la consulenza genetica ed itest genetici nella pratica clinica
TRANSCRIPT
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La Consulenza Genetica ed i test genetici nella pratica clinica
La Sindrome di Epstein, la Sindrome di Fechtner e le sindromi correlate
Marco Seri
Siena, Giovedì 24 Settembre 2009
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Un po’ di storia……
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Cataracts
Nephritis
Deafness
Macrothrombocytopenia
Leukocyte inclusion bodiesFECHTNER SYNDROME
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Cataracts
Nephritis
Deafness
Macrothrombocytopenia
Leukocyte inclusion bodies
MAY-HEGGLIN ANOMALYSEBASTIAN SYNDROME
FECHTNER SYNDROME
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In MHA, the “Döhle-like bodies” consist of an amorphous area of cytoplasm
containing clusters of ribosomes oriented along parallel microfilaments
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In SBS, the “Döhle-like bodies” are composed of highly dispersed
filaments and few ribosomes
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D22S278
D22S426
D22S283 4.10 q11-q13
Lod score θθθθ=0.0
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Z82217
Z95114
AL031426
Z82215
AL022302
AL022313
AL031845
Z70289
FTNS/MHA critical region: ca. 480000 bp
TNF-inducible
protein CG12-1
APOL2
APOL
MYH9 TXN2
dJ1119A7. 5
E1F3S7
dJ1119A7.3
CACNG2
D22S683
Cen Tel
D22S278
D22S1173
D22S283
D22S426
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Il gene MYH9 codifica per la catena pesante della miosina non muscolare di tipo IIA (NMMHC-IIA) che è espressa in alcuni tessuti quali piastrine, rene, leucociti e coclea.
Altre miosine non convenzionali sono associate a malattie nell’uomo che mostrano sordità o difetti dell’occhio.
Miosina VIIA Usher syndrome type IB (sordità congenita, areflessia vestibolare e retinite pigmentosa progressiva)DFNB2 (sordità recessiva non sindromica)
Miosina XV DFNB3 (sordità recessiva non sindromica)
Miosina VI Snell's waltzer deafness: modello murino DFNA22 (sordità dominante non sindromica)
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Cataracts
Nephritis
Deafness
Macrothrombocytopenia
Leukocyte inclusion bodies
EPS
TEIN
SYNDROME
MAY-HEGGLIN ANOMALYSEBASTIAN SYNDROME
FECHTNER SYNDROME
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Sarcomeric, smooth muscle and nonmuscle class II myosins
COOH
Exameric enzymes
light chain
heavy chain
COOH
Coiled coil• assembly of bipolar myosin filaments
Motor domain• ATP hydrolysis• actin binding
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Cataracts
Nephritis
Deafness
Macrothrombocytopenia
Leukocyte inclusion bodies
EPS
TEIN
SYNDROME
MAY-HEGGLIN ANOMALYSEBASTIAN SYNDROME
FECHTNER SYNDROME
DFNA17
MYH9-RELATED DISEASE
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•Recurrent mutations (codons 96, 702, 1155, 1165, 1424, 1841, 1933) represents 88% of all mutations identified up to date in the MYH9 gene.
•Deletions and missense or frameshift mutations resulting in a truncated protein have never been described.
•The presence of missense or frameshift mutations have been described only in the last exon, coding for the C-terminal tail which is essential to assemble the molecule in bipolar filaments, suggesting that the dominant negative effect is the pathogenetic mechanism.
•However, it is still being debated if the pathogenetic mechanism is a Dominant Negative effect due to the altered dimer formation orrather if it is Haploinsufficiency.
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A locus presents Haploinsufficiency if more genic product than the one obtained by a single gene allele is necessary for
the expression of the normal phenotype.
The Dominant Negative effect takes place when the genic product of the mutated allele affects the function of the wild type one.
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La patogenesi
Aploinsufficienza?Effetto Dominante Negativo?
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Kunishima S et al. “Immunofluorescence analysis of neutrophil Nonmuscle Myosin Heavy Chain-A in MYH9 disorders: association of subcellular localization with MYH9 mutations” Laboratory Investigation (2003)
Deutsch S et al.”Asp1424Asn MYH9 mutation results in an unstable protein responsible for the phenotypes in May-Hegglin anomaly/Fechtner syndrome”Hemostasis, Thrombosis, and Vascular Biology (2003)
Takubo T et al.”Expression of non muscle type myosin heavy polypeptide 9 (MYH9) in mammalin cells” European Journal of Histochemistry (2003)
Franke JD et al. “Rod mutations associated with MYH9-related disorders disrupt non-muscle myosin-IIA assembly” Blood (2004)
Pecci A et al. “Pathogenetic mechanisms of hematological abnormalities of patients with MYH9 mutations” Human Molecular Genetics (2005)
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It seems that several genetic mechanisms take place in the pathogenesis of the disease in different tissues.
