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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 2

CHCHD2: a rare genetic risk

factor of Parkinson’s disease

in the Caucasian population

2

/EdZKhd/KE

'ĞŶĞƟĐĚŝƐĐŽǀĞƌŝĞƐŚĂǀĞůĞŶƚƐƵƉƉŽƌƚƚŽƚŚĞŐƌŽǁŝŶŐĐŽŶƐĞŶƐƵƐƚŚĂƚƉƌŽƚĞŝŶĂŐŐƌĞŐĂƟŽŶ͕ ŝŵƉĂŝƌŵĞŶƚŝŶŽdžŝĚĂƟǀĞƐƚƌĞƐƐ͕ĂŶĚŵŝƚŽĐŚŽŶĚƌŝĂůĚLJƐĨƵŶĐƟŽŶĂƌĞŝŵƉŽƌƚĂŶƚƉĂƚŚǁĂLJƐŝŶ Parkinson’s disease (PD).1 _{/ŶĂŐƌĞĞŵĞŶƚǁŝƚŚƚŚŝƐŚLJƉŽƚŚĞƐŝƐ͕&ƵŶĂLJĂŵĂĂŶĚĐŽůůĞĂŐƵĞƐ}2

ƌĞƉŽƌƚĂŚĞƚĞƌŽnjLJŐŽƵƐŵƵƚĂƟŽŶ;Ɖ͘dŚƌϲϭ/ůĞͿŝŶCHCHD2, which is suggested to be involved ŝŶŵŝƚŽĐŚŽŶĚƌŝĂůƌĞƐƉŝƌĂƟŽŶ͘dŚŝƐǀĂƌŝĂŶƚǁĂƐĚĞƚĞĐƚĞĚŝŶĂůĂƌŐĞŵƵůƟͲŐĞŶĞƌĂƟŽŶ:ĂƉĂŶĞƐĞ family with an autosomal dominant form of PD.dŚĞƐĐƌĞĞŶŝŶŐŽĨĂĚĚŝƟŽŶĂůϯϯϬŝŶĚĞdž ĐĂƐĞƐǁŝƚŚĂƵƚŽƐŽŵĂůĚŽŵŝŶĂŶƚWŽďƐĞƌǀĞĚĂŶŽƚŚĞƌŶŽŶƐLJŶŽŶLJŵŽƵƐ;Ɖ͘ƌŐϭϰϱ'ůŶͿĂŶĚ ĂƐƉůŝĐĞƐŝƚĞǀĂƌŝĂŶƚ;Đ͘ϯϬϬнϱ'хͿ͘>ŝŬĞǁŝƐĞ͕ƐĞƋƵĞŶĐŝŶŐŽĨƐƉŽƌĂĚŝĐWĐĂƐĞƐŝĚĞŶƟĮĞĚĂ ŶƵŵďĞƌŽĨƉƵƚĂƟǀĞƌŝƐŬǀĂƌŝĂŶƚƐŝŶCHCHD2 ŝŶƚŚĞƐĂŵĞƉŽƉƵůĂƟŽŶ͘ EŽǀĞů ŐĞŶĞƟĐ ĨĂĐƚŽƌƐ ƐƵĐŚ ĂƐ CHCHD2 ƌĞƋƵŝƌĞ ƌĞƉůŝĐĂƟŽŶ͕ ďŽƚŚ ǁŝƚŚŝŶ ƚŚĞ ĚŝƐĐŽǀĞƌLJƉŽƉƵůĂƟŽŶĂŶĚŝŶĚŝīĞƌĞŶƚĞƚŚŶŝĐƉŽƉƵůĂƟŽŶƐ͘'ĞŶŽƚLJƉŝŶŐƚŚĞŽďƐĞƌǀĞĚƐŝĂŶ ǀĂƌŝĂŶƚƐǁŽƵůĚďĞĂĐŽƐƚͲĞīĞĐƟǀĞŽƉƟŽŶ͕LJĞƚŽƚŚĞƌŵƵƚĂƟŽŶƐĂƌĞŵŝƐƐĞĚ͘ƐƚŚĞŝĚĞŶƟĮĞĚ ǀĂƌŝĂŶƚƐĂƌĞƌĂƌĞĂŶĚŽďƐĞƌǀĞĚŝŶĂŶŽŶͲƵƌŽƉĞĂŶƉŽƉƵůĂƟŽŶ͕ŝƚŝƐŝŵƉŽƌƚĂŶƚƚŽĞdžƉůŽƌĞ ĨŽƌ ŽƚŚĞƌ ůŝŬĞůLJ ƉĂƚŚŽŐĞŶŝĐ ŵƵƚĂƟŽŶƐ͕ ŝŶ ĂĚĚŝƟŽŶ ƚŽ ƚŚĞ ŝĚĞŶƟĐĂů ǀĂƌŝĂŶƚƐ͘ dŚĞƌĞĨŽƌĞ͕ ƐĞƋƵĞŶĐŝŶŐĚĂƚĂŝƐƉƌĞĨĞƌƌĞĚ͘tŚŽůĞĞdžŽŵĞƐĞƋƵĞŶĐŝŶŐ;t^ͿĚĂƚĂŝƐĂŐƌĞĂƚƐŽƵƌĐĞƚŽ ĨƵƌƚŚĞƌ ŝŶǀĞƐƟŐĂƚĞ ŶŽǀĞů ŐĞŶĞƐ ƌĞĐĞŶƚůLJ ƉƵďůŝƐŚĞĚ ŝŶ ƚŚĞ ŐĞŶŽŵŝĐƐ ƌĞƐĞĂƌĐŚ ĮĞůĚ͘ dŚĞ t^ĚĂƚĂƐĞƚŽĨƚŚĞ/ŶƚĞƌŶĂƟŽŶĂůWĂƌŬŝŶƐŽŶ͛ƐŝƐĞĂƐĞ'ĞŶŽŵŝĐƐŽŶƐŽƌƟƵŵŝƐŽĨƐƵĸĐŝĞŶƚ ƐŝnjĞƚŽƉĞƌĨŽƌŵǀĂůƵĂďůĞĞdžĂŵŝŶĂƟŽŶƐƚŽĚĞƚĞƌŵŝŶĞƚŚĞĞdžƚĞŶƚŽĨƌĞƉůŝĐĂƟŽŶ͘tĞƵƐĞĚ ƚŚŝƐ ĚĂƚĂƐĞƚ͕ ƚŽŐĞƚŚĞƌ ǁŝƚŚ ĚĂƚĂ ŐĞŶĞƌĂƚĞĚ ďLJ ƚŚĞ ĂƌƌĂLJͲďĂƐĞĚ ĞdžŽŵĞ EĞƵƌŽy ĐŚŝƉ͕ ƚŽ establish the pathogenicity of CHCHD2 in individuals of western European ancestry.

