Identificazione di geni responsabili di malattie genetiche: l`esempio

Identificazione di geni
responsabili di malattie
genetiche: l’esempio del braccio
corto del cromosoma X
Brunella Franco, TIGEM and
Universita’ “Federico II, Naples,
Italy
[email protected]
MAP= LINEAR ORDER OF KNOWN ELEMENTS
PHYSICAL MAP
GENETIC MAP
- Polymorphic markers
GENE MAP
- Genes
- DNA clones
SEQUENCE MAP
- A,C, T,G
CYTOGENETIC MAP
- Chromosome bands
DISEASE MAP
- Disease loci
Mappe fisiche
•  Yeast artificial chromosomes (YAC)
500-1000kb
•  Bacterial artificial chromosomes
(BAC) 120-150kb
•  P-1 derived artificial chromosomes
(PAC) 120-150kb
•  Cosmidi 40-60kb
•  Fagi 10-20kb
Mappe genetiche
•  Stabiliscono la posizione di markers
polimorfici sul genoma
•  Sono basate sulle distanze genetiche
ottenute con lo studio della frequenza di
ricombinazione
Timeline of large-scale genomic analyses
GENE IDENTIFICATION
MUTATION
IDENTIFICATION
MOLECULAR
DIAGNOSIS
PROTEIN
FUNCTION
GENE
EXPRESSION
DRUG
THERAPY
GENE
THERAPY
General strategies for
identifying human disease
genes
•  Functional cloning
•  Positional cloning
•  Position-independent candidate gene
approaches
•  Positional candidate gene approaches
Biochemical
basis known
Purificatio Function
n protein al test
product
Unknown
biochemical
basis
Positional
Cloning
Insights from
Animal models
Candidate genes
Functional
Cloning
Chromosomal
localization
Positional
candidate,
candidate
gene
approach
Functional cloning
This approach relies on the knowledge of the
function of an unidentified disease gene in order to
identify the responsible gene.
Two approaches are used:
A) those which depend on the availability of the purified
gene product
B) and those for which a functional assay is required
(Complementation test).
This approach has been useful in only a few cases.
Functional cloning
Complementation test
Complementation of the pathological phenotype
observed in cells with DNA fragments or normal
human chromosomes
E.g.: Cloning of the gene for Fanconi Anemia
and Multiple Sulfatase deficiency
Analisi di complementazione
FA-A
FA-A
FA-B
Fusione
Fusione
FA
fenotipo
wt
fenotipo
Clonaggio Funzionale
Libreria di
cDNA
Trasfezione
Selezione con
MMC e DEB
Cellule FA
Isolamento del
DNA episomale
Gene FA
MULTIPLE SULFATASE DEFICIENCY
CLINICAL FEATURES: A combination of features of all
individual sulfatase deficiencies (various types of
mucopolysaccharidoses, metachromatic
leukodystrophy, ichthyosis etc…)
INHERITANCE PATTERN: Autosomal recessive
BIOCHEMICAL FEATURE: Deficiency of all measured
sulfatase activities (attivita delle solfatasi e’
misurabile nelle cellule)
Problems:
-
Protein involved unknown
-
No patient with chromosomal
abnormality
-
No familial cases suitable to perform
linkage mapping
Microcell Mediated Chromosome Transfer
DONOR CELLS:
Mouse/human monochromosome hybrids
(HPRT deficient with Hygrom. resistant human
chromosomes 1 to 22)
Micronucleation and enucleation
MMCT
x
x
MSD cells
XMMCT
γ irradiation
selection
Microcells
selection
MSD cells
Double selection of hybrid clones
with Hygromicin B and HAT
Complementation test: ARSA, ARSB, and ARSC enzymatic assay
Microsatellites analysis
identification of the complementing chromosome
or of the minimal chromosome complementing region
The gene mutated in MSD maps to chromosome 3p26
D3S3630
D3S2397
3p26.2
3p26.1
2.4 Mb
ILR5A
TRNT1
LOC1185
KIAA1497
SUMF1
SETMAR
AK075459
IPTR1
Cosma MP, Pepe S, Annunziata I, Newbold RF, Grompe M, Parenti G, Ballabio A. Cell 2003
Functional cloning
Purification of the protein product
A) Gene fragments are obtained through
sequencing after isolation of the protein
product
E.g.,: cloning of the gene for Hemophilia A
(FVIII)
B) Use of Specific antibodies
E.g.,: cloning of the gene for PKU due to
X-linked
icthyosis due
to STS
deficiency
CLINICAL FEATURES
•  scaly skin (ichthyosis)
•  hypogonadotropic
hypogonadism
•  inability to smell
(anosmia)
CLONING OF STEROID SULFATASE (STS) cDNA
cDNA SOURCE: Library prepared from human placenta mRNA in λGT11
vector
PROBE USED: Anti-STS polyclonal antibodies
NUMBER OF P.F.U. SCREENED = 100,000
NUMBER OF POSITIVE RECOMBINANTS AFTER IMMUNOSCREENING = 6
two positive clones were mapping to the X chromosome
one to the Xp22.3
region
STS
Ballabio A, et al. “Isolation and characterization of a steroid sulfatase cDNA
clone:
genomic deletions in patients with X-chromosome-linked ichthyosis”.
