Analysis of the PINK1 Gene in a Large Cohort of Cases with Parkinson Disease

ORIGINAL CONTRIBUTION Analysis of the PINK1 Gene in a Large Cohort of Cases With Parkinson Disease

Ekaterina Rogaeva, PhD; Janel Johnson, BS; Anthony E. Lang, MD; Cindy Gulick, BS; Katrina Gwinn-Hardy, MD; Toshitaka Kawarai, MD; Christine Sato, MS; Angharad Morgan, PhD; John Werner, BS; Robert Nussbaum, MD; Agnes Petit, PhD; Michael S. Okun, MD; Aideen McInerney, MS; Ronald Mandel, MD; Justus L. Groen; Hubert H. Fernandez, MD; Ron Postuma, MD; Kelly D. Foote, MD; Shabnam Salehi-Rad, MS; Yan Liang, PhD; Sharon Reimsnider, PhD; Anurag Tandon, PhD; John Hardy, PhD; Peter St George-Hyslop, MD; Andrew B. Singleton, PhD

Background: Mutations in the PTEN-induced kinase tion frequencies were evaluated in additional controls (100 (PINK1) gene located within the PARK6 locus on white and 50 Filipino subjects). chromosome 1p35-p36 have recently been identified in patients with recessive early-onset Parkinson Results: We identified 27 variants, including the first disease. reported compound heterozygous mutation (Glu240Lys and Leu489Pro) and a homozygous Leu347Pro mu- Objective: To assess the prevalence of PINK1 muta- tation in 2 unrelated young-onset Parkinson disease tions within a series of early- and late-onset Parkinson patients. disease patients living in North America. Conclusion: Autosomal recessive mutations in PINK1 Design: All coding exons of the PINK1 gene were se- are a rare cause of young-onset Parkinson disease. quenced in a series of 289 Parkinson disease patients and 80 neurologically normal control subjects; the muta- Arch Neurol. 2004;61:1898-1904

ARKINSON DISEASE (PD) IS A logues) (Figure 1). PINK1 is transcrip- neurodegenerative disorder tionally transactivated by the phosphates that is characterized by pro- and tensin homologue (PTEN) gene, an gressive dysfunction of move- oncogene involved in several signal trans- ment due to the predominant duction pathways.9-11 PINK1 shows vari- degenerationP of dopaminergic neurons in able levels of expression in different can- the basal ganglia.1 Although familial PD cer cell types9,10; however, it is not clear represents less than 10% of all cases, par- whether the connection between PINK1 and kinsonism-causing mutations have been PTEN is relevant to PD. Preliminary cell cul- defined in at least 4 different genes: SNCA,2 ture investigations using epitope-tagged, parkin,3 DJ1,4 and possibly UCHL1.5 Sev- overexpressed exogenous PINK1 have sug- eral additional PD loci have been identi- gested that PINK1 is located in mitochon- fied by genetic linkage methods,6 includ- dria and may exert a protective effect on the ing the PARK6 locus on chromosome cell, which is abrogated by mutations, re- 1p35-p36, which is responsible for a resulting in an increased susceptibility to cel- cessive early-onset form of disease.7 lular stress.8 Valente and colleagues8 have recently The prevalence of PINK1 mutations in reported that mutations in the PTEN- PD, however, remains unknown. We now induced kinase (PINK1) gene cause the describe the results of the first popula- PARK6 form of disease. In 3 PD families, tion study, to our knowledge, of PINK1 they discovered a homozygous nonsense within a series of early- and late-onset PD mutation (Trp437stop) and a homozy- patients living in North America. We re- gous Gly309Asp mutation. port several novel PINK1 variants, includ- PINK1 encodes a putative serine- ing a compound heterozygous mutation Author Affiliations are listed at threonine protein kinase, which is highly (Glu240Lys and Leu489Pro) and a ho- the end of this article. conserved in evolution (approximately mozygous substitution (Leu347Pro) in 2 Financial Disclosure: None. 75%-85% identity in mammalian ortho- PD patients.

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©2004 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/29/2021 1. M A V R Q A L G R G L Q L G R A L L L R F T G K P G R A Y G L ------G R P G P 2. M A V R Q A L G R G L Q L G R A L L L R F A P K P G P L F G W ------G K P G P 3. M A V R Q A L G R G L Q L G R A L L L R F A P K P G P V S G W ------G K P G P 4. M S F R L L T T R L Y K H G R L L V Q N Y L K R D I H V S N V W T G P - - T E S T K V N K I N E T L R H D L R I N D F R 5. M S V R L L T V R L I K H G R Y I L R S Y C K R D I H A N I L D Q N Q L K T R S K R G F P L P S T A A N V L R T T P Q Q 6. - - - - M S M K R F G K A A Y R I A N E L V A K G G R L P I F ------Q R F L P 7. - - - - M S M K R F G K A A Y R I A N E F V A R S G R I S I F ------Q R I L P

