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Cracking the code-ing sequence for Parkinson’s disease

Jansen, I.E.

2017

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Jansen, I. E. (2017). Cracking the code-ing sequence for Parkinson’s disease.

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CHAPTER 6

Discovery and functional

prioritization of Parkinson’s

disease candidate genes from

large-scale whole exome sequencing

6

ABSTRACT

Background͗tŚŽůĞͲĞdžŽŵĞƐĞƋƵĞŶĐŝŶŐ;t^ͿŚĂƐďĞĞŶƐƵĐĐĞƐƐĨƵůŝŶŝĚĞŶƟĨLJŝŶŐ

ŐĞŶĞƐƚŚĂƚĐĂƵƐĞĨĂŵŝůŝĂůWĂƌŬŝŶƐŽŶ͛ƐĚŝƐĞĂƐĞ;WͿ͘,ŽǁĞǀĞƌ͕ƵŶƟůŶŽǁƚŚŝƐĂƉƉƌŽĂĐŚ has not been deployed to study large cohorts of unrelated subjects. To discover ƌĂƌĞWƐƵƐĐĞƉƟďŝůŝƚLJǀĂƌŝĂŶƚƐ͕ǁĞƉĞƌĨŽƌŵĞĚt^ŝŶϭ͕ϭϰϴƵŶƌĞůĂƚĞĚĐĂƐĞƐĂŶĚ ϱϬϯĐŽŶƚƌŽůƐƵďũĞĐƚƐ͘ĂŶĚŝĚĂƚĞŐĞŶĞƐǁĞƌĞƐƵďƐĞƋƵĞŶƚůLJǀĂůŝĚĂƚĞĚĨŽƌĨƵŶĐƟŽŶƐ ƌĞůĞǀĂŶƚƚŽWĂƌŬŝŶƐŽŶ͛ƐĚŝƐĞĂƐĞďĂƐĞĚŽŶƉĂƌĂůůĞůZEͲŝŶƚĞƌĨĞƌĞŶĐĞ;ZEŝͿƐĐƌĞĞŶƐ in human cell culture, and Drosophila and C. elegans models.

ZĞƐƵůƚƐ͗ ƐƐƵŵŝŶŐ ĂƵƚŽƐŽŵĂů ƌĞĐĞƐƐŝǀĞ ŝŶŚĞƌŝƚĂŶĐĞ͕ ǁĞ ŝĚĞŶƟĨLJ Ϯϳ ŐĞŶĞƐ ƚŚĂƚ ŚĂǀĞ ŚŽŵŽnjLJŐŽƵƐ Žƌ ĐŽŵƉŽƵŶĚ ŚĞƚĞƌŽnjLJŐŽƵƐ ůŽƐƐͲŽĨͲĨƵŶĐƟŽŶ ǀĂƌŝĂŶƚƐ ŝŶ W ĐĂƐĞƐ͘ĞĮŶŝƟǀĞƌĞƉůŝĐĂƟŽŶĂŶĚĐŽŶĮƌŵĂƟŽŶŽĨƚŚĞƐĞĮŶĚŝŶŐƐǁĞƌĞŚŝŶĚĞƌĞĚďLJ ƉŽƚĞŶƟĂůŚĞƚĞƌŽŐĞŶĞŝƚLJĂŶĚďLJƚŚĞƌĂƌŝƚLJŽĨƚŚĞŝŵƉůŝĐĂƚĞĚĂůůĞůĞƐ͘tĞƚŚĞƌĞĨŽƌĞ ůŽŽŬĞĚ ĨŽƌ ƉŽƚĞŶƟĂů ŐĞŶĞƟĐ ŝŶƚĞƌĂĐƟŽŶƐ ǁŝƚŚ ĞƐƚĂďůŝƐŚĞĚ W ŵĞĐŚĂŶŝƐŵƐ͘ &ŽůůŽǁŝŶŐZEŝͲŵĞĚŝĂƚĞĚŬŶŽĐŬĚŽǁŶ͕ϭϱŽĨƚŚĞŐĞŶĞƐŵŽĚƵůĂƚĞĚŵŝƚŽĐŚŽŶĚƌŝĂů ĚLJŶĂŵŝĐƐ ŝŶ ŚƵŵĂŶ ŶĞƵƌŽŶĂů ĐƵůƚƵƌĞƐ͕ ĂŶĚ ϰ ĐĂŶĚŝĚĂƚĞƐ ĞŶŚĂŶĐĞĚ ɲͲƐLJŶƵĐůĞŝŶ ŝŶĚƵĐĞĚ ŶĞƵƌŽĚĞŐĞŶĞƌĂƟŽŶ ŝŶ Drosophila. Based on complementary analyses ŝŶ ŝŶĚĞƉĞŶĚĞŶƚ ŚƵŵĂŶ ĚĂƚĂƐĞƚƐ͕ ϱ ĨƵŶĐƟŽŶĂůůLJ ǀĂůŝĚĂƚĞĚ ŐĞŶĞƐͶGPATCH2L,

h,Z&ϭWϭ>, PTPRH, ARSB and VPS13CͶĂůƐŽ ƐŚŽǁĞĚ ĞǀŝĚĞŶĐĞ ĐŽŶƐŝƐƚĞŶƚ ǁŝƚŚ

ŐĞŶĞƟĐƌĞƉůŝĐĂƟŽŶ͘

ŽŶĐůƵƐŝŽŶƐ: LJŝŶƚĞŐƌĂƟŶŐŚƵŵĂŶŐĞŶĞƟĐĂŶĚĨƵŶĐƟŽŶĂůĞǀŝĚĞŶĐĞ͕ǁĞŝĚĞŶƟĨLJ

6

/EdZKhd/KE

EĞdžƚŐĞŶĞƌĂƟŽŶƐĞƋƵĞŶĐŝŶŐĂƉƉƌŽĂĐŚĞƐŚĂǀĞƌĞĐĞŶƚůLJĂĐĐĞůĞƌĂƚĞĚƚŚĞŝĚĞŶƟĮĐĂƟŽŶŽĨ variants responsible for familial Parkinson’s disease (PD).1-4_{tŚŝůĞĂƉŽƐŝƟǀĞĨĂŵŝůLJŚŝƐƚŽƌLJ}

ŝƐĐŽŵŵŽŶŝŶW͕ůĂƌŐĞ͕ŵƵůƟŐĞŶĞƌĂƟŽŶĂůƉĞĚŝŐƌĞĞƐ͕ĞƐƉĞĐŝĂůůLJǁŝƚŚĂǀĂŝůĂďůĞEĂŶĚ ĐůŝŶŝĐĂů ĞǀĂůƵĂƟŽŶƐ ƌĞŵĂŝŶ ĞdžĐĞƉƟŽŶĂů͕ ŚŝŶĚĞƌŝŶŐ ƉƌŽŐƌĞƐƐ ŝŶ ƵŶƌĂǀĞůŝŶŐ ƚŚĞ ŐĞŶĞƟĐ ƵŶĚĞƌƉŝŶŶŝŶŐƐ͘/ŵƉŽƌƚĂŶƚůLJ͕ƐĞǀĞƌĂůŐĞŶĞƐŝŶŝƟĂůůLJĚŝƐĐŽǀĞƌĞĚƚŽĐĂƵƐĞWŝŶĨĂŵŝůŝĞƐ͕ƐƵĐŚ as >ZZ<Ϯ͕'ĂŶĚWZ<ϮͬƉĂƌŬŝŶ, were subsequently discovered with surprisingly high frequency in “sporadic” PD cohorts.5,6_{dŽ ĚĂƚĞ͕ůĂƌŐĞƉŽƉƵůĂƟŽŶ ƐĂŵƉůĞƐ ŽĨ ŝŶĚŝǀŝĚƵĂůƐ}

ǁŝƚŚWŚĂǀĞƉƌŝŵĂƌŝůLJĐŽŶƚƌŝďƵƚĞĚƚŽƚŚĞĚŝƐĐŽǀĞƌLJŽĨĐŽŵŵŽŶǀĂƌŝĂŶƚƐƵƐĐĞƉƟďŝůŝƚLJůŽĐŝ͕ ďĂƐĞĚŽŶŐĞŶŽŵĞͲǁŝĚĞĂƐƐŽĐŝĂƟŽŶƐƚƵĚŝĞƐŽĨĐĂƐĞͬĐŽŶƚƌŽůĐŽŚŽƌƚƐ͘7_{dŚĞǀĂƌŝĂŶƚƐŝĚĞŶƟĮĞĚ}

ďLJ ŐĞŶŽŵĞͲǁŝĚĞ ĂƐƐŽĐŝĂƟŽŶ ƐƚƵĚŝĞƐ ;'t^Ϳ ŚĂǀĞ ŵŽĚĞƐƚ ĞīĞĐƚ ƐŝnjĞƐ ĂŶĚ ĐŽůůĞĐƟǀĞůLJ ĨĂŝůƚŽĂĐĐŽƵŶƚĨŽƌĐƵƌƌĞŶƚĞƐƟŵĂƚĞƐŽĨWŚĞƌŝƚĂďŝůŝƚLJ͘8,9 _{Considering the above, it seems}

ůŝŬĞůLJƚŚĂƚĂĚĚŝƟŽŶĂůůĞƐƐĐŽŵŵŽŶĂůůĞůĞƐ͕ǁŝƚŚůĂƌŐĞƌĞīĞĐƚƐŝnjĞƐĐŽŶƚƌŝďƵƚĞƚŽWƌŝƐŬŝŶ ƚŚĞƉŽƉƵůĂƟŽŶ͕ĂŶĚŶĞdžƚŐĞŶĞƌĂƟŽŶƐĞƋƵĞŶĐŝŶŐŝƐŽŶĞƉƌŽŵŝƐŝŶŐĂƉƉƌŽĂĐŚƚŽŝĚĞŶƟĨLJ ƐƵĐŚĂůůĞůĞƐ͘ĞƐƉŝƚĞƌĞĐĞŶƚƐƵĐĐĞƐƐĞƐŝŶŽƚŚĞƌŶĞƵƌŽĚĞŐĞŶĞƌĂƟǀĞĚŝƐĞĂƐĞǁŝƚŚĐŽŵƉůĞdž ŐĞŶĞƟĐĞƟŽůŽŐŝĞƐ͕ŝŶĐůƵĚŝŶŐůnjŚĞŝŵĞƌ͛ƐĚŝƐĞĂƐĞ10-12 _{and amyotrophic lateral sclerosis,}13, 14

sequencing has yet to be deployed in large, unrelated PD case/control samples for rare variant discovery.

dŚĞ ƐƵĐĐĞƐƐĨƵů ĚŝƐĐŽǀĞƌLJ ŽĨ ƌĂƌĞ ǀĂƌŝĂŶƚ ƌŝƐŬ ĂůůĞůĞƐ ŝŶ ƉŽƉƵůĂƟŽŶͲďĂƐĞĚ W ƐĂŵƉůĞƐĨĂĐĞƐĂŶƵŵďĞƌŽĨƉŽƚĞŶƟĂůĐŚĂůůĞŶŐĞƐ͘WĞƌŚĂƉƐŵŽƐƚŝŵƉŽƌƚĂŶƚůLJ͕ĂŶĂůLJƐĞƐŽĨ ƌĂƌĞǀĂƌŝĂŶƚƐŝŶůĂƌŐĞĨĂŵŝůLJƉĞĚŝŐƌĞĞƐŝƐŐƌĞĂƚůLJĨĂĐŝůŝƚĂƚĞĚďLJƐĞŐƌĞŐĂƟŽŶĂŶĂůLJƐŝƐǁŚŝĐŚ is not possible in cohorts of unrelated individuals, leading to an increased number of ĐĂŶĚŝĚĂƚĞ ǀĂƌŝĂŶƚƐ ƚŽ ĐŽŶƐŝĚĞƌ͘ ƐƐƵŵƉƟŽŶƐ ŽĨ Ă ƌĞĐĞƐƐŝǀĞ ŝŶŚĞƌŝƚĂŶĐĞ ŵŽĚĞů ĂŶĚ ƚŚĞ ĂƉƉůŝĐĂƟŽŶŽĨƐƚƌŝŶŐĞŶƚĮůƚĞƌƐ͕ƐƵĐŚĂƐĐŽŶƐŝĚĞƌĂƟŽŶŽĨŽŶůLJƐƚƌŽŶŐůLJĚĂŵĂŐŝŶŐ͕ůŽƐƐͲŽĨͲ ĨƵŶĐƟŽŶ;>Ž&ͿǀĂƌŝĂŶƚƐ͕ŝƐŽŶĞƉŽƚĞŶƟĂůƐŽůƵƟŽŶ͕ďƵƚƚŚŝƐŝƐůŝŬĞůLJƚŽŵŝƐƐŵĂŶLJŝŵƉŽƌƚĂŶƚ ǀĂƌŝĂŶƚƐ͕ ŝŶĐůƵĚŝŶŐ ĚŽŵŝŶĂŶƚůLJͲĂĐƟŶŐ ĂůůĞůĞƐ͘ &ƵƌƚŚĞƌ͕ W ŝƐ ĐŚĂƌĂĐƚĞƌŝnjĞĚ ďLJ ĞdžƚĞŶƐŝǀĞ ŐĞŶŝĐĂŶĚĂůůĞůŝĐŚĞƚĞƌŽŐĞŶĞŝƚLJĂŶĚĞdžƚƌĞŵĞůLJůĂƌŐĞĐŽŚŽƌƚƐŵĂLJďĞƌĞƋƵŝƌĞĚƚŽĚŽĐƵŵĞŶƚ ƐƵĸĐŝĞŶƚŶƵŵďĞƌƐŽĨĐĂƐĞƐƚŽĨĂĐŝůŝƚĂƚĞŵĞĂŶŝŶŐĨƵůƐƚĂƟƐƟĐĂůĐŽŵƉĂƌŝƐŽŶƐ͘15 _{Lastly, as PD} ŝƐ;ŝͿĐŽŵŵŽŶ;ΕϭͲϯйƉƌĞǀĂůĞŶĐĞͿ͕;ŝŝͿƐƚƌŽŶŐůLJĂŐĞͲĚĞƉĞŶĚĞŶƚ͕ĂŶĚ;ŝŝŝͿŽŌĞŶƉƌĞĐĞĚĞĚ ďLJ Ă ƉƌŽůŽŶŐĞĚ ƉƌĞƐLJŵƉƚŽŵĂƟĐ Žƌ ŵŝŶŝŵĂůůLJ ƐLJŵƉƚŽŵĂƟĐ ƉŚĂƐĞ͕ ǁĞ ŵĂLJ ĞdžƉĞĐƚ ƚŽ ĮŶĚ ƚƌƵůLJ ƉĂƚŚŽŐĞŶŝĐ ƌĂƌĞ ǀĂƌŝĂŶƚƐ͕ ŝŶĐůƵĚŝŶŐ ƚŚŽƐĞ ǁŝƚŚ ůĂƌŐĞ ĞīĞĐƚ ƐŝnjĞƐ͕ ŝŶ ͞ĐŽŶƚƌŽů͟ ĐŽŚŽƌƚƐŽĨĂĚƵůƚƐ;ĚƵĞƚŽƵŶƌĞĐŽŐŶŝnjĞĚŽƌĞĂƌůLJĚŝƐĞĂƐĞƐƚĂŐĞƐǁŝƚŚŵŝŶŝŵĂůƐLJŵƉƚŽŵƐͿ͘ dŚĞƌĞĨŽƌĞ͕ŐŝǀĞŶƚŚĞŽĐĐƵƌƌĞŶĐĞŽĨƌĂƌĞǀĂƌŝĂŶƚƐ͕ŝŶĐůƵĚŝŶŐƉŽƚĞŶƟĂůůLJĚĂŵĂŐŝŶŐǀĂƌŝĂŶƚƐ in most genomes of presumably healthy individuals,16_{ŝƚŵĂLJďĞĚŝĸĐƵůƚƚŽŝĚĞŶƟĨLJŐĞŶĞƐͬ}