The difficult accessibility of the other tissues involved in the disease (e.g. inner ear and kidneys) makes it extremely difficult to understand the molecular
mechanism leading to the disease.
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MYH9
nephritis
deafness cataracts
leukocyte inclusions
giant platelets
thrombocytopenia
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Protocollo diagnostico
• Striscio di sangue
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Control
Platelet
s
Platelet
s
MGG
mAb agai nst
NMMHC- IIA
Immunoistochimica
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A B
Immunofluorescenza
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Se positivo analisi degli esoni contenenti le
mutazioni ricorrenti, (1, 16, 24, 25, 26, 30,
38, 40) quindi analisi delle altre parti del
gene
Se negativo non viene effettuata la diagnosi
molecolare ma…..
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…….è sempre importante inviare il paziente
alla CONSULENZA GENETICA
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Acknowledge
University of BolognaDr E. PanzaProf M.Seri
University of PaviaDr A. PecciProf C. Balduini
University of GenovaDr.ssa M. MariniProf R. Ravazzolo
University of TriesteDr.ssa F. Di BariProf.ssa A. Savoia
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Setting up an in vitro model to study the effect of MYH9 mutations
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CLONING OF THE ENTIRE MYH9 cDNA
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SCHEMA CLONAGGIO MIOSINA 9
Frammento A Frammento B Frammento C
2338 bp 1675 bp 1865 bp
Aat II Eco RI
MYH9 RT3 MYH9 RTMYH9 RT2
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NMMHC-IIA (red) GST (green)
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COOH
Motor domain Coiled coil
COOH
heavy chains
lightchains
N93K A95T
S96L
R1933X
R1165L
T1155I
S1114P
R702H
R705H
R702C
5828delG
5779delC5774delA
E1841K
R1165C
Del L1205-Q1207
D1424N
D1424H
D1424Y
K371N
381 16 2610 25 4030
MHA
SBS
MHA/SBS
DFNA17
FTNS
EPTS
FTNS/EPTS
E1945X
24
Del E1066-A1072
D1424H
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NMMHC-IIA (red)
Transfection of COS cells with wild-type MYH9 cDNA
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NMMHC-IIA (red)
Transfection of COS cells with D1424H MYH9 cDNA
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NMMHC-IIA (red)
Transfection of COS cells with D1424H MYH9 cDNA
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NMMHC-IIA (red)
Transfection of COS cells with D1424H MYH9 cDNA
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FLAG (D1424H) V5 (WT)
The coThe co--trasfection, revealed by the double FLAG/V5 labelling is good trasfection, revealed by the double FLAG/V5 labelling is good
Cotransfection experiments with MYH9 WT and D1424H
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C D
A B
x
Flag-Tag V5-Tag
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FLAG (D1424H) V5 (WT)
FLAG (mutant) signal has a strong preferential localization in tFLAG (mutant) signal has a strong preferential localization in the subhe sub--membrane regionmembrane regionrather than in the cytoplasm, rather than in the cytoplasm, V5 (WT) signal has a more homogeneous distribution among the suV5 (WT) signal has a more homogeneous distribution among the subb--membrane region membrane region And the cytoplasmAnd the cytoplasm
Cotransfection experiments with MYH9 WT and D1424H
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TRANSFECTED CELLS
A
WILD-TYPE NMMHC-IIA D1424H NMMHC-IIA
B CPERIPHERAL BLOOD
D E F
CO-TRANSFECTED CELLS WILD-TYPE NMMHC-IIAD1424H NMMHC-IIA MERGING
G H I
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In co-transfected cells the mutant MYH9 showed adistribution similar to that observed in cells transfectedwith mutant MYH9 alone, with part of the Flag signal diffused in the cytoplasm and part organized in rodlike aggregates.
Aggregates were globally less frequent than in cells transfected with D1424H cDNA alone.
Confocal analysis demonstrates that WT signal presented a high degree of co-localization with the mutant signal diffused in the cytoplasm, thus suggesting that transfectedWT and D1424H molecules interact in these cells.
