Parkin, DJ-1, PINK1, LRRK2, and VPS35. Unique variants

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1 JOURNAL OF NEUROCHEMISTRY (Suppl. 1) doi: /jnc.13593, *Laboratory of Neurogenetics, National Institute on Aging, Bethesda, Maryland, USA German Center for Neurodegenerative Diseases (DZNE)-T ubingen, T ubingen, Germany Abstract Parkinson s disease is a common, progressive neurodegenerative disorder, affecting 3% of those older than 75 years of age. Clinically, Parkinson s disease (PD) is associated with resting tremor, postural instability, rigidity, bradykinesia, and a good response to levodopa therapy. Over the last 15 years, numerous studies have confirmed that genetic factors contribute to the complex pathogenesis of PD. Highly penetrant mutations producing rare, monogenic forms of the disease have been discovered in singular genes such as SNCA, Parkin, DJ-1, PINK 1, LRRK2, and VPS35. Unique variants with incomplete penetrance in LRRK2 and GBA have been shown to be strong risk factors for PD in certain populations. Additionally, over 20 common variants with small effect sizes This article is part of a special issue on Parkinson disease. are now recognized to modulate the risk for PD. Investigating Mendelian forms of PD has provided precious insight into the pathophysiology that underlies the more common idiopathic form of disease; however, no treatment methodologies have developed. Furthermore, for identified common risk alleles, the functional basis underlying risk principally remains unknown. The challenge over the next decade will be to strengthen the findings delivered through genetic discovery by assessing the direct, biological consequences of risk variants in tandem with additional high-content, integrated datasets. Keywords: Parkinsons Disease, genetics, mendelian, risk alleles. J. Neurochem. (2016) 139 (Suppl. 1), Substantial progress has been made in understanding the genetics of Parkinson s disease (PD). Highly penetrant mutations producing rare, monogenic forms of the disease have been discovered in singular genes such as SNCA, Parkin, DJ-1, PINK1, LRRK2, and VPS35. Unique variants with incomplete penetrance in LRRK2 and GBA have been shown to be strong risk factors for PD in certain populations. In addition, over 20 common variants with small effect sizes have been shown to modulate the risk for PD. Wholegenome approaches have clearly aided our quest to comprehend the role genetics plays in the pathogenesis of PD. We now detect Mendelian mutations in segregating families in a straightforward manner and we also have the ability to assess how common variability may play a role in disease. Parkinson s disease was long thought to be a sporadic disorder without genetic causation. However, in 1997, mutations responsible for the disease were identified in the alphasynuclein gene (SNCA) (Polymeropoulos et al. 1997a,b). This landmark discovery revealed the first indisputable, heritable component of PD and launched years of significant research into the genetics of PD. Quickly following detection of the first mutations in SNCA, additional genetic links were identified at Received November 13, 2015; revised manuscript received January 25, 2016; accepted February 9, Address correspondence and reprint requests to Andrew B. Singleton. Laboratory of Neurogenetics, National Institute on Aging, Bethesda, MD, USA. singleta@mail.nih.gov Abbreviations used: , tyrosine 3-monooxygenase/tryptophan 5- monooxygenase activation protein; APOE, apolipoprotein E; AR, autosomal recessive; ARJP, autosomal recessive juvenile parkinsonism; ATP13A2, ATPase type 13A2; BAG5, BCL2-associated athanogene 5; BST1, bone marrow stromal cell antigen 1; CDCV, common disease common variant; CDRV, common disease rare variant; CFH, complement factor H; COR, C terminus of ROC; CTSD, cathepsin D; DaT, dopamine transporters; DNA, deoxyribonucleic acid; EOPD, early-onset Parkinson s disease; FBXO7, F-box only protein 7; GAK, cyclin G-associated kinase; GBA, glucocerebrocidase; GCH1, GTP cyclohydrolase 1; GWA, genomewide association; HLA-DRB5, major histocompatibility complex class II, DR beta 5; ips, induced pluripotent stem cells; LB, Lewy body; LRRK2, leucine-rich repeat kinase 2; MAPT, microtubule associated protein tau; MRI, magnetic resonance imaging; NUCKS1, nuclear casein kinase and cyclin-dependent kinase substrate 1; OR, odds ratio; PARK2, Parkin; PARK7, protein deglycase DJ-1; PD, Parkinson s disease; PINK1, PTENinduced kinase 1; PLA2GB, phospholipase A2, group VI; RAB7L1, Rab-7- like protein 1; ROC, Ras of complex proteins; SLC41A1, solute carrier family 41 (magnesium transporter), member 1; SNCA, a-synuclein; SNP, single-nucleotide polymorphism; SYT11, synaptotagmin 11; TGN, trans- Golgi network; TH, tyrosine hydroxylase; Ubl, ubiquitin-like domain; VPS35, vacuolar protein sorting

2 60 D. G. Hernandez et al. two novel chromosomal regions and linkage of SNCA was excluded in > 200 PD families (Munoz et al. 1997; Scott et al. 1997, 1999; Farrer et al. 1998; Kitada et al. 1998). Therefore, by 1998, it was evident that PD was a genetically heterogeneous disease. Several genes have since been linked to inherited forms of parkinsonism and several monogenic forms of the disease and numerous genetic risk factors have been identified. However, it has taken many years of study to begin understanding the underlying gene functions and molecular mechanisms that lead to disease. In this review, we first provide a brief overview of the monogenic forms of disease and then move forward to discuss how our view of PD etiology has matured since 1997 to now include risk alleles. Monogenic forms of Parkinson s disease Understanding the monogenic forms of PD provides insight more broadly into the genetic architecture of this disease, and as described later, there appears to be overlap in the genes that contain disease-causing mutations and those that contain risk variants (Table 1). Mutations in three genes, SNCA (PARK1; encoding a-synuclein), LRRK2 (PARK8; encoding dardarin), and VPS35 (encoding vacuolar protein sorting 35) have been shown to cause autosomal dominant forms of PD. Mutations in six other genes, PINK1 (PARK6; PTEN-induced kinase 1), DJ- 1 (PARK7), Parkin (PARK2), ATP13A2 (PARK9), FBXO7, and PLA2GB have been shown to cause autosomal recessive PD and/ or parkinsonism. The mutations in these genes, with the exception of LRRK2, cause PD in a small subset of patients. All known