Dd,K^

tŚŽůĞĞdžŽŵĞƐĞƋƵĞŶĐŝŶŐĚĂƚĂƐĞƚ tĞŝŶǀĞƐƟŐĂƚĞĚƚŚĞƉƌĞƐĞŶĐĞĂŶĚŐĞŶĞƟĐďƵƌĚĞŶŽĨƉƵƚĂƟǀĞƉĂƚŚŽŐĞŶŝĐƌĂƌĞǀĂƌŝĂŶƚƐŝŶ CHCHD2 ďLJĞdžƉůŽƌŝŶŐƚŚĞ/W't^ĚĂƚĂƐĞƚĐŽŵƉƌŝƐŝŶŐϭ͕ϮϰϯWĐĂƐĞƐ;ĂǀĞƌĂŐĞĂŐĞŽĨ ŽŶƐĞƚϰϮ͘ϯLJĞĂƌƐͿĂŶĚϰϳϮĐŽŶƚƌŽůƐ͘^ĂŵƉůĞůŝďƌĂƌŝĞƐǁĞƌĞƉƌĞƉĂƌĞĚǁŝƚŚZŽĐŚĞEŝŵďůĞŐĞŶ Žƌ/ůůƵŵŝŶĂĞdžŽŵĞĐĂƉƚƵƌĞŬŝƚƐ͕ĂŶĚƐƵďũĞĐƚĞĚƚŽϭϬϬͲďƉƉĂŝƌĞĚͲĞŶĚƐĞƋƵĞŶĐŝŶŐŽŶƚŚĞ Illumina HiSeq2000. The reads were aligned using BWA-MEM 0.7.9a to the reference ŐĞŶŽŵĞ;h^ŚŐϭϵͿ͘3_{'d<ϯ͘džĂŶĚEEKsZǁĞƌĞƵƐĞĚƚŽĐĂůů͕ƋƵĂůŝƚLJͲďĂƐĞĚĮůƚĞƌ͕ĂŶĚ}

annotate variants.4,5_{&ŽƌĨƵŶĐƟŽŶĂůƉƌĞĚŝĐƟŽŶƐ͕ϲĚŝīĞƌĞŶƚĂůŐŽƌŝƚŚŵƐǁĞƌĞƵƐĞĚ͗^/&d}6_,

PhyloP7_{, Polyphen}8_͕>Zd9_{͕DƵƚĂƟŽŶdĂƐƚĞƌ}10 _{and CADD}11_{. Variants were only considered for}

subsequent analyses if targeted by both capture kits.

EĞƵƌŽyĞdžŽŵĞĂƌƌĂLJĚĂƚĂƐĞƚ

CHCHD2 and PD

2

The NeuroX array12_{ĐŽŶƚĂŝŶƐĂƉƉƌŽdžŝŵĂƚĞůLJϮϰϬ͕ϬϬϬǀĂƌŝĂŶƚƐŽĨƐƚĂŶĚĂƌĚ/ůůƵŵŝŶĂĞdžŽŵĞ}

ĐŽŶƚĞŶƚĂŶĚĂƉƉƌŽdžŝŵĂƚĞůLJϮϰ͕ϬϬϬǀĂƌŝĂŶƚƐŝŵƉůŝĞĚƚŽďĞĂƐƐŽĐŝĂƚĞĚƚŽŶĞƵƌŽĚĞŐĞŶĞƌĂƟǀĞ diseases.

'ĞŶĞƟĐĂŶĂůLJƐĞƐ

Both for the WES and the NeuroX dataset, variants were only considered when passing ƐƚĂŶĚĂƌĚǀĂƌŝĂŶƚƋƵĂůŝƚLJĐŽŶƚƌŽůƐƚĞƉƐ;ǀĂƌŝĂŶƚĞdžĐůƵƐŝŽŶǁŚĞŶŵŝƐƐŝŶŐŐĞŶŽƚLJƉĞƌĂƚĞхϱй͕ Hardy Weinberg equilibrium p-value < 1e-6 _{and non-random missingness by phenotype}