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These findings suggest that the NMMHC-IIA mutated in position 1424 is able to interact with the WT form in living cells, despitepart of the mutant protein precipitates in non functional aggregates.
The fact that NMMHC-IIA mutated in position 1424 retains the ability to interact with WT protein represents the basis for it to exert a dominant negative biochemical effect on the normal protein.
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COOH
Motor domain Coiled coil
COOH
heavy chains
lightchains
N93K A95T
S96L
R1933X
R1165L
T1155I
S1114P
R702H
R705H
R702C
5828delG
5779delC5774delA
E1841K
R1165C
Del L1205-Q1207
D1424N
D1424H
D1424Y
K371N
381 16 2610 25 4030
MHA
SBS
MHA/SBS
DFNA17
FTNS
EPTS
FTNS/EPTS
E1945X
24
Del E1066-A1072
D1424H
E1841KR1933X
R702C
R702H
D1424N
R1165C
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Malattie Mendeliane
Malattie Multifattoriali
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MYH9 is associated with nondiabetic end-stage renal disease in African Americans (Nature Genetics 2008)
MYH9 is a major-effect risk gene for focal segmental glomerulosclerosis (Nature Genetics 2008)
Genome-wide linkage analysis of serum creatinine in three isolated European populations (Kidney International 2009)
Polymorphisms in the Nonmuscle Myosin Heavy Chain 9 gene (MYH9) are associated with albuminuria in hypertensive African Americans (American Journal of Nephrology 2009)
Polymorphisms in the Nonmuscle Myosin Heavy Chain 9 gene (MYH9) are strongly associated to end stage renal disease hystorically attribuited to hypertension in African Americans (Kidney International 2009)
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From Nat. Genet. 24:333-335; 2000
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Figure 1
C
A
B
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Two-Hybrid System
MYH9 (NMMHC-IIA) – MYH10 (NMMHC-IIB)
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Future prospectives........
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COOH
Motor domain Coiled coil
COOH
heavy chains
lightchains
WT
R1933X
Y1460X
W828X
∆∆∆∆451X
Deletion constructs
In collaboration with Professor Francesco Blasi, San Raffaele, Milano,Italy.
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WT K371N R702H R702C R1165C D1424H D1424N E1841K E1945X R1933X
Entry Clone 201
(Kanamicina)x x x
Entry Clone 207
(Gentamicina)x x - x - x - x x x
pDEST 26 6xHIS
(Ampicillina)x
pDEST 27 GST
(Ampicillina)x
pcDNA3.1/nV5 DEST-V5
(Ampicillina)x x x
pDEST-FLAG (Kanamicina) x x x x
pDEST53GFP
(Ampicillina)x x x x x x
VECTORS, MUTANTS and MYH9 CLONES
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A B C
D E F
WILD-TYPE NMMHC-IIA D1424H NMMHC-IIA
TRANSFECTED CELL LIN
ES
PERIPHERAL BLO
OD
Panza E, Pecci A, Marini M, Giacopelli F, BozziV, Seri M,Balduini C, Ravazzolo R.“Transfection of the mutant MYH9 cDNA reproduces the most typical cellular phenotype of MYH9-related disease in different cell lines”Pathogenetics 2008 Dec 1;1(1):5.
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FLAG (mutant) V5 (WT)
In some cells the FLAG (mutant) signal has a high irregular distIn some cells the FLAG (mutant) signal has a high irregular distribution, and form ribution, and form structures similar to filaments and spots, while the V5 (WT) sigstructures similar to filaments and spots, while the V5 (WT) signal is more nal is more regularly distributed (microscopic spots uniformly distributed iregularly distributed (microscopic spots uniformly distributed in the cytoplasm). n the cytoplasm).
Cotransfection experiments with MYH9 WT and D1424H
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FLAG (mutata) V5 (WT)
Some aggregates are evident with antiSome aggregates are evident with anti--FLAG (mutant) antibody;FLAG (mutant) antibody;these aggregates are more evident in FLAGpos / V5neg cells orthese aggregates are more evident in FLAGpos / V5neg cells orin cells with weak V5 signal, where WT has not been trasfected oin cells with weak V5 signal, where WT has not been trasfected or seems to be poorly r seems to be poorly expressed.expressed.