monogenic forms of PD combined explain only about 30% of familial and 3 5% of sporadic cases (Kumar et al. 2011). Autosomal dominant Parkinson s disease Alpha-synuclein Using traditional linkage mapping, the first mutation underlying PD in SNCA (Ala53Thr in exon 4) was discovered in a large Italian family and subsequently identified in three Greek families with familial PD (Polymeropoulos et al. 1997b). The primary Greek families found to harbor the p.a53t mutation originated from a very small geographical area in southern Greece. Eight additional families, located in central and southwestern Greece, were also confirmed to have mutations in a-synuclein, suggesting the presence of a founder mutation (Athanassiadou et al. 1999; Spira et al. 2001). A decade later, two Korean and one Swedish family were shown to have the same mutation (Ki et al. 2007; Choi et al. 2008; Puschmann et al. 2009). Shortly following the discovery of SNCA mutations causing a rare familial form of PD, Spillantini and colleagues determined that a-synuclein was a major constituent of Lewy bodies, the pathological hallmark of PD (Spillantini et al. 1997). This was a profound finding in the brains of typical sporadic PD patients that distinctly tied together the etiology Table 1 Loci involved in monogenic forms of Parkinson s disease (PD) and risk loci identified prior to the advent of genome wide association studies. Gray loci for which only questionable evidence exists Locus Gene Protein Model Park1 SNCA a-synuclein Autosomal dominant Park2 PARK2 Parkin Autosomal recessive Park3 Unknown Unknown Autosomal dominant Park4 SNCA a-synuclein Autosomal dominant Park5 UCHL1 Ubiquitin c terminal Autosomal dominant hydrolase Park6 PINK1 Pten-induced putative Autosomal recessive kinase 1 Park7 PARK7 DJ-1 Autosomal recessive Park8 LRRK2 Leucine-rich repeat Autosomal dominant kinase 2 Park9 ATP13A2 Lysosomal type 5 Autosomal recessive ATPase Park10 Unknown Unknown Risk locus Park11 GIGYF2 GRB interacting Autosomal dominant GYF protein 2 Park12 Unknown Unknown X-linked Park13 HTRA2 HTRA serine Autosomal dominant peptidase 2 Park14 PLA2G6 Phospholipase A2 Autosomal recessive Park15 FBXO7 F-box only protein 7 Autosomal recessive Park17 VPS35 Vacuolar protein Autosomal dominant sorting 35 Park18 EIF4G1 Eukaryotic translation Autosomal dominant initiation factor 4 gamma 1 Park19 DNAJC16 DNAJ/HSP40 homolog Autosomal recessive subfamily C member 6 SNCA a-synuclein Risk locus LRRK2 Leucine-rich repeat Risk locus kinase 2 GBA Glucocerebrocidase Risk locus and pathogenesis of rare familial forms of PD with sporadic cases. Understanding the crucial link between the two disease phenotypes ultimately established that examination of rare forms of familial PD, even those that differed clinically and neuropathologically from typical PD, was pertinent to the study of the common form. Mutations in SNCA are rare. As of yet, only five (p.a30p, p.e46k, p.h50q, p.g51d, p.a53t) different autosomal dominant, missense mutations have been discovered in a- synuclein along with duplications and triplications of the complete gene (Klein and Schlossmacher 2006). The first identified missense mutation, p.a53t is the most frequent and has been found in seven families throughout the world. The remaining four missense mutations were found in only one family each: p.a30p was reported in a German family with autosomal dominant PD and p.e46k in a Spanish

3 Genetics in Parkinson disease 61 family from the Basque country (Kruger et al. 1998; Zarranz et al. 2004). Triplication of the entire genomic region containing SNCA was first discovered in 2003 and has since been reported as a cause of disease in several families (Singleton et al. 2003; Farrer et al. 2004; Ibanez et al. 2009). However, duplications of the entire coding region of SNCA have been reported as a more common cause of disease in families and apparently sporadic cases (Chartier- Harlin et al. 2004; Ibanez et al. 2004, 2009; Nishioka et al. 2006; Fuchs et al. 2007; Ikeuchi et al. 2008; Troiano et al. 2008; Uchiyama et al. 2008). The clinical phenotype associated with SNCA mutations consists of progressive L-DOPA responsive parkinsonism with cognitive decline, autonomic dysfunction, and dementia. The average age of onset for those patients with the p.a53t mutation is 46 years of age, which is significantly younger than typical sporadic PD and the disease is fully penetrant (Papapetropoulos et al. 2001). In contrast, families with the A30P mutation have an age of onset that is slightly later (age 52) and the disease is not fully penetrant (Kruger et al. 1998), while the p.e46k mutation causes dominant PD and Lewy body dementia with symptoms beginning between the ages of 50 and 65 years with dementia presenting within 2 years of diagnosis (Zarranz et al. 2004). Genomic duplications and triplications at the SNCA locus cause early-onset PD with the age of onset and severity of the disease phenotype correlating with the SNCA copy number, suggesting a gene-dose effect. Triplication of a-synuclein causes a fully penetrant early onset Parkinson s disease (EOPD) that is rapidly progressive, dopa-responsive parkinsonism, and is accompanied or followed by dementia. Clinical presentation ranges widely from severe idiopathic PD to PD with dementia or diffuse Lewy body disease. PD patients with duplication of SNCA, therefore generating three copies of the gene, develop the disease about a decade later than those with four copies of synuclein and the disease course, while still aggressive, is generally more benign (Singleton et al. 2003; Farrer et al. 2004; Singleton and Gwinn-Hardy 2004; Nishioka et al. 2006; Ross et al. 2008a). Although mutations in SNCA are a clear cause of PD and the presence of a-synuclein aggregates is a hallmark of PD, the normal function of a-synuclein remains poorly understood. SNCA encodes a small 140 amino acid protein that exists in a range of quaternary states from monomeric, to low molecular weight oligomers, to the high molecular weight amyloid fibrils that are found in Lewy bodies. Alpha-synuclein is mainly found in the cytosol where it is bound to lipid rafts in an interaction that is required for it association with the synapse (Fortin et al. 2004). It has been reported that a-synuclein interacts with members of both the Rab and SNARE families, pointing to a role in vesicular trafficking (Gitler et al. 2008; Burre et al. 2010; Thayanidhi et al. 