1. A - - - A G C V R G E R P G W A A G P G A E P R R V G L G L P N ------R L R F F R Q S V A G L A A R L 2. A - - - A A W G R G E R P G R V V S P G A Q P R P V G L P L P D ------R Y R F F R Q S V A G L A A R I 3. G - - - A A W G R G E R P G R V S S P G A Q P R P L G L P L P D ------R Y R F F R Q S V A G L A A R I 4. ------N N S F L R F G N Q A R R L F I D N V L N R V T N P Y 5. A - - - A K S V V N V V P R T I N S P S G S P F N G S G S S P T S S S G I F R V G Q H A R K L F I D N I L S R V T T T Y 6. R I F P A T Y N L G V H V V L ------K ------K A P F P R Q N A L R I A R L V 7. R I F P A T H N L G V H V V L ------K ------R S P F P R Q N A L R I A R L V

1. Q R Q F V V R A W - - - - - G C A G P C G R A V F L A F G L G L G L I E E K Q A E S R R A V S A C Q E I Q - A I F T Q K 2. Q R Q F M V R A R - - - - - G G A G P C G R A V F L A F G L G L G L I E E K Q A E G R R A A S A C Q E I Q - A I F T Q K 3. Q R Q F V V R A R - - - - - G G A G P C G R A V F L A F G L G L G L I E E K Q A E S R R A A S A C Q E I Q - A I F T Q K 4. S V D L R L Q A T K K L L Y G D S T P F F A L V G V S L A S G D G V L - - - - T K N D E L E A V C W E I R H A M S N F Q 5. S E D L R Q R A T R K L F F G D S A P F F A L I G V S L A S G S G V L - - - - S K E D E L E G V C W E I R E A A S R L Q 6. T R H G R V F R P - - - - - F S S V I I E R H R F Q N Q N D W R R K F Q P I R K E L P R N V D L V E R I R - Q I F G N S 7. T R H G R F F R P - - - - - F S S V I I E R H R F Q N K D D W R H K L E P L R K Q Q S K S V D L V E R I K - Q I F G N S

1. S K P G P D P L D T R R L Q G F R L E E Y L I G Q S I G K G C S A A V Y E A T M P T L P Q N L E ------2. T K R V S D P L D T R C W Q G F R L E D Y L I G Q A I G K G C N A A V Y E A T M P T L P Q H L E ------3. N K Q V S D P L D T R R W Q G F R L E D Y L I G Q A I G K G C N A A V Y E A T M P T L P Q H L E ------4. Q K V G E K D I E S R L D E E F G I E N L N I G K P I A K G C S A V V Y A A S L K E S T V S D N D D M N C N D I H N S K 5. N A W N H D E I S D T L D S K F T I D D L E I G P P I A K G C A A V V Y A A D F K K D V A S D G A S L H T D A Q P Q A T 6. L R Y N E D - L K S T E W P N - R I D S Y E F G E F L G Q G C N A A V Y S A R L A N S D A E S S G ------7. V R Y N E D - L K S S E W P N - R V D S Y E F G E F L G Q G C N A A V Y S A K L A N S D I E I S ------

1. ------2. ------3. ------4. S S - - - S N G K C P N E A W T G D E P E L S G Q R S D G I S P L L L P E R T A S E A F H Q M M N E S N A Q T R T N N N 5. P A F A P N S W S T H E M M S P L Q N M S R F V H N F G G S V D N V F H Y S Q P S A A S D F V G A Q S R E Q D Q R H H - 6. ------7. ------

1. ------V T K S T G L L P G R G P G T S A P G E G Q E R A A G A P A F P L A I K M M W N I S A G S S S E A 2. ------K A K H L G L I - G K G P D V V L K G A D G E Q A P G T P T F P F A I K M M W N I S A G S S S E A 3. ------K A K H L G L L - G K G P D V V S K G A D G E Q A P G A P A F P F A I K M M W N I S A G S S S E A 4. R K V R F N S E T R I R T M S E N S A S N H E T I G H Y Q P D S I E Q D Y T I E E Y P L A L K M M F N Y D I Q N N A M A 5. - - E Q Q Q H Q N Q E Q E Q H Q N Q E P S S S A F N V T S P A N S N I N S S V D S Y P L A L K M M F N Y D I Q S N A L S 6. ------N T H Y G A G F - N E V T N I L A E I P P V S K V - A Q K K F P L A I K L M F N F E H D R D G D A 7. ------N T K Y G A G F - N E V T N I L A E M P P V S K V - I E K K Y P L A I K L M F N F E H D R D G D A

1. I L - N T M S Q E L V P A S R V A - - L A G E Y G A V T Y R K S K R G P K Q L A P H P N I I R V L R A F T S S V P L L P 2. I L - S K M S Q E L V P A S R V A - - L D G E Y G A V T Y R R S R D G P K Q L A P H P N I I R V F R A F T S S V P L L P 3. I L - S K M S Q E L V P A S R M A - - L D G E Y G A V T Y R R S R D G P K Q L A P H P N I I R V F R A F T S S V P L L P 4. I L - K A M Y R E T V P A K R R T - - V D - N A - - - W E K S L M E K T N F V P P H P N I V E M Y G V F C D Q V P D L S 5. I L - R A M Y K E T V P A R Q R G - - M N - E A A D E W E R L L Q N Q T V H L P R H P N I V C M F G F F C D E V R N F P 6. H L L K S M G N E L A P Y P N A A K L L N G Q M G ------T F R P L P A K H P N V V R I Q T A F I D S L K V L P 7. H L W S S M G N E L A P Y P N A A K L L N G R M G ------N F K P L P A K H P N V V R I Q T A F V D S L K V L P

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METHODS In addition, 50 Filipino controls were used to assess the population-specific allele frequency of the 347Pro variant.

