NUBPL MUTATIONS LINK PARKINSON’S DISEASE AND OTHER MOVEMENT DISORDERS TO RECESSIVE COMPLEX I DEFICIENCY P.S. Eis1, B. Schüle2, S. Kim2, V.E. Kimonis3, J.W. Langston2, E. Hatchwell1

1Population Diagnostics, Inc., Melville, NY, USA 2The Parkinson’s Institute and Clinical Center, Sunnyvale, CA, USA

3Division of Genetics and Metabolism, Department of Pediatrics, University of California, Irvine, Children’s Hospital of Orange County, Orange, CA, USA

POPULATION DIAGNOSTICS, INC.

ABSTRACT

FIGURE 1 – PD patient has same CNV as first reported patient with NUBPL CID (A) Genome-wide CNV analysis of 466 PD cases revealed a chromosomal rearrangement in a female patient diagnosed with sporadic PD (onset at age 65). This mutation 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 an Fe/S protein [Sheftel et al. 2009]. The deleted region also results in complete loss or disruption of HEATR5A, DTD2, and GPR33 (limited information is available for these 3 genes).

Discovery of NUBPL as a Candidate PD Gene Genome-wide CNV Analysis

METHODS

ACKNOWLEDGEMENTS & REFERENCES

DISCUSSION & CONCLUSIONS

ACKNOWLEDGEMENTS We are indebted to the PD patients participating in this study for their commitment to help move the research towards a cure. We are also grateful to the Complex I deficiency families for publicly sharing their medical odysseys [spoonergirls.com; hopeforkatherinebelle.com ] and family histories, and their commitment to research on NUBPL for the benefit of neurodevelopmental and neurodegenerative d isorders.

REFERENCES Calvo SE et al. High-throughput, pooled sequencing identifies mutations in NUBPL and FOXRED1 in human complex I deficiency. Nat Genet. 2010 Oct;42(10):851-8. PMID: 20818383 Gautier CA et al. Mitochondrial dysfunctions in Parkinson's d isease. Rev Neurol (Paris). 2013 Oct 9 PMID: 24119854 Kevelam SH et al. NUBPL mutations in patients with complex I deficiency and a d istinct MRI pattern. Neurology. 2013 Apr 23;80(17):1577-83. PMID: 23553477 Lek M. et al. Analysis of protein-coding genetic variation in 60,706 humans. Nature. 2016 Aug 17;536(7616):285-91. PMID: 27535533 Prasad A. et al. A d iscovery resource of rare copy number variations in ind ividuals with autism spectrum d isorder. G3 (Bethesda). 2012 Dec;2(12):1665-85. PMID: 23275889 Schapira AH et al. Mitochondrial complex I deficiency in Parkinson's d isease. Lancet. 1989 Jun 3;1(8649):1269. PMID: 2566813 Schapira AH Glucocerebrosidase and Parkinson d isease: Recent advances. Mol Cell Neurosci. 2015 May;66(Pt A):37-42. PMID: 25802027 Sheftel AD et al. Human ind1, an iron-sulfur cluster assembly factor for respiratory complex I. Mol Cell Biol. 2009 Nov;29(22):6059-73. PMID: 19752196 Sidransky E et al. Multicenter analysis of glucocerebrosidase mutations in Parkinson's d isease. N Engl J Med . 2009 Oct 22;361(17):1651-61. PMID: 19846850 Tenisch EV et al. Massive and exclusive pontocerebellar damage in mitochondrial d isease and NUBPL mutations. Neurology. 2012 Jul 24;79(4):391. PMID: 22826544 Tucker EJ et al. Next-generation sequencing in molecular d iagnosis: NUBPL mutations highlight the challenges of variant detection and interpretation. Hum Mutat. 2012 Feb;33(2):411-8. PMID: 22072591

RESULTS

ADD footnotes

Scalechr14:

200 kb hg18

30,900,000 31,000,000 31,100,000 31,200,000 31,300,000 31,400,000 31,500,000HEATR5A

DTD2GPR33

GPR33

NUBPLNUBPL

NUBPL

A

B

0.8

1.6

0.0

-0.8

-1.6

Log 2

ratio

2 3

1 Cop

y nu

mbe

r

Chromosome 14 genome position (hg18)