2010). Consistent with its localization at the synapse, Nemani et al. (2010) showed that over-expression of human SNCA in mice inhibits synaptic transmission. Interestingly, they also observed synaptic inhibition when over-expressing the PD-linked mutations p.a53t and p.e46k, but not with the p.a30p mutation. It has been suggested through experiments using over-expression of human SNCA mutants in rats, that mutants favoring low molecular weight oligomers that bind membranes (p.e35k and pe57k) cause more dopaminergic neuron death in the substantia nigra than those that quickly form fibrils (WT, p.a30p, and p.a53t) (Winner et al. 2011). This mechanism is somewhat controversial, as the mutants that increase cell death are not found in familial PD cases, and it does not account for neurodegeneration caused by duplication or triplication of the SNCA locus or for disease caused by familial mutants that quickly form fibrils. These studies point to an important role for SNCA in intracellular trafficking, membrane interaction, and synaptic activity; but, more work must be done to fully understand the mechanisms underlying disease and normal gene function. Leucine-rich repeat kinase 2 In addition to SNCA, autosomal dominant PD-causing mutations have been found in the gene encoding Leucine-rich repeat kinase 2 (LRRK2). Linkage of PD to a region on chromosome 12 was originally mapped in a large Japanese family with autosomal dominant, late-onset PD showing incomplete penetrance (Funayama et al. 2002). Within two years, the locus was verified and further delineated in several European families (Zimprich et al. 2004). In 2004, two groups performed positional cloning identifying mutations in LRRK2 as the root cause of chromosome 12-linked PD (Paisan-Ruiz et al. 2004; Zimprich et al. 2004). Mutations in LRRK2 are now recognized as the most common known cause of familial PD. To date, more than 100 distinct missense and non-sense mutations have been reported in LRRK2 (Rubio et al. 2012); however, only for a small minority is there overwhelming proof of pathogenicity (p.r1441c/g/h, p.y1699c, p.s1761r, p.i2012t, p.g2019s, and p.i2020t) (Healy et al. 2008; Aasly et al. 2010; Nuytemans et al. 2010; Bardien et al. 2011; Lorenzo-Betancor et al. 2012). These pathogenic modifications are clustered in exons encoding the Ras of complex proteins (ROC), C-terminus of ROC, or kinase domains of the protein. The most well-studied mutation, p.g2019s, is common across many populations and has been identified in up to 42% of familial cases, depending on the ethnic background (Healy et al. 2008; Correia Guedes et al. 2010). It is frequent in North African, Middle Eastern and Ashkenazi Jewish PD patients, and it is believed that most LRRK2 p.g2019s mutation carriers are from a common founder originating in North Africa and spreading with the Jewish diaspora (Brice 2005; Farrer et al. 2005; Kachergus et al. 2005; Lesage et al. 2006a,b; Ozelius et al. 2006; Healy et al. 2008). However, pg2019s remains rare in Asian populations, where it accounts for less than 1% of LRRK2 mutations (Tan et al. 2005, 2010). Importantly, p.g2019s is also detected in

4 62 D. G. Hernandez et al. sporadic PD cases. The mutation is seen in approximately 2% of sporadic cases in Northern European and US populations and up to 10% of sporadic cases worldwide (Bardien et al. 2011). The penetrance of the p.g2019s mutation is age dependent and varies from 28% at 59 years, to 51% at 60 years, to 74% at 79 years of age. Overall, mutations in LRRK2 are the most common known genetic cause of late-onset PD and are found in both autosomal dominant and sporadic cases. LRRK2 mutations are found in ~ 10% of patients with autosomal dominant familial PD (Khan et al. 2005; Johnson et al. 2007; Paisan- Ruiz et al. 2008; Lesage et al. 2009), 3.6% of patients with sporadic PD and 1.8% of healthy controls (Lesage et al. 2006a). Phenotypically, LRRK2 mutation carriers are essentially indistinguishable from sporadic PD (Haugarvoll and Wszolek 2009) demonstrating mid to late onset of disease around 60 years of age, with a slow progression and a good response to levodopa therapy. Dementia in individuals with LRRK2 mutations is rare. Neuropathologic features are consistent with typical PD showing Lewy bodies (LB) in the brainstem and loss of dopaminergic neurons in the substantia nigra (Haugarvoll and Wszolek 2009). LRRK2 encodes a large, multidomain protein of 2527 amino acids in the ROCO protein family (Greggio et al. 2009; Li et al. 2009). It is widely expressed in the brain tissue and localized to LBs in the brainstem where it is associated with the endoplasmic reticulum of dopaminergic neurons (Alegre-Abarrategui et al. 2008; Vitte et al. 2010). The GTPase ROC and C terminus of ROC domains as well as the Ser/Thr kinase domain forms an enzymatic core that characterizes the LRRK2 protein and contains the proven autosomal dominant pathogenic mutations. The most common p.g2019s mutation has been shown to consistently increase kinase activity (Greggio 2012). The flanking ankryn, leucine rich repeat and WD40 domains allow for interaction with numerous other proteins including , BAG5, GAK, and Rab7l1 (Nichols et al. 2010; MacLeod et al. 2013; Beilina et al. 2014). LRRK2 has been implicated in numerous cellular processes including autophagy, cytoskeletal dynamics, kinase cascades, mitochondrial function, and vesicular trafficking (MacLeod et al. 2006; Gloeckner et al. 2009; Parisiadou et al. 2009; Mortiboys et al. 2010; Niu et al. 2012; Bravo-San Pedro et al. 2013; Manzoni et al. 2013; Orenstein et al. 2013; Beilina et al. 2014). The many diverse activities of LRRK2 prohibit a clear view of its exact physiological functions, but investigations are ongoing to create a framework in which its role in PD can be better understood. The identification of LRRK2 mutations has proven to be a landmark discovery that has profoundly impacted our understanding of Parkinson s disease. Vacuolar protein sorting 35 In 2011, Zimprich and colleagues were the first to use nextgeneration sequencing methods to detect a PD causing gene. They identified a mutation in vacuolar protein sorting 35 homolog gene (VPS35, encoding vacuolar protein sorting 35) in a family from Austria with 16 affected individuals, as a cause of late-onset autosomal dominantly inherited parkinsonism. Exomes from a pair of affected second cousins were compared to generate a list of rare, shared heterozygous