p-value < 1e-5_{Ϳ͘DŝŶŽƌĂůůĞůĞĨƌĞƋƵĞŶĐŝĞƐǁĞƌĞĚĞƚĞƌŵŝŶĞĚƉĞƌŝŶĚŝǀŝĚƵĂůǀĂƌŝĂŶƚ͘^ƚĂƟƐƟĐĂů} ĐŽŵƉĂƌŝƐŽŶƐ ǁĞƌĞ ƉĞƌĨŽƌŵĞĚ ŝĨ ĂƉƉůŝĐĂďůĞ͕ ǁŚŝĐŚ ĚĞƉĞŶĚƐ ŽŶ ƚŚĞ ƐĂŵƉůĞ ƐŝnjĞ ĂŶĚ ǀĂƌŝĂŶƚĨƌĞƋƵĞŶĐLJ͘&ŽƌƚŚĞĞdžŽŵĞĚĂƚĂƐĞƚ͕ƚŚĞƐĞƋƵĞŶĐĞŬĞƌŶĞůĂƐƐŽĐŝĂƟŽŶƚĞƐƚ;^<dͿ13 ǁĂƐĂƉƉůŝĞĚƚŽƉĞƌĨŽƌŵĂŐĞŶĞͲďĂƐĞĚĂƐƐŽĐŝĂƟŽŶĂŶĂůLJƐŝƐ͘dŚŝƐƌĂƌĞǀĂƌŝĂŶƚĂŐŐƌĞŐĂƟŽŶ ĂƐƐŽĐŝĂƟŽŶƚĞƐƚĂĐĐŽƵŶƚĞĚĨŽƌƚŚĞƉƵƚĂƟǀĞĐŽŶĨŽƵŶĚŝŶŐĨĂĐƚŽƌƐŐĞŶĚĞƌ͕ĐŽƵŶƚƌLJŽĨŽƌŝŐŝŶ͕ ĐĂƉƚƵƌĞŬŝƚƚLJƉĞ͕ƉŽƌƟŽŶŽĨƚŚĞĞdžŽŵĞƚŚĂƚŚĂƐďĞĞŶĐĂƉƚƵƌĞĚĨŽƌĂƚůĞĂƐƚϭϬdžĂŶĚƚŚĞĮƌƐƚ ϰD^ĐŽŵƉŽŶĞŶƚƐ͘dŚĞ^<dĂŶĂůLJƐŝƐǁĂƐƉĞƌĨŽƌŵĞĚŽŶĂůůǀĂƌŝĂŶƚƐŝĚĞŶƟĮĞĚŝŶCHCHD2 ŽƌĐŽĚŝŶŐǀĂƌŝĂŶƚƐŽŶůLJ͘&ŽƌƚŚĞEĞƵƌŽyĚĂƚĂƐĞƚ͕ůŽŐŝƐƟĐƌĞŐƌĞƐƐŝŽŶǁĂƐƉĞƌĨŽƌŵĞĚƚŚĂƚ ĚĞƚĞƌŵŝŶĞĚ ƚŚĞ ĞīĞĐƚ ĞƐƟŵĂƚĞ ŽĨ ƚǁŽ CHCHD2 ĐŽĚŝŶŐ ǀĂƌŝĂŶƚ͕ ǁŚŝůĞ ĐŽƌƌĞĐƟŶŐ ĨŽƌ ĐŽǀĂƌŝĂƚĞƐŐĞŶĚĞƌ͕ĐŽƵŶƚƌLJŽĨŽƌŝŐŝŶĂŶĚƚŚĞĮƌƐƚϰD^ĐŽŵƉŽŶĞŶƚƐ͘ dĂďůĞϭ͘ CHCHD2ǀĂƌŝĂŶƚƐŝŶ/W'ĐŽŚŽƌƚ͘

Change dbSNP141 ExAC Path.* &ƵŶĂLJĂŵĂƉĂƉĞƌƕ

ĐE AA hZ EAS pred. &Ăŵ͘W ^ƉŽƌ͘W

WŽƐŝƟŽŶ;ŚŐϭϵͿ (NM_016139) (n=340) (n=517) chr7:56174129 ͲϮϯ'х hdZϱ rs368503740 0·00006 0·0 NA ·· ·· chr7:56174118 ͲϭϮхd hdZϱ rs112876794 0·0008 0·0 NA ·· ·· chr7:56174102 ϱхd Pro2Leu rs142444896 0·004 0·008 b,d,e,f 0·035 0·018 chr7:56174067 ϰϬхd Pro14Ser rs137965562 0·0001 0·005 b,e,f ·· ·· chr7:56172125 ϵϰ'х Ala32Thr rs145190179 0·00006 0·0006 b,d,e,f ·· ·· chr7:56172118 ϭϬϭхd Pro34Leu rs371198317 0·0003 0·0 a,b,d,e,f ·· ·· chr7:56171981 Ϯϯϴх' Ile80Val rs149119842 0·00003 0·0 f ·· ·· chr7:56169498 +46Tins hdZϯ rs35957514 0·0002 0·0 NA ·· ·· chr7:56169469 нϳϱdх hdZϯ unknown ND ND NA ·· ·· džсdžŽŵĞŐŐƌĞŐĂƟŽŶŽŶƐŽƌƟƵŵ͖сĂŵŝŶŽĂĐŝĚ͖hZсƵƌŽƉĞĂŶƉŽƉƵůĂƟŽŶ͖^сĂƐƚƐŝĂŶƉŽƉƵůĂƟŽŶ͖&Ăŵ͘сĨĂŵŝůŝĂů͖ WсWĂƌŬŝŶƐŽŶ͛ƐĚŝƐĞĂƐĞ͖^ƉŽƌ͘сƐƉŽƌĂĚŝĐ͖ŽŶƚƌ͘сĐŽŶƚƌŽůƐ͖KZсŽĚĚƐƌĂƟŽ͖hdZсƵŶƚƌĂŶƐůĂƚĞĚƌĞŐŝŽŶ͖ŝŶƐсŝŶƐĞƌƟŽŶ͖EсŶŽĚĂƚĂ ĂǀĂŝůĂďůĞŝŶĚĂƚĂďĂƐĞ͖EсŶŽƚĂƉƉůŝĐĂďůĞĨŽƌŶŽŶͲĞdžŽŶŝĐǀĂƌŝĂŶƚƐ͖ͼͼсǀĂƌŝĂŶƚŶŽƚƉƌĞƐĞŶƚŝŶƚŚŝƐĚĂƚĂƐĞƚ͘ΎWĂƚŚ͘ƉƌĞĚ͘ĚŝƐƉůĂLJƐ ǁŚŝĐŚŽĨĂůŐŽƌŝƚŚŵƐƉƌĞĚŝĐƚƉĂƚŚŽŐĞŶŝĐŝƚLJ͗Ăс^/&d6_{, b=PhyloP}7_{, c=Polyphen2}8_͕Ěс>Zd9_{͕ĞсDƵƚĂƟŽŶdĂƐƚĞƌ}10_{, f=CADD}11_{. ƕKŶůLJ}