6

/Ŷ ƚŚŝƐ ĐŽůůĂďŽƌĂƟǀĞ ƐƚƵĚLJ ŽĨ ƚŚĞ /ŶƚĞƌŶĂƟŽŶĂů WĂƌŬŝŶƐŽŶ͛Ɛ ŝƐĞĂƐĞ 'ĞŶŽŵŝĐƐ ŽŶƐŽƌƟƵŵ;/W'Ϳ͕ǁĞƌĞƉŽƌƚƚŚĞƌĞƐƵůƚƐŽĨǁŚŽůĞĞdžŽŵĞƐĞƋƵĞŶĐŝŶŐ;t^ͿŝŶϭ͕ϭϰϴ WĐĂƐĞƐ͕ƚŚĞůĂƌŐĞƐƚƐƵĐŚĐŽŚŽƌƚĞdžĂŵŝŶĞĚƚŽĚĂƚĞ͘ŽŶƐŝƐƚĞŶƚǁŝƚŚƚŚĞLJŽƵŶŐĞƌĂŐĞŽĨ WŽŶƐĞƚŝŶƚŚŝƐĐŽŚŽƌƚ͕ǁŚŝĐŚŝƐŽŌĞŶĂƐƐŽĐŝĂƚĞĚǁŝƚŚĂƌĞĐĞƐƐŝǀĞŝŶŚĞƌŝƚĂŶĐĞ͕17-19 _{and to} ƉƌŝŽƌŝƟnjĞĐĂŶĚŝĚĂƚĞŐĞŶĞƐͬǀĂƌŝĂŶƚƐĨŽƌŝŶŝƟĂůŝŶǀĞƐƟŐĂƟŽŶ͕ŽƵƌĂŶĂůLJƐŝƐĨŽĐƵƐĞƐŽŶŐĞŶĞƐ ǁŝƚŚŚŽŵŽnjLJŐŽƵƐŽƌĐŽŵƉŽƵŶĚŚĞƚĞƌŽnjLJŐŽƵƐ>Ž&ǀĂƌŝĂŶƚƐ͘tĞĨƵƌƚŚĞƌĐŽƵƉůĞƚŚĞŚƵŵĂŶ ŐĞŶĞƟĐ ƐƚƵĚŝĞƐ ǁŝƚŚ ĨƵŶĐƟŽŶĂů ƐĐƌĞĞŶŝŶŐ ŝŶ ŵĂŵŵĂůŝĂŶ ĐĞůů ĐƵůƚƵƌĞ ĂŶĚ ŝŶǀĞƌƚĞďƌĂƚĞ ĂŶŝŵĂůŵŽĚĞůƐ͕ƐƵĐĐĞƐƐĨƵůůLJŝĚĞŶƟĨLJŝŶŐƚŚŽƐĞĐĂŶĚŝĚĂƚĞŐĞŶĞƐƐŚŽǁŝŶŐŝŶƚĞƌĂĐƟŽŶƐǁŝƚŚ ĞƐƚĂďůŝƐŚĞĚWŵĞĐŚĂŶŝƐŵƐ͕ŝŶĐůƵĚŝŶŐŵŝƚŽĐŚŽŶĚƌŝĂůĚLJŶĂŵŝĐƐĂŶĚɲͲƐLJŶƵĐůĞŝŶŵĞĚŝĂƚĞĚ ŶĞƵƌŽĚĞŐĞŶĞƌĂƟŽŶ͘ůƚŚŽƵŐŚŶŽƐƵĸĐŝĞŶƚůLJƉŽǁĞƌĞĚĞdžŽŵĞĚĂƚĂƐĞƚǁĂƐĂǀĂŝůĂďůĞĨŽƌ ĚĞĮŶŝƟǀĞƌĞƉůŝĐĂƟŽŶ͕ŚƵŵĂŶŐĞŶĞƟĐǀĂůŝĚĂƟŽŶǁĂƐƵŶĚĞƌƚĂŬĞŶŝŶƐĞǀĞƌĂůŝŶĚĞƉĞŶĚĞŶƚ ĚĂƚĂƐĞƚƐ͘KƵƌŝŶƚĞŐƌĂƚĞĚĂƉƉƌŽĂĐŚŝĚĞŶƟĮĞƐĮǀĞƐƚƌŽŶŐĐĂŶĚŝĚĂƚĞWƐƵƐĐĞƉƟďŝůŝƚLJŐĞŶĞƐ ǁŽƌƚŚLJŽĨĨƵƌƚŚĞƌŝŶǀĞƐƟŐĂƟŽŶ͕ĂŶĚĞdžĞŵƉůŝĮĞƐĂƉŽǁĞƌĨƵůƐƚƌĂƚĞŐLJǁŝƚŚƉŽƚĞŶƟĂůďƌŽĂĚ applicability to the follow up of future rare variant studies in PD and other neurologic ĚŝƐŽƌĚĞƌƐǁŝƚŚĐŽŵƉůĞdžŐĞŶĞƟĐĞƟŽůŽŐŝĞƐ͘

Z^h>d^

ŝƐĐŽǀĞƌLJŽĨƌĞĐĞƐƐŝǀĞůŽƐƐͲŽĨͲĨƵŶĐƟŽŶǀĂƌŝĂŶƚƐĨƌŽŵWĞdžŽŵĞƐ

 ƚŽƚĂů ŽĨ ϵϮϬ͕ϴϵϲ ǀĂƌŝĂŶƚƐ ;ϵϯ͘Ϯй ƐŝŶŐůĞ ŶƵĐůĞŽƟĚĞ ǀĂƌŝĂŶƚƐ ĂŶĚ ϲ͘ϴй ŝŶƐĞƌƟŽŶƐ ĂŶĚ ĚĞůĞƟŽŶƐͿǁĞƌĞĐĂůůĞĚŝŶĂt^ĚĂƚĂƐĞƚŽĨϭ͕ϲϱϭƐƵďũĞĐƚƐ͕ŝŶĐůƵĚŝŶŐϭ͕ϭϰϴLJŽƵŶŐͲŽŶƐĞƚ PD cases (average age of onset 40.6 years, range 5-56 years) and 503 control subjects with European ancestry. As our cohort has an average age at onset below 45 years, ǁĞĨŽĐƵƐĞĚŽƵƌƐĞĂƌĐŚŽŶŚŽŵŽnjLJŐŽƵƐĂŶĚƉƵƚĂƟǀĞĐŽŵƉŽƵŶĚŚĞƚĞƌŽnjLJŐŽƵƐǀĂƌŝĂŶƚƐ͕ consistent with a recessive inheritance model. Although most PD cases were prescreened ĨŽƌŵƵƚĂƟŽŶƐŝŶĞƐƚĂďůŝƐŚĞĚWŐĞŶĞƐ͕ǁĞŝĚĞŶƟĮĞĚϮƐƵďũĞĐƚƐǁŝƚŚŚŽŵŽnjLJŐŽƵƐĞdžŽŶŝĐ variants in parkin and PINK1 ;^ƵƉƉůĞŵĞŶƚĂƌLJdĂďůĞϭͿ͘/ŶŽƌĚĞƌƚŽŝĚĞŶƟĨLJŶŽǀĞůWŐĞŶĞ ĐĂŶĚŝĚĂƚĞƐ ǁĞ ĨŽĐƵƐĞĚ ŽŶ ǀĂƌŝĂŶƚƐ ƚŚĂƚ ĂƌĞ ƌĂƌĞ ŝŶ ĐŽŶƚƌŽů ƉŽƉƵůĂƟŽŶƐ͘ ŽŶƐŝĚĞƌŝŶŐ the worldwide prevalence for PD (0.041% in individuals aged 40-49 years),20 _{we used a}

6

ŐŐƌĞŐĂƟŽŶŽŶƐŽƌƟƵŵ;džͿͿĂŶĚƚŚĞƌĞĨŽƌĞŝŵƉůŝĐĂƚĞĚƚŽďĞŶŽǀĞů͘džĐĞƉƚŝŶƚŚĞĐĂƐĞ of ARSB͕ƚŚĞϮϲŽƚŚĞƌŐĞŶĞƐŚĂƌďŽƌ>Ž&ǀĂƌŝĂŶƚƐŝŶŽŶůLJĂƐŝŶŐůĞĐĂƐĞ͕ĐŽŶƐŝƐƚĞŶƚǁŝƚŚƚŚĞ ŚLJƉŽƚŚĞƐŝƐƚŚĂƚŶŽǀĞůƌĞĐĞƐƐŝǀĞWĂůůĞůĞƐŵĂLJĐŽŶƐŝƐƚŽĨŵĂŶLJƌĂƌĞ͕͞ƉƌŝǀĂƚĞ͟ŵƵƚĂƟŽŶƐ͘ &ŽƵƌWĐĂƐĞƐŝŶŽƵƌĐŽŚŽƌƚǁĞƌĞŝĚĞŶƟĮĞĚǁŝƚŚĂ>Ž&ǀĂƌŝĂŶƚŝŶƚŚĞARSB gene, in which ŵƵƚĂƟŽŶƐŚĂǀĞƉƌĞǀŝŽƵƐůLJďĞĞŶůŝŶŬĞĚǁŝƚŚƚŚĞƌĞĐĞƐƐŝǀĞůLJƐŽƐŽŵĂůƐƚŽƌĂŐĞĚŝƐŽƌĚĞƌ͕DW^ s/;ĂůƐŽĐĂůůĞĚDĂƌŽƚĞĂƵdžͲ>ĂŵLJ^LJŶĚƌŽŵĞͿ͘ůůϰŝŶĚŝǀŝĚƵĂůĐĂƐĞƐ͕ĂůŽŶŐǁŝƚŚϭĐŽŶƚƌŽů ƐƵďũĞĐƚ͕ ǁĞƌĞ ŚŽŵŽnjLJŐŽƵƐ ĨŽƌ Ă ǀĂƌŝĂŶƚ ;ƌƐϭϯϴϮϳϵϬϮϬͿ ƉƌĞĚŝĐƚĞĚ ƚŽ ĚŝƐƌƵƉƚ ƐƉůŝĐŝŶŐ͘ High-quality calls: 920,896 variants Autosomal exonic: 446,139 variants D&чϭй͗ 386,078 variants Loss-of-Function: 5,906 variants Homozygous freq. cases > controls: 18 genes Case-specific putative CH: 9 genes Stop codon: 17 genes Splicing: 1 gene LoF + miss: 3 genes 2x LoF: 6 genes C St Annotation RefSeq & UCSC

Annotation 1000G, ESP6500 & ExAC

Manual curation IGV Count with KGGSeq

6

ůƚŚŽƵŐŚƚŚŝƐǀĂƌŝĂŶƚŝƐŶĞŝƚŚĞƌƌĞƉŽƌƚĞĚŝŶdžŶŽƌǁĂƐĨƌĞƋƵĞŶĐLJŝŶĨŽƌŵĂƟŽŶĂǀĂŝůĂďůĞ ĨƌŽŵ Ěď^EW͕ ƚŚĞ D& ǁĂƐ Ϭ͘Ϭϲϱ ŝŶ ŽƵƌ ĐŽŚŽƌƚ ;D&_CASESсϬ͘Ϭϳϯ͕ D&_KEdZK>^=0.052,

pсϬ͘ϬϱϰͿ͘ůƚŚŽƵŐŚƌĞůĂƟǀĞůLJĨƌĞƋƵĞŶƚŝŶŽƵƌĐŽŶƚƌŽůĚĂƚĂƐĞƚ;D&хϭйͿ͕ǁĞŚĂǀĞƌĞƚĂŝŶĞĚ ŝƚ ĂŵŽŶŐ ŽƵƌ ƌĞƐƵůƚƐ͕ ďĂƐĞĚ ŽŶ ϯ ĐŽŶƐŝĚĞƌĂƟŽŶƐ͘ &ŝƌƐƚ͕ ŝŶĨŽƌŵĂƟŽŶ ǁĂƐ ŶŽƚ ƉƌĞƐĞŶƚ ŝŶ Ěď^EW͕džŽƌ^WϲϱϬϬ͕ǁŚŝĐŚǁĂƐƚŚĞďĂƐŝƐĨŽƌĂƉƉůLJŝŶŐƚŚŝƐĨƌĞƋƵĞŶĐLJĮůƚĞƌŝŶĂůůŽƚŚĞƌ ĐĂƐĞƐ͘ ^ĞĐŽŶĚ͕ Ăƚ ůĞĂƐƚ ŽŶĞ ŽĨ ƚŚĞ ŚŽŵŽnjLJŐŽƵƐ ŝŶĚŝǀŝĚƵĂůƐ ŚĂĚ ĐůŝŶŝĐĂů ŵĂŶŝĨĞƐƚĂƟŽŶƐ ĐŽŶƐŝƐƚĞŶƚǁŝƚŚDW^s/͕ƐƵƉƉŽƌƟŶŐƉŽƚĞŶƟĂůƉĂƚŚŽŐĞŶŝĐŝƚLJŽĨƚŚŝƐĂůůĞůĞ;ƐĞĞŝƐĐƵƐƐŝŽŶͿ͘ >ĂƐƚůLJ͕ĂƐĚĞƚĂŝůĞĚďĞůŽǁ͕ŽƵƌĨƵŶĐƟŽŶĂůƐƚƵĚŝĞƐŝĚĞŶƟĨLJůŝŶŬƐďĞƚǁĞĞŶŵĂŶŝƉƵůĂƟŽŶŽĨ Z^ĂŶĚĐĞůůƵůĂƌͬŽƌŐĂŶŝƐŵĂůƉŚĞŶŽƚLJƉĞƐĐŽŶƐŝƐƚĞŶƚǁŝƚŚĂƉŽƚĞŶƟĂůƌŽůĞŝŶW͘ KĨ ŶŽƚĞ͕ ǁŚŝůĞ ƚŚĞ ĂŶĂůLJƐĞƐ ŽĨ ƚŚĞ /W' t^ ĚĂƚĂƐĞƚ ĂŶĚ ƐƵďƐĞƋƵĞŶƚ ǁŽƌŬ ĚĞƐĐƌŝďĞĚŚĞƌĞǁĞƌĞŝŶƉƌŽŐƌĞƐƐ͕ĂŶŝŶĚĞƉĞŶĚĞŶƚĨĂŵŝůLJͲďĂƐĞĚƐĞƋƵĞŶĐŝŶŐƐƚƵĚLJŝĚĞŶƟĮĞĚ

VPS13C as a cause of autosomal recessive parkinsonism.21_{ ůƚŚŽƵŐŚ ƚŚĞ ƐŝŶŐůĞ /W'}

ƐƵďũĞĐƚ ǁŝƚŚ ĐŽŵƉŽƵŶĚ ŚĞƚĞƌŽnjLJŐŽƵƐ VPS13C >Ž& ĂůůĞůĞƐ ǁĂƐ ƉƵďůŝƐŚĞĚ ĂƐ Ă ƌĞƉůŝĐĂƚĞ case in that work, we retained it among the 27 candidates described here, since it was independently carried forward for all analyses detailed below.

dŽůĞƌĂďŝůŝƚLJŽĨŐĞŶĞůŽƐƐͲŽĨͲĨƵŶĐƟŽŶŝŶŚƵŵĂŶƐĂŶĚĂŶŝŵĂůŵŽĚĞůƐ dŚĞ͞ƚŽůĞƌĂďŝůŝƚLJ͟ŽĨƌĞĐĞƐƐŝǀĞ>Ž&ŐĞŶŽƚLJƉĞƐŚĂƐŝŵƉŽƌƚĂŶƚŝŵƉůŝĐĂƟŽŶƐĨŽƌƵŶĚĞƌƐƚĂŶĚŝŶŐ ƚŚĞ ŐĞŶĞƟĐ ďĂƐŝƐ ŽĨ ĂĚƵůƚͲŽŶƐĞƚ͕ ĂŐĞͲŝŶŇƵĞŶĐĞĚ ĚŝƐŽƌĚĞƌƐ ƐƵĐŚ ĂƐ W͘ Ɛ ŵŽƐƚ ŽĨ ƚŚĞ ŝĚĞŶƟĮĞĚŚŽŵŽnjLJŐŽƵƐĂŶĚƉƵƚĂƟǀĞĐŽŵƉŽƵŶĚŚĞƚĞƌŽnjLJŐŽƵƐ>Ž&ŐĞŶŽƚLJƉĞƐĂƌĞďĂƐĞĚŽŶ ĂƐŝŶŐůĞŝŶĚŝǀŝĚƵĂů͕ǁĞĂůƐŽĞdžĂŵŝŶĞĚĨŽƌƚŚĞŝƌŽĐĐƵƌƌĞŶĐĞŝŶĂůĂƌŐĞ͕ƌĞĐĞŶƚůLJƉƵďůŝƐŚĞĚ study16_{ ŽĨ ƉƌĞĚŝĐƚĞĚ ĐŽŵƉůĞƚĞ ŐĞŶĞ ŬŶŽĐŬͲŽƵƚƐ ŝŶ ƚŚĞ /ĐĞůĂŶĚŝĐ ƉŽƉƵůĂƟŽŶ͕ ŝŶĐůƵĚŝŶŐ}

104,220 subjects with imputed genotypes, based on whole genome sequencing from a ƐƵďƐĞƚŽĨϮ͕ϯϲϯŝŶĚŝǀŝĚƵĂůƐ͘dŚĞ/ĐĞůĂŶĚŝĐƉŽƉƵůĂƟŽŶŝƐĞŶƌŝĐŚĞĚĨŽƌƌĂƌĞĚŝƐĞĂƐĞͲĐĂƵƐŝŶŐ ŵƵƚĂƟŽŶƐǁŝƚŚĂƌĞĐĞƐƐŝǀĞŝŶŚĞƌŝƚĂŶĐĞƉĂƩĞƌŶ͕ŐŝǀĞŶĂƐƚƌŽŶŐĨŽƵŶĚĞƌĞīĞĐƚĂŶĚŶŽŶͲ ƌĂŶĚŽŵŵĂƟŶŐƉĂƩĞƌŶƐ͘dǁĞůǀĞŽĨƚŚĞǀĂƌŝĂŶƚƐƚŚĂƚǁĞŝĚĞŶƟĮĞĚĂƌĞĂůƐŽƉƌĞƐĞŶƚŝŶƚŚĞ /ĐĞůĂŶĚŝĐƐƚƵĚLJ;^ƵƉƉůĞŵĞŶƚĂƌLJdĂďůĞϮͿ͖ŚŽǁĞǀĞƌ͕ƚŚĞŽďƐĞƌǀĞĚŚŽŵŽnjLJŐŽƚĞĨƌĞƋƵĞŶĐŝĞƐ ĂƌĞŶŽƚƐƵĸĐŝĞŶƚůLJŚŝŐŚƚŽĐŽŶĮĚĞŶƚůLJĞdžĐůƵĚĞƚŚĞŵĂƐƉŽƐƐŝďůĞWŐĞŶĞƐĂŶĚŝŵƉŽƌƚĂŶƚůLJ͕ ĚĞƚĂŝůĞĚƉŚĞŶŽƚLJƉŝĐĚĂƚĂŝƐŶŽƚƉƵďůŝĐĂůůLJĂǀĂŝůĂďůĞĨŽƌƚŚĞƐĞƐƵďũĞĐƚƐ͘&ŽƌĞdžĂŵƉůĞ͕Ϯϵ /ĐĞůĂŶĚŝĐ ƐƵďũĞĐƚƐ ĂƌĞ ƌĞƉŽƌƚĞĚ ŚŽŵŽnjLJŐŽƵƐ ĨŽƌ ƚŚĞ ŝĚĞŶƟĐĂů PTCHD3 stopgain variant ;Đ͘ϭϰϮϲd͕Ɖ͘ZϰϳϲyͿĂƐƚŚĞƐŝŶŐůĞWĐĂƐĞŝŶŽƵƌt^ƐƚƵĚLJ͘,ŽǁĞǀĞƌ͕ƚŚŝƐŝƐŽŶůLJϬ͘ϬϮϴй of the total sample set and below the reported prevalence of young-onset PD (0.041%).

