1. 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)

2. 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

3. amorph or hypomorph (1) deletion disruption of the gene structure
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

4. Point mutations, which involve alteration in a single base pair, and small deletions or insertions generally directly affect the function of only one gene

5. amorph or hypomorph (2) frame-shift in translation nonsense mutation
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

7. Loss of function mutations in the PAX3 gene (Waardenburg s.)

9. conserved motifs at or near the intron ends.
Classical splicing:
conserved motifs at or near the intron ends.

12. hypermorph trisomia duplication amplification (cancer)
Chromatin derepression (FSH)
trasposition under a strong promoter
leukemia
overactivity of an abnormal protein

13. neomorph generation of chimeric proteins duplication
amplification (cancer)
missense mutations
inclusion of coding cryptic exons
usage of alternative ORFs
overactivity of an abnormal protein

14. antimorph missense mutations inclusion of coding cryptic exons
usage of alternative ORFs

19. Gene conversion

20. 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.

21. 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..

22. 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

23. Sequencing problems FALSE POSITIVE FALSE NEGATIVE
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