Báo cáo khoa học: LRRK2 in Parkinson’s disease: genetic and clinical studies from patients ppt

MINIREVIEW LRRK2 in Parkinson’s disease: genetic and clinical studies from patients Udhaya Kumari 1,2 and E. K. Tan 1,2 1 Department of Neurology, Singapore General Hospital, Singapore, Singapore 2 National Neuroscience Institute, Duke-NUS Graduate Medical School, Singapore, Singapore Introduction Parkinson’s disease (PD), a chronic progressive neuro- degenerative movement disorder, is characterized clini- cally by resting tremor, rigidity, bradykinesia and postural instability. This condition was first described in 1817 by James Parkinson in his seminal paper ‘the shaking palsy’. Although PD predominantly affects older individuals,  10% of people with the disease are under the age of 40 years [1]. The neuropathologi- Keywords LRRK2; mutations; Parkinson’s disease; penetrance; polymorphisms Correspondence E K. Tan, Department of Neurology, Singapore General Hospital, Outram Road, Singapore 169608, Singapore Fax: +65 6220 3321 Tel: +65 6326 5003 E-mail: gnrtek@sgh.com.sg (Received 30 May 2009, revised 27 July 2009, accepted 6 August 2009) doi:10.1111/j.1742-4658.2009.07344.x Mutations in leucine-rich repeat kinase 2 (LRRK2) (PARK8) are associ- ated with both familial and sporadic forms of Parkinson’s disease. Most studies have shown that LRRK2 mutations may explain between 5% and 13% of familial and 1–5% of sporadic Parkinson’s disease. Importantly, a common recurrent mutation (G2019S) located in the kinase domain has been reported across most ethnic populations, with the highest prevalence among Ashkenazi Jews and North African Arabs. A recent worldwide meta-analysis pooling data from 24 populations reported a higher occur- rence of G2019S in southern than in northern European countries and the penetrance is estimated to be  75% at the age of 79 years. The R1441 ‘hotspot’ amino acid codon residue (G ⁄ H ⁄ C) in the Ras of com- plex proteins domain is the second most common site of pathogenic LRRK2 substitutions after G2019S, with most carriers developing symp- toms by the age of 75 years. Two polymorphic variants found almost exclusively among Asians (G2385R and R1628P) have been shown to increase the Parkinson’s disease risk by approximately two-fold. The mutational event associated with R1628P is more recent, occurring  2500 years ago, compared to estimates of 4000 years for G2385R carri- ers. LRRK2 mutation carriers generally simulate late onset Parkinson’s disease and present with the usual typical clinical features. Genetic testing for G2019S in sporadic late-onset Parkinson’s disease can be considered in some situations and may be useful in populations with high carrier sta- tus. The identification of asymptomatic mutation and risk variant carriers provides a unique opportunity for recruiting these subjects in potential neuroprotective trials and longitudinal studies to identify biomarkers of neurodegeneration. Abbreviations ANK, ankryn; ARM, armadillo; COR, C-terminal of ROC; LRRK2, leucine-rich repeat kinase 2; PD, Parkinson’s disease; PET, positron emission tomography; ROC, Ras of complex proteins. FEBS Journal 276 (2009) 6455–6463 ª 2009 The Authors Journal compilation ª 2009 FEBS 6455 cal hallmarks are characterized by a progressive and profound loss of neuromelanin-containing dopaminer- gic neurons in the substantia nigra pars compacta with the presence of eosinophillic, intracytoplasmic and pro- teinaceous inclusions termed as Lewy bodies and dys- trophic Lewy neurites in surviving neurons [2]. For many decades, the relative influence of genes and envi- ronmental agents on the pathophysiology of PD has been debated. However, subsequent to the discovery of a-synuclein as a causative gene in 1997, there is increas- ing recognition that genes play an important role in the disease, particularly in familial cases. At present, many causative genes and susceptibility loci have been identified (Table 1). Many of these PD-associated genes affect both familial and sporadic forms and are found in a number of different ethnic populations. The discovery and subsequent identification of the gene for leucine-rich repeat kinase 2 (LRRK2) (PARK8)asa causative PD gene has significantly contributed to our understanding of not only the eitopathology of the condition, but also provides information that could