tĞĂĚĚŝƟŽŶĂůůLJĞdžĂŵŝŶĞĚĨŽƌƚŚĞƉƌĞƐĞŶĐĞŽĨŽƚŚĞƌ>Ž&ǀĂƌŝĂŶƚƐǁŝƚŚĂƌĞĐĞƐƐŝǀĞ ŝŶŚĞƌŝƚĂŶĐĞ ƉĂƩĞƌŶ ŝŶ ŽƵƌ ŝŵƉůŝĐĂƚĞĚ ĐĂŶĚŝĚĂƚĞ ŐĞŶĞƐ ;^ƵƉƉůĞŵĞŶƚĂƌLJ dĂďůĞ ϮͿ͘ &Žƌ Ă ƐƵďƐĞƚŽĨŐĞŶĞƐ͕ǁĞŝŶĚĞĞĚŝĚĞŶƟĮĞĚƐĞǀĞƌĂůǀĂƌŝĂŶƚƐǁŝƚŚƉĂƌƟĐƵůĂƌůLJŚŝŐŚŚŽŵŽnjLJŐŽƚĞ frequencies including OR7G3 (9.16%), SSPO (9.38%) and PTCHD3 (16.55%). This is ĐŽŶƐŝƐƚĞŶƚ ǁŝƚŚ ƉƌŝŽƌ ƌĞƉŽƌƚƐ ĚĞƐĐƌŝďŝŶŐ Ă ŚŽŵŽnjLJŐŽƵƐ ĚĞůĞƟŽŶ ĐŽǀĞƌŝŶŐ PTCHD3 in ĂƉƉĂƌĞŶƚůLJŚĞĂůƚŚLJŝŶĚŝǀŝĚƵĂůƐ͕ĐŽŶƐŝƐƚĞŶƚǁŝƚŚĂŶŽŶͲĞƐƐĞŶƟĂůƌŽůĞ͘22 _{Assuming that the}

6

WES data, their high variant frequency makes these genes unlikely to be highly penetrant PD risk loci. ,ƵŵĂŶŐĞŶĞƐŚĂƌďŽƌŝŶŐŚŽŵŽnjLJŐŽƵƐ>Ž&ǀĂƌŝĂŶƚƐͶĞƐƉĞĐŝĂůůLJƚŚŽƐĞŽďƐĞƌǀĞĚ ƌĞĐƵƌƌĞŶƚůLJ ŝŶ ůĂƌŐĞ ƉŽƉƵůĂƟŽŶͲďĂƐĞĚ ĚĂƚĂƐĞƚƐ͕ ƉŽƚĞŶƟĂůůLJ ŝĚĞŶƟĨLJ ŐĞŶĞƐ ƚŚĂƚ ĂƌĞ ĚŝƐƉĞŶƐĂďůĞ ĨŽƌ ĨĞƚĂů ĂŶĚ ƐƵďƐĞƋƵĞŶƚ ĐŚŝůĚ ĚĞǀĞůŽƉŵĞŶƚ͘ 'ŝǀĞŶ ƚŚĞ ůŝŵŝƚĞĚ ŚƵŵĂŶ ƉŚĞŶŽƚLJƉŝĐŝŶĨŽƌŵĂƟŽŶĂǀĂŝůĂďůĞ͕ǁĞĨƵƌƚŚĞƌŝŶǀĞƐƟŐĂƚĞĚƚŚĞƉŽƚĞŶƟĂůƚŽůĞƌĂďŝůŝƚLJĨŽƌƚŚĞ ŝŵƉůŝĐĂƚĞĚŐĞŶĞƐƵƐŝŶŐĂĐƌŽƐƐͲƐƉĞĐŝĞƐĂƉƉƌŽĂĐŚ͕ƉĞƌĨŽƌŵŝŶŐƐLJƐƚĞŵĂƟĐ>Ž&ĂŶĂůLJƐŝƐŝŶ the nematode, C. elegans. KƵƚŽĨƚŚĞϮϳĐĂŶĚŝĚĂƚĞŐĞŶĞƐŝĚĞŶƟĮĞĚŝŶŽƵƌt^ĂŶĂůLJƐŝƐ͕ 10 were well conserved in the C. elegans ŐĞŶŽŵĞ͕ ĂŶĚ ϵ ŚĂĚ ƌĞĂĚŝůLJ ĂǀĂŝůĂďůĞ ZEͲ ŝŶƚĞƌĨĞƌĞŶĐĞ;ZEŝͿƌĞĂŐĞŶƚƐĨŽƌ>Ž&ƐĐƌĞĞŶŝŶŐ;ƐĞĞDĞƚŚŽĚƐͿ͘ĂĐŚŐĞŶĞǁĂƐƚĂƌŐĞƚĞĚ ĨŽƌŬŶŽĐŬĚŽǁŶƵƐŝŶŐZEŝĂŶĚǁĞĂƐƐĞƐƐĞĚĨŽƌĚĞǀĞůŽƉŵĞŶƚĂůůĞƚŚĂůŝƚLJĂŶĚƐƵƌǀŝǀĂů͘dŚĞ ƌĞƐƵůƚƐŽĨƚŚĞƐĞƐƚƵĚŝĞƐ͕ĂůŽŶŐǁŝƚŚŽƚŚĞƌ>Ž&ĚĂƚĂĨƌŽŵƉƵďůŝĐĚĂƚĂďĂƐĞƐ͕ĂƌĞĂǀĂŝůĂďůĞ ŝŶ^ƵƉƉůĞŵĞŶƚĂƌLJdĂďůĞϯ͘<ŶŽĐŬĚŽǁŶŽĨŚŽŵŽůŽŐƐŽĨDIS3 (ĚŝƐͲϯ), KALRN (unc-73), and

PTCHD3 (ptr-10) resulted in developmental arrest and/or reduced survival in C. elegans.

Notably, homologs of KALRN and DIS3 are also associated with reduced viability following ŐĞŶĞƟĐĚŝƐƌƵƉƟŽŶŝŶďŽƚŚDrosophila23,24 _{and mice.}25, 26_{dŚƵƐ͕ƚŚĞƐĞƌĞƐƵůƚƐĂƌĞƉŽƚĞŶƟĂůůLJ}

ĐŽŶƐŝƐƚĞŶƚǁŝƚŚĐŽŶƐĞƌǀĞĚ͕ĞĂƌůLJĂŶĚͬŽƌĞƐƐĞŶƟĂůĚĞǀĞůŽƉŵĞŶƚĂůƌŽůĞƐĨŽƌƚŚĞƐĞŐĞŶĞƐĂŶĚ ƚŚĞĂďƐĞŶĐĞŽĨŝŶĚŝǀŝĚƵĂůƐŚĂƌďŽƌŝŶŐŚŽŵŽnjLJŐŽƵƐ>Ž&ǀĂƌŝĂŶƚƐŝŶƚŚĞ/ĐĞůĂŶĚŝĐĐŽŚŽƌƚ͘16 ^ŝŶĐĞƚŚĞŚƵŵĂŶŐĞŶŽŵĞĐŽŶƚĂŝŶƐŵƵůƟƉůĞŐĞŶĞƉĂƌĂůŽŐƐĨŽƌKALRN and PTCHD3, ŐĞŶĞƟĐƌĞĚƵŶĚĂŶĐLJŵŝŐŚƚĂĐĐŽƵŶƚĨŽƌŚŽǁ>Ž&ŵŝŐŚƚďĞƚŽůĞƌĂƚĞĚŝŶŚƵŵĂŶƐďƵƚŶŽƚ ŝŶƐŝŵƉůĞĂŶŝŵĂůŵŽĚĞůƐ͘ůƚĞƌŶĂƟǀĞůLJ͕ŝƚŝƐƉŽƐƐŝďůĞƚŚĂƚƚŚĞĂůůĞůŝĐǀĂƌŝĂŶƚƐŝŵƉůŝĐĂƚĞĚ ŝŶŽƵƌWt^ĐŽŚŽƌƚĂŶĚ/ĐĞůĂŶĚŝĐƐƚƵĚLJŵŝŐŚƚŶŽƚĐĂƵƐĞĂĐŽŵƉůĞƚĞ>Ž&;i.e.ŐĞŶĞƟĐ ŶƵůůͿĚĞƐƉŝƚĞƚŚĞĂůŐŽƌŝƚŚŵŝĐƉƌĞĚŝĐƟŽŶƐ͕ŝŶƐƚĞĂĚĐĂƵƐŝŶŐŽŶůLJĂƉĂƌƟĂůůŽƐƐͲŽĨͲĨƵŶĐƟŽŶ͘ EĞǀĞƌƚŚĞůĞƐƐ͕ƚŚĞƐĞĐƌŽƐƐͲƐƉĞĐŝĞƐĐŽŵƉĂƌŝƐŽŶƐƐƵŐŐĞƐƚĞƐƐĞŶƟĂůĂŶĚĞĂƌůLJĚĞǀĞůŽƉŵĞŶƚĂů roles for homologs of PTCHD3, DIS3 and KALRN, and ŝŶĨŽƌŵŝŶŐ ŽƵƌ ĐŽŶƐŝĚĞƌĂƟŽŶ ŽĨ ƉŽƚĞŶƟĂůĐŽŶƚƌŝďƵƟŽŶƚŽĂĚƵůƚͲŽŶƐĞƚĚŝƐŽƌĚĞƌƐ͕ƐƵĐŚĂƐW͘

sĂƌŝĂŶƚĂŐŐƌĞŐĂƟŽŶĂŶĂůLJƐĞƐ

6

KƵƌ ĂŶĂůLJƐĞƐ ŽĨ >Ž& ǀĂƌŝĂŶƚƐ ŝŶ W ĞdžŽŵĞƐ ŝĚĞŶƟĨLJ Ă ŶƵŵďĞƌ ŽĨ ƉƌŽŵŝƐŝŶŐ ĐĂŶĚŝĚĂƚĞŐĞŶĞƐ͘,ŽǁĞǀĞƌ͕ĞǀĞŶƚŚŽƵŐŚĂƉŽƐŝƟǀĞĨĂŵŝůLJŚŝƐƚŽƌLJǁĂƐŽďƐĞƌǀĞĚĨŽƌĂůŵŽƐƚ ϰϬйŽĨƚŚĞĐĂƐĞƐ͕ƐĞŐƌĞŐĂƟŽŶĂŶĂůLJƐŝƐŽĨƚŚĞǀĂƌŝĂŶƚƐŝŶĨĂŵŝůŝĞƐŝƐŶŽƚĨĞĂƐŝďůĞ͕ĂƐE ƐĂŵƉůĞƐ ĂƌĞ ŶŽƚ ĂǀĂŝůĂďůĞ ĨƌŽŵ ĂĚĚŝƟŽŶĂů ĨĂŵŝůLJ ŵĞŵďĞƌƐ͘ &ƵƌƚŚĞƌ͕ ƐŝŶĐĞ ŵŽƐƚ ŽĨ ƚŚĞ genes implicated contribute to single or few cases, we are unable to perform meaningful ƐƚĂƟƐƟĐĂůĐŽŵƉĂƌŝƐŽŶƐ͕ďĂƐĞĚŽŶƚŚĞůŝŵŝƚĞĚŶƵŵďĞƌƐŽĨ>Ž&ǀĂƌŝĂŶƚƐŝĚĞŶƟĮĞĚďLJt^ ŝŶĐĂƐĞƐǀƐ͘ĐŽŶƚƌŽůƐ͘ƐĂŶĂůƚĞƌŶĂƟǀĞƐƚƌĂƚĞŐLJǁĞƚŚĞƌĞĨŽƌĞĚĞƉůŽLJĞĚĂĐŽŵďŝŶĂƟŽŶŽĨ ĐĞůůͲďĂƐĞĚĂŶĚŵŽĚĞůŽƌŐĂŶŝƐŵĨƵŶĐƟŽŶĂůƐĐƌĞĞŶƐƚŽĚĞĮŶĞƉŽƚĞŶƟĂůůŝŶŬƐďĞƚǁĞĞŶƚŚĞ ϮϳĐĂŶĚŝĚĂƚĞŐĞŶĞƐ;dĂďůĞϭͿĂŶĚǁĞůůͲĞƐƚĂďůŝƐŚĞĚŵĞĐŚĂŶŝƐŵƐŽĨWƐƵƐĐĞƉƟďŝůŝƚLJĂŶĚ ƉĂƚŚŽŐĞŶĞƐŝƐ͕ŝŶĐůƵĚŝŶŐ;ϭͿŵŝƚŽĐŚŽŶĚƌŝĂůŚĞĂůƚŚĂŶĚ;ϮͿɲͲƐLJŶƵĐůĞŝŶŵĞĚŝĂƚĞĚƚŽdžŝĐŝƚLJ͘ &ƵŶĐƟŽŶĂůƉƌŝŽƌŝƟnjĂƟŽŶ͗ŵŝƚŽĐŚŽŶĚƌŝĂůŚĞĂůƚŚ ůƚŚŽƵŐŚ ƚŚĞ ŵĞĐŚĂŶŝƐŵ ŽĨ ŶĞƵƌŽĚĞŐĞŶĞƌĂƟŽŶ ŝŶ W ƌĞŵĂŝŶƐ ŝŶĐŽŵƉůĞƚĞůLJ ĚĞĮŶĞĚ ĂŶĚ ŵĂLJ ďĞ ŚĞƚĞƌŽŐĞŶĞŽƵƐ͕ ŵŝƚŽĐŚŽŶĚƌŝĂů ĚLJƐĨƵŶĐƟŽŶ ŚĂƐ ďĞĞŶ ƉƌŽƉŽƐĞĚ ƚŽ ƉůĂLJ ĂŶ ŝŵƉŽƌƚĂŶƚ ƌŽůĞ ƉĂƌƟĐƵůĂƌůLJ ŝŶ LJŽƵŶŐ ŽŶƐĞƚ W͘27-29 _{Notably, ƉĂƌŬŝŶ ;WZ<ϮͿ͕ :Ͳϭ and} PINK1, associated with autosomal recessive, juvenile-onset Parkinsonism, have roles in

mitochondrial dynamics and quality control.30_{^ƉĞĐŝĮĐĂůůLJ͕WĂƌŬŝŶŝƐĂŶϯƵďŝƋƵŝƟŶůŝŐĂƐĞ}

ĂŶĚƌĞĐƌƵŝƚĞĚƐĞůĞĐƟǀĞůLJƚŽĚLJƐĨƵŶĐƟŽŶĂůŵŝƚŽĐŚŽŶĚƌŝĂǁŝƚŚĂůŽǁŵĞŵďƌĂŶĞƉŽƚĞŶƟĂů͘31

&ƵƌƚŚĞƌ͕ƚŚĞŶĞƵƌŽƚŽdžŝĐŝƚLJŽĨɲͲƐLJŶƵĐůĞŝŶ͕ƚŚĞƉƌŝŵĂƌLJĐŽŶƐƟƚƵĞŶƚŽĨ>ĞǁLJďŽĚLJŝŶĐůƵƐŝŽŶƐ in PD, has also been linked to mitochondrial injury.32_{ tĞ ƚŚĞƌĞĨŽƌĞ ŚLJƉŽƚŚĞƐŝnjĞĚ ƚŚĂƚ}

>Ž& ŝŶ ĐĂŶĚŝĚĂƚĞ ŐĞŶĞƐ ŝĚĞŶƟĮĞĚ ĨƌŽŵ ŽƵƌ ĂŶĂůLJƐĞƐ ŽĨ t^͕ ŵŝŐŚƚ ƐŝŵŝůĂƌůLJ ŝŵƉĂĐƚ ŵŝƚŽĐŚŽŶĚƌŝĂ͕ĐŽŶƐŝƐƚĞŶƚǁŝƚŚƌŽůĞƐŝŶWƐƵƐĐĞƉƟďŝůŝƚLJ͘

dŚĞƌĞĨŽƌĞ ǁĞ ƋƵĂŶƟĮĞĚ ŵŝƚŽĐŚŽŶĚƌŝĂů ŵŽƌƉŚŽůŽŐLJ ĂŌĞƌ ŐĞŶĞ ŬŶŽĐŬĚŽǁŶ ŝŶ ;ϮͿͲDϭϳ ŶĞƵƌŽďůĂƐƚŽŵĂ ĐĞůůƐ ďLJ ĞdžĂŵŝŶŝŶŐ ϯ ƉĂƌĂŵĞƚĞƌƐ ĐŽŵŵŽŶůLJ ƵƐĞĚ ĨŽƌ ƋƵĂŶƟĮĐĂƟŽŶ ŽĨ ŵŝƚŽĐŚŽŶĚƌŝĂů ŵŽƌƉŚŽůŽŐLJ͗ ŵŝƚŽĐŚŽŶĚƌŝĂů ŶƵŵďĞƌ͕ ĂdžŝĂů ůĞŶŐƚŚ ƌĂƟŽ͕ and roundness.33_{ĞůůƐƚƌĂŶƐĚƵĐĞĚǁŝƚŚƚŚĞƐŚZEĞŶĐŽĚŝŶŐĂƐĐƌĂŵďůĞĚƐĞƋƵĞŶĐĞǁĞƌĞ}

ƵƐĞĚĨŽƌŶŽƌŵĂůŝnjĂƟŽŶĂŶĚƉŽƐŝƟǀĞĐŽŶƚƌŽůƐĨŽƌŵŝƚŽĐŚŽŶĚƌŝĂůŵŽƌƉŚŽůŽŐLJǁĞƌĞŝŶĐůƵĚĞĚ ŝŶĞĂĐŚĞdžƉĞƌŝŵĞŶƚ͘&ŽƌĞdžĂŵƉůĞ͕ŬŶŽĐŬĚŽǁŶŽĨƚŚĞŵŝƚŽĐŚŽŶĚƌŝĂůĮƐƐŝŽŶŐĞŶĞĚLJŶĂŵŝŶ 1-like (DNM1LͿ͕ Ă ƉŽƐŝƟǀĞ ĐŽŶƚƌŽů͕ ƌĞƐƵůƚƐ ŝŶ ĞůŽŶŐĂƚĞĚ ŵŝƚŽĐŚŽŶĚƌŝĂ ĂŶĚ ƚŚĞƌĞĨŽƌĞ ĚĞĐƌĞĂƐĞƐŵŝƚŽĐŚŽŶĚƌŝĂůĂdžŝĂůůĞŶŐƚŚƌĂƟŽĂŶĚƌŽƵŶĚŶĞƐƐ;&ŝŐƵƌĞϮ͕Ϳ͘34_{<ŶŽĐŬĚŽǁŶŽĨ}

