acids, of which four are asparagine-linked glycosylation sites.90,91
Although ARSG has been molecularly characterized, and dog17 and mouse8 models of MPS IIIE exist, ARSG deficiency durante humans has yet to be uncovered. These model organisms present with ataxia,17,92 which is only additionally observed durante a New Zealand Huntaway dog model of MPS IIIA.13
Transcriptional regulation of the causative genes for MPS III
The expression of most lysosomal genes is mediated by the transcription factor EB (TFEB), a member of the microphthalmia-associated transcription factor E subfamily of basic helix-loop-helix transcription factors.93 This functions by binding to the Coordinated Lysosomal Expression and Regulation (CLEAR) element (a DNA motif consisting of the palindromic consensus sequence GTCACGTGAC) of associated genes, and increasing their transcription.93 Stresses such as starvation and lysosomal storage induce TFEB translocation to the nucleus, thus facilitating transcriptional activity, and contributing to an increase durante CLEAR-containing lysosomal genes and lysosomal biogenesis durante general.93–96
This phenomenon has been demonstrated to involve the regulatory multi-subunit containing mammalian target of rapamycin complex 1 (mTORC1). Sopra normal conditions (for instance, durante the presence of full nutrients and basal lysosomal function), a complex including vacuolar H+ (V)-ATPase, the Ragulator complex, and Rag GTPases is durante an active state. Sopra being so, it recruits mTORC1 to the lysosomal surface, where mTORC1 durante turn becomes activated and phosphorylates TFEB, thus maintaining TFEB durante the cytoplasm durante an inactive form. However, durante conditions of cellular , such as starvation increased lysosomal storage, the Rag GTPases are inactivated, mTORC1 is detached from the lysosome, and TFEB is mai longer phosphorylated. Consequently, TFEB translocates to the nucleus, thus facilitating its capacity as a transcription factor and increasing the expression of CLEAR element containing genes.97–101
The genes for MPS IIIA, MPS IIIB, MPS IIID, and MPS IIIE each contain at least two CLEAR elements (listed durante Table 4) and their mRNA levels are increased durante cell culture upon exogenous TFEB expression, and reduced corso miRNA-mediated inhibition of TFEB.93
Table 4 CLEAR element sequences present durante the genes for MPS III
: Location refers to the position of the CLEAR element relative to the transcription start site, where the A of the ATG translation initiation start site of the coding sequence is nucleotide +1. Note that the gene for MPS IIIC (HGSNAT) does not contain any CLEAR elements. © 2014 The American Society for Biochemistry and Molecular Biology. Adapted from Moskot M, Montefusco S, Jakóbkiewicz-Banecka J, et al. The phytoestrogen genistein modulates lysosomal metabolism and transcription factor EB (TFEB) activation. . 2014;289:17054–17069.103
Abbreviations: MPS, mucopolysaccharidosis; NAGLU, α–acetylglucosaminidase; HGSNAT, heparan acetyl CoA: α-glucosaminide -acetyltransferase; GNS, -acetylglucosamine 6-sulfatase; ARSG, arylsulfatase G; CLEAR, Coordinated Lysosomal Expression and Regulation; SGSH, N-sulfoglucosamine sulfohydrolase.
Interestingly, HGSNAT does not contain a CLEAR element, and there is mai evidence to suggest that it is directly regulated by TFEB.93,102–104 Furthermore, the mechanisms of HGSNAT transcriptional activation have not been characterized. However, the 5′ untranslated region of HGSNAT has been demonstrated to contain a SINE-VNTR-Alu (SVA) – B element insertion.105 Although its influence HGSNAT expression requires further investigation, SVA elements are known to be non-autonomous, hominid-specific, non-long terminal repeat (LTR) retrotransposons that possess the capacity to sporadically generate disease-causing insertions.106
Laboratory diagnosis of MPS III
The detection of heparan sulfate durante the urine of suspected MPS III patients, , more specifically, the GAGs within, is often the first biochemical step durante diagnosis.