potentially influence clinical management. LRRK2 is a large (280 kDa) multidomain protein, with pathogenic mutations distributed throughout its length, although there is a degree of clustering within the enzymatic domains. The gene encompasses 144 kb, with an ORF consisting of 7581 bp (in 51 exons) and its encoded protein is unusually large (2527 amino acids). It is a multidomain protein comprising (from N-terminal to C-terminal), armadillo (ARM), ankryn (ANK), LRR, Ras of complex proteins (ROC), C-ter- minal of ROC (COR), mitogen-activated protein kinase kinase kinase and WD40 domains. The presence of the four protein–protein interaction domains (ARM, ANK, LRR and WD40) strongly suggests a role of LRRK2 in protein complex formation. Discovery of LRRK2 as a cause of PD In 2002, Funayama et al. [3] identified a novel locus on chromosome 12p11.2–q13.1 that co-segregates with autosomal dominant parkinsonism in a family from Sagamihara (a region in Japan) consisting of 31 indi- viduals from four generations. Two large families with autosomal-dominant late-onset parkinsonism, family A (German–Canadian) and family D (Western Nebraska) are also linked to this PARK8 locus. In 2004, missense LRRK2 mutations were identified in family A (Y1699C) and in family D (R1441C). Sixteen individu- als (eight unaffected and eight affected) in family A were genotyped and all affected were heterozygous for the mutation and all the unaffected aged over 60 years Table 1. Genes and loci linked with PD. Locus Gene Chromosome Inheritance ⁄ clinical phenotype PARK1 ⁄ PARK4 a-synuclein 4q21 AD and sporadic ⁄ early onset PD PARK2 Parkin 6q25.2-q27 AR and sporadic ⁄ early onset PD PARK3 Unknown 2p13 AD ⁄ late onset PD No causative gene identified PARK5 UCH-L1 4p14 AD ⁄ late onset PD Reported in a PD sibling pair PARK6 PINK1 1p35-p36 AR and sporadic ⁄ early onset PD PARK7 DJ-1 1p36 AR ⁄ early onset PD PARK8 LRRK2 12p11.2-q13.1 AD and sporadic ⁄ late onset PD PARK9 ATP13A2 1P36 AR ⁄ early onset PD PARK10 Unknown 1P32 ?AD PARK11 GIGYF2 2q36-q37 AD ⁄ late onset PD Pathogenicity uncertain PARK12 Unknown Xq21-q25 Unknown No causative gene identified PARK13 HTRA2 2p13 Unknown Pathogenicity uncertain PARK14 PLA2G6 22q13.1 AR ⁄ L-dopa responsive dystonia-parkinsonism Awaiting more data PARK15 FBX07 22q12-q13 AR ⁄ parkinsonism–pyramidal syndrome Awaiting more data AD, Autosomal dominant; AR, autosomal recessive; UCHL1, ubiquitin carboxy-terminal hydrolase L1; PINK1, PTEN-induced kinase 1; ATP13A2, ATPase type 13A2; GIGYF2, GRB10-interacting GYF protein 2; HTRA2, HtrA serine peptidase 2; PLA2G6, group VI phospholipase A2; FBX07, F-box protein 7. Genetic and clinical studies of LRRK2 U. Kumari and E. K. Tan 6456 FEBS Journal 276 (2009) 6455–6463 ª 2009 The Authors Journal compilation ª 2009 FEBS did not have the mutation. In family D, 34 individuals were genotyped (ten affected and 24 unaffected). All affected were heterozygous for R1441C mutation and 24 clinically unaffected were genotyped; only two older than 60 years of age were mutation carriers. These individuals are considered to be at risk [4]. At the same time, another study revealed missense mutations segre- gates with PARK8-linked PD in five families from England and Spain. The authors named the protein dardarin because dardar is derived from the Basque (i.e. where families of LRRK2 are found) word for tremor, and it was a common symptom [5]. Identification of a common LRRK2 mutation (G2019S) In 2005, three concurrent reports identified a LRRK2 mutation (G2019S, which produces a glycine to serine amino acid substitution at codon 2019) to be common in both familial and sporadic PD [6–8]. Nichols et al. [6] analyzed 767 affected individuals from 358 multi- plex families and revealed that 5% were either hetero- zygotes or homozygotes for the mutation. Di Fonzo et al. [7] found 6.6% of unrelated families from Italy, Portugal and Brazil with PD and with autosomal dom- inant inheritance harbor the mutation. Gilks et al. [8] analyzed 482 sporadic PD patients and reported 1.6% of them as having the mutation. These three reports highlighted, for the first time, that a common recurrent mutation can be a cause of both sporadic and familial PD and consequently set the stage for numerous genetic screening studies for this mutation worldwide [9]. Prevalence of LRRK2 G2019S mutation Most