ϭϯŐĞŶĞƐƐŚŽǁĂƐŝŐŶŝĮĐĂŶƚĞīĞĐƚŽŶĂƚůĞĂƐƚŽŶĞŽĨƚŚĞϯƉĂƌĂŵĞƚĞƌƐ;^ƵƉƉůĞŵĞŶƚĂƌLJ dĂďůĞϱ͖^ƵƉƉůĞŵĞŶƚĂƌLJ&ŝŐƵƌĞϭͿ͘GPATCH2L shows the largest increase in mitochondrial roundness, while h,Z&ϭWϭ>ĚŝƐƉůĂLJƐƚŚĞůĂƌŐĞƐƚĚĞĐƌĞĂƐĞ;&ŝŐƵƌĞϮ͕Ϳ͘

6

A C D B Scr transduced DNM1L shRNA

Scr transduced UHRF1BP1L shRNA

6

A

C

D B

Figure 3. ,ŝŐŚ ĐŽŶƚĞŶƚ ĂƐƐĂLJ ĨŽƌ WĂƌŬŝŶ dƌĂŶƐůŽĐĂƟŽŶ͘ īĞĐƚ ŽĨ W/E<ϭ ƐŚZE ; ĂŶĚ Ϳ ĂŶĚ

6

ŽƵƌĂƐƐĂLJ͘WͲŝŶĚƵĐĞĚWĂƌŬŝŶĂĐĐƵŵƵůĂƟŽŶǁĂƐĂƐƐĞƐƐĞĚďLJŚŝŐŚͲĐŽŶƚĞŶƚŵŝĐƌŽƐĐŽƉLJ ĂŶĚĂƵƚŽŵĂƚĞĚŝŵĂŐĞĂŶĂůLJƐŝƐĨŽůůŽǁŝŶŐƐLJƐƚĞŵĂƟĐƐŚZEͲŬŶŽĐŬĚŽǁŶŽĨŽƵƌϮϳĐĂŶĚŝĚĂƚĞ ŐĞŶĞƐ;&ŝŐƵƌĞϯͿ͘ĂƐĞĚŽŶƐƚƌŝŶŐĞŶƚĐƌŝƚĞƌŝĂ;ƐĞĞDĞƚŚŽĚƐͿ͕ϲŐĞŶĞƐƐŝŐŶŝĮĐĂŶƚůLJŵŽĚŝĮĞĚ WĂƌŬŝŶƚƌĂŶƐůŽĐĂƟŽŶ;&ŝŐƵƌĞϯĂŶĚϯ͖^ƵƉƉůĞŵĞŶƚĂƌLJ&ŝŐƵƌĞϮ͖^ƵƉƉůĞŵĞŶƚĂƌLJdĂďůĞϱͿ͕ including 4 genes (GPATCH2L, PTCHD3, SVOPL and E&ϱϰϯͿǁŝƚŚĐŽŶƐŝƐƚĞŶƚĂĐƟǀŝƟĞƐŝŶ ďŽƚŚƚŚĞŵŝƚŽĐŚŽŶĚƌŝĂůŵŽƌƉŚŽůŽŐLJĂŶĚWĂƌŬŝŶƚƌĂŶƐůŽĐĂƟŽŶĂƐƐĂLJƐ͘ &ƵŶĐƟŽŶĂůƉƌŝŽƌŝƟnjĂƟŽŶ͗ɲͲ^LJŶƵĐůĞŝŶŵĞĚŝĂƚĞĚƚŽdžŝĐŝƚLJ  ǁĞĂůƚŚ ŽĨ ĞǀŝĚĞŶĐĞ ĂůƐŽ ƐƵƉƉŽƌƚƐ Ă ĐĞŶƚƌĂů ƌŽůĞ ĨŽƌ ɲͲƐLJŶƵĐůĞŝŶͲŵĞĚŝĂƚĞĚ ƚŽdžŝĐŝƚLJ ŝŶ WƉĂƚŚŽŐĞŶĞƐŝƐ͘ɲͲƐLJŶƵĐůĞŝŶĂŐŐƌĞŐĂƚĞƐ͕ƚĞƌŵĞĚ>ĞǁLJďŽĚŝĞƐ͕ĂƌĞƚŚĞĚĞĮŶŝŶŐĚŝƐĞĂƐĞ ƉĂƚŚŽůŽŐLJ͕ĂŶĚďŽƚŚɲͲƐLJŶƵĐůĞŝŶŐĞŶĞ;SNCAͿŵƵƚĂƟŽŶƐ͕ůŽĐƵƐŵƵůƟƉůŝĐĂƟŽŶ͕ĂŶĚƉƌŽŵŽƚĞƌ ƉŽůLJŵŽƌƉŚŝƐŵƐĂƌĞĂƐƐŽĐŝĂƚĞĚǁŝƚŚWƐƵƐĐĞƉƟďŝůŝƚLJ͘5_{&ƵƌƚŚĞƌ͕ĞdžƉƌĞƐƐŝŽŶŽĨɲͲƐLJŶƵĐůĞŝŶ} ŝŶ ŶƵŵĞƌŽƵƐ ĂŶŝŵĂů ŵŽĚĞůƐ ŝŶĐůƵĚŝŶŐ ŝŶ ƚŚĞ ĨƌƵŝƚ ŇLJ͕39-41 _{Drosophila melanogaster,} ƌĞĐĂƉŝƚƵůĂƚĞƐĨĞĂƚƵƌĞƐŽĨWͲƌĞůĂƚĞĚŶĞƵƌŽĚĞŐĞŶĞƌĂƟǀĞƉĂƚŚŽůŽŐLJ͘dƌĂŶƐŐĞŶŝĐĞdžƉƌĞƐƐŝŽŶ ŽĨɲͲƐLJŶƵĐůĞŝŶŝŶƚŚĞŇLJƌĞƟŶĂůĞĂĚƐƚŽŶĞƵƌŽƚŽdžŝĐĐŚĂŶŐĞƐ39_{ĂŶĚŝƐĂŵĞŶĂďůĞĨŽƌĚĞƚĞĐƟŽŶ} ŽĨ ŐĞŶĞƟĐ ŵŽĚŝĮĞƌƐ͘42, 43_{ 'ĞŶĞƟĐ ŵĂŶŝƉƵůĂƟŽŶ ŽĨ ĞƐƚĂďůŝƐŚĞĚ W ƐƵƐĐĞƉƟďŝůŝƚLJ ŐĞŶĞƐ͕}

including PARK244, 45 _{and VPS35,}46_{ŵŽĚƵůĂƚĞɲͲƐLJŶƵĐůĞŝŶƚŽdžŝĐŝƚLJŝŶƚƌĂŶƐŐĞŶŝĐŇŝĞƐ͕ƐŝŵŝůĂƌ}

ƚŽĮŶĚŝŶŐƐŝŶŵĂŵŵĂůŝĂŶŵŽĚĞůƐ͘44, 47_{tĞƚŚĞƌĞĨŽƌĞŚLJƉŽƚŚĞƐŝnjĞĚƚŚĂƚ>Ž&ŝŶŚŽŵŽůŽŐƐŽĨ}

ŶŽǀĞůWŐĞŶĞƐŵĂLJƐŝŵŝůĂƌůLJĞŶŚĂŶĐĞɲͲƐLJŶƵĐůĞŝŶͲŝŶĚƵĐĞĚƌĞƟŶĂůĚĞŐĞŶĞƌĂƟŽŶ͘

KƵƚŽĨƚŚĞϮϳĐĂŶĚŝĚĂƚĞŐĞŶĞƐŝŵƉůŝĐĂƚĞĚďLJŽƵƌt^ĂŶĂůLJƐĞƐ͕ϭϯǁĞƌĞǁĞůůͲ conserved in Drosophila ;^ƵƉƉůĞŵĞŶƚĂƌLJdĂďůĞϲͿ͘ǀĂŝůĂďůĞZEŝƐƚŽĐŬƐƚĂƌŐĞƟŶŐĞĂĐŚ ŽĨƚŚĞϭϴŇLJŚŽŵŽůŽŐƐ;ƐŽŵĞŐĞŶĞƐŚĂĚŵƵůƟƉůĞĐŽŶƐĞƌǀĞĚƉĂƌĂůŽŐƐͿǁĞƌĞĐƌŽƐƐĞĚƚŽ ŇŝĞƐ ŝŶ ǁŚŝĐŚ ƚŚĞ ŚƵŵĂŶ ɲͲƐLJŶƵĐůĞŝŶ ƚƌĂŶƐŐĞŶĞ ǁĂƐ ĚŝƌĞĐƚĞĚ ƚŽ ĂĚƵůƚ ƉŚŽƚŽƌĞĐĞƉƚŽƌƐ using the ZŚŽĚŽƉƐŝŶϭͲ'>ϰ;ZŚϭͿ driver (ZŚϭхɲͲƐLJŶƵĐůĞŝŶ).48_{&ŽƌƌĂƉŝĚƐĐƌĞĞŶŝŶŐ͕ƌĞƟŶĂů}

6

ĞĂĐŚ ŽĨ ƚŚĞƐĞ ŐĞŶĞƐ ǁĞƌĞ ƚĂƌŐĞƚĞĚ ƵŶĚĞƌ ƐŝŵŝůĂƌ ĞdžƉĞƌŝŵĞŶƚĂů ĐŽŶĚŝƟŽŶƐ ;Rh1>RNAi), ďƵƚ ŝŶĚĞƉĞŶĚĞŶƚ ŽĨ ɲͲƐLJŶƵĐůĞŝŶ ĞdžƉƌĞƐƐŝŽŶ͕ ǁĞ ĚŝĚ ŶŽƚ ŽďƐĞƌǀĞ ĂŶLJ ƐŝŐŶŝĮĐĂŶƚ ƌĞƟŶĂů ƉĂƚŚŽůŽŐLJŝŶϭϱĚĂLJͲŽůĚĂŶŝŵĂůƐ;&ŝŐƵƌĞϱͿ͘dŚĞƌĞĨŽƌĞ͕ǁŝƚŚŝŶƚŚĞDrosophilaɲͲƐLJŶƵĐůĞŝŶ ƚƌĂŶƐŐĞŶŝĐŵŽĚĞůƐLJƐƚĞŵ͕ƚŚĞŝŵƉůŝĐĂƚĞĚ>Ž&ĞŶŚĂŶĐĞƌƐĂƉƉĞĂƌĐŽŶƐŝƐƚĞŶƚǁŝƚŚƐLJŶĞƌŐŝƐƟĐ ;ŶŽŶͲĂĚĚŝƟǀĞͿ ĞīĞĐƚƐ ŽŶ ɲͲƐLJŶƵĐůĞŝŶʹŵĞĚŝĂƚĞĚ ƌĞƟŶĂů ĚĞŐĞŶĞƌĂƟŽŶ͘ ^ŝŶĐĞ ŝŶĐƌĞĂƐĞĚ ɲͲƐLJŶƵĐůĞŝŶĞdžƉƌĞƐƐŝŽŶůĞǀĞůƐĂƌĞŽŶĞŝŵƉŽƌƚĂŶƚŵĞĐŚĂŶŝƐŵŽĨWƐƵƐĐĞƉƟďŝůŝƚLJ͕5 _western ďůŽƚƐǁĞƌĞƉĞƌĨŽƌŵĞĚƚŽĚĞƚĞƌŵŝŶĞǁŚĞƚŚĞƌĂŶLJŽĨƚŚĞŝĚĞŶƟĮĞĚŐĞŶĞƟĐĞŶŚĂŶĐĞƌƐĂůƚĞƌ ɲͲƐLJŶƵĐůĞŝŶƉƌŽƚĞŝŶůĞǀĞůƐ͘,ŽǁĞǀĞƌ͕ĨŽůůŽǁŝŶŐZEŝͲŵĞĚŝĂƚĞĚŬŶŽĐŬĚŽǁŶ͕ŶŽŶĞůĞĚƚŽ ƐŝŐŶŝĮĐĂŶƚĐŚĂŶŐĞƐ;^ƵƉƉůĞŵĞŶƚĂƌLJ&ŝŐƵƌĞϯͿ͘dŚƵƐ͕ǁĞŚLJƉŽƚŚĞƐŝnjĞƉŽƚĞŶƟĂůŝŶƚĞƌĂĐƟŽŶƐ ǁŝƚŚ ŵŽƌĞ ĚŽǁŶƐƚƌĞĂŵ ŵĞĐŚĂŶŝƐŵƐ ŽĨ ɲͲƐLJŶƵĐůĞŝŶ ŶĞƵƌŽƚŽdžŝĐŝƚLJ͘ &Žƌ ϯ ŽƵƚ ŽĨ ƚŚĞ ϰ candidate enhancers (ARSB, VPS13C, PTPRHͿ͕ĂǀĂŝůĂďůĞƐŝZEƐƉĞƌŵŝƩĞĚĂĚĚŝƟŽŶĂůƚĞƐƟŶŐ ŽĨŐĞŶĞŚŽŵŽůŽŐƐĂƐĐĂŶĚŝĚĂƚĞŵŽĚŝĮĞƌƐŝŶĂŶĞƐƚĂďůŝƐŚĞĚC. elegans ŵŽĚĞůŽĨɲͲƐLJŶƵĐůĞŝŶ ƚŽdžŝĐŝƚLJ͘49_{,ŽǁĞǀĞƌ͕ŶŽƐŝŐŶŝĮĐĂŶƚĚŝīĞƌĞŶĐĞƐǁĞƌĞĚĞƚĞĐƚĞĚŝŶƚŚĞɲͲƐLJŶƵĐůĞŝŶͲŝŶĚƵĐĞĚ}

locomotor phenotype observed in 1 week old worms following knockdown of these genes ;^ƵƉƉůĞŵĞŶƚĂƌLJ&ŝŐƵƌĞϰͿ͘tĞƐƉĞĐƵůĂƚĞƚŚĂƚƚŚĞƐĞĐŽŶƚƌĂĚŝĐƚŽƌLJƌĞƐƵůƚƐŵŝŐŚƚƐƚĞŵĨƌŽŵ ĚŝīĞƌĞŶĐĞƐŝŶĂƐƐĂLJƐĞŶƐŝƟǀŝƚLJĂŶĚͬŽƌƟƐƐƵĞͲƐƉĞĐŝĮĐƚŽdžŝĐŵĞĐŚĂŶŝƐŵƐĂƐƚŚĞŇLJĂŶĚǁŽƌŵ ŵŽĚĞůƐĂƌĞďĂƐĞĚŽŶɲͲƐLJŶƵĐůĞŝŶĞdžƉƌĞƐƐŝŽŶŝŶƚŚĞƌĞƟŶĂǀĞƌƐƵƐŵƵƐĐůĞ͕ƌĞƐƉĞĐƟǀĞůLJ͘

KĨƚŚĞϰŐĞŶĞƐĚŝƐĐŽǀĞƌĞĚƚŽŝŶƚĞƌĂĐƚǁŝƚŚɲͲƐLJŶƵĐůĞŝŶƚŽdžŝĐŝƚLJŝŶDrosophila, we ǁĞƌĞĂďůĞƚŽŽďƚĂŝŶĂĚĚŝƟŽŶĂůŐĞŶĞƟĐƌĞĂŐĞŶƚƐ͕ŝŶĐůƵĚŝŶŐĐůĂƐƐŝĐĂů>Ž&ĂůůĞůĞƐ͕ĨŽƌƚŚĞϮ homologs of PTPRH, Ptp10D and WƚƉϰ͘/ŶŽƵƌƐĐƌĞĞŶ͕ϮŝŶĚĞƉĞŶĚĞŶƚZEŝůŝŶĞƐƚĂƌŐĞƟŶŐ

Ptp10D ƌŽďƵƐƚůLJ ĞŶŚĂŶĐĞĚ ɲͲƐLJŶƵĐůĞŝŶ ƚŽdžŝĐŝƚLJ͕ ďƵƚ ŽŶůLJ ϭ ŽĨ ƚŚĞ Ϯ ĂǀĂŝůĂďůĞ ůŝŶĞƐ ĨŽƌ WƚƉϰŵĞƚŽƵƌƚŚƌĞƐŚŽůĚĐƌŝƚĞƌŝĂ;^ƵƉƉůĞŵĞŶƚĂƌLJdĂďůĞϳͿ͘/ŶƚĞƌĞƐƟŶŐůLJƉƌŝŽƌƐƚƵĚŝĞƐŝŶ Drosophila suggest that Ptp10D and WƚƉϰ ĂƌĞ ƚŚĞ ƌĞƐƵůƚ ŽĨ Ă ŐĞŶĞ ĚƵƉůŝĐĂƟŽŶ ĞǀĞŶƚ͕

