NextGen Sequencing and Brain Diseases: a Pot Pourri. Guy A. Rouleau, MD, PhD, FRCP(C) University of McGill Montreal Neurological Institute

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2 NextGen Sequencing and Brain Diseases: a Pot Pourri Guy A. Rouleau, MD, PhD, FRCP(C) University of McGill Montreal Neurological Institute

3 Disclamers I have no conflicts of interests in this work I have no financial interests in this work

4 Disclamers I did none of the work

5 Our approach Clinical collection Multicenter collaboration Uniform phenotypic assessment Web based database Sequencing approach depends on genetic architecture Recessive disease Dominant Sporadic Biological validation

6 CanHSP Project Multicenter multidisciplinary project that brings together Canadian experts in the fields of neurology, genetics and molecular biology to study spastic paraplegia Started in spring 2012 Funded for a duration 5 years

7 Steering Committee Kym Boycott, Pierre Drapeau, Guy Rouleau, Oksana Suchowersky Team Coordinator Anna Szuto (Patrick A. Dion) Team Leader Guy A. Rouleau Aim1 Clinical Oksana Suchowersky François Bolduc Jean-Pierre Bouchard Nicolas Dupré Guy Rouleau Grace Yoon Aim2 Genetics Kym Boycott Jillian Parboosingh Peter Ray Guy Rouleau Aim3 Functional Pierre Drapeau François Bolduc Alex Parker Guy Rouleau Christine Vande Velde

8 Goal 1 Clinical characterisation Create a network of clinical experts in spastic paraplegia Create a multicentric and national registry of clinical information associated with a genomic bank (DNA, lymphoblastoid cell lines and fibroblast) of spastic paraplegia cases Familial Cases Sporadic Cases Establish recommendations for patient management Clinical evaluation Molecular screening Treatment

9 Goal 1 Clinical characterisation 1. Determining the patients of interest Sporadic and familial cases of spastic paraplegia, including: Cases of spastic ataxia Cases spastic paraplegia with neuropathy Any case where spastic paraplegia is the primary symptom Any case with a known molecular diagnosis, even if DNA is not available 2. Phenotyping Meeting with the CanHSP team of clinical experts to determine Inclusion / exclusion criteria pertinent clinical findings to characterize Research of pre-existing phenotyping tools: Sickkids HSP clinical data sheet Spastic Paraplegia Rating Scale (SPRS) Diagnostic form for spinocerebellar degeneration (SPATAX network) Create a standardized clinical datasheet Soft launch: Retroactive review of HSP patients charts to test the CanHSP clinical questionnaire Gather and incorporate feedback

10 Clinical datasheet

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12 Goal 1 Clinical characterisation Collecting and storing the data Secure web-based interface for one common database created by our bioinformatics team. It allows for: Effective way to curate a shared database Simultaneous access Tracking of recruitment Control of access privileges (Entering data, consulting local data, data mining) Efficient way to generate epidemiological data Access to data Each site has access to their own cohort Internal requests for data mining are submitted to the CanHSP clinical team to request access specific data from all sites External requests for data mining will be accepted at the end of the CanHSP project Recruiting sites and external collaborations At the moment, active sites include Montreal, Quebec, Ottawa, Toronto, and Edmonton/Calgary Possibility of inviting external PIs to collaborate to the CanHSP project

13 Web based interface of the CanHSP database

14 Where are we now and future directions: To date: 402 phenotyped patients entered into the CanHSP database Collection of over 182 individuals/families in the genomic bank In the next 4 years: Possibility of extending the network of recruiting sites Creation of a website for physicians and patients

15 Agenesis of Corpus Callosum: anatomically defined condition that results from disruption of the early stages of fetal callosal development Diagnosis: absence of callosal fibers, typically accompanied by a characteristic dilatation of posterior lateral ventricules (colpocephaly) Paul LK et al., 2007

16 Agenesis of Corpus Callosum Heterogeneous condition found usually as one manisfestation of a congenital syndrome and noccosionally as an isolated condition or ACC: between 1/4000 and 1/ to 5% of individuals w/ neurodevelopmental disorders Most frequent clinical findings in patients with ACC: Mental retardation (60%) Visual problemes (33%) Speech delay (29%) Seizures (25%) Feeding problems (20%)

17 Agenesis of Corpus Callosum: examples of syndromes associated with ACC Paul LK et al., 2007

18 Genetics of ACC Many different mechanisms: Mendelian, sporadic, complex Cases with isolated ACC and developmental delay with normal chromosomes have been published with apparent autosomal dominant, autosomal recessive or X-linked modes if inheritance but no causative genes identified to date

19 ACC: CCA1 pedigree - Normal mother and father with 3 affected children - Female 1: ACC confirmed by CT; normal development; IQ of Female 2: ACC by CT and MRI; normal development except for slow acquisition of walking. IQ of Male 3: ACC by MRI, respiratory problems at birth, prolonged enuresis, poor motor coordination and late language acquisition at 5. IQ of 77. I 1 2 I1: R (father unaffected) I2: R (mother unaffected) II1: R (daugther affected) II2: R (daugther affected) II II3: R (son affected) 1 2 3