studies have shown that LRRK2 mutations may explain between 5% and 13% of familial and 1–5% of sporadic PD [4–9] (Fig. 1). The variability depends on the ethnic population and the extent of genetic screening. Of greater interest is the prevalence of G2019S, which has been found to be very rare in Asia, South Africa and in some European countries, such as Poland, Greece and Germany. However, that appears as a predilection for some ethnic races. This mutation accounts for 13.3% of sporadic and 29.7% of familial PD among Ashkenazi Jews and 40.8% of sporadic and 37.0% of familial PD in North African Arabs [10,11]. The muta- tion accounts for  1–7% of familial patients from European and US origin and for 1–3% of sporadic PD from most Caucasian populations. This mutation is located in the kinase domain of the protein and may be associated with increased kinase activity [12,13]. The estimation of the penetrance of autosomal domi- nant mutations is a challenging task but it is essential for genetic counseling. Penetrance estimates are usually high when they are based on high-risk families and might not apply to the general population. The pene- trance of G2019S-associated disease increased from 17% at age 50 years to 85% at age 70 years in an initial family-based study, but varied in subsequent reports, depending on sample size, study design, inclu- sion of probands in the analysis and methods of calcu- lation. To address some of these issues, Healy et al. [14], in a recent worldwide meta-analysis, pooled data from 24 populations, involving 1045 individuals with LRRK2 mutations from 133 families. Interestingly, they found a higher occurrence of G2019S in southern than in northern European countries. The authors esti- mated the penetrance to be 28%, 51% and 74% at 59, 69 and 79 years of age, respectively. The over-riding message is that, although the penetrance clearly increases with age, it is not complete because some very elderly carriers remain free of disease. Ethnic dif- ferences for LRRK2 mutations have been reported Fig. 1. Genomic and protein structures of LRRK2. LRRK2 has 2527 amino acids and contains ARM, ANK, LRR, ROC, COR, MAP- KKK and WD40 domains. Proven pathogenic mutations are shown in red. Potentially pathogenic mutations, for which co-segrega- tion analyses were reported, are highlighted in blue. Variants of unknown significance, found in single PD patients, are highlighted in black. Risk factors are shown in red and in a yellow box. U. Kumari and E. K. Tan Genetic and clinical studies of LRRK2 FEBS Journal 276 (2009) 6455–6463 ª 2009 The Authors Journal compilation ª 2009 FEBS 6457 among Asian races. For example, the LRRK2 G2019S substitution has not been found in three independent Chinese populations involving more than 2000 study subjects, whereas it has been detected among Japanese and rarely among Indians [15–17]. Different founder haplotypes have been described in G2019S carriers. A common 193 kb genomic region, so-called haplotype 1, is shared by 95% of G2019S carriers of European, North and South African and Ashkenazi Jewish origin [18,19]. The mutation possibly arose in Ashkenazi Jews much earlier than in North African Arabs and Euro- peans several thousand years ago [20]. The frequency of this haplotype in non-G2019S Chinese carriers in both PD and controls is  30–33%, which is similar to the frequency in European noncarriers [21]. The sec- ond rare haplotype is found in a total of five families of European ancestry. The third, found primarily in Japanese carriers, has also been reported in a Turkish family [22,23]. Distinct G2019S haplotypes in different races suggest that the mutation originates from different founders in Europe and Asia. Mutational hotspot at position R1441 The R1441 ‘hotspot’ amino acid codon residues gly- cine ⁄ histidine ⁄ cysteine (G ⁄ H ⁄ C) in the ROC domain is the second most common site of pathogenic LRRK2 substitutions, after G2019S [24]. The R1441C mutation was initially found in two autosomal-dominant PD families [4]. Affected individuals reported typical PD symptoms and the mean age at onset in the first family was 65 years. Two asymptomatic mutation carriers were more than 60 years old. The phenotype of carri- ers in the second family was similar, with mean age at onset of 56 years. Interestingly, Zabetian et al. [25] reported a R1441C patient