257 Kb deletion 138 Kb duplication

NUBPL variant a Gene location

PD cases with other movement disorders c

Family history of movement disorders c R,T R,T R,T R,T P R P P T

R R

Complex I deficiency cases d

Previously reported New cases

✓ ✓

✓

✓ ✓ ✓ ✓ ✓ ? ✓ ✓ ✓

✓

✓ ✓

✓

✓ ✓

✓

✓

✓

✓

✓

1 2 4 3 5 7 8 9 10 11 6

✓

ExAC subjects b

Het / Hom

CNV [257Kb Del/138Kb Dup]

c.120C>G [p.A40=]

c.166G>A [p.G56R]

c.205_206delGT [p.V69Yfs*80]

c.311T>C [p.L104P]

c.313G>T [p.D105Y]

c.545T>C [p.V182A]

c.579A>G [p.L193F]

c.667_668ins [p.E223Afs*4]

c.693+1G>A

c.693+7G>A

c.694-18A>T

c.815-27T>C

c.815-13T>C

Exons 1-7

Exon 2

Exon 2

Exon 2

Exon 4

Exon 4

Exon 7

Exon 7

Exon 8

Intron 8

Intron 8

Intron 8

Intron 9

Intron 9

n/a

novel

16 / 0

novel

20 / 0

1 / 0

308 / 0

novel

novel

1 / 0

5 / 0

novel

316 / 2

novel

✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓

✓

Complex I Deficiency

ET

PD RLS

Essential Tremor (ET) Parkinson’s Disease (PD) Restless Legs Syndrome (RLS)

Age of Onset

Disease Severity

HYPOTHESIS NUBPL Variants Cause Autosomal Recessive (AR) and Autosomal Dominant (AD) Movement Disorders

AR disease (MIM 252010)

AD disease?

Complex I Deficiency (CID) is an autosomal recessive d isorder caused by homozygous or compound heterozygous mutations in one of at least 23 nuclear-encoded genes, which include Complex I (CI) subunits and assembly factors (MIM 252010). Parkinson’s Disease (PD ) pathology in many patients includes deficiency in CI activity (first reported in 1989). In a genome-wide PD gene d iscovery study, we performed array comparative genomic hybrid ization (aCGH) on a PD cohort of 466 patients to identify copy number variants (CNVs) impacting genes potentially causative of PD. This genome-wide screen led to the identification of one patient with a large, comp lex chromosomal rearrangement that was an apparent loss of function mutation impacting CI assembly factor NUBPL (nucleotide binding protein-like), an Fe-S protein. Mutations in NUBPL (MIM 613621) were identified as a cause of CID in 2010; and intriguingly, the first patient was reported to have a CNV identical to that found in our PD patient on one allele, and a splicing mutation (c.815-27T>C) on the other allele. Given the strong biology linking CI activity and PD, we sequenced NUBPL in the PD cohort (n = 476) and found three novel SNVs that may be causing or contributing to PD. We also observed three patients that are heterozygous for the splicing mutation (c.815-27T>C). Subsequent identification of four new patients with NUBPL CID revealed a family history, in older relatives, of PD, essential tremor (ET), or restless legs syndrome (RLS), which further supports the role of this CI assembly factor in late-onset movement d isorders. Analogous to mutations in GBA (MIM 606463) linking autosomal recessive Gaucher d isease to an increased risk of PD in GBA mutation carriers, we hypothesize that heterozygous NUBPL mutation carriers have an increased risk for late-onset movement d isorders that include PD, ET, and RLS.

NUBPL Variants Link Recessive CI Deficiency to PD, ET, and RLS Sequence Analysis of NUBPL

Table footnotes continued: d Source of genetic information (see ASHG poster 2519/T for clinical details on CID patients, #4 and

#8 represent two affected siblings in these families):

1 2 3 4 5 6 7 8-11 1-8

CID patients

PD patients

Australia Argentina Germany Canada United States Netherlands France United States United States

Country of origin Reference 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 Unpublished

FIGURE 2 – Experimental and predicted NUBPL protein expression Genotypes and disease severity correlate with NUBPL expression (0-100% corresponds to 20-100% CI activity). Protein levels were previously reported (Calvo et al. 2010; Tucker et al. 2012) or inferred for subjects with control (Wild type), c.815-27T>C heterozygous, c.815-27T>C homozygous (inferred), and c.815-27T>C + CNV compound heterozygous genotypes. Movement disorder patients (ET, PD, RLS) were found to be heterozygous carriers of pathogenic and novel NUBPL variants (see Table). c.815-27T>C homozygotes are predicted to have mild CID or a late-onset movement disorder (dashed line).

