Familial Amyloidotic Polyneuropathy Familial Amyloidotic Polyneuropathy
FAP in a nutshell
Severe peripheral neuropathy
Late onset
Familial Systemic deposition of amyloid
• Sensitive and autonomic, and later also motor dysfunction. motor dysfunction. • Simetrical, centripetal and progressive.
• mean age at onset 35 yrs.
• Autosomal dominant.
• Vascular walls, peripheral nerves, thyroid, kidney, etc. • CNS and liver spared.
Corino Andrade
FAP ‐ Genetics Familial, autosomic , dominant.
No modifier genes identified so far, but familial aggregation of subphenotypes.
Parent of origin effects: Anticipation of age at onset, increased penetrance, particularly on mother to child transmission. Still unexplained (genetic imprinting?). (g p g )
I II
III IV
?
FAP ‐ Pathology gy Generalized deposition of amyloid, with predisposition for small arteriolar walls, peripheral nerves, thyroid, kidney, etc.
CNS and liver spared.
Kidney
Severe loss of axonal fibers in later stages of the disease Peripheral nerve
II – Molecular Biology
FAP – Molecular Biology FAP Molecular Biology The fundamental constituent of amyloid in PAF is identical immunochemically to a small serum protein, transthyretin (TTR). [Costa et al., PNAS, 1978]
Bi h i l characterization Biochemical h t i ti off this thi fundamental constituent of amyloid reveals the presence of a TTR mutation, with ith a methionine thi i for valine f li substitution at position 30. [Saraiva et al., Trans Assoc Am Physicians, 1983]
FAP ATTR V30M
-20 -10 ACAGAAGTCCACTCATTCTTGGCAGGATGGCTTCTCATCGTCTGCTCCTCCTCTGCCTTGCTGGA ..........................-M--A--S--H--R--L--L--L--L--C--L--A--G-1 1 10 CTGGTATTTGTGTCTGAGGCTGGCCCTACGGGCACCGGTGAATCCAAGTGTCCTCTGATGGTCAAA -L--V--F--V--S--E--A--G--P--T--G--T--G--E--S--K--C--P--L--M--V--K20 30 GTTCTAGATGCTGTCCGAGGCAGTCCTGCCATCAATGTGGCCATGCATGTGTTCAGAAAGGCTGCT -V--L--D--A--V--R--G--S--P--A--I--N--V--A--M--H--V--F--R--K--A--A40 50 GATGACACCTGGGAGCCATTTGCCTCTGGGAAAACCAGTGAGTCTGGAGAGCTGCATGGGCTCACA -D--D--T--W--E--P--F--A--S--G--K--T--S--E--S--G--E--L--H--G--L--T60 70 80 ACTGAGGAGGAATTTGTAGAAGGGATATACAAAGTGGAAATAGACACCAAATCTTACTGGAAGGCA -T--E--E--E--F--V--E--G--I--Y--K--V--E--I--D--T--K--S--Y--W--K--A90 100 CTTGGCATCTCCCCATTCCATGAGCATGCAGAGGTGGTATTCACAGCCAACGACTCCGGCCCCCGC -L--G--I--S--P--F--H--E--H--A--E--V--V--F--T--A--N--D--S--G--P--RG S S G 110 120 CGCTACACCATTGCCGCCCTGCTGAGCCCCTACTCCTATTCCACCACGGCTGTCGTCACCAATCCC -R--Y--T--I--A--A--L--L--S--P--Y--S--Y--S--T--T--A--V--V--T--N--P127 AAGGAATGAGGGACTTCTCCTCCAGTGGACCTGAAGGACGAGGGATGGGATTTCATGTAACCAAGA -K--E--*-......................................................... GTATTCCATTTTTACTAAAGCAGTGTTTTCACCTCATATGCTATGTTAGAAGTCCAGGCAGAGACA ..................................................................
Chr 18 Chr.
ATAAAACATTCCTGTGAAAGGCACTTTTCATTCC ..................................
Amyloid Type
Precursor
Disease
AA
apoSAA SAA
SSecondary (reactive) amyloidosis d ( ti ) l id i FMF
AL
Ig light chains (κ,λ)
ATTR
Transthyretin TTR V30M TTR L111M
Senile systemic amyloidosis Polyneuropathy Cardiomiopathy
AapoAI
apoA‐I Arg 26 apoA‐I Arg 60
Polyneuropathy, Nephropathy Nephropathy
A Lys
Lysozime Thr 56, His 67
Nephropathy
AFibA
Fibrinogen Aα E526V, R554L
Nephropathy
AGel
Gelsolin Asn 187
FFA
ACys
Cystatin C Gln 68
HCHWA, Icelandic type
Aβ2M
β2‐microglobulin
Hemodialisis amyloidosis
Aβ
βPP βPP Gln 618
Alzheimer s disease Alzheimer’s disease HCHWA, Dutch type
AScr
PrPC, PrPSc, PrPCJD
Kuru, Scrapie, CJD, GSS
AIAPP
A ili Amilin
Di b Diabetes melitus, type 2 li 2
ACal
procalcitonin
Thyroid medulary carcinoma
TTR M t ti TTR Mutations Neuropathy Arg 47 Glu 18 Leu 30 Leu 33 Asn 35
Vitreous/other
Ile 50
Gly 54 Gly 54
Tyr 77
Val 107
Lys 61
Val 33
Gly 42
Ala 30 Ser 24 Thr 34 Gln 89 Ala 47
Arg 58
Cys 114 Ile 33 Ile 33 Ala 49
Pro 36 Pro 36 Val 47
Ile 50 Thr 45 Thr 45
Cardiomiopathy
Gly 18 Gly 30
Met 30
Ala 60 Pro 55 Arg 10 His 69 His 69 Ile 112 Ala 71 Arg 50 Leu 64 Ile 122 Lys 59 Ser 84 Gly 97 Asp 45 Leu 68 Ile 20
P 12 Pro 12
His 60
Met 111 Ser 125
Ser 6 Met 119
III – Epidemiology
FAP Epidemiolgy FAP ‐ 3525 TTR V30M carriers registered at Centro de Estudos de Paramiloidose, Porto (June 2008), covering i most patients i and d heathy h h carriers above 20 years of age (data from genetic counceling).
