john e. landers, ph.d. professor of neurology university ... · background of als • a...
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John E. Landers, Ph.D.
Professor of Neurology
University of Massachusetts Medical School
Background of ALS
• A progressive, fatal, disease caused by the
degeneration of motor neurons
• As motor neurons die, muscles are unable
to function and gradually become weaker
• Eventually all voluntary movement is lost
and muscles become paralyzed
• Typically develops between ages 40-70
• ~50% affected die within 3 years/~75%
within 5 years
• ~5,000 people are newly diagnosis in the
US every year
• ~30,000 total affected people in the US
• Family History of ALS
• 10% of all ALS cases
• Typically due to a single mutation
in a single gene
• Researchers have identified ~2/3 of
the genes contributing to Familial
ALS
• Stronger Genetic / Weaker
Environmental Influence
• No Family History of ALS
• 90% of ALS cases
• Multiple genes may be contributing
to your risk of ALS + Environmental
Factors
• Few Risk Genes Contributing to
ALS have been Identified
• Weaker Genetic / Stronger
Environmental Influence
ALS
Genetics Opens Several Avenues of
Research for Therapeutic Treatments
Therapeutic Treatments
Genetics
Pathways/PathogenesisGene Therapy Model Organisms
Improvements in Any of These Areas Will Facilitate
The Development of Therapeutic Treatments
• All Affected Family Members are due
to the Same Genetic Mutation
• Segregation of DNA regions to
Multiple Affected Family Members to
Narrow Genomic Region
• Typically due to a single “causative”
mutation in a single gene
• Familial ALS Genes: SOD1, FUS,
TARDBP, UBQLN2
• Identification of common SNP
alleles that are more frequent in
ALS vs. control population
• Does not necessary identifying the
contributing factor but rather an
associated marker
• Usually identify variants with small
impact of disease risk (odds ratios
< 2)
• Sporadic ALS Genes: UNC13A,
SARM1, C21orf2
Overview of Positional Cloning
2-20 MbUp to >100
Genes
Pediatr Nephrol (2007) 22:2023–2029http://portal.ccg.uni-koeln.de/ccg/index.php?id=49
Gene Mapping
GeneIdentification
MutationScreening
Require Multiple Affected
Family Members
Linkage
Analysis
Human Genome Project
Novel Approaches Needed to Identify Causative
Genes for Difficult Families
• For some disorders, families are too small for traditional positional cloning
• Late-onset disease- difficult to obtain multiple generations
• Complete penetrance of the disease is not always observed
• Results in Numerous genomic regions which are too large and expensive to sequence by Sanger sequencing
Exome sequencing has resulted in a massive boost
in gene identification for Mendelian diseases
Exome Sequencing = Technique to Deep Sequence
only the ~2% of the genome coding for protein
Perform Exome Sequencing on Affected Family Members
Identify Variants in Each Sample
Filter Variants Based on Several Criteria
Identify Causative Mutation
Wu et al. Nature 488: 499–501. (2012).
Variant identification:
► Exome sequence two affected members from two ALS families
Variant Filtering:► Observed in both family members
► Alters amino acid
►Within a linkage peak
► Absent/Rare in SNP databases
Family 1
282,792
SNPs
C71G
Family 2
382,751
SNPs
2 SNPs 3 SNPs
M114T
PFN1
Combined LOD Score of Families #1- 2 = 4.51
(Chance of observing: 30,000:1)
Wu et al. Nature 488: 499–501. (2012).
• Conversion of monomeric (G)-actin to
filamentous (F)-actin
• Poly-L-Proline Binding Domain
• Reported to bind >50 proteins
• Regulation of Actin Polymerization
(VASP, Formin)
• VCP
• SMN
• Huntingtin
W. Witke. Trends in Cell Biol. 14: 461-469 (2004).
S=Soluble
I=Insoluble
Primary Motor Neurons
N2A Cells
Wu et al. Nature 488: 499–501. (2012).
Primary Motor Neurons
Wu et al. Nature 488: 499–501. (2012).
F-actin = Filamentous Actin
G-actin = Monomeric ActinWu et al. Nature 488: 499–501. (2012).
Problem: How to Identify Genes Associated with ALS
Without Multiple Family Members?
