Patent Application: US-201113172412-A

Abstract:
a polynucleotide consisting of the base sequence of seq id no : 2 , or a complementary strand thereto , wherein the x is one of the group being defined by the bases a , c or t . a primer and a probe specific for that polynucleotide , wherein the primer and / or probe contains at least 10 consecutive nucleotides , and finally use of the probe for proving parkinsonism inheritance .

Description:
the inventors identified seven unrelated persons all having the new mutation , from 248 multiplex kindreds with dominantly inherited pd , and six further unrelated persons from three population - based series of persons with dominantly inherited pd . these 13 persons and their families made basis for the inventors &# 39 ; further work . segregation and linkage analysis provides evidence for pathogenicity and an estimate of age - associated penetrance ; haplotype analysis demonstrates the mutation originates from a common and ancient founder . the patients and controls were examined by neurologists specialized in movement disorders . a full history , including family history and neurological examination , was completed on each patient . clinical diagnosis of pd required the presence of at least two of three cardinal signs ( resting tremor , bradykinesia and rigidity ), improvement from adequate dopaminergic therapy and the absence of atypical features or other causes of parkinsonism . blood samples were taken and genomic dna was extracted using standard techniques . six families ( families 194 , 281 , 3081 , 3082 , 3083 and 3211 ) were known to have a positive lod - score for str ( short tandem repeat ) markers in the park8 locus ( zimprich et al . 2004b ). amplification of all 51 exons of the lrrk2 gene was performed by polymerase chain reaction ( pcr ) in one patient having pd , from each of these six families . all pcrs were carried out for each primer set with 20 - 50 ng of template dna in a total volume of 25 μl using a final reaction concentration of 200 μm dntp , 1 × pcr - buffer ( qiagen ), 1 × q - solution ( qiagen ), and 0 . 8 μm of each primer . one unit of taq polymerase ( qiagen ) was added to each reaction . amplification was performed using a 57 - 52 c .°- touchdown protocol over 38 cycles . the primers used for pcr amplification of lrrk2 exons and for sequencing are available on request . the nucleotide sequences of all pcr products were determined by direct sequencing . each pcr product was cleaned by using a millipore pcr purification plate . three microliters of purified pcr product was used per sequencing reaction with 1 μl of either the forward or reverse pcr primer and 1 μl of bigdye reaction mix ( applied biosystems ). electrophoresis was performed under standard conditions on an abi 3730 automated sequencer ( applied biosystems ). all sequences were obtained with both forward and reverse primers . sequences were analyzed with seqscape software version 2 . 1 . 1 ( applied biosystems ) and compared with published sequence of lrrk2 ( genbank accession no . ay792511 ). after identification of a heterozygous g2019s ( g6055a ) mutation in the proband of family 3215 ( referred to as family 3211 in zimprich et al , 2004b ), we designed a probe employing taqman chemistry on an abi7900 ( applied biosystems ) to screen for this mutation . first we examined 248 pd patients from families with a known family history , consistent with autosomal dominant transmission of a suspected causative gene . then 377 norwegian , 271 irish and 100 polish pd patients ( constituting the three population series ) were checked using this assay ; and 2260 samples of healthy persons from similar populations were also included ( 1200 us american , 550 norwegian , 330 irish and 180 polish subjects ), the latter to be used as control samples . mutations were confirmed by direct sequencing of pcr products from lrrk2 exon 41 . finally , all participating family members of lrrk2 g2019 mutation carriers ( affected and unaffected ) were screened for the mutation . by 6055 g & gt ; a or g6055a it is meant that nucleotide number 6055 of the lrrk2 gene , counted from the 5 ′ end of the polynucleotide , has changed from g ( guanine ) to a ( adenine ). this change also causes a change in the polypeptide encoded by the polynucleotide , and g2019s denotes a polynucleotide where amino acid number 2019 is changed from g ( glycine ) to s ( serine ). these shortenings are well known to persons skilled of the art . fourteen str markers were genotyped in mutation carriers and all available family members , in all 13 families , for linkage analyses and to determine whether there was a particular haplotype associated with the lrrk2 mutation . str markers were chosen to span the park8 region including d12s87 , d12s1648 , d12s2080 , d12s2194 , d12s1048 , d12s1301 and d12s1701 . lrrk2 is located between d12s2194 and d12s1048 . we also developed seven novel str markers in this region ( shown in table 1 below ) by searching for repeat polymorphisms using repeatmasker of in silico bac sequence ( ucsc human genome browser web site ). the labeling of these novel markers reflects their physical position relative to the start codon of lrrk2 . one primer of each pair was labeled with a fluorescent tag . pcr reactions were carried out on 10 - 20 ng of dna in a total volume of 15 μl with final reaction concentrations of 150 μm dntp , 1 × pcr - buffer ( qiagen ), 1 × q - solution ( qiagen ) and 0 . 