Familial Essential Tremor Gene Identification Using Exome Sequencing Anjali Shah Rutgers University, Class of 2015 Summer Undergraduate Research Fellowship Ross Lab
Familial Essential Tremor Gene Identification Using Exome Sequencing
Anjali ShahRutgers University, Class of 2015
Summer Undergraduate Research Fellowship
Ross Lab
INTRODUCTION
• Most common movement disorder• Average age of onset is 35-45 years• Main symptom is action tremor• Criteria (Movements Disorder Society):
Symmetrical Kinetic Postural Bilateral
• Motor Symptoms: Problems with gait and balance Neuropathy Rigidity
• Age is a major risk factor for ET
ENVIRONMENTAL RISK FACTORS
• Approximately 50% of ET cases are non-familial suggesting environmental risk factors play a significant role (Louis et al. 2010)
• Environmental toxins (Louis et al. 2008):• β-carboline alkaloids • Lead• Pesticides
GENETIC FACTORS• Familial inheritance is high ( 50-60%)• Various patterns of inheritance in families:
Autosomal dominant with low penetrance Multifactorial inheritance
Interplay between more than one gene and environmental factors Non- Mendelian pattern of inheritance
MA S, DAVIS TL, BLAIR MA et al. Familial essential tremor with apparent autosomal dominant inheritance: Should we also consider other inheritance modes? Mov Disord 2006;21:1368–74
LINKAGE STUDIES IN FAMILIES • Linkage studies identified 3 susceptible loci for familial ET:
• ETM1 (chromosome 3q13) (Gulcher et al. 1997)• 20 million base pairs • ~100 genes• Candidate ET gene- DRD3 (dopamine receptor D3 gene)
• ETM2 (chromosome 2p25-p22) (Higgins et al. 1997)• 41 million base pairs• ~100 genes• Candidate ET gene- HS1BP3 (heat shock1-binding protein 3)
• ETM3 (chromosome 6p23) (Shatunov et al. 2006)• 1.8 million base pairs• 7 genes
POPULATION STUDY
GWAS Study (Stefansson et al. 2009):
• 305,624 SNPs were tested for association with ET in
452 cases and 14,394 controls
• Reached the genome-wide significance level with 1
marker in Leucine-rich repeat and Ig domain
containing Nogo receptor interacting protein-1 gene
(LINGO1)
• Some studies replicate the findings and some do not.
GENOME SEQUENCING
• First human genome was fully sequenced in 2003
• It took 13 years and $2.7 billion
NOW:• Life Technologies introduced a
sequencer that can decode a human genome in one day for $1000
NEXT GENERATION EXOME SEQUENCING
• Nonsense mutation p.Q290X in FUS/TLS (fused
in sarcoma/translocated in liposarcoma)
(Merner. N et al. 2013)
• Screening of 270 ET cases revealed 2 additional
rare variants- p. P431L and p. R216C
• Both variants were found to be highly
conserved and pathogenic by bioinformatics
software.
• 2 other studies
• Parmalee et al. 2012
• Labbé et al. 2013
Did not detect any FUS variants in ET
MY PROJECT
Exome Sequencing of 2 individuals from a family
1200 shared SNPs
Narrowed down to 3
Sequenced using 3730 Sequencer of variants in Controls and ET series
Non-synonymous variants shared
Segregated in affected members of the family, not seen in controls, rare
in public data
Objective- Find a rare variant that caused ET in the family.