ĂŶĚƚŚĞƐĞŐĞŶĞƐƐŚŽǁĞǀŝĚĞŶĐĞŽĨƉĂƌƟĂůĨƵŶĐƟŽŶĂůƌĞĚƵŶĚĂŶĐLJ͕ŝŶĐůƵĚŝŶŐĨŽƌŶĞƌǀŽƵƐ system phenotypes.50_{ŽŶƐŝƐƚĞŶƚǁŝƚŚƚŚŝƐ͕ǁĞĨŽƵŶĚƚŚĂƚƚƌĂŶƐŚĞƚĞƌŽnjLJŐŽƐŝƚLJĨŽƌƐƚƌŽŶŐ} ;ŶƵůůͿĂůůĞůĞƐŽĨďŽƚŚŐĞŶĞƐĞŶŚĂŶĐĞĚɲͲƐLJŶƵĐůĞŝŶͲŝŶĚƵĐĞĚƌĞƟŶĂůĚĞŐĞŶĞƌĂƟŽŶ;WƚƉϰ1_, Ptp10D1_{ͬн͖ZŚϭͲ'Ăůϰͬн͖h^ͲɲͲƐLJŶƵĐůĞŝŶͬнͿ͖ǁŚĞƌĞĂƐŚĞƚĞƌŽnjLJŐŽƐŝƚLJĨŽƌĞŝƚŚĞƌĂůůĞůĞŝŶ} ŝƐŽůĂƟŽŶƐŚŽǁĞĚŶŽƐŝŐŶŝĮĐĂŶƚĞŶŚĂŶĐĞŵĞŶƚ;&ŝŐƵƌĞϱĂŶĚ^ƵƉƉůĞŵĞŶƚĂƌLJ&ŝŐƵƌĞϱͿ͘ 'ĞŶĞƟĐƌĞƉůŝĐĂƟŽŶŽĨĐĂŶĚŝĚĂƚĞWŐĞŶĞƐĨƌŽŵt^ tĞŶĞdžƚĞǀĂůƵĂƚĞĚŽƵƌϮϳŐĞŶĞĐĂŶĚŝĚĂƚĞƐŝŶĂĚĚŝƟŽŶĂůĂǀĂŝůĂďůĞŐĞŶĞƟĐĚĂƚĂƐĞƚƐŝŶĐůƵĚŝŶŐ ;ϭͿĂŶŝŶĚĞƉĞŶĚĞŶƚĞdžŽŵĞƐĞƋƵĞŶĐŝŶŐĚĂƚĂƐĞƚĨƌŽŵƚŚĞWĂƌŬŝŶƐŽŶWƌŽŐƌĞƐƐŝŽŶDĂƌŬĞƌƐ /ŶŝƟĂƟǀĞ ;WWD/Ϳ ƉƌŽũĞĐƚ͕51_{ ;ϮͿ Ă ǁŚŽůĞ ŐĞŶŽŵĞ ƐĞƋƵĞŶĐŝŶŐ ĚĂƚĂƐĞƚ ŝŶĐůƵĚŝŶŐ W ŝŶĚĞdž} ĐĂƐĞƐŽĨĂƵƚĐŚŐĞŶĞƟĐŝƐŽůĂƚĞďĞůŽŶŐŝŶŐƚŽƚŚĞ'ĞŶĞƟĐZĞƐĞĂƌĐŚŝŶ/ƐŽůĂƚĞĚWŽƉƵůĂƟŽŶ ;'Z/WͿƉƌŽŐƌĂŵ͕52_{;ϯͿĂŶŝŶĚĞƉĞŶĚĞŶƚEĞƵƌŽyĞdžŽŵĞĂƌƌĂLJĚĂƚĂƐĞƚ͕}7, 53 _{and (4) a large PD}

't^ĚĂƚĂƐĞƚ͘53_{tŝƚŚŝŶƚŚĞWWD/ĞdžŽŵĞĚĂƚĂƐĞƚ͕ŝŶĐůƵĚŝŶŐϰϲϮWĐĂƐĞƐĂŶĚϭϴϯĐŽŶƚƌŽůƐ͕}

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ĂƐŽďƐĞƌǀĞĚĨŽƌĂŶ/W'ĐĂƐĞ͘ůƚŚŽƵŐŚƚŚĞĂŐĞŽĨŽŶƐĞƚĚŝīĞƌƐϮϬLJĞĂƌƐďĞƚǁĞĞŶƚŚĞƐĞ ƚǁŽWĐĂƐĞƐ;ϰϳĂŶĚϲϴLJĞĂƌƐĨŽƌƚŚĞ/W'ĂŶĚWWD/ƉĂƟĞŶƚ͕ƌĞƐƉĞĐƟǀĞůLJͿ͕ƚŚĞLJƐŚĂƌĞ ƐŝŵŝůĂƌĂƐLJŵŵĞƚƌŝĐĐůŝŶŝĐĂůƐLJŵƉƚŽŵƐĂƚŽŶƐĞƚ͕ǁŚŝĐŚĂƌĞĐŚĂƌĂĐƚĞƌŝnjĞĚďLJƌĞƐƟŶŐƚƌĞŵŽƌ͕ ďƌĂĚLJŬŝŶĞƐŝĂĂŶĚƌŝŐŝĚŝƚLJ͘&ƵƌƚŚĞƌŵŽƌĞ͕ďŽƚŚWĐĂƐĞƐŚĂǀĞĂĨĂƚŚĞƌĚŝĂŐŶŽƐĞĚǁŝƚŚW͕ ŝŵƉůLJŝŶŐƚŚĞǀĂƌŝĂŶƚƚŽďĞŚŝŐŚůLJƉĞŶĞƚƌĂŶƚ͘tĞĞdžĐůƵĚĞĚƚŚĞƉŽƐƐŝďŝůŝƚLJƚŚĂƚƚŚĞƐĞƚǁŽ WĐĂƐĞƐŵŝŐŚƚďĞƌĞůĂƚĞĚďLJĐŽŵƉƵƟŶŐƉĂŝƌǁŝƐĞŐĞŶĞƟĐƌĞůĂƟŽŶƐŚŝƉƐ54 _{from common} ^EWƐ;D&шϬ͘ϬϭͿ͘EŽĞǀŝĚĞŶĐĞŽĨƌĞůĂƚĞĚŶĞƐƐǁĂƐŽďƐĞƌǀĞĚ;_jk = -0.0018). Based on dž͕ŽŶůLJŽŶĞ;Ϭ͘ϬϬϯйͿŽƵƚŽĨϯϮ͕ϲϰϳƵƌŽƉĞĂŶŝŶĚŝǀŝĚƵĂůƐŚĂƐƚŚŝƐƐĂŵĞŚŽŵŽnjLJŐŽƵƐ ǀĂƌŝĂŶƚ͘dŚĞŽďƐĞƌǀĂƟŽŶŽĨƚǁŽWĐĂƐĞƐ;Ϭ͘ϭϮйͿŽĨŽƵƌϭ͕ϲϭϬƐƚƵĚŝĞĚWƉĂƟĞŶƚƐ;ϭ͕ϭϰϴ /W't^ƉůƵƐϰϲϮWWD/t^ͿǁŝƚŚƚŚŝƐGPATCH2L ŵƵƚĂƟŽŶŝƐĐŽŶƐŝƐƚĞŶƚǁŝƚŚĂϰϬͲ ĨŽůĚĞŶƌŝĐŚŵĞŶƚŝŶŽƵƌWĐŽŚŽƌƚ͘dŚĞƐĞĐŽŶĚŐĞŶĞŚĂƌďŽƌŝŶŐĂŶŝĚĞŶƟĐĂů>Ž&ǀĂƌŝĂŶƚŝƐ &Dϴϯ͘dŚĞƉ͘'ϴϲyǀĂƌŝĂŶƚŝŶ&Dϴϯ͕ĚĞƚĞĐƚĞĚǁŝƚŚŝŶĂŶ/W'ƐƵďũĞĐƚǁŝƚŚƐƉŽƌĂĚŝĐ

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dŚŝƐŐĞŶĞĂůƐŽƐŚŽǁƐĂŶĂƐƐŽĐŝĂƟŽŶǁŝƚŚWŝŶƚŚĞ/W't^ĚĂƚĂƐĞƚǁŚĞŶƉĞƌĨŽƌŵŝŶŐ a similar burden analysis considering missense variants (see above, p с Ϭ͘ϬϭϲͿ͘ hƐŝŶŐ ƚŚĞEĞƵƌŽyĚĂƚĂƐĞƚ͕ǁĞĂĚĚŝƟŽŶĂůůLJĐŽŶĮƌŵĞĚƚŚĞĞŶƌŝĐŚŵĞŶƚŽĨƌĂƌĞPTPRH variants in subjects with PD (WES: p=0.034, NeuroX: pсϬ͘ϬϰϱͿ͘&ƵƌƚŚĞƌŵŽƌĞ͕VPS13C and ARSB ƐŚŽǁƐŝŐŶŝĮĐĂŶƚĂƐƐŽĐŝĂƟŽŶƐƚŽWǁŚĞŶĐŽŶƐŝĚĞƌŝŶŐƚŚĞũŽŝŶƚĞīĞĐƚŽĨĂůůǀĂƌŝĂŶƚƐ͕ďŽƚŚ common and rare (Supplementary Table 9).

>ĞǀĞƌĂŐŝŶŐĂǀĂŝůĂďůĞ/W''t^ĚĂƚĂ;ϭϯ͕ϳϬϴĐĂƐĞƐͬϵϱ͕ϮϴϮĐŽŶƚƌŽůƐͿǁĞŶĞdžƚ ĂƐƐĞƐƐĞĚĨŽƌƉŽƚĞŶƟĂůĐŽŵŵŽŶǀĂƌŝĂŶƚĂƐƐŽĐŝĂƟŽŶƐŝŐŶĂůƐ;p фϭdžϭϬ-4_{), using a 1Mb genomic}

window centered on each of the 27 candidate genes. Three loci (VPS13C, PCDHA9, and

TCHHL1ͿƐŚŽǁĞĚĞǀŝĚĞŶĐĞĐŽŶƐŝƐƚĞŶƚǁŝƚŚĂŶĂƐƐŽĐŝĂƟŽŶƉĞĂŬ;^ƵƉƉůĞŵĞŶƚĂƌLJ&ŝŐƵƌĞϲͿ͘

ŐĞŶŽŵĞǁŝĚĞƐŝŐŶŝĮĐĂŶƚĂƐƐŽĐŝĂƟŽŶĂƚƚŚĞVPS13C locus, was in fact recently reported7_͖

the best SNP (rs2414739, p сϯ͘ϱϵdžϭϬ-12_{ͿŵĂƉƐΕϭϱϬŬďĚŝƐƚĂůƚŽVPS13C. Based on local}

ƉĂƩĞƌŶƐ ŽĨ ůŝŶŬĂŐĞ ĚŝƐĞƋƵŝůŝďƌŝƵŵ ĚĞĮŶĞĚ ďLJ ,ĂƉŵĂƉ ;^ƵƉƉůĞŵĞŶƚĂƌLJ &ŝŐƵƌĞ ϲͿ͕ ŝƚ ŝƐ ƵŶůŝŬĞůLJƚŚĂƚƌƐϮϰϭϰϳϯϵŝƐĂƉƌŽdžLJĨŽƌƉ͘ϯϭϰϳyŽƌƐŝŵŝůĂƌ>Ž&ǀĂƌŝĂŶƚƐŝŶsW^ϭϯ͖however, ŝƚŵŝŐŚƚďĞƉŽƐƐŝďůĞƚŚĂƚƚŚĞ^EWŝŶŇƵĞŶĐĞƐ VPS13CĞdžƉƌĞƐƐŝŽŶďLJĂīĞĐƟŶŐƚŚĞůŽŶŐŶŽŶͲ ĐŽĚŝŶŐZEůŶĐͲsW^ϭϯͲϭ55_{ŝŶǁŚŝĐŚƚŚĞ^EWŝƐůŽĐĂƚĞĚ͘dŚĞŽƚŚĞƌϮĐĂŶĚŝĚĂƚĞĂƐƐŽĐŝĂƟŽŶ}

peaks, adjacent to PCDHA9 and TCHHL1, are considerably weaker signals (rs349129 = 1.40 džϭϬ-5_{ĂŶĚƌƐϳϱϮϵϱϯϱсϳ͘ϲϲdžϭϬ}-5_{͕ƌĞƐƉĞĐƟǀĞůLJͿĂŶĚŐŝǀĞŶƚŚĞĚŝƐƚĂŶĐĞƐ;ΕϱϬϬŬďͿŵĂŶLJ}

ŽƚŚĞƌĐĂŶĚŝĚĂƚĞŐĞŶĞƐĂƌĞƉŽƚĞŶƟĂůůLJŝŵƉůŝĐĂƚĞĚ͘

/ŶƐƵŵ͕ǁĞŝĚĞŶƟĨLJĂĚĚŝƟŽŶĂů ŐĞŶĞƟĐĞǀŝĚĞŶĐĞĐŽŶƐŝƐƚĞŶƚǁŝƚŚƌĞƉůŝĐĂƟŽŶĨŽƌ 7 genes ('Wd,Ϯ>͕&Dϴϯ͕ϯϲ͕h,Z&ϭWϭ>͕WdWZ,͕Z^and VPS13C) that were implicated by our WES analysis, of which 5 ('Wd,Ϯ>͕h,Z&ϭWϭ>͕WdWZ,͕ ARSB and

6

ĚĞŶŽƚĞƉŽƚĞŶƟĂůĨƵŶĐƟŽŶƐ͘h,Z&ϭWϭ>ǁĂƐĐŽͲĞdžƉƌĞƐƐĞĚǁŝƚŚSNCA, PINK1, GBA, and

ATP13A2ŝŶĂŶĞƚǁŽƌŬƐŝŐŶŝĮĐĂŶƚůLJĞŶƌŝĐŚĞĚĨŽƌŐĞŶĞƐǁŝƚŚƌŽůĞƐŝŶƐLJŶĂƉƟĐƚƌĂŶƐŵŝƐƐŝŽŶ

(pсϮ͘ϮϳdžϭϬ-11_{Ϳ ĂƐ ǁĞůů ĂƐ ĂƐƚƌŽĐLJƟĐ ;pсϴ͘ϭϴdžϭϬ}-8_{) and dopaminergic neuronal markers}

(pсϯ͘ϵϴdžϭϬ-46_{). GPATCH2LǁĂƐĐŽͲĞdžƉƌĞƐƐĞĚǁŝƚŚPARK7 in a network enriched for other}

neuronal genes (pсϯ͘ϰϭdžϭϬ-12_{) with cellular roles in metabolism of macromolecules}

(pсϯ͘ϴϮdžϭϬ-15_{). Lastly, PTPRHǁĂƐĂƐƐŝŐŶĞĚƚŽĂĐŽͲĞdžƉƌĞƐƐŝŽŶŵŽĚƵůĞŝŶĐůƵĚŝŶŐ&yϬϳ and}

enriched for oligodendrocyte markers (pсϴ͘ϲϵdžϭϬ-22_{). Importantly, the implicated modules}

ǁĞƌĞƉƌĞƐĞƌǀĞĚ;͘ƐƵŵŵĂƌLJхсϭϬͿŝŶƚŚĞŝŶĚĞƉĞŶĚĞŶƚ'ddžĚĂƚĂƐĞƚ͘

/^h^^/KE

We report the results from WES analysis in the largest PD cohort studied to date. Assuming a ƌĞĐĞƐƐŝǀĞŝŶŚĞƌŝƚĂŶĐĞŵŽĚĞů͕ǁĞŝĚĞŶƟĮĞĚϮϳĐĂŶĚŝĚĂƚĞŐĞŶĞƐŚĂƌďŽƌŝŶŐƌĂƌĞŚŽŵŽnjLJŐŽƵƐ ŽƌĐŽŵƉŽƵŶĚŚĞƚĞƌŽnjLJŐŽƵƐ>Ž&ǀĂƌŝĂŶƚƐ͘tŝƚŚƚŚĞĞdžĐĞƉƟŽŶŽĨARSB͕ǁĞĚŝĚŶŽƚŝĚĞŶƟĨLJ ƌĞĐƵƌƌĞŶƚƌĞĐĞƐƐŝǀĞĂůůĞůĞƐŝŶŵŽƌĞƚŚĂŶĂƐŝŶŐůĞWĐĂƐĞ͘dŚŝƐƌĞƐƵůƚͶƉŽƚĞŶƟĂůůLJĐŽŶƐŝƐƚĞŶƚ ǁŝƚŚ Ă ŚŝŐŚůLJ ŚĞƚĞƌŽŐĞŶĞŽƵƐ ŐĞŶĞƟĐ ĞƟŽůŽŐLJ ĨŽƌ WͶĐƌĞĂƚĞƐ ƐŝŐŶŝĮĐĂŶƚ ďĂƌƌŝĞƌƐ ĨŽƌ ƐƚĂƟƐƟĐĂůĐŽŶĮƌŵĂƟŽŶĂŶĚŐĞŶĞƟĐƌĞƉůŝĐĂƟŽŶŽĨŶŽǀĞůWƐƵƐĐĞƉƟďŝůŝƚLJůŽĐŝ͘ĚĚŝƟŽŶĂů ŐĞŶĞƟĐ ƐĂŵƉůĞƐ ǁĞƌĞ ŶŽƚ ĂǀĂŝůĂďůĞ ĨŽƌ ƐĞŐƌĞŐĂƟŽŶ ĂŶĂůLJƐŝƐ͕ ĂŶĚ ŐŝǀĞŶ ƚŚĞ ƌĂƌŝƚLJ ĂŶĚ ŚĞƚĞƌŽŐĞŶĞŝƚLJŽĨƚŚĞŝŵƉůŝĐĂƚĞĚĂůůĞůĞƐ͕ĚĞĮŶŝƟǀĞŚƵŵĂŶŐĞŶĞƟĐƌĞƉůŝĐĂƟŽŶǁŽƵůĚůŝŬĞůLJ ƌĞƋƵŝƌĞǀĞƌLJůĂƌŐĞƐĂŵƉůĞƐŝnjĞƐ͕ŝŶĐůƵĚŝŶŐŵĂŶLJƚŚŽƵƐĂŶĚƐŽĨWĐĂƐĞƐǁŝƚŚĞŝƚŚĞƌt^ ŽƌŐĞŶĞƌĞƐĞƋƵĞŶĐŝŶŐ͘tĞƚŚĞƌĞĨŽƌĞĐŽƵƉůĞĚŽƵƌt^ĂŶĂůLJƐĞƐǁŝƚŚĨƵŶĐƟŽŶĂůƐƚƵĚŝĞƐ ŝŶďŽƚŚŵĂŵŵĂůŝĂŶĐĞůůƐĂŶĚĞdžƉĞƌŝŵĞŶƚĂůĂŶŝŵĂůŵŽĚĞůƐ͕ŝŶĐůƵĚŝŶŐDrosophila and C. elegans͕ŝŶŽƌĚĞƌƚŽƉƌŝŽƌŝƟnjĞŐĞŶĞƐĨŽƌĨƵƚƵƌĞƐƚƵĚLJ͘KƵƌƌĞƐƵůƚƐŚŝŐŚůŝŐŚƚϭϱŽƵƚŽĨƚŚĞ

6

ůŽĐŝĐĂŶĚŝĚĂƚĞƐĨŽƌĨƵƌƚŚĞƌŝŶǀĞƐƟŐĂƟŽŶ͕ĂŶĚŝůůƵƐƚƌĂƚĞĂƉŽǁĞƌĨƵů͕ŝŶƚĞŐƌĂƟǀĞĚŝƐĐŽǀĞƌLJ strategy for future, large-scale PD genomic studies.

