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Mucopolysaccharides or glycosaminoglycans Dr Derakhshandeh, PhD Medical Genetics Mucopolysaccharidosis (Glycosamino-Glycan) Heterogeneous: In clinic & genetic Disease: Lysosomal enzyme defect Mucopolysaccharide are: – Protective polysaccharides – Attached to proteins: proteoglycan: – Hybrid molecules: Protein (# 5%) + polysaccharide (#95%) ! 2 polysaccharides 3 Proteoglycans are glycosaminoglycans covalently linked to serine residues of specific core proteins 4 Glycosaminoglycan (GAG) 5 Proteoglycans negatively charged compounds Can bind unspecifically to many other substances: – Growth factors – Cytokines 6 Mucopolysaccharide Definition A gel-like substance: Filling the space of most tissue: connective tissue Skin bone and cartilage body cells mucous secretions synovial fluids 7 synovial fluids The synovial membrane secretes synovial fluid 8 Mucopolysaccharidoses Genetic disorders Deficiency of enzymes necessary to breakdown mucopolysaccharides (MPS) Excessive accumulation of mucopolysaccharides in body tissues 9 Mucopolysaccharidoses Results: – many serious physical disorders – Various genetic deformities such as: skeletal deformities (especially of the face) mental retardation decreased life expectancy 10 MPS: Signs Hepatomegaly Splenomegaly Enlarged tongue Retinal pigmentation Hip dislocation Kyphosis Heart murmurs Heart valve damage from thickening 11 Examples Hunter syndrome Hurler syndrome Scheie syndrome Sanfilippo syndrome Morquio disease Maroteaux-Lamy syndrome 12 13 Hurler syndrome type I (Alpha-L-iduronate deficiency ) 14 Hurler syndrome (type I) 15 Hurler syndrome Definition type I An inherited disease (AR) Storage of abnormal quantities of this material (mucopolysaccharide) in different body tissues is responsible for the symptoms and appearance of the disease 16 Hurler syndrome type I 17 Mucopolysaccharidosis I (MPS I) Disease (Hurler, Hurler-Scheie, Scheie Syndromes) Key Symptom Images Hernia Corneal clouding Coarse facial features Claw hand 18 MPS (Type I) The children slowly develop Coarse, thick, facial features with low nasal bridge Prominent dark eyebrows Progressive stiffness Mental retardation 19 Symptoms Short stature Full lips with a thick, large tongue Increased body hair (hirsutism) Deafness Stiffness (in joints) Shortness of breath Abnormal bones of spine and claw hand 20 Causes of the Hurler syndrome Inherited as an autosomal recessive trait Metabolic defect: inability – The body's to make an enzyme: lysosomal alpha-L-iduronidase 21 The lysosomal hydrolase: alpha-L-iduronidase (IDUA) one of the enzymes in the metabolic pathway responsible for the degradation of the glycosaminoglycans: heparan sulfate and dermatan sulfate In humans: a deficiency of IDUA leads to the accumulation of glycosaminoglycans resulting in the lysosomal storage disorder mucopolysaccharidosis type I 22 incidence & and risk factors Approximately 1 in 150,000 infants are affected Newborn infants with this defect appear normal at birth By the end of the first year, signs of impending problems begin to develop 23 Tests that may indicate the syndrome Increased excretion of dermatan sulfate and heparan sulfate in the urine Absence of lysosomal alpha-Liduronidase (in cultured fibroblasts) Culture of cells from amniotic fluid obtained by amniocentesis for enzyme testing (prenatal testing) 24 Tests that may indicate the syndrome Abnormal histological staining of white blood cells called metachromasia X-ray of the skeleton X-ray of the spine X-ray of the chest ECG 25 Prevention Genetic counseling: important for parents with a family history of Hurler syndrome Prenatal diagnosis: An amniocentesis in the amniotic fluid are then cultured and the a-L-iduronidase activity in the cells is determined. 