Mucopolysaccharidoses II and III Diseases Paper

MUCOPOLYSACCHARIDOSES II AND III:
GLYCOSAMINOGLYCAN
STORAGE DISORDERS
Sarah Wattar
Connor Wagner
Nuriel Anne Voyer
Alicia Wamsley
Fahim Zubair

2
Background
The lysosomal storage disorders known as mucopolysaccharidoses (MPS) were initially
explained during the mid-1960s via chemical and radioisotope incorporation analyses1. Since then, a
great deal of scientific studies has shed light on the pathologies and clinical diagnoses of these diseases.
Within this group of disorders there are many different subtypes, but the scope of this paper will focus on
Hunter’s Syndrome (MPSII), an X-linked recessive disease, and Sanfilippo’s Syndrome (MPSIII), a
recessive autosomal disease.
Both diseases have a wide spectrum of symptoms, ranging from mild to very severe cases. Urine
analysis is the most effective tool in diagnosing these diseases, as it accurately checks for elevated
glycosaminoglycan (GAGs) levels. Disease confirmation is achieved by running a serum sample through
the polymerase chain reaction and/or performing gel electrophoresis. Although both are highly rare,
MPSII can be more commonly found in Israel and the MPSIII has a higher occurrence in the Netherlands.
Etiologically, both MPSII and III are acquired at the genetic level when infrequent random mutations occur
on key lysosomal GAG enzyme-coding genes and are heritably transmitted; no behavioral or
environmental factors have been implicated in the causation of these diseases.
Pathophysiology
MPSII and MPSIII are diseases characterized as lysosomal storage disorders that are caused by
non-functional enzymes. Mutations in particular genes, at specific loci, produce these non-functional
enzymes which are unable to catalyze the destruction and subsequent removal of GAGs. These diseases
are identifiable by the intralysosomal aggregation coupled with the elevated excretion in urine of partially
degraded GAGs. Overall, the pathophysiology inevitably leads to dysfunction in cells, tissues, and
organs.2 The over accumulation and presentation of GAGs to cells for storage directly leads to lysosomal-
swelling.3 This swelling is problematic because the lysosomes then occupy a greater area of the
cytoplasm, which can obscure other organelles and deform the nuclear outline. Over time, this swelling
alters the shape of the cells directly leading to the organomegaly.2,4 The vast majority of the phenotypical
manifestations of the diseases are simply elucidated by the improper degradation and over accumulation
of GAGs. For example, this cellular alteration of heart cells changes their shape from fusiform to round;

3
the ramification of this causes the cordinae tendinae to become thicker, interfering with normal cardiac
function and producing valvular stenosis.5,6
Although all the MPSs are grouped together as lysosomal storage disorders physiologically
caused at the chromosomal level, MPSII is uniquely transmitted in an X-linked manner. “Wilson et al.
localized the gene to Xq28,” 2,4 which when mutated leads to a deficiency in the enzyme, iduronate
sulfatase, which is pivotal in the degradation of heparan and dermatan sulfates by separating their
oxygen-linked sulfates.4 More specifically, “iduronate 2-sulfatase is part of a family that includes all
sulfatases studied to date, and they carry the posttranslational modification of a cysteine residue in the
catalytic site of 2-amino-3-propionic acid.” 3 This modification is of the utmost importance for the normal
catalytic functionality of this sulfatase.4 Without this modification, iduronate 2-sulfatase is unable to cleave
the sulfate group from “the 2- position of L-iduronic acid present in dermatan sulfate and in heparan
sulfate” see Figure 1. 4
Originally the classification of MPSII fell under two
categories, mild (MPSIIB) and severe (MPSIIA). MPSIIA
clinically manifests as Dysostosis Multiplex (abnormally-
shaped ribs, vertebrae, enlarged skull, spatulate ribs,
etc.), organomegaly, retinal degeneration, mental
retardation, and even death before 15 years of age.
Whereas MPSIIB is the antithesis, clinically presenting
normal intelligence, short stature, and a longer average
lifespan (as compared to MPSIIA).4 This classification in
more recent times has become obsolete because of the
considerable variability in the milder form. Instead it would
be true to state that MPSII is phenotypic continuum, with a
myriad of severities.2,7
Figure 14

