Haplotype analysis of Lrrk2 R1441H carriers with parkinsonism

Abstract

The Roc domain of the Lrrk2 protein harbors two pathogenic mutations which cause autosomal dominant parkinsonism (R1441C and R1441G). A third putatively pathogenic variant (R1441H) has been identified in four probands of diverse ethnicity with parkinsonism. Herein we show that the R1441H substitutions lie on different haplotypes within our patients, confirming this codon as a mutational hotspot. The absence of this variant in control subjects and the presence of two other pathogenic variants at this amino acid position collectively support the contention that R1441H is a pathogenic substitution.

Introduction

Leucine-rich repeat kinase 2 (LRRK2) variants cause familial and sporadic parkinsonism. Pathogenic substitutions are distributed across different domains of the Lrrk2 protein; Ras of complex proteins (Roc; R1441G/C), C-terminal of Roc (COR; Y1699C) and mitogen-activated protein kinase kinase kinase (MAPKKK G2019S & I2020T), and recently, substitutions in the COR (R1628P) and WD40 domain (G2385R) have been associated with disease-risk in ethnic Chinese patients [1–3].

The Roc domain contains two confirmed and one putative pathogenic variant at a single codon, arginine (R)1441. The original descriptions of Lrrk2 substitutions in parkinsonism described R1441C (LRRK2 4321C>T) and R1441G (LRRK2 4321C>G), and subsequent studies identified R1441H (LRRK2 4322G>A) [4, 5]. Although, the R1441G substitution appears to be geographically restricted to Northern Spain with evidence of a common founder, R1441C appears to be worldwide and have occurred as at least three independent mutational events [6, 7].

Four families with a history of parkinsonism have been identified to harbor R1441H [4, 5, 8, 9], and given its amino acid position and absence in over 3500 controls screened in studies to date, it is most likely the cause of their disease. However, the families are not large enough to demonstrate definitive co-segregation with disease. The four families are of diverse ethnicity (Portuguese, Greek, US and Taiwanese) and do not share a known ancestral lineage. This study set out to infer haplotypic structure for the chromosomal 12q12 region flanking LRRK2 in R1441H carriers to establish if there is any evidence of a founder-effect.

Subject and Methods

Parkinsonism was diagnosed by movement disorder neurologists, according to published criteria and family members of the index case were examined if available [10]. The institutional review boards at each institution approved the study and each participant provided signed informed consent. Genomic DNA was extracted from peripheral blood using standard protocols. Direct sequencing of exon 31 was used to verify LRRK2 4322G>A (R1441H) mutation carrier status. For the Taiwanese and US Lrrk2 R1441H carriers all 51 exons of the LRRK2 gene were sequenced with no other putative pathogenic variants observed. Adjacent genetic markers (14 single nucleotide polymorphisms (SNPs) and eight microsatellites) spanning ~6 Mb across the LRRK2 locus were selected to infer haplotype structure in families harboring the Lrrk2 R1441H substitution as previously described [7]. All PCR primers and conditions are available on request.

Results and Discussion

The inferred haplotype data suggest that the R1441H substitution has arisen on multiple independent occasions (Table). The haplotypes of the R1441H carriers of European descent show diversity which may indicate a number of independent founders (Table). Even though it appears the R1441H carriers do not have a single common founder the clinical presentation of affected carriers appears to be similar to typical Parkinson’s disease with an age at onset range of 32–64 years (median 54.5 years). All initially display levodopa responsive parkinsonism, however disease in one of the siblings from the Greek R1441H family appeared to transition into a progressive supranuclear palsy-like disorder [9]. These observations are reminiscent of one patient from a family with the Lrrk2 R1441C mutation (Family D), who displayed predominant tau rather than a-synuclein pathology, on post-mortem examination [7]. Neuropathologic studies in Lrrk2 R1441H carriers may provide further insight.

Table.

Chromosome 12q12 haplotype analysis of Lrrk2 R1441H parkinsonism patients

LRRK2 4322 G>A (R1441H) mutation is highlighted in light grey.

LRRK2	NCBI 03/06	Genetic Marker*	Portuguese	Greek	US	Taiwanese
	33,305,718	D12S2080	184	184	192	192
	38,738,008	D12S2194	261	249	253	249
	38,873,924	D12S2514	291/294	291	294	294
ex5	38,918,058	rs10878245	T	C	T	T
int5	38,918,366	rs10878246	T	T/G	T	T
ex30	38,989,178	rs7133914	G	G	A	G
ex30	38,989,254	rs11175964	G	G	A	G
int30	38,989,339	D12S2516	254	252/254	254	252
int30	38,989,419	rs11175966	A	C	C	C
ex 31 R1441H	38,990,504	rs34995376	A	A	A	A
int33	39,000,026	rs1896252	T/C	T/C	C	T
ex34	39,000,101	rs1427263	C/A	C/A	A	C
ex34	39,000,140	rs11176013	A/G	A/G	G	A
ex34	39,000,168	rs11564148	T	T/A	T	T
int34	39,000,276	rs11564205	A	A	A	A
ex43	39,028,521	rs10878405	G	G/A	G	G
int43	39,028,630	rs11176143	G	A	G	G
int45	39,034,922	D12S2518	154	154/168	154	168
ex49	39,044,919	rs3761863	T/C	T/C	C	T/C
	39,116,885	D12S2519	140	132/140	140	138
	39,120,098	D12S2520	260	254/257	257	248
	39,128,754	D12S2521	319	363	315	327

^*Microsatellite allele sizes were normalized using CEPH-control DNA (1331-01 and 1331-02) and approximate positions are determined from the NCBI March 2006 human genome assembly. Where phase is determined, only the genotype for the R1441H allele is shown. The United States (US) family report ancestry as from the United Kingdom.

The occurrence of Lrrk2 R1441H in four kindreds, the absence in >3500 healthy controls and the pathogenicity of other variants occurring at this amino acid (R1441C and R1441G) collectively support the contention that R1441H is a pathogenic substitution. Longitudinal studies of these families may provide the necessary evidence for R1441H pathogenicity with disease co-segregation, and help elucidate the pathophysiology of Lrrk2 R1441H-associated parkinsonism.