Proc Natl Acad Sci USA, 84: 4519-4523, 1987.
Male patients with Xp22 deletions and transloca
Clinical features
SS CDPX
Interstitial
deletions
Terminal
deletions,
X/Y
translocations
MRX
XLI
KAL
OA1
1
1
>100
1
2
2
1
1
1
1
~ 20
~ 10
1
Cell line (BA number)
169 311 364 59
Chromosomal abnormality
X;Y X;Y X;Y X;Y
Locus
Probe
STS
PABX
39
GM3
63
+
+
+
M115
120
+
+
+
DXS1145 YHX2-R
2
+
+
+
+
DXS31
M1A
56
+
+
+
DXS89
pTAK10
54
+
+
DXS1060 AFM205tf2
62
+
+
+
GM37
110
+
+
+
+
DXS996
AFM212xe5
61
+
+
+
EST 01879
72
+
+
+
+
M15
126
+
+
+
+
DXS1139 S232A2
3
br MP d
82
+
DXS1130 A117C12e
7
STS
STS 5'untr.
4
+
+
STS
STSexon10
5
DXS1131 GX4HL
48
+
DXS1133 X04A
6
+
+
GM57
128
+
+
+
DXS237
GMGX9
8
+
DXS1132 GX3RL
47
+
DXF22S1 G1.3a
11
sWXD600
104
+
+
+
DXS278
S232B
10
DXS1134 S232C
13-14
KAL
KALexon9
15
+
KAL
KALexon3
79
DXF22S2 G1.3b
16
+
DXS1138 B255C2-L
17
XF10H1.3
19
DXS143
dic 56
20
DXS404
P4
21
GM12
65
sWXD166
98
DXS187
pHAX5
55
DXS1137 210B5-R
22
DXS1140 113E10-R
GM6
66
DXS410
P45
23
DXS1136 61B3-L
74
DXS70
18-55
24
61 120 16
ID X;Y ID
18
ID
19
ID
20
TD
23
ID
88 139 91 115 338 75 175 304 35 46
ID X;Y X;Y ID X;Y X;Y X;Y X;Y X;Y X;Y
-
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
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+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
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+
+
+
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-
+
+
-
-
+
+
(+)
-
+
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+
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+
+
+
+
+
+
+
-
+
+
-
+
+
-
(+)
+
+
(+)
+
-
-
-
-
-
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
-
-
-
-
-
-
-
-
-
+
+
+
+
+
+
+
+
+
+
-
-
+
63
ID
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
-
71
ID
-
+
-
13
ID
+
+
+
+
+
+
+
-
-
-
+
+
+
+
+
+
+
+
+
+
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-
+
+
+
-
-
-
-
-
-
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
80
ID
+
+
+
-
57
TD
+
+
+
-
+
+
-
-
-
-
+
+
+
+
+
+
+
+
+
+
+
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+
+
+
Xp22.3 disease genes
Xp22
.3
.2
.1
SS
Short stature
CDPX
X-linked recessive chondrodysplasia punctata
MRX
X-linked mental retardation
STS
X-linked ichthyosis (STS deficiency)
KAL
X-linked Kallmann syndrome
OA1
Ocular albinism type 1
Biochemical
basis known
Purificatio Function
n protein al test
product
Unknown
biochemical
basis
Position
al
Cloning
Insights from
Animal models
Candidate genes
Functional
Cloning
Chromosomal
localization
Positional
candidate,
candidate
gene
approach
Positional cloning
Isolating the gene knowing only its
chromosomal location, without using any
information about the pathogenesis or the
biochemical function.