Mitochondrial mechanisms have been strongly implicated in PD risk and pathogenesis.28, 30_{ &ŽůůŽǁŝŶŐ ƐŚZEͲŵĞĚŝĂƚĞĚ ŬŶŽĐŬĚŽǁŶ͕ ϭϱ ĐĂŶĚŝĚĂƚĞ ƌĞĐĞƐƐŝǀĞ}

ůŽĐŝ ŝĚĞŶƟĮĞĚ ŝŶ ŽƵƌ t^ ĚĂƚĂƐĞƚ ƐŚŽǁĞĚ ĞīĞĐƚƐ ŽŶ ŵŝƚŽĐŚŽŶĚƌŝĂů ŵŽƌƉŚŽůŽŐLJ ĂŶĚ WĂƌŬŝŶƚƌĂŶƐůŽĐĂƟŽŶƚŽŵŝƚŽĐŚŽŶĚƌŝĂŝŶĐĞůůĐƵůƚƵƌĞ͘tĞĨŽĐƵƐŽƵƌŝŶŝƟĂůĚŝƐĐƵƐƐŝŽŶŽŶϯ genes, 'Wd,Ϯ>͕h,Z&ϭWϭ>͕and VPS13C͕ĨŽƌǁŚŝĐŚǁĞĚŝƐĐŽǀĞƌĞĚĂĚĚŝƟŽŶĂůŐĞŶĞƟĐ ĞǀŝĚĞŶĐĞ ĐŽŶƐŝƐƚĞŶƚ ǁŝƚŚ ƌĞƉůŝĐĂƟŽŶ ŝŶ ŝŶĚĞƉĞŶĚĞŶƚ ĐŽŚŽƌƚƐ͘ /Ŷ ƚŚĞ /W' ĐŽŚŽƌƚ͕ Ă ƐŝŶŐůĞWĐĂƐĞǁĂƐŝĚĞŶƟĮĞĚǁŝƚŚĂŚŽŵŽnjLJŐŽƵƐƐƚŽƉŐĂŝŶǀĂƌŝĂŶƚ;Ɖ͘ZϯϲϮyͿŝŶGPATCH2L, ĂŶĚĂƐĞĐŽŶĚŝŶĚŝǀŝĚƵĂůǁŝƚŚƚŚĞŝĚĞŶƟĐĂů͕ƌĂƌĞŐĞŶŽƚLJƉĞǁĂƐĚŝƐĐŽǀĞƌĞĚŝŶWWD/͘dŚŝƐ ǀĂƌŝĂŶƚŝƐƌĞƉŽƌƚĞĚǁŝƚŚĂůŽǁĨƌĞƋƵĞŶĐLJŽĨϬ͘ϬϬϯйŝŶdž͘ůƚŚŽƵŐŚŵŝŶŝŵĂůĐůŝŶŝĐĂů Žƌ ĚĞŵŽŐƌĂƉŚŝĐ ŝŶĨŽƌŵĂƟŽŶ ŝƐ ĂǀĂŝůĂďůĞ ǁŝƚŚŝŶ dž͕ ƚŚŝƐ ĮŶĚŝŶŐ ŝƐ ĐŽŵƉĂƟďůĞ ǁŝƚŚ ƉŽƉƵůĂƟŽŶ ƉƌĞǀĂůĞŶĐĞ ĞƐƟŵĂƚĞƐ ĨŽƌ W͘20_{ EĞǀĞƌƚĞŚĞůĞƐƐ͕ ŐĞŶŽƚLJƉŝŶŐ ŽĨ Ɖ͘ZϯϲϮy ŝŶ} ĂĚĚŝƟŽŶĂůůĂƌŐĞWĐĂƐĞĂŶĚĐŽŶƚƌŽůĐŽŚŽƌƚƐǁŝůůďĞƌĞƋƵŝƌĞĚƚŽĚĞĮŶŝƟǀĞůLJĞƐƚĂďůŝƐŚĂŶ ĂƐƐŽĐŝĂƟŽŶǁŝƚŚWƐƵƐĐĞƉƟďŝůŝƚLJ͘ GPATCH2L knockdown both increased mitochondrial ƌŽƵŶĚŶĞƐƐ ĂŶĚ ŝŵƉĂŝƌĞĚ WĂƌŬŝŶ ƚƌĂŶƐůŽĐĂƟŽŶ͘ dŚĞ ĞŶĐŽĚĞĚ ƉƌŽƚĞŝŶ͕ 'Wd,Ϯ>͕ ǁŚŝĐŚ ŚĂƐŶŽƚƉƌĞǀŝŽƵƐůLJďĞĞŶƐƚƵĚŝĞĚ͕ĐŽŶƚĂŝŶƐĂŐůLJĐŝŶĞͲƌŝĐŚZEͲďŝŶĚŝŶŐŵŽƟĨ͕ƚŚĞ͞'ͲƉĂƚĐŚ͟ domain.57 _{GPATCH2, a paralog of GPATCH2L͕ŝƐƵƉƌĞŐƵůĂƚĞĚŝŶĐĂŶĐĞƌĐĞůůƐ͕ůŽĐĂůŝnjĞƐƚŽƚŚĞ}

ŶƵĐůĞƵƐǁŚĞƌĞŝƚŝŶƚĞƌĂĐƚƐǁŝƚŚZEͲƉƌŽĐĞƐƐŝŶŐŵĂĐŚŝŶĞƌLJ͕ĂŶĚŵĂŶŝƉƵůĂƟŽŶŝŶĐƵůƚƵƌĞ ĂůƚĞƌƐĐĞůůƉƌŽůŝĨĞƌĂƟŽŶ͘58, 59 _{Notably, GPATCH2L is non-conserved in either the C. elegans or} Drosophila genomes, precluding study of this candidate in these models. While our results

using cellular assays implicate GPATCH2L in mitochondrial quality control mechanisms, ĨƵƌƚŚĞƌĨŽůůŽǁͲƵƉƐƚƵĚŝĞƐŝŶŵĂŵŵĂůŝĂŶŵŽĚĞůƐLJƐƚĞŵƐǁŝůůďĞŶĞĞĚĞĚƚŽĐŽŶĮƌŵĂƌŽůĞŝŶ PD pathogenesis.

Another promising gene, h,Z&ϭWϭ>, ŚĂƌďŽƌĞĚĂŚŽŵŽnjLJŐŽƵƐƐƚŽƉŐĂŝŶǀĂƌŝĂŶƚ ;Đ͘ϰϭϮϲхd͕Ɖ͘<ϭϯϳϲyͿŝŶĂƐŝŶŐůĞ/W'ĐĂƐĞ͘dŚŝƐŝƐĂŶŽǀĞůǀĂƌŝĂŶƚ͕ďĂƐĞĚŽŶŝƚƐĂďƐĞŶĐĞ ĨƌŽŵƚŚĞdžĐŽŚŽƌƚ͘ĚĚŝƟŽŶĂůƐƵƉƉŽƌƚĨŽƌh,Z&ϭWϭ>as a ďŽŶĂĮĚĞ PD locus, comes ĨƌŽŵĐŽŵƉůĞŵĞŶƚĂƌLJĂŶĂůLJƐĞƐŝŶďŽƚŚƚŚĞ/W't^ĂŶĚEĞƵƌŽyĚĂƚĂƐĞƚƐ͕ĚŽĐƵŵĞŶƟŶŐ ĂďƵƌĚĞŶŽĨƌĂƌĞŵŝƐƐĞŶƐĞĂŶĚ>Ž&ǀĂƌŝĂŶƚƐŝŶĂƐƐŽĐŝĂƟŽŶǁŝƚŚĚŝƐĞĂƐĞƌŝƐŬ͘/ŶƚŚĞh<͕

h,Z&ϭWϭ>ǁĂƐĂƐƐŽĐŝĂƚĞĚǁŝƚŚĂƐƵďƐƚĂŶƟĂŶŝŐƌĂĐŽͲĞdžƉƌĞƐƐŝŽŶŵŽĚƵůĞŝŶĐůƵĚŝŶŐďŽƚŚ SNCA and PINK1͕ ƌĞŝŶĨŽƌĐŝŶŐ ƉŽƚĞŶƟĂů ůŝŶŬƐ ǁŝƚŚ ĞƐƚĂďůŝƐŚĞĚ W ŐĞŶĞƟĐ ŵĞĐŚĂŶŝƐŵƐ͘

Indeed, h,Z&ϭWϭ> knockdown cause sharply reduced mitochondrial numbers and ĂůƚĞƌĞĚ ŵŽƌƉŚŽůŽŐLJ͘ /ŶƚĞƌĞƐƟŶŐůLJ͕ h,Z&ϭWϭ> encodes a protein bearing an amino terminal homologous to yeast VPS13, and studies in cell culture provide support for a role ŝŶƌĞƚƌŽŐƌĂĚĞƚƌĂŶƐƉŽƌƚĨƌŽŵƚŚĞĞŶĚŽƐŽŵĞƚŽƚŚĞƚƌĂŶƐͲ'ŽůŐŝŶĞƚǁŽƌŬ͘60

6

WƐƵƐĐĞƉƟďŝůŝƚLJǀĂƌŝĂŶƚƐďĂƐĞĚŽŶ't^͘7_{KƵƌĮŶĚŝŶŐƐŽĨĂƉŽƚĞŶƟĂůŵŝƚŽĐŚŽŶĚƌŝĂůƌŽůĞ}

for VPS13C͕ ĂŐƌĞĞ ǁŝƚŚ ƚŚŽƐĞ ŽĨ >ĞƐĂŐĞ Ğƚ Ăů͕͘ ǁŚŽ ĂĚĚŝƟŽŶĂůůLJ ƌĞƉŽƌƚĞĚ ƚŚĂƚ sW^ϭϯ ůŽĐĂůŝnjĞƐƚŽƚŚĞŽƵƚĞƌŵĞŵďƌĂŶĞŽĨŵŝƚŽĐŚŽŶĚƌŝĂĂŶĚ>Ž&ǁĂƐĂƐƐŽĐŝĂƚĞĚǁŝƚŚƌĞĚƵĐĞĚ ŵŝƚŽĐŚŽŶĚƌŝĂů ŵĞŵďƌĂŶĞ ƉŽƚĞŶƟĂů͕ ĨƌĂŐŵĞŶƚĂƟŽŶ͕ ĂŶĚ ŝŶĐƌĞĂƐĞĚ WĂƌŬŝŶͲĚĞƉĞŶĚĞŶƚ mitophagy. Importantly, VPS35, which causes autosomal dominant, late-onset PD, is ƐŝŵŝůĂƌůLJ ŝŶǀŽůǀĞĚ ŝŶ ĞŶĚŽƐŽŵĂů ƚƌĂĸĐŬŝŶŐ͕61 _{and has also recently been implicated}

in mitochondrial dynamics,62_{ ŝŶĐůƵĚŝŶŐ ŝŶƚĞƌĂĐƟŽŶƐ ǁŝƚŚ WĂƌŬŝŶ͘}63 _{Like h,Z&ϭWϭ>͕} VPS13C and GPATCH2L ĂƌĞĞdžƉƌĞƐƐĞĚŝŶƚŚĞďƌĂŝŶ͕ŝŶĐůƵĚŝŶŐǁŝƚŚŝŶƚŚĞƐƵďƐƚĂŶƟĂŶŝŐƌĂ͖

ŚŽǁĞǀĞƌ͕ ĂĚĚŝƟŽŶĂů ǁŽƌŬ ǁŝůů ďĞ ŶĞĞĚĞĚ ƚŽ ĚĞĮŶĞ ƚŚĞŝƌ ĨƵŶĐƟŽŶƐ͕ ŝŶĐůƵĚŝŶŐ ƉŽƚĞŶƟĂů ŝŶƚĞƌĂĐƟŽŶƐǁŝƚŚŽƚŚĞƌĞƐƚĂďůŝƐŚĞĚĚŝƐĞĂƐĞŐĞŶĞƐ;e.g. VPS35, parkin) and requirements for mitochondrial maintenance.

ĂƐĞĚŽŶĨƵŶĐƟŽŶĂůƐĐƌĞĞŶŝŶŐŝŶDrosophila, 4 candidate genes from our WES ĂŶĂůLJƐĞƐǁĞƌĞŝŵƉůŝĐĂƚĞĚĂƐ>Ž&ĞŶŚĂŶĐĞƌƐŽĨɲͲƐLJŶƵĐůĞŝŶŶĞƵƌŽƚŽdžŝĐŝƚLJ͕ǁŚŝĐŚĂůƐŽŚĂƐ a central role in PD pathogenesis. We discuss the 3 genes (VPS13C, PTPRH, and ARSB) ǁŚĞƌĞĂĚĚŝƟŽŶĂůŚƵŵĂŶŐĞŶĞƟĐĞǀŝĚĞŶĐĞƐƵƉƉŽƌƚƐƌĞƉůŝĐĂƟŽŶ͘/ŶƚĞƌĞƐƟŶŐůLJ͕ďĞƐŝĚĞƐŝƚƐ ƌĞƋƵŝƌĞŵĞŶƚ ĨŽƌ ŵŝƚŽĐŚŽŶĚƌŝĂů ŵĂŝŶƚĞŶĂŶĐĞ͕ ZEŝͲŵĞĚŝĂƚĞĚ ŬŶŽĐŬĚŽǁŶ ŽĨ Drosophila

Vps13 ĞŶŚĂŶĐĞĚ ɲͲƐLJŶƵĐůĞŝŶ ƚŽdžŝĐŝƚLJ͘ /Ŷ ƚŚĞ ƐŝŶŐůĞ ƌĞƉŽƌƚĞĚ VPS13C PD case with a

ĐŽŵƉůĞƚĞĚĂƵƚŽƉƐLJ͕ŶĞƵƌŽƉĂƚŚŽůŽŐŝĐĂůĮŶĚŝŶŐƐŝŶĐůƵĚĞĚĂďƵŶĚĂŶƚɲͲƐLJŶƵĐůĞŝŶĂŐŐƌĞŐĂƚĞƐ ŝŶ ďŽƚŚ ƚŚĞ ďƌĂŝŶƐƚĞŵ ĂŶĚ ĐŽƌƚĞdž͘21 _{Thus, VPS13C ĂŶĚ ĂƐƐŽĐŝĂƚĞĚ ĞŶĚŽƐŽŵĂů ƐŽƌƟŶŐ}

pathways (including VPS35) may represent a point of convergence for mitochondrial and ɲͲƐLJŶƵĐůĞŝŶͲŵĞĚŝĂƚĞĚWŵĞĐŚĂŶŝƐŵƐ͘ŽŶƐŝƐƚĞŶƚǁŝƚŚƚŚŝƐ͕ĞǀŝĚĞŶĐĞĨŽƌƚŚĞŝŵƉĂĐƚŽĨ ɲͲƐLJŶƵĐůĞŝŶ ƚŽdžŝĐŝƚLJŽŶŵŝƚŽĐŚŽŶĚƌŝĂŚĂƐƌĞĐĞŶƚůLJĞŵĞƌŐĞĚ͕28 _{including from studies in}

mammals.64 /Ŷ ƚŚĞ /W' t^ ĐŽŚŽƌƚ͕ Ă ƐŝŶŐůĞ W ĐĂƐĞ ǁĂƐ ĚŝƐĐŽǀĞƌĞĚ ǁŝƚŚ ĐŽŵƉŽƵŶĚ ŚĞƚĞƌŽnjLJŐŽƵƐ>Ž&ǀĂƌŝĂŶƚƐŝŶPTPRH;Đ͘Ϯϲϱϵхd͕Ɖ͘YϴϴϳyĂŶĚĐ͘ϱϵϴ'хd͕Ɖ͘ϮϬϬyͿ͘ŽƚŚ ǀĂƌŝĂŶƚƐǁĞƌĞĂůƐŽŽďƐĞƌǀĞĚĂƚůŽǁĨƌĞƋƵĞŶĐŝĞƐŝŶƚŚĞdžĚĂƚĂďĂƐĞ;Ϭ͘ϬϯϵйĂŶĚϬ͘ϬϬϯй͕ ƌĞƐƉĞĐƟǀĞůLJͿ͖ŚŽǁĞǀĞƌ͕ƚŚĞLJĞĂĐŚŵĞƚŽƵƌƉƌĞͲƐƉĞĐŝĮĞĚƚŚƌĞƐŚŽůĚŽĨфϭйďĂƐĞĚŽŶƚŚĞ ƉŽƉƵůĂƟŽŶƉƌĞǀĂůĞŶĐĞŽĨW͘ŶĐŽĚŝŶŐĂƌĞĐĞƉƚŽƌƉƌŽƚĞŝŶƚLJƌŽƐŝŶĞƉŚŽƐƉŚĂƚĂƐĞ͕PTPRH (also called SAP-1ͿǁĂƐĮƌƐƚĚŝƐĐŽǀĞƌĞĚĨŽƌŝƚƐƉŽƚĞŶƟĂůĂƐƐŽĐŝĂƟŽŶǁŝƚŚŐĂƐƚƌŽŝŶƚĞƐƟŶĂů cancers65, 66_{ĂŶĚƌĞŵĂŝŶƐƉŽŽƌůLJƐƚƵĚŝĞĚŝŶƚŚĞŶĞƌǀŽƵƐƐLJƐƚĞŵĐŽŶƚĞdžƚ͘/ŶƐƚƵĚŝĞƐŽĨďŽƚŚ}

vertebrates and invertebrates, receptor protein tyrosine phosphatases have been strongly implicated as key neural cell adhesion receptors, with roles in neurodevelopment and ƐLJŶĂƉƟĐĨƵŶĐƟŽŶ͕ĂŶĚŽƚŚĞƌŵĞŵďĞƌƐŽĨƚŚŝƐĨĂŵŝůLJŚĂǀĞďĞĞŶŝŵƉůŝĐĂƚĞĚŝŶŶƵŵĞƌŽƵƐ neuropsychiatric disorders.67 _{In Drosophila͕ZEŝͲŵĞĚŝĂƚĞĚŬŶŽĐŬĚŽǁŶŽĨƚŚĞĐŽŶƐĞƌǀĞĚ} PTPRH ortholog, Ptp10D͕ĞŶŚĂŶĐĞĚɲͲƐLJŶƵĐůĞŝŶͲƚƌŝŐŐĞƌĞĚƌĞƟŶĂůĚĞŐĞŶĞƌĂƟŽŶ͖ďƵƚǁĂƐ