coding mutations. Subsequently, only one mutation, p.d620n, was validated by Sanger sequencing, and was not found in a group of more than 650 controls. This mutation was found in every affected individual in the family and segregates with late-onset PD in a Mendelian autosomal dominant manner (Haugarvoll and Wszolek 2009). Another group, also using exome sequencing, simultaneously identified the p.d620n mutation as a cause of late-onset PD in a large Swiss family (Vilarino-Guell et al. 2011). Sequencing of the entire coding portion of VPS35 revealed a handful of other mutations in these studies (p.g51s, p.m57i, p.t82r, p.i241m, p.p316s, p.r524w, and p.l774m), however, the mutations have not been proven to be pathogenically relevant. Several thousand PD and control subjects have since been screened for the c.1858g>a (p.d620n) mutation, identifying numerous families with this form of PD. Overall, VPS35 mutations are a rare cause of PD accounting for only about 1% of familial parkinsonism and 0.2% of sporadic PD (Zimprich et al. 2011; Lesage et al. 2012; Nuytemans et al. 2013). VPS35-linked PD resembles typical idiopathic disease with a mean age of onset at 53 years, bradykinesia, resting tremor, and good response to levodopa therapy (Kumar et al. 2012). VPS35 encodes a highly conserved 796 amino acid protein. The homologous yeast gene, vps35, has been well characterized by the discovery of mutants showing abnormal vacuole sorting and secretion (Paravicini et al. 1992). These early experiments in yeast showed that Vps35p is a core member of the retromer complex, which also contains Vps26p and Vps29p, and is responsible for the retrograde transport of proteins in endosomes to the trans-golgi network (Seaman et al. 1998). Vps35p is located at the center of the complex and is required for the recognition and binding of the cytosolic domains of cargo for retrograde transport (Nothwehr et al. 2000). The human homologs of the retromer complex were later cloned and shown to function in the same endosome trans-golgi network pathway (Haft et al. 2000). A recent report links the PD-associated p.d620n mutation to dysfunction of the retromer complex by redistributing retromer bound endosomes to the perinuclear region in both cell lines and PD patient-derived fibroblasts. The study goes on to show that the mutant VPS35 protein alters the trafficking of cathepsin D, a protein that is implicated in the degradation of a-synuclein (Cullen et al. 2009; Follett et al. 2014). Although mutations in VPS35 are rare, its high level of conservation has made it one of the best understood genes associated with Mendelian PD. VPS35 is linked to the

5 Genetics in Parkinson disease 63 other autosomal dominant PD genes, SNCA and LRRK2, through endosomes and vesicular trafficking and underscores the importance of studying these pathways in health and disease. Autosomal recessive parkinsonism Mutations in six genes: PARK2 (encoding parkin), PINK1 (PARK6; PTEN-induced kinase 1), DJ-1 (PARK7), ATP13A2 (PARK9; ATPase type 13A2), PLA2G6 (PARK14; phospholipase A2, group VI), and FBX07 (PARK15; F-box only protein 7) have been shown to cause autosomal recessive (AR) PD/parkinsonism. The mutations in these genes cause early-onset PD in a small subset of patients. All known monogenic forms of PD combined explain only about 20% of early-onset PD and less than 3% of late-onset PD, although as will be discussed below, this proportion varies across ethnic groups. Parkin Parkin was the second gene identified to cause parkinsonism and the first gene decisively shown to be inherited in an AR manner. A homozygous deletion of exons 3 7 in the Parkin gene was first reported by Kitada and colleagues in Japanese families with autosomal recessive juvenile on-onset parkinsonism (ARJP); a severe, early-onset form of disease with onset often occurring before 20 years of age (Kitada et al. 1998). This study also identified four families with ARJP showing homozygous deletions of exon 4 only. Mutations in Parkin are the primary cause of ARJP and early-onset, recessive parkinsonism. Numerous unique mutations in all 12 exons of Parkin have been identified throughout various ethnic populations. These mutations consist of point mutations and exon rearrangements, including both deletions and duplications (Hedrich et al. 2001, 2004; West et al. 2002; Foroud et al. 2003; Lesage et al. 2007; Nuytemans et al. 2010). To date, approximately 147 different exonic mutations have been described of which a third are single-nucleotide changes, 13% are minor deletions, and 54% are larger deletions or duplications comprised of one or more exons (Grunewald et al. 2010). The number of disease-causing exon rearrangements in Parkin is likely still to increase as many exon rearrangements were often missed because of the labor intensive and expensive early methods required for their identification. Mutations are present in approximately 50% of patients with recessive, EOPD in the age range of 7 58 years of age and present in up to 77% of sporadic cases with disease onset younger than 20 years (Lucking et al. 2000). Key clinical features of Parkin-linked disease have been reported to include age at onset < 40 years, foot dystonia, psychiatric symptoms, and a dramatic response to treatment (Khan et al. 2003). However, these symptoms can mirror those of typical EOPD cases without Parkin mutations (Periquet et al. 2003). The pathology of Parkin disease consists of severe neuronal loss in the substantia nigra, occasional tau pathology and a distinct lack of postmortem LBs in most cases (Farrer et al. 2001; van de Warrenburg et al. 2001; Mori et al. 2003). A possible explanation for the lack of LBs, generally a pathological hallmark of PD, may be the young age of Parkin disease onset (Doherty and Hardy 2013). It is notable that no cases of juvenile-onset PD have been reported with postmortem LBs (Mizutani et al. 1991; Takahashi et al. 1994) and the rare patients with Parkin mutations and LBs have a significantly older age of disease onset (mean age of onset: without postmortem LBs = 27 years; with postmortem LBs = 46 years) (Doherty and Hardy 2013; Doherty et al. 2013). Parkin is one of the largest genes in the human genome and codes for a 465-amino acid E3 ubiquitin ligase made up of an ubiquitin-like domain and two RING domains (Scuderi et al. 2014). The ubiquitin-like domain of E3 ubiquitin ligases is required for recognizing substrates