2

>ŝŶŬĂŐĞͲĚŝƐĞƋƵŝůŝďƌŝƵŵĐĂůĐƵůĂƟŽŶƐ

dŚĞ s& ĮůĞƐ ŽĨ ƚŚĞ dŚŽƵƐĂŶĚ 'ĞŶŽŵĞƐ WƌŽũĞĐƚ WŚĂƐĞ ϯ14 _{covering CHCHCD2 were}

ĚŽǁŶůŽĂĚĞĚ ƉĞƌ ƉŽƉƵůĂƟŽŶ ƚŚƌŽƵŐŚ ƚŚĞ ŽŶůŝŶĞ ĞŶƐĞŵďůĞ ďƌŽǁƐĞƌ͘15 _{The European}

ƉŽƉƵůĂƟŽŶ ĂŶĚ ĂƐƚ ƐŝĂŶ ƉŽƉƵůĂƟŽŶƐ ĐŽŵƉƌŝƐĞĚ ϱϬϯ ĂŶĚ ϱϬϰ ƵŶƌĞůĂƚĞĚ ŝŶĚŝǀŝĚƵĂůƐ͕ ƌĞƐƉĞĐƟǀĞůLJ͘s&ƚŽŽůƐ16_{ǀϬ͘ϭ͘ϭϭĐŽŶǀĞƌƚĞĚƚŚĞs&ĮůĞƐŝŶƚŽW>/E<ĨŽƌŵĂƚƚŽŵĂŬĞƚŚĞŵ}

ĐŽŵƉĂƟďůĞ ǁŝƚŚ,ĂƉůŽǀŝĞǁ17_{ϰ͘Ϯ͘dŚĞƐƚĂŶĚĂƌĚ>ĐŽůŽƌ ƐĐŚĞŵĞ;͛ͬ>KͿǁĂƐƵƐĞĚŝŶ}

,ĂƉůŽǀŝĞǁƚŽǀŝƐƵĂůŝnjĞƚŚĞ>ƉůŽƚ͘&ŽƌƚŚĞĐŽŵƉůĞƚĞŶĞƐƐŽĨƚŚĞƉŝĐƚƵƌĞ͕ĂůůǀĂƌŝĂŶƚƐǁĞƌĞ considered, even when the minor allele frequency was 0.

Z^h>d^

Table 1 shows CHCHD2 ǀĂƌŝĂŶƚƐ ŝĚĞŶƟĮĞĚ ŝŶ ŽƵƌ ĞdžŽŵĞ ĂŶĚ EĞƵƌŽy ĚĂƚĂƐĞƚƐ͘ t^ captured 8 variants that were located in regions targeted by both capture kits, including ƚŚƌĞĞĐŽĚŝŶŐǀĂƌŝĂŶƚƐ͕ƚŚƌĞĞϱ͛hdZǀĂƌŝĂŶƚƐĂŶĚƚǁŽϯ͛hdZǀĂƌŝĂŶƚƐ͘KŶĞǀĂƌŝĂŶƚŝŶƚŚĞϱ͛ hdZƌĞŐŝŽŶǁĂƐƌĞŵŽǀĞĚĚƵĞƚŽƐŝŐŶŝĮĐĂŶƚĚŝīĞƌĞŶĐĞŝŶŵŝƐƐŝŶŐŶĞƐƐďĞƚǁĞĞŶĐĂƐĞƐĂŶĚ ĐŽŶƚƌŽůƐ͘tĞĚŝĚŶŽƚŶŽƚĞĂŶLJŽĨƚŚĞƉƵƚĂƟǀĞĐĂƵƐĂůǀĂƌŝĂŶƚƐ;Ɖ͘dŚƌϲϭ/ůĞ͕Ɖ͘ƌŐϭϰϱ'ůŶ͕ĂŶĚ Đ͘ϯϬϬнϱ'хͿĚĞƐĐƌŝďĞĚďLJ&ƵŶĂLJĂŵĂĞƚĂů͘2 _{Also, these variants are absent in the European}

ƉŽƉƵůĂƟŽŶŝŶƚŚĞƉƵďůŝĐůLJĂĐĐĞƐƐŝďůĞdžŽŵĞŐŐƌĞŐĂƟŽŶŽŶƐŽƌƟƵŵ;džͿďƌŽǁƐĞƌ͕18

ƐƵŐŐĞƐƟŶŐƚŚĂƚƚŚĞLJĂƌĞǀĞƌLJƌĂƌĞĂŶĚƐŝĂŶͲƐƉĞĐŝĮĐƌĂƚŚĞƌƚŚĂŶĂĐŽŵŵŽŶŐĞŶĞƟĐďĂƐŝƐ for Parkinson’s disease. However, in our dataset, among people of western European ĂŶĐĞƐƚƌLJ͕ƚŚƌĞĞŶŽǀĞůƉƵƚĂƟǀĞƉĂƚŚŽŐĞŶŝĐǀĂƌŝĂŶƚƐŝŶĞdžŽŶϮ;Ɖ͘ůĂϯϮdŚƌ͕Ɖ͘WƌŽϯϰ>ĞƵ͕ĂŶĚ p.Ile80Val) were noted in four of 1,243 cases (<1%). These variants are predicted to be

&ƵŶĂLJĂŵĂƉĂƉĞƌƕ /W'