26 Genetic cDNA clone encoding human IDUA (alpha-L-iduronidase) : – localize IDUA to chromosome 4p16.3 – confirmed by Southern blot analysis This localization is different from that of a previous report mapping IDUA to chromosome 22 27 28 Identification of mutations in the alpha-L-iduronidase gene (IDUA) that cause Hurler and Scheie syndromes* Chemical cleavage Direct PCR sequencing mutations for MPS-I – delG1702 – R89Q (present in 40% of Scheie syndrome alleles) *Scott et al. Am J Hum Genet. 1993 November; 53(5): 973–986. 29 ASO 30 31 32 33 Hunter syndrome type II (Sulpho-idoronide sulphatase deficiency ) 34 Hunter syndrome type II 35 Hunter syndrome type II 36 Hunter syndrome type II (Sulpho-idoronide sulphatase deficiency ) X-linked Coarse, thick, facial features Progressive stiffness decreased mental development Hepatomegaly (liver enlargement) Splenomegaly (spleen enlargement) Abnormal bone x-rays 37 Sanfilippo syndrome type III 38 Sanfilippo syndrome type III 39 Sanfilippo syndrome type III 40 Sanfilippo syndrome type III Definition Sanfilippo syndrome is one of the hereditary mucopolysaccharide storage diseases it is characterized by the absence of one of several enzymes These enzymes help the body get rid of a substance normally found outside of our cells called a mucopolysaccharide This substance is called heparan sulfate, and in Sanfilippo syndrome, large amounts of it are excreted in the urine 41 Alternative Names Mucopolysaccharidosis type III subtypes A - B – C - D Type IIIA: heparan sulfate sulfatase deficiency Type IIIB: Nacetyl-glucos-aminidasedeficiency Type IIID: N-acetyl-glucosamine-6-sulfate sulfatase deficiency 42 Sanfilippo syndrome Causes an autosomal recessive trait It is possibly the most common of the mucopolysaccharide storage diseases ! It has a relatively late onset rather than during the first year of life 43 Causes Coarse, thick, facial features Prominent dark eyebrows Progressive stiffness gait disturbances speech disturbances decreased mental development that progresses to severe mental retardation 44 Prevention Genetic counseling: important for prospective parents with a family history of Sanfilippo syndrome Prenatal diagnosis: An amniocentesis in the amniotic fluid are then cultured and the enzyme activity in the cells is determined. 45 Sanfilippo syndrome Signs and tests Hepatomegaly (liver enlargement) Splenomegaly (spleen enlargement) Corneas clear Echocardiogram may show thickened heart Abnormal bone x-rays such as thickened skull and oval vertebrae 46 Sanfilippo syndrome Signs and tests Seizures mental retardation Activities of one of the enzymes may be low in fibroblast skin cells Urine may have increased heparan sulfate Abnormal pathological staining character of white blood cells called metachromasia 47 MPS Heart valve damage from thickening MPS-coronary artery: metachromasia thickening 48 Sanfilippo syndrome Severe diarrhea or constipation Severe hearing loss Hyperactivity Aggressive and destructive behavior Poor attention Physical aggression Speech and language delay Sleep disturbance Severe intellectual impairment most often before 6 years of age Mild growth retardation Vision impairment 49 Morquio syndrome Type IV 50 Morquio syndrome Type IV Skeletal abnormality - hand 51 Skeletal abnormality: flattened vertebrae 52 53 Morquio syndrome Type IV subtypes A & B Type IVA: Galactose-6sulfatase deficiency Type IVB: b-Galactosidase deficiency 54 Features and Characteristics children with Morquio syndrome Joint stiffness Mild growth retardation Stiff joints that may not extend fully Without mental retardation ! Abnormal bone x-rays – X-ray of the skeleten – X-ray of the spine – X-ray of the chest 55 Prevention Genetic counseling: important for prospective parents with a family history of Morquio syndrome Prenatal diagnosis: An amniocentesis in the amniotic fluid are then cultured and the enzyme activity in the cells is determined. 