NUBPL genotypes

a Gray-shaded variants (p.G56R + c.815-27T>C) are present in cis. Splicing mutation c.815-27T>C (~1% frequency in European ancestry subjects) was detected in all known cases of NUBPL Complex I deficiency. Pathogenicity of c.815-27T>C is established but a deleterious contribution from G56R remains in question (see Tucker et al. 2012).

b Assessment of the frequency of the CNV (na) was performed using an in-house CNV db on apparently healthy subjects (see Methods). SNV frequencies were assessed using the Exome Aggregation Consortium (ExAC) db (exac.broadinstitute.org; Lek et al. 2016). Number of subjects reported as heterozygotes (Het) or homozygotes (Hom) are for Non-Finnish Europeans (NFE), which corresponds to the self-reported ethnicities of the patients.

c Movement disorders: P = Parkinson’s disease, R = Restless legs syndrome, T = tremor or essential tremor Family history was unavailable for CID and PD patients with blank fields.

0

20

100

80

60

40

NU

BP

L p

rote

in l

evel

(%

)

Healthy

Mild CID?

Severe CID

ET, PD, RLS

(undetectable protein level)

CONTACT INFORMATION

Peggy S. Eis, PhD, Chief Technology Officer pegeis@populationdiagnostics.com

� PD patients were found to be heterozygous carriers of pathogenic NUBPL variants (CNV and c.815-27T>C) that were previously identified

in recessive CID patients, and novel/ ultra-rare SNVs (see Table, RESULTS) that are potentially deleterious.

� A family history of later onset movement d isorders (ET, PD, RLS) was found in 5 of 5 CID families (family history was not ava ilable in the other 6 CID families). [See ASHG 2016 poster 2519/T for details on the 4 newly reported NUBPL CID patients.]

� HYPOTHESIS (see figure, left): Like GBA mutations, which cause AR Gaucher d isease but also correlate with a 5- to 20-fold increased risk of PD (Sidransky et al. 2009; Schapira 2015 review), we propose that heterozygous NUBPL mutations may modify the risk for a constellation of movement d isorders (ET, PD, RLS) and/ or cause an AD form of NUBPL d isease.

� FUTURE EXPERIMENTS: − Segregation experiments for NUBPL mutations found in CID family members. − Genetic validation in additional PD cohorts and assessment of ET and RLS cohorts to strengthen the association of NUBPL in movement d isorders. − Functional validation of novel/ u ltra-rare SNVs found in PD patients to confirm their pathogenicity and consideration of NUBPL as a new drug target.

SUBJECTS: Parkinson’s Disease (PD) Cohort (n = 466 or 476, see below) Patients provided informed written consent and the study was approved by the Parkinson’s Institute and Clinical Center ’s IRB protocol. Demographics of the cohort: 65% male, 35% female; 27% familial, 73% sporad ic; 10% early-onset (<50 years), 90% late-onset (>50 years). Complex I Deficiency (CID) Patients (see also ASHG 2016 poster 2519/ T) Family members (or their parents) provided informed written consent and they participated in the University of California approved IRB protocol.

EXPERIMENTS: Array CGH (1M feature microarray, Agilent, Santa Clara, CA) was used for genome-wide detection of CNVs in the PD cohort (n = 466, 10 experiments excluded after applying QC criteria). Sanger sequencing (NUBPL exons and flanking regions) was used to detect sequence variants in the PD cohort (n = 476). CID patient mutations were previously reported (see Table, footnote d) or identified via exome sequencin g (see poster 2519/ T).

VARIANT INTERPRETATION: PD patient CNV data was interpreted using an in -house database of control CNVs (apparently healthy males and females of European ancestry, see Prasad et al. 2012 for details). PD and CID patient SNVs were interpreted using the Exome Aggregation Consortium (ExAC) database (Lek et al. 2016). PD patient SNVs reported in the Table are: 1) not found (novel) in the ExAC db, 2) ultra -rare (<0.1%) in ExAC populations, or 3) also found in recessive CID patients (c.815-27T>C).

✓

Cohort size = 476

PD cases d

1 2 4 3 5 7 8 6 9-11