Next Generation Sequencing e sue possibili
applicazioni in campo clinico
NGS per Diagnostica
Eterogeneità Genetica
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
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.
NGS per Ricerca
Geography of exome sequencing
Schinzel-Giedion
N a tG e n e t A pr 1 0
Miller syndrome
Na t G e ne t N ov 0 9
HPMR
N a tG e n e t A u g 1 0
Kabuki syndrome
N a tG e n e t A ug 1 0
DFNB82
A m J H u m G e n e t J un 1 0
Perrault syndrome
A m J Hu m G e n e t J ul 1 0
Brain Malformation
N a tu re A u g 1 0
UNITED WE STAND
DE NOVO
COMPOUND HETEROZYGOSITY
HOMOZYGOSITY
Sindromi malformative
Malattie Mendeliane non
Sindromiche
FA2H causes SPG35 and leukodystrophy
Two affected sibs from a consanguineous family
carry a c.270+3A>T mutation in the FA2H gene
exon 1 exon 2
L
R
G
E
Q
Q
G
S
M
E
N
E
P
V
L
R
G
E
Q
Q
V
Q
P
G
R
G
P
P
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
Candidate variants filtering
Cumulative total calls: 22711
ROHs in common: 28
No 1000 genomes: 8
NS, in UTRs, SS, nonsense: 28
Cumulative no dbSNPs: 1968
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
A novel FA2H c.270+3A>T splice site mutation in an Italian consanguineous pedigree
Malattie legate al cromosoma X
TARGETED RESEQUENCING OF X CHROMOSOME EXONS
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%
I
1
2
1
2
1
2
1
2
II
1
1
2
1
2
1
2
A CASE STUDIED WITH BOTH “CLASSICAL”
AND “NEXT GENERATION” APPROACHES
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
of Limbs compatible with a balanced chromosome X inactivation pattern
POSITIONAL CLONING OF A PAK3 VARIANT
.
Magini et al., submitted
NGS-BASED DETECTION OF A PAK3 VARIANT
CHR BAND
GEN
E
hg19
Xp22.31
STS
7268094
.
.
Xp22.12
MAP3K15
.
Xq13.1
STARD8
.
Xq23
PAK3
CHANGE
19443659
67940201
110439126
SANGER
p.L515
P
+
LINKAG
E
?
-
p.P477T
+
-
p.G582fs
-
-
p.K389
N
+
+
