Transcript Mucopolysaccharides

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%) !
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polysaccharides
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Proteoglycans are
glycosaminoglycans
covalently linked to serine residues of
specific core proteins
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Glycosaminoglycan (GAG)
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Proteoglycans
negatively charged compounds
Can bind unspecifically to many other
substances:
– Growth factors
– Cytokines
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Mucopolysaccharide
Definition
 A gel-like substance:
Filling the space of most tissue:
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


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connective tissue
Skin
bone
and cartilage body cells
mucous secretions
synovial fluids
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synovial fluids
The synovial membrane secretes synovial fluid
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Mucopolysaccharidoses
Genetic disorders
Deficiency of enzymes necessary to
breakdown mucopolysaccharides (MPS)
Excessive accumulation of
mucopolysaccharides in body tissues
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Mucopolysaccharidoses
Results:
– many serious physical disorders
– Various genetic deformities such as:
skeletal deformities (especially of the
face)
mental retardation
decreased life expectancy
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MPS: Signs
Hepatomegaly
Splenomegaly
Enlarged tongue
Retinal pigmentation
Hip dislocation
Kyphosis
Heart murmurs
Heart valve damage from thickening
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Examples
Hunter syndrome
Hurler syndrome
Scheie syndrome
Sanfilippo syndrome
Morquio disease
Maroteaux-Lamy syndrome
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Hurler syndrome
type I
(Alpha-L-iduronate deficiency )
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Hurler syndrome (type I)
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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
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Hurler syndrome type I
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Mucopolysaccharidosis I (MPS I) Disease
(Hurler, Hurler-Scheie, Scheie Syndromes)
Key Symptom Images
Hernia
Corneal
clouding
Coarse
facial
features
Claw hand
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MPS (Type I)
The children slowly develop
Coarse, thick, facial features
with low nasal bridge
Prominent dark eyebrows
Progressive stiffness
Mental retardation
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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
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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
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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
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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
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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)
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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
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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.
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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
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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.
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ASO
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Hunter syndrome type II
(Sulpho-idoronide sulphatase deficiency )
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Hunter syndrome type
II
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Hunter syndrome type II
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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
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Sanfilippo syndrome type III
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Sanfilippo syndrome type
III
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Sanfilippo syndrome type III
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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
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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
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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
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Causes
Coarse, thick, facial features
Prominent dark eyebrows
Progressive stiffness
gait disturbances
speech disturbances
decreased mental development
that progresses to severe
mental retardation
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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.
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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
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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
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MPS
Heart valve damage from thickening
MPS-coronary artery:
metachromasia
thickening
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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
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Morquio syndrome Type IV
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Morquio syndrome Type
IV
Skeletal abnormality - hand
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Skeletal abnormality: flattened
vertebrae
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Morquio syndrome Type IV
subtypes A & B
Type IVA: Galactose-6sulfatase deficiency
Type IVB: b-Galactosidase
deficiency
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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
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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.
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Maroteaux-Lamy syndrome
Type V
(N-Acetyl-galactose-amin-4sulfatase (Arylsulfatase B)
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Maroteaux-Lamy syndrome TypeV
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Maroteaux-Lamy syndrome TypeV
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Features and Characteristics
Maroteaux-Lamy syndrome
Coarse facial
features
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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.
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gene or point
mutations:
mutations where
changes are at
molecular level
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Methods for detection
of known mutations
Methods for detection
of unknown mutations
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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
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 Genomic DNA sequencing
ARMS
Amplification Refractory
Mutation System
Allele Specific PCR (ASPCR)
PCR Amplification of Specific
Alleles (PASA)
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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
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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
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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
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Modifications/Adaptions to the
original ARMS methodology
Multiplex ARMS
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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
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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
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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
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PCR-RFLP
1
2
UD -/+
3
M
4
-/-
LSV
UD
5
+/+
6
7
+/+
-/-
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Methods for detection of
unknown mutations
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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)
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Methods for unknown mutations
(diagnostic methods)
These methods are relatively
simple, but still require:
experience and skill to
perform.
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DGGE
Denaturing gradient gel electrophoresis
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DGGE
is often used in diagnostic
laboratories
non-radioactive tracers and
detects almost all mutations
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DGGE
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SSCP
single strand conformation
polymorphism
 simplicity
 clearly by heteroduplex analysis (HA)
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SSCP Analysis
BRCA1 Exon 15, 4650delCA
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Pedigree of a selected
family with breast cancer
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SSCP
Analysi
s
BRCA1,
Exon 20,
Nt 5382
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SSCP Analysis
Exon 11pi BRCA1 MS R1347G
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