Molecular Diagnosis

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Molecular Diagnosis

• Genetic diseases
• Single gene mutation
• Chromosome abnormalities (Downs syndrome)
• Multi-factorial inheritance
• Mitochondrial DNA mutations
• Infectious agents
• Cancers
• Single nucleotide polymorphism and diseases

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Molecular diagnosisGenetic testing

• Reference Book
• Human Molecular Genetics
• 3rd Edition
• ISBN 0-8153-4184-9

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Introduction

• The choice of materials to test
• DNA
• RNA
• Protein
• Three possible questions
• Gene testing has to be targeted.
• Does the patient have any mutation in this
  particular gene that may cause disease.
• Does the patient have a 3-base deletion of the
  codon for F508 in his CFTR gene?

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RNA has advantages and disadvantages

• Advantages
• Without introns
• RT-PCR can detect aberrant splicing
• Disadvantages
• Less convenient to obtain and work with
• Handle with extreme care and process rapidly to
  avoid degradation
• Gene of interest may not be expressed in readily
  accessible tissues
• Many mutation results in mRNA instability

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Genetic diseasesSingle gene mutation

• Autosomal dominant disorders
• e.g. Huntingtons disease (CAG)n
• Autosomal recessive disorders
• e.g. b- thalassemia
• Sex-linage inheritance disorders
• X-linked dominant
• ?????
• ?????? ????
• ??????
• ??????????heterozygotes ????
• ?fragile X
• X-linked recessive
• ????????
• ????
• ????
• ?DMD????

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Molecular basis of hereditary diseases

• Huntingtons disease
• (CAG)n
• Myotonic dystrophy
• (CTG)n or (CCTG)n
• Fragile X chromosomes
• (CGG)n
• Cystic fibrosis
• (single nucleotide change of CFTR)
• Duchenne muscular dystrophy
• (65 deletion, 30 non-sense mutation)
• Sickle cell anemia
• (b chain 6th amino acid E? V substitution)
• b-thalassemia
• Diminished (b or b)or absent (b0 ) of b-globin

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Laboratory diagnosis of trinucleotide repeat
diseases

• Huntington disease (CAG)n
• polyglutamine repeat
• A fragment is amplified by PCR, PAGE, silver
  stain
• lane 1,2,6,10 are normal, others are affected
  people
• Myotonic dystrophy (large expansion)
• Southern blot, bands of 9,10 kB are normal
• Fragile X (CGG)n
• Southern blot, DNA is digested with EclXI EcoRI
• The DNA of the inactivated female is methylated
• EclXI is sensitive to methylation (do not
  cut)
• Hybridize to Ox1.9 probe

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• The protein made by the Huntington's gene is
  called huntingtin
• Huntingtin indirectly leads to nerve cell damage
  and toxicity is through the formation of protein
  aggregates and neuronal inclusions.

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Huntingtons disease
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Isolation of Huntingtons gene
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FISH
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Detection of trinucleotide repeat diseases I
(Huntington disease)
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Myotonic dystrophy (??????)

• Type I, Type II, and congenital type

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Myotonic dystrophy
DM1 and CMyD are caused by an abnormal
trinucleotide (CTG) repeat expansion in the DM1
locus on chromosome 19q13.3. DM2 is caused by
an abnormal tetranucleotide (CCTG) repeat
expansion in the DM2 locus on chromosome 3q21.
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Detection of trinucleotide repeat diseases II
(Myotonic dystrophy)
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Fragile X chromosome

• Characterizied by satellite regions visible at
  the ends of metaphase chromosomes. These are due
  to a long series of CGG triplet repeats. (due to
  backward slippage of daughter strand)
• prominent and elongated ears and long face.
• Most of the affected males have mental
  retardation, and their testes are larger than
  normal.