with sporadic PD with onset at age 61 years and all nine siblings were asymp- tomatic even though they were more than 60 years old. These initial observations suggest that the pene- trance of R1441C can be highly variable. Recently, in a worldwide pooled analysis involving 33 affected and 15 unaffected R1441C mutation carriers, the demographics and clinical features of LRRK2 carriers were found to be generally similar to idiopathic PD [14]. More than 90% had developed symptoms by 75 years of age. Four independent founders for the R1441C mutation have also been reported [26]. The apparent high penetrance in this pooled analysis needs to be interpretated with caution because this is not a population-based study. Although R1441C is found in different ethnic races, R1441G is most common in the Basque Country ( 20%) and is rare outside of Northern Spain [27–29]. A common founder for R1441G carriers was found to date back to the 7th Century in Northern Spain [29]. R1441H has been described in four probands of diverse ethni- cities [30]. Polymorphic variants G2385R variant The discovery of polymorphic risk variants is unex- pected because few of genetic variants linked to PD have been consistently replicated [31]. Evidence of an association of LRRK2 polymorphic variants with PD was reported in 2005 when a haplotype that increases disease risk when present in two copies was identified among sporadic Chinese PD population in Singapore [32]. However, other studies did not reveal association with any LRRK2 haplotypes in Caucasians, suggesting the existence of ethnic-specific differences [33–35]. In 2005, Mata et al. [24,36] reported a G2835R variant a PD family from Taiwan and it was thought that it could be a pathogenic mutation. However, Tan et al. [37] and Di Fonzo et al. [38] subsequently found that the G2385R variant is a common polymorphism and increases the risk of PD in Singaporean and Taiwan populations. This association has been consistently replicated among Chinese and Japanese populations with an average carrier rate of  9% in PD and 4% in controls [37,39–41]. The population attributable risk for the heterozygous genotype is 4%. It has been sug- gested that the G2385R variant possibly originates from one common ancestor in China 4000 years ago [40]. Thus far, LRRK2 G2385R appears absent in Caucasian subjects [41]. Interestingly, the G2385R var- iant has not been shown to be a risk factor for PD among Indians and Malays in Singapore or to be asso- ciated with other neurodegenerative conditions such as Alzheimer’s disease [42]. The LRRK2 G2385R is located in the WD40 domain, and the base substitu- tion alters the net positive charge of the WD40 domain. Because WD40 domain is involved in mediat- ing protein–protein interactions, the LRRK2 G2385R variant may impact on interactions with substrates and ⁄ or regulatory proteins. Preliminary studies suggest that it may lead to decreased kinase activity and be pro-apoptoptic under cellular stresses [12,41]. The clin- ical features of G2385R carriers are similar to noncar- riers, although those individuals with familial PD appear to have a higher carrier rate [43]. Recently, a large-scale pooled analysis involving multiple Asian centers revealed that the G2385R variant lowers the age of onset of PD [44]. However, because almost all the G2385R carriers are heterozygotes, the additive Genetic and clinical studies of LRRK2 U. Kumari and E. K. Tan 6458 FEBS Journal 276 (2009) 6455–6463 ª 2009 The Authors Journal compilation ª 2009 FEBS effect of carrying two copies of the variant could not be meaningfully determined. R1628P variant R1628P is the second risk factor to be identified in the Han Chinese population after the G2385R because investigators found that it increased the risk of PD among Chinese in Taiwan and Singapore [45,46]. LRRK2 R1628P is located in the COR domain and is evolutionarily conserved across species, highlighting the importance of the residue (arginine) to protein function. The LRRK2 R1628P variant has not been detected in Indian subjects and does not appear to be associated with risk among individuals of Malay eth- nicity [47]. It is possibly rare or absent among whites and, interestingly, has not been detected in Japanese patients. The phenotype of carriers appears similar to idiopathic PD [45,48]. It would appear that the muta- tional