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Next Generation Sequencing e sue possibili applicazioni in campo - - PowerPoint PPT Presentation
Next Generation Sequencing e sue possibili applicazioni in campo - - PowerPoint PPT Presentation
Next Generation Sequencing e sue possibili applicazioni in campo clinico NGS per Diagnostica Eterogeneit Genetica Il concetto classico di Eterogeneit genetica implica il fatto che la stessa condizione pu essere causata da mutazioni in
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Il concetto classico di Eterogeneità genetica implica il fatto che la stessa condizione può essere causata da mutazioni in geni differenti e questo sta divenendo un problema consistente in Genetica Medica Per alcune malattie il grado di eterogeneità genetica è tale che ci sono più di 100 geni che possono causare la stessa malattia
Eterogeneità Genetica
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La Retinite Pigmentosa (RP) è un gruppo eterogeneo di malattie ereditarie caratterizzato dalla progressiva perdita della vista, cecità notturna e depositi di pigmento nella retina.
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NGS per Ricerca
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Geography of exome sequencing
Miller syndrome Na t G e ne t N ov 0 9 HPMR Na tG e ne t Aug 1 0 Kabuki syndrome Na tG e ne t Aug 1 0 Perrault syndrome A m J Hum G e ne t J ul 1 0 Brain Malformation Na ture Aug 1 0 DFNB82 A m J Hum G e ne t J un 1 0 Schinzel-Giedion Na tG e ne t Apr 1 0
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UNITED WE STAND
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DE NOVO
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COMPOUND HETEROZYGOSITY
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HOMOZYGOSITY
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Sindromi malformative
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Malattie Mendeliane non Sindromiche
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FA2H causes SPG35 and leukodystrophy
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L R G E Q Q V Q P G R G P P L R G E Q Q G S M E N E P V
exon 1 exon 2
Two affected sibs from a consanguineous family carry a c.270+3A>T mutation in the FA2H gene
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Single Nucleotide Variants SNVs Sib1 Sib 2 Validated 20967 22651 Transition to transversion ratio 2.91 2.89 In dbSNP 19727 (94%) 21237 (94%) Heterozygous in dbSNP 10372 11159 Homozygous in dbSNP 9355 10078 Not in dbSNP 1240 (6%) 1414 (6%) Heterozygous not in dbSNP 1086 1234 Homozygous not in dbSNP 130 154 180
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Candidate variants filtering
Cumulative total calls: 22711 Cumulative no dbSNPs: 1968 ROHs in common: 28 NS, in UTRs, SS, nonsense: 28 No 1000 genomes: 8 2 NS (1 gene) damaging by at least one prediction tool (SNP&GO, Polyphen, SIFT) 1 altering SS by at least one prediction tool (NetGene2, GDB) +1bp del
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A novel FA2H c.270+3A>T splice site mutation in an Italian consanguineous pedigree
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Malattie legate al cromosoma X
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1 2 1 I II 1 2 1 2 1 2 1 2 1 2 1 2
Length (Mb) ~ 150 CCDS entries 1153 RefSeq entries 2036 OMIM loci/genes 716 Agilent SureSelect human X chromosome probes 7664 Probe/CCDS genomic regions overlap 95% Probe/RefSeq genomic regions overlap 94%
TARGETED RESEQUENCING OF X CHROMOSOME EXONS TARGETED RESEQUENCING OF X CHROMOSOME EXONS
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X-linked recurrence of a previously undescribed neurocutaneous phenotype Individuals III-2 and III-4: microcephaly, neurodevelopmental delay, ichthyosis, epilepsy, tetraparesis and ACC Male fetus III-3: ACC callosum and hyperkeratosis of epidermis with desquamation at the autoptic examination after voluntary abortion Individuals I-2, II-2 and II-3: mild ichthyosis limited to the extensor surfaces
- f Limbs compatible with a balanced chromosome X inactivation pattern
A CASE STUDIED WITH BOTH “CLASSICAL” A CASE STUDIED WITH BOTH “CLASSICAL” AND “NEXT GENERATION” APPROACHES AND “NEXT GENERATION” APPROACHES
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POSITIONAL CLONING OF A POSITIONAL CLONING OF A PAK3 PAK3 VARIANT VARIANT
Magini et al., submitted
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Xp22.31 STS 7268094 p.L515 P +
- CHR BAND
GEN E hg19 CHANGE SANGER LINKAG E
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Xq13.1 STARD8 67940201 p.G582fs
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Xp22.12 MAP3K15 19443659 p.P477T +
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Xq23 PAK3 110439126 p.K389 N + +