Cotransfection experiments with MYH9 WT and D1424H
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FLAG (mutant) V5 (WT)
Some feeble aggregates are evident with the antibody antiSome feeble aggregates are evident with the antibody anti--FLAG (mutant) also in someFLAG (mutant) also in somecells cocells co--transfected FLAGpos / V5pos with strong V5 signal; transfected FLAGpos / V5pos with strong V5 signal; In these cells aggregates are not visible with antiIn these cells aggregates are not visible with anti--V5 (WT) antibody.V5 (WT) antibody.
Cotrasfection experiments with MYH9 WT and D1424H
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FLAG (mutant) V5 (WT)
Cotransfection experiments with MYH9 WT and D1424H
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FLAG (mutant) V5 (WT)
Cotransfection experiments with MYH9 WT and D1424H
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FLAG (mutant) V5 (WT)
Cotransfection experiments with MYH9 WT and D1424H
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�GST signal is not a good tag to follow WT MYH9
�6xHis is not a good tag to follow mutant MYH9
MYH9 WT MYH9 D1424H
Cotransfection experiments in COS7 cell linesCotransfection experiments in COS7 cell lines
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MYH9 D1424H
MYH9 WT
�V5 is good to follow WT MYH9
Cotransfection experiments in COS7 cell linesCotransfection experiments in COS7 cell lines
but still, the overall system is not good enough......
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Looking for a new expression vector........
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Gateway Converting System KitGateway Converting System Kit
FLAG
pCMVpCMV--FLAGFLAG--DESTDEST
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The genotype-phenotype correlation leads to the concept that
the MYH9-RD phenotype results from the joint effect of MYH9
mutations and unknown enviromental and/or genetic factors, such as
multiple gene products.
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21 Families
6FTNS 6EPTS 4MHA 1SBS 4MHA/SBS
47 Patients
38 familial cases 9 sporadic
16FTNS 3FTNS6EPTS 3EPTS9MHA 1MHA3SBS4MHA/SBS 2MHA/SBS
19FTNS 8EPTS 10MHA 3SBS 6MHA/SBS
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MYH9 Mutations Exon FTNS EPTS MHA SBS MHA/SBS
N93K 1 1 case
R702C 16 1 case 2 case 1 case 1 case
R702H 16 3 cases
K910Q 21 1 caso*
Del E1066-A1072 24 1 case
T1155I 25 1 case
R1165C 26 1 case
D1424H 30 3 cases*
D1424H 30 1 case
E1841K 38 1 case
R1933X 40 1 case
E1945X 40 1 case
* same patient
MYH9 mutations are present in 19 out of 21 patients analyzed
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COOH
Motor domain Coiled coil
COOH
heavy chains
lightchains
N93K A95T
S96L
R1933X
R1165L
T1155I
S1114P
R702H
R705H
R702C
5828delG
5779delC5774delA
E1841K
R1165C
Del L1205-Q1207
D1424N
D1424H
D1424Y
K371N
381 16 2610 25 4030
MHA
SBS
MHA/SBS
DFNA17
FTNS
EPTS
FTNS/EPTS
E1945X
24
Del E1066-A1072
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•Alcune mutazioni sono ricorrenti 96 (S->L); 702 (R->C, R->H); 1155 (T->I); 1165 (R->C, R->L); 1424 (D->H, D->N); 1841 (E->K); 1933 (R->X)
•Mutazioni Nonsenso e frameshift colpiscono solo l’ultimo esone del gene MYH9 gene (R1933X, E1945X, 5774delA, 5779delC and 5828delG)
•Solo delezioni “in frame” di pochi aminoacidi sono state identificate nel gene MYH9 (del L1205-Q1207; del E1066-A1072 )
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geni
modificatori
FTNS-EPTS
R1933XMHA-SBS
E1841K
R702C
R702HFTNS-EPTS
D1424H
MHA-SBST1155I
R1165C
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Control
Neutrop
hils
Platelet
s
Neutrop
hils
Platelet
s
MGG
mAb agai nst
NMMHC- IIA
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A
E
B C
F G
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•Pertanto la classificazione di FTNS, EPTS, MHA and SBS come malattie separate appare non corretta ed i criteri clinici utilizzati per questa classificazione vanno riconsiderati.
•Queste malattie mostrano uno spettro continuo di sintomi dalla macrotrombocitopenia, alle inclusioni nei polimorfonucleati fino alla sordità neurosensoriale, alla cataratta ed infine alla nefrite.
•Noi proponiamo così che queste patologie siano considerate come un’unica entità clinica da chiamare “MYH9-related disease”.