Contr. Fisher exact Exome sequencing NeuroX

CHCHD2 and PD

2

ƉĂƚŚŽŐĞŶŝĐ;dĂďůĞϭͿĂŶĚƚŚĞĂīĞĐƚĞĚĂŵŝŶŽĂĐŝĚƐĂƌĞĐŽŶƐĞƌǀĞĚĚŽǁŶƚŽƌŽĚĞŶƚƐ͘dŚŝƐ ĐŽŶƐĞƌǀĞĚƐĞƋƵĞŶĐĞƚŽŐĞƚŚĞƌǁŝƚŚƚŚĞůĂĐŬŽĨŝĚĞŶƟĮĐĂƟŽŶŽĨĞdžŽŶŝĐǀĂƌŝĂŶƚƐŝŶĐŽŶƚƌŽůƐ ŝŶĚŝĐĂƚĞƚŚĂƚǀĂƌŝĂƟŽŶŝŶCHCHD2 might be a rare risk factor in people of western European ĂŶĐĞƐƚƌLJ͘dŚĞďƵƌĚĞŶĂŶĂůLJƐŝƐŽĨƌĂƌĞǀĂƌŝĂŶƚƐĚŝĚŶŽƚƐŚŽǁĂƐƐŽĐŝĂƟŽŶǁŝƚŚWĂƌŬŝŶƐŽŶ͛Ɛ ĚŝƐĞĂƐĞŝŶƚŚĞĞdžŽŵĞĚĂƚĂƐĞƚ͕ŶĞŝƚŚĞƌĐŽŶƐŝĚĞƌŝŶŐƚŚĞϯĞdžŽŶŝĐǀĂƌŝĂŶƚƐŽŶůLJ;p = 0.28), ŶŽƌǁŚĞŶƚĞƐƟŶŐĨŽƌƚŚĞũŽŝŶƚĞīĞĐƚŽĨĂůůϳǀĂƌŝĂŶƚƐŝŶĐůƵĚŝŶŐƚŚĞhdZǀĂƌŝĂŶƚƐ;p = 0.24).

dŚĞ ĨŽƵƌ ƉĂƟĞŶƚƐ ǁŝƚŚ WĂƌŬŝŶƐŽŶ͛Ɛ ĚŝƐĞĂƐĞ ǁŝƚŚ ƚŚƌĞĞ ŶŽǀĞů ǀĂƌŝĂŶƚƐ ǁĞƌĞ ƚǁŽ ŝŶĚŝǀŝĚƵĂůƐ ĨƌŽŵ ƚŚĞ h^ ĂŶĚ ƚǁŽ ĨƌŽŵ &ƌĂŶĐĞ͘ dŚĞ h^ ĐĂƐĞƐ ǁĞƌĞ ĚŝĂŐŶŽƐĞĚ Ăƚ age 39 years and 51 years with asymmetric onset and showed typical symptoms such ĂƐďƌĂĚLJŬŝŶĞƐŝĂĂŶĚ͕ŝŶŽŶĞƉĂƟĞŶƚ͕ƌĞƐƟŶŐƚƌĞŵŽƌ͘ůƚŚŽƵŐŚƚŚĞƐĞƚǁŽƉĂƟĞŶƚƐǁĞƌĞ reported to have familial Parkinson’s disease, we did not have access to family members ƚŽĮŶĚŽƵƚǁŚĞƚŚĞƌƚŚĞǀĂƌŝĂŶƚĐŽƐĞŐƌĞŐĂƚĞĚǁŝƚŚƚŚĞĚŝƐĞĂƐĞ͘dŚĞƚǁŽ&ƌĞŶĐŚƉĂƟĞŶƚƐ were isolated cases with an age at onset of 20 years and 39 years.

hƐŝŶŐEĞƵƌŽyĚĂƚĂ͕ǁĞĐĂƉƚƵƌĞĚƚǁŽǀĂƌŝĂŶƚƐŝŶŽƵƌĂƵĐĂƐŝĂŶƉŽƉƵůĂƟŽŶ;dĂďůĞ ϭͿ͘KŶĞŽĨƚŚĞƐĞǀĂƌŝĂŶƚƐ͕ƌƐϭϰϮϰϰϰϴϵϲ͕ǁĂƐƌĞƉŽƌƚĞĚďLJ&ƵŶĂLJĂŵĂĂŶĚĐŽůůĞĂŐƵĞƐ2 _to ďĞƐŝŐŶŝĮĐĂŶƚůLJĂƐƐŽĐŝĂƚĞĚǁŝƚŚWĂƌŬŝŶƐŽŶ͛ƐĚŝƐĞĂƐĞŝŶƚŚĞ:ĂƉĂŶĞƐĞƉŽƉƵůĂƟŽŶ͘ůƚŚŽƵŐŚ ǁĞĞƐƟŵĂƚĞĚƚŚĂƚŽƵƌEĞƵƌŽyĚĞƐŝŐŶŚĂƐϵϵйƉŽǁĞƌƚŽĚĞƚĞĐƚǀĂƌŝĂŶƚƐǁŝƚŚĂŶĂůůĞůĞ ĨƌĞƋƵĞŶĐLJŽĨϬ͘ϰй;ƵƌŽƉĞĂŶĨƌĞƋƵĞŶĐLJŝŶdžͿĂŶĚĂŶŽĚĚƐƌĂƟŽŽĨϰ͘ϲϵ͕ǁĞĐŽƵůĚŶŽƚ replicate (p сϬ͘ϴϵͿƚŚŝƐĂƐƐŽĐŝĂƟŽŶǁŝƚŚŝŶŽƵƌǁĞƐƚĞƌŶƵƌŽƉĞĂŶƉŽƉƵůĂƟŽŶ͘dŚĞƐĞĐŽŶĚ variant, not observed in the original CHCHD2 ƉƵďůŝĐĂƟŽŶ͕ĂůƐŽůĂĐŬĞĚĂŶĂƐƐŽĐŝĂƟŽŶƚŽW (p = 0.74).