SUBJECTS GENETIC ANALYSIS Informed written consent was obtained from all individuals in- Total RNA and genomic DNA were isolated from blood samples volved in the study. Standard neurological clinical examination using QIAGEN extraction kits (Valencia, Calif). Mutations in was performed on all participants recruited from several North parkin and DJ1 had been excluded in all patients as previously American clinics (the diagnosis of PD was based on published described.4,13 The entire open reading frame of PINK1 was se- criteria12). The sample characteristics are summarized in Table 1. quenced in all patients and in 80 NIH controls. The 8 exons The 209 unrelated subjects collected at the National Institutes with their exon-intron boundaries were amplified using poly- of Health (NIH) were diagnosed as having PD by neurologists merase chain reaction conditions (Table 2). For proband 4685, from the NIH and the University of Florida; 41% of the patients the reverse transcription–polymerase chain reaction products had an early onset (Ͻ50 years of age). The second set of 80 un- of PINK1 were amplified with primers placed in exons 1 and related subjects was collected at the Movement Disorders Cen- 8, they were cloned using a TA cloning kit (Invitrogen, Carls- tre of the Toronto Western Hospital (the subjects were prese- bad, Calif), and then 3 randomly selected clones were selected for having an early onset and a positive family history). quenced. Mutations were detected by direct inspection of the Age- and sex-matched normal control subjects were recruited fluorescent chromatographs and analysis using SeqScape soft- from the same population, including 100 individuals from Toronto ware version 1.0 (Applied Biosystems, Foster City, Calif). The and 80 from NIH sites. The NIH controls were completely frequencies of the Glu240Lys and Leu489Pro mutations were sequenced, while the Toronto controls were used to estimate evaluated in 100 controls using SNaPshot (Applied Biosys- the frequency of the mutations found in the probands. tems). The frequency of the Leu347Pro in Filipino controls was

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1. G A L V D Y P D V L - P S R L H P E G L G H G R T L F L V M K N Y P C T L R Q Y L C V N T P S P R L A A M M L L Q L L E 2. G A L A D Y P D M L - P P H Y Y P E G L G H G R T L F L V M K N Y P C T L R Q Y L E E Q T P S S R L A T M M T L Q L L E 3. G A L A D Y P D M L - P P H Y Y P E G L G H G R T L F L V M K N Y P C T L R Q Y L E E Q T P S S R L A T M M T L Q L L E 4. M S A T L Y P M A L - P Q R L N P Q G Y G R N M S L F L L M K R Y N I N L K D Y L R Q P G V D M R T R I L L F A Q L L E 5. D G H L L Y P V A Q - P Q R I N P Q G Y G R N M S L Y L L M K R Y D H S L R G L L D S Q D L S T R N R I L L L A Q M L E 6. D A I E R Y P D A L H T A R W Y E S I A S E P K T M Y V V M R R Y R Q T L H E Y V W T R H R N Y W T G R V I I A Q L L E 7. D A L E R Y P D A L H T A R W Y E S I A S Q P K T M Y V V M R R Y R Q T L H D Y V W T H H R N Y W T G R V M V A Q L L E

1. G V D H L V Q Q G I A H R D L K S D N I L V E L D - P D G C P W L V I A D F G C C L A D E S I G L Q L P F S S W Y V D R 2. G V D H L V Q Q G I A H R D L K S D N I L V E W D - S D G C P W L V I S D F G C C L A D Q H V G L R L P F N S S S V E R 3. G V D H L V Q Q G I A H R D L K S D N I L V E W D - S D G C P W L V I S D F G C C L A D E R V G L Q L P F N S S S V E R 4. A V A H L N R H G V S H R D I K S D N I L I E L R - P N M P P T L V L T D F G C C I A D K R H G L R I P Y T S D E I D K 5. A V N H L S R H G V A H R D L K S D N V L I E L Q - D D A A P V L V L S D F G C C L A D K V H G L R L P Y V S H D V D K 6. A C T L L H K H K V A Q R D M K S D N I L L E Y D F D D E I P Q L V V A D F G C A L A C D - - N W Q V D Y E S D E V S L 7. A C T Y L H K H K V S Q R D M K S D N I L L E Y D L D D E I P Q L V I A D F G C A L A S D - - D W T V L Y E S D D V S L