•L’espressività variabile della malattia può essere poi spiegata dall’effetto congiunto di specifiche mutazioni, di fattori ambientalicosì come dalla presenza di "modifier genes”.
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Cataracts
Nephritis
Deafness
Macrothrombocytopenia
Leukocyte inclusion bodies
EPS
TEIN
SYNDROME
MAY-HEGGLIN ANOMALYSEBASTIAN SYNDROME
FECHTNER SYNDROME
DFNA17
MYH9-RELATED DISEASE
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•Per meglio definire lo spettro delle malattie da mutazioni nel gene MYH9, abbiamo studiato 3 famiglie aggiuntive che mostrano fenotipi caratterizzati da trombocitopenia associata a vari livelli con sordità, anomalie oculari e nefrite e pertanto in parte sovrapponibili al quadro clinico presente nei pazienti con mutazioni nel gene MYH9.
•L’analisi di linkage e/o l’analisi di sequenza ha escluso un coinvolgimento del gene MYH9 come responsabile delle malattie presenti in queste famiglie.
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Macrothrombocytopenia
Hearing loss
Macrothrombocytopenia
Bilateral Cataracts
Hearing loss
Macrothrombocytopenia
Hearing loss
Nephritis
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•Infine abbiamo raccolto un gruppo di 10 pazienti Alport nei quali era stata ipotizzata una trasmissione autosomica dominante e senza mutazioni nei geni del collagene di tipo 4 (COL4).
•L’analisi di questi casi tramite sequenziamento diretto della regione codificante del gene MYH9 ci ha consentito di escludere un coinvolgimento di questo gene nella patogenesi delle forme dominanti di Sindrome di Alport.
•Tutti questi dati dimostrano che le malattie da mutazioni nel gene MYH9 presentano un fenotipo definito che ha come caratteristica fondamentale la presenza delle inclusioni nei leucociti rilevabili attraverso la microscopia ottica o tramite esperimenti di immunocitochimica.
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Cosa stiamo facendo:
Sistema del doppio ibrido per trovare proteine ineragenti con la MYH9
Sistema del doppio ibrido mirato per proteine candidate
Esperimenti di coimmunoprecipitazione
Esperimenti di trasfezione del gene MYH9 wild-type e mutato
Studi di immunocitochimica su tessuti di pazienti
Modello animale
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CLONAGGIO cDNA MIOSINA NON MUSCOLARE 9
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NMMHC-A (red) GST (green)
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NMMHC-A (red)
Transfection of COS cells with wild-type MYH9 cDNA
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NMMHC-A (red)
Transfection of COS cells with wild-type MYH9 cDNA
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NMMHC-A (red)
Transfection of COS cells with D1424H MYH9 cDNA
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NMMHC-A (red)
Transfection of COS cells with D1424H MYH9 cDNA
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NMMHC-A (red)
Transfection of COS cells with D1424H MYH9 cDNA
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A
B
C
D
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E
F
G
H
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A
B
C
D
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•Il gene codificante per la podocina (NPHS2) è stato recentemente identificato come responsabile di una sindrome nefrosica autosomica recessiva resistente agli steroidi.
•Il gene NPHS1 che codifica per la nefrina è stato identificato essere coinvolto nella forma congenita di sindrome nefrosica del tipo Finnico.
•La nefrina è il componente maggiore dello "slit-diaphragm" presente tra i processi interdigitati dei podociti ed è stato ipotizzato che questa proteina èimportante per mantenere la struttura di questi processi.
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•Recentemente è stato dimostrato che la nefrina rappresenta una molecola di segnale ed i segnali indotti dalla nefrina sono enormemente amplificati dalla podocina che si lega alla coda citoplasmatica della nefrina.
•Infine è stato dimostrato di recente che la nefrina ancora lo "slit-diaphragm" all'actina nel citoscheletro
•Queste due proteine giocano così un ruolo cruciale nella funzione di filtrazione della barriera glomerulare e quando mutate determinano una disfunzione del podocita e la presenza di proteinuria.
