POPULATION DIAGNOSTICS, INC.
– a gene discover. company Our knowledge of what causes a disease accelerates the systema6c discovery and development of diagnos6cs and therapeu6cs. Keystone Symposium poster presenta6on: Parkinson’s Disease: Gene6cs, Mechanisms and Therapeu6cs March 3, 2014 1 Genetic Variation in Mitochondrial Complex I Genes
are Associated with Parkinson’s Disease
Peggy S. Eis1, Eli Hatchwell1, J. William Langston2, and Birgitt Schuele2
1Population
2The
Diagnostics, Inc., Melville, NY, USA
Parkinson’s Institute and Clinical Center, Sunnyvale, CA, USA
www.populationdiagnostics.com
2 ABSTRACT
Our genome-wide Parkinson’s Disease (PD) gene discovery study provides the first genetic evidence that rare
variants impacting Complex I (NADH dehydrogenase) genes may be causing mitochondrial dysfunction in PD
patients. Complex I (CI) deficiency was linked to PD over 20 years ago [Schapira et al. 1989] and PD-causing
genes (LRRK2, PARK2, PARK7, PINK1, SNCA) are known to be involved in mitochondrial dysfunction [Gautier et
al. 2013]. However, no mutations have previously been identified in nuclear CI genes, although there have been
some reports of links to mitochondrial DNA variants [OMIM 556500] in PD patients. Our gene discovery study on
~470 PD cases (27% familial, 73% sporadic) was performed using array CGH and novel CNV analysis methods,
which provides superior signal-to-noise and resolution as compared to prior CNV studies using SNP genotyping
microarray data. Our approach – the CNV Beacon method – provides a dramatic reduction in the genome search
space to enable rapid, cost-effective discovery of genes that are causative or contributing to complex diseases
such as Parkinson’s and Alzheimer’s. Once CNV-identified genes are found, targeted sequencing is performed to
reveal additional variants (e.g., SNVs) that are associated with the disease. We will report on three CI genes found
to have loss-of-function copy number variants (CNVs) in a subset of PD patients, including sequencing data for
one CI gene known to cause CI deficiency in pediatric patients via an autosomal recessive mechanism. Our
hypothesis is that heterozygous carriers of autosomal-recessive mutations in CI genes have an increased risk for
PD, as has analogously been found for the disease-causing genes GBA [Gaucher’s disease; OMIM 606463] and
NPC1 [Niemann-Pick disease; OMIM 257220], both of which are associated with PD.
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3 GENE DISCOVERY PLATFORM
GENE DISCOVERY PLATFORM A Common, Complex Disease is a Collection of Rare Diseases
Complex disease cohort
!  Complex(diseases(are(50090%(heritable(but(only(1010%(
of(gene<c(causes(are(known.((
!  Dissec<on(of(heterogeneous(complex(diseases(into(
gene<c(subtypes(will(enable(development(of(targeted,(
preven<ve(therapies(and(pre0symptoma<c(Dx(tes<ng.(
Type 1
Type 2
Type 3
Type 4
Type 5
FIGURE 1 Dissection of Complex Diseases into Rare Subtypes.
Complex diseases are a collection of rare subtypes that manifest with common symptoms. While genetic background (modifier
genes) and environment contributes to disease onset and progression, rare variants of high effect (e.g., Odds Ratios of 5 - 50) are
the predominant causes of the disorder. For example, for PD, the schematic of Types 1-5 would correspond to LRRK2, PARK2,
PARK7, PINK1, and SNCA, but the list of rare subtypes due to rare variants is growing [reviewed by Puschmann 2013].
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4 GENE DISCOVERY PLATFORM Reducing the Genome Search Space
Gene discovery via copy number variants (CNVs)
Disease
cohort
Unknown disease gene
Pathogenic CNV causes disease &
is a “beacon” of the disease gene
NVE
vs. platform
CNV
analysis
CNV
Candidate disease gene
Sequencing
analysis
Sequenced CNV Beacon gene
reveals full mutational spectrum
CNVs, indels, SNVs…
Validated disease gene
FIGURE 2 The CNV Beacon® Method
Nearly all disease genes have a mutational spectrum that includes CNVs in addition to SNVs, etc (e.g., disease-causing CNVs
have been found for SNCA, APP, and BRCA1). Thus, finding a CNV in a disease cohort that is absent or present at lower
frequency in controls (‘Normal’ subjects) enables discovery of candidate disease genes, which are then sequenced to identify
additional mutations that validate a gene as causative. Like SNVs, most CNVs are benign, but interpretation of disease cohort
CNV data requires a database of only 1,000 Normal genomes for gene discovery. Population Diagnostics (PDx) has created a
Normal CNV database and proprietary algorithms – the Normal Variation Engine® (NVE) platform – to perform rapid, low-cost
disease gene discovery studies (e.g., Parkinson’s, Alzheimer’s, Autism, and Endometriosis).
