Diagnostica Molecolare - completo

Emoglobinopatie e talassemie

Dip. Area Critica Medico Chirurgica, Firenze

Mutazioni: delezioni

Processamento dell'mRNA e mutazioni

Traduzione e mutazioni

Emoglobina = tetramero di 4 catene globiniche + 4 gruppi eme

Ferro + protoporfirina IX

αξ βε γδ

prenatale nascita 3 mesi 6 mesi

ξ2ε2 α2β2 Gower 1 HbA: 95%

α2ε2 α2β2 Gower 2 (glic) 3%

HbA1c: ξ2γ2 α2δ2

HbA2: Portland 2% β4 α2γ2

HbH <1%

HbF: γ4

Classificazione dell'emoglobinopatie e talassemie: alterazioni strutturali dell'Hb:

• anomala polimerizzazione Hb (HbS)
• anomala cristallizzazione Hb (HbC)
• emoglobine instabili
• emoglobine con affinità per l'O2

(policitemia);↓• emoglobine con affinita’ per l’O2 (cianosi);difetti quantitativi nella produzione delle catene globinicheα-talassemia• β-talassemia• δβ-talassemia, γδβ-talassemia, αβ-talasemia•persistenza di Hb fetale (HbF)• pancellulare• eterocellulareemoglobinopatie acquisite• meta-emoglobinemia• sulfo-emoglobinemia• carbossi-emoglobinemia• incremento HbF (chemioterapia, ripresa midollare, mielodisplasie)β β- talassemia: difetto produzione catene globinicheαeccesso catene globiniche HbA HbFHbA2 Xβ -talassemia major XXXβ -talassemia intermedia(tipo 2 e tipo 3) XXβ -talassemia minor Xβ -tratto talassemicoβ -talassemia eterozigote eccessoβcateneridotta quantita Hb per RBC Ridotta produzione(ipocromia) di RBC fragiliridotta produzione RBC maturi(iporigenerazione)ridotta sopravvivenza RBC sequestrazione

anisopoichilocitosis

plenica

anemia

splenomegalia

ittero

trasfusioni

ipersplenismo

calcoli biliari

aumentato

difettoso accumulo Fe

assorbimento Fe

utilizzo Fe

emocromatosi

cirrosi

endocrinopatie

cardiomiopatia

α α-talassemia = difetto produzione catene globiniche

β eccesso produzione catene globiniche X

α tratto -talassemico tipo 2

portatore sano X X

omozigote Xα tratto -talassemico X X

tipo 1 eterozigote X X

malattia da HbH X X X

idrope fetale X X

eccesso precipitazione corpi inclusi nei α α progenitori eritroidi

catene catene

ridotta quantita Hb per RBC

eritropoiesi inefficace maturazione (ipocromia) a livello midollare di pochi RBC difettosi

ridotta produzione RBC maturi (iporigenerazione)

ridotta sopravvivenza RBC sequestrazione

anisopoichilocitosisplenica

ipossia tessutale

anemia splenomegalia

ittero

iperproduzione Epo

trasfusioni

ipersplenismo

calcoli biliari

spansione aumentato difettoso accumulo Fe

emopoiesi assorbimento Fe utilizzo Fe emocromatosi

deformita’

ossea cirrosifratture endocrinopatiedeficit folati emopoiesi extramidollare cardiomiopatia

Inherited connective tissue disorders

• Bethlem myopathy
• Ullrich congenital muscular dystrophy
• Marfan syndrome

Dip. Area Critica Medico Chirurgica, Firenze

BETHLEM MYOPATHY

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

• Congenital 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

Dip. Area Critica Medico Chirurgica, Firenze

Bethlem myopathy

Dip. Area Critica Medico Chirurgica, Firenze

Ullrich Scleroatonic Muscular Dystrophy

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

Proximal joint contractures

Hyperextensibility of distal joints resembling Ehlers Danlos syndrome

Normal

intelligence

Consanguineity among unaffected parents

Dip. Area Critica Medico Chirurgica, Firenze

Dip. Area Critica Medico Chirurgica, Firenze

A Japanese patient with Ullrich scleroatonic muscular dystrophy

28nts delCOL6A2 Exon 18 Dip. Area Critica Medico Chirurgica, Firenze

Collagen type VI

Chromosome Gene cDNA Chain

21q22.3 3.5 kbCOL6A1 a1

21q22.3 COL6A2 4.2 kb a2

2q37 COL6A3 10kb a3

Dip. Area Critica Medico Chirurgica, Firenze

Type VI Collagen

EM glycoprotein 140 kDa

Component of 100 nm beaded microfibrils

Tissue Distribution

Present in most soft connective tissues

Skin

Vessel wall

Tendon

Cartilage

Internal organs

Cardiovascular system

Uterus

Neuromuscular junction

Cornea

Lung

Dip. Area Critica Medico Chirurgica, Firenze

RNA and DNA analysis in Bethlem myopathy

Skin biopsy

Blood withdrawal

Genomic DNA extraction

Fibroblast cells culture

Segregation analysis

RNA extraction

RT-PCR PCR amplification of single exons

Heteroduplex analysis

Direct sequencing

Dip. Area Critica Medico Chirurgica, Firenze

DOMINANT AND RECESSIVE

COL6 MUTATIONS IN BM AND UCMD

THα 1 (VI) C1 C2N1

THα 2 (VI) C2C1N1

THα N10-N13 (VI) C1 C2 C3 C4 C5= BM dominant mutations

* = common mutations=UCMD recessive =UCMD dominant

Dip. Area Critica Medico Chirurgica, Firenze

mutations mutations

Istochemical analysis on muscular biopsy

Immunofluorescent analysis on fibroblast culture

Ullrich patients analysis Dip. Area Critica Medico Chirurgica, Firenze

Northern blot Immunoprecipitationnt ntrolol ntrolr tie tietie ntnt nt

Pa Co PaCo PaCo

COL6A3 α α3(VI) 3(VI)

