Transcript Distrofie muscolari dei cingoli

Different classes of mutations –
mutation detection
Vincenzo Nigro
Dipartimento di Patologia
Generale, Seconda Università
degli Studi di Napoli
Telethon Institute of Genetics and
Medicine (TIGEM)
 For
mutations other than point mutations,
sex biases in the mutation rate are very
variable. However, small deletions are
more frequent in females.
 The total rate of new deleterious mutations
for all genes is estimated to be about three
per zygote. This value is uncertain, but it is
likely that the number is greater than one.
the number of male germ-cell divisions
Relative frequency of de novo achondroplasia for
different paternal ages
The effect of an allele
 null
or amorph = no product
 hypomorph
= reduced amount / activity
 hypermorph
= increased amount / activity
 neomorph
= novel product / activity
 antimorph
= antagonistic product / activity
amorph or hypomorph (1)
 deletion


the entire gene
part of the gene
 disruption

of the gene structure
by insertion, inversion, translocation
 promoter
inactivation
 mRNA destabilization
 splicing mutation


inactivating donor/acceptor
activating criptic splice sites
Point mutations,
which involve
alteration in a single
base pair, and small
deletions or
insertions generally
directly affect the
function of only one
gene
amorph or hypomorph (2)
 frame-shift


in translation
by insertion of n+1 or n+2 bases into the
coding sequence
by deletion of n+1 or n+2 bases into the
coding sequence
 nonsense
mutation
 missense mutation / aa deletion



essential / conserved amino acid
defect in post-transcriptional processing
defect in cellular localization
Loss of function mutations in the
PAX3 gene (Waardenburg s.)
Classical splicing:
conserved motifs at or near the intron ends.
hypermorph
 trisomia
 duplication
 amplification
(cancer)
 Chromatin derepression (FSH)
 trasposition under a strong promoter

leukemia
 overactivity
of an abnormal protein
neomorph
 generation
of chimeric proteins
 duplication
 amplification
(cancer)
 missense mutations
 inclusion of coding cryptic exons
 usage of alternative ORFs
 overactivity of an abnormal protein
antimorph

missense mutations
 inclusion of coding cryptic exons
 usage of alternative ORFs
Gene conversion
Human Gene Mutation Database
 The
Human Gene Mutation Database
(HGMD)
 Locus-Specific Mutation Databases
 Springer LINK: Human Genetics Mutations Submission Form
 Nomenclature for the description of
sequence variations (mutation
nomenclature)

nucleotides are designated by the bases (in upper case); A (adenine), C
(cytosine), G (guanine) and T (thymidine)

nucleotide numbering;
 nucleotide +1 is the A of the ATG-translation initiation codon,
the nucleotide 5' to +1 is numbered -1; there is no base 0
 non-coding regions;
• the nucleotide 5' of the ATG-translation initiation codon is
-1
• the nucleotide 3' of the translation termination codon is *1
 intronic nucleotides;
• beginning of the intron: the number of the last
nucleotide of the preceeding exon, a plus sign and the
position in the intron, e.g. 77+1G, 77+2T (when the exon
number is known, IVS1+1G, IVS1+2T)
• end of the intron: the number of the first nucleotide of
the following exon, a minus sign and the position
upstream in the intron, e.g. 78-2A, 78-1G (when the exon
number is known, IVS1-2A, IVS1-2G)


Description of nucleotide changes
substitutions are designated by a “>”-character
 76A>C denotes that at nucleotide 76 a A is changed
to a C
 88+1G>T (alternatively IVS2+1G>T) denotes the G to
T substitution at nucleotide +1of intron 2, relative to
the cDNA positioned between nucleotides 88 and 89
 89-2A>C (alternativelyIVS2-2A>C) denotes the A to C
substitution at nucleotide -2 of intron 2, relative to the
cDNA positioned between nucleotides 88 and 89



deletions are designated by "del" after the nucleotide(s)
flanking the deletion site
 76_78del (alternatively 76_78delACT) denotes a ACT
deletion from nucleotides 76 to 78
 82_83del (alternatively 82_83delTG) denotes a TG
deletion in the sequence ACTTTGTGCC (A is
nucleotide 76) to ACTTTGCC
insertions are designated by "ins" after the nucleotides
flanking the insertion site, followed by the nucleotides
inserted
NOTE: as separator the "^"-character is sometimes used
but this is not recommened (e.g. 83^84insTG)
 76_77insT denotes that a T was inserted between
nucleotides 76 and 77
variability of short sequence repeats, e.g. in
ACTGTGTGCC (A is nt 1991), are designated as
1993(TG)3-6 with nucleotide 1993 containing the first TGdinucleotide which is found repeated 3 to 6 times in the
population.

