CADASIL

Molecular genetics and genetic counseling

E. Tournier-Lasserve Genetics Lariboisire AP-HP Hospital Lab, U740 Inserm/Paris7 University Research lab

CADASIL, a long journey to establish its definition

Phenotype AD GOM Linkage chr19 Notch3 gene Stereotyped mutations

CADASIL locus

Clinical and MRI features of an ischaemic SADB Autosomal dominant ischaemic SADB Highly specific EM marker: Granular Osmiophilic Material Linkage to chromosome 19p13, first molecular marker Gene identification and mutation spectrum establishment
Stroke1991/1993; Nat Genet 1993 Nature 1996; Lancet 1997

NOTCH3, gene and protein

NOTCH3 gene / 33 Exons encoding a 2321 aminoacids single pass transmembrane receptor Furin cleavage at S1 site Large extra cellular domain including 24 EGF-like motifs, each containing 6 cystein residues

Membrane tethered intracellular domain

Gene

14

24

33

34 EGFR

LNR TM

ANK

PEST

Full length receptor

Heterodimer

S1 S2 S3

Joutel et al, Nature, 1996

Highly stereotyped NOTCH3 mutations in CADASIL patients

50 index patients included (criteria: ischaemic familial SVD + Linkage to chr 19 and/or GOM on skin biopsy in 31 of these families) Screening of all 33 exons of NOTCH3 using SSCP and heteroduplex screening followed by sequencing of any detected variant 45 index patients heterozygous for a missense mutation leading to an odd number of cystein residues in one of the 24 EGF motifs encoded by exons 2-24 Cosegregation with the affected phenotype in the families of these 45 index 5 index patients including 1 patient with GOM and 1 patient from a linked family did not show any variant using SSCP/HeteroD. Sanger sequencing of all exons detected a cystein mutation in both patients

Joutel et al, Lancet 1997

Confirmation of this highly stereotyped nature of CADASIL mutations in large series of biopsy proven CADASIL patients

Dichgans group: 125 EM biopsy proven CADASIL patients / Mutation identified in 120 / in all cases mutations led to an odd number of cystein residues whatever the mechanism (missense, small in frame deletions taking a Cys residue)

Kalimos group: 131 EM biopsy proven CADASIL patients / Mutations identified in 131 / in all cases mutations led to an odd number of cystein residues whatever the mechanism (missense, duplication involving a cys residue)

All Mutations in CADASIL biopsy proven patients lead to an odd number of cystein residues within one of the EGF-like motifs

Peters et al, Arch Neurol 2005 Tikka et al, 2009

Strong clustering of CADASIL mutations in exons 3-4 / EGF1-5

Joutel et al 1997: 69 % patients have a mutation en Exons 3 and 4 (EGF1-5) Peters et al 2005: 67,5 % in exons 3-4 (EGF1-5) Federico et al 2005 review: 62,5 % in exons 3-4 (EGF1-5)

Joutel et al, Lancet 1997

Screening of NOTCH3 in SADB patients Why and How ?

Importance of a diagnostic test to avoid mis-diagnosis or no diagnosis

Importance for genetic counseling

Need for a highly sensitive and highly specific diagnostic test

1993-2003:

1993: linkage analysis possible only in large families and/or skin biopsy search for GOM
1996: gene identification / sequencing available but cost +++ / combination of exons 3-4 sequencing and EM skin biopsy study 2001: combination of exons 3-4 sequencing and skin biopsy immunostaining (Notch3 Ab)

2003-2013: Extensive sequencing of NOTCH3 EGF-like motifs

2003-2013 NOTCH3 sequencing experience in Lariboisire Yield of screening for CADASIL mutations
2721 index patients referred Lacunar infarcts + leukoaraiosis Cognitive defects + leukoaraiosis Familial or sporadic

361 index patients with an odd number of cystein residues

13 % of referred index patients are CADASIL patients

Identification of a CADASIL type mutation in a SADB patient Implications for the patient and his/her family
Importance of a diagnostic test to avoid mis-diagnosis in the index patient : 20 % of CADASIL patients were previously diagnosed as having multiple sclerosis and received inadequate treatments. Others were diagnosed as Alzheimers disease patients or psychiatric patients.