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Fragile X chromosomes
Fragile X chromosomes are characterizied by
satellite regions visible at the ends of
metaphase chromosomes. Fragile X is associated
with Martin-Bell Syndrome, the most common form
of inherited predisposition to mental retardation
A locus on the human X chromosome contains such a
stretch of nucleotides in which the triplet CGG
is repeated 5-50X
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Detection of trinucleotide repeat diseases III
(Fragile X)
F Fully expanded and methylated NM
Methylated X do not cut with EclXI P
Unmethylated premutation N X in normal male
and active normal female
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Scanning a gene for mutation

• Methods based on sequencing
• Methods based on detecting mismatches or
  hetero-duplexes
• Methods based on single-strand conformation
  analysis (SSCP)
• Methods based on translation
• Methods for detecting deletion
• Methods for detecting DNA methylation

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Cystic fibrosis

• The defect in a single gene results in the
  production of abnormally viscous mucus secretions
  causing recurrent chest infections, pancreatic
  insufficiency, malabsorption of food and
  intestinal obstruction in the newborn (meconium
  ileus).
• Almost all are single nucleotide mutation in CFTR
  gene.

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CF and DMD pose rather different sets of problem
for DNA diagnosis
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Diagram of how cystic fibrosis is inherited
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Methods based on sequencing

• Sequencing is becoming cheaper and easier (high
  throughput) ? meta PCR of a large gene
• Alternative methods are because they are cheaper
  (e.g. SSCP), quicker (e.g.dHPLC), or give some
  special information (PTT and quantitative PCR),
• PTT is (protein truncation test)
• Exon average 145 bp, a sequencing run 500-800 bp.
  ? meta-PCR

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Meta-PCR (exon-linking PCR)
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Mutation detection by sequencing
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Methods based on detecting hetero-duplexes

• Most mutations occurs in heterozygous form.
• Heteroduplex can be formed simply by heating the
  heterozygous test PCR product to denature and
  then to cooling slowly.
• For homozygous or X-linked ? add some reference
  wild type DNA
• Heteroduplexes have abnormal motility on
  nondenaturing gels.

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Mutation scanning by dHPLC
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Scanning the CTFR gene for mutationsby SSCP,
heteroduplex and DGGE (denaturing gradient gel
electrophoresis)
SSCP
DGGE
Heteroduplex variants
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• Heteroduplex and SSCP analysis
• Upper panel (SSCP)
• Single stranded DNA run more slowly in the same
  gel
• Lane 1,2 wild type
• Lane 3-8 variants
• Lower panel (Heteroduplex)
• Heteroduplex in lane 3-8
• Denaturing gradient gel electrophoresis (DGGE)
• Exon PCR of CFTR in 9 urea-formaldehyde
  denaturants
• Usually splits into many sub-bands
• Subject A has variant in amplicon 6
• Subject B has variants in amplicon 17 and 24.

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Methods based on detecting mismatches

• Chemical cleavage of mismatch (CCM)
• Cleavage by osmium tetraoxide (very toxic)
• Cleavage by KMnO4
• Enzymatic cleavage of mismatch (ECM)
• Endonulcease VII
• T4 resovase
• Only dHPLC is widely used in major diagnostic
  laboratory

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Mutation scanning by chemical (hydroxylamine)
cleavage of mismatches
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Methods based on translation

• PTT is a specific test for frameshift, splice
  site or nonsense mutations that create a
  premature termination codon.
• It is not useful in mutation like cystic fibrosis
  where mutations are not truncated.
• It reveal approximate location of any mutation
• Technical problems???

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DMD mutation scanning using PTT(protein
truncation test)
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Duchenne Muscular Dystrophy (???????? )

• The etiological cause of DMD is the genetic
  mutation of the dystrophin gene that lead to the
  absence or diminution of dystrophin protein in
  muscle cells.
• Dystrophin gene is the largest gene so far
  identified, which spans approximately 2.5 million
  base pairs on the X-chromosome

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CF and DMD pose rather different sets of problem
for DNA diagnosis
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Roles of Dystrophin in striatal muscle cell

• Dystrophin is an enormous rod-like protein (427
  kDa) localized beneath the inner surface of
  plasma membrane of mature striatal muscle cell
  (myofibers), both skeletal and cardiac.
• The N-terminal domain binds to the F-actin of
  cytoskeletal structures, while the C-terminal
  cysteine-rich domain along with the distal
  C-terminus, anchors to the plasma membrane
  through dystrophin-associated (DAP) glycoprotein
  complexes.
• Thus, dystrophin crosslinks and stabilizes the
  cell membrane and the cytoskeletal structures.
• In Duchenne muscular dystrophy muscle, the
  absence of dystrophin also leads to the
  diminution of the DAP complex including
  dystroglycans and sarcoglycans.