20 Hypothesis: ACC: CCA1 pedigree recessive: homozygous mutations, compound heterozygous de novo Methodology: Whole-exome sequencing on DNA extracted from patient blood sample Targeted capture: SureSelect v4xt 51MB (Agilent) Sequencing: Illumina HiSeq 2000 v3 (3 individuals/lane) Bioinformatics analysis: sequence reads trimming and QC using FastX, mapping and alignment using BWA In-house data pipeline based on GATK for realignment around indels, duplicates removal and base quality recalibration. GATK also used for SNP/INDEL calling and finally Annovar for variant annotation Segregation: 3 probands Non-family controls: in-house exomes, public databases (Exome Variant server, )

21 Exome sequencing quality obtained per sample name sample info Alignment stats variant stats PED.id capture kit project % target >=10x % target >=20x average calls total calls coverage >=20x S12959 CCA1 V4 ACC 97,8% 93,0% 100, S12960 CCA1 V4 ACC 98,4% 95,1% 126, S12961 CCA1 V4 ACC 98,9% 96,2% 134, Average 98,4% 94,8% 120,

22 Compound heterozygous analysis Average number of variants obtained per patient after successive filtering steps initial number of variants fam aff w/variant (3) non fam CTR w/variant (10/965) public databases (<0.01) variant function variant type several hits in same gene Series

23 Validation step (Sanger sequencing) Eight variants in 4 different genes; ONE gene with compound heterozygous mutations Chr Position Ref Mut Var Gene Variant Type Detailed Annotation Mutation confirmed in: T C SNP CDK5RAP2 nonsyn CDK5RAP2:NM_018249:exon24:c.A3695G:p.N1232S R , R , R , R (Father) C G SNP CDK5RAP2 nonsyn CDK5RAP2:NM_018249:exon4:c.G280C:p.G94R R , R , R , R (Mother) C T SNP ADAMTS14 nonsyn ADAMTS14:NM_080722:exon6:c.C988T:p.R330C R , R , R , R (Father) G A SNP ADAMTS14 nonsyn ADAMTS14:NM_080722:exon21:c.G3097A:p.D1033N R , R , R , R (Father) G C SNP IFIH1 nonsyn IFIH1:NM_022168:exon15:c.C2863G:p.Q955E R , R , R , R (Mother) A C SNP IFIH1 nonsyn IFIH1:NM_022168:exon12:c.T2414G:p.I805S R , R , R , R (Mother) C G SNP OR5K1 nonsyn OR5K1:NM_ :exon1:c.C753G:p.F251L R , R , R , R (Father) G T SNP OR5K1 nonsyn OR5K1:NM_ :exon1:c.G774T:p.M258I R , R , R , R (Father)

24 Segregation analysis of CDK5RAP2 variants CDK5RAP2:NM_ exon24:c.a3695g p.n1232s I 1 2 Wt/N1232S Wt/G94R CDK5RAP2:NM_ exon4:c.g280c p.g94r II Wt/N1232S Wt/G94R Wt/N1232S Wt/G94R Wt/N1232S Wt/G94R I1: R (father unaffected) I2: R (mother unaffected) II1: R (daugther affected) II2: R (daugther affected) II3: R (son affected)

25 Bioinformatic analysis N1232S SIFT: Tolerated Polyphen: possibly damaging G4R SIFT: damaging Polyphen: probably damaging

26 CDK5RAP2= MCPH3 (Autosomal recessive primary microcephaly) MCPH Protein Gene Chromosome Ref. MCPH1 Microcephalin MCPH1 8p23 [28, 29] MCPH2 WD-repeat containing protein 62 WDR62 19q13.12 [30 32] MCPH3 Cyclin dependent kinase 5 regulatory subunit-associated protein 2 CDK5RAP2 9q33.3 [17, 27] MCPH4 MCPH5 MCPH6 Centrosomal protein of 152 kda Abnormal spindlelike, microcephaly associated Centromeric protein J CEP152 15q21.1 [33, 34] ASPM 1q31 [35, 36] CENPJ 13q12.2 [17, 37] MCPH7 SCL/TAL1 interrupting locus STIL 1p32 [38] Kraemer N., 2011 MCPH is a rare autosomal recessive neurodevelopmental disorder reduced brain size and mental retardation The reduced brain size is believed to result from assymmetric division of neuronal progenitor cells, causing reduced number of neurons Only 2 mutations reported in 2 Pakistani microcephaly pedigrees: Y82X and R1334SfsX5

27 CDK5RAP2 Gene Hypothesis that these are hypomorphic alleles that lead to a milder neurodevelopmental disease Need to demonstrate the functional effects of our compound het mutations