No additional affected members are available
for ~90% of FALS DNA collected
• Exome sequence of individual cases will not
identify causative genes:
• ~80 Novel non-synonymous variants
• ~800 Rare (<0.5%) non-synonymous variants
Linkage Studies
GWAS
Nature 461, 747-753 (2009)
Linkage Studies
GWAS
Rare Variant Association Analysis
For Every Gene, Compare the Frequency of All Rare Deleterious Variantsin a Cohort of Cases and Controls
Nature 461, 747-753 (2009)
ALS Patient
DNA Sequences
Control
DNA Sequences
Sequence #
1.
2.
3.
4.
5.
1.
2.
3.
4.
5.
Variant
ALS Patient
DNA Sequences
Control
DNA Sequences
Sequence #
1.
2.
3.
4.
5.
1.
2.
3.
4.
5.
Gene #1 Gene #2
ALS Patient
DNA Sequences
Control
DNA Sequences
Sequence #
1.
2.
3.
4.
5.
1.
2.
3.
4.
5.
Gene #1 Gene #2
ALS Patient
DNA Sequences
Control
DNA Sequences
Sequence #
1.
2.
3.
4.
5.
1.
2.
3.
4.
5.
Gene #1 Gene #2
ALS Patient
DNA Sequences
Control
DNA Sequences
Sequence #
1.
2.
3.
4.
5.
1.
2.
3.
4.
5.
Gene #1 Gene #2
ALS Patient
DNA Sequences
Control
DNA Sequences
Sequence #
1.
2.
3.
4.
5.
1.
2.
3.
4.
5.
Gene #1 Gene #2
ALS Patient
DNA Sequences
Control
DNA Sequences
Sequence #
1.
2.
3.
4.
5.
1.
2.
3.
4.
5.
Gene #1 Gene #2
ALS Patient
DNA Sequences
Control
DNA Sequences
Sequence #
1.
2.
3.
4.
5.
1.
2.
3.
4.
5.
Gene #1 Gene #2
Variants in ALS: 3
Variants in Controls: 4
No Differences Observed in
ALS versus controls
ALS Patient
DNA Sequences
Control
DNA Sequences
Sequence #
1.
2.
3.
4.
5.
1.
2.
3.
4.
5.
Gene #1 Gene #2
Variants in ALS: 3 3
Variants in Controls: 4 1
Higher Rate of Variants
in ALS versus controls
Rare Variant Association (RVA) Analysis
• Advantages
• Does not require related family members for analysis
• Can identify risk factors of greater effect than through GWAS
• Multiple software packages available to allow for
weighting/filtering variants by:
• Allele frequency
• Functional annotations
• Both positive and negative effects on phenotypes
• Software packages: CAST, CMC, VT, SKAT
Rare Variant Association (RVA) Analysis
• Disadvantages
• Although, associated genes can be identified, it does not necessarily identified those mutations that are pathogenic
• May require large sample sizes to identify associated genes based on genetic contribution
• Samples must be exome or whole genome sequenced
• Many different parameters are established by user• Allele frequency threshold to define as “rare variant”• Various packages/methods of statistical testing• Many different methods of defining a variant as “deleterious”• Application of multiple parameters increases the level of multiple test
correction to be applied
PolyPhen-2 FATHMM CADD
LRT MetaLR MetaSVM
phyloP PROVEAN MutationAssessor
SIFT SiPhy MutationTaster
Exome-wide Rare Variant Analysis To Identify
Novel Familial ALS Associated Gene
• Familial ALS Exomes:Australia 68
Belgium 13
Canada 39
Germany 202
Ireland 18
Italy 151
Netherlands 47
Turkey 131
UK 216
USA 448
Total 1376 Exomes (1,022 pass QC)
(~90% devoid of known mutations)
• Control Exomes: 13,883 Exomes
(7,315 pass QC)
Optimization of Parameters for Rare
Variant Analysis Using Known ALS Genes
1) Analysis performed on 10 Known ALS