4
Similar to MPSII, MPSIII also disrupts the cascade-like process in which particular GAGs are
removed, but differs in the genetic origination— the chromosomal locus.4 MPSIII is also classified by 4
subtypes, each having a different enzyme instrumental in the removal of GAGs; the types and
problematic enzymes are as follows: MPSIIIA (Heparan N-sulfatase), MPSIIIB (α-N-Acetyl-
glucosaminidase), MPSIIIC (Acetyl-CoA:α-glucosaminide acetyltransferase), and lastly MPSIIID (N-
Acetylglucosamine 6-Sulfatase), as seen in figure 2.4
All of the subtypes of MPSIII involve the
same biochemical pathway, only differing in the
individual reaction that is disrupted by the non-
functional enzyme involved in the stepwise
catabolism of the GAGs.2,4 “Heparan Sulfate consists
of glucuronic acid and L-iduronic acid residues,
some of which are sulfated, alternating with α-linked
glucosamine residues. The latter are either sulfated
or acetylated on the amino group and may be
sulfated on the 6-hydroxyl.” 8 While the common
characteristics are generally known, there can be
considerable variability in the sulfation, and
proportions of glucuronic acid and L-iduronic acid
between different “species” of heparan sulfate or on
a sulfate chain.8 In unaffected individuals, this allows
many species of heparan sulfate to complete a myriad of functions including the activation of growth
factors by binding onto them, which could be linked to the diverse clinical presentation in afflicted
persons.9 The clinical presentations of this disease differ slightly compared to other MPS diseases with
the onset of disease typically occurring between the ages of 2 and 6. Severe mental deterioration typically
presented in patients by 6 to 10 years of age. Other symptoms include mild somatic manifestations
Figure 24

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(which is uncharacteristic of MPS), hyperactivity, and mild hepatosplenomegaly.2 Death follows soon after
in the 20’s and/or 30’s.2
Diagnosis
Today, there exist many different diagnostic tools to aid health professionals in diagnosing and
treating various diseases, and this is the case for MPS II/III as well. MPSII is caused by a mutation in the
gene encoding for iduronate sulfatase (IDS), and comes with a wide spectrum of symptoms. Changes to
one’s diet cannot prevent disease progression, but limiting milk, sugar and dairy products has helped
some individuals experiencing excessive mucus.10
Once MPSII has reached the brain and symptoms begin to present themselves, the most
effective diagnostic tool is to perform a urinalysis to examine one’s GAG levels.11 Because the mutations
associated with the MPS diseases result in the stoppage of the main GAG catabolic pathway, at a certain
stage in MPSII/III the alternative pathways of GAG catabolism are activated. This results in partially-
degraded GAGs that eventually gets excreted out in urine. Hence, when testing urine from a MPS patient,
the concentration of partially-degraded GAGs is considerably higher than that of a healthy patient.11 This
can be detected by running a urinalysis and assessing the GAG levels in one’s urine. A first morning urine
sample is used for the determination of GAGs in the urine. One method that can be used is the
colorimetric method which yields semi-quantitative results, while another is use of ultraviolet-visible
spectrophotometry which results in quantitative data. If these tests show excess GAG concentrations
being excreted in the urine, the clinician can perform an enzymatic assay of a blood sample on a dried
blood spot absorbent to assess the functionality of IDS.11 By doing so, one is able to conclude whether
the GAG enzymes are functioning properly (low levels of GAGs in urine), indicating an unaffected patient,
or if at least one is deficient (showing higher levels of GAGs because there is a lack of that enzyme to
degrade them), indicating a diseased patient. High levels can be interpreted via the colorimetric method,
where a color change signifies an increased amount of GAGs. Using methylene blue as a marker, when
mixed with the urine sample, normal GAG levels are indicated by the lack of color change (vial stays
blue); whereas, an increased amount of GAGs is evidenced with a blue to purple color change.11 The
ultraviolet-visible spectrophotometry determination of urinary GAGs is performed by preparing dilutions of
the urine and then adding methylene blue. The samples are then all tested to measure their absorbance

