29 Oct 2004

Biochemists, cell and molecular biologists, start your engines! You now have another Parkinson protein challenge. The gene that causes PARK8 Parkinson disease has been cloned by a team of researchers from Spain, England, and the United States. As described in a 22 October article in the early, online Neuron, the protein product "dardarin" may be a kinase, which should whet the appetites of the protein phosphorylation crowd.

Based on factors such as age-of-onset and clinical presentation, researchers have divided familial parkinsonism into distinct, numbered disorders—about 10 to date. These categories have been productively married with modern genetics to identify mutations in four different proteins that lead to inherited forms of PD: PARK1/α-synuclein, PARK2/parkin, PARK6/PINK (see ARF related news story), and PARK7/DJ-1 (see ARF related news story).

PARK8, identified only in 2002 (Funayama et al., 2002) is an especially attractive prize. Unlike many of the other familial parkinsonian conditions, which begin early in life, PARK 8 resembles sporadic Parkinson disease: It strikes late in life, and its clinical phenotype is relatively uniform and similar to the sporadic disease. Although the clinical phenotype of PARK8 is relatively uniform, its pathology may vary widely.

The research team that identified the gene responsible for PARK8 comprised Andy Singleton and his colleagues at National Institute on Aging in Bethesda, Maryland, Jordi Pérez-Tur and colleagues at the València Institute of Biomedicine and other institutions in Spain, and Nick Wood and colleagues at the Institute of Neurology in London and other institutions in England. These researchers had previously identified four families from Spain's Basque region and one from England with PARK8. In the current study, they were able to reduce the area of chromosome 12 originally identified by Funayama and colleagues (12p11.2-q13.1) to a short 2.6 Mb region containing only 11 genes. Among these, they discovered two separate missense mutations—one segregating with affected members in the Basque families and another within the English family—in a gene encoding a 2482 a.a. protein the authors chose to name "dardarin," from the Basque word dardara, meaning tremor.

Neither the Basque family variant, R1396G, nor the English, Y1654C, was found in chromosomes of non-PD control populations in North America (1300 subjects) or Spain (160 Basque subjects). Interestingly, however, the R1396G mutation was found in eight percent of 137 Basque PD patients (30 familial, 107 sporadic) unrelated to the four kindreds studied here. Because PARK8 has a late onset, and some potential patients may die before the disease manifests itself, this raises the possibility that more sporadic cases, at least in the Basque population, are in fact R1396G PARK8. Dardarin appears to contain a kinase domain, a RAS domain, a WD40 domain and a leucine-rich repeat. A 9Kb species seems to be predominant, though there may be splice variants. The authors confirmed that dardarin is expressed throughout adult human brain.

Does the presence of the kinase domain mean that dardarin could play a role in the phosphorylation of α-synuclein or tau? If this were true, the authors speculate, "[T]he description of myriad PARK8 pathological phenotypes ranging from pure nigral degeneration to pathologies with varied protein deposition, including synucleinopathy and tauopathy, is intriguing and these data raise the possibility that dardarin may present a link between these key molecules."—Hakon Heimer

Comments

- Thomas Gasser
  University of Tübingen
- Posted: 09 Nov 2004
The identification of genes for Mendelian forms of Parkinson disease (PD) has greatly advanced the understanding of the molecular pathogenesis of this common neurodegenerative disorder. The discovery of mutations in the gene for α-synuclein (SNCA; PARK1) in dominantly inherited PD and of three genes, parkin, DJ1, PINK1 (PARK2, 6, and 7, respectively) causing recessive, early onset parkinsonism have been major milestones in recent years.

A gene causing an autosomal-dominant, late-onset form of parkinsonism, originally mapped in a large Japanese family, PARK8 on chromosome 12q12, (Funayama et al., 2002), is the latest addition to this list, and may prove to be particularly interesting.

Recently, our group, consisting of researchers at the Hertie-Institute for Clinical Brain Research in Tubingen, Germany, the GSF Research Center for Environment and Health in Neuherberg, Germany, and at the Mayo Clinic, Jacksonville, Florida, showed that two large families with autosomal-dominant late-onset parkinsonism (Families A and D) are linked to the PARK8 locus (OMIM# 607060) (Zimprich et al., 2004).