The general approach is to to built physical and genetic
maps of the critical region, refine the subchromosomal
localization, and then identify genes in the region to
investigate as
candidate genes. Positional cloning
remains ardous, and is
becoming unnecessary as
information accumulates which allows a positional
candidate gene approach.
Positional cloning
Definition of the critical region
Linkage analysis
Loss of heterozygosity (LOH)
Chromosomal abnormalities
Positional cloning. Definition of the
critical region
Linkage analysis
On the basis of the linkage studies a disease
gene is assigned to a cromosomal region by
virtue of the cosegregation with a locus located
to the same chromosomal region.
Drawback: Availability of adequate families
and
informative markers
Positional cloning. Definition of the
critical region
Chromosomal abnormalities:
•  Translocations and inversions
•  Deletions and duplications
Karyotyping
Fluorescent in situ hybridization
(FISH)
Array CGH
Molecular studies
Positional cloning. Identification of
candidate genes within the critical region
Transcription maps
CpG islands
Zoo Blotting
cDNA selection
Exon trapping
DNA sequencing
ESTs search
Genome
project.
Databases
searching
Xp22.3 disease genes
Xp22
.3
.2
.1
SS
Short stature
CDPX
X-linked recessive chondrodysplasia punctata
MRX
X-linked mental retardation
STS
X-linked ichthyosis (STS deficiency)
KAL
X-linked Kallmann syndrome
OA1
Ocular albinism type 1
Male patients with Xp22 deletions and translocations
Clinical features
SS CDPX
Interstitial
deletions
Terminal
deletions,
X/Y
translocations
MRX
XLI
KAL
OA1
1
1
>100
1
2
2
1
1
1
1
~ 20
~ 10
1
KALLMANN SYNDROME
MAIN CLINICAL
FEATURES
hypogonadotropic hypogonadism due
to Gn-RH deficiency and inability to
smell (anosmia)
ADDITIONAL
FINDINGS
unilateral renal aplasia or hypoplasia
and neurological abnormalities
INHERITANCE
PATTERN
X-linked recessive, autosomal recessive
and autosomal dominant
CLINICAL FEATURES
•  hypogonadotropic
hypogonadism
•  inability to smell
(anosmia)
Kallman Syndrome Critical Region defined by
deletion mapping (1989-1991)
S232C
T
E
L
YAC2R
50 Kb
KAL
t(X;Y) 2
t(X;Y) 1
C
E
N
Junction fragment
Zoo blotting
with the junction
fragment
Franco B, et al. “A gene deleted in Kallmann's syndrome shares homology with neural
cell adhesion and axonal path-finding molecules”. Nature 353: 529-536, 1991.