The traditional method of urinary GAG quantification involves the use of dimethylmethylene blue. Here, dimethylmethylene blue associates with sulfated GAG and the absorbance of the complex at a wavelength of 520 nm can be measured a spectrophotometer.107,108 An elevation durante urinary GAG will suggest a form of MPS.
Subsequent analysis can permit a further characterization of the accumulating product. High-resolution cellulose acetate electrophoresis of urinary samples can separate and identify distinct GAGs, thus providing an indication of MPS III from other forms.109 Additionally, qualitative gradient polyacrylamide gel electrophoretic separation can confirm increased secretion of particular heparan sulfate chains, thus diagnosing the particular MPS III subtype.110
Furthermore, methods comprising tandem mass spectrometry are now being developed. One involves quantification after derivatization of sulfated acetylhexosamine and sulfated acetylhexosamine-uronic acid.111 This method identifies specific oligosaccharides that accumulate as a result of a specific enzyme deficiency that act at the non-reducing end of heparan sulfate oligosaccharides.34,111,112 This can identify all MPS subtypes, except for MPS IIIB and MPS IIIC. Additionally, extraction and depolymerization of heparan sulfate using methanolysis under acidic conditions, and analysis of the subsequent desulfated disaccharide reaction products by liquid chromatography/tandem mass spectrometry can be used.113,114 This has the disadvantage that it cannot differentiate between MPS III types, but is simple and quick to run and thus a good first screen esame. A liquid chromatography/tandem mass spectrometry-based technique that exploits the unique nature of the non-reducing end residue of the accumulated GAG upon the deficiency of a particular enzyme durante the pathway has also been developed.115
Two types of artificial substrates that can be catalytically processed by the enzymes durante skin fibroblasts and leucocytes otherwise deficient durante MPS IIIA to MPS IIID are available: those that are radiolabeled116–119 and those that are fluorogenic.120–123 The basis of radiolabeled substrates is that the relevant enzymatic activity alters the charge of the substrate, thus permitting chromatographic separation. Regarding the latter, a fluorescent 4-methylumbelliferone product is released upon successful completion of the reaction, and can be detected by employing a fluorometric device.
An important fact of the fluorogenic substrate for the MPS IIIC assay is that HGSNAT acetylates it to a non-fluorescing intermediate, which is durante turn hydrolyzed to the fluorescing form by β-hexosaminidase; so the presence of the latter durante cells that are being evaluated is therefore imperative.122 Also, for MPS IIIA and MPS IIID, regardless of the chosen substrate, activity of a second sulfatase should be tested, durante order to categorically exclude multiple sulfatase deficiency.
Genomic DNA sequencing
This is achieved by employing oligonucleotide primers that span the intron/exon junctions of the exons of the gene of interest using the polymerase chain reaction with genomic DNA as the template. Products can be sequenced and cross-referenced to the tenore wild-type sequence and known disease-causing mutations and polymorphisms.
This is the definitive method for diagnosing not only disease but also carrier status (the latter is particularly difficult to detect corso GAG analysis and activity assays as the levels between carriers and unaffected controls are likely to be similar). However, this is assuming the mutation occurs within one of the boundaries that are being sequenced. Any changes beyond the scope of the amplifying oligonucleotides durante concept cannot be screened. Also, if an entire exon is absent durante heterozygosity, this cannot be detected corso tenore qualitative genomic DNA sequencing (which will still have the alternative allele as a template). This would justify the development and application of quantitative techniques for mutation screening, particularly for MPS IIID, where there is a proportionally higher occurrence of large rearrangements.30
Future diagnostic methods
Two motivations for the creation of new screening techniques are decreasing the age at which it can be applied, and increasing output and/ decreasing cost, either by a simpler and more rapid method to detect a particular disease, by an increased scope durante the number of diseases that can be diagnosed using the same esame.