56 Maroteaux-Lamy syndrome Type V (N-Acetyl-galactose-amin-4sulfatase (Arylsulfatase B) 57 Maroteaux-Lamy syndrome TypeV 58 Maroteaux-Lamy syndrome TypeV 59 Features and Characteristics Maroteaux-Lamy syndrome Coarse facial features 60 Treatment At the present time, there is no cure for MPS disorders. Enzyme replacement therapy and gene therapy are the two treatments that researchers have been focusing on to eventually cure MPS diseases. There are a number of research institutions around the world working on finding a cure for the MPS diseases including facilities in the United States, Canada, England, and Australia. 61 gene or point mutations: mutations where changes are at molecular level 62 Methods for detection of known mutations Methods for detection of unknown mutations 63 Methods for detection of known mutations PCR and size separation eg. DMD PCR and restriction enzyme digestion eg. SMN exon 7 & 8 Allele specific amplification (ASA) Allele refactory mutation system (ARMS) eg. CF Allele specific oligonucleotide hybridisation (ASO) Dot Blot eg. CF DNA chips eg. Brca1 64 Genomic DNA sequencing ARMS Amplification Refractory Mutation System Allele Specific PCR (ASPCR) PCR Amplification of Specific Alleles (PASA) 65 ARMS Two complementary reactions one contains a primer specific for the normal allele the other contains one for the mutant allele both have a common primer one PCR primer perfectly matches one allelic variant of the target but is mismatched to the other mismatch is located at/near 3' end of primer 66 ARMS genotyping is based on: whether there is amplification in one or both reactions band in normal reaction: only indicates normal allele band in mutant reaction: only indicates mutant allele bands in both reactions indicate a heterozygote 67 Uses Population screening rapid (1 working day) inexpensive non-isotopic Used for testing for B-thalassaemia Cystic Fibrosis alpha-1-antitrysin sickle-cell anaemia Phenylketonuria Apolipoprotein E, etc 68 Modifications/Adaptions to the original ARMS methodology Multiplex ARMS 69 Multiplex ARMS Many genetic diseases have more than one mutation often closely spaced eg CF (over 900 mutations) now known - majority are rare but some a relatively common eg F508, G551D 70 To set up multiplex ARMS Determine commonest mutations in the respective population develop the muliplex ARMS for the commonest mutations validate the results of the multiplex test on samples with known mutations determined via another methology 71 72 Molecular Analysis of Iranian Patients with Duchenne/Becker Muscular Dystrophies. S Kheradmand kia 1, *DD Farhud 1, S Zeinali 2, AR Mowjoodi 2H Najmabadi 3,F Pourfarzad 3, P Derakhshandeh Iranian J Publ Health, Vol. 32, No. 3, pp.47-53, 2003 Multiplex PCR Multiplex PCR Set A Set B Set C 73 74 PCR-RFLP 1 2 UD -/+ 3 M 4 -/- LSV UD 5 +/+ 6 7 +/+ -/- 75 Methods for detection of unknown mutations 76 detection of unknown mutations Small Mutations Physical methods Denaturing gradient gel electrophoresis (DGGE) eg. DMD, Thal Single stranded conformation polymorphism analysis (SSCP) Heteroduplex analysis (HA) 77 Methods for unknown mutations (diagnostic methods) These methods are relatively simple, but still require: experience and skill to perform. 78 DGGE Denaturing gradient gel electrophoresis 79 DGGE is often used in diagnostic laboratories non-radioactive tracers and detects almost all mutations 80 DGGE 81 SSCP single strand conformation polymorphism simplicity clearly by heteroduplex analysis (HA) 82 SSCP Analysis BRCA1 Exon 15, 4650delCA 83 Pedigree of a selected family with breast cancer 84 SSCP Analysi s BRCA1, Exon 20, Nt 5382 85 SSCP Analysis Exon 11pi BRCA1 MS R1347G 86