Genes using 308 different combinations of
parameters:- 7 Allele Frequencies
- 44 Functional Filters
2) For each analysis, plotted how many
genes could be detected at different P-
value thresholds
3) Determined the optimal set of parameter
by measuring the area under the curve
4) No one test is best for all genes
Cases: n=1,022
Controls: n=7,315
Known Genes Tested: SOD1, FUS,
TARDBP, VCP, VAPB, TUBA4A,
TBK1, OPTN, PFN1, UBQLN2
FATHMM
MAF < 0.001
Kenna et al. Nature Gen. 48: 1037-1042
Rare Variant Analysis Using
Optimized Parameter Settings
1) Analysis detected 7 of 10 Known
ALS Genes with P values <5 x 10-4
2) One gene, NEK1, detected with a
significant P value
OR=8.2; P= 1.7 x 10-6)
OPTN, VAPB, PFN1 displayed less
significant P-values (ALS genes lower in
frequency in the population)
Cases: n=1,022
Controls: n=7,315Kenna et al. Nature Gen. 48: 1037-1042
Signal From FALS Rare Variant Analysis
is Primarily Driven by NEK1 LOF Variants
ALS Cases (n=1,022): 12 LOF Variants (1.17%)
Controls (n=7,315): 14 LOF (0.19%)
P = 7.3 x 10-7 Odds Ratio: 8.9
p.I10T(1)
p.Y30C(1)
c.217+1C>T*(1)
p.R91X(1)
p.R91Q(1)
p.N93D(1)
p.V98I(3)
p.I105T(1)
p.A115S(1)
p.Q132R*(1)
p.R161X(1)
p.R161X(1)
p.D185G(1)
p.L193F(1)
p.E204G(1)
p.V223M(1)
p.Y229C(12)
p.N250S(1)
p.R261H(17)
p.R261H(52)
c.878+1C>G*(1)
p.I308M(1)
p.A313T(1)
p.K337N(1)
p.A341T(7)
p.A341T(36)
p.T344A(1)
p.R356K(1)
p.D379E(4)
p.Q380E*(1)
p.L384S(1)
c.1153+3T>G*(1)
p.Q393X(1)
p.W409X(1)
p.W409X(1)
p.R440Q(1)
p.E462V(1)
p.A463V(105)
p.A463V(790)
p.G478A(1)
c.1446+1C>A*(1)
c.877− 1C >A *(1)
p.K511Q(1)
p.A512V(1)
p.Q525K(1)
p.R540Q(1)
p.M545T(1)
p.R550X(1)
c.1683+2A>G*(1)
p.F597I(1)
p.N598S(2)
p.N598S(2)
p.E610K(1)
p.A626T(165)
p.A626T(1119)
p.R630H(1)
p.V646I(1)
p.K648E(1)
p.K648E(2)
p.K654M(1)
p.S681F(1)
p.P682L(1)
p.Q696X(1)
p.V704I(2)
p.V713M(3)
p.V713M(19)
p.R742C(6)
p.N745K(10)
p.N745K(77)
p.E752G(216)
p.E752G(1520)
p.H769R(2)
p.H775Y(2)
p.D784E(1)
p.G791R(1)
p.G792D(1)
p.G792D(1)
p.G825V(1)
p.G825V(1)
p.P835L(2)
p.A846T(1)
p.K870N(1)
c.2 6 12 − 2 T >C *(1)
c.2 45 8− 2 T >C *(1)
p.Q911E(1)
p.K939E(1)
p.C945Y(1)
p.Q962R(1)
p.L986F(1)
p.P993A(1)
p.Q1031R(1)
p.S1036X(3)
p.S1036X(5)
p.T1065A(5)
p.M1075L(1)
p.T1096S(1)
p.T1096S(1)
p.D1099H(1)
p.E1142Q(1)
p.R1146K(1)
p.D1180A(1)
p.F1211S(1)
p.H1265Y(1)
p.I1270S(1)
p.D1283G(1)
p.N1284D(2)
FALS (n=1,022)
Controls (n=7,315)
Domain key
PKD
Coiled−coil
a
Variants observed in
ALS
Variants observed
in Controls
Kenna et al. Nature Gen. 48: 1037-1042
ALS Cases (n=2,303): 23 LOF Variants
Controls (n=1,059): 0 LOF Variants
P = 1.5 x 10-4
p.K3E(1)
p.I10T(1)
p.V64A(1)
p.N93D(1)
p.E158fs(1)
p.D185G(1)
p.F220fs(1)
p.S224F(1)
p.Y229C(1)
p.R232H(1)
p.P243A(1)
p.R261C(1)
p.R261H(39)
p.R261H(6)
c.877− 1C>A*(1)
p.H330fs(1)
p.A341T(7)
p.A341T(9)
p.Q343X(1)
p.R356K(1)
p.D379E(4)
p.K422E(1)
p.Y443C(1)
p.E444D(1)
p.A463V(277)
p.A463V(124)
p.R484H(2)
p.R540X(1)
p.P569R(1)
p.N598S(1)
p.E624K(1)
p.A626T(362)
p.A626T(152)
p.V646I(1)
p.R655fs(1)
p.E662fs(2)
p.V675I(1)
p.P682L(3)
p.P682L(1)
p.V704I(3)
p.V704I(2)
p.V713M(1)
p.R742C(2)
p.R742C(2)
p.N745K(36)
p.N745K(14)
p.E752G(458)
p.E752G(222)
p.S761fs(1)
p.G792D(1)
p.K841E(1)
p.Y871fs(1)