1. G G N G C L M A P E V S T A R P G P R A V I D Y S K A D A W A V G A I A Y E I F G L V N P F Y ------G Q G 2. G G N G S L M A P E V S T A H S G P S A V I D Y S K A D T W A V G A I A Y E I F G L A N P F Y ------G Q G 3. G G N G S L M A P E V S T A H S G P H A V I D Y S K A D T W A V G A I A Y E I F G L A N P F Y ------G Q G 4. G G N V A L M A P E I I E Q L P G T F A M L N Y T K A D L W A C G A I A Y E I F G S N N P F Y S ------D V 5. G G N A A L M A P E I F N T M P G P F A V L N Y G K A D L W A C G A L A Y E I F G N R N P F Y S S S G G M A R E R G E M 6. G G N A K T K A P E I A T A V P G K N V K V N F E M A D T W A A G G L S Y E V L T R S N P F Y ------7. G G N T K T R A P E I A T A V P G K N K K V N F E M A D T W A A G G L S Y E I L T R S N P F Y ------

1. K A H L E S R S Y Q E A Q L P A L P E S V P P D V R Q L V R A L L Q R E A S K R P S A R V A A N V L H L S L W G E H - - 2. S A H L E S R S Y Q E A Q L P E M P K S V P P E A R R L V R S L L Q R E A S K R P S A R L A A N V L H L S L W G E H - - 3. S A H L E S R S Y Q E A Q L P E M P K S V P P E T R Q L V R S L L Q R E A N K R P S A R I A A N V L H L S L W G E H - - 4. N S A L K N T T Y E E D M L P A M D Q N V P R L I Q C L V Q N I L Q R N P S K R L S P D I A A N V V Q L F L W S P S S W 5. T L S L R N S D Y R Q D Q L P P M S D A C P P L L Q Q L V Y N I L N P N P S K R V S P D I A A N V V Q L F L W A P S N W 6. - K L L D T A T Y Q E S E L P A L P S R V N F V A R D V I F D L L K R D P N E R V K P N I A A N A L N L S L F R M G E D 7. - K H L D T A T Y E E S Q L P A L P S R V N F V T R D V I F D L L K R N P N E R V K P N T A A N A V N L S L F R M G E D

1. ------I L A L K N L K L D K M V G W L L Q Q S A A T L L - - A N R L T E K C C V 2. ------L L A L K N L K L D K M I A W L L Q Q S A A T L L - - A D R L R E K S C V 3. ------L L A L K N L K L D K M I A W L L Q Q S A A T L L - - A D R L R E K S C V 4. L R D R Y V P S S N E I L Q W L L S L T T K I L C E ------G P L R V T P D - - - - - G T M G R R T Y T 5. L K A G G M P N S P E I L Q W L L S L T T K I M C E ------G R P Q M G A G L M P V A S C G N R R A Y V 6. V K Q M M E K C G I S Q M T T L L A G S S K V L S Q K I N S R L D K V M N L I T A E T I M A N L - - A P H L I S R A - - 7. V R Q M M E K C G I S Q M S T L L A G S T K V L S Q K F N S H L D K V I N L I T A E T I I S N L - - A P H L I S R A - -

1. E T K M K M L F L A N L E C E T L C Q A ------A L L L C S W R A A L - - - - - 2. E T K L Q M L F L A N L E C E A L C Q A ------A L L L S S W R A A P - - - - - 3. E T K L Q M L F L A N L E C E A L C Q A ------A L L L S S W R A A P - - - - - 4. E Y L L I A S F L T R V R L E R I K R A L D W ------I H N V N A ------5. E Y L L I C S F L A R A R L R R I R G A L N W ------I Q N V V A ------6. E R Q L R A T F L S R M N R E D I W R S L Q Y F F P A G V Q L D T P A T S S D C L E T I S S L M S S F S N D S E N Y E K 7. E R Q L R A T F L S R M N R E D I W Q S L K Y F F P P G V P L N T P A T S S D C F E S I S S L I S S L S N G S Q D F E M

1. ------2. ------3. ------4. ------5. ------6. Q Q K P A K N G Y N N V P L L L R N V I R T D A D G I N G I V H R V R S K 7. Q K Q P A R N G Y N N V P I L L R H V I R T N S D G I D G I V H R V R S K

Figure 1. Multiple alignment of PINK1 orthologues using clustal format for T-Coffee version 1.41. Mutations found in patients 4685 and 521-1 are highlighted (serine-threonine protein kinase domains are underlined for human orthologue). Line 1, Homo sapiens AAH28215 GI:20381329. Line 2, Mus musculus NP_081156 GI:28076909. Line 3, Rattus norvegicus XP_216565 GI:34872205. Line 4, Anopheles gambiae XP_313587 GI:31209241. Line 5, Drosophila melanogaster NP_572340 GI:24640183. Line 6, Caenorhabditis elegans NP_495017.1 GI:17532823. Line 7, Caenorhabditis briggsae CAE59093 GI:39596866.