CHCHD2 and PD

2

/^h^^/KE

LJĞdžƉůŽƌŝŶŐƚǁŽŝŶĚĞƉĞŶĚĞŶƚŐĞŶĞƟĐĞdžŽŵĞĚĂƚĂƐĞƚƐŽĨƚŚĞ/W'͕ǁĞĂŝŵĞĚƚŽĞƐƚĂďůŝƐŚ ƚŚĞŝŶŇƵĞŶĐĞŽĨCHCHD2 ǀĂƌŝĂŶƚƐŝŶWĂƌŬŝŶƐŽŶ͛ƐĚŝƐĞĂƐĞƉĂƟĞŶƚƐǁŝƚŚǁĞƐƚĞƌŶƵƌŽƉĞĂŶ ĂŶĐĞƐƚƌLJ͘tĞŝĚĞŶƟĨLJƚŚƌĞĞŶŽŶƐLJŶŽŶŵŽƵƐǀĂƌŝĂŶƚƐƚŚĂƚĂƌĞƉƌĞĚŝĐƚĞĚƚŽŚĂǀĞĂĚĂŵĂŐŝŶŐ ĞīĞĐƚ ŽŶ ƚŚĞ ĨƵŶĐƟŽŶ ŽĨ ƚŚĞ ŐĞŶĞ͘ dŚĞƐĞ ƉƵƚĂƟǀĞ ƌŝƐŬ ǀĂƌŝĂŶƚƐ ŝŶ CHCHD2 ĞŵƉŚĂƐŝnjĞ ƚŚĞĐƵƌƌĞŶƚƚŚŝŶŬŝŶŐƚŚĂƚŵŝƚŽĐŚŽŶĚƌŝĂůĚLJƐĨƵŶĐƟŽŶƉůĂLJƐĂŶŝŵƉŽƌƚĂŶƚƉĂƌƚŝŶW͘/ŶƚŚŝƐ ƌĞŐĂƌĚ͕ĚLJƐƌĞŐƵůĂƟŽŶŽĨW/E<ϭͬƉĂƌŬŝŶƉĂƚŚǁĂLJ͕ǁŚŝĐŚŝƐĞƐƉĞĐŝĂůůLJĂƐƐŽĐŝĂƚĞĚƚŽLJŽƵŶŐͲ onset PD, might result in damaged mitochondria.1_{/ŶƚĞƌĞƐƟŶŐůLJ͕ƚŚƌĞĞƉĂƟĞŶƚƐĐĂƌƌLJŝŶŐĂŶ}

ĞdžŽŶŝĐCHCHD2 variant in our cohort were diagnosed with PD at an early age (< 45 years). dŚĞ ĨƌĞƋƵĞŶĐLJ ŽĨ ŶĞǁůLJ ŝĚĞŶƟĮĞĚ ƉƵƚĂƟǀĞ ǀĂƌŝĂŶƚƐ ŝŶ ŽƵƌ ĞdžŽŵĞ ƐĞƋƵĞŶĐŝŶŐ ĚĂƚĂƐĞƚ ƌĂŶŐĞĚ ĨƌŽŵ Ϭ͘Ϭϰй ƚŽ Ϭ͘Ϭϴй͕ ŚŝŐŚĞƌ ƚŚĂŶ ƚŚĂƚ ƌĞƉŽƌƚĞĚ ŝŶ dž ;Ϭ͘ϬϬϯй ƚŽ 0.03%),18_{ǁŚŝĐŚƐƵŐŐĞƐƚƚŚĂƚƚŚĞLJĂƌĞůŝŬĞůLJƚŽďĞƌĂƌĞƌŝƐŬǀĂƌŝĂŶƚƐĨŽƌWŝŶƉŽƉƵůĂƟŽŶƐŽĨ} ƵƌŽƉĞĂŶĂŶĐĞƐƚƌLJ͘,ŽǁĞǀĞƌ͕ƚŚĞƌĞǁĂƐŶŽĂƐƐŽĐŝĂƟŽŶĨŽƌƚŚĞƚǁŽŵŽƌĞĐŽŵŵŽŶǀĂƌŝĂŶƚƐ ƚŚĂƚǁĞƌĞĐĂƉƚƵƌĞĚďLJƚŚĞEĞƵƌŽyŐĞŶŽƚLJƉŝŶŐĂƌƌĂLJ͘KŶĞŽĨƚŚĞƐĞƚǁŽEĞƵƌŽyǀĂƌŝĂŶƚƐ͕ ƌƐϭϯϳϵϲϱϱϲϮ͕ǁĂƐƌĞƉŽƌƚĞĚĂƐƐŝŐŶŝĮĐĂŶƚůLJĂƐƐŽĐŝĂƚĞĚƚŽWďLJ&ƵŶĂLJĂŵĂĞƚĂů͘KƵƌ> ƉĂƩĞƌŶĂŶĂůLJƐĞƐƌĞǀĞĂůĞĚƚŚĂƚƚŚŝƐǀĂƌŝĂŶƚŝƐŽŶƚŚĞƐĂŵĞŚĂƉůŽƚLJƉĞĂƐǀĂƌŝĂŶƚƐƌƐϴϰϬϲ ĂŶĚƌƐϭϮϰϰϴϵϲ͕ďŽƚŚŝŶƚŚĞƵƌŽƉĞĂŶĂƐƚŚĞĂƐƚͲƐŝĂŶƉŽƉƵůĂƟŽŶ͘/ƚŝƐƚŚĞƌĞĨŽƌĞŚŝŐŚůLJ ƉƌŽďĂďůĞ͕ƚŚĂƚƚŚĞƐĞƚǁŽĂĚĚŝƟŽŶĂůǀĂƌŝĂŶƚƐǁŽƵůĚĂůƐŽůĂĐŬĂŶĂƐƐŽĐŝĂƟŽŶŝŶŽƵƌ/W' NeuroX cohort, if they would have been genotyped. We therefore conclude that in our /W'ĐŽŚŽƌƚCHCHD2 is not a common risk factor for PD, which is in agreement with the ƌĞƐƵůƚƐŽĨĂ:ĂƉĂŶĞƐĞ't^ĂŶĚƚŚĞŵŽƐƚƌĞĐĞŶƚĂŶĚůĂƌŐĞƐƚWŵĞƚĂͲĂŶĂůLJƐŝƐ͘19,20