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levels (and seeing the differences in absorbance between 520 and 600 nm).11 The enzyme assay done to
test the functionality of IDS is done on blood with filter paper. “This determination is based on the reaction
of a substrate containing 4-methylumbelliferone-iduronate-2-suphatase to the test sample in a first
incubation and then adding the enzyme α-iduronidase in a second incubation, releasing fluorescent 4-
methylumbelliferone, thus measuring the fluorescence emitted with the fluorimeter (360-450nm)”11
Because the symptoms progress very slowly with onset, MPS is hard to confirm once a diagnosis has
been made.
Confirmation can be done through some form of genetic analysis, such as PCR. By mapping out
one’s genetic makeup, qualified lab personnel would be able to see exactly where the mutation is, what it
is affecting (whether it is the enzyme responsible for the degradation of GAGs or not), and thus whether
the patient is burdened with MPS or not.
Another confirmation test can be running a gel electrophoresis test.10 “The urinary GAGs from the
different MPS displayed distinct patterns on gradient-PAGE and further confirmation of MPS types and
subtypes was demonstrated by an electrophoretic shift in the banding pattern after digestion with the
appropriate MPS enzyme.”10 Refer to figure 3 to see the results of a gel electrophoresis test with ‘QC’
representing a negative control, and MPSI-MPSVII being represented on the following wells.14 As you can
see the patterns are distinctly different for a patient who has MPS versus one who does not, making gel
electrophoresis a good confirmatory test. Gel electrophoresis can even differentiate between multiple
subtypes of MPS. Another great diagnostic tool would be to perform an amniocentesis and testing the
prenatal fluid for the abnormalities related to MPS.12 This way, genetic mapping (PCR) could be
performed on the prenatal fluid and the
disease could be caught at the earliest
stage possible.
MPSIII can be diagnosed similarly
to MPSII using a urine or blood test, looking
for an above average amount of GAG
hexuronic acids in the urine, and can be
Figure 314

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confirmed by a PCR analysis, as well as by running gel electrophoresis.
Epidemiology
MPSII affects people of all nationalities, but the disease is more prevalent in Canada, the Middle
East and the Northwestern European region of the world. Most cases arise from people in the Jewish
community, specifically the Ashkenazis and Moroccans. Theorists have hypothesized that during the
beginning stages of pregnancy, the MPSII allele is predominantly favored in Israelites. Studies have
shown that the Israelites’ genetics do not have the new mutation that would prevent the disease from
being passed down from the dominant carrier.15 Studies on male populations show the ratio of incidences
in male births of MPSII are as follows: Israel- 1/34,000, United Kingdom- 1/132,000, British Columbia-
1/111,000, and Northern Ireland- 1/72,000.4 The time period of these incidences were from 1969-1975.4.
These studies are likely to be underestimated due to insufficient certainty. The life expectancy is fairly
high for people with MPSII. They can usually expect to live approximately six decades, but death could
occur in teenage and early adulthood patients.
The four subtypes of MPSIII can specifically be located in the Netherlands, British Columbia, and
Northern Ireland. The Netherlands exhibits type A, B, and C with a total ratio of 1/73,000 in total live births
combined. British Columbia displays only type A which has a total ratio of 1/324,000 in total live births.
And Northern Ireland shows a higher rate of type A and B with a ratio of 1/280,000 in total live births
combined. The study of MPSIII in the Netherlands found 40 probands out of 6 million births (1/150,000)
between 1945 and 1969, but this result, not very different from those of the British Columbia and Northern
Ireland surveys, was corrected for an ascertainment probability of 0.488 to give 1/73,000.4 The
probands—defined as the first person in a family to show the signs of the genetic disorder—do not show
high death rates.4
Etiology
MPSII and MPSIII, in addition to the other clinically-discovered MPS subtypes, fall into the
broader general family of inborn metabolic diseases known as GAG storage disorders. Both disease
subtypes feature entirely genetic etiologies, inherited from parent to child.2 Uniquely, only MPSII has been
found to be attributed to detrimental mutations on a particular locus on the X sex chromosome.16
Conversely, all the other MPSs (including MPSIII) are now recognized as recessive disorders arising out