A chorionic villus sample taken at 9–10 weeks gestation cultured cells from 14–16 weeks amniocentesis can be tested for MPS III-causing mutations corso genomic DNA sequencing enzymatic activity assays.124 Increased heparan sulfate, although less reliably, has also been detected using electrophoresis durante amniotic fluid taken at 16 weeks gestation from a pregnancy involving an MPS IIIA fetus.125 Sopra each case, the main disadvantage is the intrusiveness of the method of sample collection.
Therefore, steps are being taken to establish multi-tasked newborn screening strategies for MPS III (and other lysosomal storage disorders), whether corso detection of protein, activity, GAG levels. Recent developments have included: measuring the protein amounts of multiple specific enzymes durante dried blood spots using a multiplex immune-quantification assay, including sulfamidase and NAGLU;126,127 the synthesis of substrates for the enzymes deficient durante MPS IIIA to MPS IIID that measure the reaction products durante an durante vitro activity assay corso tandem mass spectrometry;128 and the use of tandem mass spectrometry to measure and characterize the accumulated GAGs durante dried blood spots after digestion into disaccharides.129
Another advance is whole genome exome sequencing, which would, durante theory, permit screening of the entire genome protein-coding region. These may possess similar caveats as genomic DNA sequencing of a selected gene but could also provide a tenore method to esame any subject for all sequencing abnormalities.
Treatments durante development for MPS III
Paio to the lack of a therapy to rescue the primary insult and/ pathophysiology of MPS III, present efforts are generally directed toward regulating behavior and sleep disturbances. However, a number of therapies for MPS III are being developed and evaluated.
Enzyme replacement therapy
The ability for sulfamidase, NAGLU, and GNS to be secreted from and taken up by the lysosomes within cells has provided the basis for the foundation of therapies for MPS III.
The major obstacle thus far durante the establishment of enzyme replacement therapy (ERT) is the inability of these same proteins to traversone the blood brain barrier (BBB). Injection of human recombinant sulfamidase directly into the brain130 into cerebrospinal fluid corso the cerebellomedullary cistern131 of MPS IIIA mice has been successful durante alleviating a number of symptoms but has also emphasized the importance of early detection account of the irreversibility of certain (particularly neurodegenerative) aspects of the pathophysiology.
The problem of potential invasiveness of targeting the brain has been grappled by engineering forms of sulfamidase to traversone the BBB. For instance, a mixture of increasing the parte of recombinant sulfamidase and modifying the chemical structure to remove mannose-6-phosphate glycans has been attempted.132 Despite significantly increasing the effectiveness upon systemic delivery into the MPS IIIA mouse outside the CNS, it was less effective durante the brain.
The use of gene therapy is an extension of ERT insofar as it attempts to introduce the coding sequence of the protein of interest into the cells of the patient corso the use of a viral vector, rather than synthesize it durante and purify it from another host. Manipulated cells will not only themselves possess enzymatic activity, but will also secrete it into circulation to be taken up by un-altered cells.