In these kindreds, we identified missense mutations in a novel gene, leucine-rich repeat kinase 2 (LRRK2), which cosegregate with disease (Family A; Y1699C, 5096A>G and Family D; R1441C, 4321C>T). Screening of 44 additional families revealed two further missense and one putative splice site mutation, all in families with typical late-onset PD compatible with a dominant transmission (I1122V; 3364A>G), (I2020T; 6059T>C), and (L1114L; 3342A>G). Affected individuals in one additional family were found to carry the same mutation as Family D (R1441C, 4321C>T).

We determined the coding sequence and exon composition of LRRK2 and found that the gene spans a genomic region of 144 kb, with 51 exons encoding 2,527 amino acids. Our sequence analysis also revealed several intronic, silent, and coding polymorphisms that do not appear to segregate with disease and which are found at an appreciable frequency in controls (>one percent), including R1514Q; 4541G>A, M1646T; 4937T>C, and N2081D; 6241A>G. The LRRK2-sequence, and its variants are deposited in Genbank under accession number AY792511. We have since established that coding changes are relatively frequent, and penetrant, in autosomal dominant, familial, and sporadic, community-based, late-onset Parkinson disease in the U.S. (>1.5% of cases, unpublished data). Our report appears in the November 18 issue of Neuron. In the same issue, Paisan-Ruiz et al. publish two mutations, one in a British family, and one in a group of related Basque families in the same gene, which they named dardarin. Interestingly, the mutation they found in the British family is identical to the one found by us in Family A, while the founder mutation in the Basque families affects the very same nucleotide that is altered in Family D, though it causes a different amino-acid change. It should be noted that the designation of the position of the mutations differs in the two publications, since Paisan-Ruiz et al. have used the in silico prediction of the gene, which is incomplete, rather than the actual cDNA sequence. LRRK2 is a novel member of the recently defined ROCO protein family (Bosgraaf and Van Haastert, 2003) as predicted in silico by the presence of two conserved domains, (i) a ROC (Ras in complex proteins) domain that belongs to the Ras/GTPase superfamily and a COR domain (C-terminal of Roc) that is also characteristic for this protein family. Three further conserved domains can be identified: (i) a leucine-rich repeat (LRR), (ii) a tyrosine kinase catalytic domain (TyrKc), and (iii) a WD40 domain. The sequence of this newly identified gene, therefore, suggests multiple functions; it is unclear which domain or domains are related to neurodegeneration. Remarkably, we found that affected individuals with LRRK2 mutations exhibit strikingly variable pathologic changes, representing aspects of several of the major neurodegenerative diseases. The common feature of the neuropathology of six affected individuals from families A and D who have come to autopsy is neuronal loss and gliosis in the substantia nigra, which is the likely pathologic substrate of parkinsonism. In three of the individuals (two from Family A and one from Family D) there was non-specific loss of dopaminergic neurons, with ubiquitin-immunoreactive cytoplasmic and nuclear inclusions. Two individuals (both from Family D) had Lewy body pathology. In one case, Lewy bodies were relatively restricted to brainstem nuclei as in typical PD. In the other case, there was widespread α-synuclein pathology, including Lewy bodies and Lewy neurites, in a pattern typical of diffuse Lewy body disease. In the final individual, there was tau immunoreactive neuronal and glial lesions that morphologically resembled PSP.

Pathogenic mutations in LRRK2 thus appear to be central to etiology of not just one, but several neurodegenerative diseases, including those associated with α-synuclein and tau pathology, and clinical parkinsonism.

References:

Bosgraaf L, Van Haastert PJ. Roc, a Ras/GTPase domain in complex proteins. Biochim Biophys Acta. 2003 Dec 7;1643(1-3):5-10. PubMed.

Funayama M, Hasegawa K, Kowa H, Saito M, Tsuji S, Obata F. A new locus for Parkinson's disease (PARK8) maps to chromosome 12p11.2-q13.1. Ann Neurol. 2002 Mar;51(3):296-301. PubMed.

Zimprich A, Müller-Myhsok B, Farrer M, Leitner P, Sharma M, Hulihan M, Lockhart P, Strongosky A, Kachergus J, Calne DB, Stoessl J, Uitti RJ, Pfeiffer RF, Trenkwalder C, Homann N, Ott E, Wenzel K, Asmus F, Hardy J, Wszolek Z, Gasser T. The PARK8 locus in autosomal dominant parkinsonism: confirmation of linkage and further delineation of the disease-containing interval. Am J Hum Genet. 2004 Jan;74(1):11-9. PubMed.

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PARK8 is Cloned: Introducing…"Dardarin"

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