8
of mutations on certain autosomal genes.4 Each of the subclasses associated with MPSII or III,
respectively, are similar in how they all lead to the deficiency of a singular key lysosomal enzyme
principally contributing to the breakdown of (a) certain GAG(s).4 Each of these GAG-catabolizing
enzymes comprise a system of ten enzymes working in a mechanistic manner to degrade the GAGs by
promoting the lysosome’s ability to do so within various cells.2 The single enzyme deficiency caused by
the varying mutations effected by mild-severe MPS II or one of the four subclasses of MPSIII on its
lysosomal enzyme-encoding gene hence functionally halts further catabolism of the aforementioned
GAGs; in turn, this leads to the pathophysiologies discussed above.4
Whether it manifest as a more or less severe clinical form based on the degree of mutation that
occurs, MPSII is characterized by a mutation occurring on the iduronate-2-sulfatase gene, particularly
localized at the gene address Xq28.16 The mutation directly causes a loss in lysosomal enzyme iduronate
sulfatase; the lack of this enzyme not only prevents the first biochemical step needed for the destruction
of the dermatan sulfate, but also the first step for the breakdown of heparan sulfate as well (both are
GAGs).4 It is generally difficult to correlate disease phenotype with a mutated gene’s genotype for any of
the MPS types due to lack of clear 1:1 association between the mutated genotype and clinical
phenotype.2 However, a well-cited study for MPSII has indicated via Southern blotting analysis that
patients with major mutations in the Xq28 gene—including gene rearrangements and partial/full gene
deletion—invariably present the severe form of the disease.16 In the study, this was compared against
patients who only featured small mutations in the MPSII gene—nonsense, missense, and small base pair
deletions—who predominately show the mild form of the disease.16
In the case of MPSIII, each of the subclasses of the disease demonstrates a different single
enzyme deficiency that affects a different step of the enzymatic breakdown cascade for the GAG,
heparan sulfate.2 For all except MPSIIIC, reliable animal models able to acquire the MPSIII A, B, and D
disease subtypes in genes and mutations homologous to humans have provided evidence for the now
well-known, separate genetic etiologies of MPSIII A, B, and D.4 MPSIIIA, considered the most severe
disease form out of the four subtypes2, is characterized by a mutation in the gene on 17q25.3 (named
MPS3A) which leads to the deficiency in heparan N-sulfatase.4 This lysosomal enzyme catalyzes the third
step of heparan sulfate’s catabolic pathway.4 Secondly, MPSIIIB features a mutation in the NAGLU gene

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located in 17q21, which encodes for alpha-N-acetyl glucosaminidase; this enzyme allows for the fifth step
of the catabolic pathway of heparan sulfate to occur.4 Notably, for MPSIIIC, the gene encoding for the
enzyme implicated in this disease subclass has yet to be identified via molecular approaches; however,
the enzyme affected is known: acetyl-CoA:α-glucosaminide acetyltransferase.2 This novel GAG
enzyme—given that it is not a hydrolase like the other MPS III enzymes—catalyzes the fourth step of
heparan sulfate’s breakdown.4 Lastly, MPSIII D is attributed to an enzyme deficiency in N-
acetylglucosamine 6-sulfatase, effecting the very last step in this GAG’s catabolism; the encoding gene
for this enzyme finds itself in chromosomal locus 12q14.4
Prognosis
The current prognoses for these disorders are grave. There is no known cure available; however,
there exist treatments that can help with the quality of life and progression of the disease. Early detection
of the disease and multidisciplinary management improves the quality of life.12 Physical therapy and daily
exercises may delay joint problems and improve the ability to move. Surgery to remove one’s tonsils and
adenoid may improve breathing among affected individuals with obstructive airways. Also sleep analysis
could help a patient suffering from sleep apnea.12
Gene therapy using a retroviral vector may be effective. Viral vectors are derived from
adenovirus, adeno-associated virus (AAV), retrovirus or herpes simplex virus (HSV), and involve
replacement of some or all of the gene that encode viral protein with the therapeutic gene.14 This gene
therapy can be implemented in two ways. In-vivo and involves a systemic injection of vector into a
peripheral, artery or portal vein and/or a localized injection into a specific organ like a muscle. Ex-vivo can
be done by removing certain cells, such as hematopoietic stem cells, and infusions of the modified cells
back in the patients.12
Initially, treatment was attempted by doing hematopoietic stem cells transplantation (HSCT). The
HSCT did prove to be somewhat effective in reconstructing the functionality of the defective enzymes,
allowing the levels of GAGs to decrease in urine. It also plays a minimal role in decreased spleen and
liver volumes, as well as diminished facial coarsening and improved joint mobility as well as respiratory
function.12 The overall results of HSCT however were considerably disappointing, especially at the
neurological level. Treatment then moved to enzyme replacement therapy (ERT), which was much more

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effective in treating MPSII. ERT was proven to be effective as well as safe as a treatment for MPSII, as
well as MPSIII.2 ERT is usually done by injecting an intravenous infusion into the patient of the enzyme
that is defective. By replacing the defective enzyme with the working replacement enzyme, the body’s
ability to remove the GAGs is enhanced and the patient shows better outcomes.2
For MPSIII, the most effective treatment is currently gene therapy (substrate deprivation therapy).
In this method of treatment, siRNAs are injected and used to reduce GAG synthesis. This method is most
effective for MPSIII A. Type B has no confirmed treatment but cell-mediated therapy, enzyme
enhancement therapy, substrate deprivation therapy, and viral gene therapy are all possible prospective
fields of treatment; the most effective being gene therapy.2 Gene therapy treatment for type B includes
injecting adeno-associated virus (AAV) vectors into the brain coding for NAGLU (NAGLU being the gene
mutated by MPSIII B) as well as a gene delivery lentiviral (LV) vector for NAGLU.2 All the subtypes of the
MPSIII (A, B, C, and D) are very similar in their phenotypes and symptoms, and can be treated most
effectively with gene therapy. Interestingly, however, the most effective treatment for MPSII was ERT.2