and the RINGbox is needed for interaction with its specific E2 ubiquitinconjugating enzyme. Shimura and colleagues have demonstrated that the E2 enzyme associated with Parkin is UbcH7 and that a PD-associated missense mutation (p.t240r) in the first RING domain is sufficient to disrupt this interaction (Shimura et al. 2000). Narendra and colleagues demonstrated that Parkin has a generally cytosolic localization except in the context of mitochondrial damage when it is phosphorylated by PINK1, another gene associated with ARJP, and translocated to the surface of unhealthy mitochondria to ubiquitinate mitochondrial membrane proteins (Narendra et al. 2008, 2010). These ubiquitinated proteins signal to the cell that the damaged mitochondria must undergo mitophagy. The prevalence of Parkin mutations has implicated mitochondrial quality control in the pathogenesis of PD indicating that multiple pathways lead to different parkinsonian phenotypes. PTEN-induced putative kinase 1 The PARK6 locus was first mapped to a 12.5-centimorgan (cm) region on chromosome 1p35-p36 in a large consanguineous family from Sicily (Valente et al. 2001). In 2004, Valente and colleagues identified two homozygous mutations in the PTEN-induced putative kinase 1 (PINK1) gene. A p.g309d missense mutation and a p.w437x truncating mutation were found in a Spanish family and two Italian families, respectively. Both Italian families share a common haplotype, demonstrating a shared ancestry (Valente et al. 2004). Since the discovery of PINK1 in PD, more than 10 different mutations have been associated with EOPD. The combination of homozygous and compound heterozygous loss of function mutations in PINK1 is the second most common cause of autosomal recessive EOPD, predicted to be present in about 3.7% of patients (Healy et al. 2004; Valente

6 64 D. G. Hernandez et al. et al. 2004; Bonifati et al. 2005; Klein et al. 2005a; Li et al. 2005; Marongiu et al. 2007). The clinical phenotype of PINK1-related PD strongly resembles levodopa responsive, classic idiopathic PD with no reports of dementia (Ibanez et al. 2006). PINK1 encodes a 581 amino acid Ser/Thr kinase that localizes to the mitochondria (Valente et al. 2004). As previously mentioned, PINK1 phosphorylates Parkin to regulate mitophagy of damaged mitochondria (Narendra et al. 2010; Kane et al. 2014). Additionally, over-expression of wild-type PINK1, but not the disease associated mutation p.g309d, has been demonstrated to rescue stress-induced apoptotic death via the mitophagy pathway (Valente et al. 2004). The convergence of two genes in the mitophagy pathway confirms that it is essential to neuronal health and survival and may be a good therapeutic target in a subset of EOPD patients. DJ-1 The PARK7 locus on chromosome 1p was established through the discovery of a consanguineous pedigree from the Netherlands with autosomal recessive PD. In 2003, Bonifati and colleagues identified recessively inherited missense and exonic deletions in DJ-1 in two European families making it the third gene associated with Autosomal Recessive Parkinsons Disease (ARPD) (Bonifati et al. 2003). Homozygosity mapping and positional cloning was performed on consanguineous pedigrees from a genetically isolated population in the Northern Netherlands revealing a homozygous deletion of several exons in DJ-1 causing disease. Subsequently, a missense mutation in a highly conserved residue (p.l166p) of DJ-1 was found to cause disease in an Italian ARPD family. Mutations in DJ-1 are extremely rare, identified in < 1% of early-onset PD cases. The mutations are found in both homozygous and compound heterozygous states, resulting in loss of protein function. Phenotypically, DJ-1 mutations cause levodopa responsive disease onset in the mid-twenties, resembling Parkin and PINK1 linked forms (Mitsumoto and Nakagawa 2001; Mitsumoto et al. 2001; Abou-Sleiman et al. 2003; Bonifati et al. 2003; Canet- Aviles et al. 2004; Pankratz et al. 2006; Zhou et al. 2006). The DJ-1 gene spans 24 kb in length and includes eight exons encoding a protein of 189 amino acids that was first identified in cancer (Nagakubo et al. 1997). DJ-1 belongs to the pepidase C56 family of proteins and has been reported to protect cells against oxidative stress and to play a role in maintaining normal dopaminergic function in the nigrostriatal pathway (Canet-Aviles et al. 2004; Kinumi et al. 2004; Martinat et al. 2004; Taira et al. 2004; Takahashi-Niki et al. 2004; Menzies et al. 2005; Meulener et al. 2005). Besides the importance of DJ-1 in dopamine neurotransmission and signaling, it has been reported to have multiple functions associated with PD pathogenesis, such as chaperone activity and the ability to inhibit a-synuclein aggregation, which is thought to be a key event in Lewy body formation (Shendelman et al. 2004). It has also been suggested that DJ-1 may be involved in transcriptional regulation of neuroprotective or anti-apoptotic genes (Xu et al. 2005). Discovery of two sibs with EOPD that have inactivating heterozygous mutations in DJ-1 and PINK1 led to the suggestion that these two proteins interact to protect the cell from stress-induced apoptosis (Tang et al. 2006). More recent work has linked DJ-1 to the Parkin/PINK1 pathway again through transcriptional regulation of PINK1 (Requejo- Aguilar et al. 2015). ATP13A2, PLA2G6, and FBXO7 More rare, recessively inherited forms of PD are caused by mutations in three genes: ATP13A2 (ATPase type 13A2), PLA2G6 (phospholipase A2, group VI), and FBXO7 (F-box only protein 7). Mutations in ATP13A2 cause the rare, juvenile-onset disorder Kufor Rakeb syndrome, which is characterized by a lower response to levodopa and additional atypical features of disease such as dystonia and supranuclear palsy (Ramirez et al. 2006; Bruggemann et al. 2010). At least 11 families with numerous different missense, nonsense, and deletion mutations in ATP13A2 have been identified to date. Phenotypic severity is highly variable between patients and appears to be related to the type of mutation inherited (Park et al. 2015). ATP13A2 encodes 1175 amino acid member of the P5 family of ATPases. ATP13A2 is a multifunctional protein with 10 transmembrane domains that is hypothesized to play a role in both of the previously described PD pathways, endosome lysosome dynamics (Dehay et al. 2012; Usenovic et al. 2012), and mitochondrial health (Grunewald et al. 2012; Gusdon et al. 2012), as well