6 μm of each primer , with 1 unit of taq polymerase ( qiagen ). amplification was performed using a 57 - 52 ° c .- touchdown protocol over 38 cycles . the pcr product for each marker was diluted by a factor of 10 to 100 with water . one microliter was then added to 10 μl of hi - di formamide and rox size standard . all samples were run on an abi 3100 genetic analyzer , and results were analyzed using genescan 3 . 7 and genotyper 3 . 7 software ( applied biosystems ). since population allele frequencies were not available from the ceph database , these have been estimated by genotyping 95 unrelated caucasian subjects , a population based series from the united states ( shown in table 2 below ). multipoint nonparametric lod scores for all families were calculated using genehunter - plus ( kong and cox 1997 ). the frequency of the deleterious allele was set at 0 . 0001 , and empirically determined allele frequencies were employed . the map positions for each marker were taken from rutgers combined linkage - physical map version 1 . 0 ( map - o - mat web site ). the three loci d12s2080 , d12s2194 and d12s1301 are very tightly linked , with no observed recombinants in the database or within our genotyped families , and thus inter - marker distances were assigned as 0 . 01 cm . chromosome 12 haplotypes in the park8 region were established for those families in which chromosome phase for mutation - carrying individuals could be deduced , thereby determining which alleles co - segregated with the lrrk2 g2019s mutation in each family . for those affected individuals in whom the associated allele for a marker could not be determined , both alleles are given . the age - dependent penetrance was estimated as the probability of a gene carrier becoming affected , at a given age , within the 13 families . the number of affected mutation carriers , for each decade , was divided by the total number of affected individuals , plus the number of unaffected carriers within that range . for some affected family members no dna was available and only historical data on the disease course was obtained . these individuals were excluded from penetrance calculations . as mentioned previously , we identified 13 affected probands ( i . e . 13 patients ) who carry a heterozygous g6055a mutation in exon 41 of the lrrk2 gene . the mutation leads to a g2019s amino acid substitution of a highly conserved residue within the predicted activation loop of the mapkkk ( mitogen - activated protein kinase kinase kinase ) domain ( fig1 ). after genotyping a total of 42 additional family members , 22 additional subjects were found to carry the mutation , seven with a diagnosis of pd ( shown in table 3 below ). one affected member of family p - 089 did not carry the mutation and , for the purposes of this study , was considered a phenocopy and excluded from further analyses . seven families originated from norway , three were from the united states , two from ireland , and one was from poland . one family from the united states descended from russian / rumania , and another from italy . for only one family ( family 111 ), the ethnic origin was unknown . the lrrk2 g2019s mutation segregates with disease in all kindreds , consistent with autosomal dominant transmission . to ensure patient confidentiality , simplified versions of the family pedigrees are presented in fig2 . there was no evidence of the mutation in the 2260 control samples . age at onset of clinical symptoms was quite variable , even within the same family . family 1120 , a family from the united states , had both the earliest and latest age at onset for a patient . the youngest affected subject had an onset at 39 years , whereas the oldest carrier presented with initial symptoms at 78 years . where recorded , most lrrk2 g2019s carriers have late - onset disease (& gt ; 50 years at onset ). the mean age at onset of affected mutation carriers was 56 . 8 years ( range 39 - 78 years , n = 19 ). unaffected carriers have a mean age of 53 . 9 years ( range 26 - 74 years , n = 14 ). the penetrance of the mutation was found to be highly age - dependent , increasing from 17 % at the age of 50 to 85 % at the age of 70 ( fig4 ). evidence for linkage ( the statistical burden of proof that this mutation causes disease ) to the park8 locus was found across families , with a combined maximum multipoint lod score of 2 . 41 [ for all 14 markers ], corresponding to a p value of 4 . 3 . × 10 − 4 as only a defined chromosomal region was investigated , rather than a genome - wide search , this lod score exceeds that required for significance , p = 0 . 