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7
T
A
7
T
A
7
T
A
7
T
A
7
T
A
7
T
A
7
T
A
7
T
A
7
T
A
7
T
A
7
T
A
7
T
A
7
T
A
3
C
A
7
T
A
3
C
A
7
T
A
3
C
A
7
T
A
7
C
A
7
T
A
7
T
A
7
T
A
7
T
A
3
C
A
7
C
A
7
T
A
3
T
A
7
T
A
9
C
A
7
C
A
7
T
A
3
C
G
1
C
A
7
T
A
9
C
A
7
T
A
3
C
G
7
T
A
7
T
A
NPHS2P
1- Intron 1 (301-309)
2- 951T>C
4- 1038A>G
7
C
A
7
C
A
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Marco Seri Simone GangarossaRoberto CusanoEmanuele Panza Bianca Rocca
Monica Marini Raffaele LandolfiRoberto Ravazzolo
Nicola BizzarroFilomena Di BariAnna Savoia Paola Malatesta
Maria CapriaAlessandro PecciPatrizia Noris Pasi A. KoivistoUmberto MagriniCarlo L. Balduini Ilaria Meloni
Alessandra RenieriMaria SavinoMaria Del Vecchio Luisa MurerMaria d’ApolitoLeopoldo Zelante Giovanni Banfi
Gianluca Caridi Michele PurrelloGian Marco Ghiggeri
Carmine PecoraroSaverio Sartore
Achille Iolascon
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A B
C D
Normal platelets
Normal PMN
Giant
platelet
PMN with
Dohle-like
body
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Myh9 Myh10
Esempio 1. L’anticorpo anti-Myh9e quello anti-Myh10 sembrano riconoscere due sistemi di filamenti apparentemente differenti. Myh9 forma
filamenti grossolani uniformemente distribuiti in tutto il citoplasma della cellula, dalla regione perinucleare a quella corticale, dove
definiscono bene i confini cellulari; appaiono orientati lungo l’asse longitudinale dei prolungamenti e lungo le linee di forza. Myh10 forma
filamenti più sottili, che appaiono differenti innanzitutto perché possono essere orientati in maniera diversa, come nel nella parte inferiore della
cellula; inoltre sono presenti solo in alcune zone di citoplasma e non disegnano mai l’intero profilo della cellula – in particolare non sono mai
presenti fino alla zona corticale, che è sempre delimitata da Myh9.
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La sovrapposizione dei due segnali sullo stesso piano dimostra meglio questi aspetti; si osserva come il segnale rosso e quello verde
siano in parte separati, me si intuiscono anche dei punti di sovrapposizione, contrassegnati da un segnale giallo.
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Esempio 2. Idem. Notare come Myh9 disegni sempre bene il profilo e l’impalcatura della cellula e Myh10 corra solo in alcune
zone di citoplasma
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Esempio 2, sovrapposizione, idem.
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Esempio di proiezione ortogonale della fluorescenza rilevata su un determinato piano confocale sull’asse z. A destra della linea
tratteggiata è riportata la proiezione della fluorescenza rilevata lungo la linea centrale verticale sull’asse y (in pratica x e z sono fisse,
viene rilevata la fluorescenza lungo y); sotto la linea tratteggiata è riportata la proiezione della fluorescenza rilevata lungo la linea cen
-trale orizzontale sull’asse x (z e y fisse).
Si osservi come sono presenti zone di segnale rosso (filamenti Myh9), zone di segnale verde (filamenti Myh10) zone buie (spazio fra
i filamenti), ma anche zone con un chiaro segnale giallo (zone di colocalizzazione), indicate dalle frecce.
y
x
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Altro esempio, idem.
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Altra metodica per effettuare lo studio di colocaliz-
zazione.
Tracciando una retta su un piano confocale viene
rilevata e confrontata l’intensità di segnale lungo la
retta per ciascun canale.
Qui si vede benissimo che su questo piano i due
maggiori picchi di fluorescenza nel verde - che
identificano due singole fibre di myh10 che passano
sul piano-, corrispondono con precisione nanometrica
ad altrettanti picchi di fluorescenza nel rosso,
identificando quindi due punti in cui le due strutture
vengono a contatto.
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An accurate clinical re-evaluation of patients allows to observe that patients carrying MYH9 mutations show a continuous spectrum ofclinical manifestation without evidence to support the differentiation indistinct diseases.
The only two findings observed in all patients are the macrothrombocytopenia and the abnormal distribution of NMMHC-IIAwithin leukocytes.
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Control
Platelet
s
Platelet
s
MGG
mAb agai nst
NMMHC- IIA
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Biochemical mechanisms through which mutations affect the NMMHC-IIA function are not completely understood.
About 70% of the families have mutations in the C-terminal tailand 88% of them affect only 4 residues (1165, 1424, 1841, 1933)
representing less than 1% of all the possible residues involved.