p.S902G(1)
p.L920X(1)
p.S1036X(10)
p.T1065A(3)
p.L1091fs(1)
p.E1129G(1)
p.N1189K(1)
p.N1284D(2)
SALS (n=2,303)
Controls (n=1,059)
Domain key
PKD
Coiled-coil
bVariants observed in
ALS
Variants observed in Controls
Increased LOF Variants in NEK1 Protein
Replicates in Sporadic ALS
Analysis of ALS within an Isolated
Community in the Netherlands
Population: <25,000
Genome Sequencing Was
Performed on Four ALS Patients
• Patients in isolated
communities may be the
result of the homozygosity
of a genetic mutation
• The region of the mutation
can be identified by
identifying regions of
homozygosity of affected
patients
• Similar approach used to
identify FUS and OPTN
Kenna et al. Nature Gen. 48: 1037-1042
Homozygosity Mapping Identifies NEK1
As a Candidate Risk Factor for ALS
Identified 4 Rare Non-synonymous Variants Within Regions of Homozygosity
R261H Variant within NEK1 Remained and Replicated as a Risk Factor for ALS
Kenna et al. Nature Gen. 48: 1037-1042
ALS
Cases
#
Controls
%
Cases
%
ControlsOdds Ratio P
7,194 11,732 1.62 0.70 2.41 1.2 x 10-7
NEK1 Displays Several Functional Properties
• NEK1 is a primary regulatory of the formation of non-motile primary
cilium
• Cilia are microtubule-based sensory organelles present on the surface
of non-proliferating cells, including neurons.
• ~90% of Ciliopathies display neurobehavioural or neurodevelopmental
deficits
• Homozygotes for NEK1 LOF cause Short Rib Polydactyly Syndrome
Type II
• Characterized by constricted thoracic cage, short ribs, shortened tubular
bones
• Fibroblast from patients display defect of cilia formation and
cytoskeleton microtubule architecture
• Disruption of NEK family members disrupts neuronal morphology,
neurite outgrowth, microtubule stability and microtubule dynamics
• Regulates DNA damage repair through interactions with C21ORF2
(recently identified as a GWAS hit)
Improvements Towards the Identification of Novel Familial
ALS Genes Using Rare Variant Association Analysis
• Compared the Number of Variants in Every Gene Within:
• 1,138 Index Familial ALS (FALS) Exomes (one sample per family)
• Increased from 1,022 FALS Exomes
• 19,494 Control Exomes
• Increased from 7,315 control exomes
• Restricted to Variants:
• Loss of Function (LOF) Mutations (nonsense or splice altering)
• Last 2 ALS genes (NEK1, TBK1) displayed LOF mutations
• Minor Allele Frequency < 0.1%
• Exome-Wide Analysis
• 11,472 genes passed all QC parameters
Loss of Function (LOF) Rare Variant Analysis
Identifies KIF5A as an ALS Gene
1,138 Index Familial ALS (FALS) Cases, 19,494 Controls
Nonsense/Splice Altering Variants
• 6/1,138 FALS Cases vs 3/19,494 Controls
• Odds Ratio = 32.1; P = 5.6 x 10-7
• Segregation observed in two siblings for one variant and a
one sibling from a second variant
• Three heavy chain isoforms (KIFA5A, KIF5B,
KIF5C) are all expressed in neurons
• Hetero/homodimerize through Stalk Domain
• Axonal Transport many cargos by binding to
distinct adaptor proteins
• KIF5 transports RNA and RNA binding proteins
– Includes ALS proteins FUS and hnRNPA1
• Transports Neurofilaments and Mitochondria
– Abnormal NFs, Mitochondrial dysfunction and Impaired
