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)
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
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..
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