11
Works Cited
[1] Joseph C. Fratantoni, Clara W. Hall, Elizabeth F. Neufield. The Defect in Hurler’s and Hunter’s
Syndromes: Faulty Degradation of Mucopolysaccharidoses. Proceedings of National Academies of
Sciences. 1968;60(2):699-706.
[2] Maria Francisca Coutinho, Lúcia Lacerda, Sandra Alves. Glycosaminoglycan Storage Disorders: A
Review. Biochemistry Research International. 2011;2012:1-16.
[3] Bernard Schmidt, Thorston Selmer, Arnd Ingendoh, Kurt von Figurat. A novel amino acid modification
in sulfatases that is defective in multiple sulfatase deficiency. Cell. 1995;82(2):271-278.
[4] Elizabeth F. Neufield and Joseph Muenzer. The Mucopolysaccharidoses. In: C. R. Scriver, A.
L. Beaudet, W. S. Sly, and D. Valle, editors. The Metabolic & Molecular Bases of Inherited Diseases.
New York: McGraw-Hill; 2001. p. 3421-3452.
[5] Eva Piotrowiska, Joanna Jakòbkiewicz-Banecka, Sylvia Baranska, Anna Tylki-Seymanska, Barbara
Czartoryska, Allcja Wegrzyn, et al. Genistein-Mediated inhibition synthesis as a basis for gene
expression targeted isoflavone therapy for mucopolysaccharidoses. European Journal of Human
Genetics. 2006;14(7): 846-852.
[6] M. Haskins, M. Casal, N. M. Ellinwood, J. Melniczek, H. Mazrier, and U. Giger. Animal models for
mucopolysaccharidoses and their clinical relevance. Acta Paediatrica Suppl. 2002;439:88-97.
[7] J. E. Wraith, M. Scerpa, M. Beck, O. A. Bodamer, L. D. Meirleir, N. Guffon, et al.
Mucopolysaccharidosis type II (Hunter Syndrome): a clinical review and recommendations for
treatment in the era of enzyme replacement therapy. European Journal of Pediatrics. 2008;167:267-
277.
[8] Kjellen, L., Lindahl, U. Proteoglycans: Structures and Interactions. Annual Review of Biochemistry.
1991;60:443-475.
[9] Conrad HE. Heparin-binding proteins. New York:Academic Press;1998.
[10] S. Byers, T. Rozaklis, L.K. Brumfield, E. Ranieri, J.J. Hopwood. Glycosaminoglycan Accumulation
and Excretion in the Mucopolysaccharidoses: Characterization and Basis of a Diagnostic Test for
MPS. Molecular Genetics and Metabolism. 1998;65(4):282-290.

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[11] M. Piraud, S. Boyer, M. Mathieu, I. Marie. Diagnosis of Mucopolysaccharidoses in a Clinically
Selected Population by urinary glycosaminoglycan analysis: A study of 2,000 Urine Sample. Clinica
Chimica Acta. 1993;221(1,2):171-181.
[12] Robert L. Mango, Lingfei Xu, Mark S, Sands Carole, Gabriela Vogler, Tobias Seiler, et al. Neonatal
retroviral vector-mediated hepatic gene therapy reduces bone, joint, and cartilage disease in
mucopolysaccharidosis VII mice and dogs. Molecular Genetics and Metabolism. 2004;82(1):4-19.
[13] T. Wood, O.A. Bodamer, Maira Graeff Burin, et al. Expert Recommendations for the Laboratory
Diagnosis of MPS VI: Molecular Genetics and Metabolism. 2012;106(1):73-821.
[14] R C Nowinski, E F Hays. Oncogenicity of AKR endogenous leukemia viruses. Journal of Virology.
1978;27(1):13–18.
[15] E. Bensimonschiff, J. Zlotogora, D. Abeliovich, M. Zeigler, G. Bach. Hunter Syndrome among Jews
in Israel. Biomedicine & Pharmacotherapy. 1994;48(8-9):381-384.
[16] J J Hopwood, S Bungee, C P Morris, et al. Molecular basis of mucopolysaccharidosis type II:
mutations in the iduronate-2-sulphatase gene. Human Mutation. 1993;2:435-442.

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