The residence of 97% of these carriers is known. Northern coastal areas most heavily affected.
Number of live patients > 1000. g y p National registry implementation under way.
PAF ‐ distribuição d de portadores / nut3 t d / t3 100 ou mais (4) 60 a 99 (3) 40 a 59 (3) 30 a 39 (3) 20 a a 29 (2) 10 a 19 (2) 0 a 9 (11)
Cávado (733) Grande Porto G d P (1206)
Junho de 2008
Baixo Vouga (102) Baixo Mondego (261)
Grande Lisboa G d Li b (291) Península de Setubal (90)
Serra da Estrela (78) (72) Cova da Beira
FAP Prevalence Studies FAP ‐ Prevalence Studies Northern Portugal, one study only (HIEF) Alves IL et al. Human Mutation 1997, 9(3), 226‐33 , ( ),
In 5000 individuals: • • • • • • •
Met 30 Met 119 Met 30/Met 119 Asn 90 Ile 122 Unknown Thr 190
8 35 1 12 1 3 1
FAP Prevalence Studies FAP ‐ Prevalence Studies
Northern Sweden – endemic counties (ELISA) Holmgren et al. J Med Genet 1994, 31, 351‐4
In 1276 individuals: • Met 30 16 • Met 30/Met 30 3 • Other mutations not looked for
FAP – World Distribution FAP
© 2001 National Geographic Society
The Travels of a Gene?
© 2001 National Geographic Society
III - Diagnosis
Histopathology Biopsies • Nerve and skin • Fat aspirate p • Other
The basis of diagnosis in old times (before genetic testing) It is rarely positive in asymptomatic carriers
ATTR V30M – Molecular Testingg 30 ·· AGTCCTGCCATCAATGTGGCCGTGCATGTG ·· ·· -S--P--A--I--N--V--A--V--H--V- ··
↓ ·· AGTCCTGCCATCAATGTGGCCATGCATGTG ·· ·· -S--P--A--I--N--V--A--M--H--V- ··
ATTR Diagnosis – DNA Sequencing ATTR Diagnosis DNA Sequencing
Alternatives
TTR V28M
• SSCP • DHPLC • etc. t
IV - Treatment
FAP - Treatment ea e Before the advent of liver transplantation, FAP was an incurable disease.
Liver transplantation is a primitive a primitive form of gene therapy, gene therapy and is effective because > 90% of human TTR is produced in the liver.
Only current alternative: Prevention [PND, DGPI] [PND DGPI]
PAF – Prevenção Primária PAF Prevenção Primária Aconselhamento genético • Diagnóstico pré‐natal
• Diagnóstico pré‐implantação g p p ç
DESENVOLVIMENTO EMBRIONÁRIO PRÉ-IMPLANTAÇÃO É
1º Dia
Dia 1-2
Dia 2
Dia 3
•
1º e 2º glóbulos polares
•
1 a 2 bl blastómeros, ó ao 3º di dia
•
Células da trofoectoderme de blastocistos, ao 5º dia
BIÓPSIA
Dia 4
Dia 5
Dia 6-7
Dia 6
PROCESSO DE FIV • Exames preliminares ♀ ♂ • Estimulação: ≥ 9 ovócitos • ICSI - não contaminação
ez células él l s d da granulosa l s
ABERTURA DA ZONA PELÚCIDA • Solução ácida de Tyrode (pH 2.2) • Laser • Dissecção parcial
*Meio sem Ca2+/Mg2+
TRANSFERÊNCIA DE EMBRIÕES 4º ou 5º dia após punção folicular
TAXA DE GRAVIDEZ CLÍNICA EM DGPI 22,4%
Prevenção Secundária Prevenção Secundária • Inibição da formação de amilóide • Esta Estabilizadores za or s do o ttetrâmero trâm ro de TTR • Remoção da proteína circulante
• Remoção dos depósitos • Antagonistas dos glicosaminoglicanos
Fib il é Fibrilogénese ‐ I Estudos realizados in vitro com proteínas purificadas. purificadas
Estes estudos mostraram que é relativamente fácil q obter fibras com as características genéricas das fibras de amilóide a partir de um grande número de proteínas ou fragmentos proteicos.
Fib il é Fibrilogénese ‐ II A fibrilogénese passa quase sempre pela formação de um intermediário, que pode formar fibras de imediato, ou a partir de agregados de tipo amorfo.
ATTR - Fibrilogenesis
Native tetramer
pH <3
pH 3
pH 5.3
Rearranjed tetramer
Early model, based on acidic denaturation
A-state
Amyloid fibrils
SAP
GAGs
Serag et al. Nature Struct Biol 9: 734-9, 2002
ATTR Citotoxicity Mechanisms ATTR ‐ Citotoxicity Mechanisms
Hou, X. et al. FEBS Journal 2007, 274 (7), 1637-50.
Kadowaki et al., J Chem Neuroanatomy 28 (2004) 93–100
FAP – C Clinical ca Trials as
DMSO
CEP
II/III (?)
1984
Apheresis
CEP
I/II
2001
CPHPC (Ro 63‐8695)
CAAPP / Roche
II/III
2002
Fx‐1006A
FoldRx
II/III
2009
Difl i l Diflunisal
BUMC
II/III
2010
Doxycyclin
IBMC
?
?