Founder mutation in Parkinson's disease presented on 12/10/08
- 1. Founder mutation p.R1441C in the leucine-rich repeat kinase 2 gene in Belgian Parkinson’s disease patients Presented by: Pearl Pfiester 12.10.08
- 2. LRRK2 Gene LRRK2 = leucine-rich repeat kinase 2 gene Contains two domains: Roc and Kinase Gene location: 12q12
- 3. Why study LRRK2 Gene? To date, six genes identified contribute to hereditary Parkinson’s Disease (PD) Mutations in LRRK2 reported in 5% familial patients and 1-2% of sporadic mutations globally 22 putative pathogenic mutations identified 2 nd most common pathogenic mutation: p.R1441C Functional assays show increased levels of kinase activity in vitro for LRRK2 mutations
- 4. Objective Identify mutations and prevalence in LRRK2 linked to Parkinson’s disease in Belgian patients with similar phenotypes
- 5. Signal Transduction Ligand binds to receptor G protein dissociates from receptor One G protein subunit activates adenylate cyclase that converts ATP to cAMP cAMP activates PKA (protein kinase A) to phosphorylate proteins
- 6. Subjects 304 Belgian PD patients that were residents of Flanders 18.1% had positive family history Control group: 278 Belgiums with no clinical evidence of PD
- 7. Gene Sequencing PCR Amplified: 10 exons of Roc and Kinase domains of LRRK2 exons 29-31 and 38-44 Amplified strands were purified, sequenced, and analyzed by the ABI3730 DNA Analyzer technology The novel variants were confirmed and tested in the control group by pyrosequencing Using the UCSC Genome Browser, conserved evolutionary sequences can be compared between organisms The effect of mutations at the protein level was predicted using the SIFT program
- 8. Haplotype Analysis 28 LRRK2 intragenic and flanking markers were genotyped The markers were analyzed in the control group to determine the frequency of the haplotype The microsatellite markers were fluorescently labeled by PCR Amplification and then analyzed
- 9. Results The amplified exons in LRRK2 found 10 heterozygous carriers in the Belgian PD patients All had positive family history of PD Frequency for heterozygous mutations in LRRK2 is 3.29% Only 6 patients out of the 10 carriers had the same mutation of p.R1441C so the frequency is 1.97% The other 4 patients had novel missense mutations The authors confirmed frequencies of five known SNPs from another European study
- 10. Results Reported and novel coding mutations in the LRRK2 Roc and kinase domains. Symbols: red stars = putative pathogenic missense mutations detected in the Flanders-Belgian PD population (6 out of 10 carriers) green stars = novel rare variants (4 out of 10 carriers) yellow stars = known polymorphisms
- 11. Data on protein conservation of all coding mutations in the LRRK2 Roc and kinase domains
- 12. Results for p.R1441C Of the 6 carriers with p.R1441C mutation, 5 had common tremor as initial onset The other carrier had rigidity
- 13. Results 10 Carriers found total 6 had same mutation of p.R1441C 4 had novel missense mutation
- 14. Results for p.R1441C Based on the markers in families DR75, DR99, and DR135, the authors were able to find a disease haplotype Probands in the other 3 families all shared alleles for several markers within the inferred disease haplotype So the haplotype was supported Combining allele and haplotype data, a shared segment of 438 kb between centromeric D12S2194 and telomeric D12S1301 was found This shared haplotype was not present in the control group
- 16. Results: Allele and Haplotype Analysis for p.R1441C
- 17. Results for Novel Mutations
- 18. Results for Novel Mutations
- 19. Conclusion Found 5 mutations in 10 carrier patients: R1441C, Y2189C, R1325Q, K1468E, and R1483Q The estimated frequency for overall LRRK2 mutations is 3.29%. p.R1441C mutation was present on one disease haplotype Supports founder effects involved Comparisons between Italian patients showed independence Particular codon must be a hotspot because three other mutated codons have been reported. Phenotypes: 5 had tremor whereas the other patient experienced rigidity Not all genetic studies reported detailed phenotypes Low penetrance Are the 4 novel missense mutations pathogenic or benign polymorphisms? Need functional assays to understand mutation’s role for disease and to determine more accurate frequencies for benign and pathogenic polymorphisms in LRRK2 In conclusion, 5 missense mutations were found where 4 were novel in important conserved regions. This paper showed that p.R1441C is a frequent cause in the Flanders region of Belgium. Pathogenicity and function of the gene and mutations is yet to be determined.