Axonal Transport are all commonly observed in ALS
• Mutations in KIF5A identified in hereditary spastic paraplegia
(SPG10) and Charcot-Marie-Tooth, type 2 disease
Kinesin Family Member 5A (KIF5A)
SPG10/CMT2p.Y63C, p.D73N, p.R162W, p.M198T,
p.S202N, p.S203C, p.R204Q, p.R204W, p.V231L, p.D232N, p.G235E,
p.E251K, p.K253N, p.K256del, p.N256S, p.K257N, p.S258L, p.L259Q, p.Y276C, p.P278L, p.R280H, p.R280C, p.R280L, p.R323W, p.A361V, p.E755K
ALSp.Pro986Leu**, c.2993-3C>T, p.Arg1007Gly, p.Arg1007Lys,c.3020+1G>A, c.3020+2T>A,c.3020+3A>G, p.Asp996fs,
p.Asn999fs, p.Asn997fs,c.2993-1G>A, p.Asn999del
Motor DomainMicrotubule Binding, Kinesin Motor
(9-327)
Stalk
Heavy Chain Dimerization
(331-906)
Tail
Cargo Binding
(907-1032)
https://www.youtube.com/watch?v=y-uuk4Pr2i8
CAGGAAATGCCACAGATATCAATGACAATAGGTA
ConsensusSplice Sequence:
G N A T D I N D N R
T A G A A A G
Normal Splice mRNA:
Normal Protein:
Mutant Splice mRNA:
Mutant Protein:
Normal Splice
RNA
B.
C.
A.
MDNGNATDINDNRSDLPCGYEAEDQAKLFPLHQETAAS MDNGVTCRVAMRLRTRPSFSLSTKRQQPANLPHPRLHTCTFSF
26 27 28
26 27 28 26 28
Transla: onStop
AG ... Exon 27 AG GT...GC ACT A A G
Mutant Splice
Transla: onStop
100
200
(+) RT (-) RT
Co
ntr
ol
#1
Co
ntr
ol
#2
AL
S
Wa
ter
Co
ntr
ol
#1
Co
ntr
ol
#2
AL
S
Wa
ter
Co
ntr
ol
#1
Co
ntr
ol
#2
AL
S
Wa
ter
Co
ntr
ol
#1
Co
ntr
ol
#2
AL
S
Wa
ter
(+) RT (-) RTD.
Variants Identified in FALS are Predicted
Bioinformatically to Create a Truncated Protein
28 Coding Exons
29 Total Exons
1032 Amino Acids
a.a. 998-1007
• Deletes 34 C-terminal a.a. and adds novel 39 a.a. sequence
• Confirmed by RT-PCR using Patient-Derived RNA
• The C-terminal region functions to bind cargo
Analysis of Small Insertions/Deletions (indels)
Reveals Additional LOF Variants in FALS
Position Variant Exon cDNA Description Predicted Exon
Skipping
Control Variants
57,963,470 A>G 11 c.1117+4A>G 3' Splice Junction P
57,966,423 C>T 15 c.1630C>T p.Arg544* -
57,976,884 G>C 28 c.3021G>C 5' Splice Junction N
FALS Variants 57,975,729 GA>A 26 c.2987delA p.Asp996fs -
57,976,382 C>T 27 c.2993-3C>T 5' Splice Junction Y
57,976,385 GA>G 27 c.2996delA p.Asn999fs -
57,976,411 A>G 27 c.3019A>G p.Arg1007Gly Y
57,976,412 G>A 27 c.3020G>A p.Arg1007Lys Y
57,976,413 G>A 27 c.3020+1G>A 3' Splice Junction Y
57,976,414 T>A 27 c.3020+2T>A 3' Splice Junction Y
57,976,415 A>G 27 c.3020+3A>G 3' Splice Junction Y
• 2/1,138 FALS Cases vs 0/19,494 Controls (indels)
• Combined results: 8/1,138 FALS Cases vs 3/19,494 Controls
• Combined Odds Ratio = 41.2; P = 3.8 x 10-9
GWAS Identifies an Association of the
KIF5A Region and Sporadic ALS Risk
• C9orf72, TBK1, UNC13a, C21orf2, TNIP1 Previously Identified
• KIF5A: Pro986Leu (Odds Ratio = 1.38; P = 6.43 x 10-10)
Cohort Size: 20,806 Cases 59,804 Controls
10,031,630 genotyped and imputed variants tested for
association
Replication Studies Confirm Pro986Leu
as a Risk Factor for ALS
Replication samples contributed in part by:
1) Project Mine
2) New York Genome Center
3) CReATe Consortium
4) AnswerALS
5) Genomic Translation for ALS Care Consortium
Odds Ratio = 1.38; P = 2.31 x 10-12
KIF5A Loss of Function Mutations Display Higher
Prevalence in Familial vs. Sporadic ALS