Xp22.3 disease genes
Xp22
.3
.2
.1
SS
Short stature
CDPX
X-linked recessive chondrodysplasia punctata
MRX
X-linked mental retardation
STS
X-linked ichthyosis (STS deficiency)
KAL
X-linked Kallmann syndrome
OA1
Ocular albinism type 1
X-Linked recessive
Chondrodysplasia Punctata (CDPX)
CLINICAL
FEATURES
PATHOGENENESIS
Unknown
Facial dysmorphism (nasal
hypoplasia), epiphyseal stippling
in early infancy, distal phalangeal
hypoplasia
PHYSICAL MAP OF THE CDPX REGION (1992-1994)
Xpter
BA311
705-4
DXF28F1
455R
511L
455-2
19H-2 DXS284 19H-1 455L
NB6R
19H12 (440Kb)
NB6F12 (320Kb)
455B10 (290Kb)
YACs
511A5 (480Kb)
481C6 (290kb)
18
Cosmids
Exon trapping
products
32
76
66
75
58
48
96
357L
BA126d
PABX
BA169
BA364
CDPX (650-700kb)
21-L
Alignment of exon 76.15 with
sulfatases
3
36
30
20
5
44
23
26
6
51
RPNVVLLLTDDQD-EVLGGMT
ALNVLLIIVDDLR-PSLGCYG
PPNILLLLMDDMGWGDLGVYG
PPNIVLIFADDLGYGDLGCYG
PPNILLIFADDLGYGDLGSYG
PPHLVFLLADDLGWNDVGFHG
PPHLVFVLADDLGWNDVSFHG
RPNIILVMADDLGIGDPGCYG
GPNFLLIMADDLGIGDLGXYG
KPNILLIMADDLGTGDLGCYG
KPNVVLLVADDMGSGDLTSYG
KPNVVVFLLDDVGWMDVGFNG
RPNVIVIIADDMGYSDISPFG
G6S
IDS
Gal6S
ARSA
ARSA m.musc.
ARSB
ARSB cat
ARSC
ARSC rat
Exon 76.15
SU-ARS
ARS-A/B coli
ARS-A/B Kleb.
PHYSICAL MAP OF THE CDPX REGION
Xpter
BA311
705-4
DXF28F1
455R
511L
455-2
19H-2 DXS284 19H-1 455L
NB6R
19H12 (440Kb)
NB6F12 (320Kb)
455B10 (290Kb)
YACs
511A5 (480Kb)
481C6 (290kb)
18
Cosmids
Genes
32
76
66
75
58
48
ARSD ARSE
96
ARSF
357L
BA126d
PABX
BA169
BA364
CDPX (650-700kb)
21-L
ARSE mutations in CDPX
Patient
Nucl eoti de
Change
BA 440
BA 337
BA 339
BA 396
BA 383
G
G
G
G
G
C
A
C
T
C
A mi no A ci d
Substi tuti on
A rg12
Gl y117
A rg111
Gl y137
Gl y245
Ser
A rg
Pro
Val
A rg
Exon
2
4
4
4
5
Franco B, et al. “A cluster of sulfatase genes on Xp22.3: mutations in
chondrodysplasia punctata (CDPX) and implications for warfarin Embryopathy”.
Cell 81: 1-20, 1995.
Xp22.3 disease genes
Xp22
.3
.2
.1
SS
Short stature
CDPX
X-linked recessive chondrodysplasia punctata
MRX
X-linked mental retardation
STS
X-linked ichthyosis (STS deficiency)
KAL
X-linked Kallmann syndrome
OA1
Ocular albinism type 1
Nettleship-Falls type ocular albinism
(OA1)
•  Inheritance : X-linked
•  Prevalence: 1:50,000
•  Eyes : Albino pupillary reflex ,Depigmented fundus and prominent
choroidal vessels, Foveal hypoplasia, Nystagmus,
Photophobia,Impaired vision, Impaired stereoscopic vision
•  Skin : Normal pigmentation
•  Histological analysis :
Abnormally giant melanosomes, called macromelanosomes
•  Optic nerve defect :
Reduction of ispilateral component of the optic tract
Positional cloning of the gene
responsible
for OA1
CEN
TEL
0
0.1
0.2
0.3
0.4
0.5
0.6
131D2
0.7
0.8
0.9
1
Mb
YACS
A118A8
187H6
COSMIDS
OA1 APXL
BA127
BA163
BA38
BA199
OA1 CRITICAL REGION
PATIENTS
COMPARISON OF MELANOSOMAL SIZE
IN WILD-TYPE AND Oa1 MUTANT RPE
P5
P7
Incerti B, Oa1 knock-out: new insights on the pathogenesis of ocular
albinism type 1. Hum Mol Genet. 2000 Nov 22;9(19):2781-8.