ŶŽƚ ĂƐƐŽĐŝĂƚĞĚ ǁŝƚŚ ƐƵďƐƚĂŶƟĂů ŶĞƵƌŽƚŽdžŝĐŝƚLJ ŝŶĚĞƉĞŶĚĞŶƚ ŽĨ ɲͲƐLJŶƵĐůĞŝŶ ĞdžƉƌĞƐƐŝŽŶ͘

Ptp10D ŵƵƚĂŶƚ ŇŝĞƐ ĂƌĞ ĂůƐŽ ǀŝĂďůĞ ĂŶĚ ĨĞƌƟůĞ ďƵƚ ĚĞŵŽŶƐƚƌĂƚĞ ůŽŶŐͲƚĞƌŵ ŵĞŵŽƌLJ

ĚĞĮĐŝƚƐŝŶďĞŚĂǀŝŽƌĂůĂƐƐĂLJƐ͘68 _{More recent studies further implicated Ptp10D in}

6

ĐŽŶƐŝƐƚĞŶƚǁŝƚŚŽƵƌĮŶĚŝŶŐƐƚŚĂƚŚƵŵĂŶPTPRH ƉĂƌƟĐŝƉĂƚĞƐŝŶĂƐƵďƐƚĂŶƟĂŶŝŐƌĂŐĞŶĞ ĐŽͲĞdžƉƌĞƐƐŝŽŶ ŶĞƚǁŽƌŬ ƐƚƌŽŶŐůLJ ĞŶƌŝĐŚĞĚ ĨŽƌ ŽůŝŐŽĚĞŶĚƌŽĐLJƚĞ ŵĂƌŬĞƌƐ͘ ĞƐŝĚĞƐ ŽƵƌ ĚŝƐĐŽǀĞƌLJŽĨŚŽŵŽnjLJŐŽƵƐ>Ž&ŝŶPTPRH͕ĨƵƌƚŚĞƌĂŶĂůLJƐĞƐŽĨƚŚĞ/W't^ĚĂƚĂƐĞƚ͕ĂŶĚ ƚŚĞƐƵďƐƚĂŶƟĂůůLJůĂƌŐĞƌ͕ŝŶĚĞƉĞŶĚĞŶƚEĞƵƌŽyĐŽŚŽƌƚ͕ŝŵƉůŝĐĂƚĞĂďƵƌĚĞŶŽĨƌĂƌĞǀĂƌŝĂŶƚƐ ĂƚƚŚŝƐůŽĐƵƐŝŶĂƐƐŽĐŝĂƟŽŶǁŝƚŚWƐƵƐĐĞƉƟďŝůŝƚLJ͘ ɲͲƐLJŶƵĐůĞŝŶͲŝŶĚƵĐĞĚ ŶĞƵƌŽĚĞŐĞŶĞƌĂƟŽŶ ǁĂƐ ĂůƐŽ ĞŶŚĂŶĐĞĚ ďLJ ŬŶŽĐŬĚŽǁŶ ŽĨ

CG32191, a Drosophila homolog of ARSB͘ZEŝƚƌĂŶƐŐĞŶŝĐůŝŶĞƐƚĂƌŐĞƟŶŐϯŽƚŚĞƌĐŽŶƐĞƌǀĞĚ

ŇLJARSB ŚŽŵŽůŽŐƐƐŚŽǁĞĚĐŽŶƐŝƐƚĞŶƚŝŶƚĞƌĂĐƟŽŶƐǁŝƚŚɲͲƐLJŶƵĐůĞŝŶ;^ƵƉƉůĞŵĞŶƚĂƌLJdĂďůĞ ϲĂŶĚϳͿ͘/ŶƚŚĞ/W'ĐŽŚŽƌƚ͕ǁĞĚŝƐĐŽǀĞƌĞĚϰWĐĂƐĞƐŚŽŵŽnjLJŐŽƵƐĨŽƌĂǀĂƌŝĂŶƚƉƌĞĚŝĐƚĞĚ ƚŽ ĚŝƐƌƵƉƚ ƐƉůŝĐŝŶŐ ŽĨ ĞdžŽŶƐ ϭ ĂŶĚ Ϯ ŝŶ ARSB͘ ůƚŚŽƵŐŚ ƚŚĞ ŝĚĞŶƟĮĞĚ ǀĂƌŝĂŶƚ ŚĂƐ ŶŽƚ ƉƌĞǀŝŽƵƐůLJďĞĞŶĚŽĐƵŵĞŶƚĞĚŝŶdž͕ǁĞŝĚĞŶƟĮĞĚĂƐŝŶŐůĞ/W'ĐŽŶƚƌŽůŚŽŵŽnjLJŐŽƚĞ͘ ĚĚŝƟŽŶĂůĞǀŝĚĞŶĐĞƐƵƉƉŽƌƟŶŐĂƐƐŽĐŝĂƟŽŶŽĨƚŚĞARSBŐĞŶĞǁŝƚŚWƐƵƐĐĞƉƟďŝůŝƚLJĐŽŵĞƐ from burden analysis in the independent NeuroX cohort. The surprisingly common ARSB ƐƉůŝĐŝŶŐǀĂƌŝĂŶƚ;ƌƐϭϯϴϮϳϵϬϮϬ͕D&сϬ͘ϬϲϱŝŶ/W'ͿŝƐĂƐŝŶŐůĞŶƵĐůĞŽƟĚĞŝŶƐĞƌƟŽŶĂůůĞůĞ ǁŝƚŚŝŶ Ă ƉŽůLJͲ ƌĞƉĞĂƚ͕ ǁŚŝĐŚ ǁĞ ƐƉĞĐƵůĂƚĞ ŵŝŐŚƚ ůĞĂĚ ƚŽ ŝŶĞĸĐŝĞŶƚ ĐĂƉƚƵƌĞ ŝŶ ƉƌŝŽƌ t^ĂŶĚƉŽƐƐŝďůLJĞdžƉůĂŝŶƚŚĞĂďƐĞŶĐĞŽĨƚŚŝƐǀĂƌŝĂŶƚĨƌŽŵdžĂŶĚƚŚĞϭϬϬϬ'ĞŶŽŵĞƐ ƉƌŽũĞĐƚƌĞĨĞƌĞŶĐĞ͘ůůϰWĐĂƐĞƐŝŶŽƵƌĚĂƚĂǁŝƚŚƚŚĞŚŽŵŽnjLJŐŽƵƐARSB splicing variant ǁĞƌĞ ĐŽŶĮƌŵĞĚ ďLJ ^ĂŶŐĞƌ ƐĞƋƵĞŶĐŝŶŐ͘ /ŶƚƌŝŐƵŝŶŐůLJ, ŵƵƚĂƟŽŶƐ ŝŶ ARSB, encoding the ůLJƐŽƐŽŵĂůĞŶnjLJŵĞƌLJůƐƵůĨĂƚĂƐĞ͕ĂƌĞĂƐƐŽĐŝĂƚĞĚǁŝƚŚƚŚĞƌĞĐĞƐƐŝǀĞůLJƐŽƐŽŵĞĚŝƐŽƌĚĞƌ͕ DƵĐŽƉŽůLJƐĂĐĐŚĂƌŝĚŽƐŝƐƚLJƉĞs/;DW^s/͕ĂůƐŽĐĂůůĞĚDĂƌŽƚĞĂƵdžͲ>ĂŵLJƐLJŶĚƌŽŵĞͿ͕ŝŶǁŚŝĐŚ the glycosaminoglycan, dermatan sulfate, accumulates causing skeletal dysplasia and ŽƚŚĞƌ ŚĞƚĞƌŽŐĞŶĞŽƵƐ ŵĂŶŝĨĞƐƚĂƟŽŶƐ͘70_{ ^ƵďƐƚƌĂƚĞ ĂĐĐƵŵƵůĂƟŽŶ ĂŶĚ ĂƐƐŽĐŝĂƚĞĚ ĐĞůůƵůĂƌ}

stress has been reported to induce markers of impaired autophagy and mitochondrial ĚLJƐĨƵŶĐƟŽŶ ŝŶ ARSB ĚĞĮĐŝĞŶƚ ĮďƌŽďůĂƐƚƐ ĨƌŽŵ DW^ s/ ƉĂƟĞŶƚƐ͕ ĂƐ ŝŶ ŽƚŚĞƌ ůLJƐŽƐŽŵĂů disorders.71, 72_{ /ŵƉŽƌƚĂŶƚůLJ͕ DĂƌŽƚĞĂƵdžͲ>ĂŵLJ ĐĂŶ ďĞ ĐŚĂƌĂĐƚĞƌŝnjĞĚ ďLJ ŵŝŶŝŵĂů Žƌ ĞǀĞŶ}

absent clinical signs, leading to incidental discovery or diagnosis in adulthood, and such ŵŝůĚ ƉŚĞŶŽƚLJƉĞƐ ŚĂǀĞ ďĞĞŶ ƐƵŐŐĞƐƚĞĚ ƚŽ ĂĐĐŽŵƉĂŶLJ ƉĂƌƟĂů >Ž& ǁŝƚŚ ƉƌĞƐĞƌǀĞĚ ůŽǁͲ ůĞǀĞůZ^ĞŶnjLJŵĂƟĐĂĐƟǀŝƚLJ͘70, 73, 74_{^ŝŵŝůĂƌŐĞŶŽƚLJƉĞͲƉŚĞŶŽƚLJƉĞƌĞůĂƟŽŶƐŚŝƉƐŚĂǀĞďĞĞŶ}

ĚŽĐƵŵĞŶƚĞĚĨŽƌŽƚŚĞƌůLJƐŽƐŽŵĂůͲƐƚŽƌĂŐĞĚŝƐŽƌĚĞƌƐ͕ŝŶĐůƵĚŝŶŐ'ĂƵĐŚĞƌĚŝƐĞĂƐĞ͕ǁŚŝĐŚŚĂƐ established links with PD risk.75, 76_{tŚŝůĞĂĨƵůůĂĐĐŽƵŶƟŶŐŝƐŽƵƚƐŝĚĞƚŚĞƐĐŽƉĞŽĨƚŚŝƐƐƚƵĚLJ͕}

ĂƚůĞĂƐƚϭŽĨƚŚĞϯ/W'ĐĂƐĞƐĨŽƌǁŚŝĐŚƌĞĐŽƌĚƐǁĞƌĞĂǀĂŝůĂďůĞƌĞǀĞĂůĞĚĐůŝŶŝĐĂůĨĞĂƚƵƌĞƐ ƉŽƚĞŶƟĂůůLJŽǀĞƌůĂƉƉŝŶŐǁŝƚŚDW^s/͘

6

ƚŚĞ ƉŽƐƐŝďŝůŝƚLJ ŽĨ ĚŽŵŝŶĂŶƚůLJͲĂĐƟŶŐ ĂůůĞůĞƐ͖ ŚŽǁĞǀĞƌ͕ ƚŚŝƐ ƐƵďƐƚĂŶƟĂůůLJ ŝŶĐƌĞĂƐĞƐ ƚŚĞ ŶƵŵďĞƌŽĨǀĂƌŝĂŶƚƐƚŽĐŽŶƐŝĚĞƌĂŶĚĂůƐŽƉŽƚĞŶƟĂůůLJĐŽŵƉůŝĐĂƚĞƐĨƵŶĐƟŽŶĂůƐƚƵĚŝĞƐ;ŝ͘Ğ͘ ĐŽŵƉĂƌĞĚ ǁŝƚŚ ůŽƐƐͲŽĨͲĨƵŶĐƟŽŶ ƐĐƌĞĞŶŝŶŐ ƵƐŝŶŐ ZEͲŝŶƚĞƌĨĞƌĞŶĐĞͿ͘ KƵƌ ƐƚƵĚLJ ĚĞƐŝŐŶ ĞdžĐůƵĚĞĚĐŽŶƐŝĚĞƌĂƟŽŶŽĨŵĂŶLJŶŽŶͲƐLJŶŽŶLJŵŽƵƐǀĂƌŝĂŶƚƐƚŚĂƚĐŽƵůĚƉŽƚĞŶƟĂůůLJĐĂƵƐĞ ůŽƐƐͲ;ŽƌŐĂŝŶͲͿŽĨŐĞŶĞĨƵŶĐƟŽŶ͕ĂůŽŶŐǁŝƚŚĐĞƌƚĂŝŶŶŽŶͲƚƌƵŶĐĂƟŶŐ͕ĨƌĂŵĞƐŚŝŌŝŶŐĂůůĞůĞƐ ;ƐĞĞDĞƚŚŽĚƐͿ͘ǀĞŶǁŝƚŚĨĂŝƌůLJƐƚƌŝŶŐĞŶƚĐƌŝƚĞƌŝĂĨŽƌǀĂƌŝĂŶƚĮůƚĞƌŝŶŐĂŶĚƚŚĞĂƐƐƵŵƉƟŽŶ ŽĨ ƌĞĐĞƐƐŝǀĞ ŝŶŚĞƌŝƚĂŶĐĞ͕ ǁĞ ĨŽƵŶĚ ĞǀŝĚĞŶĐĞ ĨŽƌ ƐƵďƐƚĂŶƟĂů ĞƟŽůŽŐŝĐ ŚĞƚĞƌŽŐĞŶĞŝƚLJ͘ /ŵƉƌŽǀĞĚĐŽŶĮĚĞŶĐĞĨŽƌĚŝƐĐŽǀĞƌLJŽĨWĐĂƵƐĂůǀĂƌŝĂŶƚƐǁŝůůůŝŬĞůLJĐŽŵĞĨƌŽŵWt^ ĐŽŚŽƌƚƐǁŝƚŚƐŝŐŶŝĮĐĂŶƚůLJĞŶŚĂŶĐĞĚƐĂŵƉůĞƐŝnjĞƐ͕ĂƐǁĞůůĂƐŝŶĐƌĞĂƐĞĚŶƵŵďĞƌƐŽĨĂĚƵůƚ ĐŽŶƚƌŽůƐ͕ ŝŶĐůƵĚŝŶŐ ƚŚŽƐĞ ǁŝƚŚ ĐĂƌĞĨƵů ŶĞƵƌŽůŽŐŝĐĂů ĂƐƐĞƐƐŵĞŶƚƐ ƚŽ ĞdžĐůƵĚĞ ŵŝůĚ W ƐLJŵƉƚŽŵƐ͘/ŶĚĞĞĚ͕ǁŚŝůĞŵŽƐƚŽĨƚŚĞǀĂƌŝĂŶƚƐŝŵƉůŝĐĂƚĞĚďLJƚŚĞ/W't^ĐŽŚŽƌƚǁĞƌĞ ƌĞƉƌĞƐĞŶƚĞĚ Ăƚ ůŽǁ ĨƌĞƋƵĞŶĐŝĞƐ ǁŝƚŚŝŶ ƚŚĞ ůĂƌŐĞƐƚ ĂǀĂŝůĂďůĞ ƉƵďůŝĐ ĚĂƚĂďĂƐĞ͕ dž77, 78_͖ ŚŽǁĞǀĞƌ͕ǁĞŚĂǀĞŶŽŝŶĨŽƌŵĂƟŽŶĂďŽƵƚƉŽƚĞŶƟĂůWŵĂŶŝĨĞƐƚĂƟŽŶƐŝŶƐƵĐŚŝŶĚŝǀŝĚƵĂůƐŽƌ even subject age.