COL6A2a

COL6A1 α

COL6A2 2(VI)α α1(VI)+α2(VI) α1(VI)

mRNA nonsense mediated decay of the aberrant splicings Medium Cells

Ullrich patients analysis Dip. Area Critica Medico Chirurgica, Firenze

MARFAN SYNDROME

• Marfan syndrome (MFS) is an inherited connective tissue disorder with an autosomal dominant transmission

• Prevalence: 1/ 5,000-10,000

• 25% sporadic cases

• Clinical phenotype: high inter-familial and intra-familial variability

Basic Mechanisms in Aortic Aneurysms: Genetic aspects

Monogenic disorders: mostly autosomal dominant syndrome

• Marfan (MFS)
• Pectus carinatum Ch 17p24-p25 FBN1 (90%)
• Ch 3p24-p25 TGFBR2 (<5%)
• Ch 9q33-q34 TGFBR1 (<5%)

Ectopia lentis

Dural Ectasia

Scoliosis

Centro Marfan, Firenze

DNA analysis in Marfan syndrome

1. Blood withdrawal
2. Genomic DNA extraction from blood
3. PCR amplification of exons
4. Heteroduplex analysis
5. Direct sequencing

Centro Marfan, Firenze

RNA ANALYSIS IN MARFAN SYNDROME

1. Skin biopsy
2. Fibroblast cell culture
3. RNA extraction from cells
4. Reverse Transcriptase PCR (RT-PCR)
5. Heteroduplex analysis
6. Direct sequencing

Centro Marfan, Firenze

FBN1 mutations

Marfan database

1. Our data

SOSC OS EL TAA S-GN SC

Pepe G. et al, JMCC 1997; Clin Genet 2001, Molec Vis 2008 Attanasio et al., Clin Genet 2008

Centro Marfan, Firenze

Mutation Screening Analysis of 103 Marfan patients

Type of Mutation n(%)

• 50 (61%) missense
• 2 (2%) same sense
• 13 (16%) nonsense
• 3 (4%) small

insertionssmall deletions 8 (10%)donor splice site 6 (7%)82 (100%)

Detected mutations

Centro Marfan, Firenze Attanasio M et al,Clin Genet 2008

Fibrillin-1 qualitative andquantitative defects

Alleles Monomers Multimers Phenotype

NormalSevereMildPTC Dietz et al, 1992

Centro Marfan, Firenze

Genetic aspects

Major genes

Genes that, mutated, can cause clinical manife-stations corresponding to a monogenic disorder

MFS - FBN1

More major genes, mutated, can be responsibleof a single monogenic disorder

FBN1 - MFS - TGFBR2

Dip. Area Critica Medico Chirurgica,Firenze

Genetic aspects

Major genes

One major gene, mutated, can be responsiblefor several monogenic disorders

F-TAAF-AAA SGSFBN1MFS MASSF-EL nMFS Dip. Area Critica Medico Chirurgica,Firenze

Genetic aspects

Modifier genes

Genes that, mutated, modify the clinicalphenotype of a patient (with a monogenicdisorder) by increasing or decreasing theseverity also of single/few manifestation/sα β/β Thalassemia - >Intermedia thalassemia

Genes in which

single specific mutations can be responsible of a single clinical manifestation in multifactorial disorders

Dip. Area Critica Medico Chirurgica, Firenze

Genetic aspects

Modifier genes

Genes that, mutated, modify the clinical phenotype of a patient (with a monogenic disorder) by increasing or decreasing the severity also of single/few manifestation/s

MFS (CVS)- MTHFR -> Homocysteine

Genes in which single specific mutations can be responsible of a single clinical manifestation in multifactorial disorders

Dip. Area Critica Medico Chirurgica, Firenze

ELASTIC LAMINA

FENESTRATION

MMP-2?

HYPERHOMO ELASTOLYSIS

CYSTEINEMIA PEPTIDES

VSMC PROLIFERATION VASCULAR

MIGRATION OCCLUSION

Clin Med Cardiol Fi Genetic aspects

Genes increasing susceptibility or predisposition

Genes that, mutated, increase susceptibility or predisposition to multifactorial - multigenic disorders

When many mut genes are present in One family the disorder can appear in more generations.

Dip. Area Critica Medico Chirurgica, Firenze

Genes

increasingsusceptibility or predisposition

When many mutated genes arepresent in one family the disordercan appear in more generations.

AAA Dip. Area Critica Medico Chirurgica,Firenze

Thoracic Aortic Aneurysm (TAA)

Thoracic Aortic Dissection (TAD)

TAA + TAD

Major genes:

FBN1 <10% of patients

TGFBR2 ≈5%

Dip. Area Critica Medico Chirurgica,Firenze

Thoracic Aortic Aneurysm (TAA)

Thoracic Aortic Dissection (TAD)

TAA + TAD

Chromosomal localization of majorgenes mapped by linkage analysis:

Ch 11q23.2-q24

Ch 5q13-q14

Ch 3p24-p25

Dip. Ar