Surace EM, et al.,
“Amelioration of both functional and
morphological abnormalities in the retina
of a mouse model of ocular albinism
following AAV-mediated gene transfer”.
Mol Ther. 2005 Oct;12(4):652-8.
Male patients with Xp22 deletions and transloca
Clinical features
SS CDPX
Interstitial
deletions
Terminal
deletions,
X/Y
translocations
MRX
XLI
KAL
OA1
1
1
>100
1
2
2
1
1
1
1
~ 20
~ 10
1 BK
CLINICAL FEATURES
•  Short stature
•  Nasal hypoplasia
•  Focal calcif. of cartilage
SS
CDPX
•  Distal phalangeal hyp
•  Mental retardation
•  Scaly skin (ichthyosis)
•  Hypog hypogonadism
•  Anosmia
MRX
XLI
KAL
•  Impaired visual acuity
•  Nystagmus, strabismus
•  Photophobia
OA1
Analysis of a terminal Xp22.3 deletion in a
patient with six monogenic disorders:
implications for the mapping of X linked
ocular albinism
A Meindl, D Hosenfeld, W Brückl, S Schuffenhauer,
J Jenderny, A Bacskulin, H-C Oppermann, O. Swensson,
P Bouloux, T Meitinger
Positional cloning. Identification of
candidate genes within the critical region
Transcription maps
CpG islands
Zoo Blotting
cDNA selection
Exon trapping
DNA sequencing
ESTs search
Genome
project.
Databases
searching
The death of Positional Cloning:
“THE POSITIONAL CANDIDATE GENE
APPROACH”
Positional candidate gene
approach
Once a disease has been mapped, it
is
possible to use database
searches to identify candidate genes.
Positional candidate gene
approach
Position dependent
Position independent
Position-independent
candidate gene approach
A candidate gene for a human disorder may be
suggested without any knowledge of the
chromosomal location. This can happen if the
phenotype resembles another phenotype in
animals or humans for which the gene is know, or
if the molecular pathogenesis suggests that the
gene may be a member of a known gene family.
Such approaches have only rarely been succesful,
and have been overtaken by a positional candidate
gene approaches
E.g.: cloning of the genes responsible for spinocerebellar
ataxia 8 (SCA8, Kobb et al 1999); rodhopsin for Retinitis
pigmentosa, Waardenburg syndrome type 2 (MITF, Hughs et
al 1994) and IV (SOX10, Pingault et al., 1998)
Positional candidate gene
approach
Position dependent
Position independent
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=O
GENES
A
B
C
Tissue specificity
Imprinting
Sequence domains
Instability
D
E
Developmental expression
Mouse synteny
DISEASES
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a
b
c
d
e
Anticipation
Developmental defect
Tissue involved
Biochemical defect
Mouse model
Imprinting
Positional Candidate
Gene Approach
CANDIDATE DISEASES
CANDIDATE GENES
Biochemical defect
Sequence domains
Tissue involved
Expression pattern
Imprinted inheritance
Imprinted expression
Developmental defect
Developmental expression
Mouse model
Mouse syntheny
ORAL-FACIAL-DIGITAL SYNDROME TYPE I
(MIM 311200)
• 
OFDI is characterized by malformations of the face,
oral cavity and digits. Distinctive clinical signs are
polycystic kidneys and skin and hair anomalies
(alopecia and miliary skin lesions)
•  Inheritance: X-linked dominant, male lethal
•  Occurrence: Possibly 1:250.000
Identification of the gene responsible for OFDI
DXS85
MID1
3
Xp22 2
19.8 cM
MSL3L1
1
MAEG
RAB9
Cxorf-5 (71-7A)
Xq22
OFD1
Pirin
STK9
DXS7105
Ferrante MI et al. “Identification of the gene for oral-facial-digital type I (OFDI)
syndrome”. Am. J. Hum. Genet. 68: 569-576, 2001.