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5 GENE DISCOVERY PLATFORM Advantages of the CNV Beacon Method:
!  CNVs provide >1,000-fold reduction in genome search space as compared to SNVs.
!  Due to their size range (~1Kb to 1Mb), CNVs are more likely to result in loss or gain of gene function/expression.
!  Intronic and intergenic regions that harbor regulatory loci (e.g., transcription factor binding sites) can be readily
interrogated and interpreted as compared to SNV-based data (exome and whole genome studies).
!  CNV-identified genes can be sequenced using a variety of candidate gene methods or they an be assessed in
existing exome and whole genome data sets (targeted interpretation).
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6 GENE DISCOVERY PLATFORM PD Gene Discovery Study
Results:
CNVs!found!in!PD!cases!and!not!in!
controls!ranged!from!2Kb!to!20Mb.!
PD Cohort – 467 cases
9,162 distinct CNV-subregions
(76,008 redundant CNVs)
PDx’s NVE® Data – 1,005 Normals
14,693 distinct CNV-subregions
(162,316 redundant CNVs)
NVE interpretation
benign vs. pathogenic CNVs
!
In!addi=on!to!NUBPL!and!2!other!
Complex!I!genes,!genes!were!found!with!
the!following!PDRrelevant!biology:!!!
! 
PD-associated
39 CNV-subregions
! 
p-value <0.05 (FDR adjusted)
! 
! 
Inter-genic
17 CNV-subregions
Intronic
13 CNV-subregions
Exonic
9 CNV-subregions
! 
! 
! 
! 
21 PD-associated genes
OR values: 0.05 – 130.1
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! 
apoptosis,!autophagy!
cell!signaling!(e.g.,!NOS,!Ras,!Wnt)!
dopaminergic!func=on!
mitochondrial!dysfunc=on!
neuroinflamma=on!
neuroprotec=ve!factors!
neurotransmi@er!receptors,!ion!channels!
oxida=ve!stress!
ubiqui=n/proteasome!pathway!!!!
7 COMPLEX I DEFICIENCY Genome-wide CNV Analysis – PD Cohort (n = 478)
Discovery of NUBPL as a candidate PD gene
A
00
,0
00
31
,5
60
,0
00
31
,5
20
,0
00
31
,4
80
,0
00
31
,4
40
,0
00
31
,4
00
,0
00
60
,3
31
31
,3
20
,0
00
,0
00
31
,2
80
,0
00
31
,2
40
,0
00
31
,2
00
,0
00
31
,1
60
,0
00
31
,1
20
,0
00
31
,0
80
,0
00
31
,0
40
,0
00
31
,0
00
,0
00
30
,9
60
,0
00
20
,0
30
,9
80
,8
30
30
,8
40
,0
00
00
,0
00
,8
30
30
,7
60
,0
00
Chromosome 14 genome position (hg18)
Log2 ratio
1.6
0.8
0.0
-0.8
-1.6
B
Scale
chr14:
30,900,000
Copy
number
HEATR5A
31,000,000
GPR33
DTD2
GPR33
200 kb
31,100,000
NUBPL
NUBPL
hg18
31,200,000
31,300,000
31,400,000
31,500,000
NUBPL
3
2
1
240 Kb deletion
137 Kb duplication
FIGURE 3 Complex I (CI) assembly factor gene NUBPL is disrupted.
(A) Genome-wide CNV analysis revealed a chromosomal rearrangement in a female patient diagnosed with sporadic PD (onset
at age 65). This large mutation, with an estimated frequency of 0.2% (1 in 468 PD cases) was confirmed (data not shown) to be
identical to one of two mutations found in the first reported case of CI deficiency due to loss of function mutations in NUBPL
[Calvo et al. 2010; Tucker et al. 2012]. (B) UCSC Genome Browser (hg18) view shows the deleted (red bar) and duplicated (blue
bar) regions of the rearrangement disrupt NUBPL (Ind1), a CI assembly factor that is also an Fe/S protein [Sheftel et al. 2009].