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Symptoms of Duchenne Muscular Dystrophy

• Enlarged calves and progressive muscle weakness
  and failure are the hallmark signs.
• Specific early clinical symptoms can include
• Delayed onset of walking
• Difficulty in performing a standing jump
• A waddled (????) walk
• Difficulty in getting up from the floor.
• Specific late clinical symptoms can include
• Difficulty in getting up from a chair
• The loss of ability to normally climb stairs
• A very wide gaited walk with balance problems

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Methods for detecting deletion

• Homozygous or hemizygous deletion are simple to
  detect (must R/O technical failure)
• Heterozygous deletion of one or more whole exons
  are not detectable when genomic DNA is amplified
  exon by exon
• (SSCP and heterduplex ? negative because of
  whole exon deletion)
• Two or more multiplex PCR reactions will reveal
  98 of the deletions.

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Multiplex screen for dystrophin deletions in males
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Quantitative gene dosage to detect heterozygous
deletion in genomic DNA

• Real time PCR
• By cleavage of a sequence specific
  oligonucleotide labeled with a dye and a quencher
• MAPH (Multiplex Amplifiable Probe Hybridization)
• Genomic DNA is spotted onto a tiny membrane
• Hybridize to a mixture of 40 probes each
  specific for an exon of a gene
• after wash ? multiplex PCR
• exons can be distinguished by the length of the
  PCR products

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During PCR the labeled oligonucleotide hybridizes
to the target sequence and the 5'-dye is removed
by 5' 3'-exonuclease activity of Taq. The
fluorescence of the donor is no longer quenched
and can be measured.
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Multiplex amplifiable probe hybridization (MAPH)
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Detection of DNA methylation

• Excessive or deficient methylation of CpG is a
  common pathogenic mechanism in cancer and
  imprinted gene expression
• Restriction enzyme HpaII cuts only unmethylated
  CCGG whereas MspI cuts any CCGG
• Bisulfite seqeuncing ? when single stranded DNA
  is treated with sodium bisulfite, C ? U but 5-MeC
  is not converted to U

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Restriction enzyme that recognizes DNA methylation
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Testing for a specified sequence change

• Is much simpler problem than scanning a gene for
  presence of any mutation.
• Diagnosis of dieases with limited allelic
  heterogeneity (see table 18-4)
• Diagnosis within a family
• SNP genotyping ? to test a DNA sample for a
  pre-defined sequence variant.

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Many simple methods are available for genotyping
a specified variant

• Allele specific oligonucleotide (ASO)
• Allele specific PCR amplification
• Amplification Refractory Mutation System (ARMS)
• Oligonucleotide ligation assay (OLA)
• Oligonucleotide array for mutation detection
• By hybridization to DNA array (e.g. Affimetrix
  array)
• Arrayed Primer Extension (APEX)

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Sickle cell anemia

• B chain 6th amino acid E? V substitution results
  in polymerization of deoxy form within the red
  cells
• The replacement of A by T at the 17th nucleotide
  of the gene for the beta chain of hemoglobin
  changes the codon GAG (E) to GTG (V).
• The sickled-shape cells block microcirculation
• Stasis ? hypoxia and ischemic infarction of
  liver, kidney, heart, bone, nervous system
• Hemolytic anemia or even DIC

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ASO
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Amplification Refractory mutation system (ARMS)
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Multiplex ARMS to detect cystic fibrosis
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Introducing an artificial diagnostic restriction
site
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Mutant has no ScaI site
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Oligonucleotide ligation assay (OLA)

• Two oligonucleotides are constructed that
  hybridize to adjacent sequences in the target,
  with join sited at the position of the mutation.
• DNA ligase will not join the two oligonucleotide
  unless they are perfectly hybridized.

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Oligonucleotide ligation assay (OLA) using OLA
to test 31 hot spots in CFTR
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