No consistent correlations have been identified between the 27 different MYH9 mutations identified so far and the variable
clinical evaluation of the disease.
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Understanding genotype-phenotype correlation
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Collection of new cases
“Italian registry for MYH9-Related Disease”Pavia, Italy.
Molecular analysis of MYH9 exon 1, 15, 16, 25, 26, 30, 38, 40. Bologna, Italy.
150 cases up to date (February 2008)
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This study included 108 patients with MYH9-RD confirmed by molecular analysis, diagnosed by the collaborative groups from Junuary 2001 to December 2006.
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Five new mutations identified; three missense and two single base deletions
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•La Sindrome di Fechtner (FTNS; MIM 153640) è una malattia autosomica dominante descritta per la prima volta da Peterson et al. (1985) che è stata considerata come una variante della Sindrome di Alport.
•Infatti è caratterizzata da nefrite, sordità neurosensoriale ed anomalie dell’occhio ma mostra anche caratteristiche aggiuntive come la macrotrombocitopenia e corpi inclusi all’interno dei polomorfonucleati chiamati “Döhle-like bodies”.
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•L’anomalia di May-Hegglin (MHA; OMIM 155100) condivide con la FTNS la triade clinica di trombocitopenia, piastrine giganti e le caratteristiche inclusioni leucocitiche.
•La Sindrome di Sebastian (SBS; MIM 605249) ècaratterizzata dalle stesse alterazioni ematologiche ma si differenzia dalla MHA sulla base delle immagini ultrastrutturali dei “Döhle-like bodies”.
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MHA/SBSFTNS
38
E1841K
1 16 26
R1165CN93K R702C
30
D1424H
40
R1933X
Motor domain Coil coiled domainACD
Assembly competence domain
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Missense K C C H K
93 702 1165 1424 1841
| | | | |
HsnmIIa (P35579) --ELTCLNEASVL---VLEGIRICRQG---ELRSKREQEVN---QQELDDLLVDL---QVRRTEKKLKD--
XlnmIIa (AAC83556) --ELACLNEASVL---VLEGIRICRQG---ELRTKREQEVT---QQELDDISVDL---QVRRTEKKLKD--
RnnmIIa (AAA74950) --ELTCLNEASVL---VLEGIRICRQG---ELRSKREQEVS---QQELDDLLVDL---QVRRAEKKLKD--
RnNeur (S21801) --ELTCLNEASVL---VLEGIRICRQG---ELRSKREQEVN---QQELDDLLVDL---QVRRTEKKLKD--
GgnmII (P14105) --ELTCLNEASVL---VLEGIRICRQG---ELRSKREQEVT---QQELDDIAVDL---QVRRAEKKLKD--
HsnmIIb (P35580) --ELTCLNEASVL---VLEGIRICRQG---ELRTKREQEVA---QQELDDLTVDL---LVRRTEKKLKE--
XlnmIIb (AAA49915) --ELTCLNEASVL---VLEGIRICRQG---ELRTKREQEVA---QQELDDLMVDL---LVRRTEKKLKE--
RnnmIIb (AAF61445) --ELTCLNEASVL---VLEGIRICRQG---ELRTKREQEVA---QQELDDLTVDL---LVRRTEKKLKE--
BtnmIIb (BAA36494) --ELTCLNEASVL---VLEGIRICRQG---ELRTKREQEVA---QQELDDLLVDL---LVRRTEKKLKE--
GgsmII (P10587) --ELTCLNEASVL---VLEGIRICRQG---ELRAKREQEVT---QQELDDLVVDL---TLRQKDKKLKD--
OcsmII (P35748) --ELTCLNEASVL---VLEGIRICRQG---ELRAKREQEVT---QQELDDLVVDL---ALKQRDKKLKE--
DmnmII (Q99323) --ELTCLNEASVL---VLEGIRICRQG---ELRSKREQELA---QSELEDATIEL---ANRKMDKKIKE--
Cenmy1 (CAA99841) --MLTCLNEASVL---VLEGIRICRQG---QLKAKRDEEYA---IQEAEDVQKEL---TLRRMETKMAE--
Cenmy2 (AAA83339) --ELTYLNEASVL---VLEGIRICRQG---DLMSRKDEEVN---QQELEDSSMEL---AARRLEKRLND--
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•La Sindrome di Epstein (EPTS-OMIM 153650) è una malattia ereditaria che si differenzia dalla FTNS solo per l’assenza dei “Döhle-like bodies” nei granulociti e per l’assenza della cataratta bilaterale.