evaluated using a restriction digest assay. Exon 5 was ampli- We also found 2 cases (from the Toronto set) in fied as already described, it was digested overnight with 0.3 U whom both alleles of PINK1 were mutated. In proband of HpaII at 37°C, and the resulting restriction fragments were 4685 (PD:1 family), we found a compound heterozy- resolved on a 1.5% agarose gel. gous nonsynonymous mutation (Figure 2): a heterozygous G→A mutation in exon 3 at genomic nucleotide RESULTS position 6480 (GAG→AAG) and a heterozygous t→C mutation in exon 7 at position 15754 (CTG→CCG) GENETIC STUDIES (NT 004610). The G→A 6480 mutation causes the substitution of glutamic acid to lysine at codon 240 Genomic DNA sequence analysis of the entire open frame (Glu240Lys). The t→C 15754 mutation causes the sub- of PINK1, the untranslated region, and all 5Ј and 3Ј intron- stitution of leucine to proline at codon 489 (Leu489Pro). exon boundaries was performed on 289 unrelated sub- The Glu240Lys and Leu489Pro mutations are in trans to jects with disease and on 80 neurologically normal con- one another, which was established by cloning and se- trol subjects. We identified 27 sequence variations quencing of reverse transcription–polymerase chain re- (Table 3), including 10 common sequence variations and action products and by mutation analysis of family mem- 8 single heterozygous substitutions with an allele fre- bers. The Glu240Lys and Leu489Pro mutations are quency of less than 1% that alter the amino acid sequence unique to the PD:1 family and are not present in 360 nor- (observed in 8 patients and 5 controls). mal chromosomes.

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Toronto Data Set National Institutes of Health Data Set

PD Controls PD Controls Sample Characteristic (n = 80) (n = 100) (n = 209) (n = 80) Age at onset, mean (range), y 41.2 (14-50) 65.2* 54.2 (18-83) 63.8* Sex Female 41% 59% 42% 54% Male 59% 41% 58% 45% Positive family history With 40 (50%) . . . 96 (46%) . . . Without 21 (26%) . . . 108 (52%) . . . Unknown 19 (24%) . . . 5 (2%) . . . White 91% 96% 79% 94%

*Age at examination.

Table 2. Primers Used for PINK1 Analysis*

Primer Name Exon Primer Sequence Fragment, Base Pair Annealing Temperature, °C Analysis 2733 Forward 1 5Ј-GCCCCAAGTTTGTTGTGAC 501 63.5 Sequencing 2769 Reverse 5Ј-CTCCGCTCGGCTTAGGAC 2765 Forward 2 5Ј-GTGGGGTTTCTGACCTCTCA 550 59 Sequencing 2736 Reverse 5Ј-GGCACCTTTCCTGTGGATAA 2737 Forward 3 5Ј-TACAGGCAGGGCTTACAAGG 250 59 Sequencing 2738 Reverse 5Ј-GCTGAGGACATAAGTGATGGA 2739 Forward 4 5Ј-GTGTTGGTGTGGCCTTAGGT 373 59 Sequencing 2740 Reverse 5Ј-TGACCCTGTTCTCCAAAACC 2741 Forward 5 5Ј-ACGTATTGGGAGTCGTCGAT 299 59 Sequencing 2742 Reverse 5Ј-CCTGAAGAGTCAGTCCTAAATGC 2743 Forward 6 5Ј-TGCTGGTGGCTTTAGTAGGG 293 59 Sequencing 2744 Reverse 5Ј-ACAAGGCATCGAGTCTCCTG 2745 Forward 7 5Ј-CTCAAGCTCTGGGTTCCTTG 435 59 Sequencing 2746 Reverse 5Ј-CCCAAACCTGACCTTCACTC 2776 Forward 8 5Ј-GGGTAGAGGAAGAATTGGGTTG 529 59 Sequencing 2777 Reverse 5Ј-AGTTCTTCCATTTGCCAAGC 2767 Forward 1 5Ј-CCTAACCGTCTCCGCTTCTT 1677 59 RT-PCR 2775 Reverse 8 5Ј-CACCCCAGAGGCTTAGATGA 2778 Detection 3 5Ј-AAGCCATCTTGAACACAATGAGCCAG NA NA SNaPshot 2779 Detection 7 5Ј-AGACGTGAGACAGTTGGTGAGGGCAC NA NA SNaPshot

Abbreviations: NA, not applicable; RT-PCR, reverse transcription–polymerase chain reaction.