7/27/2013
Diagnosis 1 = ET (or postural tremor) Diagnosis 2 = ET (or post. tremor)
FAMILY 324
7/27/2013
Diagnosis 1 = ET (or postural tremor) Diagnosis 2 = ET (or post. tremor)
FAMILY 324
Sent for exome sequencing
EXOME SEQUENCING
• Objective- To find novel, non-synonymous variants that segregated within the affected family members and that were not seen frequently in public databases
• 20,000 variants in each person
• 1200 shared variants
• 71 Non-synonymous variants shared between the 2
affected family members
• 18 variants were either Sequenced or Genotyped
Chr Gene Base change AA
ET ET ET ET ET ?dx ?dx U U U U U U UControls
(%)EVS MAF
(%)s_31 s_8 8 18 20 24 22 32 33 58 64 19 43 7
12 DDX55 C > A S450R 012 P2RX7 G > A R264H 0 0.04712 KNTC1 G > T V1012L 0 0.03715 DENND4A T > C S1839G 010 AGAP6 G > A R565Q 0.2213 MIPEP A > G L197P 015 AGPHD1 T > A Y145X 0.039 KIAA2026 G > C T834R 0
12 POLE C > T V1016M 0 0.11612 NOC4L G > A R346H 0 0.01220 C20orf96 C > G S117T 0.0118 TCEB3C T > G E356A 015 CEP152 A > G W867R 09 FAM22G C > T P457S 0.07
15 DMXL2 C > A A2372S 4 FRG1 C > T P91S 1 HRNR G > C A2706G 7 PMS2L5 C > T R9X
Carrier
Non-carrier
VARIANTS IDENTIFIED
Chr Gene Base change AA
ET ET ET ET ET ?dx ?dx U U U U U U UControls
(%)EVS MAF
(%)s_31 s_8 8 18 20 24 22 32 33 58 64 19 43 7
12 DDX55 C > A S450R 012 P2RX7 G > A R264H 0 0.04712 KNTC1 G > T V1012L 0 0.03715 DENND4A T > C S1839G 010 AGAP6 G > A R565Q 0.2213 MIPEP A > G L197P 015 AGPHD1 T > A Y145X 0.039 KIAA2026 G > C T834R 0
12 POLE C > T V1016M 0 0.11612 NOC4L G > A R346H 0 0.01220 C20orf96 C > G S117T 0.0118 TCEB3C T > G E356A 015 CEP152 A > G W867R 09 FAM22G C > T P457S 0.07
15 DMXL2 C > A A2372S 4 FRG1 C > T P91S 1 HRNR G > C A2706G 7 PMS2L5 C > T R9X
Carrier
Non-carrier
VARIANTS IDENTIFIED
7/27/2013
Diagnosis 1 = ET (or postural tremor) Diagnosis 2 = ET (or post. tremor)
Carrier
Non- carrier
DDX55 Ser450Arg (rs143479625)
DDX55 SUMMARY
• C>A base change Serine to Arginine AA change
• Sequenced in:
ET Cases(N=267)Controls(N=282)
No positives
7/27/2013
Diagnosis 1 = ET (or postural tremor) Diagnosis 2 = ET (or post. tremor)
Carrier
Non- carrier
KIAA2026 Thr834Arg rs191841054
KIAA2026 SUMMARY
• C>G base change Threonine to Arginine AA change
• Sequenced in:
ET Cases(N=267)Controls(N=282)
No positives
• C>T base change Threonine to Isoleucine
AA change at the same position
• Sequenced in Controls and ET Cases
• 1 positive ET Case (Familial Case)
Control- CC
Family Het- CG
ET Het- CT
7/27/2013
Diagnosis 1 = ET (or postural tremor) Diagnosis 2 = ET (or post. tremor)
Carrier
Non- carrier
POLE V1016M (rs147692158)
POLE SUMMARY
• G>A base change Valine to Methionine AA change
• Sequenced in:
ET Cases(N=267)Controls(N=282)
1 positive in ET series (Sporadic case)
Control- GG
ET Het- GA
CANDIDATE GENES
• DDX55• DEAD box protein 55 • Chromosome 12• Encodes for DEAD box protein (ASP-ALA-GLU-
ALA)• Alteration of RNA secondary structure• Ribosome and spliceosome assembly
• KIAA2026• Uncharacterized KIAA protein• Chromosome 9
• POLE• DNA polymerase epsilon catalytic subunit A enzyme• Chromosome 12
FUTURE STUDIES
• Genotype more ET samples for these variants
• Sequence the whole genomic region to see if there are other variants in the promoter or intronic regions that could be risk variants
• Functional study of the associated variants1. Role of the mutated variants in cells 2. Create an in-vivo model
ACKNOWLEDGEMENTS
Entire Ross Lab: Owen RossAlexandra OrtolazaSruti RayaproluRonnie WaltonCatherine LabbéKotaro Ogaki
SURF Coordinators:David AusejoNell Robinson
Benefactors:David A. and Linda B. Stein