Importance to avoid mis diagnosis and /or long and costly investigations in the affected relatives of the index patient

2003-2013: 208 relatives (> 95 % of them being clinically affected) of the 361 index patients were referred for diagnostic and shown to be mutated

Importance to answer the questions of patients and families and genetic counseling

Genetic counseling
Answering questions of patients and family members Presymptomatic testing (PT): caution +++ screening an adult asymptomatic member for a CADASIL mutation requires important precautions Experienced multidisciplinary team (neurologist / geneticist / psychologist) and established multi-step procedure including follow-up after testing Lariboisire 2007-2010 experience in presymptomatic testing:

33 asymptomatic relatives of an affected member came for PT

11 / 33 reached the final step 6 were mutated

Presymptomatic screening rarely requested / high drop-out during PT

procedure / importance of providing care post testing
Reyes, Kurtz et al, J Neurol 2012

2003-2013 NOTCH3 sequencing experience in Lariboisire Other rare variants in NOTCH3

2721 index patients referred for NOTCH3 screening

361 CADASIL index patients Odd number od cystein

And in addition
76 index patients (2.8 %) Rare missense mutation which do not affect the number of cystein

9 index patients (0.3 %) Mutation leading to a stop codon

Are these rare missense variants polymorphisms or deleterious variants ?

Rare NOTCH3 missense variants sparing cystein

Several families reported as CADASIL families in the past few years

Do patients with rare non cystein missense mutations have GOM ? A systematic survey

19 patients referred for NOTCH3 screening

PATIENT P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17 P18 P19 AA MUTATION I658T A564T G536V A40V R764H L925V P81R A1057V L295R A1342D W1028S D524N R1120Q V560M R767H R680H R61W G73R G73R NT MUTATION* 2051T>C 1768G>A 1685G>T 197CT 2378G>A 2851C>G 320G>C 3248C>T 962T>C 4103C>A 3161G>C 1648G>A 3737G>A 1756G>A 2378G>A 2117G>A 259C>T 295G>C 295G>C Exon Skin Biopsy 13 YES 11 YES 11 YES 2 YES 15 YES 17 YES 4 YES 20 YES 8 YES 24 YES 19 YES 10 YES 22 YES 11 YES 15 YES 13 YES 3 YES 3 YES 3 YES

Sequencing of EGF 2-24 and no mutation leading to an odd number of cysteins Non cystein missense mutations absent from all databases (15 mutations) or with a frequency < 1/ 2000 (3 mutations) Skin biopsy: EM search for GOM

Pathologists blind to mutation status

427 vascular sections including 101 arteriolar, 263 venous, 63 capillary walls 10-24 vessels / patient
Amador, Mine, Prieto et al / Lariboisire Chapon, Debout et al / CHU de Caen

90 % of patients with rare non cystein mutations do not have GOM

Skin biopsy analysis allows to eliminate CADASIL in such patients

PATIENT

AA MUTATION

NT MUTATION*

Exon

Skin Biopsy

P1
P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13

I658T
A564T G536V A40V R764H L925V P81R A1057V L295R A1342D W1028S D524N R1120Q

2051T>C
1768G>A 1685G>T 197CT 2378G>A 2851C>G 320G>C 3248C>T 962T>C 4103C>A 3161G>C 1648G>A 3737G>A

13 No
11 No 11 No 2 No 15 No 17 No 4 No 20 No 8 No 24 No 19 No 10 No 22 No

P14
P15 P16 P17 P18 P19

V560M
R767H R680H R61W G73R G73R

1756G>A
2378G>A 2117G>A 259C>T 295G>C 295G>C

11 No
15 No 13 No 3 No 3 Yes 3 Yes

Amador, Mine, Prieto et al / Lariboisire Chapon, Debout et al / CHU de Caen

2 out of 19 patients with rare non cystein mutations have GOM (10 %)
Both of these unrelated index patients have a G73R mutation
Skin

