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 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

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 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

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

conserved motifs at or near the intron ends. 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 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