event associated with LRRK2 R1628P is more recent, occurring  2500 years ago, compared to estimates of 4000 years for carriers of the LRRK2 G2385R variant [46]. Very few subjects carry both G2385R and R1628P risk alleles. However, the esti- mated population attributable risk of R1628P and G2385R variants is  10% [48]. The functional activi- ties of R1628P have yet to be determined. Phenotype–genotype correlation On the basis of findings of a multicenter pooled analy- sis, it is quite clear that R1441C, G2019S and other mutational carriers share a common phenotype with idiopathic PD [14,26]. Thus, LRRK2 carriers simulate late onset PD and present with the usual typical PD clinical features. These observations challenge the clas- sification of ‘idiopathic PD’. Although long-term longi- tudinal data are not available, Healy et al. [14], in their worldwide pooled analysis, reported that both motor symptoms and nonmotor symptoms (e.g. cogni- tion) of LRRK2 carriers appear to be milder than those of idiopathic PD. Phenotype studies Positron emission tomography (PET), by providing quantitative information on dopaminergic function, is useful for the in vivo investigation of PD. [ 18 F]6-fluoro- l-dopa uptake correlates with the number of nigral dopamine neurons in humans and in animal models of PD [49]. Thus, PET and other functional imaging modalities provide a useful means to monitor nigral integrity in LRRK2 asymptomatic and symptomatic carriers. In one of the earliest functional imaging stud- ies on LRRK2 carriers, Adams et al. [50] reported that abnormalities on functional imaging studies are quite similar between LRRK2 carriers and sporadic PD. More recently, a multitracer PET study was carried out in asymptomatic members of the kindred from family D (R1441C) with some of them rescanned 2–3 years apart [49]. Worsening of PET markers over time was greater compared to healthy controls for some of the carriers. This suggests that progressive dopaminergic dysfunction occurs in pre-symptomatic members of the LRRK2 kindred. The identification of these individuals could provide an opportunity for potential early neuroprotective interventions. Interestingly, transcranial sonography studies in LRRK2 carriers revealed that substantia nigra echoge- nicity was greater compared to controls but smaller than in idiopathic PD [51]. However, it remains specu- lative as to whether iron has a different pathophysio- logical role in LRRK2 carriers than in idiopathic PD. Hyposmia is a common finding in majority of PD patients. Using the University of Pennsylvania Smell Test, one study showed that the mean test score in G2019S parkinsonian carriers was lower than that in healthy controls, but no different in patients with PD [52]. Two asymptomatic G2019S carriers had a normal smell test. This test cannot differentiate LRRK2 carriers from idiopathic PD. Myocardial 123 I-metaiod- obenzylguanidine (which assesses postganglionic sym- pathetic cardiac innervation) uptake is decreased in most PD patients. Quattrone et al. [53] showed that 50% of G2019S carriers compared to all the patients with idiopathic PD had impaired 123 I-metaiodobenzyl- guanidine uptake. This suggests that G2019S carriers may not be a homogenous entity. Taken together, the current limited clinical studies of LRRK2 mutation carriers appear to suggest that, although clinically inseparable, there may be subtle differences between these carriers with respect to idiopathic PD and further investigations should be considered. It is unclear how the variable pathology associated with G2019S muta- tions influences the phenotypic features. Genetic testing for G2019S A genetic test can help confirm or exclude a suspected genetic disease. The test can also help determine the risk of developing the disorder for an individual. The recent discovery of the common LRRK2 G2019S mutation provides an opportunity for testing in fami- lies with autosomal-dominant pattern inheritance, as well as in some cases of sporadic PD. However, the clinical utility of such testing would require careful U. Kumari and E. K. Tan Genetic and clinical studies of LRRK2 FEBS Journal 276 (2009) 6455–6463 ª 2009 The Authors Journal compilation ª 2009 FEBS 6459 evaluation of the potential risks and benefits of testing and the availability of treatment options to manage those at risk. However, the