(MUCOPOLYSACCHARIDES)

Glycosaminoglycans (GAGs) are heteropolysaccharides, long unbranched polymers consisting of a repeating disaccharide unit The repeating unit consists of an amino sugar (either N-acetyl glucosamine or N-acetylgalactosamine) along with a uronic sugar (either D-glucuronic acid or L-iduronic acid) or Glycosaminoglycans are highly polar and attract water; aminosugars hydroxyl residues may be bound to sulfate groups, increasing negative charge density Glycosaminoglycans can also be linked to extracellular , in Hyaluronic acid consists of D-glucuronic acid and N- acetylglucosamine repeating units Hyaluronates have molecular masses higher than 106 daltons (50000 repeating units) Hyaluronates are found in the and in joints (sinovial tissue and fluid), tendons and cartilage Other GAGs are shorter than hyaluronate and are covalently linked to proteins consists of repeating GlcA- GalNAc units Chondroitin sulfate is found in cartilage, tendons, ligaments and in the aortic wall Keratan sulfate is a linear polymer The basic repeating disaccharide unit within keratan sulfate is 3Galβ1-4GlcNAcβ1. keratan sulfate is found in the cornea, in bones, cartilage, hair, nails, horns and hooves

Heparin is a natural anticoagulant with a very high negative charge density

• At least 7 different mucopolysaccharidosis are known, caused by a deficiency of one of the involved glycosaminoglycans degradation • Glycosaminoglycans accumulate and are excreted with urines • Rate of incidence of all mucopolysaccharidosis 1:25000

• All mucopolysaccharidosis cause bones alteration and, with one exception, mental retardation

• Early death is caused by liver or kidney failure and by cardiovascular symptoms DISEASE DEFICIENCY MPS I () α-L-

MPS II () Iduronate 2-sulfate sulfatase

MPS III (San Filippo A syndrome) heparan sulfatase

MPS IV () galactosamine 6-sulfate/ β- galactosidase MPS V (Sindrome di Scheie) α-L-iduronidase

MPS VI (Maroteaux-Lamy syndrome) Aril sulfatase B

MPS VII () β glucuronidase Type I mucopolysaccaridosis (Hurler syndrome)

• MPS I is caused by a deficiency of -iduronidase • It is inherited as an autosomal recessive trait • Rate of incidence 1:100000 • Partially degraded dermatan sulfate and accumulate mainly in fibroblasts • MPS I is classified into 3 different syndromes according to disease severity: 1. Scheie syndrome 2. Hurler-Scheie syndrome 3. Hurler syndrome

Symptoms

Untreated patients die within the first 10 years of life, because of respiratory complications or heart failure Characteristic facial features (gargoyle) of MPS I children

Neutrofiles are filled with granules, consisting of accumulated GAGs

A monocyte from a Hurler syndrome patient, showing a a big inclusion and some smaller granules Electron micrograph of a brain from a MPS I patient, showing accumulated GAGs (zebra bodies) Diagnosis

• Newborns are generally bigger than average • MPS I is usually diagnosed between 6 and 24 months of age, when CNS is already compromised • Growth is faster during the first year, then it slows down, stopping between 2 and 4 years of age • Heparan sulfate and dermatan sulfate can be detected in urines • Diagnosis is confirmed by enzyme activity assays on cultured fibroblasts • Prenatal diagnosis can be performed either on chorionic villi or through amniocentesis Therapy

• Enzyme replacement therapy – with recombinant iduronidase (Aldurazyme), shows improvement of non neurological symptoms

• Bone marrow (or umbilical cord cells) graft – improves all symptoms, except corneal clouding and skeletal abnormalities. Neurodegeneration can be stopped.