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•Gli esperimenti di immunocitochimica hanno messo in evidenza deipatterns di immunoreattività che è possibile correlare con le diverse mutazioni nel gene MYH9.
•La mutazione D1424H è associata con un singolo aggregato di medie/larghe dimensioni nel citoplasma dei granulociti talvolta associato con alcuni aggregati minori. I pazienti che presentanoquesta mutazione mostrano anche una distribuzione negativa ed irregolare della miosina nelle piastrine.
•Al contrario, le mutazioni al codone 702, normalmente identificate nei pazienti EPTS, determinano la presenza nei granulociti neutrofili di diversi microaggregati di dimensioni molto piccole spiegando cosìla difficoltà di rilevare la presenza delle inclusioni attraverso una colorazione con May-Grünwald-Giemsa. Le piastrine dei pazienti che presentano una mutazione al codone 702 mostrano un pattern diffuso.
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•Macrotrombocitopenia è sempre presente.
•Anche nei pazienti EPTS si dimostra tramite esperimenti di immunocitochimica la presenza di aggregati di miosina nei leucociti.
•La presenza di ipoacusia è stata accertata nei pazienti indipendentemente dalla diagnosi iniziale di FTNS, EPTS or MHA-SBS ed in particolare nel 82% dei pazienti affetti da SBS o MHA.
•La cataratta bilaterale è stata identificata in 11 pazienti e sorprendentemente in 3 pazienti con una diagnosi di MHA-SBS.
•Una proteinuria massiva associata a ESRF è stata rilevata in 5 pazienti FTNS ed in 2 EPTS. Tuttavia una microematuria ricorrente e bassi livelli di proteinuria sono stati riscontratianche in casi MHA-SBS
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•Le mutazioni ricorrenti (codoni 96, 702, 1155, 1165, 1424, 1841, 1933) rappresentano una percentuale dell’ 85% di tutte le mutazioni fino ad oggi identificate nel gene MYH9.
•Delezioni e mutazioni nonsenso o frameshift che risultano in unaprematura terminazione della proteina MYH9 non sono mai state descritte.
•La presenza di mutazioni nonsenso e frameshift solo nell’ultimo esone codificante per la parte C-terminale della proteina che contiene il dominio deputato ad assemblare le molecole di MYH9 in filamenti bipolari, suggeriscono come meccanismo patogeneticoun effetto dominante negativo.
•Tuttavia se il meccanismo patogenetico di queste malattie sia determinato da un meccanismo dominante negativo causato dalla alterata formazione di dimeri di miosina oppure dipenda da una aploinsufficienza resta ancora da dimostrare.
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Kunishima S et al. “Immunofluorescence analysis of neutrophil Nonmuscle Myosin Heavy Chain-A in MYH9 disorders: association of subcellular localization with MYH9 mutations” Laboratory Investigation (2003)
Deutsch S et al.”Asp1424Asn MYH9 mutation results in an unstable protein responsible for the phenotypes in May-Hegglin anomaly/Fechtner syndrome”Hemostasis, Thrombosis, and Vascular Biology (2003)
Takubo T et al.”Expression of non muscle type myosin heavy polypeptide 9 (MYH9) in mammalin cells” European Journal of Histochemistry (2003)
Franke JD et al. “Rod mutations associated with MYH9-related disorders disrupt non-muscle myosin-IIA assembly” Blood (2004)
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all subjects with mutations in the motor domainof NMMHC-IIA present with severe thrombocytopenia and develop nephritis and deafness before the age of 40;
while subjects with mutations in the tail domain have much lower risk ofnoncongenital complications and significantly higher platelet counts.
Mutations at residue 1933 do not induce kidney damage or cataracts and cause deafness only in the elderly;
mutations at residue 702 result in severe thrombocytopenia and producenephritis and deafness at a juvenile age;
alterations at residue 1424 or 1841 result in intermediate clinical pictures.
The work demonstrates that:
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MYH9-related disease (MYH9-RD) is a rare autosomal dominant disorder caused by mutation in MYH9, the gene coding for the heavy chain of nonmuscle myosin IIA (NMMHC-IIA).
All patients present from birth with macrothrombocytopenia,but in infancy or adult life some of them develop sensorineural deafness, presenile cataracts, and/or progressive nephritis leading to end-stage renal failure.
COOH
Motor domain Coiled coil
COOH
heavy chains
lightchains