Co-delivery of sulfamidase and SUMF1 corso intraventricular injection of a recombinant adeno-associated virus (AAV) that drives the expression of both coding sequences resulted durante increased sulfamidase activity durante the mouse brain; a decrease durante lysosomal storage and microglial activation and an enhancement of motor and cognitive capabilities were also observed.133 This strategy is now at the human clinical trial stage.134 Moreover, a therapy using a similar approach and with comparable results for MPS IIIB is being developed based upon successful and efficacious AAV-mediated human NAGLU delivery durante MPS IIIB mice and Schipperke dog models.135,136
A recent advance has attempted to address a number of issues (repeated administration, invasiveness of delivery methods, and inability of the normal enzyme to traversone the BBB) with one solution.137 Sulfamidase engineered to be fused to both the signal peptide of the highly secreted iduronate-2-sulfatase protein and the BBB binding domain of apolipoprotein B was included durante an AAV-based vector designed to target the liver, thus allowing it to serve as a “factory” for perpetual systemic delivery of the modified enzyme. Adult MPS IIIA mice then intravascularly received this once. Not only did the signal peptide enhance secretion, but also the BBB binding domain permitted rescue of sulfamidase activity durante the brain, reduction of neuropathology, and a restoration of behavior.137
Genistein is an isoflavone purified from soy that exhibits an ability to veterano GAG levels. Although the overall mechanism by which this occurs has not been entirely clarified, its properties as a kinase inhibitor serve to induce TFEB transcriptional activity.103,104 For instance, it would seem that inhibition of the mTORC1-mediated phosphorylation of TFEB by genistein results durante the translocation of TFEB to the nucleus from the cytoplasm, an increase durante the expression of CLEAR-containing lysosomal genes, and an improvement durante the removal of lysosomal storage products.103,104
Supplementation of growth medium with genistein resulted durante reduction durante GAG levels durante skin fibroblasts derived from MPS IIIA and MPS IIIB patients.138 Moreover, treatment of an MPS IIIB mouse model showed an initial reduction durante GAG storage139 and an improved clinical outcome when higher doses were provided.140 Most importantly, a pilot study involving five MPS IIIA and five MPS IIIB patients were administered with a genistein-enriched soy isoflavone extract for 12 months, which reduced urinary GAG levels, enhanced hair morphology, and improved behavior.141
Substrate deprivation therapy
Sopra the context of MPS III, the purpose of this strategy is to veterano the levels of heparan sulfate containing GAGs by decreasing the amounts that are initially generated. Treatment of MPS IIIA mice with the non-specific inhibitor of GAG synthesis Rhodamine B resulted durante reduced urinary GAG, as well as levels durante the liver and the brain, and improved learning and memory as determined by vater maze, with mai long-term adverse effects.142–144
Development of a therapy for MPS IIIC
The rescue of HGSNAT activity has its own challenges. As with all disorders caused by faulty multi-pass transmembrane protein expression, approaches that rescue deficiencies of soluble proteins that can be secreted and taken up (such as ERT) are not applicable to HGSNAT deficiency. Some success has been achieved with molecular chaperones to rescue HGSNAT activity.83,145 These are typically reversible competitive inhibitors of the enzyme durante question, which are administered at sub-inhibiting concentrations. The basis of this is that the inhibitor is capable of associating with its interacting site and aiding the structural restoration of the misfolded protein. Sopra doing so, it restores the biochemical deficiency, which ultimately manifests as the disease durante question.146 Modified U1 spliceosomal RNAs that recognize mutated donor sites of selected splice-site mutants and restore RNA splicing of HGSNAT have also been evaluated.145 Yet, such therapies will even durante theory be successful durante restoring only a subset of mutants (durante these cases, missense and donor splice site, respectively). This presents a conundrum not just for MPS IIIC, but all disorders arising from multi-pass transmembrane protein expression deficiencies.
All the genes whose deficiencies cause MPS III have been discovered, and their gene products have been biochemically characterized to varying degrees. Furthermore, a number of genetic and biochemical diagnostic methods have been developed and adopted by diagnostic laboratories. Although MPS III disorders are rare, they are sufficiently debilitating to patients and challenging to parents and carers to warrant attention and research. Despite the current lack of a completely effective therapy for MPS III, there is much promise durante therapy development, to the point that clinical trials to evaluate intrathecally-delivered ERT durante MPS IIIA and gene therapy for MPS IIIA and MPS IIIB patients are durante either clinical trial being planned.
The author is grateful to Sophie Lazenkas and Prof John Hopwood (Lysosomal Diseases Research Unit, South Australian Health and Medical Research Institute, Adelaide, Australia) for critical reading of this manuscript.
The author reports mai conflicts of interest durante this work.
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