• Skeletal abnormalities can be surgically corrected

• therapy has been successifully tested on animal models MUCOPOLYSACCARIDOSIS TYPE II (HUNTER DISEASE)

First described in 1917 by C. Hunter Hunter disease is caused by a deficiency of iduronate-2- sulfatase, removing sulfate groups from dermatan and heparan sulfates Heparan and/or dermatan sulfate accumulate mainly in respiratory airways (orofarynx and the tracheobronchial tree)

Iduronate-2-sulfatase gene is located on X Rate of incidence – 1:100000 A few girls with Hunter disease have been described

SYMPTOMS

• Obstructive airways disease (sleep apneas) • Hearing loss • Cardiomegaly, cardyomiopaty • Hepatosplenomegaly • Skeletal abnormalities, stiffened joints • Carpal tunnel syndrome • Corneal clouding, sight loss • Hyperactivity and aggressive behaviour • Progressive neurodegeneration • Patients usually die in their twenties, because of respiratory complications or heart failure

Diagnosis

• Hunter disease is normally diagnosed between 4 and 8 years of age • Patients show short stature, arthropathy, hepatosplenomegaly, facial coarseness and skin rashes • Mental retardation appears between 18 and 24 months

• Diagnosis is performed through: • GAGs assay in urines • Enzyme activity assay in leukocytes and fibroblasts (and chorionic villi prenatal diagnosis) • Molecular gene analysis is essential to recognize mutation carriers Monocytes filled with granules of accumulated GAGs A 3D model of human Iduronate- 2 sulfatase

Iduronate-2 sulfatase is a 550 aa

Various types of mutations have been found, mostly single aa substitutions

Genotype-phenotype correlation is low Therapy

• Symptomatological therapy: skeletal abnormalities can be surgically corrected • Heart valves can be substituted • Chronic rhinitis and airways infections can improve following tonsillectomy and adenoidectomy • ERT – Elaprase and Idursulfase (ricombinant human enzymes) are commercially available, but are not able to prevent or improve cognitive regression • Bone marrow graft does not solve cognitive regression and is not often used in Hunter disease treatment Type IV mucopolysaccaridosis (Morquio syndrome)

• autosomal recessive trait • It is a rare form of dwarfism first described in 1929 by l. Morquio in Montevideo and by J. F. Brailsford in Birmingham • Keratan sulfate is the accumulated metabolite and is excreted in urines • 2 types of Morquio syndrome are known: • Type A – caused by a deficiency of galactosamine 6- sulfatase • Type B – caused by a deficiency of β-galactosidase • Rate of incidence – ranging from 1: 300000 to 1:500000 Symptoms and signs

• Keratan sulfate accumulates in lysosomes and leads to skeletal, muscle and cartilage abnormalities

• Patients show:

• Coarse facial features • Large head (macrocephaly) • Bell shaped chest • Kyphoscoliosis • Hypermobile joints • Knock-knees • Abnormal development of bones, including the spine • Short stature • Heart and liver enlargement • Aortic regurgitation • Widely spaced teeth

Diagnosis

• Morquio syndrome is usually diagnosed around 2-3 years of life (the first symptoms are bone abnormalities and loose joints)

• Diagnosis is confirmed by keratan sulfate assay in urines

• Enzyme activity assay in fibroblasts allows to discriminate between Morquio Type A and Type B

• β-galactosidase deficiency is also responsible for GM1 gangliosidosis (GM1 accumulates in the nervous system)

• In Morquio Type B neurological functions are normal Morquio disease Type A (deficiency of galactosamine 6- sulfatase)

• Galactosamine-6-sulfatase gene is located on chromosome 16 (and consists of 14 exons)

• More than 150 mutations have been identified (3 are most frequent)

• Galactosamine-6-sulfatase is a dimer, consisting of 60 kDa subunits

Morquio disease Type B (deficiency of -galactosidase) GLB1 gene

β-galactosidase (GLB1) gene is located on 102 mutations have been identified: some reduce affinity for GM1, others impair binding to keratan sulfate. Different domains are involved in binding of each substrate Therapy

• Symptomatological therapy: skeletal abnormalities and aortic rigurgitation can be surgically corrected

• ERT – Recombinant elosulfase α (Vimizin) is now available, although life-threatening allergic reactions have occurred in some patients

• No gene therapy protocol is available so far SIALIDOSIS

• Sialidosis are caused by a deficiency of lysosomal sialidase NEU1 • NEU1 is associated to β galactosidase and PPCA in a ternary complex • NEU1 is also involved in galactosialidosis, due to PPCA deficiency • 2 types of sialidosis are known: • Type I sialidosis – a non dysmorfic mild form, with a late onset. It involves gastrointestinal abnormalities, sight impairment, myoclonus, cherry red spot • Type II sialidosis – dysmorfic, severe form, with an early onset; symptoms include mental retardation, short stature, hepatosplenomegaly, multiple dysostosis. • Rate of incidence – 1: 2000000 Electron micrograph of lung cells from a sialidosis patients: vacuoles filled with accumulated sialoglycoproteins and sialoglycolipids