feasibility of diagnostic and predictive testing of PD is not as simple as it appears to be. Many questions have been raised, including the sensitivity and specificity of the test and reliability of the laboratory carrying the test [54]. Spe- cific to G2019S testing, its incomplete penetrance com- plicates pre-symptomatic genetic testing. For other LRRK2 mutations, there are questions regarding its actual pathogenicity. Some investigators have argued that such testing should be carried out under a research setting rather than as part of a clinical service because it will help remove concerns regarding insur- ability and other social issues. Genetic testing should preferably be supported by a multidisciplinary team with expertise in handling pre-testing and post-testing related problems. A recent study has demonstrated that the relationship between the level of genetic knowledge and the attitude towards the potential risks and benefits of predictive genetic testing in PD may be influenced by racial and cultural differences and, thus, this has to be taken into consideration in counseling programs [55]. For LRRK2 testing, there is a lack of available sci- entific information with respect to advising subjects on their prognosis and the result will not alter the man- agement of the disease. The large size of the LRRK2 makes it impractical to provide comprehensive screen- ing. Furthermore, the pathogenicity of many of the putative heterozygous LRRK2 mutations is unclear because many of them have been described in single patients and no segregation data in affected families are available. Nevertheless, testing for G2019S in spo- radic late-onset PD can be considered in some situa- tions and may be useful in populations with high carrier status [56]. It will be useful if professional bodies come together to set up guidelines and help provide advice to both patients and the public. Future directions The discovery of the gene for LRRK2 as a causative gene in PD is both extremely important and exciting because LRRK2 mutations are the most common cause of familial PD and a common mutation and two common polymorphic risk variants have been identi- fied. Furthermore, the varied prevalence of causative mutations and risk variants across different ethnic populations suggest that, besides a common founder effect, epigenetic or other factors such as environmen- tal or lifestyle factors may be important. Multicenter studies to determine the prevalence, penetrance and phenotype–genotype correlation for the various reported LRRK2 mutations, and gene–environmental interaction would be needed. As the the gene for LRRK2 is large, studies that report direct sequence analysis of the entire gene and copy number analysis are still limited. Thus, familial segregation analysis and functional studies to determine which ones are truly pathogenic are needed. Additional studies to determine haplotype structure, population and ethnic differences and identification of new risk variants will also be use- ful. The identification of asymptomatic mutation and risk variant carriers provides a unique opportunity in the field because these subjects are ideal candidates for potential neuroprotective trials and longitudinal studies to identify biomarkers of neurodegeneration. Clinical, genetic and biological information gathered from genetic underpinnings of PD will hopefully be trans- lated into better treatment for patients. Acknowledgement Supported by Singapore Millennium Foundation, National Medical Research Council, Biomedical Research Council, Duke-NUS Graduate Medical School and SingHealth Services. References 1 Gandhi PN, Chen SG & Wilson-Delfosse AL (2009) Leucine-rich repeat kinase 2 (LRRK2): a key player in the pathogenesis of Parkinson’s disease. J Neurosci Res 87, 1283–1295. 2 Santpere G & Ferrer I (2009) LRRK2 and neurodegen- eration. 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Tan Genetic and clinical studies of LRRK2 FEBS Journal 276 (2009) 6455–6463 ª 2009 The Authors Journal compilation ª 2009 FEBS 6463 . MINIREVIEW LRRK2 in Parkinson’s disease: genetic and clinical studies from patients Udhaya Kumari 1,2 and E. K. Tan 1,2 1 Department of Neurology, Singapore. Test for LRRK2 mutations in patients with Parkinson’s disease. Pract Neurol 8, 381–385. U. Kumari and E. K. Tan Genetic and clinical studies of LRRK2 FEBS