28 RNA Stability and Splicing RNA extraction on patients cells, cdna synthesis and sequencing

29 Sequencing results of exons 1 to 6 in cdna from patients lymphoblastoid cells Mutations are detected in patients cdna (G94R)

30 Sequencing results of exons 22 to 25 in cdna from patients lymphoblastoid cells Mutations are detected in patients cdna (N1232S)

31 ACC project: next steps Genetics: We have in the lab: 649 ACC patients (191 affected), however, most have ACCPN with loss of KCC3 function ± 15 cases which are KCC3- but only 3-4 cases that have no major neuropathic symptomes, only colpocephalia Possible collaboration with Dr Sherr for screening of CDK5RAP2 in additional patients with the same phenotype Additional collaboration??? Molecular characterization of the mutations Cell biology and binding experiments based on know protein functions Animal model: rescue experiments in zebrafish

32 Essential Tremor (ET) ET is one of the commonest neurological disorders. It is characterized by postural tremor that worsens with movement. Mainly affects the upper limbs, and less commonly affects the head, voice, face, tongue, trunk and lower limbs. The condition may cause a functional disability or even incapacitation. Thought to involve cerebellar degeneration

33 Genetics of Essential Tremor (ET) Linkage studies identified three loci, but no causative genes Hereditary essential tremor-1 (ETM1) 3q13.3 (1997) 16 Icelandic families Parametric LOD score (assuming an autosomal dominant model) = 3.71 ETM2-2p25-p22 Large American family of Czech descent A maximum lod score of 5.92 was obtained for the locus D2S272. ETM3-6p23. Two unrelated North American families. First family: a maximal nonparametric lod score of 3.28 Second family showed a maximal score of Together, maximal lod score of at D6S1630 and D6S1605 Recently, common LINGO1 variants have been associated with ET but the significance remains unclear.

34 Essential Tremor (ET) Our lab recruited ~80 French Canadian families with ET. Linkage studies have been performed on large families but results are inconclusive. Likely because of diagnostic uncertainties. To overcome the diagnostic barrier we carried out exome sequencing using only individuals with: a definite ET diagnosis An age of onset under 40

35 FET1 family Individuals with a definite diagnosis are represented by symbols filled completely black. Individuals with a probable diagnosis of ET have symbols with a small black square in the bottom left corner. Individuals with a possible diagnosis of ET have symbols with a small black square in the top right corner.

36 FET1 Exome analysis -CCDS bp covered by Aligent exome probe kit = 28,845,296 -Bp covered greater than or equal to 1X = 21,469,388 (74% of Aligent kit) -Bp covered greater than or equal to 20X = 14,145,512 (49% of Aligent kit) -Total number of reads = 26,023,097 -Looked for variants shared by all affected individuals. -Mutation frequency needs to be above the threshold followed for segregation analysis. -This project, 20X coverage and 20% MF ).

37 FET1 Exome analysis Individual FET1 Clinical Status Number of CCDS base pairs actually covered with the A gilent exome probe kit Base pairs covered greater than or equal to 1X Number Percentage in Aligent probe kit Base pairs covered greater than or equal to 20X Number Percentage in Aligent probe kit Number of uniquely mapped reads IV:6 affected 21,357,223 74% 13,577,170 47% 25,563,340 III:9 affected 21,178,474 73% 13,493,797 47% 28,622,732 II:13 affected 28,845,296 21,255,995 74% 13,590,471 47% 26,741,248 II:6 affected 21,653,639 75% 14,417,379 50% 22,655,426 II:5 control 21,901,610 76% 15,648,745 54% 26,532,741 Average - 28,845,296 21,469,388 74% 14,145,512 49% 26,023,097

38 Diagnosis of ET in FET1 and FUS mutation status Diagnosis Under 40 yrs Over 40 yrs Unknown age Total Mutation Carriers Percentage Definite % Probable % Possible % Clinically unaffected %

39 Schematic of ET sequencing variants within the FUS protein

40 ET and ALS truncating FUS mutations In ALS mutations are clustered to nuclear localization signal (NLS) in C-terminal. The mutations may gain an aberrant function or toxic property, or lose a particular function. In ET p.q290x is located in the predicted nuclear export signal (NES) in the middle of the protein. No ALS mutations have been reported in the NES. Likely leads to complete loss of function and no residual fuction Hypothesis: ET results from a LOF while ALS results from a GOF Predicts that ALS truncating mutations, but not ET truncating mutations, should give a protein

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42 Protein expression of ET and ALS FUS truncating mutants -150 FUS β-tubulin truncated FUS

43 Acknowledgements Rouleau lab : Patrick A Dion Loubna Jouan Nancy Merner Anne Noreau Anna Szuto Alexandre Dionne- Laporte Dan Spiegelman Edouard Henrion Ousmane Diallo Clinical Collaborators: François Bolduc Jean-Pierre Bouchard Kym Boycott Sylvain Chouinard Nicolas Dupre Oksana Suchowersky Grace Yoon

44 Questions?