NEU1 is a 48 kDa protein, with a 6 blades β barrel (superbarrel) structure

Pompe disease

• Pompe disease is a progressive, often fatal, disease, caused by a deficiency of lysosomal acid α-glucosidase • It is inherited as an autosomal recessive trait • Pompe disease is often considered a heart disease, because rate of cardiomyopathy/cardiomegaly is high in children

• Pompe disease can be classified as: 1. A lysosomal storage diseases (LSD) 2. A neuromuscolar disorder 3. A glycogen storage disease

• There 2 types of Pompe disease: an infantile onset, severe form (residual enzyme activity ‹1%) and late onset, chronic form (residual enzyme activity ‹40%) Pompe Disease Incidence (95% CI) Subtype Infantile-onset 1/138,000 (1/43,000-1/536,000) Late-onset 1/57,000 (1/27,000-1/128,000)

Overall incidence* 1/40,000 (1/17,000-1/100,000)

Higher incidence has been found in Taiwan and in Austria Pompe disease was first described by dutch pathologist Johannes Cassianus Pompe in a 7 years old child with heart hypertrophy and massive “vacuolar” glycogen storage in all tissues In 1963 Hers discovered GAA and established its lysosomal localization Acid α-glucosidase (GAA) is essential for glycogen degradation in lysosomes

GAA is glycosilated in the ER (high mannose oligosaccharides), mannose-6-phosphate is added in the Golgi and N-terminal and C-terminal sequences are cleaved Symptoms and signs

• Infantile onset form: • Cardiac involvement: cardiomyopaty/cardiomegaly, heart failure • Hypotonia, motor development delay • Recurrent airways infections, respiratory weakness, death occurs due to cardiorespiratory failure • Feeding problems, failure to thrive, hepatosplenomegaly, megaloglossia

• Late onset form: • Prominent skeletal involvement: progressive muscle weakness, muscular pain • Gastrointestinal abnormalities • Respiratory distress, sleep apneas, dyspneas, airways infections Respiratory distress

Scapular and paraspinal muscles atrophy

Glycogen build-up inside lysosomes affects muscular tissues: the heart in infantile onset forms, the skeletal muscle in late-onset forms

Myofibrils are progressively substituted by glycogen Muscle is progressively destroyed Diagnosis

• α-glucosidase activity is assayed, using the artificial substrate MUG (methyl-umbellilferyl glucopyranoside), in leukocytes, fibroblasts (from muscle biopsy), and dry blood spots

• genetic analysis is essential to identify carriers

• Prenatal diagnosis can be performed on chorionic villi or amniocentesis • α-glucosidase encoding gene (17q25) consists of 19 exons, corresponding to 20kb

• About 180 mutations have been identified: mainly missense, nonsense and splice-site mutations (15%), as well as small deletions (in exon 18) and insertions

• Missense mutations are more common in late onset forms Allele Mutation Ethnic Group Frequency Phenotype Asp645Glu Taiwanese* 0.8 Infantile Arg854X African- 0.5 Infantile American del525T Dutch 0.34 Infantile del exon 18 Dutch 0.23 Infantile IVS 1-13 Caucasian 0.4-0.6 Late t>g splice 3D structure of α- glucosidase from Thermotoga maritima THERAPY

• ERT was first set up in the sixties with Aspergillus niger and placental α-glucosidase; the recombinant human enzyme produced in CHO cells is now used (MYOzyme and LUmizyme)

• ERT has reduced mortality, controlling heart symptoms, but is ineffective against skeletal muscle degeration

• Gene therapy is feasible; adenovirus have been used as vectors.

• Molecular chaperones –iminosugars are found effective in some forms of Pompe disease

Gene therapy timeline for Pompe disease – adenovirus showed the lowest number of immune responses in patients 2011 –first clinical trial (intramuscular injections on 5 3- 14 years old children dipendent on mechanical ventilation). ERT was continued throughout the study. Results showed that therapy is safe and most patients tolerated longer periods of unassisted breathing