• Additional 9,046 ALS cases (predominantly sporadic) and 1,955
controls were screened for LOF variants
• 2 LOF variants (0.022%) Identified in sporadic ALS cases
–c.2989delA p.Asn997fs
–c.2993-1G>A 5' Splice Junction
• No LOF variants were identified in controls
• Rate is much lower (~30 fold) than the observed rate in FALS
cohort (0.703%)
• Suggests LOF variants display a high penetrance
ALS Associated Mutations in KIF5A
are Distinct from SPG10/CMT2 Mutations
SPG10/CMT2p.Y63C, p.D73N, p.R162W, p.M198T,
p.S202N, p.S203C, p.R204Q, p.R204W, p.V231L, p.D232N, p.G235E,
p.E251K, p.K253N, p.K256del, p.N256S, p.K257N, p.S258L, p.L259Q, p.Y276C, p.P278L, p.R280H, p.R280C, p.R280L, p.R323W, p.A361V, p.E755K
ALSp.Pro986Leu**, c.2993-3C>T, p.Arg1007Gly, p.Arg1007Lys,c.3020+1G>A, c.3020+2T>A,c.3020+3A>G, p.Asp996fs,
p.Asn999fs, p.Asn997fs,c.2993-1G>A, p.Asn999del
Motor DomainMicrotubule Binding, Kinesin Motor
(9-327)
Stalk
Heavy Chain Dimerization
(331-906)
Tail
Cargo Binding
(907-1032)
Missense
Mutations LOF
Mutations
Advantages of Whole Genome Sequencing
Over Exome Sequencing
• Exome sequencing does not cover all genes
• ENCODE Project has revealed that substantially more of the “functional genome” lies within non-coding sequences
• e.g. miRNAs, lncRNAs, promoter regions
• Exome sequencing is not suited to identify copy number variations/ indels that may contribute to human disease
• Whole genome sequencing is considerably more amenable to phasing of variants than exome sequencing
• Distinguishing cis variants and compound heterozygosity for recessive models and eQTL analysis
• Imputation of missing variants
To Comprehensively Analyze WGS,
Tens of Thousands of Cases/Controls are Required
Project MinE: An International Consortium for ALS
Whole Genome Sequence
• Goal: To sequence 15,000 ALS and 7,500
control genomes
• Consists of 17 countries:
• Australia, Belgium, Brazil, Canada, France, Germany,
Ireland, Israel, Italy, Netherlands, Portugal, Spain,
Sweden, Switzerland, Turkey, UK, USA
• Project MinE USA Directors (Oct. 2014)
• Jonathan Glass (Emory U.) / John Landers
• Approximately 35% completed
• Funding for 8,338 samples
Genetics Opens Several Avenues of Research for
Therapeutic Treatments
Therapeutic Treatments
Genetics
Pathways/PathogenesisGene Therapy Model Organisms
Improvements in Any of These Areas Will Facilitate
The Development of Therapeutic Treatments
Paralysis Beginning at ~140 days
Weight Loss
Decreased Grip Strength
Motor Neuron Loss
5X Overexpression of
No Phenotype in WT Overexpression
Transgenic Mice Expressing Mutant PFN1
Represent a Mouse Model for ALS
Additional Efforts to Develop
Mouse Models for ALS
• Several Mouse Models are Currently in Development
In Collaboration with The Jackson Laboratory
• Mouse Models are Based on Genes Identified by Our
Laboratory
• Profilin-1 (PFN1)
• Tubulin, alpha 4A (TUBA4A)
• NEK1
• KIF5A
• For Each Gene, Inducible Transgenic and CRISPR
Models are in Development for Multiple Mutations
Numerous Pathways are Impaired in the Motor
Neurons of ALS Patients
Ferraiuolo…Shaw, Nature Reviews Neurology (2011) 7:616
Genetics Pinpoints the Primary Causes and