Mutation analysis in OFDI patients
843 delTT
857delG*
875-876del
837-838delAA*
400-403del
432insT
*702delA
788insG
*702 insA
380-2c>g
311-312insG
*312delG
*294-312del
*432insT
11+2t>c
2
E97K
Q83X
R41X
S74F
H81D
*A79T
3
1173insA
1190-1194del
*1193 insT
412+2del
IVS5-10t>g
312+2del
1
5
6
Q113X
Y226X
7
1268-1272del
1318delC
1409delA
1452-1458del
*1071-1078del
IVS10-2a>t
603 insA
4
*1411+1 g>a
2055delT
1757delG
2038 insA
2175delC
1978-1979delTC
1887-1888insAT
8
9
K291X
10 10a 11
12
13
14
15
16 17 18 19 20 21 22 23
Q474X
*R367Q S434R
*Q275X
Q152X
*N352K
New Mutations
* PKD
Mutations reported by other groups
Possibile uso dell’NGS nella diagnostica delle
malattie genetiche
1)  Targeted resequencing: sequenziamento ristretto (es. un gene
molto grande o un gruppo di geni): quando si ha un forte
sospetto sull’identita’ del gene responsabile
2)  Whole Exome sequencing: sequenziamento di tutte le regioni
codificanti di tutti i geni del genoma: quando non si hanno
sospetti sul gene responsabile o per abbattere i costi del
targeted resequencing
3)  Whole Genome sequencing: tutto il genoma, ancora molto
costoso, piu’ “semplice” metodologicamente
Per arrivare al primo abbozzo (draft) di sequenziamento completo
del genoma umano sono occorsi piu’ di 10 anni a un costo di circa
3 miliardi di dollari.
HOW MANY GENES ARE THERE
IN THE HUMAN GENOME?
Nature 431, 931 - 945 (21 October 2004)
Organism complexity and gene number
Escherichia coli
Saccharomyces cerevisiae
Caenorhabditis elegans
Drosophila melanogaster
Danio rerio
Mus musculus
3,200
6,300
19,100
13,600
21,322
22,000
Homo sapiens
22,000
Fugu rubripes
Arabidopsis thaliana
Tetrahymena thermophila
26,700
25,000
27,000
Gene number does not correlate
with evolutionary status
What is the difference among a man, a
tetrahymena, a mouse and Arabidopsis thaliana?
The difference is not mainly determined by the
number of genes in the genome but rather by the
different ways these genes are used and the different
protein variants they encode
The human genome
- 46 chromosomes
- 20,000-25,000 genes
- 3.000.000.000 nucleotides
- about 2% corresponds to coding
sequences
What about the remaining 98%?
1)  Repeat sequences (about 50%)
2) Non-transcribed DNA: regulatory elements
-  the example of conserved non-coding
sequences
3) Transcribed DNA = non-coding RNAs:
-  microRNAs
HUMAN-MOUSE GENOME
COMPARISON
- About 5% of the human genome is conserved with respect
to mouse.
- Yet only about one-third of the sequence under such
selection is predicted to encode proteins
WHAT IS THE FUNCTIONAL ROLE OF
CNCs?
Nature 431, 988 - 993, 2004
PLoS Biol. 2007 Sep;5(9):e234.
CNCs and genetic diseases (1)
-Mutations in CNCs can be responsible for human genetic diseases,
mostly by genomic rearrangements (see examples below)
Am J Hum Genet. 2005 January; 76(1): 8–32.
CNCs and genetic diseases (2)
-Also point mutations in CNCs can be responsible for human genetic
Diseases
Highly conserved non-coding elements on either side of SOX9
associated with Pierre Robin sequence (an important subgroup of
autosomal dominant forms of cleft palate).
Nature Genetics 41, 359 - 364 (2009)
What about the remaining 98%?
1)  Repeat sequences (about 50%)
2) Non-transcribed DNA: regulatory elements
-  the example of conserved non-coding
sequences
3) Transcribed DNA = non-coding RNAs:
-  microRNAs
The expanding world of non-coding
RNAs
Different types of non-coding RNAs
Housekeeping
rRNA = ribosomal RNA;
tRNA = transfer RNA;
snRNA = small nuclear RNA (including spliceosomal RNA)
snoRNA = small nucleolar RNA (rRNA modification);
Regulatory
Long non-coding RNAs (including a significant fraction of Natural Antisense
Transcripts, NATs)
microRNA;
piRNAs
microRNAs
microRNAs are 21-25 nucleotide small RNAs that negatively regulate
the expression of their TARGET genes in animals. They exert this function
by binding with imperfect base pairing to target sites in the 3’-UTR of
messenger RNAs
To date, over 1000
microRNAs are annotated
in the human genome.
microRNAs regulate
various developmental and
physiological processes. A
specific function has
already been demonstrated
in limb, skin, lung and brain
development.
