Diag mol 2014 B

ANALISI POLIMORFISMI DNA

Analisi di linkage -> esclusione di geni

Fenotipi di pazienti con osteogenesi imperfetta (OI)

AR(poco comune)

AD

Deformita` da lieve a moderata.

Variab. ritardo nella crescita; cifoscoliosi.

In alc.casi

+(50%)

Bian-che

+IV

ADDeform. progress. delle ossa lunghe; deform. moder. alla nascita; statura bassa; marcata cifoscoliosi

Rara+Blu alla nasci-ta

++III

AD

AR(rara)

Letale nel periodo perinatale; costole piegate; deformita` marcate nelle ossa lunghe; platispondilia (vertebre appiattite)

--Blu+++II

ADStatura normale; niente o poche deformita`; tendenza a contusioni; ipermobilita` delle giunture.

+(50%)

RaraBlu+I

Eredita`Altre caratt. clinichePerd. udito

Dent. imperf.

Sclere

FrattureTipo

Dip. Area Critica Medico Chirurgica,Firenze

OSTEOGENESIS IMPERFECTA


I

II

III

IV

1 2

5 63 4

5 6

1 2

1 2

1 2

3 4

3 4

-/- -/+

-/--/- -/- -/-

-/-

-/+-/+

-/+ -/+-/+ -/+ -/+

-/+ -/+ -/++/+

Affected by OI

ProbandDip. Area Critica Medico Chirurgica,Firenze

Famiglia con Osteogenesi Imperfetta tipo IV dominante

RFLP: COL1A2/EcoRI

Famiglia A

Famiglia con Osteogenesi Imperfetta tipo IV dominante

Famiglia B

OI tipo I, dominante

1 RFLP: COL1A2/EcoRI2 VNTR: COL1A2/(ACT)n3 RFLP: COL1A1/EcoRI4 VNTR: COL1A1/(GCC)n

15/18 17/18

I

1234

8/10-/++/+15/18

1 2 3

1 2

8/10-/+-/+15/17

9/10+/+-/-18/19

I

1234

8/9-/+-/+15/18

8/9-/+-/-17/18


BETHLEM MYOPATHY

1996

By linkage analysis: association between Bethlem myopathy and a) COL6A1-COL6A2 on chromosome 21q 22.3 (Jobsis et al.,) b) COL6A3 on cromosome 2q37 (Speer et al.,)

Detection of the two first mutations in COL6A1 and COL6A2 in three unrelated Dutch families (Jobsis et al.,)

Detection of the first mutations in COL6A3 in a French-Canadian family (Pan et al.,)

1998


BETHLEM MYOPATHY

Autosomal dominant inherited disease producing a mild neuromuscular disorder characterized by:

Proximal skeletal muscle weakness and wasting

Multiple joint contractures (neck, elbows, ankles and the interphalangeal finger joints)

It has a slow, progressive benign course although some of the patients end up on a wheel chair after the age of 50

Age of onset: from prenatal period to adulthood


Dept. of Internal Medicine, Tor Vergata University, ROME-ITALY

Bethlem miopathy


RNA and DNA analysis in Bethlem myopathy

Skin biopsyFibroblast cells cultureRNA extractionRT-PCR

Heteroduplex analysisDirect sequencing

Blood withdrawal

Segregation analysisPCR amplification of single exons

Genomic DNA extraction


Immunofluorescence of fibroblasts of a Bethlem patient

Cont

rol

B

D C

A

Patien

t

Type I collagen Type VI collagen


Northern

Immunoprecipitation

Northern and immunoprecipitation analysis


C P C P C P C P

Cell Medium

1 2 3 4 5 6 7 8

2( I )

1( I )

1( VI )

C P

3( VI )

1( VI ) 2( VI )