A second unrelated proband (521-1) of Filipino an- proband (at age 67) possesses the Leu489Pro mutation cestry was found to have a homozygous T→C substitu- (Figure 2B). tion in exon 5 at nucleotide position 12186, leading to Proband 521-1 is 67 years old and first noticed subtle an amino acid substitution of leucine to proline at codon right hand tremor early in the fourth decade of life. Ten 347 (Leu347Pro) (Figure 3B). This variant was not iden- years later, the patient developed stiffness, slowness, and tified in any of the sequenced control samples; how- pain on the right side, with freezing of gait, and was di- ever, 3 heterozygous carriers of this variation were found agnosed as having PD at age 55 years. The patient ini- among 50 Filipino control subjects. tially had an excellent response to levodopa-carbidopa but during the last 10 years has developed dyskinesia, CLINICAL FEATURES fluctuations, and cramping while not receiving levodopa- carbidopa. Cognition, eye movements, and autonomic Proband 4685 is 73 years old and developed typical, slowly function are normal. The proband’s family history dis- progressive PD at age 30, with good response to levodopa- plays an autosomal recessive mode of inheritance of PD carbidopa therapy. The proband’s family history dis- (2 siblings of the proband, who are not available for study, plays an autosomal recessive mode of inheritance of PD are reported to have PD) (Figure 3A). (Figure 2A). Both of the proband’s parents, who died af- ter age 70, did not have PD. Of 9 siblings, 3 developed PD (2 affected siblings are not available for study but are COMMENT reported to have had a similar course). The proband’s off- spring (unaffected at age 42) has inherited only the Our results support the prior suggestion that mutations Glu240Lys substitution, and an unaffected sibling of the in PINK1 are associated with recessive familial PD.8 In a

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No. of Variants Found Genomic Exon Substitution Nucleotide Position Codon PD Controls Comments 1C→T 283 Leu63Leu† Common Novel 2C→G 4323 Intronic† Common rs2298297 2 Deletion CT 4323-4324 Intronic† Common Novel 2G→A 4381 Intronic† Common rs2298298 3A→G 6453 Glu231Gly 1 0 Novel 3A→T 6466 Asn235Ile 0 1 Novel 3A→C 6470 Thr236Thr 1 0 Novel 3G→A 6480 Glu240Lys 1 0 Novel 4A→G 11046 Arg263Gly 0 1 Novel 4T→G 11147 Pro296Pro 1 0 Novel 4C→T 11207 Leu316Leu 2 0 Novel 4A→T 11211 Met318Leu 1 0 Novel 5 Deletion C 12086 Intronic 1 0 Novel 5G→A 12101 Intronic† Common rs3131713 5G→A 12161 Ala339Thr 2 0 Novel 5G→A 12164 Ala340Thr† Common rs3738136 5T→C 12186 Leu347Pro 1 3‡ Novel 5G→C 12237 Asp362His 0 1 Novel 6T→C 15100 Asp391Asp 0 1 Novel 6C→T 15221 Intronic† Common Novel 7C→T 15562 Pro425Ser 1 0 Novel 7G→A 15715 Glu476Lys 1 1 Novel 7T→C 15754 Leu489Pro 1 0 Novel 8A→C 17053 Asn521Thr† Common rs1043424 8A→G 17219 Ser576Ser 0 1 Novel 3Ј UTR T→A 17274 UTR† Common rs686658 3Ј UTR G→A 17277 UTR† Common Novel

Abbreviations: PD, Parkinson disease; UTR, untranslated region. *Homozygous or compound heterozygous mutations are in boldface. †Allele frequency is greater than 5% in patients and controls. ‡This variant was identified in 3 of 100 Filipino control chromosomes and was not present in 160 white control chromosomes.

data set of 289 patients, we found 2 probands in fami- onymous polymorphisms that affect nonconserved resi- lies with autosomal recessive inheritance: a patient with dues and that are present as low-frequency (Ͻ30%) alleles a compound heterozygous mutation (Glu240Lys and in PD-affected and non–PD-affected subjects (Table 3). Leu489Pro) and a patient with a homozygous Leu347Pro The present study does not have sufficient statistical power substitution. to discern whether some or all of these alleles are en- These substitutions were not found in homozygous riched in the PD-affected subjects and therefore might or heterozygous states in the initial series of 180 represent weaker-risk alleles for increased susceptibil- healthy controls. The Leu347Pro, which was identified ity to “less penetrant” late-onset forms of disease. Fu- as a homozygous change in a Filipino patient with ture studies in larger case-control data sets will be re- parkinsonism, was found as a heterozygous change in quired to address this issue. 3 of 50 Filipino control subjects. In the absence of seg- In support of the notion that heterozygous muta- regation data, the pathogenicity of these variants tions in genes causing recessive forms of early-onset PD remains unclear. In support of a role for these variants can also cause late-onset PD, there have been several late- in disease, all 3 are within the kinase domain, and all onset PD families with a single parkin mutation.14 How- change evolutionarily conserved amino acids (Figure 2). ever, the evidence linking these variants to disease has Biological plausibility may be strengthened by the avail- been equivocal, and a study15 has shown a high fre- ability of a kinase assay for this protein. The identifica- quency (approximately 3%) of heterozygous patho- tion of the 347Pro variant in 3% of Filipino control genic parkin mutations in neurologically healthy con- chromosomes suggests 2 possibilities: this change is a trols. It will be interesting to evaluate the nigrostriatal benign rare variant present in the Filipino population, dopamine system in the subjects with heterozygous PINK1 or this alteration is pathogenic when homozygous. If variants to assess the potential subclinical alterations in the latter were true, a Filipino allele frequency of 3% striatal dopamine function. Such alterations have been would suggest a population prevalence of approxi- found in patients heterozygous for parkin mutations, sug- mately 1 in 4000 for disease caused by a homozygous gesting that partial loss of function of the gene can cause 347Pro substitution. subclinical reduction in dopaminergic function.16 Our survey also uncovered several intronic and ex- It is conceivable that there may be further mutations onic sequence variants, including 2 common nonsyn- in the PINK1 gene within the series of patients exam-