insertion/deletions (indels) are descibed as a deletion
followed by an insertion after the nucleotides afected
 112_117delinsTG (alternatively 112_117delAGGTCAinsTG
or 112_117>TG) denotes the replacement of nucleotides
112 to 117 (AGGTCA) by TG

duplications are designated by "dup" after the nucleotides
flanking the duplication site,
 77_79dupCTG denotes that the nucleotides 77 to 79 were
duplicated

inversions are designated by "inv" after the nucleotides
flanking the inversion site
 203_506inv (or 203_506inv304) denotes that the 304
nucleotides from position 203 to 506 have been inverted

changes in different alleles (e.g. in recessive diseases)
are described as "[change allele 1] + [change allele 2]"
 [76A>C] + [76A>C] denotes a homozygous A to C
change at nucleotide 76
 [76A>C] + [?] denotes a A to C change at nucleotide
76 in one allele and an unknown change in the other
allele

two variations in one allele are described as "[first
change + second change]"
 [76A>C + 83G>C] denotes an A to C change at
nucleotide 76 and a G to C change at nucleotide 83 in
the same allele
NOTE: current recommendations use the ";"-character
as a separator (i.e. [76A>C; 83G>C])

A pedigree of digenic inheritance showing how
retinitis pigmentosa occurs only in individuals
who have inherited one mutation in each of
ROM1 and RDS. Heterozygotes for either
mutant allele are asymptomatic
Triallelic inheritance
In the consanguineous pedigree NFB14 both the affected
(03) and the unaffected (04) individuals carry the same
mutation (A242S) in the Bardet–Biedl syndrome gene, BBS6.
Only the affected sibling is homozygous for a nonsense
mutation (Y24X; X indicates a stop codon) in BBS2.
Triallelic inheritance
Three mutations at two loci are necessary for pathogenesis
in this pedigree, as the affected sibling (03) has three
nonsense mutations (Q147X in BBS6, and Y24X and Q59X in
BBS2) and the unaffected sibling (05) has two nonsense
BBS2 mutations, but is wild-type for BBS6..
A similar model involving proteins B and D,
which are members of the same multi-subunit
complex but do not interact directly
Non-allelic complementation
Mutations at one locus (mutated proteins are indicated by
asterisks) are not sufficient to disrupt the formation of the
complex between proteins A and B, although the strength of the
interaction might be reduced (dashed line). A further mutation in
protein B causes disruption of the complex (red cross), resulting
in a detectable phenotype
DNA analysis

Today, in most laboratories the identification of
unknown mutations in candidate genes, causing
human diseases, is performed through manual
scanning of PCR products in affected individuals,
often with accurate preliminary selection

Tomorrow, after the identification of all human genes
and the sequencing of the genome, DNA mutation
scanning in the population will have a significant role
in identifying sequence variations among
individuals
Sequencing

With the ongoing reduction of costs
(today about 5€/run), direct automated
sequencing of PCR products has already
been successfully applied for mutation
detection.

Sequencing is often thought of as the
'gold standard' for mutation detection.

This perception is distorted due to the fact
that this is the only method of mutation
identification, but this does not mean it is
the best for mutation detection
Sequencing problems

FALSE POSITIVE
when searching for heterozygous DNA
differences there are a number of potential
mutations, together with sequence artifacts,
compressions and differences in peak
intensities that must be re-checked by
sequencing with additional primers and
increased costs


FALSE NEGATIVE
loss of information farther away or closer to the
primer

sequencing does not detect a minority of
mutant molecules in a wild-type environment

Strategy for mutation detection
 The
gene is known or unknown?
 Which is the size of the gene?
 How many patients must be examined?
 Expected mutations are dominant or
recessive?
 Mutations have already been identified in
this gene?
 There are other members of the same gene
families (or pseudogenes) in the genome?
Dimension of the mutation detection study
Number of
patients
Gene size
X
Number of
controls
General strategy for mutation detection
screening
of recurrent
mutations
frequent
mutations
are known?
YES
mutations
are identified?
NO
NO
YES
SEQUENCING
mutation
scanning
5’ OH
5’ OH
each primer allele specific contains:
•an obligate mismatch in the last but two 3’- OH base
•a specific mismatch in the last 3’- OH base
5’ OH
5’ OH
Multiplex ARMS
MIX 1
Wt 1
Mut 2
Wt 3
Mut 4
Wt 5
Mut 6
Wt 7
Mut 8
Wt 9
Mut 10
Wt 11
Mut 12
MIX 1
*
MIX 2
MIX 2
Mut 1
Wt 2
Mut 3
Wt 4
Mut 5
Wt 6
Mut 7
Wt 8
Mut 9
Wt 10
Mut 11
Wt 12
Current mutation detection
techniques

SSCP (single strand conformation polymorphism)

HA (heteroduplex analysis)

CCM (chemical cleavage of mismatch)

CSGE (conformation sensitive gel
electrophoresis)

DGGE (denaturing gradient gel electrophoresis)

DHPLC (denaturing HPLC)

PTT (protein truncation test)

direct sequencing
SSCP
(single-strand conformation polymorphism)

Single-stranded DNA when placed in a nondenaturing solution folds into a specific structure
determined by its sequence
 Differences as little as 1 base can generate
different conformations
 This is visualized by a difference in
electrophoretic mobility of at least one strand
 Structure of ss DNA changes under different
physical and chemical conditions e.g.
temperature, ionic strength, presence of
denaturing agents, etc.
SSCP
single strand conformation polymorphism