as in protecting cells from metal (Mn 2+ and Zn 2+ ) induced toxicity (Gitler et al. 2009; Kong et al. 2014). These studies also indicate that ATP13A2 is present in Lewy bodies of PD patients (Dehay et al. 2012) and over-expression of ATP13A2 rescues neurons from a-synuclein accumulation (Gitler et al. 2009; Kong et al. 2014). Mutations in ATP13A2 are a very rare cause of parkinsonism, but these mutations represent an important link between autophagy lysosome function, mitochondrial health and neurodegeneration. Mutations in the gene PLA2G6 cause autosomal recessive, levodopa-responsive parkinsonism with dystonia (Doherty et al. 2013). Brain iron accumulation is found in most but not all affected individuals (Morgan et al. 2006; Paisan-Ruiz et al. 2009; Sina et al. 2009). PLA2G6 mutations have also been associated with neurodegeneration with brain iron accumulation and Karak syndrome, both of which are forms of severe infantile or childhood neurodegeneration (Mubaidin et al. 2003; Malik et al. 2008). PD associated with PLA2G6 is caused by the homozygous or compound heterozygous inheritance of various missense mutations (Paisan-Ruiz et al. 2009; Sina et al. 2009; Yoshino et al.

7 Genetics in Parkinson disease ). PLA2G6 encodes an 806 amino acid protein called phospholipase a2 group VI with seven ankryn repeats, a lipase domain and a calmodulin-binding domain (Doherty et al. 2013). This enzyme hydrolyzes glycerophospholipids to produce free fatty acids and 2-lysophospholipids (Balsinde and Balboa 2005). Recent studies in Drosophila have shown that knockout of the PLA2G6 homolog results in mitochondrial dysfunction and neurodegeneration. Fibroblasts from a patient with the PD-associated mutation p.r747w also showed similarly impaired mitochondria (Kinghorn et al. 2015). Although mutations in PLA2G6 are rare, they present an interesting link of a PD gene to other more severe forms of neurodegeneration that merits further study. Shojaee and colleagues first identified mutations in FBXO7 through linkage mapping followed by gene sequencing in an Iranian family with AR recessive juvenile-onset parkinsonian-pyramidal syndrome (Shojaee et al. 2008). The affected individuals in this family first demonstrated early-onset spastic paraplegia and then later displayed dopa-responsive parkinsonism. Individuals from two additional families with similar symptoms were later shown to have three different mutations in FBXO7 in either the homozygous or compound heterozygous state (Di Fonzo et al. 2009). FBXO7 encodes F-box only protein 7 and is made up of 443 amino acids. It has been shown to directly interact with Parkin and PINK1 in mitochondrial maintenance and mitophagy (Burchell et al. 2013; Zhou et al. 2015). These studies further demonstrate that FBXO7 aids in translocation of Parkin to the mitochondria in response to cell stress and a PD-associated mutation (p.t22m) leads to mislocalization of FBXO7 to the cytosol. Mutations in FBXO7 are a rare cause of ARJP, but are found in the heterozygous state across many populations. Genetic risk Complex diseases such as PD likely arise because of a combination of many genetic, environmental, and lifestyle factors. There are two principle theories regarding the genetic component of complex disease: the common disease rare variant (CDRV) hypothesis and the common disease common variant (CDCV) hypothesis. At the inception of these theories, there was much discussion regarding the likely validity of these hypotheses, and in particular much argument in favor of one over the other; however, it is important to note that these two hypotheses are not mutually exclusive, and it is likely that any complex disease will contain both genetic components. The CDRV hypothesis speculates that a contributing risk component for complex disease will be rare genetic variants; these are strictly defined as any genetic allele with a frequency of 1% or less. The CDRV hypothesis suggests that low frequency variants are abundant in the human population and due to recent population growth, there has been little opportunity for removal of these functional but rare variants through natural selection. Therefore, deleterious rare alleles are likely to exist. This phenomenon may be particularly pronounced in late-onset diseases, where selective pressures, mainly driven through traits that occur before reproductive age is reached, do not apply. A primary limitation in the investigation of the CDRV hypothesis has been a technical one; it has traditionally been extremely difficult to identify rare variants in suitably powered sample series. However, there is now a great deal of interest in the investigation of the CDRV hypothesis because of the affordability of second-generation sequencing methods to identify these rare alleles in large groups of individuals. This is one of the fastest growing fields of disease genetics and has had some initial success (Guerreiro et al. 2013; Jonsson et al. 2013). However, the rarity of these alleles requires very large sample numbers to detect significant effects and therefore it is expensive to execute well-powered studies. As a consequence, the CDRV hypothesis remains largely untested in most common disorders, a situation that will inevitably change over the next few years as next-generation sequencing technologies become cheaper and more accessible to labs across the world. The CDCV hypothesis posits that a significant proportion of risk for common diseases is mediated through common genetic variants (i.e. variants present at greater than 1% allele frequency within a population) (Reich and Lander 2001; Pritchard and Cox 2002). By definition, these variants are common and therefore will have been within the population for a significant amount of time. Thus, conversely to rare variants, highly functional, deleterious alleles are more likely to have been selected out of the population. Therefore, the CDCV hypothesis accepts that the effect of individual common alleles on any deleterious trait is likely to be quite small, but numerous such common alleles may contribute to that trait; so, collectively the contribution of common alleles to a trait may be substantial. Testing of the CDCV has been well established, primarily because the technology available to genotype a large number of common variants has been available and relatively affordable since ~ This has led to an extremely large number of identified common risk loci as evidenced by the large catalog of positive genome wide association studies ( Common genetic risk factors in Parkinson s disease During recent years, several susceptibility genes and numerous risk loci associated with PD have been identified. While success in this regard has been driven by largely unbiased genome-wide studies, some limited success was achieved through candidate gene-based assessments in three genes: SNCA, LRRK2, and GBA. Alpha-synuclein As previously noted, families with rare mutations in SNCA enabled the novel discovery of a genetic link to PD