A B


Protein analysis

BETHLEM FAMILY BG:COL6A3 and COL6A2 gene exclusion

I

III

IV

II

A3P1

A2P1 A2P1

1 2

3 4 5

6 8 9 10 11 12

13 14 15 16 17 18

13/18

T/C 3/4

18/18

C/C 1/3

13/18

T/C 3/4

13/18

C/C 4/3

14/17

C/C 1/2

7

8/14

C/C 2/5

13/18

C/C 4/1

13/18

C/C 4/1

8/14

C/C 2/2

18/18

T/C 3/1

13/17

C/C

4/1

13/14

C/C

4/1 13/14

C/C

1/2

13/14

C/C

5/1

14/18

C/C

2/4

14/18

C/C

4/2

affected

A3P1 A2P1 A2P1

A3P1 A2P1 A2P1


Chromatograms from DHPLC analysis

A - control B Ins 2 bp C SNS


SITO DONATORE IN 5'

SITO ACCETTORE IN 3'Centro Marfan,Firenze

Genomic DNA cDNA

COL6A1 IVS3+1 G->A mutation activates a donor splice site inside Exon3 causing the deletion of 66 nts (in frame deletion)

3 4

GGG/gtgagt

a

CAA/gtactt (cryptic donor site)

AG/gt agt C A

g a

Canonical site in mammals


COL6A1 mutations

7 6 8 8 6

Genomic DNA cDNA

gt ac (2)(1)

14 13 15 15 13 gt ac (1)(6)

3 4 g

a (4)

-66 14 6

Ser

X

GlyArg


MUTAZIONI DI SPLICING

Mutazioni di tipo quantitativo

50% 50%

Centro Marfan,Firenze

GT/AG

Perdita di un esone interoRitenzione di un introne

10%-50% 50%

Mutazione negli altri nucleotididei siti donatori o accettori

Attivazione di siti criptici esonici o intronici

Totale Parziale

COL6A1 cDNA mutations

14 13 15

Genomic DNA

12 11 10 9 8 IVS 8 IVS 14

15 14 13 Normal cDNA 12 11 10 9 8

15 8 MutatedcDNA 252nts in frame deletion


Collagensstructure, function, and biosynthesis


Mutaz. in un allele

2(I) 1(I)

saranno presenti nel

50% 75%

Eterotrimerialterati


Espressione diversa delle mutazioni in base alla struttura della molecola o alla sovrastruttura finale

Collagen VI mutations in Bethlem Myopathy

N1 C1 C2 C

1

2

N10 N9 N8 N7 N6 N5 N4 N3 N2 N1 C1 C2 C3 C5 C

3 C4

G1679E G2056R

G271S(2)

IVS3, +1GA, pdelEx3

G305V IVS11, -1GA, PTC IVS14, +1GA, delEx14 (1+5)

IVS14, +2TC, delEx14

N1 C1 C2 C

S348X

G275R

IVS7, +1GA, delEx7 (2)

IVS7, +2AG, delEx7 K122R

G341D

D621N

G341V


Ullrich Scleroatonic Muscular Dystrophy

Recessive congenital muscular distrophy (CMD) affecting connective tissue and muscle

Proximal joint contractures

Hyperextensibility of distal joints resembling Ehlers Danlos syndrome

Consanguineity among unaffected parents

Normal intelligence


COL6A2 Exon 18

28nts del

A Japanese patient with Ullrich scleroatonic muscular dystrophy


Type VI collagen in the muscle of an Ullrich patient

Co P Dip. Area Critica Medico Chirurgica,Firenze

A

B

C

D

E

F

Family B

Fath

er

Mot

her

Aff

ecte

d so

n


Type VI Collageninteractions

N10 N9 N8 N7 N6 N5 N4 N3 N2

N1

N1

C1 C2TH

C1 C2TH C4C3 C5

C

C

C

1(VI)

2(VI)

3(VI)

NG2

?

Perlecan/BM

BG

Undulin

Decorin

Fibronectin

ColIV

ColIMAGPwWF

Hyaluronic Acid; Heparin)Dip. Area Critica Medico Chirurgica,Firenze

Diag mol 2014 B

Documents

Transcript of Diag mol 2014 B