Transcript degradation or
translational repression
Biogenesis and function of microRNAs
Pri-miRNA= primary microRNA
Pre-miRNa= precursor microRNA
miRNA= mature microRNA
MicroRNA regulate various
developmental and physiological
processes. A specific function has
already been demonstrated in limb,
skin, lung and brain development.
From He & Hannon (2004) Nature Rev Gen 5, 522-531
Genomic organization of microRNAs
MicroRNAs can be:
a)  intragenic, i.e., located within transcriptional units (host genes), more frequently in their
intronic regions and more rarely within exonic regions
Host gene b) intergenic.
miRNA gene 1 gene 2 miRNA -The majority of microRNAs are intragenic (over 50%)
-the expression profiles of intragenic miRNAs are highly correlated to those of their corresponding
host genes, in most cases
miR-184
miRNAs can have diverse expression patterns, similar to proteincoding genes
Karali et al, Invest Ophthalmol Vis Sci., 48(2): 509-15 (2007).
More than 1000 microRNAs identified in mouse and humans
http://microrna.sanger.ac.uk/sequences/
microRNAs
To understand their biological role it is necessary to identify their targets
and define their expression pattern
How to identify microRNA targets?
By computational approaches, how?
Several computational approaches have been developed for the prediction
of miRNA targets based on SEED analysis.
However, none of them can be considered completely reliable all predictions
must be experimentally validated.
microRNA
Possibili ruoli biologici
Processi di sviluppo e differenziamento cellulare
Evidenze in vitro
HeLA cells
Transfection with
miR-1 (muscleenriched)
Up-regulation of
muscle-specific
genes
HeLA cells
Transfection with
miR-1 (muscleenriched)
Transfection with
miR-124 (neuronenriched)
Up-regulation of
neuron-specific
genes
Down-regulation
of direct targets
Up-regulation of
muscle-specific
genes
microRNA
Possibili ruoli biologici
Processi di sviluppo e differenziamento
Evidenze in vivo
Sovraespressione di miR-1
mediante generazione di topi
transgenici
miR-1
Hand2
Alterazione del corretto
sviluppo dei cardiomiociti
Regolazione del
diferenziamento
dei cardiomiociti
microRNA
Possibili ruoli biologici
Funzione di tessuti maturi
Un microRNA, specificamente espresso
nelle isole pancreatiche, miR-375,
controlla la secrezione di insulina
modulando l’espressione del gene target
miotrofina.
Nature 432: 226-30, 2004
L’inattivazione del microRNA miR-1-2
(mediante la generazione di topi
knockout) determina difetti della
conduzione elettrica riscontrabili mediante
analisi elettrocardiografica.
Cell 129: 303-17, 2007
microRNA in malattie genetiche
I microRNA possono avere un ruolo nelle patologie umane?
1)  Cancro
2)  Malattie monogeniche
3)  Malattie multifattoriali
Ruolo dei miRNA nel cancro
microRNA e cancro
I profili di espressione dei miRNA sono
alterati nelle cellule neoplastiche rispetto
alle corrispondenti cellule normali
Calin and Croce, Nature Rev Cancer 2006
microRNA e cancro
Le miRNA signatures consentono
un’accurata classificazione dei tumori
•  tipo cellulare
•  diagnosi
•  staging
•  prognosi
•  risposta alle terapie
Lu et al. Nature 2005
microRNA e cancro
Aumentata espressione del cluster di microRNA 17-92 in varie forme di cancro
(linfomi, medulloblastomi, etc.)
microRNA e cancro
Cell Cycle 9:6, 1031-1036; March 15, 2010
Azione esercitata tramite regolazione, come target diretti degli oncogeni c-Met,
Notch-1 and Notch-2.