8 66 D. G. Hernandez et al. (Polymeropoulos et al. 1997b). Following this hallmark finding, Kruger and colleagues examined common variability within SNCA to establish whether the gene was also associated with risk for the sporadic form of PD (Kruger et al. 1999). Kruger s study initially reported that APOE genotype, a major risk factor for late-onset Alzheimer s disease (Chouraki and Seshadri 2014), interacted with a variable dinucleotide repeat within SNCA. The combination of the APOE4 allele and NACP allele 1 of the SNCA promoter polymorphism were shown to be significantly different between sporadic PD patients and controls. PD patients presenting this genotype had a 12.8-fold increased relative risk for developing PD over the course of their lives. Unfortunately, this interaction between SNCA and APOE genotypes has not been replicated; however, Maraganore and colleagues later demonstrated variability of risk for PD between SNCA promoter alleles using meta-analysis of existing dinucleotide repeat sequence alleles at the SNCA promoter (REP1) genotype data (Maraganore et al. 2006). Maraganore showed an unequivocal association between genetic variability within the SNCA locus and PD (Maraganore et al. 2006). Since then, association of Parkinson s disease with SNCA has been overwhelmingly established in genome-wide association (GWA) studies identifying a handful of significant single-nucleotide polymorphisms and revealing more about the architecture of genetic risk at this locus. Leucine-rich repeat kinase 2 As with SNCA, subsequent to the identification of LRRK2 mutations as a cause of monogenic PD (Gitler et al. 2008; Burre et al. 2010) common variability across LRRK2 was examined in several populations. Within Asian populations, the variant p.g2385r was first identified as a cause of PD (Mata et al. 2005). However, this variant was present in 5% of the population and later shown to be a risk allele that doubled the risk of PD in individuals (Di Fonzo et al. 2006). This finding was replicated extensively in Asian populations including those from Singapore, Taiwan, China, Korea, and Japan (Farrer et al. 2007; Funayama et al. 2007; Li et al. 2007; Tan et al. 2007a,b, 2009; Chan et al. 2008; Choi et al. 2008; Zabetian et al. 2009; Kim et al. 2010; Wang et al. 2012; Fu et al. 2013). An additional variant described in 2008, p.r1628p was also shown to be associated with a ~ 2 fold increase risk for developing PD and has been replicated in several Asian populations including Thai, Chinese, and Taiwanese populations (Ross et al. 2008b; Pulkes et al. 2011; Wu et al. 2012; Fu et al. 2013; Wu-Chou et al. 2013). Several additional variants within LRRK2 have been assessed and have varying levels of support for association with risk for PD (Ross et al. 2011; Wu et al. 2012). The LRRK2 locus is repeatedly identified by GWA studies in Caucasian populations, but the exact causative variants remain elusive, requiring a deeper analysis of the locus. Glucocerebrosidase Remarkably, thorough clinical observation rather than previously known genetic association, lead to the discovery of PD risk variants within the gene encoding glucocerebrocidase (GBA); a gene long tied to the autosomal recessive lysosomal storage disorder, Gaucher s disease (Tsuji et al. 1987). Tayebi and colleagues observed that a portion of Gaucher s disease patients manifested with parkinsonism, compelling an early hypothesis that GBA deficiency may lead to a predisposition to parkinsonism (Tayebi et al. 2001, 2003). A year later Aharon-Peretz and colleagues were able to show that inheritance of a single mutation in GBA increased the risk for PD (Aharon-Peretz et al. 2004). Further, meta-analysis of existing data was later used to show that in Ashkenazi Jewish populations, the frequency of two common mutations in GBA (p.n370s and p.l444p) was 15% in PD and 3% in controls; whereas non-ashkenazi Jewish populations demonstrated a much lower 3% frequency of these mutations in cases and < 1% in controls (Sidransky et al. 2009). Overall, these data indicate that a single-heterozygous mutation in GBA escalates the risk for PD ~ 5 fold, while remaining inadequate to cause Gaucher s disease. These two variants have also been linked to risk for dementia with Lewy bodies and PD with dementia (Nalls et al. 2013). The function of GBA is well established as an integral enzyme required for the breakdown of glucocerebroside to ceramide. After the association of GBA with PD, Mazzulli and colleagues showed that the knockdown of GBA in cortical neurons leads to the accumulation of a-synuclein. They further demonstrated an increase in a-synuclein in Gaucher patient induced pluripotent stem cells-derived dopaminergic neurons; however, they did not see significant accumulation of huntingtin or tau, indicating that GBA has a preference for a-synuclein (Mazzulli et al. 2011). Multiple studies have shown that glucocerebrosidase activity is lower in patients carrying GBA mutations and in sporadic PD cases, suggesting a broader role for GBA in pathogenesis (Gegg et al. 2012; Alcalay et al. 2015). Identifying risk through genome-wide association GWA studies have been applied as a means of identifying risk loci since the first successful published GWA study in 2005, identifying CFH (complement factor H) polymorphisms as a significant risk factor for age-related macular degeneration (Klein et al. 2005b). PD is no exception to this trend, with a long history of investigation using GWA (Polymeropoulos et al. 1997b; Fung et al. 2006; Pankratz et al. 2009; Satake et al. 2009; Simon-Sanchez et al. 2009, 2011; Edwards et al. 2010; Hamza et al. 2010; Consortium U.K.P.s.D., 2011). Early GWA efforts in PD failed to convincingly identify risk loci as these studies were, like many other studies at the time, of low power, only examining ~ 300 cases (Maraganore et al. 2005; Fung et al. 2006).