01 ( lander and kruglyak 1995 ). a positive lod score was found in all families where more then one affected subject was genotyped ( table 3 ). all affected members from the different families , except the individual in family p - 089 who did not carry the mutation , appear to share a common haplotype on chromosome 12 the lrrk2 gene locus ( fig3 ). haplotypes can be established with certainty in nine of the families , and all mutation carriers in these families share alleles for four str markers and 4 single nucleotide polymorphisms ( snps ) in the lrrk2 gene locus . these markers are lrrk2 d12s2516 , d12s2518 , d12s2519 , d12s2520 and snps rs7966550 , rs1427263 , rs11176013 , rs11564148 . for the remaining families , the number of available samples from relatives was not sufficient to determine phase . however , the genotypes in these cases are consistent with a common lrrk2 g2019s allele . d12s2516 is located in intron 29 and d12s2518 is located in intron 44 of the lrrk2 gene , whereas the two other shared markers are positioned 3 ′ of the gene . using the physical position of the shared and non - shared markers , the size of the shared haplotype is between 145 kb and 154 kb . we have identified a novel lrrk2 mutation , g2019s , which co - segregates with autosomal dominant parkinsonism in 13 kindreds originating from several european populations . positive lod scores were obtained in multiplex families , and combined they provide significant support for the park8 locus . lrrk2 g2019s mutation was absent in a large number of control subjects , and of similar ethnicity . the number of families linked to lrrk2 in this and previous studies now explains the majority of genetically defined autosomal dominant parkinsonism . the mean age at onset of affected lrrk2 g2019s carriers was 56 . 8 years , and comparable to that of patients in other families linked to park8 ( funayama et al . 2002 ; paisan - ruiz et al . 2004 ; zimprich et al . 2004a ). the majority of patients present with late - onset disease , indistinguishable from typical idiopathic pd . disease penetrance is age - dependent , and increases in a linear fashion from 17 % at the age of 50 to 85 % at the age of 70 . age is the single most consistent risk factor for development of pd and other neurodegenerative disorders ( lang and lozano 1998 ), and an important risk factor in lrrk2 associated parkinsonism . interestingly , age at onset was variable in this study , both within and between different families , suggesting other susceptibility factors , environmental or genetic , may influence the phenotype . although our findings clearly indicate that lrrk2 mutations account for a substantial proportion of familial late - onset parkinsonism , historically , cross - sectional twin studies have not supported a genetic etiology for late - onset pd ( tanner et al . 1999 ; wirdefeldt et al . 2004 ). the age - associated penetrance of lrrk2 mutations provides some explanation as even large and well designed twin studies are underpowered to detect incompletely penetrant mutations ( simon et al . 2002 ). lrrk2 mutations were also found in apparently sporadic pd patients ; three of the patients in this study did not have any known affected first - or second - degree relatives . however , a caveat of age - dependent penetrance is that carriers may die of other diseases , before manifesting or being diagnosed with pd . thus , it seems difficult to separate sporadic and familial pd , or to hypothesize environmental causes to be more important in one group and genetic causes more prominent in the other . in light of these results , a family history of parkinsonism , previously considered an exclusion criterion for a diagnosis of pd , must be reconsidered ( hughes et al . 1992 ). lrrk2 is a member of the recently defined roco protein family ( bosgraaf and van haastert 2003 ). in human , mouse and rat , members of the roco protein family have five conserved domains ( fig1 ). the kinase domain belongs to the mapkkk subfamily of kinases . the active sites of all kinases are located in a cleft between an n - terminal and a c - terminal lobe , typically covered by an ‘ activation loop ’, in an inactive conformation . the activation loop must undergo crucial structural changes to allow access to peptide substrates and to orientate key catalytic amino acids ( huse and kuriyan 2002 ). in different kinases , the activation loop starts and ends with the conserved residues asp - phe - gly ( dfg ) and ala - pro - glu ( ape ), respectively ( dibb et al . 2004 ). of note , the lrrk2 g2019s substitution changes a highly conserved amino acid at the start of this loop ( fig5 ). in a german family we previously described , an 12020t mutation is located in an adjacent codon ( zimprich et al . 2004a ). in other kinases , oncogenic mutations in residues within the activation loop of the kinase domain have an activating effect ( davies et al . 2002 ), thus we postulate lrrk2 g2019s and 12020t mutations may have an effect on its kinase activity . the age of an allele may be estimated from the genetic variation among different copies ( intra - allelic variation ), or from its frequency ( slatkin and rannala 2000 ). however , the local recombination rate on chromosome 12q12 is unknown , as is the frequency of the g2019s mutation in the general population . nevertheless , at centromeres there is generally a dearth in recombination ; indeed no crossovers have been observed between lrrk2 flanking markers d12s2194 and d12s1048 in our studies , or within ceph families ( map - o - mat web site ). the physical size of the shared haplotype is also small , between 145 kb and 154 kb , and the allele is widespread in families from several european populations . hence , the mutation is likely to be ancient and may be relatively common in specific populations . these data suggest a substantial proportion of late - onset pd will have a genetic basis . the physical position of markers is from ncbi build 34 . accession numbers and urls for data presented herein are as follows : online mendelian inheritance in man ( omim ), world wide web at ncbi . nlm . nih . gov / omim / map - 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