2

ϭ͘ dƌŝŶŚ:͕&ĂƌƌĞƌD͘ĚǀĂŶĐĞƐŝŶƚŚĞŐĞŶĞƟĐƐ of Parkinson disease. Nature reviews

NeurologyϮϬϭϯ͖ϵ;ϴͿ͗ϰϰϱͲϱϰ͘

Ϯ͘ &ƵŶĂLJĂŵĂD͕KŚĞ<͕ŵŽd͕ĞƚĂů͘,,Ϯ ŵƵƚĂƟŽŶƐ ŝŶ ĂƵƚŽƐŽŵĂů ĚŽŵŝŶĂŶƚ ůĂƚĞͲ onset Parkinson’s disease: a genome-wide linkage and sequencing study. Lancet

neurology 2015.

ϯ͘ >ŝ ,͕ ƵƌďŝŶ Z͘ &ĂƐƚ ĂŶĚ ĂĐĐƵƌĂƚĞ ƐŚŽƌƚ read alignment with Burrows-Wheeler

transform. ŝŽŝŶĨŽƌŵĂƟĐƐ ;KdžĨŽƌĚ͕

ŶŐůĂŶĚͿϮϬϬϵ͖Ϯϱ;ϭϰͿ͗ϭϳϱϰͲϲϬ͘

ϰ͘ DĐ<ĞŶŶĂ͕,ĂŶŶĂD͕ĂŶŬƐ͕ĞƚĂů͘dŚĞ 'ĞŶŽŵĞ ŶĂůLJƐŝƐ dŽŽůŬŝƚ͗ Ă DĂƉZĞĚƵĐĞ ĨƌĂŵĞǁŽƌŬ ĨŽƌ ĂŶĂůLJnjŝŶŐ ŶĞdžƚͲŐĞŶĞƌĂƟŽŶ DNA sequencing data. Genome research ϮϬϭϬ͖ϮϬ;ϵͿ͗ϭϮϵϳͲϯϬϯ͘

ϱ͘ tĂŶŐ <͕ >ŝ D͕ ,ĂŬŽŶĂƌƐŽŶ ,͘ EEKsZ͗ ĨƵŶĐƟŽŶĂů ĂŶŶŽƚĂƟŽŶ ŽĨ ŐĞŶĞƟĐ ǀĂƌŝĂŶƚƐ from high-throughput sequencing data.

EƵĐůĞŝĐĂĐŝĚƐƌĞƐĞĂƌĐŚϮϬϭϬ͖ϯϴ;ϭϲͿ͗Ğϭϲϰ͘

ϲ͘ <ƵŵĂƌ W͕ ,ĞŶŝŬŽī ^͕ EŐ W͘ WƌĞĚŝĐƟŶŐ ƚŚĞ ĞīĞĐƚƐ ŽĨ ĐŽĚŝŶŐ ŶŽŶͲƐLJŶŽŶLJŵŽƵƐ ǀĂƌŝĂŶƚƐŽŶƉƌŽƚĞŝŶĨƵŶĐƟŽŶƵƐŝŶŐƚŚĞ^/&d algorithm. Nature protocols ϮϬϬϵ͖ ϰ;ϳͿ͗ 1073-81.

ϳ͘ WŽůůĂƌĚ <^͕ ,ƵďŝƐnj D:͕ ZŽƐĞŶďůŽŽŵ <Z͕ ^ŝĞƉĞů ͘ ĞƚĞĐƟŽŶ ŽĨ ŶŽŶŶĞƵƚƌĂů

ƐƵďƐƟƚƵƟŽŶ ƌĂƚĞƐ ŽŶ ŵĂŵŵĂůŝĂŶ

phylogenies. Genome research ϮϬϭϬ͖ 20(1): 110-21. ϴ͘ ĚnjŚƵďĞŝ /͕ ^ĐŚŵŝĚƚ ^͕ WĞƐŚŬŝŶ >͕ Ğƚ Ăů͘  ŵĞƚŚŽĚ ĂŶĚ ƐĞƌǀĞƌ ĨŽƌ ƉƌĞĚŝĐƟŶŐ ĚĂŵĂŐŝŶŐ ŵŝƐƐĞŶƐĞ ŵƵƚĂƟŽŶƐ͘ Nature ŵĞƚŚŽĚƐϮϬϭϬ͖ϳ;ϰͿ͗ϮϰϴͲϵ͘ ϵ͘ ŚƵŶ^͕&ĂLJ:͘/ĚĞŶƟĮĐĂƟŽŶŽĨĚĞůĞƚĞƌŝŽƵƐ ŵƵƚĂƟŽŶƐ ǁŝƚŚŝŶ ƚŚƌĞĞ ŚƵŵĂŶ ŐĞŶŽŵĞƐ͘ Genome researchϮϬϬϵ͖ϭϵ;ϵͿ͗ϭϱϱϯͲϲϭ͘ ϭϬ͘ ^ĐŚǁĂƌnj :D͕ ZŽĚĞůƐƉĞƌŐĞƌ ͕ ^ĐŚƵĞůŬĞ D͕ ^ĞĞůŽǁ ͘ DƵƚĂƟŽŶdĂƐƚĞƌ ĞǀĂůƵĂƚĞƐ ĚŝƐĞĂƐĞͲĐĂƵƐŝŶŐ ƉŽƚĞŶƟĂů ŽĨ ƐĞƋƵĞŶĐĞ ĂůƚĞƌĂƟŽŶƐ͘ EĂƚƵƌĞ ŵĞƚŚŽĚƐ ϮϬϭϬ͖ ϳ;ϴͿ͗ 575-6. ϭϭ͘ <ŝƌĐŚĞƌ D͕ tŝƩĞŶ D͕ :ĂŝŶ W͕ K͛ZŽĂŬ :͕ ŽŽƉĞƌ 'D͘  ŐĞŶĞƌĂů ĨƌĂŵĞǁŽƌŬ ĨŽƌ ĞƐƟŵĂƟŶŐ ƚŚĞ ƌĞůĂƟǀĞ ƉĂƚŚŽŐĞŶŝĐŝƚLJ ŽĨ ŚƵŵĂŶŐĞŶĞƟĐǀĂƌŝĂŶƚƐ͘ϮϬϭϰ͖ϰϲ;ϯͿ͗ϯϭϬͲϱ͘ ϭϮ͘ EĂůůƐ D͕ ƌĂƐ :͕ ,ĞƌŶĂŶĚĞnj '͕ Ğƚ Ăů͘ EĞƵƌŽy͕ Ă ĨĂƐƚ ĂŶĚ ĞĸĐŝĞŶƚ ŐĞŶŽƚLJƉŝŶŐ ƉůĂƞŽƌŵ ĨŽƌ ŝŶǀĞƐƟŐĂƟŽŶ ŽĨ ŶĞƵƌŽĚĞŐĞŶĞƌĂƟǀĞĚŝƐĞĂƐĞƐ͘Neurobiology of aging 2014.