Pathways for Human Disease
Eykens & Robberecht, Advances in Genomics and Genetics(2015) 5:327
• RNA Processing
• FUS
• TDP-43
• hnRNPA1
• Protein Degradation
• UBQLN2
• VCP
• OPTN
• Cytoskeletal Dynamics
• PFN1
• TUBA4A
• KIF5A
Cytoskeletal Dynamics, RNA Processing and Protein
Degradation May Represent Overlapping Pathways in ALS
Cytoskeletal
Dynamics
RNA
Processing
Protein
Degradation
Motor
Neuron
Death
Future Directions
• Continued Efforts in ALS Genetics
• Collaborative Efforts in Whole Genome Sequencing with
numerous groups
• Development of Mouse Models in Collaboration with Jackson
Laboratory
• Profilin-1 (PFN1)
• Tubulin, alpha 4A (TUBA4A)
• NEK1
• KIF5A
• Development of Therapeutics for ALS Using High Throughput
Screening of Small Compounds and Biological Compounds
• Obtaining Compound Libraries from Several Sources
• Developing Novel Methods to Screen Knockdown
Expression of All Genes for Therapeutics
• Using Mouse Models to Validate Therapeutics for ALS
Acknowledgements
UMass Medical School
Kevin Kenna
Brendan Kenna
Aoife Kenna
Pamela Keagle
Janice Dominov
Robert Brown
National Institutes of Health
Bryan Traynor
Aude Nicolas
Alan Renton
Faraz Faghri
Ruth Chia
University of Milan
Nicola Ticozzi
Vincenzo Silani
University of Turin
Adriano Chiò
Emory University
Jonathan Glass
Umea University
Peter Andersen
University Medical Centre
Utrecht
Jan Veldink
Leonard van den Berg
King’s College London
Ammar Al-Chalabi
Chris Shaw
Jackson Laboratory
Cat Lutz
Project Mine
Guy Rouleau
Nazli Basak
Karen Morrison
Kevin Talbot
Markus Weber
Martin Turner
Michael A. Van Es
Orla Hardiman
Pamela J. Shaw
Philip Van Damme
Russell L. McLaughlin
Vincenzo Silani
Wim Robberecht
University of Helsinki
Pentti Tienari
Merck Research Labatories
David Stone
Answer ALS
Jeff Rothstein
Steve Finkbeiner
CReATe Consortium
Michael Benatar
J. Paul Taylor
NYGC
Hemali Phatnani
Genomic Translation for ALS Care
Matt Harms
David Goldstein
SLAGEN
ITALSGEN
Funding
ALS Association (Lucie Bruijn)
NIH / NINDS
Packard Center for ALS Research
• Three heavy chain isoforms (KIFA5A, KIF5B,
KIF5C) are all expressed in neurons
• Hetero/homodimerize through Stalk Domain
• Axonal Transport many cargos by binding to
distinct adaptor proteins
• KIF5 transports RNA and RNA binding proteins
– Includes ALS proteins FUS and hnRNPA1
• Transports Neurofilaments and Mitochondria
– Abnormal NFs, Mitochondrial dysfunction and Impaired
Axonal Transport are all commonly observed in ALS
• Mutations in KIF5A identified in hereditary spastic paraplegia
(SPG10) and Charcot-Marie-Tooth, type 2 disease
Kinesin Family Member 5A (KIF5A)
SPG10/CMT2p.Y63C, p.D73N, p.R162W, p.M198T,
p.S202N, p.S203C, p.R204Q, p.R204W, p.V231L, p.D232N, p.G235E,
p.E251K, p.K253N, p.K256del, p.N256S, p.K257N, p.S258L, p.L259Q, p.Y276C, p.P278L, p.R280H, p.R280C, p.R280L, p.R323W, p.A361V, p.E755K
ALSp.Pro986Leu**, c.2993-3C>T, p.Arg1007Gly, p.Arg1007Lys,c.3020+1G>A, c.3020+2T>A,c.3020+3A>G, p.Asp996fs,
p.Asn999fs, p.Asn997fs,c.2993-1G>A, p.Asn999del
Motor DomainMicrotubule Binding, Kinesin Motor
(9-327)
Stalk
Heavy Chain Dimerization
(331-906)
Tail
Cargo Binding
(907-1032)