PATIENT P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17 P18 P19

AA MUTATION I658T A564T G536V A40V R764H L925V P81R A1057V L295R A1342D W1028S D524N R1120Q V560M R767H R680H R61W G73R G73R

NT MUTATION* 2051T>C 1768G>A 1685G>T 197CT 2378G>A 2851C>G 320G>C 3248C>T 962T>C 4103C>A 3161G>C 1648G>A 3737G>A 1756G>A 2378G>A 2117G>A 259C>T 295G>C 295G>C

Exon Biopsy 13 No 11 No 11 No 2 No 15 No 17 No 4 No 20 No 8 No 24 No 19 No 10 No 22 No 11 No 15 No 13 No 3 No 3 Yes 3 Yes

2 patients without a mutation affecting the number of cystein residues (EGF 2-24 sequenced) These 2 patients have typical GOM and are therefore true CADASIL patients Is G73R causing CADASIL and if so, what is the mechanism ? Or have we missed the true CADASIL mutation ?

Is G73R causing CADASIL and if so, what is the mechanism ? Or have we missed the true CADASIL mutation ?
Three main hypotheses: 1- R73G leads per se, without a need for changing the number of cystein residues to CADASIL and GOM by a so far totally unknown mechanism (conformational change ? Interaction of this aa with a component of basal membrane ?) 2- R73G leads by a splicing effect to an abormally spliced NOTCH3 mRNA having an odd number of cystein residues 3- R73G mutation is in tight linkage disequilibrium with a true CADASIL mutation undetecble by exonic genomic sequencing ie a deep intronic mutation leading to abnormal splicing or a genomic deletion taking off an odd number of cystein. Strategy and preliminary results Sequencing of cDNA should provide the answer for hypotheses 2 and 3. Extraction of NOTCH3 mRNA from skin fibroblasts (P19) / sequencing of the NOTCH3 cDNA Search for deletions using copy number analysis for all exons encoding EGF motifs No deletion detected so far and no additional mutation in the cDNA but part of the cDNA has not yet been sequenced.

2003-2013 NOTCH3 sequencing experience in Lariboisire Other rare variants in NOTCH3

2721 index patients referred for NOTCH3 screening

361 CADASIL index patients Odd number od cystein

And in addition
76 index patients (2.8 %) Rare missense mutations which do not affect the number of cystein

9 index patients (0.3 %) Mutations leading to a stop codon

Are these stop codons rare variants polymorphisms or deleterious variants ?

Are NOTCH3 stop codons polymorphic or deleterious variants ? Phenotype(s) of NOTCH3 stop codon carriers ?

Stop codons in a mRNA leads in most cases to mRNA degradation

A patient heterozygous for a stop codon is therefore haploinsufficient (half dose protein) A few stop codons however, depending of their location, lead to abnormal splicing

Three main hypotheses: 1 - NOTCH3 stop codons could be polymorphic, non deleterious variants (fortuitous discovery) 2 - NOTCH3 stop codons could be deleterious variants leading, at least for some of them, to abnormal splicing and an odd number of cystein residues and true CADASIL 3 - NOTCH3 stop codons could be deleterious variants causing haploinsufficiency of the notch3 receptor which would lead to a novel SVD phenotype, yet to be described. Strategy Skin biopsy should answer hypothesis n3 RT-qPCR quantification of NOTCH3 mRNA from skin fibroblasts or muscle should clarify the existence of an haploinsufficiency Once haploinsufficiency is established, a cosegregation analysis of the stop codon with the SVD phenotype is required to establish its causality

Are NOTCH3 stop codons polymorphic or deleterious variants ?