I microRNA possono avere un ruolo nelle malattie
monogeniche? microRNA and genetic diseases
Genomic rearrangements
involving miRNA
Mutations in miRNA mature sequences
pri-miRNA
Allelic ablation of specific miRNA
*
Ex: miR-361 in Choroideremia
microRNA
miRNA
*
Target
mRNA 3’UTR
pre-miRNA
involvement
in human genetic
disorders
Mutations in miRNA
target sites
miRNA
(A)n
Target
mRNA 3’UTR
*
Imperfect Target Recognition
Ex: REEP1 in Hereditary Spastic Paraplegia
Ex: miR-96 in DFNA40
(A)n
Genomic rearrangements involving miRNAs
Nature genetics, doi:10.1038/ng.915
Phenotype: microcephaly, short stature and digital
abnormalities
microRNA and genetic diseases
Genomic rearrangements involving miRNA in monogenic diseases are more numerous
than commonly believed
Common aneuploidy syndromes involving microRNAs
PathoGenetics 2009, 2:7
microRNA and genetic diseases
Genomic rearrangements
involving miRNA
Mutations in miRNA mature sequences
pri-miRNA
Allelic ablation of specific miRNA
*
Ex: miR-361 in Choroideremia
microRNA
miRNA
*
Target
mRNA 3’UTR
pre-miRNA
involvement
in human genetic
disorders
Mutations in miRNA
target sites
miRNA
(A)n
Target
mRNA 3’UTR
*
Imperfect Target Recognition
Ex: REEP1 in Hereditary Spastic Paraplegia
Ex: miR-96 in DFNA40
(A)n
microRNA and genetic diseases
Mutation in the microRNA mature sequence
Two different point mutations in the seed region of human miR-96 are
responsible for nonsyndromic progressive hearing loss
microRNA e malattie genetiche
Mutation in the microRNA mature sequence
Am J Hum Genet. 2011 Oct 11.
microRNA and genetic diseases
Genomic rearrangements
involving miRNA
Mutations in miRNA mature sequences
pri-miRNA
Allelic ablation of specific miRNA
*
Ex: miR-361 in Choroideremia
microRNA
miRNA
*
Target
mRNA 3’UTR
pre-miRNA
involvement
in human genetic
disorders
Mutations in miRNA
target sites
miRNA
(A)n
Target
mRNA 3’UTR
*
Imperfect Target Recognition
Ex: REEP1 in Hereditary Spastic Paraplegia
Ex: miR-96 in DFNA40
(A)n
microRNA and genetic diseases
Mutations in miRNA target sites
Two different possibilities:
1)  A mutation can affect a pre-existing microRNA target site
(removal or increase of a physiological inhibition)
2)  A mutation can create a novel microRNA target site
(“pathological” inhibition)
MicroRNAs and genetic disease
1) Mutations affecting a pre-existing microRNA target site
(removal or increase of a physiological inhibition)
Two different point mutations in the 3'-UTR of the REEP1 gene
which have been associated with an autosomal dominant form of
hereditary spastic paraplegia (SPG31) alter the sequence of a
predicted target site for miR-140
microRNA e malattie genetiche
Mutazioni in siti di target per microRNA
Human Molecular
Genetics, 2010, 19:
2015–2027
MicroRNAs and genetic disease
2) Mutations creating a novel microRNA target site
(“pathological” inhibition)
Nature Genetics, 38: 813-18 (2006)
Ipertrofia muscolare in questo ceppo di pecora (mutante spontaneo)
Identificazione, tramite analisi di linkage, del locus responsabile, comprendente il gene
della miostatina (azione inibitoria sulla crescita delle cellule muscolari)
Analisi di mutazione: nessuna mutazione nella sequenza codificante della miostatina
MA riscontro di una mutazione puntiforme nel 3’-UTR che crea un aberrante sito di
target per il microRNA miR-1, altamente espresso in muscolo.
Questa mutazione determina inibizione della miostatina e conseguente ipertrofia muscolare