9 Genetics in Parkinson disease 67 However, in 2009, two collaborative studies examining Caucasian and Asian subjects were the first to reveal genome-wide significant risk alleles for PD (Satake et al. 2009; Simon-Sanchez et al. 2009). The Caucasian study identified risk loci at SNCA and MAPT (encoding microtubule associated protein tau) and provided supporting evidence for association at LRRK2 and PARK16, a locus in a large LD block that includes NUCKS1 (nuclear casein kinase and cyclindependent kinase substrate 1), RAB7L1 (RAB7, member RAS oncogene family-like 1), and SLC41A1 [solute carrier family 41 (magnesium transporter), member 1] (Simon-Sanchez et al. 2009). The study in Asian subjects revealed association at SNCA, LRRK2, PARK16, and BST1 (bone marrow stromal cell antigen 1) (Satake et al. 2009). Over the next several years, these loci were replicated and two additional risk loci were nominated at HLA-DRB5 (major histocompatibility complex class II, DR beta 5) and GAK (cyclin G associated kinase) (Pankratz et al. 2009; Hamza et al. 2010). A clear trend then followed, which was the use of meta-analysis of extant data sets to provide greater resolution in the identification of risk loci. The most recent work identified or confirmed 28 independent diseaseassociated risk loci (Table 2) (Nalls et al. 2014). Notably, this includes GCH1 (encoding the enzyme GTP cyclohydrolase 1) that catalyzes the first step in the synthesis of tetrahydrobiopterin. This chemical is in turn a required cofactor for tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis. In the context of PD, there are loci which appear to contain more than one risk allele and this variation in risk extends not only to the identification of independent risk alleles but also to different types of risk allele. The loci containing SNCA, HLA-DRB5, GAK, and SYT11 (synaptotagmin 11) each contain two identified common risk alleles that act independently of one another (Nalls et al. 2014). In addition, the SNCA and LRRK2 loci contain multiple types of risk allele. Both genes contain missense mutations that cause disease and both genes also contain non-coding variability that imparts moderate risk for disease, presumably through modulating expression levels. In addition, LRRK2 contains common protein coding changes that increase risk for disease and SNCA gene multiplications can be causal for PD. The notion of multiple types of risk allele at the same locus or the pleomorphic risk locus hypothesis, suggests that genes containing mutations that cause disease are involved in the pathogenesis of typical, apparently sporadic disease, presumably through an overlapping mechanism (Singleton and Hardy 2011). As with GWA detected loci in other complex diseases, the effect sizes for each of these loci are individually modest (Polymeropoulos et al. 1997b; Fung et al. 2006; Pankratz et al. 2009; Satake et al. 2009; Simon-Sanchez et al. 2009, 2011; Edwards et al. 2010; Hamza et al. 2010; Consortium U.K.P.s.D., 2011) (Table 2). Notably, however, the risk Table 2 Risk loci for PD identified through GWA. Loci in gray indicate a second risk allele that confers an effect independent of a primary risk allele at the same locus SNP Chr Base pair Nominated gene OR rs GBA/SYT rs GBA/SYT rs RAB7L1/NUCKS rs SIPA1L rs ACMSD/TMEM rs STK rs MCCC rs TMEM175/GAK/DGKQ rs TMEM175/GAK/DGKQ rs BST rs FAM47E/SCARB rs SNCA 0.76 rs SNCA rs HLA-DQB rs HLA-DQB rs GPNMB 1.11 rs FGF rs INPP5F rs MIR rs LRRK rs CCDC rs GCH rs VPS13C rs BCKDK/STX1B rs SREBF/RAI rs MAPT rs RIT rs DDRGK GWA, genome wide association; OR, odds ratio; PD, Parkinson s disease; SNP, single-nucleotide polymorphism. conferred by these alleles, when summed in an individual can be considerable. The functional and mechanistic underpinnings of these risk associations remain to be unraveled; however, early reports are beginning to appear in the literature. For example, mass spectrometry analysis has shown that the risk loci GAK and RAB7L1 interact with LRRK2 implicating these risk loci in an already known PD pathway (Beilina et al. 2014). Additionally, MAPT aggregates have been reported to be present in both neurons and glia of patients with various forms of parkinsonism (Ludolph et al. 2009). Genetics has given us a great starting point to target future studies of PD; however, there is much work to be done to truly understand the molecular mechanisms and present therapeutic strategies. The future Over the last fifteen years substantial progress has been made in comprehending the role genetics plays in the pathogenesis of PD. Fully penetrant mutations in the genes SNCA, LRRK2,