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