^ŝŶĐĞŶŽƐŝŶŐůĞĐĞůůƵůĂƌŽƌĂŶŝŵĂůĞdžƉĞƌŝŵĞŶƚĂůŵŽĚĞůŝƐĞdžƉĞĐƚĞĚƚŽƵŶŝǀĞƌƐĂůůLJ ƌĞĐĂƉŝƚƵůĂƚĞĂůůƉŽƚĞŶƟĂůĨĂĐĞƚƐŽĨĚŝƐĞĂƐĞďŝŽůŽŐLJ͕ǁĞŶŽƚĞƚŚĂƚƚŚĞĞŵƉůŽLJĞĚĨƵŶĐƟŽŶĂů ƐĐƌĞĞŶŝŶŐĂƐƐĂLJƐĂƌĞƉŽƚĞŶƟĂůůLJůŝĂďůĞƚŽĨĂůƐĞŶĞŐĂƟǀĞŽƌƉŽƐŝƟǀĞĮŶĚŝŶŐƐ͘/ŵƉŽƌƚĂŶƚůLJ͕ ĞdžƉĞƌŝŵĞŶƚĂů ĞǀŝĚĞŶĐĞ ŽĨ Ă ŐĞŶĞƟĐ ŝŶƚĞƌĂĐƟŽŶ ǁŝƚŚ ĞŝƚŚĞƌ ŵŝƚŽĐŚŽŶĚƌŝĂů ĚLJŶĂŵŝĐƐ Žƌ ɲͲƐLJŶƵĐůĞŝŶŵĞĚŝĂƚĞĚŶĞƵƌŽŶĂůŝŶũƵƌLJŝŶŽƵƌƐĐƌĞĞŶŝŶŐĂƐƐĂLJƐĐĂŶŶŽƚŝŶŝƐŽůĂƟŽŶĐŽŶĮƌŵ ĂƌŽůĞŝŶĚŝƐĞĂƐĞĐĂƵƐĂƟŽŶ͕ďƵƚƌĂƚŚĞƌƐĞƌǀĞƐƚŽƉƌŝŽƌŝƟnjĞŐĞŶĞƐĨŽƌĨƵƚƵƌĞŝŶǀĞƐƟŐĂƟŽŶ͘ KƵƚ ŽĨ ƚŚĞ Ϯϳ ĐĂŶĚŝĚĂƚĞ ŐĞŶĞƐ ŝŵƉůŝĐĂƚĞĚ ŝŶ ƚŚĞ /W' t^ ĚŝƐĐŽǀĞƌLJ ĂŶĂůLJƐŝƐ͕ ϭϰ ǁĞƌĞŝŶƐƵĸĐŝĞŶƚůLJĐŽŶƐĞƌǀĞĚĨŽƌĨŽůůŽǁͲƵƉŝŶɲͲƐLJŶƵĐůĞŝŶƚƌĂŶƐŐĞŶŝĐŇŝĞƐ͘tŚŝůĞƐŝŵƉůĞ animal models, including Drosophila or C. elegans͕ŚĂǀĞŵĂĚĞŝŵƉŽƌƚĂŶƚĐŽŶƚƌŝďƵƟŽŶƐ ƚŽ ŽƵƌ ƵŶĚĞƌƐƚĂŶĚŝŶŐ ŽĨ W ƉĂƚŚŽŐĞŶĞƐŝƐ͕ ƐĞůĞĐƚĞĚ ŵĞĐŚĂŶŝƐŵƐ͕ ƐƵĐŚ ĂƐ ƚŚĞ ƉŽƚĞŶƟĂů ƌŽůĞŽĨĂĚĂƉƟǀĞŝŵŵƵŶŝƚLJŽƌďĂƐĂůŐĂŶŐůŝĂĐŝƌĐƵŝƚĚLJƐĨƵŶĐƟŽŶ͕ĐĂŶŶŽƚďĞĂĚĚƌĞƐƐĞĚŝŶ invertebrates.79, 80_{tĞǁĞƌĞƵŶĂďůĞƚŽĐŽŶĮƌŵŽƵƌĮŶĚŝŶŐƐĨƌŽŵDrosophila in a published} C. elegans ŵŽĚĞůŽĨɲͲƐLJŶƵĐůĞŝŶƚŽdžŝĐŝƚLJ͘/ŶƚŚĞĨƵƚƵƌĞ͕ŝƚǁŝůůĂůƐŽďĞŝŵƉŽƌƚĂŶƚƚŽĞdžĂŵŝŶĞ

6

KE>h^/KE

tĞ ŚĂǀĞ ŝĚĞŶƟĮĞĚ ϱ ĞdžĐĞůůĞŶƚ W ŐĞŶĞ ĐĂŶĚŝĚĂƚĞƐ ;'Wd,Ϯ>͕ h,Z&ϭWϭ>͕ WdWZ,͕

ARSB and VPS13CͿ͕ŚĂƌďŽƌŝŶŐŚŽŵŽnjLJŐŽƵƐŽƌĐŽŵƉŽƵŶĚŚĞƚĞƌŽnjLJŐŽƵƐ>Ž&ǀĂƌŝĂŶƚƐŝŶW

ĞdžŽŵĞƐ͕ ĚĞŵŽŶƐƚƌĂƟŶŐ ĨƵŶĐƟŽŶĂů ŝŶƚĞƌĂĐƟŽŶƐ ǁŝƚŚ ŵŝƚŽĐŚŽŶĚƌŝĂů ĂŶĚͬŽƌ ɲͲƐLJŶƵĐůĞŝŶͲ ŵĞĚŝĂƚĞĚŵĞĐŚĂŶŝƐŵƐ͕ĂŶĚƐƵƉƉŽƌƚĞĚďLJĞǀŝĚĞŶĐĞŽĨƌĞƉůŝĐĂƟŽŶŝŶŝŶĚĞƉĞŶĚĞŶƚŚƵŵĂŶ datasets. The recent report21_{ŽĨĂĚĚŝƟŽŶĂůWĨĂŵŝůŝĞƐƐĞŐƌĞŐĂƟŶŐ>Ž&ŵƵƚĂƟŽŶƐŝŶVPS13C}

ĂůŽŶŐǁŝƚŚŽƚŚĞƌĞdžƉĞƌŝŵĞŶƚƐƐƵƉƉŽƌƟŶŐĂƌŽůĞŝŶŵŝƚŽĐŚŽŶĚƌŝĂůŵĞĐŚĂŶŝƐŵƐƐŝŐŶŝĮĐĂŶƚůLJ strengthens the evidence in support of this gene in PD, and validates our overall approach. dŚĞƐĞůŽĐŝĂƌĞǁĞůůͲƐƵŝƚĞĚĨŽƌĨƵƌƚŚĞƌĨƵƚƵƌĞĞīŽƌƚƐĚŝƌĞĐƚĞĚĂƚŚƵŵĂŶŐĞŶĞƟĐƌĞƉůŝĐĂƟŽŶ ĂŶĚŝŶͲĚĞƉƚŚĨƵŶĐƟŽŶĂůĚŝƐƐĞĐƟŽŶ͘tĞĂůƐŽŵĂŬĞĂǀĂŝůĂďůĞƌĞƐƵůƚƐ͕ŝŶĐůƵĚŝŶŐĮŶĚŝŶŐƐĨƌŽŵ ŚƵŵĂŶŐĞŶĞƟĐĂŶĂůLJƐĞƐĂŶĚĨƵŶĐƟŽŶĂůƐƚƵĚŝĞƐŝŶŵŽƐƚĐĂƐĞƐ͕ŽŶϮϮŽƚŚĞƌƉƌŽŵŝƐŝŶŐůŽĐŝ͘ dŚĞƐĞĚĂƚĂǁŝůůƐĞƌǀĞĂƐĂǀĂůƵĂďůĞƌĞĨĞƌĞŶĐĞĨŽƌŽŶŐŽŝŶŐĂŶĚĨƵƚƵƌĞWŐĞŶĞƟĐƐƚƵĚŝĞƐ͘ DŽƌĞďƌŽĂĚůLJ͕ŽƵƌĂƉƉƌŽĂĐŚŽĨŝŶƚĞŐƌĂƟŶŐŚŝŐŚͲƚŚƌŽƵŐŚƉƵƚƐĞƋƵĞŶĐŝŶŐŝŶWĐĂƐĞͬĐŽŶƚƌŽů ĐŽŚŽƌƚƐ ǁŝƚŚ ƉĂƌĂůůĞů ƐLJƐƚĞŵĂƟĐ ƐĐƌĞĞŶŝŶŐ ŝŶ ĐĞůůƐ ĂŶĚ ŵŽĚĞů ŽƌŐĂŶŝƐŵƐ ĨŽƌ ĨƵŶĐƟŽŶĂů ƉƌŝŽƌŝƟnjĂƟŽŶĞdžĞŵƉůŝĮĞƐĂƉŽǁĞƌĨƵůĞdžƉĞƌŝŵĞŶƚĂůƐƚƌĂƚĞŐLJǁŝƚŚŐƌĞĂƚƉƌŽŵŝƐĞĨŽƌĨƵƚƵƌĞ genomic studies of PD and other human disorders.

Dd,K^

'ĞŶĞƟĐĂŶĂůLJƐĞƐ Whole exome sequencing

tŚŽůĞĞdžŽŵĞƐĞƋƵĞŶĐŝŶŐ;t^ͿǁĂƐƉĞƌĨŽƌŵĞĚŽŶϭ͕ϭϰϴWĐĂƐĞƐĂŶĚϱϬϯŶĞƵƌŽůŽŐŝĐĂůůLJ ŚĞĂůƚŚLJĐŽŶƚƌŽůƐŽĨƵƌŽƉĞĂŶĚĞƐĐĞŶƚ͘ůůƐƵďũĞĐƚƐƉƌŽǀŝĚĞĚǁƌŝƩĞŶŝŶĨŽƌŵĞĚĐŽŶƐĞŶƚ͘ ZĞůĞǀĂŶƚůŽĐĂůĞƚŚŝĐĂůĐŽŵŵŝƩĞĞƐĨŽƌŵĞĚŝĐĂůƌĞƐĞĂƌĐŚĂƉƉƌŽǀĞĚƉĂƌƟĐŝƉĂƟŽŶŝŶŐĞŶĞƟĐ ƐƚƵĚŝĞƐ͘ /Ĩ W ƉĂƟĞŶƚƐ ǁĞƌĞ ƉƌĞƐĐƌĞĞŶĞĚ ĨŽƌ ŬŶŽǁŶ ƉĂƚŚŽŐĞŶŝĐ ŵƵƚĂƟŽŶƐ͕ ƚŚĞLJ ǁĞƌĞ ĞdžĐůƵĚĞĚĨŽƌĞdžŽŵĞƐĞƋƵĞŶĐŝŶŐǁŚĞŶŚĂǀŝŶŐƐƵĐŚĂǀĂƌŝĂŶƚ͘dŚĞĐĂƐĞƐǁĞƌĞĚŝĂŐŶŽƐĞĚ ǁŝƚŚ W Ăƚ Ă ƌĞůĂƟǀĞůLJ LJŽƵŶŐ ĂǀĞƌĂŐĞ ĂŐĞ ŽĨ ϰϬ͘ϲ LJĞĂƌƐ ;ƌĂŶŐĞ ϲͲϱϲ LJĞĂƌƐͿ͕ ŽĨ ǁŚŝĐŚ ĂƉƉƌŽdžŝŵĂƚĞůLJϯϳйƌĞƉŽƌƚĞĚĂƉŽƐŝƟǀĞĨĂŵŝůLJŚŝƐƚŽƌLJ͘dŚĞŶĞƵƌŽůŽŐŝĐĂůůLJŚĞĂůƚŚLJĐŽŶƚƌŽůƐ ĂƌĞ ŽŶ ĂǀĞƌĂŐĞ ϰϴ͘Ϯ LJĞĂƌƐ ŽĨ ĂŐĞ ;ƌĂŶŐĞ ϭϬͲϵϳ LJĞĂƌƐͿ͘  ŵŽƌĞ ĞdžƚĞŶƐŝǀĞ ŽǀĞƌǀŝĞǁ ŽĨ ĚĞŵŽŐƌĂƉŚŝĐŝŶĨŽƌŵĂƟŽŶŝƐƌĞƉŽƌƚĞĚŝŶ^ƵƉƉůĞŵĞŶƚĂƌLJ&ŝŐƵƌĞϴ͘ ƵĞƚŽŝŵƉƌŽǀĞŵĞŶƚƐŽĨƚŚĞĞdžŽŵĞƐĞƋƵĞŶĐŝŶŐƉƌŽƚŽĐŽůŽǀĞƌƟŵĞ͕ƚŚĞĞdžŽŵĞ ƐĂŵƉůĞůŝďƌĂƌŝĞƐǁĞƌĞƉƌĞƉĂƌĞĚǁŝƚŚĚŝīĞƌĞŶƚĐĂƉƚƵƌĞŬŝƚƐ͘&ŽƌƚŚŝƐƐƚƵĚLJƚŚƌĞĞĚŝīĞƌĞŶƚ ĐĂƉƚƵƌĞŬŝƚƐǁĞƌĞƵƐĞĚ͗/ůůƵŵŝŶĂdƌƵ^ĞƋ;^ĂŶŝĞŐŽ͕ĂůŝĨŽƌŶŝĂͿ;ϲϮDďƚĂƌŐĞƚͿ͕ZŽĐŚĞ;ĂƐĞů͕ ^ĐŚǁŝƚnjĞƌůĂŶĚͿEŝŵďůĞŐĞŶ^ĞƋĂƉ;ϰϰ͘ϭDďƚĂƌŐĞƚͿĂŶĚŐŝůĞŶƚ;^ĂŶƚĂůĂƌĂ͕ĂůŝĨŽƌŶŝĂͿ ^ƵƌĞ^ĞůĞĐƚ;ϯϳ͘ϲDďƚĂƌŐĞƚͿ͕ǁŚŝĐŚĐĂƉƚƵƌĞĚϵϲй͕ϴϭйĂŶĚϳϭйŽĨƚŚĞƚĂƌŐĞƚĞĚĞdžŽŵĞĂƚ ůĞĂƐƚϭϬƟŵĞƐƌĞƐƉĞĐƟǀĞůLJ;^ƵƉƉůĞŵĞŶƚĂƌLJdĂďůĞϭϭͿ͘džŽŵĞůŝďƌĂƌŝĞƐǁĞƌĞƐĞƋƵĞŶĐĞĚŽŶĂ HiSeq 2000 (Illumina, San Diego, California). The Burrows Wheeler Aligner MEM v0.7.9.a 81

6

ŚŐϭϵ͘ tĞ ĐĂůůĞĚ ƚŚĞ ƐŝŶŐůĞ ŶƵĐůĞŽƟĚĞ ǀĂƌŝĂŶƚƐ ;^EsƐͿ ĂŶĚ ŝŶƐĞƌƟŽŶƐͬĚĞůĞƟŽŶƐ ;ŝŶĚĞůƐͿ ĨŽƌĂůůƐĂŵƉůĞƐƐŝŵƵůƚĂŶĞŽƵƐůLJƵƐŝŶŐ'ĞŶŽŵĞŶĂůLJƐŝƐdŽŽůŬŝƚ;'d<Ϳϯ͘dž͕82 _{followed by} ƚŚĞĞdžĐůƵƐŝŽŶŽĨůŽǁͲƋƵĂůŝƚLJǀĂƌŝĂŶƚĐĂůůƐŶŽƚƉĂƐƐŝŶŐƚŚĞĚĞĨĂƵůƚ'd<ĮůƚĞƌƐ͘/ŶĚŝǀŝĚƵĂů ŐĞŶŽƚLJƉĞƐ ǁĞƌĞ ƌĞŵŽǀĞĚ ǁŝƚŚ ŐĞŶŽƚLJƉĞ ƋƵĂůŝƚLJ WŚƌĞĚͲƐĐŽƌĞƐ ďĞůŽǁ ϰϬ͘ EEKsZ83 ǁĂƐ ĂƉƉůŝĞĚ ƚŽ ĂŶŶŽƚĂƚĞ ƚŚĞ ǀĂƌŝĂŶƚƐ ǁŝƚŚ ŝŶĨŽƌŵĂƟŽŶ ĐŽŶĐĞƌŶŝŶŐ ǀĂƌŝĂŶƚ ƚLJƉĞ ;ǀĂůŝĚ ĂŶŶŽƚĂƟŽŶƐǁŚĞŶZĞĨƐĞƋŝŶĐŽŶĐŽƌĚĂŶĐĞǁŝƚŚh^Ϳ͕D&ŝŶƚŚĞŐĞŶĞƌĂůƉŽƉƵůĂƟŽŶĂŶĚ ƉƌĞĚŝĐƟŽŶƐŽĨƚŚĞǀĂƌŝĂŶƚ͛ƐĞīĞĐƚŽŶŐĞŶĞĨƵŶĐƟŽŶ͕ŝŵƉůĞŵĞŶƟŶŐ͘84 sĂƌŝĂŶƚŝĚĞŶƟĮĐĂƟŽŶŝŶ/W't^ĚĂƚĂƐĞƚ

Considering the worldwide prevalence of 0.041% for PD in the age range of 40 to 49 years,20

ǁĞƐĞůĞĐƚĞĚƌĂƌĞǀĂƌŝĂŶƚƐǁŝƚŚĂD&фϭй;ĐŽƌƌĞƐƉŽŶĚŝŶŐƚŽĂŚŽŵŽnjLJŐŽƵƐĨƌĞƋƵĞŶĐLJ ŽĨϬ͘ϬϭйͿŝŶƚŚĞƵƌŽƉĞĂŶƉŽƉƵůĂƟŽŶ͘ĞĐĂƵƐĞƚŚĞƐƉĞĐŝĮĞĚϬ͘ϬϰϭйŽĨƚŚĞƉŽƉƵůĂƟŽŶ ǁŝƚŚLJŽƵŶŐͲŽŶƐĞƚWĂƌŬŝŶƐŽŶ͛ƐĚŝƐĞĂƐĞ;zKWͿŝƐŶŽƚĐĂƵƐĞĚďLJŽŶĞƐŚĂƌĞĚŐĞŶĞƟĐĨĂĐƚŽƌ͕ ǁĞ ĞdžƉĞĐƚ Ă ŚŽŵŽnjLJŐŽƵƐ ĨƌĞƋƵĞŶĐLJ ŽĨ Ϭ͘Ϭϭй ƚŽ ďĞ ĂŶ ĂĚĞƋƵĂƚĞ ĐƵƚͲŽī͕ ǁŚŝĐŚ ǁŽƵůĚ ďĞĂďůĞƚŽĚĞƚĞƌŵŝŶĞǀĂƌŝĂŶƚƐƉƌĞƐĞŶƚŝŶĂƉƉƌŽdžŝŵĂƚĞůLJϮϱйŽĨƚŚĞzKWƉŽƉƵůĂƟŽŶ͘Ɛ ĂĐŽŵƉĂƌŝƐŽŶƚŽƚŚĞŵŽƐƚĐŽŵŵŽŶŐĞŶĞƟĐĐĂƵƐĞŽĨzKW͕parkin,85 _{the most frequent}

ŵƵƚĂƟŽŶŝƐĂŶĞdžŽŶϯĚĞůĞƟŽŶ͕ǁŚŝĐŚŚĂƐďĞĞŶŝĚĞŶƟĮĞĚŝŶϭϲ͘ϰйŽĨzKWƉĂƟĞŶƚƐ͘86

hƐŝŶŐ EEKsZ͕83_{ Ăůů ǀĂƌŝĂŶƚƐ ǁĞƌĞ ĂŶŶŽƚĂƚĞĚ ǁŝƚŚ D& ŝŶĨŽƌŵĂƟŽŶ ŽĨ ^WϲϱϬϬƐŝ}

;ƵƌŽƉĞĂŶ ŵĞƌŝĐĂŶ ƉŽƉƵůĂƟŽŶͿ͕87_{ ϭϬϬϬ 'ĞŶŽŵĞƐ WƌŽũĞĐƚ ;ƵƌŽƉĞĂŶ ƉŽƉƵůĂƟŽŶ ŽĨ}

April 2012 version)88_{ĂŶĚƚŚĞdžŽŵĞŐŐƌĞŐĂƟŽŶŽŶƐŽƌƟƵŵ;džͿďƌŽǁƐĞƌ;ŶŽŶͲ&ŝŶŝƐŚ}

ƵƌŽƉĞĂŶƉŽƉƵůĂƟŽŶͿ͘77, 78_{tŚĞŶŶŽƉƵďůŝĐĂůůĞůĞĨƌĞƋƵĞŶĐLJǁĂƐĂǀĂŝůĂďůĞĨŽƌŚŽŵŽnjLJŐŽƵƐ}