Patients and strategy

Patients and families 1996-2013: 12 unrelated index patients with stop codons 5 of these 12 patients have known SADB clinically affected relatives Strategy Skin biopsy EM study in 6 index + RT-qPCR in muscle from 2 index Clinical and MRI investigation of consenting relatives (national PHRC) Genotyping of all consenting adult relatives

Skin biopsy EM analysis and RT-qPCR

No GOMs in the index patients RT-qPCR on mRNA extracted from muscle from 2 index patients: mutated RNA=5% of normal mRNA Notch3 haploinsufficiency

Amador, Joutel, Mine, Prieto et al / Lariboisire Chapon, Debout et al / CHU de Caen

MRI of 8 symptomatic patients with NOTCH3 stop codons

F1 index, 71 yo F4 index, 68 yo

F2 index, 57 yo

F4 sister, 71 yo

F3 index, 55 yo

F5 index, 68 yo

F3 father, 72 yo

F6 index, 49 yo

MRI evolution of lesions with age

F5 index, 63 yo F5 index, 68 yo

F2 index, 51 yo

F2 index, 57 yo

F3 index, 49 yo

F3 index, 55 yo

F1 index, 60 yo

F1 index, 71 yo

Genealogical trees and mutation status

Family 4 13 genotyped members aged > 45 yo and with an MRI - 6 affected / all mutated - 7 unaffected / all not mutated One asymptomatic member aged 42 yo, with a normal MRI and mutated Cosegregation Age dependant penetrance ?

Family 3 3 genotyped members aged > 45 yo and with an MRI - 2 affected and mutated - 1 unaffected and not mutated

Cosegregation of the phenotype with the genotype but need to screen additional relatives (on going) to firmly establish cosegregation

Late onset of white matter hypersignals in stop codons carriers

F4-IV28, 42 yo, mutated F4-IV22, 56 yo, mutated

F4-III31, 42 yo, mutated

F4-III15, 63 yo, not mutated

NOTCH3 mutations, CADASIL and other SVD

CADASIL, a disease charaterized by highly stereotyped mutations leading to an odd number of cystein residues, GOM and Notch3 extracellular domain accumulation CADASIL: around 10-15 % of patients referred for molecular diagnostic in routine practice Importance of skin biopsy analysis for rare missense variants not affecting the number of cystein to sort out the rare CADASIL patients among those Stop codons in NOTCH3 do not lead to CADASIL but might be causing a novel vascular leukoencephalopathy whose phenotype has to be established with the detailed clinical and MRI investigations. Stop codons can be located anywhere in the 33 exons of NOTCH3. Importance for diagnosis Additional phenotypes caused by very rare Notch3 activating mutations affecting the heterodimerization domain (Fouillade et al) Importance for good genetic care, good genetic counseling and future therapeutic trials to establish precise criteria for Notch3 non CADASIL mutated patients.

2003-2013 NOTCH3 sequencing experience in Lariboisire

Next step: the other non NOTCH3 variants causing SADB

2721 index patients referred for NOTCH3 screening

361 CADASIL index patients (13 %) Odd number od cystein

And
76 index patients (2.8 %) Rare missense mutations which do not affect the number of cystein

9 index patients (0.3 %) Mutations leading to a stop codon

Next step What about the 84 % of SVD patients with no NOTCH3 mutation ?

CADASIL and other SADB with NOTCH3 mutations

Institutions: INSERM U740 / Univ Paris7, Genetics diagnostic facility, Lariboisire AP-HP hospital / Neurology dpt, Lariboisire AP-HP hospital / CERVCO

Support: INSERM, PHRC 2006-2009, Leducq Foundation 2007-2013, French National Plan for Rare Disease

Contributors: - A. Joutel, M. Mine, F. Riant, MDM. Amador, C. Prieto-Morin, A. Delaforge, M. Arnould

- D. Herv, H. Chabriat, MG Bousser, (Neurology Dpt, Lariboisire Hospital)

- S. Reyes, A. Kurtz, J. Ruffi, S. Hello, E. De Potter (Neurology / Genetics / CERVCO) - French neurologists: P. Labauge, L. Guyant-Marechal, M. Sevin, S. Alamowitch, S. Timsit, L. Milandre

- Claire Debout, F. Chapon (CHU de Caen)