The deleted region also results in complete loss or disruption of HEATR5A, DTD2, and GPR33.
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8 COMPLEX I DEFICIENCY NUBPL Sequencing – PD Cohort (n = 478)
TABLE 1. Comparison of CI Deficiency vs. PD cases
CI deficiency patients a
PD patients a
(previously published)
NUBPL variant c
CNV (240Kb del. + 137Kb dupl.)
e
c.120C>G [p.A40=]
c.166G>A [p.G56R]
Gene location
1
exons 1-4, 7
✓
f
exon 2
exon 2
c.313G>T [p.D105Y]
exon 4
c.579A>G [p.L193F]
exon 7
c.667_668ins CCTTGTGCTG [p.E223Afs*4]
exon 8
c.693+1G>A
intron 8
c.694-18A>T
intron 8
c.815-217T>C [p.D273Nfs*32]
intron 8
c.815-27T>C
intron 9
c.815-13T>C
intron 9
Patient origin and source of genetic information:
Country of origin
Reference
Australia
Argentina
Germany
Canada
United States
Netherlands
France
United States
Calvo et al. 2010
Kevelam et al. 2013
Kevelam et al. 2013
Kevelam et al. 2013
Kevelam et al. 2013
Kevelam et al. 2013
Tenisch et al. 2012
unpublished
United States
manuscript submitted
CI def. patients
1
2
3
4
5
6
7
8
PD patients
1-7
3
4
d
5
6
(Sanger sequenced)
7
www.populationdiagnostics.com
8
d
1
2
3
4
5
6
7
✓
exon 2
c.205_206delGT [p.V69Yfs*80]
a
2
EVSb
subjects
0
✓
✓
✓
✓
✓
✓
2 EA / 2 AA
✓
0
✓
0
✓
0
✓
0
✓
0
✓
0
✓
✓
✓
✓g
✓
✓
✓
✓
✓
✓
✓
✓
36 EA / 5 AA
0
Key Points:
1)  A distinct haplotype (c.166G>A + c.815-27T>C) is confirmed in 6 of 8 CI deficiency
cases as compared PD case IDs 5-7 (only c.815-27T>C is present).
2)  c.815-27T>C is a loss of function SNV [Tucker et al. 2012] and it is present in PD
cases (0.6%) and EVS subjects (0.9% EA, 0.3% AA).
3)  Loss of function CNVs are present at much lower frequency in PD cases (0.2%, see
Figure 3) and an unselected population (0.06%, DGV) as compared to loss of
function SNV c.815-27T>C.
4)  Novel SNVs (all n = 1) are known for 6 CI cases and were found in 3 PD cases.
5)  Three additional SNVs (data not shown), predicted to cause loss of function, were
found at higher frequency in PD cases (5 patients) as compared to EVS subjects.
9 COMPLEX I DEFICIENCY TABLE 1. Comparison of CI Deficiency vs. PD cases
Footnotes continued:
b
Variants in an unselected population are reported from the Exome Variant Server (EVS), an NHLBI ESP database hosted at http://evs.gs.washington.edu/EVS/;
EA = European American, AA = African American
c
Variants for RefSeq NM_025152; except for patient 2, all CI deficiency patients are compound heterozygotes; PD patients and EVS subjects are heterozygotes;
patients/subjects with the same variant are blue-highlighted and novel variants (not found in EVS, dbSNP, DGV or 1,005 PDx controls) are in red (✓)
d
2 affected siblings for IDs 4 and 8; mutations for ID 8 patients are unknown/unpublished, see http://spoonerbrain.blogspot.com/
e
To date, the only CNV known to cause CI deficiency (also present in 1 PD case, see Fig. 1); it was not found in 1,005 PDx controls or the
Database of Genomic Variants (DGV), which lists 7/11,267 subjects (African, Asian, European ancestry) with a CNV impacting an exon
f
c.166G>A does not appear to contribute to the phenotype but c.815-27T>C, which occurs on the same haplotype, is functionally validated to cause
exon skipping, which results in lower wt mRNA and protein [Tucker et al. 2012]
g
Patient is likely homozygous for c.815-27T>C (parental DNA unavailable to confirm)
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10 COMPLEX I DEFICIENCY Genome-wide CNV Analysis – PD Cohort (n = 478)
Additional Complex I Genes with Rare Variants
Complex I gene
Complex I gene
!  Involved in complex I formation/function
Genes
!  REST (NRSF) neuronal deficient mice are
more vulnerable to MPTP [Yu et al. 2012].