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4685 5794 521-1 Leu489Pro Leu489Pro Homozygous Glu240Lys Leu347Pro

B 5809 Glu240Lys B

4685 521-1

C C A G C T T C T G G C T T G G A G C A G T G C C C

A G C T G C C G G A A G G

5809

C C A G C T T C T G G C T T G G A G C A G T G C C C Wild-type

5794

A G C T G C T G G A A G G C C A G C T C C T G G C T T G G A G C A G T G C C C Exon 5 Exon 7 CTG CCG CTG CCG Leu347Pro Leu489Pro

Figure 3. The pedigree structure of the PD (Parkinson disease):2 family (A). Figure 2. The pedigree structure of the PD (Parkinson disease):1 family (A). Affected individuals are shown as solid symbols, and the arrow points to the Affected individuals are shown as solid symbols, and the arrow points to the proband on whom complete sequencing analysis was performed. B, DNA proband on whom complete sequencing analysis was performed. The sex of sequence fluorescent chromatogram of the PINK1 Leu347Pro mutation individuals has been masked to protect family confidentiality. B, DNA observed in proband 521-1 shown with a wild-type sequence (the sequence sequence fluorescent chromatogram of the PINK1 mutations observed in 3 around the mutation site is shown, with the arrow pointing to a homozygous PD:1 family members available for study (the sequence around the mutation substitution). site is shown, with arrows pointing to heterozygous substitutions).

ined herein (the next step in the analysis of PINK1 should Accepted for Publication: September 13, 2004. include a quantitative polymerase chain reaction to evalu- Author Affiliations: Centre for Research in Neurode- ate the presence of a heterozygous deletion or inser- generative Diseases (Drs Rogaeva, Kawarai, Morgan, Petit, tion). The fact that mutations in PINK1 constitute a rare Liang, Tandon, and St George-Hyslop; Mss Sato and cause for early-onset parkinsonism does not negate a role Salehi-Rad; and Mr Groen) and Division of Neurology for this gene in the pathogenesis of PD. The localization (Dr Rogaeva), Department of Medicine, and Movement of PINK1 in mitochondria8 provides a potential link with Disorders Centre, Toronto Western Hospital (Drs Lang prior theories of mitochondrial deficits in PD; however, and Postuma), University of Toronto, and Division of Neu- further studies are needed to establish the subcellular, rology, Department of Medicine, University Health Net- cellular, and tissue distribution of PINK1 protein in work (Drs Lang and St George-Hyslop), Toronto, On- healthy tissues and in brain tissue affected by PD. tario; Molecular Genetics Section (Mss Johnson and Several lines of evidence suggest that PINK1 is likely to Gulick and Dr Singleton) and Laboratory of Neuroge- be a functional kinase: bioinformatic analysis suggests that netics (Drs Hardy and Singleton), National Institute on residues Gly193 to Leu507 comprise the catalytic kinase Aging, Neurogenetics Branch, National Institute of Neu- domain, and biochemical results suggest that PINK1 is ca- rological Disorders and Stroke (Dr Gwinn-Hardy and Mr pable of autophosphorylation.9 Finally, all reported PINK1 Werner), and Genetic Diseases Research Branch, Na- mutations affect the kinase domain. Because kinases are at- tional Human Genome Research Institute (Dr Nuss- tractive therapeutic targets, this genetic discovery may iden- baum and Ms McInerney), National Institutes of Health, tify novel therapeutic opportunities. Bethesda, Md; and Movement Disorders Center, Depart-