13. Wu MC, Lee S, Cai T, Li Y, Boehnke M, Lin y͘ ZĂƌĞͲǀĂƌŝĂŶƚ ĂƐƐŽĐŝĂƟŽŶ ƚĞƐƟŶŐ ĨŽƌ sequencing data with the sequence kernel ĂƐƐŽĐŝĂƟŽŶ ƚĞƐƚ͘ ŵĞƌŝĐĂŶ ũŽƵƌŶĂů ŽĨ ŚƵŵĂŶŐĞŶĞƟĐƐϮϬϭϭ͖ϴϵ;ϭͿ͗ϴϮͲϵϯ͘ ϭϰ͘ ƵƚŽŶ ͕ ƌŽŽŬƐ >͕ ƵƌďŝŶ ZD͕ Ğƚ Ăů͘  ŐůŽďĂů ƌĞĨĞƌĞŶĐĞ ĨŽƌ ŚƵŵĂŶ ŐĞŶĞƟĐ ǀĂƌŝĂƟŽŶ͘NatureϮϬϭϱ͖ϱϮϲ;ϳϱϳϭͿ͗ϲϴͲϳϰ͘ ϭϱ͘ zĂƚĞƐ ͕ ŬĂŶŶŝ t͕ ŵŽĚĞ DZ͕ Ğƚ Ăů͘ Ensembl 2016. EƵĐůĞŝĐ ĂĐŝĚƐ ƌĞƐĞĂƌĐŚ ϮϬϭϲ͖ϰϰ;ϭͿ͗ϳϭϬͲϲ͘ ϭϲ͘ ĂŶĞĐĞŬ W͕ ƵƚŽŶ ͕ ďĞĐĂƐŝƐ '͕ Ğƚ Ăů͘ dŚĞ ǀĂƌŝĂŶƚ ĐĂůů ĨŽƌŵĂƚ ĂŶĚ s&ƚŽŽůƐ͘ ŝŽŝŶĨŽƌŵĂƟĐƐ ;KdžĨŽƌĚ͕ ŶŐůĂŶĚͿ ϮϬϭϭ͖ 27(15): 2156-8. ϭϳ͘ ĂƌƌĞƩ :͕ &ƌLJ ͕ DĂůůĞƌ :͕ ĂůLJ D:͘ ,ĂƉůŽǀŝĞǁ͗ ĂŶĂůLJƐŝƐ ĂŶĚ ǀŝƐƵĂůŝnjĂƟŽŶ ŽĨ LD and haplotype maps. ŝŽŝŶĨŽƌŵĂƟĐƐ

;KdžĨŽƌĚ͕ŶŐůĂŶĚͿϮϬϬϱ͖Ϯϭ;ϮͿ͗ϮϲϯͲϱ͘ ϭϴ͘ džŽŵĞŐŐƌĞŐĂƟŽŶŽŶƐŽƌƟƵŵ;džͿ͕ D ;hZ>͗ ŚƩƉ͗ͬͬĞdžĂĐ͘ďƌŽĂĚŝŶƐƟƚƵƚĞ͘ŽƌŐͿ [April 2015]. 2015. ϭϵ͘ ^ĂƚĂŬĞ t͕ EĂŬĂďĂLJĂƐŚŝ z͕ DŝnjƵƚĂ /͕ Ğƚ Ăů͘ 'ĞŶŽŵĞͲǁŝĚĞĂƐƐŽĐŝĂƟŽŶƐƚƵĚLJŝĚĞŶƟĮĞƐ ĐŽŵŵŽŶ ǀĂƌŝĂŶƚƐ Ăƚ ĨŽƵƌ ůŽĐŝ ĂƐ ŐĞŶĞƟĐ risk factors for Parkinson’s disease. Nature

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ϮϬ͘ EĂůůƐD͕WĂŶŬƌĂƚnjE͕>ŝůůD͕ĞƚĂů͘>ĂƌŐĞͲ scale meta-analysis of genome-wide ĂƐƐŽĐŝĂƟŽŶĚĂƚĂŝĚĞŶƟĮĞƐƐŝdžŶĞǁƌŝƐŬůŽĐŝ for Parkinson’s disease. EĂƚƵƌĞ ŐĞŶĞƟĐƐ ϮϬϭϰ͖ϰϲ;ϵͿ͗ϵϴϵͲϵϯ͘