Log2 ratio
1.6
1 familial PD case vs. 1 control (OR = 2.15)
0.8
0.0
-0.8
-1.6
4.6 Kb deletion
Deletion comprises a transcription factor
binding site, including NRSF/REST
Log2 ratio
1.6
0.8
0.0
-0.8
-1.6
4.6Kb loss detected on a 1M
CGH array is validated on a
custom CGH array
FIGURE 4 Complex I gene with an intronic CNV encompassing a transcription factor binding site.
Genome-wide CNV analysis revealed a small intronic CNV in a male patient diagnosed with familial PD. The deletion maps to a
transcription factor binding site (ENCODE ChipSeq data track, UCSC genome browser) for 4 transcription factors, including PDrelevant TFs REST (NRSF), GATA1, and GATA3 [Polanski et al. 2010; Scherzer et al. 2008; Yu et al. 2012].
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11 COMPLEX I DEFICIENCY Genome-wide CNV Analysis – PD Cohort (n = 478)
Additional Complex I Genes with Rare Variants
Oxidative stress response gene
Complex I gene
Log2 ratio
Genes
1.6
0.8
0.0
-0.8
-1.6
86 Kb deletion
FIGURE 5 Complex I gene with a loss of function deletion.
Genome-wide CNV analysis revealed an 86Kb CNV in a male patient diagnosed with sporadic PD. The deletion disrupts two
genes, a Complex I gene and an oxidative stress response gene with links to aging and neurodegeneration.
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12 MITCHONDRIAL DYSFUNCTION Mitochondrial function involves
known PD-causing genes
LRRK2
SNCA
PARK2
PARK7
PINK1
NUBPL
PD vs. 0 Ctrl
1 CNV (Fig. 1)
3 SNVs (Table 1)
CI
Gene 1
1 PD vs. 1 Ctrl
CI
Gene 2
1 PD vs. 0 Ctrl
Adapted from Knott et al. 2008
COX
COX
COX
subunit
subunit
subunit
1 PD vs. 0 Ctrl
2 PD vs. 0 Ctrl
1 PD vs. 1 Ctrl
FIGURE 6 An expanding contribution of genetics in mitochondrial dysfunction for PD.
All 5 known genes that cause PD by the presence of rare loss or gain of function mutations (via autosomal recessive or dominant
mechanisms) have well-established roles in mitochondrial dysfunction. Novel candidate PD genes found in our CNV/sequencing
study (red text / arrows) are implicated on the basis of CNVs that impact a gene at higher frequency in our PD cohort vs. PDx
controls (see Fig. 2) and novel SNVs not found in ~6,500 controls (EVS subjects, see Table 1). In addition to NUBPL and two
other Complex I genes (see Figs. 3-5), novel rare variants (CNVs) were also found in three Complex IV genes (COX family, data
not shown).
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13 CONCLUSIONS !  A genetic basis is established for Complex I deficiency (NUBPL) as a subtype of PD.
!  An increased risk of PD in heterozygous carriers of autosomal recessive disorder gene
mutations was established with GBA [Sidransky et al. 2009] and is further supported by a
recent report on NPC1 [Kluenemann et al. 2013] and the discovery of loss of function
mutations in NUBPL and 2 other CI genes in the present study.
!  CNVs provide a >1,000-fold reduction in genome search space that enables efficient, costeffective discovery of disease genes, including discovery in intronic and intergenic regions.
!  Future experiments:
− Genetic validation in additional PD cohorts to strengthen the association of CI genes with PD.
− Functional validation of novel, PD-associated variants to confirm their role in PD pathology, such as
with cellular models [Chung et al. 2013], and potential as drug targets.
ACKNOWLEDGEMENTS
We are indebted to the patients and healthy donors participating in this study for their commitment to help moving the
research towards a cure. The PD cohort used in the study was supported by The Parkinson Alliance (BS, JWL).
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15