Sensitivity
150-bp fragment > 85%
400-bp fragment > 60% (75% with two gels)
Detects both missense and nonsense mutations
Post PCR time: 36-72 hours
(gel preparation, loading and run; autoradiography,
analysis of results)
Use of radioactivity preferred
No special equipment required
DNA or mRNA as starting templates
SSCP

The simplest and fastest PCR product
screening techniques, like SSCP (single
strand conformation polymorphism) often
gives unsatisfactory results for its low
sensitivities (when testing G/C-rich and/or long
PCR fragments, when using one condition of gel)

The recurrence of false negatives may
invalidate the screening efforts, since
mutations can be

truly absent

unnoticed in any of the fragments under study
Thus, it could be necessary to re-screen
all samples using a different technique
SSCP
Variations of SSCP
DOVAM-S
Detection of virtually all mutations
 Selected 5 different SSCP conditions with
different buffers and gel matrices
ddF
Dideoxy fingerprinting
 Sequencing followed by non-denaturing
electrophoresis
Mutation detection by
heteroduplex analysis:
the mutant DNA must
first be hybridized with
the wild-type DNA
to form a mixture of
two homoduplexes and
two heteroduplexes
Heteroduplex analysis
Variations of HA
CSGE
conformation sensitive gel electrophoresis
 Mildly denaturing conditions induce
conformational changes (bends) in ds DNA
 This increase differential migration patterns for
homo- and heteroduplexes
UHG
universal heteroduplex generator
 with multiple mismatches to have an higher
chance of detection
CSGE
conformation sensitive gel electrophoresis

Sensitivity
300-bp fragment > 95%
500-bp fragment > 80%
 Detects both missense and nonsense mutations
 Post PCR time: 24-36 hours
(gel preparation, loading and run; staining,
analysis of results)
 Use of radioactivity not necessary
 No special equipment required
 DNA or mRNA as starting templates
DGGE
denaturing gradient gel electrophoresis






Sensitivity
300-bp fragment > 98%
500-bp fragment > 90%
Detects both missense and nonsense mutations
Post PCR time: 24-36 hours
(gel preparation, loading and run; staining, analysis
of results)
Use of radioactivity excluded
Special equipment required
Cumbersome preparation of the gel
DGGE

The sensitivity is adequate, but the set-up work
load is much heavier

DGGE is thus well suited for the repetitive
analysis of a given DNA region, following a
careful optimization

The ideal application for DGGE is the diagnosis
of a monogenic disorder in many patients by
testing a small gene (i.e., beta globin)

For most research projects this technique is
unsatisfactory, since too many PCR products
must be optimized to test a few genes
Chemical cleavage of mismatch
PTT
protein truncation test

Sensitivity
1000-bp fragment > 85%
 Detects only nonsense mutations
 Post PCR time: 48-72 hours
(translation/trascription, gel preparation, loading and
run, analysis of results)
 Use of 35S radioactivity
 No special equipment required
 mRNA as starting template
Applications of PTT
(% of truncating mutations)

Polycystic Kidney Disease PKD1
95%

Familial Adenomatous Polyposis APC
95%

Ataxia telangiectasia ATM
90%

Hereditary breast and ovarian cancer BRCA1-2
90%

Duchenne Muscular Dystrophy DMD
90%?

Fanconi anemia FAA
80%

Hereditary non-polyposis colorectal cancer hMSH1-2 70%-80%

Neurofibromatosis type 2 NF2
65%

Hunter Syndrome IDS
50%

Neurofibromatosis type 1 NF1
50%

Cystic Fibrosis CFTR
15%
Dimension of the mutation detection study
Number of
patients
Gene size
X
Number of
additional
controls
Number of
controls Number of
additional
controls
Number of
additional
controls
TMHA DHPLC






temperature modulated heteroduplex analysis
denaturing HPLC
Fully automatic
Sensitivity for a 300-bp fragment: > 99%
Detects both missense and nonsense
mutations
Post PCR time: 3-40 minutes
(annealing of samples, machine set up,
analysis of results)
Use of radioactivity excluded
Requires a special expensive device
DHPLC strategy

Integrated analysis by PCR and DHPLC of
all DNA samples from both isolated and
familial cases of muscular dystrophy
 It is convenient to carry out simultaneous
analysis of many samples, including
controls
 All DNA are checked for mutations in a
single DNA fragment, then we proceed to
study another fragment
 The costs of the analysis can be reduced to
1/10 of the cheapest sequencing procedure
with comparable sensitivity
PLATE B
PLATE A
POOLED PLATES
A+B
DHPLC analysis
Case 1
 The
gene is known
 It is composed of 5 small size exons
 There are 10 patients, sons of
consanguineous parents
 Expected mutations are homozygous
 Mutations have never been identified in this
gene
 There is no other member of the same gene
families (or pseudogenes) in the genome
Case 2
 The
gene is known
 The putative function of the gene product is
to serve as a transcription factor
 Expected mutations are dominant
 Mutations have never been identified in this
gene
 There are other members of the same gene
families (or pseudogenes) in the genome