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©2004 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/29/2021 ments of Neurology, Neurosurgery, and Psychiatry, Uni- shi Trust, Tokyo, Japan (Dr Kawarai); and the National versity of Florida, Gainesville (Drs Okun, Mandel, Fer- Institutes of Health, Bethesda, Md. nandez, Foote, and Reimsnider). Acknowledgment: We thank the patients and their fami- Correspondence: Andrew B. Singleton, PhD, Molecular lies for taking part in this research. Genetics Section, National Institute on Aging, National Institutes of Health, 35 Convent Dr, Room 1A1015, Mail Stop Code 3707, Bethesda, MD 20892 (singleta@mail REFERENCES .nih.gov). Author Contributions: Study concept and design (Rogaeva, 1. Lang AE, Lozano AM. Parkinson’s disease: second of two parts. N Engl J Med. 1998;339:1130-1143. Nussbaum, Foote, Hardy, St George-Hyslop, and Singleton); 2. Polymeropoulos MH, Lavedan C, Leroy E, et al. Mutation in the ␣-synuclein gene acquisition of data (Rogaeva, Johnson, Lang, Gulick, Gwinn- identified in families with Parkinson’s disease. Science. 1997;276:2045-2047. Hardy, Sato, Werner, Nussbaum, Petit, Okun, McInerney, 3. Kitada T, Asakawa S, Hattori N, et al. Mutations in the parkin gene cause auto- Mandel, Groen, Fernandez, Foote, Reimsnider, St George- somal recessive juvenile parkinsonism. Nature. 1998;392:605-608. Hyslop, and Singleton); analysis and interpretation of data 4. Bonifati V, Rizzu P, van Baren MJ, et al. Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism. Science. 2003;299:256-259. (Rogaeva, Johnson, Gulick, Kawarai, Morgan, Postuma, 5. Leroy E, Boyer R, Auburger G, et al. The ubiquitin pathway in Parkinson’s disease. Salehi-Rad, Liang, Tandon, St George-Hyslop, and Single- Nature. 1998;395:451-452. ton); drafting of the manuscript (Rogaeva, Lang, Kawarai, 6. Gwinn-Hardy K. Genetics of parkinsonism. Mov Disord. 2002;17:645-656. Morgan, Hardy, St George-Hyslop, and Singleton); criti- 7. Valente EM, Bentivoglio AR, Dixon PH, et al. Localization of a novel locus for autosomal recessive early-onset parkinsonism, PARK6, on human chromo- cal revision of the manuscript for important intellectual con- some 1p35-p36. Am J Hum Genet. 2001;68:895-900. tent (Rogaeva, Johnson, Gulick, Gwinn-Hardy, Sato, 8. Valente EM, Abou-Sleiman PM, Caputo V, et al. Hereditary early-onset Parkin- Werner, Nussbaum, Petit, Okun, McInerney, Mandel, son’s disease caused by mutations in PINK1. Science. 2004;304:1158-1160. Groen, Fernandez, Postuma, Foote, Salehi-Rad, Liang, 9. Nakajima A, Kataoka K, Hong M, Sakaguchi M, Huh NH. BRPK, a novel protein Reimsnider, Tandon, St George-Hyslop, and Single- kinase showing increased expression in mouse cancer cell lines with higher meta- static potential. Cancer Lett. 2003;201:195-201. ton); statistical analysis (Singleton); obtained funding 10. Unoki M, Nakamura Y. Growth-suppressive effects of BPOZ and EGR2, two genes (Rogaeva, Nussbaum, St George-Hyslop, and Single- involved in the PTEN signaling pathway. Oncogene. 2001;20:4457-4465. ton); administrative, technical, and material support 11. Gary DS, Mattson MP. PTEN regulates Akt kinase activity in hippocampal neu- (Johnson, Lang, Gwinn-Hardy, Kawarai, Sato, Morgan, rons and increases their sensitivity to glutamate and apoptosis. Neuromolecular Med. 2002;2:261-269. Werner, Petit, Okun, McInerney, Groen, Postuma, Foote, 12. Hughes AJ, Daniel SE, Lees AJ. Improved accuracy of clinical diagnosis of Lewy Salehi-Rad, Liang, Tandon, Hardy, and Singleton); study body Parkinson’s disease. Neurology. 2001;57:1497-1499. supervision (Rogaeva, Nussbaum, Mandel, Reimsnider, 13. Hattori N, Kitada T, Matsumine H, et al. Molecular genetic analysis of a novel Hardy, St George-Hyslop, and Singleton). Drs Rogaeva parkin gene in Japanese families with autosomal recessive juvenile parkinson- and Singleton contributed equally to this article. ism: evidence for variable homozygous deletions in the parkin gene in affected individuals. Ann Neurol. 1998;44:935-941. Funding/Support: This work was supported by grants 14. West A, Periquet M, Lincoln S, et al. Complex relationship between parkin mu- from the Canadian Institutes of Health Research, Ot- tations and Parkinson disease [published correction appears in Am J Med Genet. tawa, Ontario; the Alzheimer Society of Ontario, Toronto; 2002;114:992]. Am J Med Genet. 2002;114:584-591. the Howard Hughes Medical Institute, Chevy Chase, Md 15. Lincoln SJ, Maraganore DM, Lesnick TG, et al. Parkin variants in North Ameri- can Parkinson’s disease: cases and controls. Mov Disord. 2003;18:1306-1311. (Dr St George-Hyslop); a Center of Excellence award (Dr 16. Hilker R, Klein C, Ghaemi M, et al. Positron emission tomographic analysis of Lang); the National Parkinson Foundation, Miami, Fla; the nigrostriatal dopaminergic system in familial parkinsonism associated with a Connaught grant, Toronto (Dr Rogaeva); the Nakabaya- mutations in the parkin gene. Ann Neurol. 2001;49:367-376.

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